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Review On-Chip Detection of the Biomarkers for Neurodegenerative Diseases: Technologies and Prospects

Chao Song 1, Suya Que 2, Lucas Heimer 1 and Long Que 1,*

1 Electrical and Computer Engineering Department, Iowa State University, Ames, IA 50011, USA; [email protected] (C.S.); [email protected] (L.H.) 2 Ames High School, Ames, IA 50010, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-515-294-6951



Received: 13 April 2020; Accepted: 23 June 2020; Published: 28 June 2020


Abstract: Alzheimer’s disease (AD), Parkinson’s disease (PD) and glaucoma are all regarded as neurodegenerative diseases (neuro-DDs) because these diseases are highly related to the degeneration loss of functions and death of neurons with aging. The conventional diagnostic methods such as neuroimaging for these diseases are not only expensive but also time-consuming, resulting in significant financial burdens for patients and public health challenge for nations around the world. Hence early detection of neuro-DDs in a cost-effective and rapid manner is critically needed. For the past decades, some chip-based detection technologies have been developed to address this challenge, showing great potential in achieving point-of-care (POC) diagnostics of neuro-DDs. In this review, chip-based detection of neuro-DDs’ biomarkers enabled by different transducing mechanisms is evaluated.

Keywords: neurodegenerative diseases; on-chip detection; point-of-care diagnostics

1. Introduction Worldwide, the elderly population is growing. Within the next 30 years, 30 percent or more of the population will be aged 60 or more all over the world. In the US, the percentage of people age 65 or more is expected to reach 20 percent by 2030 [1]. Elderly people are more likely to be affected by diseases such as glaucoma, hypertension, Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), cardiac disease and cancers. Of these, glaucoma, AD, PD and HD can be classified as neurodegenerative diseases (neuro-DDs) due to degeneration, loss of functions and death of neurons with aging. Currently, these diseases are usually incurable, leading to the progressive loss of structure or function of neurons, and eventually the death of neurons. Diagnosis and treatment of these diseases at their early stages are critical to improve the quality of life for the elderly [2–7]. The ultimate goal of diagnosing preclinical neuro-DDs is to be able to apply some interventions at their early stages, which may postpone, mitigate or even avoid the upcoming neurological impairment that would occur if the conditions are left unattended. However, current clinic diagnostic methods for neuro-DDs are expensive and time-consuming, requiring skilled personnel to operate some sophisticated equipment. Hence, it has been becoming increasingly and critically important to develop simple-to-use, inexpensive, convenient yet reliable diagnostic methods such as chip-based point-of-care (POC) diagnostics. POC diagnostic methods are simple medical tests that can be carried out at the bedside or source-limited settings. Toward this goal, chip-based sensing module is one of the key elements since it can potentially facilitate the monolithically fabrication of the sample preparation module, the signal reading module, the data analysis module and the data transmission module on a single

Micromachines 2020, 11, 629; doi:10.3390/mi11070629 www.mdpi.com/journal/micromachines Micromachines 2020, 11, 629 2 of 27 chip. Currently, while a variety of chip-based sensing platforms have been developed, but in many cases, the reading of transducing signals still requires off-chip instruments, not to mention the data analysis and transmission. For instance, a fluorescence microscope is needed for measuring fluorescent signals from chip-based fluorescence sensors, and an atomic force microscope (AFM), as a standard technique for monitoring micro-cantilever deflection, is required for monitoring the displacement or the resonant frequency of the MEMS cantilever sensors. Clearly, both instruments cannot be readily miniaturized or made portable, resulting in a challenge for their POC applications. Hence, there are still quite a few technical challenges ahead to achieve chip-sensing based POC diagnostics for neuro-DDs. It should be also noted that due to the advances of microelectronics and photonics, it has been becoming increasingly promising to co-fabricate the signal reading elements on the sensor chip [8,9].

1.1. Neuro-DDs and Major Diagnosis Methods

1.1.1. Alzheimer’s Disease AD, a chronic progressive neurodegenerative disease, is the most prevalent type of dementia. Its most common early symptom is short-term memory loss. As the disease progresses, symptoms include problems with language, disorientation (including the likelihood of getting lost), mood swings, loss of motivation, lack of managing self-care and behavior issues. The AD mechanisms have been well established, the main features of the disease are dysfunction of neurons, loss of connections between neurons in close proximity, and subsequent death of neurons [10–12]. Neuron death is attributed mainly to the deposition of Aβ42 protein fibril aggregates onto neurons [13]. Three forms of Aβ42 protein exist in human cerebrospinal fluid (CSF): monomer, oligomer and fibrillar in composition. The monomer form of Aβ42 is dissolved in the CSF, circulating in the system. The oligomer Aβ42 is the bonding form or crosslinked form of monomer Aβ42, which can also be dissolved in CSF and can potentially be crosslinked, forming insoluble fibrillar Aβ42. The insoluble fibrillar Aβ42 deposited onto neurons can disrupt connections between adjacent neurons. Once these connections are lost, neurons die, forming large areas of plaque in the brain. After formation of oligomers and fibrillar Aβ42, the concentration of monomer Aβ42 in CSF is thus decreased. Another phenomenon of AD is the significantly increased concentration of the T-tau biomarker in CSF and blood [14,15]. T-tau consists of proteins that can stabilize microtubules, existing mainly in neural cells of the central nervous system. T-tau, like Aβ42, is considered toxic to neurons. The tau hypothesis proposes that excessive or abnormal phosphorylation of tau in CSF may result in the transformation of normal adult tau into the paired helical filament-tau and neurofibrillary tangles (NFT) [16]. The accumulation of hyperphosphorylated tau in neurons is believed to lead to neurofibrillary degeneration [17]. Such tangles are toxic to cells, resulting in cell death and cognitive decline. Tau can also be toxic to neurons by accumulation inside the cells, through a process involving enzyme stimulated phosphorylation of tau. For the past decades, Aβ42, T-tau and phosphorylated tau (P-tau) have been widely regarded as the promising clinic biomarkers for AD [18–20]. Clinically relevant levels of Aβ42 and T-tau in CSF are 382.2 102.0 pg/mL and at least ± >300 pg/mL, respectively [12,14]. Current diagnostic methods for AD are based on neuron imaging including magnetic resonance imaging (MRI), positron emission tomography (PET) and single photon emission computed tomography (SPECT) [21]. They are expensive and time-consuming for diagnosing AD. In addition, the challenges for these methods are for increased sensitivity and specificity in the diagnosis of AD. Hence, new sensitive, inexpensive and rapid diagnostics methods are urgently needed, particularly in the early stages of AD or before symptoms occur.

1.1.2. Parkinson’s Disease PD is another chronic progressive neurodegenerative disease, which is the second most common neurodegenerative disorder, affecting 1–2% of the population over the age of 65 years [22]. The loss of dopaminergic neurons in the brain region, which is known as substantia nigra (SN), usually results Micromachines 2020, 11, 629 3 of 27 in bradykinesia, rigidity, tremor and postural instability in the patients [23]. One of the pathological features of PD is 50–70% neuronal loss in the SN region. Neuronal inclusions include α-synuclein protein, which are located both in the neuronal cell body (Lewy bodies) and neurites (Lewy neurites) [24]. It has been proposed that oligomers of α-synuclein are the toxic species, which cause the neuronal death in the early stages of PD [25]. Increasing evidence has shown that dysfunctional regulation and misfolding of α-synuclein in Lewy bodies involve in the pathogenesis of PD [25,26]. The monomeric and the aggregated forms of α-synuclein proteins are found in CSF and serum. Formation of plaques due to the aggregation of the α-synuclein causes degeneration of neurons, making it a promising biomarker for early diagnosis and prognosis monitoring of PD [27]. Clearly routine test of α-synuclein in the blood/serum is a more realistic than the test of α-synuclein in CSF for diagnosing PD since collecting CSF requires an invasive procedure. However, α-synuclein levels in the blood from PD patients and healthy individuals are diverse and even contradictory to each other. Specifically, while the clinical relevant levels of α-synuclein in the blood of healthy people is in a range from 77 to 12 ng/mL [28,29], but the elevated levels of α-synuclein from PD patients have been reported by some research [28,30], the decreased levels of α-synuclein from PD patients by other research [31]. More efforts need to be devoted to resolving or explaining the contradict observations. PD is usually diagnosed based on medical history, a review of signs and symptoms, and a neurological and physical examination of the patients. Patients may be also examined by a specific single-photon emission computerized tomography SPECT scan called a dopamine transporter (DAT) scan [32]. The detection of the promising biomarker such as α-synuclein in serum or CSF may provide a complementary diagnosis of PD to improve the diagnostic accuracy. Toward this goal, chip-based detection including paper-based lateral flow assay [33] is an ideal option.

1.1.3. Glaucoma Glaucoma is one type of eye disease that results in damage to the optic nerve and vision loss due to high ocular pressure in the eye. Recent research has shown that glaucoma is also a kind of neuron degenerative disease [34–36]. The disease, which affects about 2 million people in the U.S., occurs more commonly among older people. Screening for glaucoma is usually performed as part of a standard eye examination by an optometrist. Testing for glaucoma should include measurements of the intraocular pressure (IOP) via tonometry, anterior chamber angle examination and examination of the optic nerve for any visible damage. A formal visual field test should be performed. The retinal nerve fiber layer can be assessed with imaging techniques such as optical coherence tomography, scanning laser polarimetry and scanning laser ophthalmoscopy. Current glaucoma screening techniques including IOP measurement have poor sensitivity and are ineffective for early diagnosis of primary glaucoma (PG) [37,38]. Given these limited screening methods, there is a great need for new biomarkers of early PG. Among many possible biomarkers [39,40], of a particular interest is the role of cytokines, which is involved in oxidative stress and inflammation [41]. However, in order to measure, screen and validate the biomarkers, tears have to be obtained from patients and then analyzed using a high-sensitivity enzyme-linked immunosorbent assay (ELISA) and mass spectrometry (MS) [42,43]. These widely used methods for tear-biomarker analysis are expensive, time-consuming and require the collection of tears from the patient. Given the limited amount of tears, the procedure of tear-sampling may be challenging and inconvenient to the patient. Hence, sensors that can monitor the biomarkers in tears in situ are very attractive. In this review, we will only focus on the chip-based detection methods based on MEMS and nanotechnologies [44–47] for monitoring some promising protein biomarkers of AD, PD and glaucoma disease. Micromachines 2020, 11, 629 4 of 27

2. Sensors for Neuro-DDs Biomarker Detection

2.1. Review of Some Chip-Based Sensing Technologies Over the past decades, numerous efforts have been undertaken to develop highly sensitive and Micromachines 2020, 11, x FOR PEER REVIEW 4 of 27 selective sensors for medical diagnosis, disease monitoring, drug discovery, detection of environmental pollutantsterms of transducing and biological mechanisms agents [48]. have Among been them, widely three used. categories The transducing of sensors in mechanisms terms of transducing include mechanismselectrical, mechanical have been and widely optical used. responses. The transducing For instance, mechanisms electrical includetransduction electrical, is enabled mechanical by carbon and opticalnanotubes responses. (CNTs) For or silicon instance, nanowi electricalres (NWs) transduction where isthe enabled electrical by cond carbonuctance nanotubes of the (CNTs) CNTs or silicon nanowiresNWs is modulated (NWs) where upon the the electrical binding conductancebetween the ofre theceptors CNTs (antibodies-Abs) or silicon NWs isimmobilized modulated uponon them the bindingand the betweenprobes (antigens-Ags; the receptors (antibodies-Abs) Figure 1a) [49,50]. immobilized Mechanical on transduction them and the is probes achieved (antigens-Ags; by MEMS Figurecantilevers1a) [ 49(Figure,50]. Mechanical 1b) [51,52] transduction where the isbindin achievedg between by MEMS antigens cantilevers to an (Figure antibody-immobilized1b) [ 51,52] where thecantilever binding surface between changes antigens the to ancantilever’s antibody-immobilized surface stress, cantilever resulting surface in its changesbending the and cantilever’s resonant surfacefrequency stress, shifting. resulting in its bending and resonant frequency shifting.

Figure 1. Two non-optical representative sensing technical platforms: (a) nano-electronics- nanowires (NWs)(NWs) andand ((bb)) micromicro/nano-mechanics-cantilevers./nano-mechanics-cantilevers. ReproducedReproduced from reference [[49]49] withwith permissionpermission fromfrom SpringerSpringer Nature.Nature.

Optical sensingsensing techniquestechniques are basedbased onon variousvarious sensingsensing mechanismsmechanisms includingincluding chemiluminescence,chemiluminescence, fluorescence,fluorescence, light absorption and scattering,scattering, reflectance,reflectance, interference,interference, surfacesurface plasmonplasmon resonanceresonance (SPR) (SPR) and and Raman Raman scattering. scattering. Of these, Of these, fluorescence fluorescence is currently is currently the major the detection major techniquedetection technique in bioscience, in bioscience, chemistry, chemistry, life science, life pharmaceutical science, pharmaceutical and medical and research. medical However,research. attachingHowever, extrinsic attaching tags extrinsic such as tags fluorophores such as fluoroph or quantumores or dots quantum to biomolecules dots to biomolecules for large scale for studies large canscale often studies be can tedious, often expensivebe tedious, and expensive not easily and generalizable.not easily generalizable. Furthermore, Furthermore, this labeling this labeling process mayprocess perturb may orperturb even changeor even their change properties. their properties This is particularly. This is particularly relevant when relevant studying when properties studying ofproperties proteins of [48 proteins,53–55]. [48,53–55]. Subtle changes Subtle inchanges the binding in the abindingffinities affinities and associated and associated kinetics ofkinetics protein of molecules,protein molecules, either by either addition by ofaddi antion extrinsic of antag extrinsic or through tag or tag-induced through tag-induced conformational conformational changes in proteinchanges molecules, in protein can molecules, have a profound can have influence a profound on someinfluence functions on some of protein function molecules.s of protein One molecules. example isOne the example study of is the the recognition study of the of recognition stereo-chemically of stereo-chemically modified double-stranded modified double-stranded DNA (dsDNA) DNA by specialized(dsDNA) by proteins specialized in a proteins living system in a living using system fluorescence-based using fluorescence-based sensing [56 sensing]. Thus [56]. the toolsThus forthe real-timetools for real-time label-free label-free detection, detection, as an emerging as an em techniqueerging technique requiring furtherrequiring development, further development, are becoming are increasinglybecoming increasingly attractive since attractive no significant since no samplesignific preparationant sample suchpreparation as the attachment such as the of attachment fluorophores of orfluorophores quantumdots or quantum to the sample, dots to isthe needed. sample, The is n majoreeded. label-free The major techniques label-free bytechniques optical meansby optical are propagationmeans are propagation surface plasmon surface resonance plasmon resonance (SPR; Figure (SPR;2a) [Figure57], Raman 2a) [57], spectroscopy Raman spectroscopy [58,59], localized [58,59], SPRlocalized (L-SPR) SPR [60 (L-SPR)], liquid [60], core ringliquid resonator core ring technology resonator (Figure technology2b) [ 61 (Figure], photonic 2b) crystal[61], photonic nanostructures crystal (Figurenanostructures2c) [ 53,62 (Figure,63] and interferometer-enabled2c) [53,62,63] and inte biosensorsrferometer-enabled including Mach–Zehnderbiosensors including interferometric Mach– (MZI)Zehnder biosensor interferometric [64], bimodal (MZI) waveguide biosensor [64], interferometric bimodal waveguide (BiMW) biosensor interferometric (Figure (BiMW)2d) [ 8,65 biosensor], Young interferometer (YI) biosensor [66,67], Fabry Pérot interferometric (FPI) biosensor [68] and the nanopore (Figure 2d) [8,65], Young interferometer− (YI) biosensor [66,67], Fabry−Pérot interferometric (FPI) thinbiosensor film-based [68] reflectometricand the nanopore interference thin film-based spectroscopic reflectometric (RIfS) sensors interference [69–72]. spectroscopic (RIfS) sensors [69–72]. For all aforementioned label-free optical biosensors, they can also be categorized based on their specific optical characteristics of transduction: (1) Evanescent-wave biosensors (Figure 2e), in which the transducing signals are achieved by the interactions between the biomolecules and the evanescent wave on the sensing surface, include optical resonators (liquid core ring resonators (LCORR), photonic crystal, etc.), interferometer-enabled biosensors (MZI sensors, FPI sensors, BiMW sensors, YI sensors and RIfS biosensors), SPR sensors, L-SPR sensors, etc. (2) Light scattering biosensors, in which the transducing signals are generated due to the electromagnetic (EM) scattering by the Micromachines 2020, 11, x FOR PEER REVIEW 5 of 27

biomolecules and nanoscale structures on the sensing surface, include Raman spectroscopy sensors, SERS sensors (Figure 2f), etc. A SPR sensor shown in Figure 2a is usually used in the laboratory environment to detect biological and chemical species. Surface plasmon (SP) can be excited at a metal and dielectric interface by a monochromatic or near-monochromatic optical source. The fields associated with the SPR extend into the medium adjacent to the interface and decay exponentially away from it. Penetration into the medium is in the range of <200 nm [57]. Consequently, the SP is very sensitive to the changes in the environment near the interface, and therefore is used as a sensing probe. Upon the excitation of the SPR, a valley in reflectance from the interface occurs. The position of the valley shifts to a different angle of incidence if there are any changes in the local environment at the interface. Raman spectroscopy is a powerful tool for analyzing the chemical composition of materials [73]. It offers information about the material’s electronic and vibrational structure and its distinctive chemical fingerprint for different materials, making it especially attractive for ultra-selective analysis. The surface enhanced Raman spectroscopy (SERS) effect was discovered in the 1970s [74]. The Raman signal of pyridine dramatically increases when absorbed on a roughened Ag electrode (or SERS substrate). The high loss of optical power is a big challenge for maintaining the high sensitivity. Liquid core ring resonators (LCORR) technology (Figure 2b) was demonstrated for multiplexed biosensing by placing LCORR in contact with multiple antiresonant reflecting optical waveguides (ARROWs) [61]. It utilizes the ARROWs to excite the whispering gallery modes of a LCORR sensor. A photonic crystal-based biosensing device is illustrated in Figure 2c [62]. It consists of a silicon waveguide with a 1D photonic crystal microcavity (side resonator) that is adjacent to the waveguide, Micromachineswhich is2020 evanescently, 11, 629 coupled to each other. A change in the refractive index of the near field region 5 of 27 surrounding the optical cavity results in a shift of the resonant wavelength.

Figure 2. (a) A light is beamed upon a metal film through a prism and the reflected beam image shows a dark line due to surface plasmon resonance (SPR). The intensity profile of the reflected beam exhibits a dip or minimal intensity at the resonance angle. A SPR experiment measures the position shift of the dip (the angle shift) upon molecular adsorption, and this shift represents the adsorption kinetics when plotted as a function of time; (b) liquid core ring resonator (LCORR) sensor; (c) photonic crystal-based biosensor; (d) bimodal waveguide interferometric (BiMWI) biosensor; (e) evanescent field biosensor and (f) surface enhanced Raman spectroscopy (SERS) sensor. Reproduced from references [61,62,66,71] with permission from AIP Publishing, Optical Society of America and Royal Society of Chemistry.

For all aforementioned label-free optical biosensors, they can also be categorized based on their specific optical characteristics of transduction: (1) Evanescent-wave biosensors (Figure2e), in which the transducing signals are achieved by the interactions between the biomolecules and the evanescent wave on the sensing surface, include optical resonators (liquid core ring resonators (LCORR), photonic crystal, etc.), interferometer-enabled biosensors (MZI sensors, FPI sensors, BiMW sensors, YI sensors and RIfS biosensors), SPR sensors, L-SPR sensors, etc. (2) Light scattering biosensors, in which the transducing signals are generated due to the electromagnetic (EM) scattering by the biomolecules and nanoscale structures on the sensing surface, include Raman spectroscopy sensors, SERS sensors (Figure2f), etc. A SPR sensor shown in Figure2a is usually used in the laboratory environment to detect biological and chemical species. Surface plasmon (SP) can be excited at a metal and dielectric interface by a monochromatic or near-monochromatic optical source. The fields associated with the SPR extend into the medium adjacent to the interface and decay exponentially away from it. Penetration into the medium is in the range of <200 nm [57]. Consequently, the SP is very sensitive to the changes in the environment near the interface, and therefore is used as a sensing probe. Upon the excitation of the SPR, a valley in reflectance from the interface occurs. The position of the valley shifts to a different angle of incidence if there are any changes in the local environment at the interface. Raman spectroscopy is a Micromachines 2020, 11, 629 6 of 27 powerful tool for analyzing the chemical composition of materials [73]. It offers information about the material’s electronic and vibrational structure and its distinctive chemical fingerprint for different materials, making it especially attractive for ultra-selective analysis. The surface enhanced Raman spectroscopy (SERS) effect was discovered in the 1970s [74]. The Raman signal of pyridine dramatically increases when absorbed on a roughened Ag electrode (or SERS substrate). The high loss of optical power is a big challenge for maintaining the high sensitivity. Liquid core ring resonators (LCORR) technology (Figure2b) was demonstrated for multiplexed biosensing by placing LCORR in contact Micromachines 2020, 11, x FOR PEER REVIEW 6 of 27 with multiple antiresonant reflecting optical waveguides (ARROWs) [61]. It utilizes the ARROWs to excite theFigure whispering 2. (a) A light gallery is beamed modes upon a metal of a film LCORR through sensor.a prism and A the photonic reflected beam crystal-based image shows biosensing device is illustrateda dark line due in to Figure surface2 plasmonc [ 62]. resonance It consists (SPR). of Th ae siliconintensity profile waveguide of the reflected with beam a 1D exhibits photonic crystal microcavitya (sidedip or resonator)minimal intensity that at is the adjacent resonance to angle. the waveguide,A SPR experiment which measures is evanescently the position shift coupled of to each the dip (the angle shift) upon molecular adsorption, and this shift represents the adsorption kinetics other. A changewhen plottedin the as refractive a function of index time; ( ofb) liquid the near core ring field reso regionnator (LCORR) surrounding sensor; ( thec) photonic optical crystal- cavity results in a shift of thebased resonant biosensor; wavelength. (d) bimodal waveguide interferometric (BiMWI) biosensor; (e) evanescent field In the followingbiosensor and sections, (f) surface someenhanced representative Raman spectroscopy sensors (SERS) based sensor. on Reproduc optical,ed from electrical references or mechanical transducing[61,62,66,71] signals for with detecting permission biomarkers from AIP Publishing, of AD, Optical PD and Society glaucoma of America are and detailed. Royal Society of Chemistry. 2.2. AD Biomarker Detection In the following sections, some representative sensors based on optical, electrical or mechanical transducing signals for detecting biomarkers of AD, PD and glaucoma are detailed. Silicon/silicon oxide (Si/SiO2) based sensor: This chip (Figure3) is prepared by depositing a layer of SiO on a silicon wafer [75]. When the thickness of SiO layer is properly selected (100 nm for this 2 2.2. AD Biomarker Detection 2 chip), the emission of any fluorophore of choice can potentially be enhanced due to the constructive interference,Silicon/silicon resulting in oxide significant (Si/SiO2) improvementsbased sensor: This in chip detection (Figure 3) sensitivity. is prepared by Briefly, depositing to detect a layer A β1–42 of SiO2 on a silicon wafer [75]. When the thickness of SiO2 layer is properly selected (100 nm for this and Aβ1–39, the capturing antibodies against Aβ are firstly immobilized on the chip. The chip is then chip), the emission of any fluorophore of choice can potentially be enhanced due to the constructive blockedinterference, with 50 mM resulting ethanolamine in significant solution improvements in 1 M TRISin detection/HCl pH sensitivity. 9 for 1 hBriefly, to mitigate to detect the Aβ non-specific1–42 binding,and followed Aβ1–39, the by capturing rigorous antibodies rinsing with against water Aβ are and firstly drying immobilized under a on stream the chip. of The nitrogen. chip is then Aβ 1–42 or Aβ1–39blocked diluted with in artificial50 mM ethanolamine cerebrospinal solution fluid in (CSF)1 M TRIS/HCl is applied pH 9 on for the 1 h to chip mitigate for incubation. the non-specific Thereafter, the chipbinding, was rigorously followed by washed rigorous with rinsing washing with water bu ffander drying (0.05 M under Tris a/HCl stream pH of 9,nitrogen. 0.25 M A NaCl,β1–42 or 0.05% v/v Aβ1–39 diluted in artificial cerebrospinal fluid (CSF) is applied on the chip for incubation. Thereafter, Tween 20) for 10 min with stirring and then rinsed with water. Biotin-labeled secondary antibody at the chip was rigorously washed with washing buffer (0.05 M Tris/HCl pH 9, 0.25 M NaCl, 0.05% v/v µ 1 g/mLTween in PBS 20) was for 10 applied min with on stirring the chip and for then 1 h rinsed incubation. with water. After Biotin-labeled being washed secondary with PBSantibody and at water for 10 min each,1 µg/mL the in Cyanine PBS was applied 3 labeled on withthe chip streptavidin for 1 h incubation. at 2 µg After/mL being in PBS washed was applied with PBS and and incubatedwater for 1 h, followedfor 10 min by anothereach, the Cyanine round of 3 labeled rinsing with with streptavidin PBS and waterat 2 µg/mL for 10in PBS min was each. applied Finally, and incubated the fluorescence imagesfor and 1 intensitiesh, followed by were another obtained round by of arinsing ProScanArray with PBS and scanner water (PerkinElmer,for 10 min each. Boston, Finally, MA,the USA). It is anticipatedfluorescence that images all these and intensities steps can were be significantly obtained by a simplified,ProScanArray potentially scanner (PerkinElmer, made automatically Boston, and MA, USA). It is anticipated that all these steps can be significantly simplified, potentially made thus more user-friendly by integrating this chip with a microfluidic interface [76]. automatically and thus more user-friendly by integrating this chip with a microfluidic interface [76].

Figure 3.FigureFluorescence 3. Fluorescence images images for fordetecting detecting A Aβ42β42 with with concentrations concentrations from 0 to from 100 ng/mL 0 to 100 using ng SC-/mL using SC-D17D17 (upper (upper array) array) or or Cov-12F4 Cov-12F4 (lower(lower array) array) as as th thee capture capture antibody antibody and Cov-6E10 and Cov-6E10 as the detection as the detection antibody. Reproduced from reference [75] with permission from Elsevier. antibody. Reproduced from reference [75] with permission from Elsevier. Some representative measurements are shown in Figure 3. In these experiments, in order to identify the capturing antibodies to achieve the highest signal intensity and specificity for Aβ42 and Aβ39, SC-D17, NT-11H3, NT-8G7, Cov-4G8 and Cov-12F4 are tested as the capturing antibodies. It has been found that antibodies SC-D17 and Cov-12F4 offer higher signal intensity and specificity for Micromachines 2020, 11, 629 7 of 27

Some representative measurements are shown in Figure3. In these experiments, in order to identify the capturing antibodies to achieve the highest signal intensity and specificity for Aβ42 and β AMicromachines39, SC-D17, 2020, NT-11H3,11, x FOR PEER NT-8G7, REVIEW Cov-4G8 and Cov-12F4 are tested as the capturing antibodies.7 of 27 It has been found that antibodies SC-D17 and Cov-12F4 offer higher signal intensity and specificity forAβ42. Aβ Using42. Using these these two antibodies two antibodies as capturing as capturing antibo antibodies,dies, the fluorescence the fluorescence results resultsof arrays of for arrays the fordetection the detection of 0, 0.1, of 0.5, 0, 0.1,1, 2, 0.5,5, 20, 1, 50 2, and 5, 20, 100 50 ng/mL and 100 of ngAβ/mL42 in of ACSF Aβ42 after in ACSF 2 h of after dynamic 2 h of incubation dynamic incubationare shown arein Figure shown 3. in It Figurehas been3. It found has been that found the optimum that the optimumconditions conditions for detecting for detectingAβ42 are Ato βuse42 areCov-12F4/Cov-6E10 to use Cov-12F4 /matchedCov-6E10 antibody matched pair antibody and 2 h pair of dynamic and 2 h ofincubation. dynamic Due incubation. to the fluorescence Due to the fluorescencesignals enhanced signals by enhancedthe constructive by the interference constructive effe interferencect of the silicon/silicon effect of the siliconoxide thin/silicon film, oxide the limit- thin film,of-detection the limit-of-detection (LOD) of Aβ42 (LOD) in ACSF of Awasβ42 improved in ACSF was to 73.07 improved pg/mL. to 73.07 pg/mL. SERS enabled sensor:sensor: surface-enhanced Raman spectroscopy (SERS) was adopted to measure AAβ42 and T-tau dissolveddissolved inin CSFCSF [[77–79].77–79]. One type of the SERS chip was enabled by core-shellcore-shell nanoparticle attached 2D hybrid graphene oxide based multifunctional nanoplatform (Figure 44))[ [80].80]. It waswas found found that that the the core-shell core-shell nanoparticle nanoparticle was very was e ffveryective effective in enhancing in enhancing Raman signal Raman by generatingsignal by electromagneticgenerating electromagnetic field hot spot. field Graphene hot spot. oxide Graphene can chemically oxide can enhance chemically the Raman enhance signal the byRaman influencing signal theby influencing aromatic molecule the aromatic interaction molecule with interaction a large surface with area.a large surface area.

Figure 4.4.( a()a Schematic) Schematic of plasmonic-magneticof plasmonic-magnetic hybrid hybrid graphene graphene oxide-enabled oxide-enabled surface-enhanced surface-enhanced Raman spectroscopyRaman spectroscopy (SERS) platform(SERS) platform for detecting for detecting AD biomarkers AD biomarkers and (b and) measured (b) measured SERS amideSERS amide I band I intensityband intensity from Afromβ conjugated Aβ conjugated nanoplatform nanoplatform changes changes with with concentration concentration between between 0 and 0 6and pg /6mL. pg/mL. The limit-of-detectionThe limit-of-detection (LOD) (LOD) can be can as lowbe as 500low fg as/mL. 500 Reproduced fg/mL. Reproduced from reference from [80reference] with permission [80] with frompermission American from Chemical American Society. Chemical Society.

Results (Figure4 4b)b) showshow thatthat muchmuch lowerlower concentrationsconcentrations cancan bebe detecteddetected byby thisthis typetype ofof SERSSERS chip than with the ELISA kit (0.312 ngng/mL/mL forfor AAββ42, 0.15 ngng/mL/mL for T-tau). It has been demonstrated as low as 100 fgfg/mL/mL forfor bothboth AAββ42 and T-tau cancan bebe detected.detected. Optical LSPR biosensor: localized surface plasmon resonance (LSPR) has also been utilized to detect amyloid-β derived didiffusibleffusible ligandsligands (ADDLs).(ADDLs). The LSPR nanosensor (Figure5 5a)a) isis fabricatedfabricated by nanosphere lithography [[81].81]. Usually LSPR leads to extraordinary light absorption or reflectionreflection at specificspecific wavelengths.wavelengths. As As a result,a result, the the extraordinary extraordinary light light absorption absorption or reflection or reflection caused acaused light intensity a light peakintensity or dip peak in or the dip broadband in the broadband spectrum. spectrum. The peak The or peak dip in or the dip spectrum in the spectrum was highly was highly related related to the refractiveto the refractive index, shape,index, sizeshape, and size environmental and environmen conditionstal conditions of the substrate of the material.substrate UV-vismaterial. extinction UV-vis measurementsextinction measurements from the sensor from the were sensor collected were collec by anted optical by an fiber optical coupled fiber coupled to a spectrometer to a spectrometer (Ocean Optics,(Ocean Dunedin,Optics, Dunedin, FL, USA; FL, Figure USA;5 a).Figure 5a). Micromachines 2020, 11, 629 8 of 27 Micromachines 2020, 11, x FOR PEER REVIEW 8 of 27

Micromachines 2020, 11, x FOR PEER REVIEW 8 of 27

FigureFigure 5. ( a5.) ( Sketcha) Sketch and and experimental experimental setupsetup for for localized localized surface surface plasmon plasmon resonance resonance (LSPR) (LSPR) based based sensorssensors and and (b) ( someb) some experimental experimental resultsresults of of amyloid- amyloid-β βderivedderived diffusible diffusible ligands ligands (ADDLs) (ADDLs) detection. detection. ReproducedReproducedFigure 5. from (a from) Sketch reference reference and [experimental81 [81]] with with permission perm setupission for from fromlocalized American American surface Chemical Chemicalplasmon Society. resonance Society. (LSPR) based sensors and (b) some experimental results of amyloid-β derived diffusible ligands (ADDLs) detection. UsingUsingReproduced a sandwich a sandwich from assay, reference assay, this this [81] type type with ofof perm nanosensornanosensorission from cancan American quantitatively quantitatively Chemical determine Society. determine the concentration the concentration of ADDLof ADDL (Figure (Figure5b), 5b), providing providing a a unique unique methodmethod for analyzing analyzing the the aggregation aggregation mechanism mechanism of this of this putativeputative ADUsing AD pathogen a pathogen sandwich at at physiologically assay, physiologically this type of relevantrelevant nanosensor monomer monomer can quantitatively concentrations. concentrations. determine Experiments Experiments the concentration found found that that 12 −1 theof bindingADDL (Figure constants 5b), of providing two ADDL a uniqueepitopes method to the specific for analyzing anti-ADDL the aggregation antibodies were mechanism 7.3 × 10 of M this12 1 the binding constants of two ADDL epitopes to the specific anti-ADDL antibodies were 7.3 10 M− andputative 9.5 × AD108 pathogenM−1, respectively. at physiologically The LOD relevant of this monomertype of LSPR concentrations. sensor for Experimentsdetecting ADDL found× was that and 9.5 108 M 1, respectively. The LOD of this type of LSPR sensor for detecting ADDL was conservativelythe ×binding constants− estimated of totwo be ADDL 10 pM. epitopes to the specific anti-ADDL antibodies were 7.3 × 1012 M−1 conservatively estimated to be 10 pM. andArrayed 9.5 × 10 nanopore-based8 M−1, respectively. sensor: The usingLOD theof thisunique type optical of LSPR property sensor of for the detecting nanopore ADDL thin film- was basedArrayedconservatively RIfS nanopore-based sensor estimated for label-free to sensor: be 10biodetection pM. using the [82,83], unique a chip optical consisting property of ofarrayed the nanopore sensors fabricated thin film-based RIfSfrom sensor theArrayed for anodic label-free nanopore-based aluminum biodetection oxide sensor: (AAO) [82 using ,nanopore83], athe chip unique th consistingin film optical on a ofglassproperty arrayed substrate of sensorsthe has nanopore been fabricated developed thin film- from the anodicasbased shown aluminum RIfS in Figuresensor oxide 6afor (AAO)[84]. label-free The nanopore A βbiodetection42 antibody thin film or[82,83], T-tau on a aantibody glasschip consisting substrate were immobilized hasof arrayed been developed onsensors the chemically fabricated as shown in Figurefunctionalizedfrom6a [ 84the]. anodic The sensing A aluminumβ42 antibody surface oxide (Figure or (AAO) T-tau 6b).antibody nanopore Upon the wereth bindingin film immobilized on of a the glass biomarker onsubstrate the chemically to has the been antibody, developed functionalized the sensingreflectedas shown surface optical in (FigureFigure signal 6a6b). [84].(interference Upon The A theβ42 fringes) bindingantibody from oforthe T-tauthe biomarkersensor antibody will weshift. tore the immobilizedThe antibody, shift of the onthe opticalthe reflected chemically signaloptical signalwasfunctionalized (interference used as the sensingtransducing fringes) surface from signal. (Figure the sensorThese 6b). sensors willUpon shift. theon thebinding The chip shift ofwere the of theused biomarker optical as a reference to signal the antibody, wasfor control used theas the experiments, for detection of Aβ42, T-tau and the mixture of Aβ42 and T-tau, respectively. transducingreflected signal. optical signal These (interference sensors on thefringes) chip from were the used sensor as will a reference shift. The for shift control of the optical experiments, signal for was used as the transducing signal. These sensors on the chip were used as a reference for control detection of Aβ42, T-tau and the mixture of Aβ42 and T-tau, respectively. experiments, for detection of Aβ42, T-tau and the mixture of Aβ42 and T-tau, respectively.

Figure 6. (a) Photo of fabricated sensors on chip for detecting Aβ42 and T-tau; (b) sketch showing the functionalized sensor surface for detecting biomarkers; (c) measured transducing signals of Aβ42 in FigureAFigureβ42-T-tau 6. (a 6.) Photo(a mixture) Photo of ofusing fabricated fabricated an Aβ sensors42-sensor sensors on on and chip chip (d )for measured for detecting detecting transducing Aβ42 A βand42 T-tau; andsignals T-tau; (b of) sketch T-tau (b) sketchinshowing Aβ420T- showing the the functionalizedtau-mixturefunctionalized using sensor sensor a T-tau-sensor. surface surface for Reproduced fordetecting detecting biomarkers; from biomarkers; reference (c) measured [84] ( cwith) measured transducing permission transducing fromsignals Elsevier. of Aβ signals42 in of Aβ42A inβ42-T-tau Aβ42-T-tau mixture mixture using an using Aβ42-sensor an Aβ42-sensor and (d) measured and (d )transducing measured signals transducing of T-tau signals in Aβ420T- of T-tau in Aβtau-mixture420T-tau-mixture using a T-tau-sensor. using a T-tau-sensor. Reproduced from Reproduced reference [84] from with reference permission [84 from] with Elsevier. permission from Elsevier. MicromachinesMicromachines2020 2020,,11 11,, 629 x FOR PEER REVIEW 99 ofof 2727

The detection of Aβ42 and T-tau in both buffer and in CSF for AD was carried out. Typical detectionThe detection time was ofless A thanβ42 and20 min T-tau after in a both biom buarkerffer was and applied in CSF foron the AD functionalized was carried out. nanosensor. Typical detectionThe procedure time was for lessthe thandetection 20 min was after much a biomarker faster than was neuroimaging applied on the methods functionalized such as nanosensor. magnetic Theresonant procedure imaging for the (MRI). detection It was was found much that faster as low than as neuroimaging 7.8 pg/mL of methods Aβ42 in suchbuffer as and magnetic 15.6 pg/mL resonant of imagingT-tau in (MRI).buffer can It was be foundreadily that detected as low with as 7.8 high pg/mL specificity of Aβ42 and in bu veryffer good and 15.6 repeatability. pg/mL of T-tau Furthermore, in buffer canthe bedetection readily of detected both A withβ42 and high T-tau specificity spiked and into very CSF good samples repeatability. was demonstrated Furthermore, as shown the detection in Figure of both6c,d. ABasedβ42 and on T-tauthese spikedmeasurements, into CSF the samples feasibility was demonstratedto monitor these as shown biomarkers in Figure in clinical6c,d. Based samples on thesewas demonstrated measurements, using the feasibility the sensors. to monitorIt is anticipate these biomarkersd that the detection in clinical of samples clinic CSF was samples demonstrated can be usingreadily the carried sensors. out It when is anticipated available thatafter the the detection cellular components of clinic CSF in samples the samples can be are readily removed. carried out whenAn available interdigitated after the electrode cellular componentsof the gold (IDE-Au) in the samples sensor: are An removed. electrochemical immunosensor for detectingAn interdigitated Aβ42 was developed. electrode The of thefabricated gold (IDE-Au) IDE-Au sensor sensor: isAn shown electrochemical in Figure 7a,b immunosensor [85]. First, the forAβ42 detecting antibody Aβ 42was was immobilized developed. Theonto fabricated the sensor IDE-Au surface, sensor which is shown was in Figuremodified7a,b by [ 85 ].a First,dithiobissuccinimidyl the Aβ42 antibody propionate was immobilized self-assembled onto monolayer the sensor (DTSP-SAM), surface, which followed was modified by applying by a dithiobissuccinimidylAβ42 on the sensor. Second, propionate the electrochemical self-assembled impedance monolayer spectroscopy (DTSP-SAM), (EIS) followed was carried by applying out to Amonitorβ42 on the impedance sensor. Second, change the during electrochemical the bonding impedance process of spectroscopy Aβ42 antibody (EIS) and was A carriedβ42. Based out on to monitorthe EIS measurements, the impedance changethe developed during theIDE-Au bonding immunosensor process of A hadβ42 a antibody high sensitivity and Aβ 42.of 11 Based kΩ/M on with the EISa regression measurements, coefficient the developedof 0.99 as shown IDE-Au in immunosensor Figure 7c. In addition, had a high this sensitivity sensor was of 11selective kΩ/M withwith aa regressionlimit of detection coefficient (LOD) of 0.99 of as10 shownpM and in had Figure a detection7c. In addition, range thisvarying sensor from was 10 selective pM to 100 with nM. a limit The ofshelf-life detection of (LOD)the IDE-Au of 10 pMimmunosensor and had a detection was also range evaluated. varying It from was 10found pM tothat 100 the nM. sensor The shelf-lifewas stable of thefor IDE-Au30 days immunosensorat 4 °C, while wasthe magnitude also evaluated. of the It waselectrochemical found that theresponse sensor reduced was stable rapidly for 30 after days at30 4days◦C, while(Figure the 7c). magnitude of the electrochemical response reduced rapidly after 30 days (Figure7c).

FigureFigure 7.7. ((aa)) PrinciplePrinciple ofof interdigitatedinterdigitated electrodeelectrode ofof thethe goldgold (IDE-Au)(IDE-Au) immunosensor;immunosensor; ((bb)) thethe systemsystem withwith IDE-AuIDE-Au basedbased immunosensorimmunosensor andand ((cc)) measuredmeasured resultsresults ofof AAββ usingusing thisthis systemsystem andand thethe stabilitystability ofof thethe IDE-AuIDE-Au basedbased immunosensor.immunosensor. ReproducedReproduced fromfrom referencereference [[85]85] withwith permissionpermission fromfrom TheThe RoyalRoyal SocietySociety ofof Chemistry.Chemistry.

Metal–semiconductorMetal–semiconductor field-efield-effectffect transistor transistor (MESFET) (MESFET) based based biosensor: biosensor: a semiconducting a semiconducting carbon nanotubecarbon nanotube (CNT) film-based (CNT) film-based biosensor biosensor with a metal with semiconductora metal semiconductor field effect field transistor effect structuretransistor (CNT-MESFET)structure (CNT-MESFET) has been developedhas been developed for detecting for detecting Aβ as shown Aβ as Figure shown8a [Figure86]. In 8a this [86]. type In ofthis senor, type anof senor, Au strip an wasAu strip fabricated was fabricated on the middle on the of middle the CNT of the film CNT channel, film channel, resulting resulting in a Schottky in a Schottky barrier formingbarrier forming at an interface at an interface between between the Au stripthe Au and strip the and CNT. the CNT. Micromachines 2020, 11, 629 10 of 27 Micromachines 2020, 11, x FOR PEER REVIEW 10 of 27

FigureFigure 8.8. (a) Sketch of metal–semiconductor field-efield-effectffect transistortransistor (MESFET)(MESFET) basedbased sensorsensor andand opticaloptical micrographsmicrographs ofof thethe carboncarbon nanotubenanotube (CNT)(CNT) filmfilm andand thethe procedureprocedure toto obtainobtain thethe sensors;sensors; ((bb)) sketchsketch showingshowing thethe surface surface functionalization functionalization process process and (andc) measured (c) measured results results of Aβ biomarkerof Aβ biomarker with MESFET with sensor.MESFET Reproduced sensor. Reproduced from reference from [referenc86] withe permission[86] with permission from Elsevier. from Elsevier.

ToTo detectdetect AAββ42,42, thethe AAββ antibodyantibody was firstfirst immobilizedimmobilized onon thethe AuAu toptop gategate ofof thethe CNT-MESFETCNT-MESFET biosensorbiosensor (Figure(Figure8 8b).b). When When the the chemical chemical linker linker/A/Aββ antibodyantibody bindsbinds withwith AAββ42,42, thethe threshold voltagevoltage ofof thethe CNT-MESFETCNT-MESFET isis changed.changed. AsAs aa result,result, thethe conductanceconductance ofof channelchannel waswas modulatedmodulated andand soso waswas thethe source-to-drainsource-to-drain currentcurrent ofof thethe CNT-MESFETCNT-MESFET (Figure(Figure8 c).8c). Hence, Hence, the the concentration concentration of of A Aββ4242 couldcould bebe determineddetermined byby measuringmeasuring itsits source-to-drainsource-to-drain current.current. A linearlinear relationshiprelationship waswas observedobserved inin thethe plotplot ofof conductanceconductance changechange versusversus AAββ4242 concentrationconcentration onon thethe semi-logarithmsemi-logarithm scalescale forfor thethe rangerange ofof 12 9 1010−−12–10–10−−9 g/mL.g/mL. It It was was demonstrated demonstrated that that as as low low as as 1 1 pg/mL pg/mL A β4242 could could be readily detected in human serumserum usingusing thethe CNT-MESFETCNT-MESFET biosensor.biosensor.

2.3. PD Biomarker Detection 2.3. PD Biomarker Detection MEMS cantilever detection of PD biomarkers: a variety of biomolecules have been detected using MEMS cantilever detection of PD biomarkers: a variety of biomolecules have been detected the MEMS cantilever device [87–89]. This type of MEMS cantilever device has also been adapted using the MEMS cantilever device [87–89]. This type of MEMS cantilever device has also been for detecting α-synuclein [90]. A functionalized cantilever for detecting α-synuclein and a reference adapted for detecting α-synuclein [90]. A functionalized cantilever for detecting α-synuclein and a cantilever are schematically shown in Figure9a. Silicon cantilever arrays (IBM Research Laboratory, reference cantilever are schematically shown in Figure 9a. Silicon cantilever arrays (IBM Research Switzerland) were used in the experiments. Each cantilever within an array was 500 µm long, 100 µm Laboratory, Switzerland) were used in the experiments. Each cantilever within an array was 500 µm long, 100 µm wide and 1 µm thick (with a 10 nm tolerance of the thickness). The arrayed cantilevers Micromachines 2020, 11, 629 11 of 27 wideMicromachines and 1 µ m2020 thick, 11, x (with FOR PEER a 10 REVIEW nm tolerance of the thickness). The arrayed cantilevers had a pitch 11 of 27 of 250 µm. The use of an array of cantilevers allowed multiple tests and in situ references in one single had a pitch of 250 µm. The use of an array of cantilevers allowed multiple tests and in situ references experiment, thereby resulting in increased throughput experiments. in one single experiment, thereby resulting in increased throughput experiments. Using the MEMS cantilevers, the aggregation of the protein α-synuclein can be detected Using the MEMS cantilevers, the aggregation of the protein α-synuclein can be detected in a in a quantitative, label-free manner. Specifically, a test cantilever was functionalized with the quantitative, label-free manner. Specifically, a test cantilever was functionalized with the dithobis(succinimidyl undecanoate) (DSU) monolayer on top side and PEG saline on back side dithobis(succinimidyl undecanoate) (DSU) monolayer on top side and PEG saline on back side (Figure9a). The DSU monolayer provides the binding sites for the α-synuclein in solution. While (Figure 9a). The DSU monolayer provides the binding sites for the α-synuclein in solution. While the thereference reference cantilever cantilever was was functionalized functionalized with with the theOH OH monolayer monolayer on the on thetop topside side and andPEG PEG saline saline on onthe the back back side side (Figure (Figure 9a).9a). Then Then the thefunctionaliz functionalizeded cantilever cantilever was operated was operated in a dynamic in a dynamic mode in mode the inpresence the presence of α of-synucleinα-synuclein monomers monomers in solution. in solution. The The response response of ofthe the test test cantilever cantilever due due to to any any nonspecificnonspecific adsorption adsorption of of protein protein to to the the either either sideside (PEG(PEG backback side or OH te terminatedrminated top top side) side) of of the the cantilevercantilever could could be be canceled canceled out out by by the the response response from from the the reference reference cantilever. cantilever. As As shown shown in in Figure Figure9b, it9b, was it found was found that approximately that approximately 6 ng of 6α -synucleinng of α-synuclein was aggregated was aggregated on the surface on the of surface the cantilever of the overcantilever a 9-h period, over a and 9-h aperiod, small amountand a small (1 ng) amount of α-synuclein (1 ng) of α was-synuclein removed was from removed the surface from by the following surface monomersby following buff monomerser through buffer the cantilever, through whichthe cantilever, were close which to conventional were close to fluorescence conventional measurements fluorescence ofmeasurementsα-synuclein aggregation of α-synuclein under aggregation similar conditions. under similar In addition, conditions. this In detection addition, method this detection requires a concentrationmethod requires of aα concentration-synuclein protein of α-synuclein 50 times protein smaller 50 than times that smaller of the than fluorescence that of the fluorescence method and potentiallymethod and off erspotentially a faster responseoffers a faster time. response time.

FigureFigure 9. 9.(a ()a) Schematic Schematic of of the the functionalized functionalized MEMSMEMS cantilevercantilever for detecting detecting αα-synuclein-synuclein and and (b (b) )bound bound massmass on on the the surface surface of of the the cantilever cantilever and and frequency frequency ofof thethe cantilevercantilever versus time. The The left left axis axis shows shows thethe bound bound mass mass on on the the cantilever, cantilever, the the right right axisaxis showsshows the correspondi correspondingng differential differential frequency frequency shift. shift. TheThe grey grey area area indicates indicates the the period period that that10 10µ µg/mLg/mL α α-synuclein-synuclein inin 20 mM sodium phosphate phosphate buffer buffer is is flowingflowing through through the the fluidic fluidic chamber chamber atat aa raterate ofof 3.33.3 µµL/min.L/min. Reproduced from from reference reference [90] [90 ]with with permissionpermission from from Hindawi Hindawi Publishing Publishing Corporation. Corporation.

SingleSingle nanopore nanopore for for detecting detecting PD PD biomarkers: biomarkers: solid-statesolid-state single nanopore nanopore has has been been extensively extensively usedused for for DNA DNA sequencing sequencing andand biomolecule detection detection by by monitoring monitoring the the changes changes of ionic of ionic current current or orconductance conductance of of the the single single nanopore nanopore for forthe thepast past years years [91–93]. [91– Detection93]. Detection of α-synuclein of α-synuclein using solid- using solid-statestate nanopore nanopore has has also also been been demonstra demonstratedted [94,95]. [94,95 A]. Aschematic schematic diagram diagram of of αα-synuclein-synuclein protein protein detectiondetection using using a a single single nanopore nanopore is is shown shown inin FigureFigure 1010a.a. A flowflow cell was separated separated by by a a 40 40 nm nm thick thick siliconsilicon nitride nitride membrane membrane with with a 20a 20 nm nm nanopore, nanopore, which which was was formed formed using using a a focused focused electron electron beam beam in TEM.in TEM. Translocation Translocation of α-synuclein of α-synuclein proteins proteins across across the nanopore the nano waspore achieved was achieved by applying by applying an electric an field,electric resulting field, resulting in characteristic in characteristic current blockade current blockade on the ionic on the current ionic trace.current The trace. current The current signal through signal thethrough nanopore the wasnanopore recorded was by recorded two Ag /byAgCl two electrodes Ag/AgCl connectedelectrodes toconnected the patch-clamp to the patch-clamp amplifier by immersingamplifier theby immersing two electrodes the two in two electrodes reservoirs in two of thereserv flowoirs cell of filledthe flow with cell an filled electrolyte with an solution electrolyte (1 M KCl,solution 10 mM (1 HEPES,M KCl, 10 pH mM 9). HEPES, pH 9). Micromachines 2020, 11, 629 12 of 27 Micromachines 2020, 11, x FOR PEER REVIEW 12 of 27

Figure 10. (a) Sketch of the experimental setup: the flow flow cell is separated by silicon nitride membrane withwith aa nanopore.nanopore. The silicon nitride membrane is coated by a layer of tween 20 molecules to mitigate non-specificnon-specific binding;binding; (b(b)) the the chemical chemical structure structure of of tween tween 20; 20; (c )(c sketch) sketch showing showing the the assemble assemble process process of tweenof tween 20 on20 hydrophobic on hydrophobic silicon silicon nitride nitride membrane membrane and the compactand the coatingcompact layer coating reduce layer irreversible reduce non-specificirreversible non-specific adsorption of adsorptionα-synuclein of oligomers α-synuclein and oligomers (d) measured and current (d) measured traces of current nanopore traces under of 100nanopore mV with underα-synuclein 100 mV samplewith α-synuclein incubated sample for 96 h incubated at pH 9. Reproducedfor 96 h at pH from 9. reference Reproduced [94] withfrom permissionreference [94] from with Springer permissi Nature.on from Springer Nature.

InIn orderorder toto mitigatemitigate/avoid/avoid non-specificnon-specific adsorptionadsorption betweenbetween αα-synuclein-synuclein andand nanoporenanopore surfacesurface soso that continuous detection detection of of αα-synuclein-synuclein translocation translocation through through the the nanopores nanopores can can be be realized, realized, a atween tween 20 20 (Figure (Figure 10b,c) 10b,c) coating coating method method on on the the SiN SiN nanopores nanopores was developed. The time-dependent kinetics ofofα α-synuclein-synuclein oligomerization oligomerization was was studied studied using using the the solid-state solid-state nanopores nanopores (Figure (Figure 10d). 10d). In the In experiments,the experiments, it was it foundwas found that four that types four of types oligomers of oligomers were formed were during formed aggregation during aggregation under controlled under incubationcontrolled incubation condition bycondition analyzing by analyzing the translocation the translocation of α-synuclein of α-synuclein incubated incubated over a range over of a times.range Inof addition,times. In the addition, effect of lipid the smalleffect unilamellar of lipid vesiclessmall unilamellar (SUVs) on α -synucleinvesicles (SUVs) oligomerization on α-synuclein process hasoligomerization been investigated, process indicating has been that investigated, the dramatic in enhancementdicating that of the the aggregationdramatic enhancement rate of α-synuclein of the resultsaggregation from therate presence of α-synuclein of 20% results 1,2-dioleoyl-sn-glycero-3-[phospho- from the presence of 20% 1,2-dioleoyl-sn-glycero-3-[phospho-l-serine] (DOPS) [96,97]. All these resultsL-serine] suggest (DOPS) that [96,97]. solid-state All these nanopores results suggest are a suitable that solid-state platform nanopores to investigate are heterogeneousa suitable platform and vitalto investigate oligomerization heterogeneous of α-synuclein and vital in situ, oligomerization thereby providing of α-synuclein important in insights situ, thereby of its aggregation providing process,important which insights is regarded of its aggregation as the key roleprocess, in the which pathogenesis is regarded of PD. as the key role in the pathogenesis of PD.Nanopore thin film sensor: Recently the nanopore thin film-based RIfS sensor fabricated on a glass substrateNanopore have been thin developed film sensor: to detectRecentlyα-synuclein the nanopore in buff thiner and film-based serum in ourRIfS lab sensor [98]. fabricated The illustration on a ofglass the substrate sensor and have its operationalbeen developed principle to detect is shown α-synuclein in Figure in 11buffera. A photoand serum of a fabricated in our lab nanopore [98]. The thinillustration film sensor of the on sensor glass and is givenits operational in Figure principle 11b. A SEMis shown image in Figure of the 11a. nanopores A photo in of the a fabricated thin-film sensornanopore is shownthin film in sensor Figure on 11 c.glass The is fabricationgiven in Figure process 11b. isA similarSEM image to that of the reported nanopores previously in the thin- [99]. Thefilm sensorsensorsurface is shown is in functionalized Figure 11c. The using fabrication EDC/NHS process chemistry is similar so that to that the antibodyreported previously for α-synuclein [99]. canThe besensor immobilized surface is on functi the surface,onalized followed using EDC/NHS by applying chemistr ethanolaminey so that (EA) the antibody on the sensor for α in-synuclein order to mitigatecan be immobilized or avoid non-specific on the surface, binding. followed by applying ethanolamine (EA) on the sensor in order to mitigate or avoid non-specific binding. Micromachines 2020, 11, 629 13 of 27 Micromachines 2020, 11, x FOR PEER REVIEW 13 of 27

FigureFigure 11.11. ((aa)) SchematicSchematic ofof thethe nanoporenanopore thinthin filmfilm sensorsensor onon glass;glass; ((bb)) photophoto ofof aa nanoporenanopore thinthin filmfilm sensorsensor fabricated fabricated on on glass; glass; ((cc)) SEMSEM imagesimages ofof thethe nanoporesnanopores inin thethe sensor;sensor; ((dd)) representativerepresentative measuredmeasured opticaloptical signalssignals (i.e., (i.e., interference interference fringe fringe shift) shift) with with increased increased concentrations concentrations of α -synucleinof α-synuclein in bu inffer buffer and (ande) measured (e) measured optical optical signals signals of α-synuclein of α-synuclein in bu ffiner buffer and spiked and spiked in serum. in serum.

ForFor allall thethe measurements,measurements, afterafter applyingapplying thethe samplessamples ofof αα-synuclein,-synuclein, thethe thinthin filmfilm sensorssensors areare placedplaced onon aa stirringstirring plateplate withwith anan incubationincubation timetime ofof 3030 min.min. ThenThen thethe sensorssensors werewere rinsedrinsed rigorouslyrigorously withwith bubufferffer to to remove remove unbounded unbounded chemicals. chemicals. OneOne representativerepresentative measurementmeasurement inin FigureFigure 11 11dd showsshows thethe shiftshift ofof opticaloptical signalsignal fromfrom thethe sensorsensor whenwhen didifferentfferent concentrationsconcentrations ofof αα-synuclein-synuclein inin bubufferffer werewere detected.detected. TheThe measuredmeasured opticaloptical signalsignal ofof αα-synuclein-synuclein inin bubufferffer isis shownshown inin FigureFigure 11 11e,e, clearlyclearly thethe shiftshift ofof thethe opticaloptical signalsignal increasedincreased withwith itsits concentration. concentration. TheThe measurementsmeasurements ofof αα-synuclein-synuclein spikedspiked inin serumserum werewere alsoalso carriedcarried outout asas displayeddisplayed inin FigureFigure 11 11e.e. AsAs shown,shown, thethe measuredmeasured signalssignals inin bubufferffer andand inin serumserum werewere veryvery consistent,consistent, indicatingindicating thethe sensorssensors hadhad veryvery goodgood specificity.specificity. InIn addition,addition, itit waswas foundfound thatthatα α-synuclein-synuclein ofof 1010 ngng/mL/mL couldcould bebe readilyreadily detecteddetected usingusing thisthis typetype ofof sensorssensors withoutwithout anyany optimization.optimization. For specificity specificity evaluati evaluation,on, two two closely closely related related proteins, proteins, A Aββ4242 and and T-tau, T-tau, were were tested. tested. It Itwas was found found the the optical optical signals signals for for A Aββ4242 and and T-tau T-tau were were significantly significantly smaller than thosethose ofof αα-synuclein-synuclein ofof thethe same same concentrations, concentrations, indicating indicating its highits high specificity specificity of the nanoporeof the nanopore thin film thin sensor film for sensor detecting for αdetecting-synuclein. α-synuclein. InterdigitatedInterdigitated electrodeelectrode (IDE)(IDE) aptameraptamer sensor:sensor: silversilver IDEIDE electrodeselectrodes fabricatedfabricated onon siliconsilicon waferwafer havehave beenbeen developeddeveloped toto detectdetect αα-synuclein-synuclein [[100].100]. DiDifferentfferent fromfrom otherother sensors,sensors, thisthis IDEIDE sensorsensor usesuses aptameraptamer asas thethe probeprobe toto detectdetect αα-synuclein-synuclein atat aa lowlow levellevel onon anan amine-modifiedamine-modified IDEIDE sensingsensing surface.surface. AA schematicschematic illustrationillustration ofof thethe IDEIDE sensorsensor forfor detectingdetecting αα-synuclein-synuclein byby anan aptamer-basedaptamer-based probeprobe isis shownshown inin FigureFigure 12 12a.a. TheThe procedureprocedure involvesinvolves thethe followingfollowing steps.steps. First,First, anan IDEIDE waswas functionalizedfunctionalized withwith (3-aminopropyl) (3-aminopropyl) triethoxysilane triethoxysilane (APTES). (APTES). Second, Second, the aptamer-goldthe aptamer-gold nanoparticles nanoparticles (aptamer-GNP) (aptamer- conjugatesGNP) conjugates were immobilized were immobilized on the on surface the surf oface the of functionalized the functionalized IDE. Thereafter,IDE. Thereafter,α-synuclein α-synuclein was detectedwas detected by the by reactionthe reaction with with aptamer. aptamer. Note Note that that in in order order to to mitigate mitigate or or avoid avoid the the non-specificnon-specific binding,binding, thethe concentrationconcentration ofof thethe aptameraptamer needsneeds toto bebe optimized to cover the entire surface of a GNP. AfterAfter identifyingidentifying thethe optimaloptimal concentrationconcentration ofof aptamer,aptamer, thethe aptamer-GNPaptamer-GNP probeprobe waswas preparedprepared andand consequentlyconsequently immobilizedimmobilized onon thethe surfacesurface ofofan an amine-modified amine-modified IDE. IDE. Micromachines 2020, 11, 629 14 of 27 Micromachines 2020, 11, x FOR PEER REVIEW 14 of 27

Figure 12.12. ((aa)) Schematic Schematic illustration illustration of of the surface func functionalizationtionalization steps of the IDE sensorsensor forfor detecting α-synuclein.-synuclein. First, First, the the surface surface is modified is modified by APTES by APTES to immobilize to immobilize the GNP-aptamer, the GNP-aptamer, then thenalpha-synuclein alpha-synuclein is detected. is detected. (b) (Theb) The measured measured responses responses of ofα-synucleinα-synuclein of of different different concentrations, concentrations, and thethe linearlinear regressionregression analysis.analysis. The limit of detection is 10 pM. ReproducedReproduced from reference [[100]100] withwith permissionpermission fromfrom HindawiHindawi PublishingPublishing Corporation.Corporation.

The limitlimit of of detection detection (LOD) (LOD) of αof-synuclein α-synuclein of the of IDE the sensor IDE sensor was evaluated was evaluated by applying by applying different concentrationsdifferent concentrations of α-synuclein of α-synuclein from 10 pM from to 1 10µM. pM In to Figure 1 µM. 12 b,Inthe Figure linear 12b, regression the linear analysis regression with aanalysis series ofwith diff aerent series concentrations of different concentrations of α-synuclein of andα-synuclein a constant and level a constant of aptamer level of was aptamer displayed. was Baseddisplayed. on the Based regression on the analysisregression and analysis a 3σ calculation, and a 3σ calculation, it was found it thatwas thefound LOD that of the the LOD IDE sensorof the wasIDE 10sensor pM. was Specific 10 pM. detection Specific of detectionα-synuclein of α-synuclein was performed was performed on the IDE on sensor. the IDE For sensor. this analysis, For this twoanalysis, proteins, two amyloid-beta proteins, amyloid-beta and tau, were and independently tau, were independently tested. It was foundtested. only It wasα-synuclein found only caused α- asynuclein current change, caused while a current the other change, control while proteins the other did not control show proteins any significant did not changes show inany current significant from thechanges baseline in current level, indicating from the thatbaselineα-synuclein level, indicating could be that specifically α-synuclein detected could by be the specifically sensor without detected any foulingby the sensor effects. without any fouling effects. SPR sensor for for αα-synuclein-synuclein detection: detection: both both SPR SPR and and LSPR-based LSPR-based sensors sensors have have been been utilized utilized for fordetecting detecting α-synucleinα-synuclein [101,102]. [101,102 As]. Asmentioned mentioned in inSection Section 2.1, 2.1 SPR, SPR imaging imaging (SPRi) (SPRi) is is a a label freefree biosensing platformplatform widelywidely usedused forfor studyingstudying biomolecularbiomolecular interactions.interactions. SPRi has also been adapted toto screenscreen forfor thethe peptoidpeptoid withwith highhigh aaffinityffinity andand specificityspecificity toto αα-synuclein-synuclein throughthrough aa combinatorialcombinatorial peptoidpeptoid librarylibrary (Figure(Figure 1313a)a) [[103].103]. In general, α-synuclein ofof a series of concentrations isis injectedinjected intointo thethe flowflow chamberchamber toto detect detect its its binding binding to to the the peptoids, peptoids, which which is is detected detected by by CCD CCD camera, camera, resulting resulting in thein the changes changes of the of reflected the reflected light. Basedlight. onBased these on measurements, these measurements, the binding the affi bindingnity was affinity determined was bydetermined analyzing by the analyzing kinetic interaction the kinetic between interaction the between peptoids the to α peptoids-synuclein. to α-synuclein. Specifically, using SPRi, peptoid α-synuclein binding peptoid-7 (ASBP-7) was identified as a probe that has high affinity and specificity to α-synuclein. Toward this goal, ASBP-7 of different concentrations ranging from 0.125 µM to 1 mM was fixed on the bare gold chip via covalent interaction, and the pure α-synuclein of 1.316 µM was then injected into the flow chamber. As shown in Figure 13b, the highest binding was achieved when the concentration of ASBP-7 was 64 µM. ASBP-7 was then used to detect PD serum. In these experiments, the serum samples from both PD patients and age-matched normal individuals were used. As shown in Figure 13c, a similar trend of the binding signal to that of the pure α-synuclein was observed, namely the binding signal increased first and then declined with the increase of the concentration of ASBP-7. When the concentration of ASBP-7 was between 32 to 512 µM, the binding signals from PD serum was significantly higher than those from normal serum (>1.0 ∆AU). Hence, with ASBP-7 in this appropriate concentration range (32–512 µM), PD could be distinguished from the control by a SPRi platform. Micromachines 2020, 11, 629 15 of 27 Micromachines 2020, 11, x FOR PEER REVIEW 15 of 27

Figure 13. (a) Schematic illustration of surface plasmon resonance imaging (SPRi) for screening the peptoidFigure 13. library. (a) Schematic (b) Measured illustration results forof surface pure α -synucleinplasmon resonance binds to di imagingfferent concentrations (SPRi) for screening of ASBP-7. the (peptoidc) Measured library. results (b) Measured for Parkinson’s results diseasefor pure (PD)α-synuclein serum andbinds for to normal different serum concentrations for healthy of people, ASBP- respectively.7. (c) Measured Clearly, results the for measurements Parkinson’s disease of PD (PD) serum serum are di andfferentiated for normal from serum the normalfor healthy ones people, using ASBP-7respectively. at a concentrationClearly, the measurements range from 32 of toPD 512 serumµM. are Error differentiated bars represent from the the standard normal ones deviation using (ASBP-7n = 3 in at (b a), concentration and n = 11 in range (c)). from Reproduced 32 to 512 from µM. reference Error bars [103 represent] withpermission the standard from deviation American (n = Chemical3 in (b), and Society. n = 11 in (c)). Reproduced from reference [103] with permission from American Chemical Society. 2.4. Glaucoma (GA) Biomarker Detection BiomarkerSpecifically, detection using SPRi, in the peptoid tear film α-synuclein can assist thebinding diagnosis peptoid-7 of early (ASBP-7) GA, which was canidentified potentially as a provideprobe that a complementary has high affinity diagnosing and specificity method to to α the-synuclein. widely used Toward IOP measurementthis goal, ASBP-7 method of fordifferent more accurateconcentrations diagnosis. ranging To date,from there0.125 areµM no to known1 mM highlywas fixed reliable on the liquid bare biomarkers gold chipfor via glaucoma covalent diagnosis,interaction, but and many the pure potential α-synuclein promising of 1.316 ones haveµM was been then identified injected [41 into–43 the]. One flow possible chamber. biomarker As shown is cytokinein Figure Interleukin13b, the highest 12 (IL-12p70) binding was since achieved recent wh studiesen the have concentration found that of the ASBP-7 mean was concentrations 64 µM. ASBP- of IL-12p707 was then in used tear film to detect were significantlyPD serum. In lower these for experi the diagnosedments, the primaryserum samples open-angle from glaucoma both PD patients (POAG) groupand age-matched compared to normal the control individuals group (3.94 were used.2.19 pg As/mL shown in control in Figure vs. 2.31 13c, 1.156a similar pg/mL trend inPOAG; of the ± ± pbinding= 0.035 signal)[21]. to This that indicates of the pure that α measuring-synuclein the was concentration observed, namely of IL12p70 the binding in the tear signal film increased can aid with first theand diagnosis then declined of early with glaucoma, the increase in of addition the concentr to IOPation measurement. of ASBP-7. When As aforementioned, the concentration while of ASBP- there are7 was many between possible 32 to protein 512 µM, biomarkers the binding in tearssignals for from glaucoma, PD serum there was are significantly very few chip-based higher than detection those methodsfrom normal to detect serum them (>1.0 that ΔAU). have Hence, been with reported. ASBP-7 In thisin this regard, appropriate soft contact concentration lens based range sensors (32–512 are aµM), particularly PD could suitable be distinguished platform from for in the situ control detection by a ofSPRi biomarkers platform. in tears [104]. There are some contact lens sensors that have been reported, but only to monitor the glucose in tears for diagnosing diabetes2.4. Glaucoma [105]. (GA) Biomarker Detection RecentlyBiomarker a contactdetection lens in the sensor tear (Figure film can 14 assista–c) forthe detecting diagnosis IL-12p70 of early GA, has beenwhich developed can potentially [106]. Theprovide biomarker a complementary is monitored diagnosing by measuring method the to optical the widely reflection used IOP signal measurement from the nanopore method thinfor more film sensoraccurate embedded diagnosis. in theTo date, contact there lens. are The no binding known of highly the biomarker reliable withliquid the biomarkers functionalized for surfaceglaucoma of thediagnosis, sensor resultsbut many in apotential change ofpromising the optical ones path have diff beenerence identified (OPD), and [41–43]. thus One the shift possible of the biomarker reflected opticalis cytokine signal Interleukin (interference 12 (IL-12p70) fringes) from since the recent sensor. studies The have higher found the concentrationthat the mean ofconcentrations the biomarker, of theIL-12p70 larger in the tear shift film of the were optical significantly signal. lower for the diagnosed primary open-angle glaucoma (POAG) group compared to the control group (3.94 ± 2.19 pg/mL in control vs. 2.31 ± 1.156 pg/mL in POAG; p = 0.035) [21]. This indicates that measuring the concentration of IL12p70 in the tear film can aid with the diagnosis of early glaucoma, in addition to IOP measurement. As aforementioned, while there are many possible protein biomarkers in tears for glaucoma, there are very few chip-based detection methods to detect them that have been reported. In this regard, soft contact lens based Micromachines 2020, 11, 629 16 of 27 Micromachines 2020, 11, x FOR PEER REVIEW 17 of 27

FigureFigure 14.14. ((aa,,bb)) ContactContact lenslens withwith embeddedembedded sensorssensors forfor detectiondetection ofof glaucomaglaucoma biomarkers;biomarkers; ((cc)) SEMSEM imagesimages showingshowing thethe nanoporenanopore inin thethe sensors;sensors; ((dd)) surfacesurface functionalizationfunctionalization ofof thethe sensorssensors onon contactcontact lens;lens; ((ee)) representativerepresentative measuredmeasured opticaloptical signalssignals fromfrom aa sensorsensor onon thethe contactcontact lenslens withwith artificialartificial teartear andand 11 pgpg/mL/mL ILIL 12p7012p70 spikedspiked inin artificialartificial teartear andand ((ff)) measuredmeasured opticaloptical signalssignals fromfrom thethe sensorsensor withwith increasedincreased concentrations concentrations of ILof 12p70IL 12p70 in artificial in artificial tear. Reproduced tear. Reproduc from referenceed from [reference106] with permission[106] with frompermission IEEE. from IEEE.

For the measurements, the surface of the contact lens sensor needs to be functionalized with As aforementioned, to our knowledge, very few chip-based sensing platforms have been the human IL-12p70 antibody first. The detailed procedure is illustrated in Figure 14d. Briefly, developed to monitor biomarkers in tears for glaucoma [109]. However, some reported chip-based the contact lens sensor surface coated with 10 nm Au is functionalized with human IL-12p70 antibody sensors including contact lens-based sensor or sensing methods, which have been used to monitor through 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysulfosuccinimide (NHS) other chemicals or biochemicals (such as lysozyme) in tears, can be potentially modified or adapted chemistry. This is followed by applying 100 µL 1 M ethanolamine (EA) to block the unoccupied to detect GA biomarkers. Note that some solution-based sensors utilizing functionalized gold HSCnanoparticles10COOH/ HSCto detect8OH biomarkers sites activated in tears by thehave EDC also/ NHS.been reported Finally, the[110], sensor which surface are beyond is rinsed the scope with theof this PBS review. buffer toIn removethe following, non-specifically a couple of adsorbed chip-based proteins. sensing Atplatforms this stage, are thedescribed. sensor is ready for measuringContact the lens-based biomarker lysozyme IL-12p70. detection: Mouse IL-12p70 Using co dilutedmmercial in artificialcontact lenses tears (TheraTears,(CLs) to collect Akron, tear Lakesamples, Forest, lysozyme IL, USA) levels with in a concentrationtears is subsequently ranging fromanalyzed 0 to 10by pg a/ mLcost-effective was applied and onto field-portable the sensor surfacereader [111]. for 2 hThe incubation procedure in to sequence, collect tear and samples the shifts is illustrated of the reflected in Figure light 15a. were After collected. CLs are As worn shown for in15 Figuremin, they 14e,f, are by taken increasing off and theimmediately concentration placed of into IL-12p70 several from capped 0 to 101.5 pgmL/mL, collection the shift tubes. increased These fromtubes 1.2 contain to 9 nm. 600 ThisµL of indicates the assay that reaction the sensitivity buffer (0.1 of the M contactsodium lens phosphate, sensor was 0.1 0.78M NaCl, nm/(pg pH/mL) 7.5, forcontaining IL-12p70 2 detection.mM sodium Note azide that as the a preservative). shift of the artificial After the tears CLs (0 pgand/mL reaction of IL-12p70) buffer wassolution 1.2 nm, are indicatingmixed thoroughly, the possible the non-specific CLs are removed binding from of the the minerals tubes. The in theremaining artificial solution tears with is then the antibodies analyzed by of IL-12p70,a mobile-phone which canbased be well-plate nulled by subtractingreader (Figure the shift15b,c) due and to image the artificial processing. tear. The same principle can be applied for other biomarkers by functionalizing different types of antibodies or aptamers on the sensor surface. Hence, the potential glaucoma biomarker in tears could be screened and validated using this contact lens sensor. As a demonstration, neural cells have been cultured on AAO nanopore thin film on polydimethylsiloxane (PDMS) [107]. It has been found that these cells can grow (spread Micromachines 2020, 11, 629 17 of 27 and divide) normally, indicating its good biocompatibility. It is anticipated that the irritation caused by the contact lens can be totally eliminated by using hydroxyethyl methacrylate (HEMA) to replace PDMS for the contact lenses. The fabrication process of contact lenses using HEMA, a copolymer widely used for soft contact lenses [108], is similar to that using PDMS. As aforementioned, to our knowledge, very few chip-based sensing platforms have been developed to monitor biomarkers in tears for glaucoma [109]. However, some reported chip-based sensors including contact lens-based sensor or sensing methods, which have been used to monitor other chemicals or biochemicals (such as lysozyme) in tears, can be potentially modified or adapted to detect GA biomarkers. Note that some solution-based sensors utilizing functionalized gold nanoparticles to detect biomarkers in tears have also been reported [110], which are beyond the scope of this review. In the following, a couple of chip-based sensing platforms are described. Contact lens-based lysozyme detection: Using commercial contact lenses (CLs) to collect tear samples, lysozyme levels in tears is subsequently analyzed by a cost-effective and field-portable reader [111]. The procedure to collect tear samples is illustrated in Figure 15a. After CLs are worn for 15 min, they are taken off and immediately placed into several capped 1.5 mL collection tubes. These tubes contain 600 µL of the assay reaction buffer (0.1 M sodium phosphate, 0.1 M NaCl, pH 7.5, containing 2 mM sodium azide as a preservative). After the CLs and reaction buffer solution are mixed thoroughly, the CLs are removed from the tubes. The remaining solution is then analyzed by a mobile-phone based well-plate reader (Figure 15b,c) and image processing. Micromachines 2020, 11, x FOR PEER REVIEW 18 of 27

FigureFigure 15. 15.(a ()a (i)–(iv)) (i)–(iv) Sketch: Sketch: commercialcommercial contact lenses lenses are are worn worn for for 15 15 min min and and then then are are removed removed andand placed placed in in a a collection collection tube tube filledfilled withwith aa reactionreaction buffer. buffer. The The contact contact lenses lenses are are washed washed in inthe the reactionreaction tube tube and and then then are are removed removed and and discarded. discarded. Of Of the the washed washed solution solution 100 100µ µLL is is mixed mixedwith with 5050µ L ofµL the of fluorescent the fluorescentMicrococcus Micrococcus lysodeikticus lysodeikticuscell cell solution solution in in an anELISA ELISA well and and monitored. monitored. (b (,cb), cThe) The schematicschematic illustration illustration of of a a mobile-phone mobile-phone basedbased well-plate reader reader and and a apicture picture of of some some contact contact lens lens casescases with with the the well-plate well-plate reader. reader. ReproducedReproduced from reference reference [111] [111 ]with with permission permission from from The The Royal Royal Society of Chemistry. Society of Chemistry.

Specifically, utilizing the CL-based mobile sensing approach, the lysozyme levels of a group of healthy participants over a two-week period were monitored. It was found that the lysozyme levels increased from 6.89 ± 2.02 to 10.72 ± 3.22 µg mL−1 (mean ± SD) for CL wearers when they played a mobile-phone game during the wear-duration, inducing an instance of digital eye-strain. In addition, a lysozyme level of 2.43 ± 1.66 µg mL−1 in a patient cohort with dry eye disease (DED) in comparison with that of 6.89 ± 2.02 µg mL−1 of healthy human participants has been observed. This effort suggests that monitoring the chemicals inside tears can be achieved by a simple tear collection method enabled by CLs, followed by a rapid, easy-to-use and cost-effective measurement system. This system can be potentially adapted to detect the GA biomarkers. Eyeglasses-based tear biosensing system: recently non-invasive measurements of tear biomarkers by integrating a microfluidic electrochemical detector into an eyeglasses nose-bridge pad has been demonstrated [112]. Different from the contact lens sensors, this tear-sensing platform is placed outside the eye region. The system is enabled by an electrochemical biosensor enclosed within a microfluidic chamber, while the supporting electronics are attached to the inner frame of the eye glasses’ inner frame. One example is the alcohol biosensor system (Figure 16). It consists of a fluidic device for collecting the tears, an AOx modified electrochemical alcohol detector, wireless electronics and the supporting eyeglasses platform. For tear collection, a strong capillary force in the inlet enabled by the super-hydrophilic membranes is designed to capture the low volume of tears, leading to the alcohol sensor for detection. Using this system, continuously monitoring the tear alcohol levels of human subjects after alcohol consumption has been carried out. The measurements of three volunteers were successfully validated by comparing to concurrent blood alcohol concentration (BAC) values. It is anticipated that this system can be potentially used to screen and validate the GA biomarkers in tears by replacing the alcohol sensor with GA biomarker sensors. Micromachines 2020, 11, 629 18 of 27

Specifically, utilizing the CL-based mobile sensing approach, the lysozyme levels of a group of healthy participants over a two-week period were monitored. It was found that the lysozyme levels increased from 6.89 2.02 to 10.72 3.22 µg mL 1 (mean SD) for CL wearers when they played a ± ± − ± mobile-phone game during the wear-duration, inducing an instance of digital eye-strain. In addition, a lysozyme level of 2.43 1.66 µg mL 1 in a patient cohort with dry eye disease (DED) in comparison ± − with that of 6.89 2.02 µg mL 1 of healthy human participants has been observed. This effort suggests ± − that monitoring the chemicals inside tears can be achieved by a simple tear collection method enabled by CLs, followed by a rapid, easy-to-use and cost-effective measurement system. This system can be potentially adapted to detect the GA biomarkers. Eyeglasses-based tear biosensing system: recently non-invasive measurements of tear biomarkers by integrating a microfluidic electrochemical detector into an eyeglasses nose-bridge pad has been demonstrated [112]. Different from the contact lens sensors, this tear-sensing platform is placed outside the eye region. The system is enabled by an electrochemical biosensor enclosed within a microfluidic chamber, while the supporting electronics are attached to the inner frame of the eye glasses’ inner frame. One example is the alcohol biosensor system (Figure 16). It consists of a fluidic device for collecting the tears, an AOx modified electrochemical alcohol detector, wireless electronics and the supporting eyeglasses platform. For tear collection, a strong capillary force in the inlet enabled by the super-hydrophilic membranes is designed to capture the low volume of tears, leading to the alcohol sensor for detection. Using this system, continuously monitoring the tear alcohol levels of human subjects after alcohol consumption has been carried out. The measurements of three volunteers were successfully validated by comparing to concurrent blood alcohol concentration (BAC) values. It is anticipated that this system can be potentially used to screen and validate the GA biomarkers in tears by replacing the alcohol sensor with GA biomarker sensors. Micromachines 2020, 11, x FOR PEER REVIEW 19 of 27

Figure 16. Eyeglasses-based tear sensing system: photo an andd schematic illustration of the eyeglasses platform consisting of the fluidicfluidic device and wirele wirelessss electronics. A sketch showing enzymatic alcohol detection and signal transduction: ( (aa)) the the baseline of the electrochemical biosensor; ( b) the current change ofof thethe sensorsensor duedue to to the the captured captured tear; tear; (c )(c the) the measured measured alcohol alcohol signal signal by by the the sensor sensor and and (d) the(d) signalthe signal after after drying drying of the of the sensor. sens Reproducedor. Reproduced from from reference reference [112 [112]] with with permission permission from from Elsevier. Elsevier. 3. Summary and Future Directions 3. Summary and Future Directions The number of people suffering from neuro-DDs has been rising due to the increasing average age The number of people suffering from neuro-DDs has been rising due to the increasing average of world population. Hence, sensitive, cost-effective, rapid and reliable diagnostic methods, ideally age of world population. Hence, sensitive, cost-effective, rapid and reliable diagnostic methods, point-of-care (POC) diagnostic methods for neuro-DDs, are greatly needed. With the discoveries of ideally point-of-care (POC) diagnostic methods for neuro-DDs, are greatly needed. With the more and more reliable protein biomarkers in serum, CSF, tear and other biofluids for neuron-DDs, discoveries of more and more reliable protein biomarkers in serum, CSF, tear and other biofluids for it is becoming increasingly possible that the conventional, expensive and time-consuming diagnostic neuron-DDs, it is becoming increasingly possible that the conventional, expensive and time- methods such as neuroimaging or the IOP measurement can be complemented with chip-base detection consuming diagnostic methods such as neuroimaging or the IOP measurement can be complemented methods. For instance, the chip-based sensors including contact-lens sensors can serve as point-of-care with chip-base detection methods. For instance, the chip-based sensors including contact-lens sensors (POC) equipment for early detection in a rapid and cost-effective manner at home. If the sensors can serve as point-of-care (POC) equipment for early detection in a rapid and cost-effective manner detect the changes of concentrations of the proteins that serve as neuro-DDs’ biomarkers, it is a good at home. If the sensors detect the changes of concentrations of the proteins that serve as neuro-DDs’ indication that further tests are required, making the neuroimaging or eye examination in hospital the biomarkers, it is a good indication that further tests are required, making the neuroimaging or eye next step for the patients’ further diagnosis and treatment. By this way, not only the financial burdens examination in hospital the next step for the patients’ further diagnosis and treatment. By this way, not only the financial burdens to the patients and the society can be significantly mitigated, but also the frequency of patients’ visiting to hospital and the diagnostic turnaround time can be reduced greatly. More importantly, when taken together, the POC and neuroimaging diagnostics may dramatically improve the diagnostic accuracy of neuro-DDs in a rapid and inexpensive manner. In this review, some chip-based detection methods for the biomarkers of neuro-DDs have been presented. Until now, not many efforts have been devoted to the chip-based diagnosis of neuro-DDs as evidenced by the limited literatures published in this field (Table 1). Basically, this field is still at its infant stage. Given the many unmet demands of health care from patients with neuro-DDs, it is anticipated that significant research and development efforts will be devoted to this field in the near future. In order to realize POC diagnostics in the future, several challenges still need to be addressed. Undoubtedly, the screening and validation of the most reliable biomarkers in serum, CSF, tears or other biofluids for neuro-DDs in biological and medical research is critical for achieving precise diagnosis and treatment. In terms of technologies, the following aspects of the technology development are equally of great importance. First, given the size of protein biomarkers for neuro- DDs is typically in the range of nanometers, and thus the nanomaterial or nanostructure-enabled sensors are the basis for achieving ultrasensitive detection [113–116], new nanomaterials and new nanofabrication technologies therefore should be explored for further improving the neuro-DDs detection. Second, for achieving early detection, the sensors need to offer sensitivity and LOD of the neuro-DDs’ biomarkers several orders of magnitude better than diagnostically and physiologically relevant levels with excellent specificity. For POC diagnostics of neuro-DDs, the costs, ease-of-use and portability of the chip-based detection also need to be examined [117–119]. It should be noted that the chip-based sensors could be fabricated significantly inexpensive and thus potentially disposable with high-volume production because of the batch-fabrication capability of micro-nano- manufacturing equipment. Most of costs eventually will result from the biomolecules such as antibodies in the bioassay. In this regard, the aptamer is emerging as an ideal candidate to replace the antibody for biodetection, given its low cost, stability and reusability [72,82]. Third, multiplexing Micromachines 2020, 11, 629 19 of 27 to the patients and the society can be significantly mitigated, but also the frequency of patients’ visiting to hospital and the diagnostic turnaround time can be reduced greatly. More importantly, when taken together, the POC and neuroimaging diagnostics may dramatically improve the diagnostic accuracy of neuro-DDs in a rapid and inexpensive manner. In this review, some chip-based detection methods for the biomarkers of neuro-DDs have been presented. Until now, not many efforts have been devoted to the chip-based diagnosis of neuro-DDs as evidenced by the limited literatures published in this field (Table1). Basically, this field is still at its infant stage. Given the many unmet demands of health care from patients with neuro-DDs, it is anticipated that significant research and development efforts will be devoted to this field in the near future. In order to realize POC diagnostics in the future, several challenges still need to be addressed. Undoubtedly, the screening and validation of the most reliable biomarkers in serum, CSF, tears or other biofluids for neuro-DDs in biological and medical research is critical for achieving precise diagnosis and treatment. In terms of technologies, the following aspects of the technology development are equally of great importance. First, given the size of protein biomarkers for neuro-DDs is typically in the range of nanometers, and thus the nanomaterial or nanostructure-enabled sensors are the basis for achieving ultrasensitive detection [113–116], new nanomaterials and new nanofabrication technologies therefore should be explored for further improving the neuro-DDs detection. Second, for achieving early detection, the sensors need to offer sensitivity and LOD of the neuro-DDs’ biomarkers several orders of magnitude better than diagnostically and physiologically relevant levels with excellent specificity. For POC diagnostics of neuro-DDs, the costs, ease-of-use and portability of the chip-based detection also need to be examined [117–119]. It should be noted that the chip-based sensors could be fabricated significantly inexpensive and thus potentially disposable with high-volume production because of the batch-fabrication capability of micro-nano-manufacturing equipment. Most of costs eventually will result from the biomolecules such as antibodies in the bioassay. In this regard, the aptamer is emerging as an ideal candidate to replace the antibody for biodetection, given its low cost, stability and reusability [72,82]. Third, multiplexing capability of the chip-based detection is also of great importance. In other words, the chip-based detection should allow simultaneous detection of a panel of biomarkers of a neurodegenerative disease, which can reduce false positives and thus achieve more reliable detection of the disease. Finally, as a routine procedure, clinic samples need to be processed for the removal of cellular components prior to detection and analysis [120]. The conventional way for sample preparation requires a centrifuge and careful pipetting techniques, which usually requires significant amount of samples and personnel time. In order to take advantages of the chip-based sensing, ideally the sample-preparation functions can be integrated on chip [121,122], thereby minimizing or even eliminating any off-chip steps. Besides the chip-based detection methods described in this review, it should be noted that some nanoparticles (NPs)-solution based sensors have also been developed and shown great promise for detecting the biomarkers of neuro-DDs [110,123–125]. It is anticipated that by integrating the NPs-solution sensors with the chip-based technologies, the sensitivity, LOD and specificity of chip-based detection for neuron-DDs can be further optimized. Micromachines 2020, 11, 629 20 of 27

Table 1. Summary of the chip-based sensing for detecting biomarkers of neurodegenerative diseases (neuro-DDs).

Biomarkers Detected for Neuro-DDs Limit-of-Detection Transducing Mechanism Advantages Limitations AD PD GA (LOD) Need fluorescent tags to samples; no microfluidic Si/SiO thin film High throughput detection; very 2 Aβ42 73.07 pg/mL for Aβ42 interface; fluorescence fluorescence sensor [75] good sensitivity microscope is needed for measurement Unsuitable for high-throughput Nanoparticle and graphene 100 fg/mL for Aβ42 No sample preparation; label-free; Aβ42, T-tau detection; SERS testing setup oxide-enabled SERS [80] 100 fg/mL for T-tau ultra-sensitivity and equipment is needed Optical Ultraviolet-visible extinction Nanostructures-enabled High throughput detection; ADDLs 10 pM for ADDLs spectroscopy is needed for L-SPR sensor [81] label-free the test Label-free; possible high SPR testing setup and SPR [101] α-Syn <1.3 µM for α-Syn throughput detection equipment is needed 7.8 pg/mL for Aβ42 Nanopore thin film sensor 15.6 pg/mL for T-tau High throughput detection; Reflectance spectroscopy is Aβ42, T-tau α-Syn IL-12p70 [84,98,106] <10 ng/mL for α-Syn label-free; very good sensitivity need for the test 2 pg/mL for IL-12p60 No microfluidic interface; Interdigitated electrode 10 pM for Aβ42 Aβ42 α-Syn Label-free; very good sensitivity Unsuitable for high-throughput (IDE)-based sensor [85,100] 10 pM for α-Syn detection CNT-MESFET: device-to-device Electrical MESFET-based sensor [86]Aβ42 1 pg/mL for Aβ42 Label-free; very good sensitivity variation and performance non-uniformity Relative expensive to fabricate Not available (NA): single single nanopore; need a setup Single nanopore sensor [94] α-Syn molecule detection for Label-free; very good sensitivity with a patch-clamp amplifier α-Syn for test Specific testing setup and MEMS cantilever sensor High throughput detection; equipment is needed for Mechanical α-Syn <6 ng for α-Syn [90] label-free; very good sensitivity monitoring resonance frequency Micromachines 2020, 11, 629 21 of 27

Author Contributions: L.Q. initiated and conceived the topic of this review. C.S., S.Q. and L.H. conducted literature search and discussions for this review article. S.Q. (High School Outreach Program at ISU) and L.H. (The First-Year Honors Program at ISU) carried out research on detection of α-synuclein using nanopore thin film sensor reported in this review article. C.S., S.Q. and L.Q. wrote and edited the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This effort was partly supported by NSF grant ECCS 1461841 and NSF grant ECCS 1610967. Acknowledgments: The authors gratefully acknowledge Pan Deng’s (Postdoc scholar at ISU, now at Chongqing University Cancer Hospital, Chongqing, China) support for this review paper. Conflicts of Interest: The authors declare no conflict of interest.

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