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Peña‑Bahamonde et al. J Nanobiotechnol (2018) 16:75 https://doi.org/10.1186/s12951-018-0400-z Journal of

REVIEW Open Access Recent advances in graphene‑based biosensor technology with applications in life sciences Janire Peña‑Bahamonde†, Hang N. Nguyen†, Sofa K. Fanourakis† and Debora F. Rodrigues*

Abstract Graphene’s unique physical structure, as well as its chemical and electrical properties, make it ideal for use in technologies. In the past years, novel sensing platforms have been proposed with pristine and modifed graphene with and polymers. Several of these platforms were used to immobilize biomolecules, such as antibod‑ ies, DNA, and to create highly sensitive and selective biosensors. Strategies to attach these biomolecules onto the surface of graphene have been employed based on its chemical composition. These methods include covalent bonding, such as the coupling of the biomolecules via the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide reactions, and physisorption. In the literature, several detection methods are employed; however, the most common is electrochemical. The main reason for researchers to use this detection approach is because this method is simple, rapid and presents good sensitivity. These biosensors can be particu‑ larly useful in life sciences and medicine since in clinical practice, biosensors with high sensitivity and specifcity can signifcantly enhance patient care, early diagnosis of diseases and pathogen detection. In this review, we will present the research conducted with , DNA molecules and, enzymes to develop biosensors that use graphene and its derivatives as scafolds to produce efective biosensors able to detect and identify a variety of diseases, pathogens, and biomolecules linked to diseases. Keywords: Nano-biosensors, Graphene, Graphene oxide, DNA, , , Detection, Pathogens

Background allow for the assessment of target biomolecules in real- Conventional sensing methods, such as lateral fow time, which would have broad clinical applications. , fuorescent microarray and electrochemi- in medicine and life sciences have been used cal methods, polymerase chain reaction (PCR)-based to monitor vitals, diagnose patients, and improve the methods, DNA microarrays, DNA sequencing tech- critical care of patients [7–10]. Due to the need for early nology, enzyme-linked immunosorbent assay (ELISA), detection and diagnosis of diseases, as well as minimally among others [1–6] require expensive reagents, high- invasive detection approaches, many novel sensors have precision instruments, and quantifcation methods to been developed. A particular focus of sensor develop- achieve highly sensitive detection. Additionally, most ment has been in miniaturization via application of of the reactions cannot be monitored quantitatively in to fabricate nanosensors. Te nano-sized real-time. Tus, novel sensors that are simple, inexpen- nature of nanomaterials and their unique chemical and sive, and possess highly specifc sensing properties would electrical properties can improve patient care by mak- ing the sensors minimally invasive and extremely sensi- tive [10]. While the sensitivity of the sensors is critical in *Correspondence: [email protected] †Janire Peña-Bahamonde, Hang N. Nguyen and Sofa K. Fanourakis detecting their target molecule, the accuracy and detec- contributed equally to this work tion limit of the sensors are also critical parameters as Department of Civil and Environmental Engineering, University they can infuence their positive and negative predictive of Houston, Houston, TX 77204‑4003, USA

© The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Peña‑Bahamonde et al. J Nanobiotechnol (2018) 16:75 Page 2 of 17

values. Typically, studies report the linear range of bio- nanomaterials are used as transducers of biosen- sensors, which can give the detection limit of the sen- sors, which are involved in converting the interactions sor. However, due to the novelty of recent sensor designs between the and the target molecules into there are no detailed reports or statistics related to accu- detectable measurements [16]. For this to occur, the racy, precision, positive, and negative predictive values of bioreceptor (molecules such as antibodies, ssDNA, and these parameters. Future studies should take into consid- enzymes) needs to be attached to the transducer surface. eration these important parameters. Te most common attachment method used for antibod- Among the nanomaterials used for nano-sensor fab- ies and ssDNA immobilization onto graphene and its rication, graphene and graphene-based nanomaterials derivatives (graphene oxide, reduced graphene oxide) is have been showing the most promise since they present EDC/NHS , while enzymes are most commonly an enhanced signal response in a variety of sensing appli- immobilized using physisorption (see Fig. 2). cations [11–13]. Furthermore, graphene-based nano- Graphene has been employed in the design of difer- materials possess high surface area and ofer excellent ent biosensors of various transduction modes because of with a variety of biomolecules, like its large surface area, electrical conductivity, high elec- antibodies, enzymes, DNA, cells, and [13]. Te tron transfer rate and capacity to immobilize diferent incorporation of such biological molecules in graphene’s molecules [17]. For instance, the conjugated structure detection scheme (Fig. 1) has allowed the development of graphene can facilitate the electron transfer between of the so-called biosensors. Tese biosensors can detect the bioreceptor and transducer, which can generate high multiple molecules, biomolecules and even cells [14, 15]. signal sensitivity for electrochemical sensors [12, 16, 18, 19]. Furthermore, graphene-based nanomaterial can act Graphene‑based nanomaterials as a biosensor as a quencher in the transducer to generate fuorescent In general terms, sensors consist of two elements: a biosensors. Studies have determined that graphene (G), receptor and a transducer (see Fig. 1). Te receptor is graphene oxide (GO), and reduced graphene oxide (rGO) the organic or inorganic material that interact specif- have a very high efciency of fuorescent quenching cally with the target molecule. Te target molecule can [20–22]. be organic, inorganic or even whole cells. Te trans- When using graphene nanomaterials for designing ducer is the part of the sensor, which converts chemical sensors, some aspects of the graphene properties afect- information into a measurable signal. Graphene-based ing the detection limit of the target molecules need to be

Fig. 1 Examples of biosensors and components on a graphene platform Peña‑Bahamonde et al. J Nanobiotechnol (2018) 16:75 Page 3 of 17

Fig. 2 Schematic of the most common attachment methods of bioreceptors, such as antibody, DNA and enzymes onto graphene surfaces

taken into consideration. For instance, diferent synthesis In this mini-review, we will briefy summarize recent batches of graphene and derivatives, as well as diferent developments on biosensor technology with graphene synthetic methods can lead to diferent properties and and graphene-based nanomaterials. More specifcally, we functionalities of the graphene-based nanomaterials in will focus on antibody, DNA and enzyme-based biosen- the biosensors. Te orientation between the G, GO or sors with applications in life sciences as well as in clini- rGO sheets and the bioreceptor can also directly afect cal settings. We aim to present conceptual advances that the selectivity and sensitivity of the biosensors. Addi- have been made in the synthesis and applications of bio- tionally, the number of layers, the functional groups and sensors for clinical diagnosis and real-time molecular oxidation states of graphene and derivatives will cause detection. diferences in the sensing performance among the sen- sors and even impact the bonding between the trans- Graphene‑based nanomaterials and antibodies ducer and bioreceptor. Te amount of functional groups Te analytical detection platforms that measure the spe- on the nanomaterials can also afect the interactions and cifc conjugation reaction between antibody and the detection limit of the target molecule. In this context, are called immunosensors. Te biocompatibility and it is often necessary to block any nonspecifc high-afnity binding of antibodies to make this sites on the nanomaterial to prevent unspecifc binding of molecule attractive for use in several felds, particularly biomolecules instead of the target molecules. Tis can be in diagnostics. Te antibody (Ab) structure is made of accomplished by coating with blocking reagents such as four polypeptide chains with a characteristic “Y” shape bovine serum albumin (BSA) [23], casein, or superblock (Fig. 3). Te chains are connected via a single disulfde [24], or treating the sensor with tween surfactant [25]. By bond. Te structure of the Ab consists of two diferent taking into consideration these limitations, biosensors of parts: the “arms” of the Ab that contain two domains, i.e. graphene-based nanomaterials can have high sensitivity/ a constant and a variable domain. Te variable domain stability as well as fast response time, potentially result- gives the selectivity of antibodies to a specifc anti- ing in advances in healthcare and diagnosis. gen. Te “body” of the Ab part consists of two diferent Peña‑Bahamonde et al. J Nanobiotechnol (2018) 16:75 Page 4 of 17

Fig. 3 Scheme of graphene modifed with antibodies for the recognition of pathogens

segments, the crystallizable fragment (Fc) and the anti- Tis method is based on the conformational changes gen-binding fragment (Fab). Te Fc and Fab contain car- produced by the biorecognition between the antibody boxyl (–COOH) and amino (–NH2) groups that bind to and the antigen. Tese nanosensors consist of a work- the target molecule with high afnity [26, 27]. Tis high- ing (where the reaction takes place) and a ref- afnity recognition to a specifc antibody–antigen reac- erence electrode (which makes the connection to the tion is mainly because of the structure, properties, and and allows the current to fow between the reactivity of the antibodies, making them excellent candi- two ). Electrochemical sensors include the dates for sensing applications. measurement of current, potential or resistance where Te versatility of functional groups of the GO surface the electrode transducer is able to detect the change in allows diferent strategies for Ab attachment. Te Ab the electrical signal caused by the binding reaction [29]. functionalization can be summarized in Table 1. Most of Tis method is selected over other immunosensor meth- the strategies to functionalize GO with antibodies involve ods since it is simple, rapid, sensitive, uses small sample functionalization via 1-ethyl-3-(3-dimethylaminopropyl) volumes, and presents good selectivity [26]. Tis method, carbodiimide hydrochloride (EDC)/N-hydroxysuccin- however, has a few limitations, such as binding afnity imide (NHS) (EDC/NHS) chemistry reaction, electro- and irreversible antigen–antibody interaction [30]. static bonding, or via 1-pyrenebutanoic acid succinimidyl Graphene-based nanomaterials on antibody biosensors ester (PASE) linker. Te functionalization via EDC/NHS ofer a broad versatility regarding pathogen detection. chemistry is the most popular and versatile method for Recently, several graphene-antibody biosensors with clin- producing biochemical conjugations. EDC is a water- ical applications have been developed for early detection soluble cross-linker agent, which allows direct biocon- of diseases (Table 1). Antibody nanosensors with G were jugation between carboxyl and amine groups. In this developed to detect E. coli [31, 32] and Zika [33]. reaction, the nucleophilic attack from the primary amine GO, on the other hand, has been employed for the detec- group from the antibody forms an amide bond with the tion of dengue virus [34], rotavirus [35] and cardiovascu- carboxyl groups on the GO surface. Tis process can lar diseases [36]. rGO has been employed to detect E. coli form conjugates between two diferent molecules with an in diferent samples [37] but with higher detection limits amide group [28]. comparing to G [31, 32] and G modifed with poly(methyl Te detection of the target molecules can be achieved methacrylate) (PMMA) [38]. More advanced research through diferent methods (see Table 1). Te most com- has shown that the modifcation of G with nanoparticles monly described method is electrochemical. Electro- can improve the sensing properties of the transductor. chemistry is a method that measures any electrical or In this context, G has been modifed with silver nano- chemical changes at the electrode/electrolyte interface. particles for the detection of Salmonella typhimurium Peña‑Bahamonde et al. J Nanobiotechnol (2018) 16:75 Page 5 of 17 [ 52 ] [ 53 ] [ 48 ] [ 36 ] [ 41 ] [ 44 ] [ 51 ] [ 46 ] [ 34 ] [ 47 ] [ 33 ] [ 50 ] [ 38 ] [ 31 ] [ 39 ] Refs. [ 40 ] [ 37 ] [ 35 ] [ 32 ] cells/mL 7 CFU/mL PFUmL 3 5 mL PYY 10 4 pg/mL 1.0 pg/mL GHRL and 0.02 pg/ 9.4 pM and 8.3 – 1.6 pg/mL 100 fg/mL 8.75 PFU/mL fg per 6 µL 0.1 fg 0.12 PFU /mL 2 nM 0.45 nM 1.60 ng/mL 10 CFU/mL 10 CFU/mL 10–10 10 CFU/mL Detection limit 3 fg/mL 10 assisted amida ‑ - assisted group of antibody was group 2 tion reaction chitosan covalent attachment to the attachment to covalent AgNPs Carbodiimide π–π interactions EDC–NHS chemistry Covalent and crosslinked via and crosslinked Covalent EDC/NHS chemistry EDC/NHS chemistry Amine functionalization Electrostatic bond Electrostatic EDC/NHS chemistry Electrostatic bond Electrostatic π–π interactions NHS surface chemistry Conjugation process Conjugation – Via PASE linker PASE Via Via PASE linker PASE Via EDC–NHS chemistry NH Antibody binding Antibody EDC–NHS chemistry 1 gp120 antibody - 1 gp120 CT GHRL and anti - PYY or H1N1) H7 - monoclonal antibodies polyclonal antibody polyclonal Rotavirus antibodies Rotavirus Anti - Anti - Monoclonal antibodies (H5N1 PAC1 antibody PAC1 polyclonal antibodies and H7 - polyclonal Cy3 antibody Cy3 adenovirus, Group II (HEV) Group Anti - adenovirus, tTG antibody Anti - tTG 4G2 monoclonal antibody Anti - Zika NS1 Antihuman IgG Ab O157:H7 antibody coli Anti E. antibody coli Anti - E. O157:H7 antibodies coli Anti - E. Anti - HIV Anti - S. typhimurium Anti - HCV antibody Antibody antibody Generic coli anti - E. enhanced Raman - - based nanosensors spectroscopy ing Photoluminescence Surface resonance Electrochemical Electrochemical Electrochemical Electrochemical immunosensor Electrochemical Surface Optoelectronic Electrochemical Electrochemical impedance Electrochemical Electrical Photoluminescence Electrical Electrical Electrical Cyclic voltammetryCyclic immunosens ‑ Electrochemical Detection methods Electrical - MB–chitosan - plasmonic shell functionalized UCNPs - functionalized UCNPs nano composites (AuNPs–G) nano composites attached hybrid oxide graphene with GQDs on AuNP embed ‑ with GQDs on AuNP ded in MWCNT nanopaper to graphene oxide graphene to silver nanoparticlessilver Graphene oxide Graphene–polypyrrole Reduced graphene oxide Reduced graphene Graphene oxide Graphene oxide Gold nanoparticle–graphene Magnetic core Graphene quantum dots Polyamidoamine dendrimer Polyamidoamine Graphene oxide Graphene Graphene oxide cellulose Graphene oxide Graphene/PMMA Graphene Graphene Peptide GO–AgNPs nano composite GO–AgNPs Graphene quantum dots with Immunosensor design Reduced graphene oxide Reduced graphene Overview of discussed graphene antibody Overview of discussed graphene Rotavirus Cholera toxin Hormones Infuenza A virus Cardiovascular diseases Cardiovascular Avian infuenza virus H7 Avian Alzheimer disease Adenovirus Celiac disease Celiac Dengue virus Zika virus Escherichia coli HIV Salmonella typhimurium Hepatitis C virus 1 Table Target Peña‑Bahamonde et al. J Nanobiotechnol (2018) 16:75 Page 6 of 17 [ 42 ] [ 43 ] [ 49 ] Refs. [ 45 ] [ 54 ] for PSMA for 0.1 fmol 0.088 pg/mL 20 fg/mL Detection limit fg/mL PSA and 4.8 fg/mL for 15 fg/mL less than 100 pg/mL covalent modifcation covalent EDC–NHS Electrostatic interactions EDC–NHS Antibody binding Antibody EDC–NHSS Non - estradiol antibody (curve) and CEA secondary antibody (Ab2) Lifeome Biolabs/Cusabio Biolabs/Cusabio Lifeome PSMA EL008782HU - 96 for carcinoembryonic antigen p53 antibodies CEA primary antibody (Ab1), Antibody RAB0331 for PSA and RAB0331 for Monoclonal antibody anti - Anti - spectroscopy Electrochemical Electrochemical Detection methods Electrochemical Electrochemical impedance Electrochemical @GO com ‑ 4 O 3 ­ Fe cyclodextrin functionalized nano particle graphene nanosheet graphene posites nano particle Magnetic Reduced graphene oxide gold oxide Reduced graphene β - Immunosensor design Graphene–PYR–NHS Reduced graphene and gold Reduced graphene (continued) Cancer 1 Table Target Peña‑Bahamonde et al. J Nanobiotechnol (2018) 16:75 Page 7 of 17

[39] and hepatitis C virus (HCV) [40]. Gold nanoparti- 56]. However, several advantages and disadvantages of cles attached to G surfaces have been employed to detect DNA biosensors have been identifed. Signifcant advan- avian infuenza virus H7, [41] and for diagnosis, progno- tages of DNA biosensors include high specifcity, ability sis, and prediction of treatment efcacy and recurrence to be used for real time analysis, to be designed as a small of cancer [42, 43]. Te modifcation of G with magnetic measurement system, and to perform multiplex measure- nanoparticles allows the early detection of Alzheimer ments of diferent targets [57, 58]. However, one of the [44] and also cancer diagnosis [45]. More complex bio- major disadvantages of DNA biosensors is that DNA can sensors modifying the surface of G with dendrimer [46], be easily degraded, thus, requiring specifc storage and polymers [47, 48] or cyclodextrin [49] have been devel- analysis conditions, such as particular media or a bufer oped to detect Celiac disease, HIV, Cholera toxin, and to keep the DNA stable and maintain its attachment to cancer. Table 1 shows in more detail the design of these the transducer. Additionally, DNA-based sensors’ efec- immunosensors, their detection method, detection limit, tiveness can be afected by changes in pH or tempera- as well as the antibody used to detect their particular tar- ture [59]. For instance, the sensitivity of DNA biosensors get molecule. Immunosensor have been developed for depends on experimental temperatures because the diferent types of microbes, such as and , hybridization event of the probe with the target mol- as well as diseases. In bacterial detection, graphene and ecules will occur at optimum temperatures to be deter- graphene oxide as sensor platforms give the lowest detec- mined prior to the deployment of the sensor. In the case tion limit (10 times less), compared to reduced graphene of pH, the current response shows the highest signal at oxide. For virus, the modifcation of graphene with gold pH 7.0, while there is almost no signal at pH below 7.0. and silver nanoparticles by covalent attachment of the Terefore, a bufer with potassium or sodium phosphate antibody allows the detection of concentrations as low is needed to enhance the efectiveness of the sensor as picograms per mL (pg/mL) of virus. In the case of [60, 61]. Despite their disadvantages, nucleic acids have detection of cancer cells, the modifcation of graphene gained increasingly more attention in the felds of biosen- oxide by functionalization with magnetic Fe­ 3O4 allows sors and biological assays for their applications in genet- to detection limits in femtograms (fg). An overall com- ics, infectious diseases, and detection of pathogens in parison among all currently available sensing platforms clinical settings [62]. In DNA biosensors using graphene- indicates that the functionalization of graphene or gra- based nanomaterials as transducers, there are two main phene oxide with silver, gold or other metal nanoparticles types of sensors: electrochemical and fuorescent sensors. and the antibody attachment via covalent bond, typically Te electrochemical sensor is based on measure- allows the lowest detection limits. ments of the change in voltage, current, or impedance Te early detection of these diseases with such sen- that can result from changes in electrochemical fac- sors can aid in diagnosis, prevention, and management tors, such as electron loss, conductivity or capacitance of the disease in ‘high-risk’ individuals, which in turn changes, which are caused by the hybridization of DNA would contribute to better management and survival of or the oxidation of adenine (A), thymine (T), cytosine patients. Many biosensors based on graphene nanoma- (C) and guanine (G) of the DNA. Te electrochemical terials have been proposed in the last few years for the signals produced by these biosensors can be detected diagnosis and real-time of the health status using cyclic voltammetry (CV), diferential pulse of patients. While the limitations of these types of sen- voltammetry (DPV) or electrochemical impedance sors (binding afnity and irreversible antigen–antibody spectroscopy (EIS) [18, 63]. In the binding) are not fully rectifed, the proposed biosensors approach, the immobilization of DNA is done via π–π exhibit very low detection limits (see Table 1), speed, sen- interactions on the surface of graphene-based nanoma- sitivity, and selectivity making these graphene-based bio- terials (Fig. 4). G edges and GO or rGO with their func- sensors ideal candidates for medical diagnostic tests. tional groups (carboxylic, hydroxyl and epoxide groups) can also be used to covalently interact with the DNA Graphene‑based nanomaterials [19, 64]. Te most common chemistry used for immobi- and deoxyribonucleic acid (DNA) lization of the DNA on graphene-based nanomaterials Deoxyribonucleic acid (DNA) has a broad range of physi- is EDC/NHS, which is described in detail in the anti- cal, chemical, and biological properties making this bio- body section. Research to improve sensitivity and selec- molecule highly suitable for biosensor technologies. tivity of electrochemical biosensors have been mostly Among the most critical properties of DNA for a bio- in the modifcation of the transducers. For instance, the sensor is its fexibility, easy synthesis, facile chemistry to original glassy carbon electrode (GCE) can be modi- attach to diverse platforms, simple regeneration and high fed with GO for the direct detection of A, T, G, and C specifcity due to unique sequences of nucleotides [55, for dsDNA or ssDNA using the DPV method at pH 7.0 Peña‑Bahamonde et al. J Nanobiotechnol (2018) 16:75 Page 8 of 17

Fig. 4 Scheme of graphene-based nanomaterials as a DNA biosensor. Electrochemical detection (a) and fuorescent detection (b)

[65]. In another study, the GCE was modifed with rGO studies to improve these features of graphene-based and DNA probes to hybridize with a target DNA to be DNA biosensors, which are summarized in Table 2. detected with either EIS or CV [25]. Tis study takes Te fuorescentDNA nanosensor is based on the advantage of the large surface area and high conductiv- hybridization of two single-stranded DNA (ssDNA). One ity of rGO. Another study investigated the DNA sensor ssDNA is labeled with a fuorescent dye, and the other using the sharp and active edges of reduced graphene is the complementary DNA corresponding to the target nanowalls (RGNW) to detect dsDNA with a sensitiv- DNA. Tis method requires optical detection; therefore ity ranging from 0.1 fM to 10 mM. In this study, the it takes advantage of the optical quenching property of authors suggest that the active edge sites of the RGNW graphene-based materials to enhance the visualization sheet could enhance the electron transfer between and detection of the target ssDNA [12]. Te immobiliza- DNA and the electrode in the DPV more uniformly tion of the fuorescent-labeled DNA can be carried out [66]. Depending on the sensing material and target, the by direct adsorption of the DNA probe on the graphene- sensor can have a wider detection range and sensitiv- based surface through the π–π interaction between ity. For example, in the case of dsDNA detection, the the ring structure of the DNA bases and the graphene best material identifed in the literature is graphene surface. nanowall, which can sense quantities as low as 0.1 fM One example of fuorescence biosensors that has been (Table 2). For ssDNA, the modifcation of reduced developed is the GO-based sensor. Tis sensor has been graphene oxide sensors with labeled ssDNA and gold produced with multicolor DNA probes for detecting dif- nanoparticles (ssDNA–AuNPs–ERGO) increases ferent sequence-specifc DNA. Tis multiplex GO-based the sensitivity to a lower detection limit of 0.005 fM DNA sensor presents low background fuorescence and (Table 2) [67]. Graphene-based DNA biosensors have excellent emission signal from specifc targets when the been investigated with focus on lowering the detection hybridization occurs [93]. Another widely use of the limits, speeding time of measurements and facilitating fuorescence sensing approach, which can also employ the fabrication process and biomedical applications. graphene-based materials, is the fuorescence resonance Terefore, there has been a large number of published energy transfer (FRET or Förster). In this detection Peña‑Bahamonde et al. J Nanobiotechnol (2018) 16:75 Page 9 of 17

Table 2 Graphene-based DNA biosensors with electrochemical detection Detected element Sensing material Detection range Refs. dsDNA Graphene nanosheets 2.0 pM to less than 10 mM [66] ssDNA Graphene nanowalls 0.1 fM to 10 mM dsDNA Epitaxial graphene 1 µM [68] BRCA1 DNA Graphene/Au 1 fM [69] 12 6 Staphylococcus aureus nuc sequence CTS–Co3O4–GR/CILE (Chitosan–Co3O4–gra‑ 1.0 10− to 1.0 10− M with the detection [70] × ×13 phene–carbon ionic liquid electrode) limit as 4.3 10− M 12 × 7 dsDNA Thionine–graphene nanocomposite (Thi–G) 1.0 10− to 1.0 10− M and low detection [71] × × 13 limit at 1.26 10− M × Survivin gene Graphene–nanostructure gold nanocompos‑ 50–5000 fM detection limit at 3.4 fM [72] ite flm glassy carbon electrode (G-3D Au/ GCE) 11 9 dsDNA [Co(phen)2(Cl)(H2O)]+ AuNPs/GR (gold–gra‑ 2.50 10− to 1.25 10− M [73] × × 12 phene) modifed electrode Detection limit at 8.33 10− M × ssDNA Graphene analogue tungsten sulfde–gra‑ 0.0–500 pM [74] phene (WS2–Gr) composite Detection limit at 0.0023 pM Multidrug resistance (MDR) DNA Nitrogen-doped graphene nanosheets func‑ Detection limit [75] 15 tionalized with Au nanoparticles (N–G/Au) 3.12 10− M × 14 6 ssDNA Nitrogen-doped graphene (NG) and ­Fe3O4 1.0 10− to 1.0 10− M [76] × × 15 nanoparticles Detection limit 3.63 10− M × 14 ssDNA of HIV-1 gene Graphene–Nafon composite flm Detection limit 2.3 10− M [77] × DNA AuNCs/GR nanobybrids and exonuclease III 0.02 fM to 20 pM [78]23 (Exo III) aided cascade target Detection limit at 0.057 fM 6 12 ssDNA Graphene and polyaniline nanowires (PANIws) 2.12 10− to 2.12 10− M [79] × ×13 modifed glassy carbon electrode Detection 3.25 10− M 6 × 12 dsDNA, ssDNA and single nucleotide poly‑ Poly(amidoamine) dendrimer (PAMAM) with 1 10− to 1 10− M [80] morphism graphene core Detection× limit× 1 pM 15 11 ssDNA Electroactive dye azophloxine functionalized 1.0 10− to 1.0 10− M [81] × × 16 graphene nanosheets (AP–GNs) Detection limit at 4.0 10− M 9 ×14 ssDNA Gold nanorods decorated GO sheets (Au 1.0 10− to 1.0 10− M [82] × × 15 NRs–GO) Detection limit at 3.5 10− M × Hepatitis B virus (HBV) GO/pencil graphite electrode (GO/PGE) 20 to 160 µg/mL [83] Detection limit 2.02 µM DNA GO–Chitosan (CHI) nano-composite 10 fM to 50 nM Detection limit 10 fM (60 s [84] hybridization times) and 100 fM at 25 °C 14 8 ssDNA ssDNA-Fe@AuNPs-AETGO 1.0 10− to 1.0 10− M [85] × × 15 Detection limit 2.0 10− M 7 12 × ssDNA rGO-graphene double-layer electrode 10− to ­10− M [86] 13 Detection limit 1.58 10− M 11 × 9 MDR1 gene Au nanoparticles/toluidine blue–graphene 1.0 10− to 1.0 10− M [87] × × 12 oxide (Au NPs/TB–GO) Detection limit 2.95 10− M 7 × 6 DNA AuNPs/ERGNO/GCE 2.0 10− to 1.0 10− M [88] × × 6 Detection limit at 1.0 10− M 17 ×13 ssDNA ssDNA–AuNPs–ERGO 1 10− M to 1 10− M [67] Detection× limit 5× aM ssDNA Gold nanoparticles decorated rGO (Au NPs/ 0.1 µM to 0.1 fM [89] rGO) Detection limit at 35 aM 12 6 Listeria monocytogenes Au/GR/CILE 1.0 10− to 1.0 10− M [90] × × 13 Detection limit 2.9 10− M 20 × 14 Amelogenin gene (AMEL) rGO modifed glassy carbon electrode (GCE/ 1.0 10− to 1.0 10− M [25] × × 21 RGO) Detection limit 3.2 10− M 13 × Methicillin-resistant Staphylococcus aureus rGO-modifed glassy carbon electrode 10− M [91] (MRSA) DNA 17 12 ssDNA Thionine functionalized rGO (Thi–rGO) 1.0 10− to 1.0 10− M [92] × × 19 Detection limit 4.28 10− M × Peña‑Bahamonde et al. J Nanobiotechnol (2018) 16:75 Page 10 of 17

method, initially, the fuorescent labeled DNA probe is measurement. Te electrochemical detection method quenched to the graphene-based nanomaterials surface takes into account the large surface area and conductiv- through FRET, making the fuorescent signal of (Fig. 4). ity of the nanomaterials. Te detection is based on the Upon hybridization of the probe with the target DNA, types and numbers of bases present in the DNA, which the fuorescent molecule is released with the dsDNA would cause the changes in electrical potential for the from the graphene surface, and the fuorescent signal measurement. Terefore, homogenous deposition of the is turned on for optical detection [16]. For instance, in probe on the graphene material is essential for accurate the efort to propose a reliable, biocompatible and scal- measurements. Also, the electrostatic potential and DNA able biosensor for HIV-1 detection, a nanocomposite of length could afect the efciency of the sensor. On the gold nanoparticles (AuNPs) and GO was synthesized and other hand, fuorescence detection can be performed in used as a quencher with the use of fuorescent carbon ssDNA or dsDNA regardless of the length of the DNA. dots (CDs) and a DNA probe, also called nano quencher. Tis method is based on the quenching and optical abil- Te FRET strategy was also used in the CDs/AuNPs/GO ity of graphene-based nanomaterials. One of the main nanoprobe. In the presence of target ssDNA, hybridiza- disadvantages of this method is that it can overestimate tion occurs, and the fuorescent signal turns on. Te pres- the fuorescence signal due to the high background fuo- ence of AuNPs on the GO nanosheets serves to quench rescence signal in some complex samples, such as serum the fuorescence of CDs in the absence of the target samples. On the other hand, the fuorescent-labeled DNA. AuNPs/GO exhibits exceptional selective and sen- probe can lose its intensity (photobleach) over time. sitive capability in the DNA biosensors [94]. Tis sensor Results of graphene-based DNA biosensor studies have has a detection limit as low as 15 fM. In the efort to fnd shown that there is still need for further investigations the best sensor, diferent composites of graphene-based related to the mechanisms of interactions between the materials have been used to achieve the desired sensitiv- DNA probe or modifed DNA probe and the graphene- ity. For instance, ssDNA can be detected with a fuores- based transducer to provide more reliable and accurate cent graphene sensor with a sensitivity as low as 0.5 pM measurements. Such studies could overcome the current using target recycling Exonuclease III (Table 3). Table 3 disadvantages of the method by lowering the detection presents the summary of other studies taking advantage limit of the current sensors. of the quenching ability of graphene-based nanomaterials to enhance or improve the fuorescent detection of DNA Graphene‑based nanomaterials and enzymes biosensors. Enzymes deserve particular attention in biosensor design In summary, the two methods seem efcient and pre- because they can be easily manipulated and have high sent low detection limits. However, each technique stability. Furthermore, these molecules are involved in has its advantages and disadvantages, which depends the metabolism of all organisms; they are reusable and mainly on the ability of immobilization of the DNA in highly selective catalysts that can discriminate between L the graphene-based nanomaterials and the method of and R enantiomers in diferent molecules. Enzymes can

Table 3 Graphene-based DNA biosensors with fuorescent detection Detected element Sensing material Detection range Refs. ssDNA GO Detection limit 200 nM [95] ssDNA GO and exonuclease III Detection limit 20 pM [96] ssDNA GO 200 nM [97] DNA and exonuclease activity GO ethidium bromide (EB) 50 to 2500 nM [98] Detection limit 32 nM Staphylococcus aureus DNA GO–DNA sensor 0.0125 to 3.125 nM [99] Detection limit at 0.00625 nM Hepatitis B virus (HBV) sequences GO/pencil graphite electrode (GO/PGE) 20 to 160 µg/mL [83] Detection limit 2.02 µM ssDNA Exonuclease III (ExoIII) and GO Detection limit 0.5 pM [100] HIV-1 gene AuNPs/GO nanocomposite 50.0 fM to 1.0 nM [101] Detection limit at 15 fM ssDNA GO 0 to 25 nM [93] Detection limit at 100 pM T antigen gene of SV40 DNA GO 40.0 to 260 nM [93] Detection limit at 14.3 nM Peña‑Bahamonde et al. J Nanobiotechnol (2018) 16:75 Page 11 of 17

catalyze a large number of reactions with high specifc- Each of these two mechanisms can create a detectable ity, efciency, and selectivity, which are essential param- electrical signal change on the sensor electrode allowing eters in sensor design [102]. However, use of enzymes in for the quantifcation of a particular analyte. In particu- sensors require modifcation or careful consideration of lar, this electrical signal is generated from the change in the type of enzyme that should be used. Enzyme stabil- current on the surface of the substrate as a direct result of ity can be problematic as higher temperatures can cause the enzyme’s activity. Enzymes catalyze redox reactions, their denaturation resulting in the loss of catalytic activ- which either produce or consume electrons, thus altering ity and reduced sensor functionality. While initially this the electrical current fowing to the detection platform. issue was addressed via the use of thermophilic enzymes, Te fundamental principle of how enzymatic biosensors nowadays, thermophilic enzymes are created or modi- work is presented in Fig. 5. While enzymes can be costly fed to become more robust using to utilize, sensors employing enzymes can detect a variety [103]. For instance, to alter enzyme properties, research- of compounds with high specifcity that would otherwise ers have used site-directed mutagenesis or chemical be difcult to detect in complex mixtures. For exam- modifcations to improve enzyme stability [103]. Alter- ple, these sensors can be particularly useful in detect- natively, with advancements in recombinant DNA tech- ing compounds such as phenols, peroxide, nology, enzymes can be manipulated rapidly by 17β-estradiol, , and bilirubin as described later in and overexpressing the desired enzyme gene [103]. Tis this section. Table 4 shows a variety of compounds capa- approach has solved several issues related to enzymatic ble of being detected by commonly immobilized enzymes stability and specifcity. and the resulting detection range achieved by each of the Advancements in enzyme-based biosensor research fabricated sensors. have resulted in improved stability while reducing enzy- Diferent molecules have been detected with enzyme- matic loss and enzyme response time [104]. It has been based nanosensors. Te most commonly used model demonstrated that the stability of enzymes is afected by enzymes utilized for the development of these sensors are pH, ionic strength, chemical inhibitors, solvent polarity, laccase and horseradish peroxidase (HRP) [109]. Tese and temperature. Te structure of graphene-based nano- enzymes are less costly, more commonly available, and materials can be an efective transducer since it allows versatile allowing them to be used to detect a high num- the direct electron transfer between enzymes and elec- ber of diferent compounds. Laccase is an oxygen-reduc- trodes [19]. Furthermore, graphene-based materials have ing enzyme, which can have a variety of applications. For been shown to be excellent substrates for increasing ther- example, a laccase-based electrochemical biosensor was mal stability, enzymatic activity, and for enzyme immobi- developed for the detection of 17β-estradiol, a natural lization [105–107]. hormone classifed as an emerging contaminant afect- Several approaches have been developed to immobilize ing humans and aquatic life [110]. Additionally, laccase enzymes onto graphene surfaces to create enzyme-based can be used for the detection of phenols and catechols biosensors. Some of the most common methods are [109, 111–113]. HRP, the other enzyme widely used for sonication, mixing, ultrasound, and cyclic voltammetry. enzyme immobilization studies, can help determine Tese methods allow the attachment of the enzymes via hydrogen peroxide concentrations even under complex adsorption, covalent bonding, or physical entrapment. To test conditions [114]. HRP has been immobilized on date, the nonspecifc binding of the enzyme to graphene porous calcium carbonate microspheres encapsulated via physical adsorption is the most common one (see with graphene capsules and presented high selectivity Table 4) since this immobilization technique is chemical- towards hydrogen peroxide. Tis sensor platform could free and straightforward. Another method used to immo- potentially be used to immobilize diferent enzymes for bilize enzymes on the nanomaterial is the EDC/NHS stable, long-term use as a biosensor [114]. Furthermore, chemistry. Tis method described earlier is also common HRP, as well as laccase, have been immobilized on a for enzymes because of its high stability and robustness. rGO–Fe3O4 based substrate [109]. Tis hybrid nanoma- Enzyme-based biosensors are typically of electrochem- terial takes advantage of the properties of rGO and the ical nature. Tis method possesses advantages over the magnetic properties of iron oxide making it an attractive others because the electrodes can sense materials pre- substrate for biosensor design. sent in the host without damaging the system. Enzyme- While HRP and laccase have been vital in enzyme bio- based electrochemical biosensors rely primarily on two sensor studies, other enzymes can be immobilized to mechanisms; one is based on the catalytic properties of create highly specifc biosensors. For example, bilirubin the enzymes (the enzyme catalyzes the analyte from its oxidase was immobilized on GO-based surfaces [115, undetectable form to a detectable form), and the other is 116]. Such biosensors can have a signifcant impact in based on enzyme activity inhibition/moderation [108]. the medical feld due to their ability to detect bilirubin, Peña‑Bahamonde et al. J Nanobiotechnol (2018) 16:75 Page 12 of 17

Table 4 Recent studies using graphene-based materials to immobilize enzymes Enzyme Immobilization Testing compound Detection method Attachment Range Refs. platform

Laccase, HRP Fe3O4–rGO – – Adsorption – [109] Laccase GO–rhodium nanopar‑ 17β-estradiol Electrochemical Donor–acceptor inter‑ 0.9–11 pM [110] ticles actions Laccase Palladium–copper Phenol Electrochemical Adsorption 0.005–1.155 mM, [111] nanocages on rGO 1.655–5.155 mM

Laccase Yolk shell Fe­ 2O3 2,6-dimethozyphenol Electrochemical Gluaraldehide reaction 0.025–750 μM [112] Laccase Graphene–cellulose Catechol Amperometric Adsorption 0.085–209.7 μM [113] microfber

Laccase MoS2 and graphene Cafeic acid Electrochemical Electrostatic interaction 0.38–100 μM [126] quantum dots

HRP CaCO3 microspheres Hydrogen peroxide Electrochemical Absorption 0.01–12 mM [114] encapsulated with a graphene capsule HRP 3D graphene/meth‑ Hydrogen peroxide Electrochemical In-situ self-polymerized 0.2 μM–1.1 mM [127] ylene blue-carbon polydopamine nanotubes Bilirubin Oxidase Electrochemically – – Adsorption – [116] reduced GO GOx ZnS–graphene Hydrogen peroxide, Electrochemical – – [117] glucose GOx Silk–graphene feld Glucose Electrical Hydrophobic interac‑ 0.1–10 mM [118] efect transistor tion GOx Nanostructured gra‑ Glucose Electrochemical Adsorption 10.0 μM–1.48 mM [119] phene with conduct‑ ing polyaniline

GOx TiO2–GO–OISL Hydrogen peroxide Electrochemical Immobilization 1–120 μM [120] GOx Chitosan/Nafon/Pt Hydrogen peroxide, 3–300 μM, [121] nanoparticle/SGGT​ glucose 0.5 μM–1 mM GOx GO modifed by amida‑ Glucose – Carbodiimide coupling – [122] tion GOx 3D GO and PANI Glucose Electrochemical – 0.07–1.10 mM [123] GOx AuPd–rGO–polyimide Hydrogen peroxide, Electrochemical Adsorption 0.004–1.0 mM, [124] glucose 0.024–4.6 mM GOx 3D graphene Glucose Electrochemical – 0.3–6 mM [125]

an essential compound for assessing liver function. ofers the highest sensitivity (down to 0.5 μM) and larg- Another enzyme with medical applications is glucose est linear range (up to 1 mM) in the detection of (GOx). Tis enzyme is highly specifc and has [121]. Tese sensing platforms show the versatility that been used to develop biosensors for the measurement graphene and its nanocomposites have regarding the of glucose levels [117–125]. Tis type of biosensor could chemistry for the detection of diferent substrates. be especially important to diabetic patients. As such, in recent years, GOx has been immobilized using difer- Conclusion ent sensing platforms, such as: zinc sulfde decorated In this mini-review, we have reported recent studies graphene [117], three dimensional graphene [125], silk describing graphene and graphene-related biosensors fbroin flm on a graphene feld efect transistor [118], with possible applications in clinical settings and life sci- nanostructured graphene-conducting polyaniline (PANI) ences. We have shown results of the reported analyti- composite [119], three-dimensional GO and polyaniline cal performance of each sensor and indicated their use (PANI) composite [123], GO and titanium oxide nano- in the life sciences and medical felds. DNA, antibody, particles modifed with an organic–inorganic supporting and enzyme-based biosensors have been presented in ligand (OISL) [120], and gold–palladium modifed poly- this study since each has its advantages and disadvan- imide/rGO flm [124], among others. Of these platforms tages. Overall, the type of sensor selected will depend the Chitosan/Nafon/Pt nanoparticle/SGGT composite on the type of application. For example, use of DNA in Peña‑Bahamonde et al. J Nanobiotechnol (2018) 16:75 Page 13 of 17

Fig. 5 Example of an enzyme biosensor

biosensing technology can be a cost-efective method for a better understanding of the and chemistry at the rapid detection of microbes, viruses, or cancer mark- the surface of graphene and the interactions with bio- ers. However, due to the vast variety of molecules present molecules at the interface will play an important role in in the body, use of antibodies or enzymes in biosensors graphene-based nanosensors. can be more efective in the detection or monitoring of Additionally, miniaturization and production of com- certain diseases. For instance, antibodies can be used for pact biosensors for diagnostic purposes is an emergent the specifc detection of viruses, such as the Zika virus, need in sensor technology since it requires development HIV, Infuenza A virus, among others. Enzymes, on the of reliable, reproducible, and cost-efective sensors with other hand, have shown to be promising in detecting glu- high accuracy, sensitivity, and specifcity. Lowering the cose levels with only small amounts of sample. Overall, cost of some of these sensors is necessary to increase usa- the incorporation of graphene and graphene-based nano- bility in remote areas for emergency uses. Furthermore, materials in biosensor technologies have shown great miniaturization of the sensors can allow rapid detection promise due to its high surface area, electrical conductiv- of virus and bacterial pathogens, as well as use in self- ity, electron transfer rate, and its capacity to immobilize monitoring biological implants to detect serious health a variety of diferent biomolecules. Te development of conditions. Te aforementioned applications in the life biosensors that are sensitive, stable, and specifc to their sciences will serve to protect lives and improve people’s target molecule and that can be processed rapidly are health. However, considerable work must still be done to promising for use in clinical settings. However, to achieve ensure, guarantee, and corroborate the biocompatibility uniform and reliable results and produce biosensors and non- of graphene-based nanomaterials such capable of being used in the medical feld, many more that their long-term use does not pose any health risk. studies need to be conducted examining the safety and reliability of the sensors. Abbreviations Although graphene is an excellent electrode material Ab: antibody; A, T, G, C: adenine, thymine, guanine, and cytosine; AD: Alzhei‑ for sensing applications in the medical feld, novel meth- mer disease; AuNPs: gold nanoparticles; BRCA1: breast cancer 1; BSA: bovine serum albumin; CD: celiac disease; CDs: carbon dots; CV: cyclic voltamme‑ ods for well-controlled synthesis and processing of gra- try; CEA: carcinoembryonic antigen; CHI: Chitosan; DNA: deoxyribonucleic phene need more attention and should be investigated in acid; dsDNA: double stranded DNA; DVP: diferential pulse voltammetry; EB: future studies. Te current chemical strategies to modify ethidium bromide; EDC/NHS: 1-ethyl-3-(3-dimethylaminopropyl) carbodiim‑ ide hydrochloride/N-hydroxysuccinimide; EIS: electrochemical impedance the surface of graphene with biomolecules are efective spectroscopy; ELISA: enzyme-linked immunosorbent assay; ExoIII: exonuclease in targeting specifc analytes. Nevertheless, the sensing III; Fab: the antigen-binding fragment; Fc: crystallizable fragment; FET: feld platform may be further refned to avoid the adsorption efect transistor; FRET: fuorescence resonance energy transfer; GCE: glassy carbon electrode; GHRL: ghrelin; GO: graphene oxide; GOx: glucose oxidase; of unwanted molecules on graphene and improve the ori- GQD: graphene quantum dot; HCV: hepatitis C virus; HIV: human immunodef‑ entation of biomolecules on graphene platforms. Hence, ciency virus; HRP: horseradish peroxidase; LOD: lower detection limit; MWCNT: Peña‑Bahamonde et al. J Nanobiotechnol (2018) 16:75 Page 14 of 17

multiwall carbon nanotube; NP: nanoparticle; OISL: organic–inorganic immunoassay for the diagnosis of cryptococcosis. Clin Infect Dis. supporting ligand; PAMAM: poly(amidoamine); PANI: polyaniline; PASE: 1-pyr‑ 2011;53:321–5. enebutanoic acid succinimidyl ester; PCR: polymerase chain reaction; PMP: 6. Holger S, Maya R, T. BT. DNA microarrays for pathogen detection. platelet-derived microparticle; PMMA: poly(methyl methacrylate); PYY: peptide Mod Tech Pathog Detect. New York: Wiley; 2015. p. 113–220. YY; RGNW: reduced graphene nanowalls; rGO: reduced graphene oxide; SGGT​ 7. Wilson CB. Sensors in medicine. West J Med. 1999;171:322. : solution-gated graphene transistor; SNP: single nucleotide polymorphism; 8. Sapsford KE, Bradburne C, Delehanty JB, Medintz IL. Sensors for ssDNA: single-stranded DNA; Thi: thionine. detecting biological agents. Mater Today. 2008;11:38–49. 9. Patolsky F, Zheng G, Lieber CM. Nanowire sensor for medicine and Authors’ contributions the life science. Nanomedicine. 2006;1:51–65. DFR coordinated the organization, content and elaboration of the manuscript. 10. Zhu Y, Murali S, Cai W, Li X, Suk JW, Potts JR, et al. Graphene and She was also responsible for editing the images and texts. JP-B compiled graphene oxide: synthesis, properties, and applications. Adv Mater. and wrote the sections: graphene-based nanomaterials as a biosensor and 2010;22:3906–24. graphene-based nanomaterials and antibodies, and conclusions. She also 11. Morales-Narváez E, Baptista-Pires L, Zamora-Gálvez A, Merkoçi A. Gra‑ assisted in the preparation of the abstract and images. HNN wrote the sec‑ phene-based biosensors: going simple. Adv Mater. 2017;29:1604905. tion Graphene-based nanomaterials and deoxyribonucleic acid (DNA), and 12. Chauhan N, Maekawa T, Kumar DNS. Graphene based biosensors— assisted in the preparation of some of the images. SKF wrote the sections: accelerating medical diagnostics to new-dimensions. J Mater Res. Abstract, Background, and Graphene-based nanomaterials and enzymes with 2017;32:2860–82. the assistance of JP-B and HNN and assisted in the creation of the images. All 13. Janegitz BC, Silva TA, Wong A, Ribovski L, Vicentini FC, Taboada Soto‑ authors read and approved the fnal manuscript. mayor MDP, et al. The application of graphene for and in vivo electrochemical biosensing. Biosens Bioelectron. 2017;89:224–33. 14. Wang Y, Li Z, Wang J, Li J, Lin Y. Graphene and graphene oxide: Acknowledgements biofunctionalization and applications in . Trends Not applicable. Biotechnol. 2011;29:205–12. 15. Liu J, Tang J, Gooding JJ. Strategies for chemical modifcation of Competing interests graphene and applications of chemically modifed graphene. J Mater The authors declare that they have no competing interests. Chem. 2012;22:12435. 16. Pumera M. Graphene in biosensing. Mater Today. 2011;14:308–15. Availability of data and materials 17. Rao CNR, Sood AK, Subrahmanyam KS, Govindaraj A. Graphene: Not applicable. the new two-dimensional nanomaterial. Angew Chemie. 2009;48:7752–77. Consent for publication 18. Park CS, Yoon H, Kwon OS. Graphene-based nanoelectronic biosen‑ All authors have read and approved this publication. sors. J Ind Eng Chem. 2016;38:13–22. 19. Kuila T, Bose S, Khanra P, Mishra AK, Kim NH, Lee JH. Recent advances Ethics approval and consent to participate in graphene-based biosensors. Biosens Bioelectron. 2011;26:4637–48. Not applicable. 20. Kasry A, Ardakani AA, Tulevski GS, Menges B, Copel M, Vyklicky L. Highly efcient fuorescence quenching with graphene. J Phys Chem Funding C. 2012;116:2858–62. This work was supported by the following funds: NPRP Grant [# 9-318-1-064] 21. Batır GG, Arık M, Caldıran Z, Turut A, Aydogan S. Synthesis and char‑ from the Qatar National Research Fund (a member of Qatar Foundation); CBET acterization of reduced graphene oxide/rhodamine 101 (rGO-Rh101) NSF Career Grant Number: 1150255; NSF BEINM Grant Number: 1705511; and nanocomposites and their heterojunction performance in rGO- the USDA National Institute of Food and , AFRI Project No. 2018- Rh101/p-Si device confguration. J Electron Mater. 2018;47:329–36. 67022-27969. The fndings achieved herein are solely the responsibility of the 22. Wu X, Xing Y, Zeng K, Huber K, Zhao JX. Study of fuorescence authors. quenching ability of graphene oxide with a layer of rigid and tunable silica spacer. Langmuir. 2018;34:603–11. 23. Cheng S, Hideshima S, Kuroiwa S, Nakanishi T, Osaka T. Label-free Publisher’s Note detection of tumor markers using feld efect transistor (FET)- Springer Nature remains neutral with regard to jurisdictional claims in pub‑ based biosensors for lung cancer diagnosis. 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