micromachines

Review Lab-on-a-Chip Systems for Aptamer-Based Biosensing

Niazul I. Khan 1 and Edward Song 1,2,*

1 Department of Electrical and Computer Engineering, University of New Hampshire, Durham, NH 03824, USA; [email protected] 2 Materials Science Program, University of New Hampshire, Durham, NH 03824, USA * Correspondence: [email protected]; Tel.: +1-603-862-5498



Received: 6 January 2020; Accepted: 17 February 2020; Published: 20 February 2020


Abstract: Aptamers are oligonucleotides or peptides that are selected from a pool of random sequences that exhibit high affinity toward a specific biomolecular species of interest. Therefore, they are ideal for use as recognition elements and ligands for binding to the target. In recent years, aptamers have gained a great deal of attention in the field of biosensing as the next-generation target receptors that could potentially replace the functions of antibodies. Consequently, it is increasingly becoming popular to integrate aptamers into a variety of sensing platforms to enhance specificity and selectivity in analyte detection. Simultaneously, as the fields of lab-on-a-chip (LOC) technology, point-of-care (POC) diagnostics, and personal medicine become topics of great interest, integration of such aptamer-based sensors with LOC devices are showing promising results as evidenced by the recent growth of literature in this area. The focus of this review article is to highlight the recent progress in aptamer-based biosensor development with emphasis on the integration between aptamers and the various forms of LOC devices including microfluidic chips and paper-based microfluidics. As aptamers are extremely versatile in terms of their utilization in different detection principles, a broad range of techniques are covered including electrochemical, optical, colorimetric, and gravimetric sensing as well as surface acoustics waves and transistor-based detection.

Keywords: Lab-on-a-chip; point-of-care; portability; miniaturization; microfluidics; soft-lithography

1. Introduction According to the IUPAC (International Union of Pure and Applied Chemistry) definition, a biosensor is “a self-contained integrated device which is capable of providing specific quantitative or semi- quantitative analytical instrumentation using a biological recognition element (biochemical receptor) which is in direct spatial contact with a transducer element [1].” In general, a biosensor can be broadly defined as a device that converts a physical, chemical or biological event into a measurable signal. As can be seen in Figure1, it consists of three main parts: (1) a biosensing element (aptamers, tissue, microorganism, organelles, cell receptors, enzymes, antibodies, proteins, etc.) which is a biologically derived material or biomimetic component that provides selectivity to the target analyte, (2) a transducer (physicochemical, optical, piezoelectric, electrochemical, etc.) that converts the resulting signal from the interaction of the analyte to the biosensing element into a measurable and quantifiable signal (in most cases electrical signal), and (3) the associated electronics or data analysis system which is primarily responsible for signal processing and user-friendly visualization of the sensing results.

Micromachines 2020, 11, 220; doi:10.3390/mi11020220 www.mdpi.com/journal/micromachines Micromachines 2020, 11, 220 2 of 30 Micromachines 2020, 11, 220 2 of 32

Figure 1. Schematic showing the operating principle of a biosensor. The plot gives a general description Figure 1. Schematic showing the operating principle of a biosensor. The plot gives a general of the sensing mechanism. description of the sensing mechanism. Aptamers have gained popularity in recent years as a target recognition element in sensing. First Aptamers have gained popularity in recent years as a target recognition element in sensing. First reported in 1990, aptamers have found applications in the fields of environmental and food safety reported in 1990, aptamers have found applications in the fields of environmental and food safety and analytics as well as therapeutics and diagnostics in the healthcare sector [2–4]. Aptamers are and analytics as well as therapeutics and diagnostics in the healthcare sector [2–4]. Aptamers are typically DNA- or RNA-based oligonucleotide sequences which are designed to bind specifically to the typically DNA- or RNA-based oligonucleotide sequences which are designed to bind specifically to target molecules. Aptamers are commonly selected through a process known as systematic evolution the target molecules. Aptamers are commonly selected through a process known as systematic of ligands by exponential enrichment (SELEX) which is an iterative process to identify high affinity evolution of ligands by exponential enrichment (SELEX) which is an iterative process to identify high aptamer sequences from a large pool (1015–1018) of randomly generated oligonucleotide sequences. affinity aptamer sequences from a large pool (1015–1018) of randomly generated oligonucleotide The first iteration begins with incubation of the target bioanalyte with a library of randomized sequences. The first iteration begins with incubation of the target bioanalyte with a library of oligonucleotide sequences in order to screen high-affinity sequences. The unbound aptamer sequences randomized oligonucleotide sequences in order to screen high-affinity sequences. The unbound are washed away, and the bound aptamers are collected and amplified through polymerase chain aptamer sequences are washed away, and the bound aptamers are collected and amplified through reaction (PCR) to regenerate the oligonucleotide library for the next iteration of SELEX [5]. polymerase chain reaction (PCR) to regenerate the oligonucleotide library for the next iteration of Various types of aptamer have been developed to recognize target species ranging from small SELEX [5]. ions to large proteins with high affinity and specificity. Furthermore, when compared with other Various types of aptamer have been developed to recognize target species ranging from small biorecognition elements such as antibodies and enzymes, aptamers possess high chemical stability, ions to large proteins with high affinity and specificity. Furthermore, when compared with other mass- producibility and reusability, long shelf-life, low-cost and small size. Because of these advantages, biorecognition elements such as antibodies and enzymes, aptamers possess high chemical stability, aptamers are often used as an integral part of biosensors leading to the creation of the term aptasensors mass-producibility and reusability, long shelf-life, low-cost and small size. Because of these to describe aptamer-based biosensors. There are different classifications of aptasensors depending on advantages, aptamers are often used as an integral part of biosensors leading to the creation of the the type of transduction mechanisms employed such as mass-based (i.e., quartz crystal microbalance term aptasensors to describe aptamer-based biosensors. There are different classifications of (QCM)) [6,7], electrochemical (amperometric, voltammetry, impedimetric) [8–18], optical [19–23] or aptasensors depending on the type of transduction mechanisms employed such as mass-based (i.e., field-effect transistor (FET)-based methods [24–26]. quartz crystal microbalance (QCM)) [6,7], electrochemical (amperometric, voltammetry, The integration of aptasensors with microfluidics offers promising solutions for addressing some impedimetric) [8–18], optical [19–23] or field-effect transistor (FET)-based methods [24–26]. pressing healthcare challenges. Alternatively known as lab-on-a-chip (LOC) technology or miniaturized The integration of aptasensors with microfluidics offers promising solutions for addressing total analysis system (µTAS), microfluidics has versatile advantages to offer in biosensing including some pressing healthcare challenges. Alternatively known as lab-on-a-chip (LOC) technology or reduced sample volume and detection time, improved sensitivity due to high surface to volume ratio, miniaturized total analysis system (µTAS), microfluidics has versatile advantages to offer in high throughput by parallel operation, portability, and disposability. In addition, microfluidics-based biosensing including reduced sample volume and detection time, improved sensitivity due to high biosensors enable real-time detection and an automated measurement process. This article reviews surface to volume ratio, high throughput by parallel operation, portability, and disposability. In recent developments (last 5 years) on LOC systems for aptamer-based biosensing. We refer the readers addition, microfluidics-based biosensors enable real-time detection and an automated measurement to other previously published review articles that cover materials exclusively on either aptamer-based process. This article reviews recent developments (last 5 years) on LOC systems for aptamer-based biosensing [27] or microfluidics-based biosensing [28–34]. Furthermore, although a LOC system biosensing. We refer the readers to other previously published review articles that cover materials typically comprises many analysis components such as sample collection, separation, filtration, mixing, exclusively on either aptamer-based biosensing [27] or microfluidics-based biosensing [28–34]. and detection to name a few, as aptamers are used primarily as receptors for target biomolecules, this Furthermore, although a LOC system typically comprises many analysis components such as sample article will focus on the sensing component of the LOC that utilizes aptamers. collection, separation, filtration, mixing, and detection to name a few, as aptamers are used primarily as2. Microfluidicsreceptors for target versus biomolecules Macrofluidics, this article will focus on the sensing component of the LOC that utilizes aptamers. Microfluidics is the manipulation of fluid in submillimeter length scale. Due to the small dimension 2.of Microfluidics microfluidic channels, versus Macrofluidics fluidic behavior deviates from the macrofluidic behavior. Some interesting and often unintuitive properties may appear on this minute scale. For example, in microscale, diffusive Microfluidics is the manipulation of fluid in submillimeter length scale. Due to the small dimension of microfluidic channels, fluidic behavior deviates from the macrofluidic behavior. Some

Micromachines 2020, 11, 220 3 of 30 mass transport dominates over convective mass transport. This is indicated by the Sherwood number Sh which represents the ratio of the convective mass transfer to the diffusive mass transfer of the system kd and is defined as [35]: Sh = D , where, k is the mass transport coefficient, d is the characteristic diameter of the channel and D is the diffusion coefficient. For macroscale systems, Sh is large which indicates that convective transport is dominant over diffusive transport. By contrast, in the microfluidic system Sh is much smaller due to the small channel geometry d, and consequently diffusive transport dominates the convective mass transport. This diffusive effect can be used in the passive mixing of two or more fluids as well as in the separation of particles based on the size in the microfluidic channel [36]. Laminar flow is another characteristic property of microfluidic systems. This is indicated by the ρvd Reynolds number (Re), which is defined as [37]: Re = η , where ρ is the mass density of the fluid, υ is the fluid velocity, and η is the dynamic viscosity of the fluid. Due to small geometric dimensions (small d) of microfluidic systems, the Reynolds number Re can be as low as 1, which means the flow is dominated by viscous forces, and the flow is considered laminar. A consequence of this flow type is that two or more layers of fluid can flow side-by-side without any mixing other than by diffusive transport of their constituent molecular and particulate components [36]. Another significant property that distinguishes microscale systems from macroscale systems is the Bond number (Bo). It is the ratio of the body forces (e.g. gravity) to the surface tension forces, ∆ραd2 and is defined as [35]: Bo = λ , where ∆ρ is the density difference of the two phases across the interface, α is the acceleration associated with the body force, and λ is the surface tension between the two fluid phases. For microscale systems, small d results in a very low Bond number, which indicates the dominance of surface tension forces over body forces.

3. Different Types of Microfluidic Aptasensors

3.1. Microfluidic Aptasensors Based on Electrochemical Detection Electrochemical biosensors provide a convenient tool for quantifying the analyte due to its direct conversion of a chemical reaction into an electrical signal. There are several classifications of electrochemical sensing such as Faradaic current-based sensing (amperometric/voltammetric), potential or charge accumulation-based sensing (potentiometric), or electrical conductivity-based sensing (conductometric). Electrochemical impedance spectroscopy (EIS), or impedimetric sensing, is also a commonly used technique where a biological or chemical event causes a change in the impedance (both resistance and reactance) at the liquid–electrode interface [38].

3.1.1. Amperometric Detection Amperometric detection is the first electrochemical technique adapted in microscale [30]. Amperometric biosensors are self-contained electrochemical devices that transduce the biological recognition events caused by the oxidation or reduction of an electroactive species into a current signal for the quantification of an analyte. The simple device architecture of this transduction approach makes it useful in low-cost portable devices for various applications including disease diagnosis and environmental monitoring [39]. He et al. used the amperometric detection technique to develop an aptamer-based label-free biosensor for the selective detection of vasopressin on a microfluidic platform to build a portable point-of-care (POC) diagnostic device [40]. As shown in Figure2, carbon nanotubes (CNTs) were deposited between two lithographically patterned gold electrodes with a gap of 10 µm on a silicon wafer. The deposited CNTswere then modified with amine-terminated aptamer using carbodiimide and N-hydroxysuccinimide (EDC/NHS) surface chemistry. Afterwards, the aptamer modified device was integrated with a polydimethylsiloxane (PDMS)-based microfluidic channel for the continuous flow of vasopressin-containing fluid. The detection was performed by measuring the current before and after vasopressin binding. The specific binding between the aptamers and vasopressin depletes electron carriers on the CNT surface and reduce the local charge distribution resulting in a decrease in current magnitude. Using this technique, a limit of detection of 43 pM was achieved which is in the same order as the physiological level of vasopressin in Micromachines 2020, 11, 220 4 of 32 Micromachines 2020, 11, 220 4 of 30 reduce the local charge distribution resulting in a decrease in current magnitude. Using this technique, a limit of detection of 43 pM was achieved which is in the same order as the physiological the bloodstream. This miniaturized portable module has the potential to be a used as a point-of-care level of vasopressin in the bloodstream. This miniaturized portable module has the potential to be a diagnostics tool for patients with excessive bleeding. used as a point-of-care diagnostics tool for patients with excessive bleeding.

Figure 2. Aptamer-based amperometric microfluidic biosensor: (A) schematic of fabrication process of Figure 2. Aptamer-based amperometric microfluidic biosensor: (A) schematic of fabrication process aptamer-based microfluidic biosensors; (B) a photo of the integrated microfluidic device; and (C) chemical of aptamer-based microfluidic biosensors; (B) a photo of the integrated microfluidic device; and (C) structures of aptamer (left) and vasopressin (right). Reprinted from [40], Copyright 2013, with permission chemical structures of aptamer (left) and vasopressin (right). Reprinted from [40], Copyright 2013, from Elsevier. with permission from Elsevier. 3.1.2. Voltammetric Detection 3.1.2. Voltammetric Detection Voltammetric sensing is a technique where the electrical potential at the working electrode is Voltammetricscanned from sensing one pre-set is a valuetechnique to another where and the the electrical electrochemical potential current at the working is recorded electrode as a function is of scannedthe from applied one potential pre-set value [41]. Unliketo another amperometric and the electrochemical technique, this current technique is recorded monitors as the a function redox activity of theacross applied a range potential of applied [41]. potentialsUnlike amperometric manifesting well-definedtechnique, this current technique peaks [32monitors]. Voltammetry the redox isone of activitythe across oldest electrochemicala range of applied techniques potentials and has revolutionizedmanifesting well-defined the field of analytical current chemistry. peaks [32]. The main Voltammetryadvantages is one of voltammetricof the oldest sensorselectrochemical are their hightechniques sensitivity, and reproducibility,has revolutionized easy the implementation field of analyticaland lowchemistry. cost. Implementing The main advantages voltammetric of sensors voltammetric was diffi sensorscult in the are past their when high computers sensitivity, were not reproducibility,readily available easy implementation to precisely control and low potential cost. Implementing scans. However, voltammetric in the present, sensors these was techniques difficult are in the increasinglypast when computers becoming were popular not readily due to availa the advancementble to precisely of portablecontrol potential computers scans. and However, their ability to in theeasily present, control these and techniques measure electricalare increasing signalsly [ 42becoming]. popular due to the advancement of portable computersChand and and Neethirajan their ability from to the easily Neethiarajan control and group measure have developed electrical ansignals aptamer-based [42]. voltammetric Chandbiosensor and forNeethirajan the sensitive from detection the Neethiarajan of norovirus group on microfluidic have developed platform an usingaptamer-based graphene-gold voltammetricnanocomposites biosensor which for the off ersensitive the benefits detection of having of norovirus enhanced on electronicmicrofluidic properties, platform high using surface- graphene-goldarea-to-volume nanocomposites ratio, and which biocompatibility offer the benefi [43].ts Built of having on a commercially enhanced electronic available properties, screen-printed high surface-area-to-volumecarbon electrode (SPCE) ratio, for their and cost-e biocompatibilityffectiveness and[43]. disposability, Built on a thecommercially PDMS-based available microfluidic screen-printeddevice has carbon two inlets, electrode a microbeads-based (SPCE) for thei filtrationr cost-effectiveness zone, a sensing and zone,disposability, and an outlet the PDMS- as shown in based Figuremicrofluidic3A. Selective device detectionhas two inlets, of norovirus a microbeads-based was accomplished filtration by immobilizingzone, a sensing the zone, ferrocene-tagged and an

Micromachines 20202020,, 1111,, 220220 55 of of 3032

outlet as shown in Figure 3A. Selective detection of norovirus was accomplished by immobilizing the viralferrocene-tagged capsid-specific viral aptamer capsid-specific onto the surface-modifiedaptamer onto the SPCE surface-modified (Figure3B). The SPCE interaction (Figure between3B). The theinteraction aptamers between and norovirus the aptamers results and in norovirus a decrease results in the in voltammetric a decrease in signalthe voltammetric from ferrocene. signal Using from diferrocene.fferential Using pulse differential voltammetry pulse (DPV), voltammetry this microfluidic-integrated (DPV), this microfluidic-integrated aptasensor was aptasensor able to detect was norovirusable to detect with norovirus a detection with limit a detection of as low aslimit 100 of pM as andlow aas detection 100 pM rangeand a fromdetection 100 pM range to 3.5 from nM. 100 pM to 3.5 nM.

Figure 3.3. MicrofluidicMicrofluidic voltammetric voltammetric aptamer-based aptamer-based detection detection of norovirus: of norovirus: Schematic Schematic of (A )of the (A PDMS) the microfluidicPDMS microfluidic chip; and chip; (B) theand processing (B) the stepsprocessing to fabricate steps theto biosensor.fabricate the Grp-AuNPs: biosensor. graphene-gold Grp-AuNPs: nanoparticlesgraphene-gold composite, nanoparticles Strp-SH: composite, thiolated Strp-SH: streptavidin, thiolated Bt-Atp-Fc: streptavidin, biotin Bt-Atp-Fc: and ferrocene biotin tagged and aptamer.ferrocene Reprintedtagged aptamer. from [43 Reprinted], Copyright from 2017, [43], with Copyright permission 2017, from with Elsevier.permission from Elsevier.

Another voltammetric aptasensor with a linear detection range from 30 pg/mL pg/mL to 10 µµg/mLg/mL was recently developeddeveloped by by Sanghavi Sanghavi et et al. al. for for the the detection detection of cortisol of cortisol in biological in biological media media (serum (serum and saliva) and onsaliva) microfluidics on microfluidics platform platform for point-of-care for point-of-car (POC)e applications (POC) applications [44]. The [44]. working The principleworking principle involves theinvolves displacement the displacement of triamcinolone of triamcinolone (a drug used (a drug for skin used treatment) for skin treatment) that were initiallythat were bound initially to cortisol bound aptamersto cortisol immobilized aptamers immobilized on gold nanoparticles. on gold Thenanoparticles. displaced triamcinoloneThe displaced was triamcinolone electrochemically was reducedelectrochemically at the graphene-coated reduced at the glassy graphene-coated carbon electrode glassy which carbon produced electrode a current which that produced was proportional a current tothat the was concentration proportional of to cortisol. the concentration This sensing of cortisol. approach This only sensing requires approach sample only volumes requires below sample one microlitervolumes below and hasone themicroliter ability toand resist has the interferences ability to resist from interferences other glucocorticoids from other in glucocorticoids the sample, and in thereforethe sample, holds and promise therefore for holds real-time promise sensing for applications.real-time sensing Moreover, applications. this method Moreover, does notthis require method a labeling,does not immobilizationrequire a labeling, or theimmobilization intermediate or washing the intermediate steps. washing steps. Electrode fouling [[45]45] often becomesbecomes a serious issue for electrochemicalelectrochemical detection. To solve this problem,problem, MatharuMatharu et al.et haveal. have developed developed a reconfigurable a reconfigurable microfluidics microfluidics platform thatplatform uses aptasensors that uses toaptasensors electrochemically to electrochemically monitor the monitor dynamics the of dyna the transformingmics of the transformi growth factorng growth TGF-β factor1 released TGF- byβ1 activatedreleased by hepatic activated stellate hepatic cells stellate over 20 cells hours over [46 ].20 This hours three-layer [46]. This microfluidic three-layer microfluidic device comprises device a microfabricatedcomprises a microfabricated gold electrode gold on el glassectrode slide, on glass a microfluidic slide, a microflu channelidic with channel microcups with microcups and a PDMS and controla PDMS layer control (Figure layer4 ).(Figure When 4). the When microcups the microcups were lowered were bylowered pneumatically by pneumatically actuating actuating the control the layer,control the layer, electrodes the electrodes were protected were protected during the during protein the adsorption protein adsorption and cell seedingand cell steps.seeding This steps. reduced This thereduced non-specific the non-specific adsorption adsorption of highly adhesiveof highly stellate adhesive cells, stellate thus preventing cells, thus electrode preventing fouling. electrode This workfouling. reported This work a 15-fold reported improvement a 15-fold in improvem the sensitivityent in compared the sensitivity to traditional compared approached to traditional with unprotectedapproached with electrodes. unprotected After seedingelectrodes. and After activation seeding of the and stellate activation cells, of square the stellate wave voltammetrycells, square (SWV)wave voltammetry was applied (SWV) to measure was applied the electron to measure transfer the which electron resulted transfer in a detectionwhich resulted limit ofin 1a ngdetection/mL. limit of 1 ng/mL.

Micromachines 2020, 11, 220 6 of 30 Micromachines 2020, 11, 220 6 of 32 Micromachines 2020, 11, 220 6 of 32

FigureFigure 4.4.Aptamer-based Aptamer-based monitoring monitoring of of the the TGF- TGF-β1 releaseβ1 release from from hepatic hepatic stellate stellate cells on cells reconfigurable on Figure 4. Aptamer-based monitoring of the TGF-β1 release from hepatic stellate cells on microfluidicreconfigurable platform: microfluidic (A) the platform: three layers (A) of the the three microfluidic layers of device. the micr Bottom:ofluidic glass device. slide Bottom: with micropatterned glass reconfigurable microfluidic platform: (A) the three layers of the microfluidic device. Bottom: glass Auslide electrodes. with micropatterned Middle: working Au electrodes. PDMS layer Middle: with fluidicworking microchannels PDMS layer andwith microcups. fluidic microchannels Top: pressurizable slide with micropatterned Au electrodes. Middle: working PDMS layer with fluidic microchannels controland microcups. PDMS layer Top: for pressurizable controlling thecontrol microcups. PDMS layer (B) Diagramfor controlling showing the themicrocups. actuation (B) of Diagram the microcups . and microcups. Top: pressurizable control PDMS layer for controlling the microcups. (B) Diagram Theshowing plotgives the actuation a general of description the microcups. of the The sensing plot mechanism.gives a general Reprinted description with permissionof the sensing from [46]. showing the actuation of the microcups. The plot gives a general description of the sensing Copyrightmechanism. 2016 Reprinted American with Chemicalpermission Society. from [ 46].https: Copyright//pubs.acs.org 2016 American/doi/10.1021 Chemical/ac502383e Society. (Further mechanism. Reprinted with permission from [46]. Copyright 2016 American Chemical Society. https://pubs.acs.org/doi/10.1021/ac502383e (Further permissions related to this material should be permissionshttps://pubs.acs.org/doi/10.1021/ac502383e related to this material should (Further be directed permissions to the ACS.) related to this material should be directed to the ACS.) 3.1.3.directed Impedimetric to the ACS.) Detection 3.1.3. Impedimetric Detection 3.1.3.An Impedimetric impedimetric Detection transduction mechanism is a label-free mechanism that works on the basis of EIS. EISAn can impedimetric analyze both transduction the resistive mechanism and capacitive is a label-free properties mechanism of the electrode that works surface on the upon basis excitation of EIS. AnEIS impedimetriccan analyze bothtransduction the resistive mechanism and capaci is a lativebel-free properties mechanism of the that electrode works onsurface the basis upon of and perturbation of the system at equilibrium with a small amplitude sinusoidal excitation signal [47]. EIS.excitation EIS can and analyze perturbation both ofthe the resistive system atand equilib capaciriumtive with properties a small amplitudeof the electrode sinusoidal surface excitation upon The resulting spectrum (Figure5A), known as the Nyquist plot, can be modeled by the Randles circuit excitationsignal [47]. and The perturbation resulting spectrum of the system (Figure at 5A),equilib knownrium aswith the a Nyquistsmall amplitude plot, can sinusoidal be modeled excitation by the assignalRandles seen [47]. in Figurecircuit The resulting5asB. Thisseen Randlesspectrumin Figure circuit (Figure 5B. isThis 5A), a valuable Randlesknown technique ascircuit the Nyquist is fora thevaluable plot, characterization, can techniquebe modeled for analysis by the the and studyRandlescharacterization, of coatings,circuit asanalysis batteries, seen andin fuel Figurestudy cells of5B. and coatings, This corrosion Randlesbatteries, phenomena circuitfuel cells is [47 anda]. valuable Thiscorrosion circuit technique phenomena consists for of [47]. athe solution characterization, analysis and study of coatings, batteries, fuel cells and corrosion phenomena [47]. resistanceThis circuit (R consistsS), a double-layer of a solution capacitance resistance (R (CS),dl a), double-layer a charge transfer capacitance resistance (Cdl), (aR chargect), and transfer the Warburg This circuit consists of a solution resistance (RS), a double-layer capacitance (Cdl), a charge transfer impedanceresistance ( (RZctW), ).and the Warburg impedance (ZW). resistance (Rct), and the Warburg impedance (ZW).

Figure 5. The Nyquist plot (A) and the corresponding Randles circuit (B). Figure 5. The Nyquist plot (A) and the corresponding Randles circuit (B). Figure 5. The Nyquist plot (A) and the corresponding Randles circuit (B). The resistor RS is inserted as a series element to reflect that all the current must pass through the The resistor RS is inserted as a series element to reflect that all the current must pass through the bulk Thesolution. resistor Moreover, RS is inserted the parallel as a series elements element ar toe introduced reflect that sinceall the there current are must two possiblepass through current the bulk solution. Moreover, the parallel elements are introduced since there are two possible current bulkpaths solution. at the electrode–solution Moreover, the parallel interface: elements one is tharee Faradaicintroduced charge since transfer there areprocess two andpossible the other current is paths at the electrode–solution interface: one is the Faradaic charge transfer process and the other pathsthe charge at the buildup electrode–solution due to the double-layerinterface: one capacitance. is the Faradaic Elements charge C transferdl and R processct are often and used the otheras the is isthedetection the charge charge parameters buildup buildup due in due tobiosensing the to thedouble-layer double-layer as they represcapacitance. capacitance.ent the Elements dielectric Elements C anddl and insulatingC Rdlct areand often Rfeaturesct are used often at as the the used as thedetection detection parameters parameters in biosensing in biosensing as they as theyrepres representent the dielectric the dielectric and insulating and insulating features features at the at the electrode–electrolyte interface, while RS and ZW depend on the bulk properties of the electrolyte and the diffusion of the redox probe, respectively [48].

Micromachines 2020, 11, 220 7 of 30

Micromachines 2020, 11, 220 7 of 32 Shin et al. have integrated an aptamer-based impedimetric biosensor with microfluidic platform electrode–electrolyte interface, while RS and ZW depend on the bulk properties of the electrolyte and to developthe diffusion an organ-on-a-chip of the redox probe, biosensor respectively for [48]. sensitive detection of a cardiac biomarker creatine kinase-muscleShin et/brain al. have (CK-MB) integrated [49 an]. aptamer-based Here, the gold impedimetric working electrode biosensor ofwith a 3-electrodemicrofluidic platform electrochemical cell (Figureto develop6A) an was organ-on-a-chip coated with biosensor a carboxy-terminated for sensitive detection thiol followedof a cardiac by biomarker an immobilization creatine of amine-linkedkinase-muscle/brain aptamers via(CK-MB) carbodiimide [49]. Here, coupling the gold working (Figure electrode6B). The of sensor a 3-electrode was then electrochemical exposed to culture mediacell samples (Figure 6A) containing was coated the with CK-MB a carboxy-terminated biomarker. Thethiol EISfollowed measurements by an immobilization in Figure of 6amine-C show that linked aptamers via carbodiimide coupling (Figure 6B). The sensor was then exposed to culture detection of the biomarkers causes a significant increase in charge transfer resistance (R ) resulting from media samples containing the CK-MB biomarker. The EIS measurements in Figure 6C showct that the reduced charge transfer between the redox probe ([Fe (CN) ]3 /4 ) and the electrode. Impedance detection of the biomarkers causes a significant increase in charge6 transfer− − resistance (Rct) resulting signalfrom (∆R ctthe) for reduced CK-MB charge was found transfer to bebetween linear inthe the redox clinically probe relevant([Fe (CN) concentrations6]3−/4−) and the electrode. from 10 pg /mL to 100 ngImpedance/mL in both signal buff er(ΔR andct) for culture CK-MB media was found samples. to be The linear sensor in the was clinically then integratedrelevant concentrations to a heart-on-a-chip cardiacfrom bioreactor 10 pg/mL as to seen 100ng/mL in Figure in both6D and buffer doxorubicin-induced and culture media samples. cardiac The damage sensor was was evaluated then by monitoringintegrated the to changes a heart-on-a-chip in the creatine cardiac kinase bioreactor concentration. as seen in Figure 6D and doxorubicin-induced cardiac damage was evaluated by monitoring the changes in the creatine kinase concentration.

FigureFigure 6. Aptamer-based 6. Aptamer-based impedimetric impedimetric biosensor on on microfluidics microfluidics platform: platform: (A) photograph (A) photograph of the of the microfabricatedmicrofabricated electrode electrode set; set; (B (B)) schematic schematic diagram diagram showing showing the theimmobilization immobilization and sensing and sensingsteps steps of theof biosensor; the biosensor; (C )(C electrochemical) electrochemical impedance impedance spectroscopy spectroscopy (EIS) measurements (EIS) measurements of the biosensor of the at biosensor at eacheach modification modification andand sensingsensing steps; steps; and and (D ()D the) the cartoon cartoon showing showing the integrated the integrated heart-on-a-chip heart-on-a-chip biosensorbiosensor module. module. Reprinted Reprinted with with permissionpermission from from [49]. [49 Copyright]. Copyright 2016 2016American American Chemical Chemical Society. Society.

NguyenNguyen et al. et haveal. have developed developed anan impedimetric microfluidic-biosensor microfluidic-biosensor that uses that the uses variation the variation in in capacitance of the Randles circuit as the sensing parameter for label-free selective detection of lung capacitance of the Randles circuit as the sensing parameter for label-free selective detection of lung carcinoma cell line (A 549) [50]. As seen in Figure 7, a coplanar two-electrode configuration was used carcinomadue to cell its simplicity line (A 549) of fabrication [50]. As seen and ease in Figure of monitoring7, a coplanar the change two-electrode in material configuration properties as the was used due toelectric its simplicity field is being of fabrication applied between and ease the of electrod monitoringes. The the gold change electrodes in material were functionalized properties aswith the electric field isaptamers being applied and EIS betweenwas performed the electrodes. at frequencies The ranging gold electrodes from 0.1 kHz were to functionalized 1 MHz to monitor with the aptamers and EISoccurrence was performed of binding atevents. frequencies The capacitive ranging response from of 0.1 the kHz impedance to 1 MHz was measured to monitor at various the occurrence cell of bindingconcentrations events. The to capacitive evaluate responsethe sensing of theperforma impedancences of was the measureddevice. A atlimit various of detection cell concentrations of 4 to evaluate the sensing performances of the device. A limit of detection of 1.5 10 cells/mL was × calculated. More recently, they have further upgraded their impedimetric biosensor by introducing gold nanoparticles into their system for enhanced sensitivity [51]. Here, the microfluidic biosensor uses 3-electrode configuration for the selective detection of the same A549 circulating tumor cells. Instead of complex EDC/NHS reaction, thiol-gold interaction was used to immobilize aptamers on the gold Micromachines 2020, 11, 220 8 of 32

1.5 × 10 cells/mL was calculated. More recently, they have further upgraded their impedimetric biosensorMicromachines by 2020introducing, 11, 220 gold nanoparticles into their system for enhanced sensitivity [51]. Here, the 8 of 30 microfluidic biosensor uses 3-electrode configuration for the selective detection of the same A549 circulating tumor cells. Instead of complex EDC/NHS reaction, thiol-gold interaction was used to surface. Both of these biosensors developed by the Jen group can find applications in POC diagnostics immobilize aptamers on the gold surface. Both of these biosensors developed by the Jen group can for early-stage detection of lung cancer. find applications in POC diagnostics for early-stage detection of lung cancer.

Figure 7. Schematic (A) and photograph (B) of the microfluidic biosensor chip. Reprinted with permission Figurefrom [507. ].Schematic (A) and photograph (B) of the microfluidic biosensor chip. Reprinted with permission from [50]. Lum et al. have also developed an impedimetric aptamer-based biosensor on microfluidic chip for specificLum detection et al. have of H5N1also developed avian influenza an impedimetric virus (AIV) aptamer-based on interdigitated biosensor gold electrode on microfluidic [52]. The aptasensorchip foris capable specific ofdetection detecting of AIVH5N1 at avian concentrations influenza asvirus low (AIV) as 0.0128 on interdigitated HAU (hemagglutinin gold electrode units) [52].in 30 The minutes aptasensorthanks to is the capable use of of interdigitated detecting AIV at microelectrode concentrations thatas low off asers 0.0128 the advantagesHAU (hemagglutinin of low ohmic units) drop, in 30 minutes thanks to the use of interdigitated microelectrode that offers the advantages of low increased signal-to-noise ratio, rapid reaction kinetics and hence low detection time. In contrast ohmic drop, increased signal-to-noise ratio, rapid reaction kinetics and hence low detection time. In to commonly used square-shaped microfluidics chamber, an oval-shaped chamber was used here. contrast to commonly used square-shaped microfluidics chamber, an oval-shaped chamber was used An oval-shaped chamber offers the advantage of minimizing the residues, such as any non-specifically here. An oval-shaped chamber offers the advantage of minimizing the residues, such as any non- adsorbed aptamers or H5N1 virus that could potentially remain in the corners of the traditional specifically adsorbed aptamers or H5N1 virus that could potentially remain in the corners of the traditionalsquare-shaped square-shaped chambers. chambers. Thus, an oval-shapedThus, an oval-shaped chamber couldchamber contribute could tocontribute the improvement to the of improvementthe detection of sensitivity. the detection This sensitivity. microfluidics-integrated This microfluidics-integrated aptasensor is aptasensor portable, is low-cost portable, and low- has the costpotential and has to the be usedpotential in rapid, to be in-fieldused in diagnostics.rapid, in-field diagnostics. AnAn interdigitated interdigitated microelectrodemicroelectrode waswas also also employed employed by by Lou Lou et al.et toal. develop to develop a novel a novel logic aptasensorlogic aptasensor(LAS) for intelligent(LAS) for detectionintelligent ofdetection cancer cellsof cancer utilizing cells a utilizing digital multimeter a digital multimeter as a simple as electrochemical a simple electrochemicalread-out[53]. IntegratedonaPDMS-basedmicrofluidicsplatform, read-out [53]. Integrated on a PDMS-based microfluidics thisbiosensorissuitableforasimultaneous platform, this biosensor is suitablemulti-analyte for a simultaneous detection. Without multi-analyte any target detection. cells, the Without two interdigitated any target cells, gold-line the two microarrays interdigitated behave as gold-lineopen circuits. microarrays However, behave in the presenceas open ofcircuits. the target Ho cells,wever, the in resistance the presence across of the the electrodes target cells, reduces the which resistancecan be measured across bythe a electrodes handheld commerciallyreduces which available can be multimeter.measured by The a sensor handheld is mass-scale commercially producible availableand reusable multimeter. enabling The low-cost sensor healthcare is mass-scale monitoring producible in POC and reusable testing and enabling clinical low-cost applications. healthcare monitoring in POC testing and clinical applications. 3.2. Microfluidic Aptasensors Based on Field-Effect Transistors (FETs) 3.2. Microfluidic Aptasensors Based on Field-Effect Transistors (FETs) Field-effect transistors (FETs) have attracted much attention in the biosensing community as they offerField-effect many advantages transistors such (FETs) as miniaturization, have attracted much low-cost, attention large-scale in the integrationbiosensing community capability withas the theyexisting offer manufacturing many advantages process such as as miniaturization, well as label-free, low-cost, rapid detection large-scale and integration highly sensitive capability detection with of theanalytes existing [25 ].manufacturing A typical FET process biosensor as well is composed as label-free, of a semiconductingrapid detection and channel highly that sensitive connects the detectionsource and of analytes the drain [25]. electrodes. A typical AnyFET adsorptionbiosensor is ofcomposed biomolecules of a semiconducting on the channel channel surface that causes a connectschangein the the source electric and field the drain that modulates electrodes. the Any gate adsorption potential, of resultingbiomolecules in a changeon the channel in thedrain surface current causes a change in the electric field that modulates the gate potential, resulting in a change in the within the channel of the FET. Such change in the drain current can be used as a detection mechanism drain current within the channel of the FET. Such change in the drain current can be used as a for the biosensors [54–58]. detection mechanism for the biosensors [54–58]. One-dimensional (1-D) nanomaterials such as silicon nanowires (SiNWs) [59], carbon nanotubes

(CNTs) [60], and polymer nanowires [61] have long been explored as channel materials in FET-based biosensors due to their several advantages such as high switching characteristics (high current on-off ratio) and large specific surface areas. But their practical applications are limited by the major challenges that include device-to-device variations and relatively high cost in large-scale fabrication [58]. On the Micromachines 2020, 11, 220 9 of 32 Micromachines 2020, 11, 220 9 of 30 One-dimensional (1-D) nanomaterials such as silicon nanowires (SiNWs) [59], carbon nanotubes (CNTs) [60], and polymer nanowires [61] have long been explored as channel materials in FET-based other hand, two-dimensional (2-D) nanomaterials such as graphene, MoS , WS , etc. are now becoming biosensors due to their several advantages such as high switching characteristics2 2 (high current on-off attractiveratio) alternatives and large specific to 1-D nanomaterialssurface areas. But as thei a conductionr practical channelapplications for are FET-based limited by biosensors the major due to their plannerchallenges structure, that include excellent device-to-device electrical variations properties, and highrelatively surface high area-to-volume cost in large-scale ratio, fabrication and easier large-scale[58]. On manufacturability. the other hand, two-dimensional Among several (2-D) 2D nanomaterials materials, graphene such as graphene, has been MoS the2, most WS2, widelyetc. are used as a promisingnow becoming FET attractive channel materialalternatives due to to1-D its nano superiormaterials physical, as a conduction chemical, channel electronic for FET-based properties as well asbiosensors its ability due to to be their grown planner in large structure, areas excellent with high electrical fidelity. properties, For example, high surface a graphene area-to-volume field-e ffect transistorratio, (GFET) and easier has large-scale been employed manufacturability. as a biosensor Among for detecting several 2D DNA materials, [62], triphosphategraphene has been [63], the bacteria, most widely used as a promising FET channel material due to its superior physical, chemical, and viruses [64,65] as well as protein biomarkers [25,66]. Integrating a GFET biosensor with a electronic properties as well as its ability to be grown in large areas with high fidelity. For example, microfluidica graphene device field-effect can produce transistor synergistic (GFET) has eff ectbeen from employed their respective as a biosensor advantages, for detecting further DNA enhancing[62], its sensingtriphosphate performances. [63], bacteria, and viruses [64,65] as well as protein biomarkers [25,66]. Integrating a Here,GFET we biosensor summarize with the a recentlymicrofluidic published device workcan pr onoduce GFET-based synergistic microfluidic effect from biosensors. their respectiveYang et al. has builtadvantages, an integrated further microfluidicenhancing its platformsensing performances. that consists of (1) analyte enrichment using aptamers, (2) isocraticHere, elution, we summarize and (3) a GFET-based the recently published nanosensor work for on the GFET-based detection of microfluidic arginine vasopressin biosensors. Yang (AVP) [67]. The workinget al. has principle built an integrated is illustrated microfluidic in Figure platform8 which that shows consists the of enrichment (1) analyte enrichment of the analyte using (AVP) aptamers, (2) isocratic elution, and (3) a GFET-based nanosensor for the detection of arginine molecules through selective capturing by aptamers (Figure8A), bu ffer washing (Figure8B), isocratic vasopressin (AVP) [67]. The working principle is illustrated in Figure 8 which shows the enrichment elutionof (Figure the analyte8C) process(AVP) molecules sequentially, through resulting selective in capturing a mixture by (known aptamers as (Figure the elute) 8A), ofbuffer the freewashing aptamers and the(Figure released 8B),AVP isocratic originally elution captured(Figure 8C) from process the samplesequentially, (“sample resulting AVP”). in a Themixture elute (known is then as incubated the with grapheneelute) of the containing free aptamers pre-functionalized and the released with AVP standard originally AVP.captured Through from the sample competitive (“sample binding betweenAVP”). standard The elute and is then sample incubated AVP, with the graphene change containing in the GFET pre-functiona currentlized measurement with standard indicates AVP. the sampleThrough AVP concentration the competitive (Figure binding8E). between This changestandard in an thed sample channel AVP, conductance the change in is the used GFET as current an e ffi cient interrogationmeasurement probe indicates for monitoring the sample the AVP presence concentration of AVP (Figure concentration. 8E). This Thechange approach in the channel suggests the conductance is used as an efficient interrogation probe for monitoring the presence of AVP effectiveness of the GFET-based integrated microfluidics for achieving label-free detection of analytes. concentration. The approach suggests the effectiveness of the GFET-based integrated microfluidics Wang etforal. achieving recently label-free used a detection similar GFET-microfluidic of analytes. Wang et platformal. recently for used measurement a similar GFET-microfluidic of aptamer-protein bindingplatform kinetics for [ 68measurement]. of aptamer-protein binding kinetics [68].

(A) (B) (C)

(D) (E)

FigureFigure 8. Schematics 8. Schematics showing showing the working the working principle principle of the of th argininee arginine vasopressin vasopressin (AVP) (AVP) detection. detection. The The sample AVP issample enriched AVPon is enriched microbead on microb surfacesead bysurfaces aptamer by aptamer binding binding (A) and (A) thenand then is washed is washed with with a a bu ffer buffer (B) followed by an increase in temperature to 55⁰C that disrupts the aptamer-AVP complexes (B) followed by an increase in temperature to 55 ◦C that disrupts the aptamer-AVP complexes and release sample AVP into a free aptamer solution (C). The mixture of the free aptamers and the released sample AVP is incubated with graphene functionalized with standard AVP (D), that induces binding of free aptamer to the standard AVP on graphene via the competitive binding process (E) and thus changing the conductance of the graphene channel. Reprinted from [67], Copyright 2015, with permission from The 28th IEEE International Conference on Micro Electro Mechanical Systems (MEMS). Micromachines 2020, 11, 220 10 of 32

and release sample AVP into a free aptamer solution (C). The mixture of the free aptamers and the released sample AVP is incubated with graphene functionalized with standard AVP (D), that induces binding of free aptamer to the standard AVP on graphene via the competitive binding process (E) and Micromachinesthus changing2020, 11 ,the 220 conductance of the graphene channel. Reprinted from [67], Copyright 2015, with10 of 30 permission from The 28th IEEE International Conference on Micro Electro Mechanical Systems (MEMS). Chan et al. have implemented a reduced graphene oxide (rGO) transistor integrated with microfluidicsChan et foral. thehave detection implemented of H5N1 a influenzareduced graphene virus gene oxide using (rGO) a flow-through transistor strategy integrated [69]. with In a traditionalmicrofluidics approach, for the detection the short targetof H5N1 capture influenza probes virus that gene are immobilized using a flow-through by the π strategyπ interactions [69]. In are a − removedtraditional from approach, the rGO the surface short bytarget washing capture after probes hybridization. that are immobilized By contrast, theby the immobilization 𝜋−𝜋 interactions section ofare the removed extended from long the capture rGO surface probe remainsby washing on the after rGO hybridization. surface leaving By thecontrast,π π stackingthe immobilization interaction − intactsection even of the after extended hybridization long capture (Figure prob9), resultinge remains in on a possiblethe rGO increase surface inleaving the LOD. the 𝜋− The𝜋 sensing stacking is monitoredinteraction byintact monitoring even after the hybridization change in the (Figure drain current. 9), resulting On the in other a possible hand, thereincrease is negligible in the LOD. change The insensing the drain is monitored current after by bindingmonitoring with the non-complementary change in the drain target current. DNA which On the confirms other hand, the selectivity there is ofnegligible the rGO cha FETnge biosensor. in the drain The rGOcurrent FET-based after binding biosensor with exhibited non-complementary a detection limit target of DNA 5 pM withwhich a typical detection time of 1 hour.

(A) (C)

(B)

Figure 9. Mechanism of extendedextended long-capturelong-capture probe probe immobilization immobilization strategy strategy to to keep keep probe probe stability stability in thein the flow flow environment: environment: (A) ( TheA) The immobilization immobilization section section of extended of extended long long capture capture probe probe still keeps still keepsπ π − stacking𝜋−𝜋 stacking interaction interaction on the on reduced the reduced graphene graphene oxide (rGO)oxide surface(rGO) surface after hybridization; after hybridization; (B) The (B PDMS) The microfluidicPDMS microfluidic integrated integrated rGO transistor rGO transistor chip; (C) chip; In flowing (C) In environment,flowing environment, short capture short probes capture with probes fully matchwith fully sequence match after sequence hybridization after hybridization with target arewi washedth target away are washed from rGO aw surface.ay from Extended rGO surface. long captureExtended probes long capture are still probes kept on are rGO still surface. kept on ReprintedrGO surface. from Reprinted [69], Copyright from [69], 2017, Copyright with permission 2017, with frompermission Elsevier. from Elsevier.

3.3. Microfluidic Microfluidic Aptasensors Based on Optical Detection Optical detection is one of the most commonly used detection principles on lab-on-a-chip-based biosensors because it ooffersffers easy interfacinginterfacing betweenbetween microfluidicmicrofluidic devices and the conventionalconventional optical-detection instruments commonly found in laboratories (such as inverted fluorescencefluorescence microscopes, digital CCD cameras, simple light-emitting diodes (LEDs),(LEDs), laser diodes and photodiode set-ups and even smartphones)smartphones) [[34].34]. Some examples of an opticaloptical detectiondetection includeinclude measurementsmeasurements based on absorbance, fluorescence, chemiluminescence, colorimetry, interferometry, and surface plasmon resonance. Micromachines 2020, 11, 220 11 of 30

3.3.1. Fluorescence Detection Among different optical detection mechanisms, fluorescence-based detection is by far the most widely used sensing technique encountered in microfluidic applications thanks to its ease of implementation. One of the common fluorescence-based techniques works on the basis of the Förster resonance energy transfer (FRET). FRET is based on the coupling between a fluorophore (visible light-emitting molecule) and a quenching molecule that absorbs visible light and emits optical energy at invisible wavelengths. Although these fluorescence biosensors offer multiple advantages such as very low detection limits, high selectivity, and availability of a wide array of fluorescence labels for tagging biomolecules, quantitative analysis can be difficult due to the interference caused by the auto-fluorescence from the microfluidic chip material in case of polymeric devices [34]. One of the most widely used nanomaterials in aptamer-based fluorescence biosensors is graphene oxide (GO). GO is a graphene nanosheet containing large amounts of oxygen atoms in the form of epoxy, hydroxyl, and carboxyl groups [70]. The main reason for its widespread use, especially in the biosensors area, is its high optical quenching ability, excellent dispersibility in aqueous media, biocompatibility and easy surface functionalization capability [71]. GO interacts with single-stranded DNA through π π stacking with the nucleotide bases and effectively quenches the fluorescence of − quantum dots (QDs) [72,73]. The quenching mechanism is based on the principle of photoinduced electron transfer or energy transfer. Neethirajan group had extensively exploited the exceptional quenching capability of GO to develop several aptamer-based biosensors on lab-on-a-chip platform [74–76]. One example is the aptamer- based biosensor on PDMS/glass microfluidic chip for simple, rapid, and sensitive food allergen (Ara h1) detection developed by Weng and Neethirajan [74]. This device utilized quantum dots-aptamer–GO complexes (QDs-aptamer-GO) as probes to undergo conformational changes upon interaction with the food allergens. As can be seen in Figure 10, in the absence of the target analyte, the fluorescence of the QDs is quenched via the FRET process between the (QDs)-aptamer probes and GO due to their self-assembly through specific π π stacking interaction, resulting in no fluorescence signal. Upon − binding the target analyte, QDs-aptamer probes are released from the GO leading to the recovery of fluorescence of QDs. The same principle was used by Weng and Neethirajan [75] to develop another aptamer-based biosensor for rapid, and sensitive detection of food allergens and food toxins. In this device, a PDMS/paper microfluidics platform was used instead of the PDMS/glass microfluidics. First proposed by Whitesides and co-workers [77], paper-based microfluidic devices, commonly referred to as microfluidic paper-based analytical devices (µPADs), has rapidly emerged as an alternative approach to the traditional microfluidic devices based on glass, silicon, PDMS or other polymeric materials. Paper is a ubiquitous material and is comprised of biocompatible porous cellulose fiber web with large surface area. Its porous nature fulfils the primary task of fluid transport through the predefined channel. Moreover, µPADs are powerless due to the capillary action of the hydrophobic channel [76]. The success of µPADs can be attributed to three main factors: low cost, simple operation, and the ability to manipulate fluid flow without any external pumps [78]. Therefore, Weng and Neethirajan [76] developed an aptamer biosensor on a paper-based microfluidic platform for sensitive detection of norovirus. Instead of using QDs, 6-carboxyfluorescein (6-FAM) was used as the aptamer label, and multi-walled carbon nanotubes (MWCNTs) and graphene oxide were used for the quencher in their devices. In the presence of the target norovirus, the labeled aptamers were released from the MWCNT surface resulting in the recovery of a fluorescence signal, which was subsequently measured by the multi-mode reader. Micromachines 2020, 11, 220 12 of 30 Micromachines 2020, 11, 220 12 of 32

Figure 10. Microfluidic fluorescence biosensor: Schematic diagram of the (A) sensing mechanism of the Figure 10. Microfluidic fluorescence biosensor: Schematic diagram of the (A) sensing mechanism of quantum dots (QDs)-aptamer-graphene oxide (GO) quenching system; and (B) microfluidic chip which the quantum dots (QDs)-aptamer-graphene oxide (GO) quenching system; and (B) microfluidic chip has two inlets for loading the QDs-aptamer-GO probe mixture and the Ara h 1 sample, respectively. whichReprinted has two from inlets [74], Copyrightfor loading 2016, the with QDs-aptamer-GO permission from probe Elsevier. mixture and the Ara h 1 sample, respectively. Reprinted from [74], Copyright 2016, with permission from Elsevier. Liang et al. have developed another fluorescent and multiplexed aptasensor for monitoring cancer cells in µFirstPADs proposed that can be by observed Whitesides by a and naked co-workers eye [79]. The [77], device paper-based utilizes quantum microfluidic dots-coated devices, mesoporous commonly referredsilica nanoparticles-labeled to as microfluidic aptamerspaper-based that areanalytic attachedal devices to the surface (µPADs), of GO. has The rapidly fluorescence emerged that wasas an alternativeinitially quenched approach via to FRET the traditional was subsequently microfluidic recovered devices upon based addition on of glass, the target silicon, cells. PDMS The unique or other polymericdesign allows materials. for alteration Paper ofis othera ubiquitous target cancer mate cellsrialby and simply is comprised utilizing the of change biocompatible of the aptamer porous celluloseand diff fibererent web colored with QDs. large With surface this technology,area. Its porous simultaneous nature fulfils detection the primary of three ditaskfferent of fluid cancer transport cells throughwas achieved the predefined with LODs channel. of 62, 70,Moreover, and 65 cells µPADs/mL forare MCF-7,powerless HL-60, due and to the K562 capillary cells, respectively. action of the hydrophobicFurthermore, channel the work [76]. contributed The success to of clinical µPADs sample can be analysis attributed with to satisfactory three main results. factors: Similarlow cost, simpleFRET-based operation, detection andmechanisms the ability wereto manipu also adoptedlate fluid by Ueno flow et al.without [80] to developany external a linear pumps array GO [78]. Therefore,aptasensor Weng for multiplexed and Neethirajan detection [76] ofdeveloped three protein an aptamer biomarkers: biosensor thrombin, on a prostatepaper-based specific microfluidic antigen platform(PSA) and for hemagglutinin sensitive detection (HA). of norovirus. Instead of using QDs, 6-carboxyfluorescein (6-FAM) Chung et al. have developed an optofluidic system that uses target specific aptamer-conjugated was used as the aptamer label, and multi-walled carbon nanotubes (MWCNTs) and graphene oxide fluorescence nanoparticle (A-FNP) for fast and continuous detection and monitoring of airborne were used for the quencher in their devices. In the presence of the target norovirus, the labeled microorganism E. Coli [81]. In this real-time and non-destructive single cell detection system, fluorescence aptamers were released from the MWCNT surface resulting in the recovery of a fluorescence signal, nanoparticles are attached to the surfaces of bacterial cells causing them to be optically excited with the which was subsequently measured by the multi-mode reader. wavelength of 540 nm. The developed system consists of a PDMS microchannel having 3 inlets (1 sample Liang et al. have developed another fluorescent and multiplexed aptasensor for monitoring flow and 2 sheath flows), 1 outlet, and an optical detector (Figure 11). The sensing was accomplished by cancerexciting cells the in A-FNP-labeled µPADs that canE. Coli be cellsobserved in the detectionby a naked zone eye with [79]. a fiber-coupledThe device utilizes laser and quantum the emitted dots- coatedfluorescence mesoporous was quantitatively silica nanoparticles-labeled measured with a apta photonmers counter. that are This attached optofluidic to the system surface resulted of GO. in The a fluorescencedetection throughput that was initially of up to ~100quenched particles via per FRET second was with subsequently 85% accuracy. recovered upon addition of the target cells. The unique design allows for alteration of other target cancer cells by simply utilizing the change of the aptamer and different colored QDs. With this technology, simultaneous detection of three different cancer cells was achieved with LODs of 62, 70, and 65 cells/mL for MCF-7, HL-60, and K562 cells, respectively. Furthermore, the work contributed to clinical sample analysis with satisfactory results. Similar FRET-based detection mechanisms were also adopted by Ueno et al. [80] to develop a linear array GO aptasensor for multiplexed detection of three protein biomarkers: thrombin, prostate specific antigen (PSA) and hemagglutinin (HA).

Micromachines 2020, 11, 220 13 of 32

Chung et al. have developed an optofluidic system that uses target specific aptamer-conjugated fluorescence nanoparticle (A-FNP) for fast and continuous detection and monitoring of airborne microorganism E. Coli [81]. In this real-time and non-destructive single cell detection system, fluorescence nanoparticles are attached to the surfaces of bacterial cells causing them to be optically excited with the wavelength of 540 nm. The developed system consists of a PDMS microchannel having 3 inlets (1 sample flow and 2 sheath flows), 1 outlet, and an optical detector (Figure 11). The sensing was accomplished by exciting the A-FNP-labeled E. Coli cells in the detection zone with a fiber-coupled laser and the emitted fluorescence was quantitatively measured with a photon counter. Micromachines 2020, 11, 220 13 of 30 This optofluidic system resulted in a detection throughput of up to ~100 particles per second with 85% accuracy.

Figure 11. Schematics of the aptamer-based optofluidicoptofluidic detection system: ( A) preparation of aptamer conjugated fluorescencefluorescence nanoparticlesnanoparticles (A-FNPs); and ( B) detection of A-FNP-bound E. Coli by the microchannel and and optical optical particle particle counter. counter. Reprinted Reprinted from from [81], [81 Copyright], Copyright 2015, 2015, with withpermission permission from fromElsevier. Elsevier.

Aptamer-based sandwichsandwich immunoassay immunoassay techniques techniques were were developed developed by by several several research research groups groups to designto design fluorescence-based fluorescence-based aptamer aptamer biosensors biosensors for rapid for sensingrapid sensing of analytes of analytes on microfluidics on microfluidics platform. Inplatform. this assay, In this two assay, aptamers two are aptamers required: are one required is the: capture one is the aptamer capture and aptamer the other and one the is other the reporter one is aptamer.the reporter The aptamer. capture aptamerThe capture is immobilized aptamer is ontoimmo abilized substrate onto and a substrate interacts withand theinteracts target with analyte. the Subsequently,target analyte. the Subsequently, reporter aptamer the whichreporter is taggedaptamer with which a certain is tagged label (i.e. with fluorophore) a certain bindslabel with(i.e. thefluorophore) captured analyte,binds with resulting the captured in a sandwich analyte, structure.resulting in For a sandwich example, Tsengstructure. et al. For used example, sandwiched Tseng aptamer-basedet al. used sandwiched assay for aptamer-ba automaticsed fluorescence assay for automatic detection fluorescen of H1N1 influenzace detection virus of H1N1 on microfluidic influenza platformvirus on microfluidic [82]. This microfluidic platform [82]. system This wasmicrofluidic designed system to automatically was designed carry to outautomatically the entire detectioncarry out processthe entire in detection less than 30process minutes in less which than is significantly30 minutes which faster is than significantly the conventional faster than viral the culture conventional method. Aviral limit culture of detection method. of A 0.032 limit hemagglutination of detection of 0.032 unit hemagglutination (HAU) was achieved unit thanks (HAU) to was the highachieved affinity thanks and highto the specificity high affinity of the H1N1-specificand high specificity aptamers. of Furthermore,the H1N1-specific this two-aptamer aptamers. Furthermore, microfluidic system this two- had approximatelyaptamer microfluidic 3 orders system of magnitude had approximately higher in sensitivity 3 orders thanof magnitude the conventional higher in serological sensitivity diagnosis. than the Liconventional et al. have also serological developed diagnosis. an integrated Li et microfluidical. have also system developed for rapid an integrated and automatic microfluidic measurements system of glycatedfor rapid hemoglobin and automatic using measurements the sandwich-based of glycated approach hemoglobin [83]. The using two parallelthe sandwich-based assays were developed approach for[83]. quantifying The two parallel glycated assays hemoglobin were anddeveloped total hemoglobin. for quantifying This assayglycated technique hemoglobin took 86% and shorter total analysishemoglobin. time This than assay the conventional technique took HPLC-based 86% shorter approach analysis as time well than as 75% the conventional less reagent consumption. HPLC-based approachMicrofluidic as well capillaryas 75% less electrophoresis reagent consumption. (MCE), a miniaturized platform of capillary electrophoresis (CE),Microfluidic can provide a simplecapillary and convenientelectrophoresis solution (MCE), for biochemical a miniaturized analysis compared platform to otherof capillary methods suchelectrophoresis as high-performance (CE), can liquidprovide chromatography a simple and (HPLC), convenient gas chromatography solution for biochemical and CE [84]. analysis Several researchcompared groups to other have exploitedmethods thissuch MCE as platformhigh-per informance conjunction liquid with fluorescencechromatography detection (HPLC), system gas to developchromatography aptamer basedand CE microfluidic [84]. Several biosensor research [85– 87groups]. For example,have exploited Pan et al. this [85 ]MCE used thisplatform platform in forconjunction rapid detection with fluorescence of tumor marker detection carcinoembryonic system to develop antigen aptamer (CEA). based A laser-inducedmicrofluidic biosensor fluorescence [85– (LIF) detection system was used to detect CEA in serum samples from colorectal cancer (CRC) patients and healthy subjects. As seen in Figure 12, a double-stranded DNA (dsDNA), which was formed by an anti-CEA aptamer and its complementary DNA (cNDA), was conjugated with a magnetic bead (MB). In the presence of the target CEA molecule in the sample, the cDNA was released and hybridized with a fluorescein amidite (FAM)-labeled DNA forming a DNA duplex. This DNA duplex triggers the selective cleavage of the FAM-labeled DNA probe by nicking endonuclease (Nb.BbvCI), resulting in a continuous release of the cDNA. The released cDNA was repeatedly hybridized with another FAM-labeled DNA that led to the generation of large numbers of FAM-labeled DNA segments, Micromachines 2020, 11, 220 14 of 32

87]. For example, Pan et al. [85] used this platform for rapid detection of tumor marker carcinoembryonic antigen (CEA). A laser-induced fluorescence (LIF) detection system was used to detect CEA in serum samples from colorectal cancer (CRC) patients and healthy subjects. As seen in Figure 12, a double-stranded DNA (dsDNA), which was formed by an anti-CEA aptamer and its complementary DNA (cNDA), was conjugated with a magnetic bead (MB). In the presence of the target CEA molecule in the sample, the cDNA was released and hybridized with a fluorescein amidite (FAM)-labeled DNA forming a DNA duplex. This DNA duplex triggers the selective cleavage of the FAM-labeledMicromachines 2020 DNA, 11, 220 probe by nicking endonuclease (Nb.BbvCI), resulting in a continuous release14 of of 30 the cDNA. The released cDNA was repeatedly hybridized with another FAM-labeled DNA that led toresulting the generation in an amplification of large numbers of the fluorescence of FAM-labeled signal. DNA Afterwards, segments, the resulting MCE laser-induced in an amplification fluorescence of (MCE-LIF)the fluorescence detection signal. system Afterwards, was able the to MCE separate laser and-induced detect thefluorescence FAM-labeled (MCE-LIF) DNA segments. detection Thesystem use wasof magnetic able to beadsseparate is toand assist detect in thethe target-induced FAM-labeled DNA strand segments. cycle that The would use lead of magnetic to an increase beads in is the to assistoptical in sensitivity. the target-induced Using this strand MCE cycle platform, that would a limit oflead detection to an increase of 68pg in/mL the was optical obtained sensitivity. with a Using linear thisrange MCE of 130pg platform,/mL–8 a nglimit/mL, of makingdetection it aof promising 68 pg/mL technologywas obtained for with use ina linear clinical range diagnosis, of 130 prognosis, pg/mL–8 ng/mL,and monitoring making it of a cancer promising progression technology in CRC. for use in clinical diagnosis, prognosis, and monitoring of cancer progression in CRC.

Figure 12. Schematic illustration of detection principle with the proposed aptamer-based microfluidic Figure 12. Schematic illustration of detection principle with the proposed aptamer-based microfluidic capillary electrophoresis (MCE) assay for amplification detection of carcinoembryonic antigen (CEA). capillary electrophoresis (MCE) assay for amplification detection of carcinoembryonic antigen (CEA). Abbreviations: S, sample reservoir; SW, sample waste reservoir; B, buffer reservoir; BW, buffer waste Abbreviations: S, sample reservoir; SW, sample waste reservoir; B, buffer reservoir; BW, buffer waste reservoir; MBs, magnetic beads. Reprinted from [85], Copyright 2015, with permission from Elsevier. reservoir; MBs, magnetic beads. Reprinted from [85], Copyright 2015, with permission from Elsevier. The MCE platform was also used by other research groups such as in Wang et al. [87] who utilized it to developThe MCE an platform aptamer-based was also fluorescence used by detectionother research of multiple groups antibiotic such as residues in Wang in theet al. field [87] of foodwho utilizedsafety screening. it to develop The an MCE aptamer-based platform was fluorescence combined with detection catalyzed of multiple hairpin assemblyantibiotic (CHA)residues as in CHA the fieldcan act of asfood a signal safety amplifier screening. without The MCE the need platform for a label,was combined which may with decrease catalyzed the performance hairpin assembly of the (CHA)aptamers as orCHA negatively can act impactas a signal the self-assemblyamplifier with ofout hairpins. the need On for the a otherlabel, hand,which He may et al.decrease combined the performanceMCE with stir of bar-assisted the aptamers sorptive or negatively extraction impact and the rolling self-assembly circle amplification of hairpins. (RCA) On the to other implement hand, Hea ratiometric et al. combined fluorescence MCE with biosensor stir bar-assisted for highly sensitive sorptive detectionextraction of and antibiotics rolling incircle food amplification [88]. Unlike (RCA)conventional to implement aptamer-based a ratiometric biosensors, fluorescence this ratiometric biosensor sensing for highly can besensitive employed detection to detect of samplesantibiotics in ina complex food [88]. matrix Unlike such conventional as foods and aptamer-based soils and can biosensors, avoid potential this ratiometric problems sensing of matrix can interference be employed or tosample-to-sample detect samples in variations. a complex matrix such as foods and soils and can avoid potential problems of matrix interference or sample-to-sample variations. 3.3.2. Colorimetric Detection One of the main advantages of colorimetric detection is that the color detection can be easily achieved with various commercial devices such as smart phones and flatbed scanners [89]. It is often considered a simple and low-cost method to measure quantitative output signal. However, its lack of sensitivity limits its usage in applications requiring high accuracy such as the detection of rare cancer cells in blood sample [53]. Recently, Zhang et al. created an aptamer-based colorimetric biosensor for achieving naked eye quantitative assay on paper, which relies on measuring the length of the colored region in a strip-like µPAD or counting the number of colorless detection microzones in a multi-zone µPAD [90]. Figure 13 shows the schematic illustration of the naked-eye aptamer-based paper Micromachines 2020, 11, 220 15 of 32

3.3.2. Colorimetric Detection One of the main advantages of colorimetric detection is that the color detection can be easily achieved with various commercial devices such as smart phones and flatbed scanners [89]. It is often considered a simple and low-cost method to measure quantitative output signal. However, its lack of sensitivity limits its usage in applications requiring high accuracy such as the detection of rare cancer cells in blood sample [53]. Recently, Zhang et al. created an aptamer-based colorimetric Micromachines 2020 11 biosensor for achieving, , 220 naked eye quantitative assay on paper, which relies on measuring the length15 of 30 of the colored region in a strip-like µPAD or counting the number of colorless detection microzones inmicrofluidic a multi-zone assay µPAD for the [90]. detection Figure of 13 adenosine shows the based schematic on the measurementillustration of of the a colored naked-eye region aptamer- as well basedas counting paper of microfluidic the colorless assay microzones for the detection in a wax-patterned of adenosine 12-zone basedµ PAD.on the For measurement test results, aof naked-eye a colored quantitativeregion as well readout as counting is performed of the colorless either by micr simplyozones measuring in a wax-patterned the length of the12-zone colored µPAD. region For of test the results,strip-like a naked-eyeµPAD or by quantitative counting the readout number is ofperfor colorlessmed either detection by simply microzones. measuring These the equipment-free length of the coloredand quantitative region of methods the strip-like hold greatµPAD potential or by counti in theng commercialization the number of colorl of aptamer-basedess detection POCmicrozones. devices Thesethat are equipment-free low-cost, portable and and quantitative user-friendly. methods hold great potential in the commercialization of aptamer-based POC devices that are low-cost, portable and user-friendly.

Figure 13. Schematic diagram illustrating the naked-eye quantitative aptamer-based assay for the detection of adenosine as a model analyte based on ( A) the the length length measurement measurement of the colored region in a strip-like microfluidic microfluidic paper-based analytical device device (µPAD) (µPAD) or (B) the counting of the colorless microzones in a wax-pattered 12-zone µPAD.µPAD. ReprintedReprinted from [[90],90], CopyrightCopyright 2016, with permission from Elsevier.

Another µµPAD-basedPAD-based colorimetric colorimetric aptasensor aptasensor was developedwas developed by Wei etby al. Wei [91] foret theal. simultaneous[91] for the simultaneousdetection of cocaine, detection adenosine, of cocaine, and adenosine, lead ion. A and target-responsive lead ion. A target-responsive hydrogel was used hydrogel as flow was regulator used asand flow controller. regulator In and the absencecontroller. of aIn target, the absence the hydrogel of a target, formed the inhydrogel the channel formed blocks in the the channel flow causing blocks a “signalthe flow o ffcausing” readout. a “signal By contrast, off” readout. in the presence By contrast of the, in target, the presence the hydrogel-free of the target, fluidic the channel hydrogel-free allows fluidicthe colorimetric channel allows indicator the tocolorimetric reach the observation indicator to spot reach producing the observation a “signal spot on” producing readout. The a “signal entire on”assay readout. can be completedThe entire withinassay can 6 minutes be completed and the wi observationthin 6 minutes canbe and made the byobservation the naked can eye be without made byany the auxiliary naked equipment.eye without The any reported auxiliary detection equipmen mechanismt. The reported offers a simple, detection cost-e mechanismffective, rapid, offers and a simple,user-friendly cost-effective, POC testing rapid, device and user-friendly to be used in variousPOC testing applications. device to be used in various applications. Nanoparticles are are often often employed employed in in colorimetric colorimetric detection detection because because of oftheir their inherent inherent optical optical or catalyticor catalytic properties. properties. Several Several works works have have been been published published on on the the colorimetric colorimetric detection detection using nanoparticles [89,92–94]. Zhao et al. used silver nanoparticles (AgNPs) to develop a naked-eye colorimetric aptasensor on glass/PDMS microfluidic platform for the simple detection of protein biomarker thrombin [89]. Surface-functionalized microfluidic channels were used as the capture platform (Figure 14). The introduction of thrombin into the channel forms a sandwich-type complex involving immobilized AgNPs. Increasing concentrations of thrombin cause an increase in the amount of aptamer-AgNPs on the complex resulting in colorimetric changes that can be observed either by a Micromachines 2020, 11, 220 16 of 32 nanoparticles [89,92–94]. Zhao et al. used silver nanoparticles (AgNPs) to develop a naked-eye colorimetric aptasensor on glass/PDMS microfluidic platform for the simple detection of protein biomarker thrombin [89]. Surface-functionalized microfluidic channels were used as the capture Micromachines 2020, 11, 220 16 of 30 platform (Figure 14). The introduction of thrombin into the channel forms a sandwich-type complex involving immobilized AgNPs. Increasing concentrations of thrombin cause an increase in the nakedamount eye of oraptamer-AgNPs a flatbed scanner. on Thrombinthe complex concentrations resulting in ofcolorimetric as low as 20 changes pM have that been can detected be observed using thiseither aptasensor by a naked device eye or without a flatbed any scanner. signal amplification. Thrombin concentrations of as low as 20 pM have been detected using this aptasensor device without any signal amplification.

Figure 14. ColorimetricColorimetric detection detection using using silver silver nanoparticles nanoparticles aptasensor: aptasensor: (A ()A schematic) schematic illustration illustration of ofcapturing capturing target target proteins proteins and and colorimetric colorimetric detection detection based based on on aptamer-AgNPs aptamer-AgNPs in in the microfluidicmicrofluidic chip; ( B) photograph of the microfluidicmicrofluidic device consistingconsisting of 7 channels for thrombin capture and colorimetric detection;detection; ( C(C) scanned) scanned picture picture of microfluidicof microfluidic chip chip after after reaction; reaction; and ( Dand) gray (D) scale gray values scale werevalues acquired were acquired on each on channel. each channel. Reprinted Reprinted from [89 from], Copyright [89], Copyright 2016, with 2016, permission with permission from Elsevier. from Elsevier. Colorimetric assays in conjunction with nucleic acid amplification have been used extensively whichColorimetric offer enhanced assays detection in conjunction capabilities. with nucleic As an acid example, amplification Lin’s group have been developed used extensively a portable glasswhich/PDMS offer microchipenhanced detection to realize capabilities. highly sensitive As an detection example, of Lin’s thrombin group protein developed by integrating a portable it withglass/PDMS rolling microchip circle amplification to realize highly (RCA) sensitive [95]. Thrombin detection was of thrombin first isolated protein using by aptamersintegrating and it with the secondaryrolling circle thrombin amplification aptamers (RCA) with [95]. a G-quadruplexThrombin was templatefirst isolated was using used aptamers to amplify and the the signal secondary using RCA.thrombin The aptamers reported detectionwith a G-quadruplex limit was 0.083 template pg/mL was measured used to from amplify thrombin the signal in plasma using and RCA. serum. The reportedA colorimetric detection limit biosensor was 0.083 for telemedicine pg/mL measur applicationed from hasthrombin been developed in plasma by and Fraser serum. et al. [96] who haveA integrated colorimetric an aptamer-tethered biosensor for telemedicine enzyme capture application (APTEC) has technique been developed on a microfluidic by Fraser deviceet al. [96] for magnet-guidedwho have integrated capture, an wash, aptamer-tethered and detection enzyme of the biomarker. capture (APTEC) Three separate technique microfluidic on a microfluidic chambers weredevice designed for magnet-guided to implement capture, the magnet-guided wash, and detectio colorimetricn of the detection biomarker. of the Three PfLDH separate protein. microfluidic In the first chamberchambers (incubation were designed chamber), to implement aptamer-coated the magn micromagneticet-guided colorimetric beads (µMBs) detection were bound of the specifically PfLDH toprotein. their target In the by first incubation chamber in a(incubation lysed sample chamber), of human aptamer-coated blood. Next, a washingmicromagnetic step was beads conducted (µMBs) in thewere second bound chamber specifically to remove to their any target unbound by incubation molecules in and a lysed other sample contaminants. of human This blood. was followed Next, a bywashing the transfer step was of the conducted aptamer- inµMB-target the second complex chamber to theto remove third chamber any unbound containing molecules the development and other reagentcontaminants. that generated This was a strongfollowed colorimetric by the transfer signal inof thethe presenceaptamer-µMB-target of PfLDH. (Figure complex 15A). to The the signalthird analysischamber wascontaining conducted the bydevelopment placing the reagent device onthat top generated of a tablet a strong device colorimetric (Figure 15B) signal displaying in the a homogeneouspresence of PfLDH. background (Figure in 15A). a concealed The signal box. analys Imagesis was were conducted captured usingby placing a smartphone the device camera on top and of coupleda tablet device with metadata (Figure 15B) containing displaying time, a homogene date, and GPSous background coordinates forin a theconc telemedicineealed box. Images application. were Atcaptured the receiver using side,a smartphone the images camera were analyzed and coupled with ImageJwith metadata software. containing time, date, and GPS coordinates for the telemedicine application. At the receiver side, the images were analyzed with ImageJ software.

Micromachines 2020, 11, 220 17 of 30 Micromachines 2020, 11, 220 17 of 32

(A) (B)

Figure 15. Microfluidic aptamer-tethered enzyme capture (APTEC) colorimetric biosensor: (A) the Figure 15. Microfluidic aptamer-tethered enzyme capture (APTEC) colorimetric biosensor: (A) the reaction scheme of the reagents and redox reaction that results in the generation of an insoluble purple reaction scheme of the reagents and redox reaction that results in the generation of an insoluble purple diformazandiformazan dye. dye. There There was was a acolor color difference difference between between positive positive and andnegative negative samples; samples; and (B and) the (B ) the smartphonesmartphone camera camera was was used used for for capturing capturing images images in a in telemedicine a telemedicine application. application. Reprinted Reprinted from [96], from [96], CopyrightCopyright 2018, 2018, with with perm permissionission from from Elsevier. Elsevier. 3.3.3. Chemiluminescence and Electrochemiluminescence-Based Detection 3.3.3. Chemiluminescence and Electrochemiluminescence-Based Detection LuminescenceLuminescence is is a a phenomenon phenomenon where where certain certain mole moleculescules emit emit light light not resulting not resulting from fromheat [97]. heat [97]. LuminescenceLuminescence can can be be of of different different types types depending depending on onthe thesource source of energy of energy or the or trigger the trigger for for luminescence,luminescence, some some examples examples being being chemiluminescence chemiluminescence (CL) (CL) and and electrochemiluminescence electrochemiluminescence (ECL). (ECL). DueDue toto their considerable advantages advantages including including sensitivity, sensitivity, selectivity, selectivity, and andlarge large linear linear quantitative quantitative range,range, luminescence-basedluminescence-based detection is becoming increasingly popular popular for for biosensing biosensing applications applications [98]. For[98]. example, For example, as noas no excitation excitation instrumentation instrumentation is required, required, the the background background noises noises in CL in signals CL signals areare significantlysignificantly reduced reduced increasing increasing the the sensitivity sensitivity compared compared to assay to assay performed performed on bench-top on bench-top equipmentequipment [99]. [99]. Chang Chang et et al. al. have have developed developed an anaptamer-based aptamer-based biosensor biosensor on a onmicrofluidic a microfluidic platform platform usingusing CLCL detectiondetection scheme for selective selective detection detection of of blood blood glycated glycated hemoglobin hemoglobin (HbA1c) (HbA1c) [99]. [99 Figure]. Figure 16 illustrates16 illustrates the the sensing sensing principle principle as as well well as as the the analysis analysis procedure procedure for for thethe integratedintegrated microfluidicmicrofluidic chip. First,chip. Hb-First, or Hb- HbA1c-specific or HbA1c-specific aptamers aptamers immobilized immobilized on on magnetic magnetic beads beads (MBs) (MBs) were were transported transported to the chamberto the chamber where where they were they incubatedwere incubated with bloodwith blood samples samples (a). Following(a). Following a washing a washing step step to removeto remove the unbound fractions by PBS buffer and aided by an external magnet (b), the target-aptamer- the unbound fractions by PBS buffer and aided by an external magnet (b), the target-aptamer-MB MB complexes were incubated with acridinium ester-labeled Hb or HbA1c specific antibodies to form complexes were incubated with acridinium ester-labeled Hb or HbA1c specific antibodies to form aptamer-antibody sandwich-like structures (c). In the following step, another washing step was aptamer-antibody sandwich-like structures (c). In the following step, another washing step was performed to remove the unbound antibodies magnetically (d). Finally, H2O2 was injected into the performedtransportation to removeunit to re-suspend the unbound the antibodiessandwich-like magnetically complex magnetic (d). Finally, beads H(e)2 Owhich2 was was injected then into thefollowed transportation by the transporting unit to re-suspendof target-aptamer-b the sandwich-likeead complex with complex acridinium magnetic to the beads NaOH (e) chamber which was thenwhere followed CL signals by thewere transporting monitored ofwith target-aptamer-bead a luminometer (f). complex Giuffrida with et acridiniumal. have reported to the NaOHa chamberchemiluminescent where CL sensor signals that were employs monitored digital withmicrofluidics a luminometer for aptamer-based (f). Giuffrida detection et al. have of human reported a chemiluminescentlysozyme [100]. With sensor this droplet that employs microfluidics-based digital microfluidics technique for that aptamer-based uses gold nanoparticles detection as of an human lysozymeenhancement [100 agent]. With for thisthe CL droplet signal, microfluidics-based a detection limit of 44.6 technique fM was achieved that uses using gold a sample nanoparticles volume as an enhancementas low as 1 µL agent and a fordetection the CL time signal, in the a detection range of limit10 minutes. of 44.6 fM was achieved using a sample volume as low as 1 µL and a detection time in the range of 10 minutes.

Micromachines 2020, 11, 220 18 of 30 Micromachines 2020, 11, 220 18 of 32

(A)

(B) (C)

FigureFigure 16. ChemiluminescentChemiluminescent aptamer-based aptamer-based microfluidic microfluidic biosensor: biosensor: schematic schematic illustration illustration of the (A) of the (workingA) working principle; principle; (B) ( (aB–)(d)a microfluidic–d) microfluidic chip chipcomposed composed of micro-components; of micro-components; and (C and) experimental (C) experimental procedure performed on the integrated microfluidic chip. (a) magnetic beads pre-coated with Hb or procedure performed on the integrated microfluidic chip. (a) magnetic beads pre-coated with Hb or HbA1c aptamers were transported to the transportation unit (the close chamber) and incubated with HbA1c aptamers were transported to the transportation unit (the close chamber) and incubated with blood samples; (b) target-aptamer-bead complexes were collected by applying an external magnetic blood samples; (b) target-aptamer-bead complexes were collected by applying an external magnetic field while washing the supernatant and non-binding substances with phosphate-buffered saline field while washing the supernatant and non-binding substances with phosphate-buffered saline (PBS); (c) the acridinium ester-labeled Hb or HbA1c specific antibodies were transported to the (PBS);transported (c) the to acridiniumthe transported ester-labeled unit and reacted Hb or with HbA1c Hb or specific HbA1c; antibodies(d) the sandwich-like were transported structures to the transportedwith target-aptamer-bead to the transported complexes unit were and collected reacted with while Hb washing or HbA1c; the supe (d)rnatant the sandwich-like and non-binding structures withsubstances target-aptamer-bead with PBS buffer; complexes (e) H2O2 was were transported collected whileto the washingtransportation the supernatant unit to re-suspend and non-binding the substancesmagnetic beads; with and PBS (f) bu theffer; target-aptamer-bead (e)H2O2 was transported complex with to thethe acridinium transportation was transported unit to re-suspend to the the magneticNaOH chamber beads; and and the ( fchemiluminescent) the target-aptamer-bead signals were complex detected with with thea luminometer. acridinium Reprinted was transported from to the[99], NaOH Copyright chamber 2015, andwith the permission chemiluminescent from Elsevier. signals were detected with a luminometer. Reprinted from [99], Copyright 2015, with permission from Elsevier. ECL is a powerful analytical technique which is gaining increased attention for the development of analyticalECL is a methods powerful using analytical µPADs technique [79]. One which example is gaining is the ECL-based increased attentionµPAD developed for the development by Ma et of analyticalal. [101]. This methods work using uses µthePADs specific [79]. recognition One example by is aptamers the ECL-based and theµPAD amplification developed strategy by Ma etof al.a [101]. Thishybridization work uses chain the specific reaction recognition (HCR) with by the aptamers ECL probe, and the Ru(phen) amplification32+ for detecting strategy ofthe a hybridizationanalyte peptides. 2+ chain reaction (HCR) with the ECL probe, Ru(phen)3 for detecting the analyte peptides. Recently,Recently, LinLin etet al. al. have have developed developed a “turn-on”a “turn-on” aptasensor aptasensor on on microfluidics microfluidics platform platform for for sensitive detectionsensitive detection of vascular of vascular endothelial endothelial growth growth factor VEGF165 factor VEGF165 that employs that employs iridium iridium (III) complex(III) complex as a novel as a novel G-quadruplex-selective luminescent probe [102]. In this work, a hairpin-structured G-quadruplex-selective luminescent probe [102]. In this work, a hairpin-structured functional nucleic functional nucleic acid (FNA) was constructed which consisted of a VEGF165 aptamer and a G- acid (FNA) was constructed which consisted of a VEGF165 aptamer and a G-quadruplex sequence as quadruplex sequence as well as its complementary sequence. Upon binding to VEGF165, the well as its complementary sequence. Upon binding to VEGF165, the functional nucleic acid rearranges functional nucleic acid rearranges its structure to form a G-quadruplex structure which is able to bind itswith structure the luminescent to form a iridium G-quadruplex (III) complex structure to report which the is analyte. able to bindThe hairpin with the structure luminescent is unable iridium to (III) complex to report the analyte. The hairpin structure is unable to bind to iridium (III) complex when

Micromachines 2020, 11, 220 19 of 32 bindMicromachines to iridium2020 (III), 11, 220complex when VEGF is absent. This luminescence-based aptasensor resulted19 ofin 30 a specific detection of VEGF165 with low background, a linear range of 0.52–78.0 nM, and a LOD of

0.17VEGFpM. is absent. This luminescence-based aptasensor resulted in a specific detection of VEGF165 with low background, a linear range of 0.52–78.0nM, and a LOD of 0.17pM. 3.3.4. Optical Interferometric Detection 3.3.4.Interferometry Optical Interferometric offers the possibility Detection of label-free, highly sensitive detection of biomolecules. In general,Interferometry interferometric off biosensingers the possibility techniques of label-free,work by measuring highly sensitive small changes detection that of biomolecules.occur in an opticalIn general, beam interferometric(sensing beam) biosensingalong its path techniques of propagation. work by When measuring any binding small changesevent takes that place, occur it in causesan optical a change beam in(sensing the refractive beam) index along along its path the ofoptical propagation. path, resulting When in any a phase binding shift event in the takes sensing place, beamit causes with arespect change to in the the reference refractive beam. index By along imaging the optical the interference path, resulting pattern in resulting a phase shiftfrom in the the projectionsensing beam of these with two respect beams, to one the can reference directly beam. moni Bytor imagingany binding the interferenceevent that occurs pattern in the resulting biosensor from [28,103].the projection of these two beams, one can directly monitor any binding event that occurs in the biosensorSong et [ 28al.,103 have]. reported an optical interferometric aptamer-based biosensor for sensitive and specificSong detection et al. haveof plant reported hormone an opticalabscisic interferometric acid (ABA) on a aptamer-based microfluidic device biosensor [104]. for As sensitivecan be seen and inspecific Figure detection 17, theof microfluidic plant hormone chip abscisic consists acid (ABA)of a onnanopore-based a microfluidic devicesensing [104 region]. As can and be seenSU-8 in microstructuresFigure 17, the microfluidic as capillary chip microfluidics consists of a nanopore-basedon both sides sensingof the regionsensing and region SU-8 microstructuresconsisting of periodicallyas capillary distributed microfluidics nanopores. on both sidesThese of nanopores the sensing are region used consistingto generate of the periodically optical interference distributed fringesnanopores. that work These as the nanopores transducing are usedsignal to for generate the biosensor. the optical Upon interference delivering the fringes samples that to work the chip, as the thetransducing capillary force signal generated for the biosensor. from the SU-8 Upon nanostructures delivering the automatically samples to the transport chip, the the capillary samples force to thegenerated nanopore from sensing the SU-8 region. nanostructures Upon specific automatically binding between transport the ABA the samples and the toaptamer, the nanopore a phase sensing shift inregion. the transducing Upon specific signal binding occurs. betweenNot only thedid ABAthis sensing and the strategy aptamer, offered a phase exceptional shift in the sensitivity transducing in ABAsignal detection, occurs. Notit also only enabled did this the sensing automation strategy of o fftheered total exceptional sensing process sensitivity without in ABA any detection, external it pumps.also enabled the automation of the total sensing process without any external pumps.

FigureFigure 17. 17. OpticalOptical interferometric interferometric detection-baseddetection-based biosensor: biosensor: (A )( schematicA) schematic of the of sensor the sensor chip; ( Bchip;,C) enlarged (B,C) enlargedview of theview SU8 of microstructures the SU8 microstructures for microfluidics for microfluidics capillary interface; capillary (D) theinterface; optical transducing(D) the optical signal transducingfrom the nanopore-sensing signal from the region.nanopore-sensing Reprinted fromregion. [104 Reprinted], Copyright from 2017, [104] with, Copyright permission 2017, from with the permission30th IEEE from International the 30th IEEE Conference International on Conference Micro Electro on Micro Mechanical Electro Systems Mechanical (MEMS). Systems (MEMS). 3.3.5. Surface Plasmon Resonance (SPR)-Based Detection 3.3.5. Surface Plasmon Resonance (SPR)-Based Detection Surface plasmon resonance (SPR)-based sensing has received increased popularity not only in Surface plasmon resonance (SPR)-based sensing has received increased popularity not only in gas sensing, but also in many other applications including in medical diagnostics, food safety, and gas sensing, but also in many other applications including in medical diagnostics, food safety, and biology [105] due to its advantages that enable label-free detection, real-time detection, high sensitivity biology [105] due to its advantages that enable label-free detection, real-time detection, high as well as its ease of preparation [106]. SPR is an optical technique that relies on the change in the sensitivity as well as its ease of preparation [106]. SPR is an optical technique that relies on the change refractive index of a metal surface on which the target molecules are immobilized. Upon incidence of in the refractive index of a metal surface on which the target molecules are immobilized. Upon light on a thin metal film at a specific angle directed by a prism, it excites a special electromagnetic incidence of light on a thin metal film at a specific angle directed by a prism, it excites a special wave known as surface plasmon at the surface of the metal. Because this incident angle is highly electromagnetic wave known as surface plasmon at the surface of the metal. Because this incident sensitive to the dielectric environment on the opposite side of the metal, any binding event on the

Micromachines 2020, 11, 220 20 of 32 angle is highly sensitive to the dielectric environment on the opposite side of the metal, any binding event on the metal surface causes the angle to shift (from I to II in the lower left-hand diagram of Figure 18A). This shift can be measured non-invasively in real-time [28,107]. Several laboratory scale SPR detection systems for immunosensing and DNA sensing has already been developed, most notably the Biocore from GE Healthcare. However, recently, efforts have been focused on reducing the size and complexity of the SPR by integrating the sensing component with microfluidics for more accurate diagnostic and automated measurements. One such exampleMicromachines is the2020 commercially, 11, 220 available Sensata Spreeta SPR [108] sensor (Figure 18B) developed20 by of 30 Waswa et al. [109] for immunological detection of E. coli O157:H7 in milk, apple juice and ground beef extract. Using the same instrument, a similar assay was designed by Wei et al. [110] to metal surface causes the angle to shift (from I to II in the lower left-hand diagram of Figure 18A). This shift can be measured non-invasively in real-time [28,107].

(A) (B)

Figure 18. Principle of surface plasmon resonance (SPR) biosensor: (A) schematic of a SPR-based Figuredetection 18. Principle method. of Reprinted surface plasmon from [107 resonance], Copyright (SPR) 2002, biosensor: with permission (A) schematic from Springer of a SPR-based Nature; and detection(B) cross-sectional method. Reprinted view of from the Sensata [107], Copyright Spreeta SPR 2002, sensor. with permission Inset shows from the photographSpringer Nature of the ; and actual (B)device. cross-sectional Reprinted view from of [the108 ],Sensata Copyright Spreeta 2003, SPR with sensor. permission Inset shows from Elsevier.the photograph of the actual device. Reprinted from [108], Copyright 2003, with permission from Elsevier. Several laboratory scale SPR detection systems for immunosensing and DNA sensing has already beenSo far, developed, significant most effort notably has been the Biocore directed from towa GErds Healthcare. developing However, metal-based recently, biosensors efforts at have visible been andfocused near-infrared on reducing optical the spectra size and [114–118]. complexity However, of the this SPR was by challenged integrating by the the sensing intrinsic component drawbacks, with suchmicrofluidics as the high forintrinsic more hydrophobicity, accurate diagnostic the high and automatedsurface inertness, measurements. and the large One electronic such example density is the of commerciallystates, presented available by the Sensata traditional Spreeta noble SPR metals [108] sensor. Therefore, (Figure researchers 18B) developed have been by Waswa in search et al. for [109 ] newfor materials immunological that can detection overcome of E. colithoseO157:H7 limitations. in milk, Graphene, apple juice an andemerging ground two-dimensional beef extract. Using material,the same offers instrument, many unique a similar opportunities assay was designed to address by Wei these et al. challenges. [110] to detect Compared Campylobacter to metals, jejune graphenein poultry supports milk. the Digital propagation microfluidics-based of surface plasmons SPR has at also infrared been spectrum implemented with torelatively further reducelow loss the andsample high confinement. volume which In is addition, particularly graphene important enab forles cases efficient where adsorption the analyte of sample biomolecules volume on is scarceits surfaceand precious due to [111its –high113]. surface-to-volume ratio and the π–π stacking interactions with the biomolecules.So far, significantFurthermore, effort an hasexte beenrnal directedgate voltage towards can developingbe used to modulate metal-based thebiosensors frequency atof visiblethe surfaceand near-infrared plasmonics in optical the infrared spectra [114spectrum–118]. However,easily [106]. this Wei was challengedet al. proposed by the a intrinsiccavity-enhanced drawbacks, infraredsuch as sensor the high based intrinsic on hydrophobicity,graphene surface the pl highasmonics surface [119], inertness, wherein and the a largecontinuous electronic and density non- of patternedstates, presented graphene bythat the excites traditional the surface noble plasmon metals. Therefore, on its surface researchers was used have as the been sensing in search medium. for new materialsIn order that to can make overcome the SPR-based those limitations. biosensors Graphene, compatible an emerging with two-dimensionallab-on-a-chip applications, material, offers microfluidicsmany unique is often opportunities integrated to addresswith the these SPR. challenges.In particular, Compared membrane to metals,based microfluidic graphene supports devices the havepropagation widely been of surface used in plasmons the field at of infrared diagnostics spectrum due with to their relatively simple low and loss flexible and high design, confinement. easy constructionIn addition, and graphene cost-effectiveness enables e[120].fficient As adsorption an example, of Chuang biomolecules et al. have on introduced its surface a due membrane- to its high basedsurface-to-volume microfluidic device ratio andintegrated the π– πwithstacking a SPR interactions sensor for with quantitative the biomolecules. detection Furthermore,of interferon an gammaexternal (IFN- gateγ) voltage[120]. This can disposable be used to modulatelow-cost microfluidic the frequency aptasensor of the surface was designed plasmonics in insuch the a infrared way thatspectrum each step easily of washing, [106]. detection, Wei et al. and proposed signal amplification a cavity-enhanced had a separate infrared fluid sensor control based mechanism. on graphene surface plasmonics [119], wherein a continuous and non-patterned graphene that excites the surface plasmon on its surface was used as the sensing medium. In order to make the SPR-based biosensors compatible with lab-on-a-chip applications, microfluidics is often integrated with the SPR. In particular, membrane based microfluidic devices have widely been used in the field of diagnostics due to their simple and flexible design, easy construction and cost-effectiveness [120]. As an example, Chuang et al. have introduced a membrane-based microfluidic device integrated with a SPR sensor for quantitative detection of interferon gamma (IFN-γ)[120]. This Micromachines 2020, 11, 220 21 of 30 disposable low-cost microfluidic aptasensor was designed in such a way that each step of washing, detection, and signal amplification had a separate fluid control mechanism. The sensing procedure was initiated by depositing the washing solution onto the transport membrane for wetting the rayon membrane. The sample solution was then flown through the sensing area and was contained in the transport membrane. Afterward, a controlled amount of streptavidin was introduced to amplify the SPR signal. Any unbound sample and streptavidin were removed through a washing step. This sensor was able to perform IFN-γ detection with a limit of detection of 10 pM within 30 minutes. Although SPR is a well-established technique with excellent sensitivity, some minor disadvantages include the requirement of a thin layer of metal (typically gold), thus raising manufacturing cost, and its strong dependence on temperature variation [28].

3.4. Microfluidic Aptasensor Based on Mass-Based Detection Gravimetric or mass-based biosensors work on the basic principle of measuring the change in the mass at the sensing surface caused by the binding of the analyte to the receptors. Most gravimetric biosensors rely on the use of piezoelectric quartz crystals which can be used in two main formats: as resonators or as surface acoustic waves [121]. The QCM biosensors have been very popular in the area of rapid detection of pathogens [122] and toxins [123] because of their multifarious advantages such as ease of use, shorter analysis time, low-cost, as well as the possibility of label-free and real-time detection. On the other hand, surface acoustic wave (SAW)-based biosensors can detect acoustic waves generated by the interdigital transducers (IDTs) which are periodic metallic bars deposited on a piezoelectric material. Upon recognition of an analyte by the immobilized receptors, the velocity of the SAW changes that produces signal by the driving electronics.

Surface Acoustic Wave (SAW)-Based Biosensor The SAW, which was first described by Lord Rayleigh, refers to the propagation of an acoustic wave along the surface of a piezoelectric material [124]. However, SAW devices could not play an important role in practical applications until White and Voltmer proposed a technique to generate a SAW using interdigital transducers (IDTs) [125] fabricated on the surface of a piezoelectric material. Application of a sinusoidal wave whose period matches the grating period of the IDTs creates a vibration beneath the IDTs generating an acoustic wave that propagates along the surface perpendicular to the direction 5 of the digits in the IDTs. As the wave velocity in the piezoelectric material is 10− times smaller than the electromagnetic wave, the wavelength of the SAW is shorter than the electromagnetic waves by 5 orders of magnitude making it a compact device [124]. SAW biosensors have been applied in clinical diagnosis due to their inherent advantages of high sensitivity, low cost, low power requirement and real-time monitoring capability. The integration of SAW technologies with microfluidics has created a new field called acoustofluidics that offers continuous, rapid and real-time monitoring of biomolecules as well as separation of microparticles based on size [126,127]. As an example, Ahmad et al. [127] have proposed a microfluidic device for size-based acoustofluidic separation of target proteins conjugated to microparticles. This platform contained a PDMS microchannel with an interdigitated transducer which was patterned on top of the piezoelectric lithium niobite (LiNbO3) substrate as a source of high-frequency SAWs. The target-specific aptamer conjugated to streptavidin-functionalized polystyrene microparticles was incubated in the sample mixture and was allowed to form the microparticle-aptamer-target complexes. (Figure 19A). Upon application of high-frequency SAWs, the acoustic radiation force caused the microparticle-aptamer-target complexes to deflect laterally and separate from the mixture to be collected at outlet 2 (Figure 19B). Micromachines 2020, 11, 220 22 of 30 Micromachines 2020, 11, 220 22 of 32

FigureFigure 19.19. SchematicSchematic illustration illustration of ofthe the acoustofluidic acoustofluidic separation separation of target of target protein protein from a from sample a sample mixture.mixture. (A(A)) AA biotin-labeled biotin-labeled aptamer aptamer conjugated conjugated to to streptavidin-functionalized streptavidin-functionalized microparticlesmicroparticles (green) specifically(green) specifically captured captured the target the biomolecules target biomolecules (blue) (blue) from afrom complex a complex mixture. mixture. (B) Microfluidic (B) Microfluidic separation ofseparation target biomolecules of target biomolecules from the non-targetfrom the non-targ ones (red)et ones using (red) surface using surface acoustic acoustic waves waves (SAWs) (SAWs) originating fromoriginating the interdigital from the interdigital transducers transducers (IDTs). (IDTs). Reprinted Reprinted with with permission permission from from [ 127[127].]. Copyright Copyright 2017 American2017 American Chemical Chemical Society. Society.

Love-waveLove-wave sensors, which which are are a special a special type type of SAW of SAW sensors, sensors, offer oincreasedffer increased surface surface sensitivity sensitivity byby reducing reducing energy dissipation dissipation of of the the acoustic acoustic wave wave into into the thefluid. fluid. They They are effective are effective in applications in applications requiringrequiring veryvery highhigh sensitivity for for detection detection of of mass mass loadings loadings in liquids in liquids [126]. [126 Zhang]. Zhang et al. et proposed al. proposed a microfluidica microfluidic aptamer-based aptamer-based love-wave love-wave biosensor biosensor for for real-time real-time monitoring monitoring of of prostate prostate specific specific antigen antigen (PSA) [126]. This SAW-based sensor consists of LiTaO3 substrate with two sets of IDTs (PSA) [126]. This SAW-based sensor consists of LiTaO3 substrate with two sets of IDTs patterned on patterned on SiO2 wave guiding layer as well as a gold film as an aptamer immobilization layer. SiO wave guiding layer as well as a gold film as an aptamer immobilization layer. Subsequently, Subsequently,2 a PDMS-based microfluidic channel was fabricated to allow liquid flow between the aIDTs. PDMS-based Two SAW microfluidic resonators were channel fabricated: was fabricated one acting to allowas a reference liquid flow and betweenthe other the as IDTs.a sensing Two SAW resonatorscomponent. were Under fabricated: this platform, one acting a detecti ason a referencelimit of 10 and mg/mL the otherwas achieved. as a sensing component. Under this platform, a detection limit of 10 mg/mL was achieved. 4. Conclusion and Outlook This review summarizes the current state-of-the-art LOC systems that utilize aptamer-based biosensors. Table1 summarizes them in terms of the device features, target analytes, matrix samples, as well as their microfluidic channel materials. The integration of aptasensors to various forms of microfluidics and paper-based analytical devices have further increased the versatility of aptamers. Although the technology seems promising for potential clinical use, there are still challenges remaining that must be addressed for it to revolutionize the healthcare and diagnostics sector. Designing and selecting a high-affinity aptamer is not a trivial task. Indeed, there are still many biomarkers of critical importance that do not have corresponding aptamers of high affinity and specificity. Therefore, the success of the LOC platforms for aptamer-based biosensing will be dependent on the discovery of new and well-functioning aptamers for the target analyte. As aptamers are generally considered reusable target receptors due to their reversible binding and releasing behavior, a crucial component in the LOC system is the washing step to remove the bound analyte molecules from the aptamers to recalibrate the device for the next measurement. Therefore, having a system that efficiently executes the washing procedures that are reliable and reproducible will greatly enhance the reusability of the LOC-based aptasensors. Moreover, an effective and fast target-removal process will be beneficial to the aptamer-based biosensors in achieving continuous and potentially real-time detection of analyte through the automated process on a chip.

Micromachines 2020, 11, 220 23 of 30

Table 1. Summary of the aptamer-based microfluidic biosensors.

Detection Principle Target Matrix Sample LOD/Linear Range Channel Material Device Features Ref. Amperometry Vasopressin Sheep serum 43 pM PDMS Change in CNT conductance. [40] 100 pM All-PDMS microfluidic chip. Voltammetry (DPV) Norovirus Bovine blood PDMS [43] 100pM–3.5 nM Disposable screen-printed carbon electrode used. 10 pg/mL, Sample volume (<1 µL). Voltammetry (SWV) Cortisol Human serum SU-8, Quartz [44] 30 pg/mL–10 µg/mL Washing steps not required. Transforming growth factor-beta Voltammetry (SWV) Human hepatic stellate cell 1 ppb PDMS Reconfigurable device prevents electrode fouling. [46] 1 (TGF-β1) Human Creatine kinase-muscle/brain embryonic stem Electrochemical Impedimetric 10 pg/mL–100 ng/mL PDMS Heart-on-a-chip cardiac bioreactor formed. [49] (CK-MB) cell-derived cardiomyocytes A549 human lung carcinoma cell 1.5 104 cells/mL, Impedimetric Buffer × PDMS Coplanar 2-electrode configuration used. [50] line 1 105—5 105 cells/mL × × A549 human lung carcinoma cell Self-assembled monolayer (SAM) of AuNPs forms the Impedimetric Whole blood sample - PDMS [51] line detection zone. 0.0128 HAU (hemagglutinin Impedimetric H5N1 Avian influenza virus Buffer PDMS Interdigital gold microelectrode formed. [52] units) T-cell acute lymphoblastic Impedimetric CCRF-CEM and Ramos cells - PDMS Simple detection with digital multimeter. [53] leukemia (ALL) On-chip resistive microheater and temperature sensor arginine vasopressin (AVP) Buffer 1 pM * PDMS [67] FET-based for temperature control. H5N1 Avian influenza virus Buffer 5 pM PDMS Applicable in flow-through sensing. [69] Homogenized Capillary-driven retarding valve helps avoid air capture Fluorescence Ara h 1 56 ng/mL PDMS [74] Biscuit sample in the microchannel. Lysozyme (343 ppb); Lysozyme, Okadaic acid, Fresh egg white, OA (0.4 ppb); PDMS/ Porous paper avoids complicated surface modification. Fluorescence Brevetoxin, Mussel tissue, [75] Brevetoxin (0.56 ppb); ß-cl paper Comparable with ELISA. ß-conglutin lupine Sausage (2.5 ppb) MWCNT: 4.4 ng/mL Spiked mussel GO: 3.3 ng/mL Works for both 1D (MWCNT) and 2D (GO) carbon Fluorescence Norovirus Paper [76] sample 13 ng/mL nanomaterials. to 13 ng/mL Optical MCF-7:62 cells/mL, HL-60:70 Fluorescence MCF-7, HL-60, and K562 Cell culture Paper Different colored QDs enabled naked eye detection. [79] cells/mL, K562: 65 cells/mL Thrombin, prostate specific Fluorescence antigen (PSA), hemagglutinin - - PDMS Enables molecular detection on solid surface. [80] (HA) [81] Fluorescence E-Coli Buffer Single cells PDMS Enables fast and continuous real-time detection.

Two-aptamer microfluidic system improves sensitivity. Fluorescence Influenza A H1N1 virus - 0.032 HAU PDMS Micropump with normally-closed valves enabled [82] efficient transportation of reagents and samples. Micromachines 2020, 11, 220 24 of 30

Table 1. Cont.

Detection Principle Target Matrix Sample LOD/Linear Range Channel Material Device Features Ref. Glycated hemoglobins Reagent consumption and analysis time reduced by 75% Fluorescence Blood - PDMS [83] (HbA1c) & Total and 86%, respectively. hemoglobin (Hb) 68 pg/mL Glass/ Fluorescence Carcinoembryonic antigen (CEA) Human serum Microchip electrophoresis (MCE). [85] 130 pg/mL–8 ng/mL PDMS Kanamycin (Kana), Kana: 0.001 ng/mL–10 ng/mL 300-fold signal amplification compared to Fluorescence Milk samples Synthetic Quartz [87] Oxytetracycline (OTC) OTC: 0.7 pg/mL–0.9 pg/mL non-amplified system. 0.3 pg/mL, Fluorescence Kanamycin (Kana) Milk and fish samples - Reduces matrix interference using ratiometric strategy. [88] 0.8 pg/mL—10 ng/mL 1.5 µM Colorimetry Adenosine Human serum Paper Naked-eye detection. [90] 1.5 µM–19.3 mM Colorimetry Cocaine, adenosine, Pb2+ Urine - Paper Naked-eye detection. [91] Naked-eye and Flatband scanner. Colorimetry Thrombin - 20 pM PDMS [89] AgNPs were used. Optical 0.083 pg/mL Colorimetry Thrombin Human blood PDMS Rolling circle amplification (RCA) used. [95] 0.1–50,000 pg/mL Smart phone/tablet detection Colorimetry PfLDH enzyme (Malaria) Human blood serum 0.01% CLEAR resin Telemedicine application [96] 3D printing system Aptamer-antibody sandwich assay. Chemiluminescence Glycated hemoglobin (HbA1c). Blood 0.65 g/dL PDMS [99] Detection time within 25 min. Droplet microfluidics. Chemiluminescence Lysozyme Human serum 44.6 fM PDMS [100] Very low sample volume of 1 µL. 8.33 pM 3D origami µPAD. Electrochemiluminescence Mucin-1 Human serum Paper [101] 25 pM–50 nM Hybridization chain reaction. 0.17 pM Highly selective. Electrochemiluminescence VEGF-165 protein DMEM cell media PDMS [102] 0.52—52.00 pM In the presence of Ir (III), no signal. Plant hormone abscisic acid SU8/ Capillary microfluidics. Optical interferometry Plant tissue 0.1 µM [104] (ABA) PDMS No external pumps required. Surface plasmon Interferon gamma (IFN-γ) Human plasma 10 pM Paper Membrane-based microfluidic disposable device. [120] resonance Prostate specific 10 ng/mL Surface acoustic wave antigen (PSA) - PDMS Interdigitated transducer. [126] 10 ng/mL—1 µg/mL Mass-based ATP Acoustic wave driven. Surface acoustic wave Thrombin buffer - PDMS [127] Interdigitated transducer. * In all cases, aptamers were used as the target receptors. Micromachines 2020, 11, 220 25 of 30

Author Contributions: Conceptualization, N.I.K. and E.S.; writing—original draft preparation, N.I.K.; writing— review and editing, E.S.; illustration, N.I.K.; supervision, E.S.; funding acquisition, E.S. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the National Science Foundation (Grant No. ECCS-1847152). Conflicts of Interest: The authors declare no conflict of interest.

References

1. Thévenot, D.R.; Toth, K.; Durst, R.A.; Wilson, G.S. Electrochemical biosensors: Recommended definitions and classification. Biosens. Bioelectron. 2001, 16, 121–131. [CrossRef] 2. Tuerk, C.; Gold, L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 1990, 249, 505–510. [CrossRef][PubMed] 3. Ellington, A.D.; Szostak, J.W. In vitro selection of RNA molecules that bind specific ligands. Nature 1990, 346, 818–822. [CrossRef][PubMed] 4. Robertson, D.L.; Joyce, G.F. Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA. Nature 1990, 344, 467–468. [CrossRef][PubMed] 5. Morales, M.A.; Mark Halpern, J. Guide to Selecting a Biorecognition Element for Biosensors. Bioconjug. Chem. 2018, 29, 3231–3239. [CrossRef][PubMed] 6. Minunni, M.; Tombelli, S.; Gullotto, A.; Luzi, E.; Mascini, M. Development of biosensors with aptamers as bio-recognition element: The case of HIV-1 Tat protein. Biosens. Bioelectron. 2004, 20, 1149–1156. [CrossRef] [PubMed] 7. Liss, M.; Petersen, B.; Wolf, H.; Prohaska, E. An Aptamer-Based Quartz Crystal Protein Biosensor. Anal. Chem. 2002, 74, 4488–4495. [CrossRef] 8. Grabowska, I.; Sharma, N.; Vasilescu, A.; Iancu, M.; Badea, G.; Boukherroub, R.; Ogale, S.; Szunerits, S. Electrochemical Aptamer-Based Biosensors for the Detection of Cardiac Biomarkers. ACS Omega 2018, 3, 12010–12018. [CrossRef] 9. Khan, N.I.; Maddaus, A.G.; Song, E. A Low-Cost Inkjet-Printed Aptamer-Based Electrochemical Biosensor for the Selective Detection of Lysozyme. Biosensors 2018, 8, 7. [CrossRef] 10. Crulhas, B.P.; Karpik, A.E.; Delella, F.K.; Castro, G.R.; Pedrosa, V.A. Electrochemical aptamer-based biosensor developed to monitor PSA and VEGF released by prostate cancer cells. Anal. Bioanal. Chem. 2017, 409, 6771–6780. [CrossRef] 11. Rohrbach, F.; Karadeniz, H.; Erdem, A.; Famulok, M.; Mayer, G. Label-free impedimetric aptasensor for lysozyme detection based on carbon nanotube-modified screen-printed electrodes. Anal. Biochem. 2012, 421, 454–459. [CrossRef][PubMed] 12. Cheng, A.K.H.; Ge, B.; Yu, H.-Z. Aptamer-Based Biosensors for Label-Free Voltammetric Detection of Lysozyme. Anal. Chem. 2007, 79, 5158–5164. [CrossRef][PubMed] 13. Li, L.-D.; Chen, Z.-B.; Zhao, H.-T.; Guo, L.; Mu, X. An aptamer-based biosensor for the detection of lysozyme with gold nanoparticles amplification. Sens. Actuators B Chem. 2010, 149, 110–115. [CrossRef] 14. Lian, Y.; He, F.; Mi, X.; Tong, F.; Shi, X. Lysozyme aptamer biosensor based on electron transfer from SWCNTs to SPQC-IDE. Sens. Actuators B Chem. 2014, 199, 377–383. [CrossRef] 15. Liang, G.; Man, Y.; Jin, X.; Pan, L.; Liu, X. Aptamer-based biosensor for label-free detection of ethanolamine by electrochemical impedance spectroscopy. Anal. Chim. Acta 2016, 936, 222–228. [CrossRef] 16. Ikebukuro, K.; Kiyohara, C.; Sode, K. Electrochemical Detection of Protein Using a Double Aptamer Sandwich. Anal. Lett. 2004, 37, 2901–2909. [CrossRef] 17. Rodriguez, M.C.; Kawde, A.-N.; Wang, J. Aptamer biosensor for label-free impedance spectroscopy detection of proteins based on recognition-induced switching of the surface charge. Chem. Commun. Camb. Engl. 2005, 34, 4267–4269. [CrossRef] 18. Zhang, Z.; Yang, W.; Wang, J.; Yang, C.; Yang, F.; Yang, X. A sensitive impedimetric thrombin aptasensor based on polyamidoamine dendrimer. Talanta 2009, 78, 1240–1245. [CrossRef] 19. Yildirim, N.; Long, F.; Gao, C.; He, M.; Shi, H.-C.; Gu, A.Z. Aptamer-Based Optical Biosensor For Rapid and Sensitive Detection of 17β-Estradiol In Water Samples. Environ. Sci. Technol. 2012, 46, 3288–3294. [CrossRef] 20. Dittmer, W.U.; Reuter, A.; Simmel, F.C. A DNA-based machine that can cyclically bind and release thrombin. Angew. Chem. Int. Ed. 2004, 43, 3550–3553. [CrossRef] Micromachines 2020, 11, 220 26 of 30

21. McCauley, T.G.; Hamaguchi, N.; Stanton, M. Aptamer-based biosensor arrays for detection and quantification of biological macromolecules. Anal. Biochem. 2003, 319, 244–250. [CrossRef] 22. Pavlov, V.; Xiao, Y.; Shlyahovsky, B.; Willner, I. Aptamer-Functionalized Au Nanoparticles for the Amplified Optical Detection of Thrombin. J. Am. Chem. Soc. 2004, 126, 11768–11769. [CrossRef][PubMed] 23. Ho, H.-A.; Leclerc, M. Optical Sensors Based on Hybrid Aptamer/Conjugated Polymer Complexes. J. Am. Chem. Soc. 2004, 126, 1384–1387. [CrossRef][PubMed] 24. Mao, S.; Yu, K.; Lu, G.; Chen, J. Highly sensitive protein sensor based on thermally-reduced graphene oxide field-effect transistor. Nano Res. 2011, 4, 921. [CrossRef] 25. Ghosh, S.; Khan, N.I.; Tsavalas, J.G.; Song, E. Selective Detection of Lysozyme Biomarker Utilizing Large Area Chemical Vapor Deposition-Grown Graphene-Based Field-Effect Transistor. Front. Bioeng. Biotechnol. 2018, 6, 29. [CrossRef] 26. Ohno, Y.; Maehashi, K.; Matsumoto, K. Label-Free Biosensors Based on Aptamer-Modified Graphene Field-Effect Transistors. J. Am. Chem. Soc. 2010, 132, 18012–18013. [CrossRef] 27. Kim, Y.S.; Raston, N.H.A.; Gu, M.B. Aptamer-based nanobiosensors. Biosens. Bioelectron. 2016, 76, 2–19. 28. Myers, F.B.; Lee, L.P. Innovations in optical microfluidic technologies for point-of-care diagnostics. Lab. Chip 2008, 8, 2015–2031. [CrossRef] 29. Jiang, Y.; Zou, S.; Cao, X. Rapid and ultra-sensitive detection of foodborne pathogens by using miniaturized microfluidic devices: A review. Anal. Methods 2016, 8, 6668–6681. [CrossRef] 30. Gencoglu, A.; Minerick, A.R. Electrochemical detection techniques in micro- and nanofluidic devices. Microfluid. Nanofluidics 2014, 17, 781–807. [CrossRef] 31. Wang, D.-S.; Fan, S.-K. Microfluidic Surface Plasmon Resonance Sensors: From Principles to Point-of-Care Applications. Sensors 2016, 16, 1175. [CrossRef][PubMed] 32. Chen, S.; Shamsi, M.H. Biosensors-on-chip: A topical review. J. Micromechan. Microeng. 2017, 27, 083001. [CrossRef] 33. Chen, X.; Zhang, S.; Han, W.; Wu, Z.; Chen, Y.; Wang, S. A review on application of graphene-based microfluidics. J. Chem. Technol. Biotechnol. 2018, 93, 3353–3363. [CrossRef] 34. Lafleur, J.P.; Jönsson, A.; Senkbeil, S.; Kutter, J.P. Recent advances in lab-on-a-chip for biosensing applications. Biosens. Bioelectron. 2016, 76, 213–233. [CrossRef][PubMed] 35. Microfluidics for Biological Applications; Tian, W.-C., Finehout, E., Eds.; Springer: New York, NY, USA, 2009; ISBN 978-0-387-09479-3. 36. Weigl, B.H.; Yager, P. Microfluidic Diffusion-Based Separation and Detection. Science 1999, 283, 346–347. [CrossRef] 37. Ansari, M.I.H.; Hassan, S.; Qurashi, A.; Khanday, F.A. Microfluidic-integrated DNA nanobiosensors. Biosens. Bioelectron. 2016, 85, 247–260. [CrossRef] 38. Farzin, L.; Shamsipur, M.; Samandari, L.; Sheibani, S. Recent advances in designing nanomaterial based biointerfaces for electrochemical biosensing cardiovascular biomarkers. J. Pharm. Biomed. Anal. 2018, 161, 344–376. [CrossRef] 39. Hammond, J.L.; Formisano, N.; Estrela, P.; Carrara, S.; Tkac, J. Electrochemical biosensors and nanobiosensors. Essays Biochem. 2016, 60, 69–80. 40. He, P.; Oncescu, V.; Lee, S.; Choi, I.; Erickson, D. Label-free electrochemical monitoring of vasopressin in aptamer-based microfluidic biosensors. Anal. Chim. Acta 2013, 759, 74–80. [CrossRef] 41. Edmonds, T.E. Voltammetric and amperometric transducers. In Chemical Sensors; Edmonds, T.E., Ed.; Springer: Dordrecht, The Netherlands, 1988; pp. 193–213. ISBN 978-94-010-9154-1. 42. Chen, A.; Shah, B. Electrochemical sensing and biosensing based on square wave voltammetry. Anal. Methods 2013, 5, 2158–2173. [CrossRef] 43. Chand, R.; Neethirajan, S. Microfluidic platform integrated with graphene-gold nano-composite aptasensor for one-step detection of norovirus. Biosens. Bioelectron. 2017, 98, 47–53. [CrossRef][PubMed] 44. Sanghavi, B.J.; Moore, J.A.; Chávez, J.L.; Hagen, J.A.; Kelley-Loughnane, N.; Chou, C.-F.; Swami, N.S. Aptamer-functionalized nanoparticles for surface immobilization-free electrochemical detection of cortisol in a microfluidic device. Biosens. Bioelectron. 2016, 78, 244–252. [CrossRef][PubMed] 45. Yang, X.; Kirsch, J.; Fergus, J.; Simonian, A. Modeling analysis of electrode fouling during electrolysis of phenolic compounds. Electrochim. Acta 2013, 94, 259–268. [CrossRef] Micromachines 2020, 11, 220 27 of 30

46. Matharu, Z.; Patel, D.; Gao, Y.; Haque, A.; Zhou, Q.; Revzin, A. Detecting Transforming Growth Factor-β Release from Liver Cells Using an Aptasensor Integrated with Microfluidics. Anal. Chem. 2014, 86, 8865–8872. [CrossRef][PubMed] 47. Guan, J.-G.; Miao, Y.-Q.; Zhang, Q.-J. Impedimetric biosensors. J. Biosci. Bioeng. 2004, 97, 219–226. [CrossRef] 48. Brosel-Oliu, S.; Uria, N.; Abramova, N.; Bratov, A. Impedimetric Sensors for Bacteria Detection. Biosens. Micro Nanoscale Appl. 2015.[CrossRef] 49. Shin, S.R.; Zhang, Y.S.; Kim, D.-J.; Manbohi, A.; Avci, H.; Silvestri, A.; Aleman, J.; Hu, N.; Kilic, T.; Keung, W.; et al. Aptamer-Based Microfluidic Electrochemical Biosensor for Monitoring Cell-Secreted Trace Cardiac Biomarkers. Anal. Chem. 2016, 88, 10019–10027. [CrossRef] 50. Nguyen, N.-V.; Yang, C.-H.; Liu, C.-J.; Kuo, C.-H.; Wu, D.-C.; Jen, C.-P. An Aptamer-Based Capacitive Sensing Platform for Specific Detection of Lung Carcinoma Cells in the Microfluidic Chip. Biosensors 2018, 8, 98. [CrossRef] 51. Nguyen, N.-V.; Jen, C.-P. Selective Detection of Human Lung Adenocarcinoma Cells Based on the Aptamer-Conjugated Self-Assembled Monolayer of Gold Nanoparticles. Micromachines 2019, 10, 195. [CrossRef] 52. Lum, J.; Wang, R.; Hargis, B.; Tung, S.; Bottje, W.; Lu, H.; Li, Y. An Impedance Aptasensor with Microfluidic Chips for Specific Detection of H5N1 Avian Influenza Virus. Sensors 2015, 15, 18565–18578. [CrossRef] 53. Lou, B.; Zhou, Z.; Du, Y.; Dong, S. Resistance-based logic aptamer sensor for CCRF-CEM and Ramos cells integrated on microfluidic chip. Electrochem. Commun. 2015, 59, 64–67. [CrossRef] 54. Lu, G.; Ocola, L.E.; Chen, J. Reduced graphene oxide for room-temperature gas sensors. Nanotechnology 2009, 20, 445502. [CrossRef][PubMed] 55. Huang, Y.-W.; Wu, C.-S.; Chuang, C.-K.; Pang, S.-T.; Pan, T.-M.; Yang, Y.-S.; Ko, F.-H. Real-Time and Label-Free Detection of the Prostate-Specific Antigen in Human Serum by a Polycrystalline Silicon Nanowire Field-Effect Transistor Biosensor. Anal. Chem. 2013, 85, 7912–7918. [CrossRef][PubMed] 56. Zhou, G.; Chang, J.; Cui, S.; Pu, H.; Wen, Z.; Chen, J. Real-Time, Selective Detection of Pb2+ in Water Using a Reduced Graphene Oxide/Gold Nanoparticle Field-Effect Transistor Device. ACS Appl. Mater. Interfaces 2014, 6, 19235–19241. [CrossRef] 57. Mao, S.; Chen, J. Graphene-based electronic biosensors. J. Mater. Res. 2017, 32, 2954–2965. [CrossRef] 58. Mao, S.; Chang, J.; Pu, H.; Lu, G.; He, Q.; Zhang, H.; Chen, J. Two-dimensional nanomaterial-based field-effect transistors for chemical and biological sensing. Chem. Soc. Rev. 2017, 46, 6872–6904. [CrossRef] 59. Cui, Y.; Wei, Q.; Park, H.; Lieber, C.M. Nanowire Nanosensors for Highly Sensitive and Selective Detection of Biological and Chemical Species. Science 2001, 293, 1289–1292. [CrossRef] 60. Cella, L.N.; Chen, W.; Myung, N.V.; Mulchandani, A. Single-Walled Carbon Nanotube-Based Chemiresistive Affinity Biosensors for Small Molecules: Ultrasensitive Glucose Detection. J. Am. Chem. Soc. 2010, 132, 5024–5026. [CrossRef] 61. Song, E.; Choi, J.-W. Conducting Polyaniline Nanowire and Its Applications in Chemiresistive Sensing. Nanomaterials 2013, 3, 498–523. [CrossRef] 62. Xu, S.; Zhan, J.; Man, B.; Jiang, S.; Yue, W.; Gao, S.; Guo, C.; Liu, H.; Li, Z.; Wang, J.; et al. Real-time reliable determination of binding kinetics of DNA hybridization using a multi-channel graphene biosensor. Nat. Commun. 2017, 8, 14902. [CrossRef] 63. Xu, S.; Man, B.; Jiang, S.; Yue, W.; Yang, C.; Liu, M.; Chen, C.; Zhang, C. Direct growth of graphene on quartz substrates for label-free detection of adenosine triphosphate. Nanotechnology 2014, 25, 165702. [CrossRef] [PubMed] 64. Huang, Y.; Dong, X.; Liu, Y.; Li, L.-J.; Chen, P. Graphene-based biosensors for detection of bacteria and their metabolic activities. J. Mater. Chem. 2011, 21, 12358–12362. [CrossRef] 65. Chen, Y.; Ren, R.; Pu, H.; Guo, X.; Chang, J.; Zhou, G.; Mao, S.; Kron, M.; Chen, J. Field-Effect Transistor Biosensor for Rapid Detection of Ebola Antigen. Sci. Rep. 2017, 7.[CrossRef][PubMed] 66. Ohno, Y.; Maehashi, K.; Inoue, K.; Matsumoto, K. Label-Free Aptamer-Based Immunoglobulin Sensors Using Graphene Field-Effect Transistors. Jpn. J. Appl. Phys. 2011, 50, 070120. 67. Yang, J.; Wang, C.; Zhu, Y.; Liu, G.; Lin, Q. A microfluidic aptasensor integrating specific enrichment with a graphene nanosensor for label-free detection of small biomolecules. In Proceedings of the 2015 28th IEEE International Conference on Micro Electro Mechanical Systems (MEMS), Estoril, Portugal, 18–22 January 2015; pp. 569–572. Micromachines 2020, 11, 220 28 of 30

68. Wang, X.; Hao, Z.; Olsen, T.R.; Zhang, W.; Lin, Q. Measurements of aptamer–protein binding kinetics using graphene field-effect transistors. Nanoscale 2019, 11, 12573–12581. [CrossRef] 69. Chan, C.; Shi, J.; Fan, Y.; Yang, M. A microfluidic flow-through chip integrated with reduced graphene oxide transistor for influenza virus gene detection. Sens. Actuators B Chem. 2017, 251, 927–933. [CrossRef] 70. Barroso-Bujans, F.; Cerveny, S.; Verdejo, R.; del Val, J.J.; Alberdi, J.M.; Alegría, A.; Colmenero, J. Permanent adsorption of organic solvents in graphite oxide and its effect on the thermal exfoliation. Carbon 2010, 48, 1079–1087. [CrossRef] 71. Lu, C.-H.; Li, J.; Qi, X.-J.; Song, X.-R.; Yang, H.-H.; Chen, X.; Chen, G.-N. Multiplex detection of nucleases by a graphene-based platform. J. Mater. Chem. 2011, 21, 10915–10919. [CrossRef] 72. He, S.; Song, B.; Li, D.; Zhu, C.; Qi, W.; Wen, Y.; Wang, L.; Song, S.; Fang, H.; Fan, C. A Graphene Nanoprobe for Rapid, Sensitive, and Multicolor Fluorescent DNA Analysis. Adv. Funct. Mater. 2010, 20, 453–459. [CrossRef] 73. Chou, S.S.; De, M.; Luo, J.; Rotello, V.M.; Huang, J.; Dravid, V.P. Nanoscale Graphene Oxide (nGO) as Artificial Receptors: Implications for Biomolecular Interactions and Sensing. J. Am. Chem. Soc. 2012, 134, 16725–16733. [CrossRef] 74. Weng, X.; Neethirajan, S. A microfluidic biosensor using graphene oxide and aptamer-functionalized quantum dots for peanut allergen detection. Biosens. Bioelectron. 2016, 85, 649–656. [CrossRef][PubMed] 75. Weng, X.; Neethirajan, S. Paper-based microfluidic aptasensor for food safety. J. Food Saf. 2018, 38, e12412. [CrossRef] 76. Weng, X.; Neethirajan, S. Aptamer-based fluorometric determination of norovirus using a paper-based microfluidic device. Microchim. Acta 2017, 184, 4545–4552. [CrossRef] 77. Martinez, A.W.; Phillips, S.T.; Butte, M.J.; Whitesides, G.M. Patterned Paper as a Platform for Inexpensive, Low-Volume, Portable Bioassays. Angew. Chem. Int. Ed. 2007, 46, 1318–1320. [CrossRef] 78. Yan, J.; Yan, M.; Ge, L.; Yu, J.; Ge, S.; Huang, J. A microfluidic origami electrochemiluminescence aptamer-device based on a porous Au-paper electrode and a phenyleneethynylene derivative. Chem. Commun. 2013, 49, 1383–1385. [CrossRef][PubMed] 79. Liang, L.; Su, M.; Li, L.; Lan, F.; Yang, G.; Ge, S.; Yu, J.; Song, X. Aptamer-based fluorescent and visual biosensor for multiplexed monitoring of cancer cells in microfluidic paper-based analytical devices. Sens. Actuators B Chem. 2016, 229, 347–354. [CrossRef] 80. Ueno, Y.; Furukawa, K.; Matsuo, K.; Inoue, S.; Hayashi, K.; Hibino, H. On-chip graphene oxide aptasensor for multiple protein detection. Anal. Chim. Acta 2015, 866, 1–9. [CrossRef] 81. Chung, J.; Kang, J.S.; Jurng, J.S.; Jung, J.H.; Kim, B.C. Fast and continuous microorganism detection using aptamer-conjugated fluorescent nanoparticles on an optofluidic platform. Biosens. Bioelectron. 2015, 67, 303–308. [CrossRef] 82. Tseng, Y.-T.; Wang, C.-H.; Chang, C.-P.; Lee, G.-B. Integrated microfluidic system for rapid detection of influenza H1N1 virus using a sandwich-based aptamer assay. Biosens. Bioelectron. 2016, 82, 105–111. [CrossRef] 83. Li, J.; Chang, K.-W.; Wang, C.-H.; Yang, C.-H.; Shiesh, S.-C.; Lee, G.-B. On-chip, aptamer-based sandwich assay for detection of glycated hemoglobins via magnetic beads. Biosens. Bioelectron. 2016, 79, 887–893. [CrossRef] 84. Nuchtavorn, N.; Suntornsuk, W.; Lunte, S.M.; Suntornsuk, L. Recent applications of microchip electrophoresis to biomedical analysis. J. Pharm. Biomed. Anal. 2015, 113, 72–96. [CrossRef][PubMed] 85. Pan, L.; Zhao, J.; Huang, Y.; Zhao, S.; Liu, Y.-M. Aptamer-based microchip electrophoresis assays for amplification detection of carcinoembryonic antigen. Clin. Chim. Acta Int. J. Clin. Chem. 2015, 450, 304–309. [CrossRef][PubMed] 86. Zhou, L.; Gan, N.; Zhou, Y.; Li, T.; Cao, Y.; Chen, Y. A label-free and universal platform for antibiotics detection based on microchip electrophoresis using aptamer probes. Talanta 2017, 167, 544–549. [CrossRef] [PubMed] 87. Wang, Y.; Gan, N.; Zhou, Y.; Li, T.; Hu, F.; Cao, Y.; Chen, Y. Novel label-free and high-throughput microchip electrophoresis platform for multiplex antibiotic residues detection based on aptamer probes and target catalyzed hairpin assembly for signal amplification. Biosens. Bioelectron. 2017, 97, 100–106. [CrossRef] Micromachines 2020, 11, 220 29 of 30

88. He, L.; Shen, Z.; Cao, Y.; Li, T.; Wu, D.; Dong, Y.; Gan, N. A microfluidic chip based ratiometric aptasensor for antibiotic detection in foods using stir bar assisted sorptive extraction and rolling circle amplification. Analyst 2019, 144, 2755–2764. [CrossRef] 89. Zhao, Y.; Liu, X.; Li, J.; Qiang, W.; Sun, L.; Li, H.; Xu, D. Microfluidic chip-based silver nanoparticles aptasensor for colorimetric detection of thrombin. Talanta 2016, 150, 81–87. [CrossRef] 90. Zhang, Y.; Gao, D.; Fan, J.; Nie, J.; Le, S.; Zhu, W.; Yang, J.; Li, J. Naked-eye quantitative aptamer-based assay on paper device. Biosens. Bioelectron. 2016, 78, 538–546. [CrossRef] 91. Wei, X.; Tian, T.; Jia, S.; Zhu, Z.; Ma, Y.; Sun, J.; Lin, Z.; Yang, C.J. Target-Responsive DNA Hydrogel Mediated “Stop-Flow” Microfluidic Paper-Based Analytic Device for Rapid, Portable and Visual Detection of Multiple Targets. Anal. Chem. 2015, 87, 4275–4282. [CrossRef] 92. Li, H.; Zhu, Y.; Dong, S.; Qiang, W.; Sun, L.; Xu, D. Fast functionalization of silver decahedral nanoparticles with aptamers for colorimetric detection of human platelet-derived growth factor-BB. Anal. Chim. Acta 2014, 829, 48–53. [CrossRef] 93. Peng, Y.; Li, L.; Mu, X.; Guo, L. Aptamer-gold nanoparticle-based colorimetric assay for the sensitive detection of thrombin. Sens. Actuators B Chem. 2013, 177, 818–825. [CrossRef] 94. Li, J.; Li, W.; Qiang, W.; Wang, X.; Li, H.; Xu, D. A non-aggregation colorimetric assay for thrombin based on catalytic properties of silver nanoparticles. Anal. Chim. Acta 2014, 807, 120–125. [CrossRef][PubMed] 95. Lin, X.; Chen, Q.; Liu, W.; Li, H.; Lin, J.-M. A portable microchip for ultrasensitive and high-throughput assay of thrombin by rolling circle amplification and hemin/G-quadruplex system. Biosens. Bioelectron. 2014, 56, 71–76. [CrossRef][PubMed] 96. Fraser, L.A.; Kinghorn, A.B.; Dirkzwager, R.M.; Liang, S.; Cheung, Y.-W.; Lim, B.; Shiu, S.C.-C.; Tang, M.S.L.; Andrew, D.; Manitta, J.; et al. A portable microfluidic Aptamer-Tethered Enzyme Capture (APTEC) biosensor for malaria diagnosis. Biosens. Bioelectron. 2018, 100, 591–596. [CrossRef][PubMed] 97. Murthy, K.V.R.; Virk, H.S. Luminescence Phenomena: An Introduction. Available online: https://www. scientific.net/DDF.347.1 (accessed on 12 September 2019). 98. Dehghani, S.; Nosrati, R.; Yousefi, M.; Nezami, A.; Soltani, F.; Taghdisi, S.M.; Abnous, K.; Alibolandi, M.; Ramezani, M. Aptamer-based biosensors and nanosensors for the detection of vascular endothelial growth factor (VEGF): A review. Biosens. Bioelectron. 2018, 110, 23–37. [CrossRef] 99. Chang, K.-W.; Li, J.; Yang, C.-H.; Shiesh, S.-C.; Lee, G.-B. An integrated microfluidic system for measurement of glycated hemoglobin levels by using an aptamer-antibody assay on magnetic beads. Biosens. Bioelectron. 2015, 68, 397–403. [CrossRef] 100. Giuffrida, M.C.; Cigliana, G.; Spoto, G. Ultrasensitive detection of lysozyme in droplet-based microfluidic devices. Biosens. Bioelectron. 2018, 104, 8–14. [CrossRef] 101. Ma, C.; Liu, H.; Zhang, L.; Li, L.; Yan, M.; Yu, J.; Song, X. Microfluidic Paper-Based Analytical Device for Sensitive Detection of Peptides Based on Specific Recognition of Aptamer and Amplification Strategy of Hybridization Chain Reaction. ChemElectroChem 2017, 4, 1744–1749. [CrossRef] 102. Lin, X.; Leung, K.-H.; Lin, L.; Lin, L.; Lin, S.; Leung, C.-H.; Ma, D.-L.; Lin, J.-M. Determination of cell metabolite VEGF165 and dynamic analysis of protein–DNA interactions by combination of microfluidic technique and luminescent switch-on probe. Biosens. Bioelectron. 2016, 79, 41–47. [CrossRef] 103. Campbell, D.P.; McCloskey, C.J. Chapter 9—Interferometric Biosensors. In Optical Biosensors; Ligler, F.S., Rowe Taitt, C.A., Eds.; Elsevier Science: Amsterdam, The Netherlands, 2002; pp. 277–304. ISBN 978-0-444-50974-1. 104. Song, C.; Chen, C.; Che, X.; Wang, W.; Que, L. Detection of plant hormone abscisic acid (ABA) using an optical aptamer-based sensor with a microfluidics capillary interface. In Proceedings of the 2017 IEEE 30th International Conference on Micro Electro Mechanical Systems (MEMS), Las Vegas, NV, USA, 22–26 January 2017; pp. 370–373. 105. Wijaya, E.; Lenaerts, C.; Maricot, S.; Hastanin, J.; Habraken, S.; Vilcot, J.-P.; Boukherroub, R.; Szunerits, S. Surface plasmon resonance-based biosensors: From the development of different SPR structures to novel surface functionalization strategies. Curr. Opin. Solid State Mater. Sci. 2011, 15, 208–224. [CrossRef] 106. Omar, N.A.S.; Fen, Y.W.; Saleviter, S.; Daniyal, W.; Anas, N.A.A.; Ramdzan, N.S.M.; Roshidi, M.D.A. Development of a Graphene-Based Surface Plasmon Resonance Optical Sensor Chip for Potential Biomedical Application. Mater. Basel Switz. 2019, 12, 1928. [CrossRef] 107. Cooper, M.A. Optical biosensors in drug discovery. Nat. Rev. Drug Discov. 2002, 1, 515–528. [CrossRef] [PubMed] Micromachines 2020, 11, 220 30 of 30

108. Chinowsky, T.M.; Quinn, J.G.; Bartholomew, D.U.; Kaiser, R.; Elkind, J.L. Performance of the Spreeta 2000 integrated surface plasmon resonance affinity sensor. Sens. Actuators B Chem. 2003, 91, 266–274. [CrossRef] 109. Waswa, J.; Irudayaraj, J.; DebRoy, C. Direct detection of E. Coli O157:H7 in selected food systems by a surface plasmon resonance biosensor. LWT Food Sci. Technol. 2007, 40, 187–192. [CrossRef] 110. Wei, D.; Oyarzabal, O.A.; Huang, T.-S.; Balasubramanian, S.; Sista, S.; Simonian, A.L. Development of a surface plasmon resonance biosensor for the identification of Campylobacter jejuni. J. Microbiol. Methods 2007, 69, 78–85. [CrossRef][PubMed] 111. Malic, L.; Veres, T.; Tabrizian, M. Two-dimensional droplet-based surface plasmon resonance imaging using electrowetting-on-dielectric microfluidics. Lab Chip 2009, 9, 473–475. [CrossRef] 112. Malic, L.; Veres, T.; Tabrizian, M. Nanostructured digital microfluidics for enhanced surface plasmon resonance imaging. Biosens. Bioelectron. 2011, 26, 2053–2059. [CrossRef] 113. Malic, L.; Veres, T.; Tabrizian, M. Biochip functionalization using electrowetting-on-dielectric digital microfluidics for surface plasmon resonance imaging detection of DNA hybridization. Biosens. Bioelectron. 2009, 24, 2218–2224. [CrossRef] 114. Verellen, N.; Van Dorpe, P.; Huang, C.; Lodewijks, K.; Vandenbosch, G.A.E.; Lagae, L.; Moshchalkov, V.V. Plasmon Line Shaping Using Nanocrosses for High Sensitivity Localized Surface Plasmon Resonance Sensing. Nano Lett. 2011, 11, 391–397. [CrossRef] 115. Liu, N.; Mesch, M.; Weiss, T.; Hentschel, M.; Giessen, H. Infrared Perfect Absorber and Its Application As Plasmonic Sensor. Nano Lett. 2010, 10, 2342–2348. [CrossRef] 116. Aksu, S.; Yanik, A.A.; Adato, R.; Artar, A.; Huang, M.; Altug, H. High-Throughput Nanofabrication of Infrared Plasmonic Nanoantenna Arrays for Vibrational Nanospectroscopy. Nano Lett. 2010, 10, 2511–2518. [CrossRef] 117. Ashley, J.; Li, S.F.Y. An aptamer based surface plasmon resonance biosensor for the detection of bovine catalase in milk. Biosens. Bioelectron. 2013, 48, 126–131. [CrossRef][PubMed] 118. Zhu, Z.; Feng, M.; Zuo, L.; Zhu, Z.; Wang, F.; Chen, L.; Li, J.; Shan, G.; Luo, S.-Z. An aptamer based surface plasmon resonance biosensor for the detection of ochratoxin A in wine and peanut oil. Biosens. Bioelectron. 2015, 65, 320–326. [CrossRef][PubMed] 119. Wei, W.; Nong, J.; Zhu, Y.; Tang, L.; Zhang, G.; Yang, J.; Huang, Y.; Wei, D. Cavity-enhanced continuous graphene plasmonic resonator for infrared sensing. Opt. Commun. 2017, 395, 147–153. [CrossRef] 120. Chuang, T.-L.; Chang, C.-C.; Chu-Su, Y.; Wei, S.-C.; Zhao, X.; Hsueh, P.-R.; Lin, C.-W. Disposable surface plasmon resonance aptasensor with membrane-based sample handling design for quantitative interferon- gamma detection. Lab Chip 2014, 14, 2968–2977. [CrossRef][PubMed] 121. Fu, Z.; Lu, Y.-C.; Lai, J.J. Recent Advances in Biosensors for Nucleic Acid and Exosome Detection. Chonnam Med. J. 2019, 55, 86–98. [CrossRef][PubMed] 122. Ozalp, V.C.; Bayramoglu, G.; Erdem, Z.; Arica, M.Y. Pathogen detection in complex samples by quartz crystal microbalance sensor coupled to aptamer functionalized core-shell type magnetic separation. Anal. Chim. Acta 2015, 853, 533–540. [CrossRef] 123. Neethirajan, S.; Ragavan, V.; Weng, X.; Chand, R. Biosensors for Sustainable Food Engineering: Challenges and Perspectives. Biosensors 2018, 8, 23. [CrossRef] 124. Liu, B.; Chen, X.; Cai, H.; Ali, M.M.; Tian, X.; Tao, L.; Yang, Y.; Ren, T. Surface acoustic wave devices for sensor applications. J. Semicond. 2016, 37, 021001. [CrossRef] 125. White, R.M.; Voltmer, F.W. Direct piezoelectric coupling to surface elastic waves. Appl. Phys. Lett. 1965, 7, 314–316. [CrossRef] 126. Zhang, F.; Li, S.; Cao, K.; Wang, P.; Su, Y.; Zhu, X.; Wan, Y. A Microfluidic Love-Wave Biosensing Device for PSA Detection Based on an Aptamer Beacon Probe. Sensors 2015, 15, 13839–13850. [CrossRef] 127. Ahmad, R.; Destgeer, G.; Afzal, M.; Park, J.; Ahmed, H.; Jung, J.H.; Park, K.; Yoon, T.-S.; Sung, H.J. Acoustic Wave-Driven Functionalized Particles for Aptamer-Based Target Biomolecule Separation. Anal. Chem. 2017, 89, 13313–13319. [CrossRef][PubMed]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).