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REVIEW ARTICLE

A review on recent advancements in electrochemical biosensingusing carbonaceous nanomaterials

Alireza Sanati1,2 & Mahsa Jalali2 & Keyvan Raeissi1 & Fathallah Karimzadeh1 & Mahshid Kharaziha1 &Sahar Sadat Mahshid3 & Sara Mahshid2

Received: 7 May 2019 /Accepted: 19 September 2019# Springer-Verlag GmbH Austria, part of Springer Nature 2019

AbstractThis review, with 201 references, describes the recent advancement in the application of carbonaceous nanomaterials as highlyconductive platforms in electrochemical biosensing. The electrochemical biosensing is described in introduction by classifyingbiosensors into catalytic-based and affinity-based biosensors and statistically demonstrates the most recent published works in eachcategory. The introduction is followed by sections on electrochemical biosensors configurations and common carbonaceousnanomaterials applied in electrochemical biosensing, including graphene and its derivatives, carbon nanotubes, mesoporous carbon,carbon nanofibers and carbon nanospheres. In the following sections, carbonaceous catalytic-based and affinity-based biosensors arediscussed in detail. In the category of catalytic-based biosensors, a comparison between enzymatic biosensors and non-enzymaticelectrochemical sensors is carried out. Regarding the affinity-based biosensors, scholarly articles related to biological elements such asantibodies, deoxyribonucleic acids (DNAs) and aptamers are discussed in separate sections. The last section discusses recentadvancements in carbonaceous screen-printed electrodes as a growing field in electrochemical biosensing. Tables are presented thatgive an overview on the diversity of analytes, type of materials and the sensors performance. Ultimately, general considerations,challenges and future perspectives in this field of science are discussed. Recent findings suggest that interests towards 2D nanostruc-tured electrodes based on graphene and its derivatives are still growing in the field of electrochemical biosensing. That is because oftheir exceptional electrical conductivity, active surface area and more convenient production methods compared to carbon nanotubes.

Keywords Aptasensors . Carbon nanotubes . Catalytic biosensors . DNA based biosensors . Electrochemical biosensors .

Enzymes . Graphene . Immunosensors

Abbreviations of various carbonaceous nanomaterials3DCNSs Three-dimensional carbon nanospheres3DG Three-dimensional graphene3DGF Three-dimensional graphene foam3DNG Three-dimensional nitrogen doped–graphene3DrGO Three-dimensional reduced graphene oxide

CQDs Carbon quantum dotsCMWCNTs Carboxylated multi-walled carbon nanotubesCNFs Carbon nanofibersCNOs Carbon nano-onionsCNTs Carbon nanotubesCNPs Carbon nanoparticlesERGO Electrochemically reduced graphene oxideGO Graphene oxideGQDs Graphene quantum dotsGr GrapheneGrF Graphene flakesGMC Graphitized mesoporous carbonsrGA Reduced graphene oxide aerogelrGO Reduced graphene oxiderGONRs Reduced graphene oxide nanoribbonsMrGO Magnetic reduced graphene oxideMWCNTs Multi-walled carbon nanotubesNG Nitrogen–doped graphene

* Sahar Sadat [email protected]

* Sara [email protected]

1 Department of Materials Engineering, Isfahan University ofTechnology, Isfahan 84156-83111, Iran

2 Department of Bioengineering, McGill University,Montreal, Quebec H3A 0E9, Canada

3 Sunnybrook Research Institute, Sunnybrook Hospital,Toronto, Ontario M4N 3M5, Canada

Microchimica Acta (2019) 186:773 https://doi.org/10.1007/s00604-019-3854-2

http://crossmark.crossref.org/dialog/?doi=10.1007/s00604-019-3854-2&domain=pdfhttp://orcid.org/0000-0002-0834-8787http://orcid.org/0000-0003-4203-819Xmailto:[email protected]:[email protected]

NGQDs Nitrogen–doped graphene quantum dotsNGNRs Nitrogen–doped graphene nanoribbonsNSWCNTs Nitrogen–doped single-walled carbon nanotubesPCNRs Porous carbon nanorodsPCNSs Porous carbon nanospheres

Introduction

Chemical sensors and biosensors are known as reliable andusually portable tools for rapid and cost-effective determina-tion of analytes that range from metal ions to organic com-pounds, pollutants, proteins, antigens, deoxyribonucleic acids(DNAs), viruses, bacteria and others. “Biosensors” are analyt-ical devices similar to chemical sensors, however incorporat-ing biological molecules for rapid and accurate detection oftarget species [1–3, 199]. Here, we have divided our investi-gation into two categories of biosensors based on the presenceof the biological recognition molecules, with a focus on elec-trochemical methods.

A biosensor usually consists of two parts: (1) A bio-logical recognition element, such as single-strand synthet-ic DNA, enzyme, antibody or different kinds of artificialbioreceptors (aptamers, plastic antibodies and etc.); (2) Asignal transducer such as electrochemical, optical, mag-netic, thermal, piezoelectric and colorimetric which trans-fers the analytical response to the analytical device [2–4].Based on a statistical analysis since 2017 (Fig. 1a), 45%of the total number of published papers in this field be-longs to electrochemical biosensors, due to their high sen-sitivity, accuracy and relatively low price [5–7]. In thisreview, we focus on the most recent electrochemical sen-sors and biosensors based on carbonaceous nanomaterials.

Considering the importance of fast and continued elec-tron transfer at the detection site, the electrical conductivityis the most important characteristics of electrochemical de-tection platforms. In this regard, high electrical conductiv-ity and biocompatibility of carbonaceous nanomaterials, aswell as their high active surface area are key parameters inelectrochemical biosensing [7–10]. In addition, uniformdistribution of carbonaceous nanomaterials on the surfaceof an electrode, forming effective binds with analytes orbioreceptors and having high charge transfer ability aresome important current challenges to reach higher stability,sensitivity and long-range linearity in this field of study.

In this review, the most recent advancements on carbo-naceous nanomaterials since 2017 and their wide applica-tion in electrochemical biosensing will be discussed. Theelectrochemical biosensors are classified into two maincategories known as catalytic- and affinity-based biosen-sors. This review provides a comprehensive coverage ofdifferent targets of interest, linear range of detection andthe limit of detection (LOD) in each category. In addition,

we discuss recently current methods of fabrication andinnovative approaches in synthesis of carbon-based elec-trochemical biosensors.

Electrochemical biosensors configurations

Electrochemical biosensors are known as potential candi-dates for environmental and health applications as well aslaboratory investigations, due to their ease of manufactur-ing and implementation. The performance of electrochem-ical biosensors is directly linked to the electrochemical/electrical response of the sensor at the interface of theelectrode and the electrolyte [11, 12].

With their conventional three-electrode cell configuration(working, counter and reference electrodes as shown inFig. 1b), the electrochemical biosensor can also be appliedon flexible and portable substrates (Fig. 1c) such as screenprinted electrodes (SPEs) [5]. SPE-biosensors introduced forthe first time in 1990s as paper-based portable biosensingdevices with extensive reduction in the cost, size and weight

Fig. 1 (a) The bar chart representing the usage fraction of different signaltransduction methods on various biosensor platforms, according toScopus data (since 2017 to August 2019). Schematic illustration of (b)conventional electrochemical three–electrode cell, and (c) screen–printedelectrode

773 Page 2 of 22 Microchim Acta (2019) 186:773

of the sensing platform [12, 13] as well as operating with a lowamount of electrolyte [14]. The electrochemical readout canbe provided by various analytical methods such as (1) amper-ometric (voltammetric) biosensors: measuring the current bycyclic voltammetry (CV), chronoamperometry (CA), differen-tial pulse voltammetry (DPV),linear sweep voltammetry(LSV) and (2) impedimetric biosensors: using electrochemicalimpedance spectroscopy technique (EIS).

Although the amperometric detection is widely adopteddue to its simplicity, high sensitivity and applicability for de-tection of different analytes [3, 6, 12, 200], the impedimetricdetection can be used as a label-free sensing method, withoutusing any electroactive specious.

Carbonaceous nanomaterials as candidateelectrodes for electrochemical biosensing

The carbonaceous nanomaterials possess unique characteris-tics such as high electrical conductivity and chemical stability,as well as large active surface area. Figure 2 shows electronmicroscopy images of different carbonaceous nanomaterials

[15–19]. The most widely used carbonaceous nanomaterialsin electrochemical biosensing are graphene (Gr) and its deriv-atives such as graphene oxide (GO) and reduced grapheneoxide (rGO), and carbon nanotubes (CNTs) including multi-walled carbon nanotubes (MWCNTs) and single-walled car-bon nanotubes (SWCNTs). From the number of the publishedworks, an increasing growth rate on Gr-based biosensors isnoticeable (Fig. 3a), which is due to their more convenient andinexpensive fabrication processes compared to CNTs.

Carbon nanotubes (CNTs)

Following the introduction ofMWCNTs (Fig. 2a) in 1991 andSWCNTs (Fig. 2b) in 1993 by Iijima [20, 21], a variety ofapplications were identified for CNTs including electrochem-ical sensors and biosensors. CNTs are described as cylindricalnanostructures (with a high aspect ratio), formed by rolling upa single Gr sheet into a single tube (SWCNTs), or severallayers of Gr sheets into a compacted cylindrical structure(MWCNTs) [22]. CNTs offer different electronic properties,featuring conductivity, semiconductivity and superconductiv-ity as well as rapid electrode kinetics [7, 23]. Therefore, CNT-based biosensors generally have higher sensitivity, lowerLOD, and faster electron transfer kinetics than traditionalcarbon-based electrodes [24]. SWCNTs are consisted of a sin-gle hollow tube with diameters between 0.4 and 2 nm, whileMWCNTs are composed of multiple concentric nanotubeswith 0.34 nm apart between layers and a final diameter rang-ing between 2 and 25 nm [25]. Both SWCNTs and MWCNTspossess a high aspect ratio due to their micrometer length andnanoscale diameter, which provides them with unique electri-cal properties and enhanced conductivity for a variety of elec-trochemical applications. This high aspect ratio as a uniquecharacteristic of one-dimensional nanomaterials, can limit theelectrical current to a specific direction and decrease the de-tection time of the sensor [26]. Accordingly, CNTs based elec-trochemical biosensors have been successfully implementedin detection of proteins, nucleonic acids and other biologicalmolecules [27, 28]. In the following sections, we discuss ap-plications of CNTs in electrochemical biosensors.

Graphene

Graphene (Gr) is a honeycomb sheet of sp2-bonded car-bon atoms having superior electron transfer capacity andoutstanding electrical conductivity (Fig. 2c) [10, 29–31].The theoretical surface to mass ratio of Gr is 2630 m2/ g,which is much larger than that of CNTs, 100 to 1000 m2/g [32]. This is encountered as an important property ofelectrochemical biosensors, which provides an elevatedactive surface area in contact with the target solution,and thus increasing the linear range of detection and thesensitivity by several orders of magnitude [5, 33].

Fig. 2 Transmission electron microscopy (TEM) images of (a)MWCNTs, (b) SWCNTs, (c) Gr, (d) carbon nano-spheres, (e) CNFs(carbon nanofibers), and (f) Field emission scanning electronmicroscopy (FE-SEM) image of mesoporous carbon (respectivelyreproduced from [15–19])

Microchim Acta (2019) 186:773 Page 3 of 22 773

Novoselov et al. [34] first obtained Gr sheets by mechanicalexfoliation of graphite in a process called scotch-tape method,which is still widely used in producing perfect-structured Grlayer(s) with relatively low yield. Chemical or thermal reduc-tion of GO is also known as conventional methods for produc-ing Gr [7], particularly with reducing agents such as Hydrazine,Hydroiodic acid, Ascorbic acid (AA), and Sodium borohydride[35, 36]. It is worth noting that graphene structures that aredoped with p-block elements, such as nitrogen, boron, sulfur,hydrogen, oxygen and/or fluorine, are widely used in electro-chemical sensors and biosensor [34, 37, 38]. Recently, three-dimensional graphene-based materials and composites, havinghigh specific surface areas, large pore volumes and fast massand electron transport, are considered as excellent candidatesfor electrochemical sensors and biosensors [39–43].

Other carbonaceous nanomaterials

Although Gr and CNTare well known in the field of biosensors,other carbonaceous nanomaterials such as carbon nanospheresand carbon nanofibers (with diameters ranged between 100 and500 nm) are recently introduced for electrochemical biosensors.Also, mesoporous carbon (including porosities between 2 and50 nm) has shown a tremendous ability for detection of smallmolecules in current electrochemical investigations. The follow-ing sections discuss methods of synthesis and application ofthese nanomaterials in electrochemical biosensors.

Carbon nanospheres

Due to enhanced electrical conductivity and high surface tovolume ratio, carbon nanospheres are another candidate in

electrochemical biosensors. Also, porous carbon nanospherescan provide excellent diffusion and transfer of electrons andsolutions, as well as adsorption of biological specious [44].

Electrochemical sensing of Malathion (an organophos-phate) with a low LOD of 0.16 ng/ mL has been achievedby using carbon nano-spheres (diameter 300 nm) in a core-shell structure with polyaniline (PANI) (Fig. 2d) [17]. In short,achieving uniform distribution of carbonaceous nanomaterialsis a common challenge in working with them. Among thesolutions that have been investigated, the core-shell structure,which enables simultaneous benefits of both materials, hasshown promising enhancements. Herein, uniform distributionof carbon was resulted from the spherical shape of carbonnano-spheres and enhanced conductivity and biocompatibilitywas resulted from PANI [17].

Carbon nanofibers

Carbon nanofibers (CNFs) are one-dimensional nanomaterialswith a large active surface area. By decreasing the diameter ofCNFs the electron transfer in only one direction is accelerated,resulting in enhanced conductivity and decreasing the detec-tion time of the biosensors/ sensors [26, 45].

A modified electrode has been reported based on CNFs/AgNPs (silver nanoparticles) for detection of triglyceride,using electrospinning technique [18]. This method resultedin an interconnected structure of CNFs with an averagediameter in the range of 200 nm. (Fig. 2e). The intercon-nected structure of CNFs was highly beneficial in provid-ing a wide linear range (0.25 to 5 mg/ mL) of detection fortriglyceride. In addition, the presence of AgNPs enhancedthe graphitization and thus the electrical conductivity of

Fig. 3 (a) The number of thepublished papers on carbon-,graphene-, and CNT- basedbiosensors according to Scopusdata (searched by keywords“carbon”, “graphene” and“CNT” + “electrochemicalbiosensor”. The fraction of theresearches conducted aroundapplications of carbonaceousnanomaterials for, (b) affinity-based comparing to catalytic-based electrochemical biosensors,(c) enzymatic comparing to non-enzymatic glucoseelectrochemical (bio)sensors(considering the publicationssince 2017 to August 2019,according to Scopus data)


CNFs [18]. Electrospinning is a well-known method toprovide a variety of interconnected CNFs with differentdiameter and porosity percentages. The CNFs forms uponheating and pyrolysis of the polymeric fibers resulted fromelectrospinning, however, controlling the electrospinningconditions and polymeric fibers characteristics to reachthe CNFs is challenging. In addition, previous investiga-tions demonstrate that surface decoration of CNFs withdifferent nanoparticles can enhance sensing behavior.

Mesoporous carbon

Nanotunel-shape structure for efficient transferring of elec-trolytes, enhanced active surface area and high adsorptionability of mesoporous materials result in interesting appli-cations in different fields of science [46]. Amongst, carbonmesoporous materials have attracted attention in the fieldof electrochemical biosensors, due to their high electricalconductivity above all other properties.

A hierarchically-porous-partially-graphitic-carbon(HPGC) membrane with three-dimensionally networkednanotunnels (Fig. 2f), has been reported for enzymatic elec-trochemical biosensing [19]. The nanotunnels with wall diam-eter of ~40–80 nm are in fact composed of partially graphiticcarbon with ordered mesoporous of ~6.5 nm in diameter. Self-polymerization of poly-dopamine (PDA) on this structure wasperformed, followed by in-situ deposition of gold nanoparti-cles (AuNPs) on the surface, which results in increasing thebiocompability of the structure and the stability of the coatedAuNPs. Finally, the glucose oxidase was immobilized on thesurface to provide the final electrode used for enzymatic sens-ing of glucose. The LOD was reported as 4.8 pM, which isfour orders of magnitude lower than that of conventionalnanostructured glucose sensors. In addition to high conductiv-ity and large surface area for functionalization of the enzyme,the HPGC structure provides fast mass transfer- an advan-tageous for fast responses to the trace amounts of analytes[19]. One of the major challenges in producing mesopo-rous carbon is developing a removable template.According to the current state of the art, silicon and poly-mer 3D structures are usually used for this aim and can beremoved in acidic media or high temperature. However,un-complete removing of such template materials can no-ticeably decrease sensitivity of the sensor.

Catalytic-based vs affinity-basedelectrochemical biosensors

Biosensors can further be divided into two main categoriesbased on the method of capture and detection of the targetanalytes: (1) catalytic-based and (2) affinity-based biosen-sors [1–3, 6]. Catalytic-based biosensors are generally

based on a catalytic process for detection of biological tar-gets; for instance, the immobilized glucose oxidase (GOx)is used for catalytic detection of glucose in individuals withdiabetes [2]. Affinity-based biosensors use biological rec-ognition elements, such as antibodies, protein receptors,biomimetic materials and nucleoid acids for high-affinityand specific capture of target molecules [47]. Due to theirhigh sensitivity, specificity and reliability, the latter catego-ry has attracted a lot of attention in the diagnosis of varioustypes of diseases and impairments such as cancers, myocar-dial infarction and bacterial infections. Figure 3b illustratesthe fraction of the published papers reporting on the affinity-based and catalytic-based electrochemical biosensors usingcarbonaceous nanomaterials.

In the following sections, we will discuss the functionalityof these electrochemical biosensors together with few exam-ples of platforms that have used from carbonaceousnanomaterials as the sensing platform.

Catalytic-based biosensors

The function of catalytic-based biosensors is mostly governedby a catalytic reaction on a catalyst surface, such as inorganicmaterials, enzymes, microorganisms, or tissues, with a redox–active target molecule. Continuous consumption of the targetmolecule within this reaction results in a transient or steady-state response that can be monitored by an electrochemicalanalyzer [1, 48–50].

Enzymatic electrochemical biosensors

Enzymes, the most common receptors in catalytic biosen-sors, bind to the target analyte through enzyme-activesites to form a complex that eventually converts the ana-lyte into the product(s) (Fig. 4a). Here, the electrochemi-cal transducer readout monitors the target consumptionduring conversion. The enzymes are selected based ontheir high-level of specificity for direct detection of thetarget. For example, glucose oxidase for glucose [51–53,201], cholesterol oxidase for cholesterol [54, 55], acetyl-cholinesterase for acetylcholine [56], tyrosinase forbisphenol [57, 58] and HRP for H2O2 [59–61]. In additionto provide the capability of catalytic-based biosensing,these enzymes can be also used in affinity-based biosen-sors as accelerators of the electrochemical reactions [62,63]. Table 1 classifies recent advancements in the field ofcatalytic-based electrochemical biosensors based on car-bon nanomaterials, in which enzymes act as a catalyst aswell as a bioreceptor for the biological targets.

H2O2 is a signaling molecule in many biological processessuch as the immune cell activation, alterations in the structureof vessels, cells death monitoring, and root growth and


stomatal closure. So, this molecule is an important target formany biosensing platforms [71, 72].

A modified core-shell structure of carbon has been de-signed for enzymatic detection of H2O2 using myoglobinenzyme [66]. The core-shell structure features MWCNTs as

the shell and the reduced graphene oxide nanoribbons(rGONRs) as the core. A layer of chitosan was immobilizedon the surface of the core-shell modified GCE electrode toionically interact with negatively charged MWCNTs andalso act as an effective site for surface immobilization ofmyoglobin enzyme for detection of H2O2.

Due to the high density of the active edges -an advantageover other carbonaceous nanomaterials [75]- the resultingcore-shell structures propose a highly efficient adsorption ofmyoglobin and thus low detection limit (1 nM) of H2O2.

Figure 5a shows the TEM image of the core-shell struc-ture of MWCNTs@rGONRs. Adsorption of nonconductivemyoglobin on the surface of the electrode increases thecharge transfer resistance on the interface of the electrode,which is obvious from the semi-circular diameter in theNyquist curve (Fig. 5b). Also, the Warburg behavior as astraight-line in the low frequencies depicts a diffusion-controlled phenomenon.

Although enzymatic biosensors can provide a suitable se-lectivity for a variety of analytes, the enzymes require longincubation periods, complicated instrumentation and showlow stability, leading to limited throughput. Therefore, manyresearchers have focused on developing non-enzymaticsensors.

Non-enzymatic electrochemical sensors as an alternativeto enzymatic electrochemical biosensors

The main drawback of enzymatic biosensors is the lack ofenough stability of the enzymes [29]. Therefore, efforts are

Table 1 Enzymatic-based electrochemical biosensors based on carbonaceous nanomaterials

Electrode Target LOD (μM) Linear range (μM) Reference

Laca-rGO-RhbNPs/GCE 17β-estradiol 5.4 × 10−6 9 × 10−7 to 11 × 10−6 [64]

AChEc-Chd-Fe2O3NPs-PEDOTe-rGO/ FTOf Acetylcholine 0.004 0.004 to 800 [56]

Tryg-rGO-Mn3O4NPs-ITOh Bisphenol 0.010 0.01 to 100 [57]

ChOxi-rGO/ GCE Cholesterol 0.5 2.5 to 25 [55]

ChOx-Ch-CeO2-NGj/ GCE Cholesterol 1.33 4 to 5000 [54]

ADHk-Au-AgNPs-P(L-Cys)l-ERGO/GCE Ethanol 0.009 0.083 to 1050 [65]

GOxm-CNTs-rGO/ GCE Glucose 2.99 3900 to 7800 [52]

GOx/ Gr Glucose 0.12 200 to 9800 [53]

HRPn-rGO–ppyNTso/ GCE H2O2 0.1 0.1 to 120 [59]

Mbp-Ch-MWCNTs@rGONRsq/ GCE H2O2 0.001 0.001 to 1625 [66]

rGO-AuNRsr/ Cu Glucose 3 10 to 7000 [67]

PtNPs-CNOss/ GCE Glucose 90 2000 to 28,000 [68]

FADt-GDHu-P2Mo18- PMAv-MWCNTs/ GCE Glucose 1000 1000 to 20,000 [69]

AuNPs-NGQDsw/ GCE Glucose 0.0033 0.01 to 5 [70]

a: laccase, b: rhodium, c: acetylcholinesterase, d: chitosan, e: poly(3,4-ethylenedioxythiophene), f: fluorine–doped tin oxide, g: tyrosinase, h: indium tinoxide, i: cholesterol oxide, j: nitrogen–doped graphene, k: alcohol dehydrogenase, l: poly(L-cysteine), m: glucose oxidase, n: horseradish peroxidase, o:polypyrrole nanotubes, p: myoglobin, q: reduced graphene oxide nanoribbons, r: gold nanorods, s: carbon nano-onions, t: flavin adenine dinucleotide, u:glucose dehydrogenase, v: 1-pyrenemethylamine, w: nitrogen-doped graphene quantum dots

Fig. 4 Schematic illustration of (a) enzymatic reaction on catalytic-basedbiosensors and (b) three different types of affinity-based biosensors(Reproduced from [47])


towards developing non-enzymatic electrochemical sensorsfor detection of chemical or biological species through theirredox activity. Non-enzymatic sensors are those that do notinvolve a biological element. Therefore, considering the def-inition of biosensors in the previous section [1, 2], they can betitled as “electrochemical sensors” rather than “biosensors”.

Glucose is one of the most common target analytes in bothenzymatic and non-enzymatic electrochemical biosensors/sensors. Figure 3c demonstrates the enhanced growth towardsnon-enzymatic electrochemical sensors as an alternative toenzymatic biosensors by focusing on electrochemical detec-tion of glucose. The figure shows the ratio of publicationsbetween 2017 and 2019 for non-enzymatic vs. enzymatic glu-cose sensors based on carbonaceous nanomaterials. In ad-dition, Table 2 lists the most recent electrochemical sen-sors based on carbonaceous nanomaterials that are usedfor detection of dopamine (DA), ascorbic acid (AA) andH2O2 and etc. These data show that interest to non-enzymatic sensors is noticeably increasing.

Comparing the results of Tables 1 and 2 shows that non-enzymatic sensors can provide an acceptable LOD and lineardetection range for a variety of analytes. In addition, it isknown that using carbonaceous nanomaterials can providespecific sensitivity towards small molecules possessing aro-matic rings such as dopamine, via π-interactions. However,understanding the mechanism behind the selectivity behaviorof the sensors based on carbonaceous materials towards othertypes of analytes requires more investigations.

Dopamine (DA), ascorbic acid (AA) and uric acid (UA) areother important target analytes, which coexist together in liv-ing systems and are significant endogenic molecules for phys-iological processes in human metabolism. Therefore, simulta-neous determination of AA, DA and UA has received a greatdeal of attention [74].

Here we discuss a non-enzymatic sensor for detectionof dopamine, based on the combination of a conductivepolymer, poly(3,4-ethylenedioxythiophene) (PEDOT),and GO coated on carbon fibers [73]. Carbon fibers(CFs) were coated on a glass substrate, followed by de-position of PEDOT/ GO using an electropolymerizationmethod. Figure 5c shows the morphology of CFs andPEDOT/ GO coated carbon fibers. An optimal thicknesswas obtained by 25 s electrodeposition of PEDOT/ GO,which exhibited 880% increase in sensitivity and 50%decrease in LOD.

Figure 5d demonstrates another platform that has come toour attention due to its unique properties for simultaneous andnon-enzymatic detection of ascorbic acid, uric acid and dopa-mine. Figure 5d also illustrates a schematic process of modi-fying GCE using electrochemical reduction of GO and poly-L-lysine. Following electropolymerization of poly-L-lysineon the surface of GCE, the surface of the electrode will bepositively charged, resulting in an electrostatic interactionwith negatively charged GO. Three individual redox peaksin CV and DPV results are clear evidence of the ability ofthe electrode to separate the electrochemical signals. This

Fig. 5 The examples ofenzymatic and non-enzymaticelectrochemical sensors: (a) TEMimage of MWCNTs@rGONRs,(b) Nyquist curves for theelectrochemical behavior of themodified GCE with chitosan(Ch), Myoglobin (Mb) andMWCNTs@rGONRs in a 0.1 MKCl solution containing 5 mM[Fe(CN)6]

3−/4-. (c) SEM image ofthe 25 s-PEDOT/ GO coatedcarbon fibers as anelectrochemical sensor fordopamine (DA), (d) schematicillustration of the simultaneousdetection of AA, DA and uric acid(UA) by a non-enzymaticelectrochemical sensor based onelectrochemically reducedgraphene oxide (ERGO) andpoly-L-lysine (PLL).(Reproduced from [66, 73, 74],respectively)


Table 2 Non-enzymatic electrochemical sensors based on carbonaceous nanomaterials

Electrode Target LOD (μM) Linear range (μM) Reference

Au-Pd-rGO/ GCE Acetaminophen 0.30 1 to 250 [75]

ERGOa-PLLb/ GCE Ascorbic acid 2.0 100 to 1200 [74]

Gr/ γ-alumina nanofibers Ascorbic acid 0.117 1 to 60 [76]

Zn-NiAl-rGO/ CE Ascorbic acid 0.0135 0.5 to 11 [77]

AuNRs-GO-MWCNTs/ GCE Ascorbic acid 0.00085 0.001 to 8000 [78]

Au-PdNPs-GrFc/ GCE Caffeic acid 0.006 30 to 93,897,000 [79]

Chd-CBe-rGO/ GCE Caffeic acid 0.00003 0.0003 to 573 [80]

rGO-PdNPs/ GCE Desipramine 0.00104 0.3 to 2.5 [81]

GO-BAMBf-Co(OH)2/ GCE Dopamine 0.4 25 to 100 [82]

rGO-CB-Ch/ GCE Dopamine 0.2 3.2 to 32 [83]

PEDOTg-GO/ CFEs Dopamine 100 – [73]

PANIh-rGO-NFi/ GCE Dopamine 24 60,000 to 180,000 [84]

rGO- PEIj/ GE Dopamine 0.02 0.05–1 [85]

AuNPs-ERGO/ ITOk Dopamine 0.015 0.02 to 200 [86]

Thermosensitive polymer- GO-MWCNTs/ GCE Dopamine 0.0042 0.06 to 4.2 and 4.2 to 18.2 [87]

CeO2-rGO/ GCE Fenitrothion 0.003 0.025 to 2.00 [88]

GQDs-CoNiAl-LDHl/ CPEm Glucose 6000 10 to 14,000 [89]

3DNiO-rGO/ GCE Glucose 0.082 9 to 1129 [90]

3DGFn-Ni0.05NPs/ GCE Glucose 4.8 15.84 to 6480 [91]

AuNCso-CuO-rGO/ GCE Glucose 0.030 0.1 to 3000 [92]

NGp-CuNS/ GCE Glucose 0.0144 2 to 22 [93]

Ni(OH)2/ CNT fiber Glucose 0.645 20 to 10,500 [94]

Ch-AuNPs-MWCNTs/ AuEq Glucose 0.5 1 to 1000 [95]

NiNPs-rGO/ GCE Glucose 0.01 0.25 to 1200 [96]

GelMAr-NiNPs-rGO/ GCE Glucose 0.005 0.15 to 10,000 [97]

GO-PANI-Fe3O4NPs/ CE Glucose 0.01 0.05 to 5000 [98]

PDAs-MnO2-IL-Grt/ GCE Guanine 0.25 10 to 300 [99]

PtNPs-CQDsu-IL-GO/ GCE H2O2 0.1 1 to 900 [100]

MoS2-Gr/ GCE H2O2 0.12 250 to16000 [101]

Pd-TNMv-rGO/ GCE H2O2 0.0025 up to 12,000 [102]

MrGOw-Nf/ GCE Lobetyolin 0.0043 0.1 to 100 [103]

3DG-CNTs/ GCE Methotrexate 0.070 0.7 to 100 [104]

Palx-Gr/ GCE Niclosamide 0.0046 0.02 to 0.99 [105]

Au-FGry/ GCE Nitrite 0.01 0.125 to 20,375.98 [106]

NWS2z-rGAaa/ GCE Nitrite 0.003 0.01 to 130 [107]

Polypyrrole-rGO/ GCE R-Mandelic acid 1500 5000 to 80,000 [108]

Au-AgNTsab-NG/ GCE Rutin 0.015 0.1 to 420 [109]

p-Melac-ERGO/ GCE Tetracycline 5 5 to 225 [110]

MnO2NS-IL-Gr/ GCE Theophylline 0.1 1 to 220 [111]

Fe3O4-SnO2-Gr/ CPE Uric acid 0.005 0.015 to 2.40 [112]

GO-TmPO4ad/ GCE Uric acid 3.73 10 to 100 [113]

Gr-PANI/ GCE Urea 5.88 10 to 200 [114]

AgNPs-NSWCNTsae/ GCE Urea 0.0047 0.066 to 20,600 [115]

3DMoS2-rGO-AuNPs/ GCE Urea 0.29 5 to 2215 [116]

a: electrochemically reduced graphene oxide, b: poly L (lysine), c: graphene flakes, d: chitosan, e: carbon black, f: bis(aminomethyl)benzene, g: poly(3,4-ethylenedioxythiophene), h: polyaniline, hi: nafion, j: polyethylenimine, k: indium tin oxide, l: lactate dehydrogenase, m: carbon paste electrode, n: threedimensional graphene foam, o: gold nanocubes, p: nitrogen–doped graphene, q: gold electrode, r: gelatin methacryloyl hydrogel, s: polydopamine, t:ionic liquid functionalized graphene, u: Carbon quantum dots, v: tert-nonyl mercaptan, w: magnetic reduced graphene oxide, x: palygorskite, y: flower–like graphene, z: nanosheets of tungsten disulfide, aa: reduced graphene oxide aerogel, ab: nanotubes, ac: polymelamine, ad: Thulium phosphate, ae:nitrogen dope–single-walled carbon nanotubes


electrode provides a non-enzymatic sensor and can simulta-neously detect DA, UA, and AA.

In short, carbonaceous nanomaterials are vastly investigat-ed as a potential platform for non-enzymatic sensors, there-fore, the development of new functional carbon nanomaterialshas a strong impact on the advancement of these sensors.

Affinity-based biosensors

Affinity-based biosensors mimicking a natural biologicalevent, utilizing the interactions of the target analytes witha biological recognition element such as proteins, antibod-ies, and synthetic DNAs, aptamers, or MolecularlyImprinted Polymers (MIPs). The resulted interaction willbe transferred to a signal by linking to a transducer [1,47]. They contribute in a variety of detection applicationsincluding diagnosis of cancers, infectious and non-infectious diseases [49, 117–122]. Fig. 3b clearly showsthe high interest towards affinity-based biosensors. Also,Fig. 4b and 6a schematically and statistically illustratedifferent types of affinity-based biosensors, which are cat-egorized based on the biological recognition elements.Accordingly, the main focus is on antibodies as the bio-logical recognition elements.

Immunosensors /antibody-based biosensors

Antibodies are proteins produced by the immune system andcontain antigen-recognition sites, which bind to their specificrelated antigens by noncovalent interactions with relativelyhigh affinity [3, 123]. The antibody/ antigen interaction isthe most important phenomenon in immunosensors.

Among different types of immunosensors, electrochemicalimmunosensors are specific, sensitive and can offer real-timeanalysis of multiple targets in an automated platform [118,119, 123]. Table 3 reports the recent advancement in this fieldbased on carbonaceous nanomaterials. Accordingly, the max-imum attention goes to the immunosensors for detection oftwo important cancer biomarkers such as carcinoembryonicantigen (CEA) and prostate-specific antigen (PSA). CEA is amulti-tumor marker for clinical diagnosis of different kinds ofcancer, such as colon tumors, breast tumors, ovarian carcino-ma, gastric, pancreatic and lung carcinomas [124–127]. Incontrast, PSA is a specific biomarker only for detection ofprostate cancer [124, 128]. The main challenge of using car-bonaceous nanomaterials for immunosensors is providing ahigh surface area to achieve low LOD and wide linear detec-tion range for biomarkers. The high surface area will provide alarge number of suitable sites on the surface for incubatingantibodies, therefore, the bioreceptors on the surface can in-teract better with the specific antigen and detect it in a widerange of concentrations. In addition, perfect electrical

conductivity is of importance as it can facilitate detection oftarget antigen in ultra-low concentrations (fM and pM).

Yang et al. [63] reported an instant platform for detection ofCEA, using horseradish peroxidase (HRP) as a redox label in asandwich array (Fig. 6b). First, functionalized nitrogen-dopedgraphene oxide was dispersed in chitosan solution and mixedwith streptavidin solution. Second, the solution was incubatedwith a GCE overnight and then dried at 4 °C. Last, biotinylatedanti-CEAwas immobilized on the surface of the modified elec-trode. Before electrochemical measurements, theimmunosensor was incubated with the solution containingCEA and HRP-anti-CEA for 30 min at room temperature atdifferent concentrations. The functionalized electrode showed alarge surface area, high electrochemical activity, high selectiv-ity and a low detection limit as 0.01 ng/ml for CEA detection.

Alpha-fetoprotein (AFP), a glycoprotein, is another impor-tant antigen with similarity to albumin and is produced by thefetal liver. AFP is considered as a biomarker in certain tumorsand liver diseases among children [148]. Wang et al. [149]investigated the effect of Cu2O, toluidine blue (TB) and GOon electrochemical detection of AFP. A modified GCE wasmade by using TB/ Cu2O/ GO, in which Cu2O and TB wereadsorbed on the surface of GO by electrostatic interactions,while GO was adsorbed electrophoretically on the surface ofthe GCE. GO is negatively charged in a wide range of pH,which is related to its functional groups such as carboxyl, andhydroxyl. As such, positively charged species such as TB andCu2O can be adsorbed on the surface of the electrode throughan electrostatic interaction with GO. The negatively chargedGO increases the specific surface of the electrode, while pos-itively charged TB and Cu2O synergistically increase redoxpeaks in the CV responses. Bovine serum albumin (BSA) wasalso used for blocking non-specific sites on the surface of themodified electrode. The sensor showed a very low LOD forAFP down to 0.1 fg/mL. In addition, biosensors based on Grand noble metal nanoparticles such as AuNPs showed prom-ising results for the detection of PSA. This is due to the syn-ergistic effect of gold and Gr nanostructures and the chemistryof gold nanostructures for ease of functionalization by thecapturing probes and preparing them to bind to the targetmolecules [133, 137, 150]. According to the literatures, theelectrochemical biosensors based on immunoassays are most-ly used for detection of cancer. The most commonly usedtarget biomarkers are PSA, AFP and CEA antigens, whichare the topic of interest in this review. This is due to the im-portance of monitoring these biomarkers in low concentra-tions and the ability of carbonaceous nanomaterials-based bio-sensors in ultrasensitive detection.

DNA-based biosensors

Deoxyribonucleic acid (DNA), a nucleic acid-based compo-nent incorporating the genetic instructions, is a biorecognition


Table 3 Some electrochemical immunosensors based on carbonaceous nanomaterials

Electrode Target LOD Linear range Reference

Tba-Cu2ONWsb-GO/ GCE AFPc 0.1 fg/ mL 0.001 pg/ mL to 100 ng/ mL [129]

Tb-Au-Fe3O4 NPs-rGO/ GCE AFP 2.7 fg/ mL 1.0 × 10−5 ng/ mL to 10.0 ng/ mL [130]

CoS2-Gr-AuNPs/ GCE CA 15–3d 0.03 U/ mL 0.1 to 150 U / mL [131]

AgNPs-MoS2-rGO/ GCE CEA 1.6 fg/mL 0.01 pg/ mL to 100 ng/ mL [132]PDAe-rGO- PVPf-IrgNPs/ GCE CEA 0.23 pg/ mL 0.5 pg/ mL to 5 ng/ mL [133]Streptavidin- NGh/ GCE CEA 0.01 ng/ mL 0.02 to 12 ng/ mL [63]NGNRsi-Fe-MOFsj-AuNPs/ GCE Galectin-3 33.33 fg/ mL 100 fg/ mL to 50 ng/ mL [134]AgNPs-GQDs/ GCE Hepatitis C virus 3 fg/ mL 0.05 pg/ mL to 60 ng/ mL [135]rGO/ glass chip Influenza Viruses 0.5 PFU/ mL 1 to 104 PFU/ mL [136]Gr-CuO NPs/ AuE E. coli o157: h7 3.8 CFU/ mL 10 CFU/ mL to 108 CFU/ mL [58]AuNPs-AgNPs-Cu2O- NGQDs

k/ GCE PSA 0.003 pg/ mL 0.01 pg/ mL to 100 ng/mL [137]GQDs-AuNPs/ AuE PSA 1.8 pg/ mL 2 to 9 pg/ mL [138]AuNPs-rGO/ GCE PSA 2 pg/ mL 25 fg/ mL to 36 ng/ mL [139]CMWCNTsl-WNFsm/ GCE cTnIn 0.04 ng/ mL 0.5 to 100 ng/ mL [140]N-So co-doped rGO-Au@AgNCp/ GCE cTnI 33 fg/ mL 100 fg/ mL to 250 ng/ mL [141]rGO-CuS / GE CA 15–3 0.3 U/ mL 1 to 150 U/ mL [142]3DG-AuNPs/ GCE CYFRA 21-1 100 pg/ mL 0.25 to 800 ng/ mL [143]ERGO-P3CAq/ GCE BRCA1 3 fM 10 fM to 0.1 μM [144]AgPtNRs-rGO/ GCE CEA 1.43 fg/ mL 5 fg/ mL to 50 ng/ mL [145]AuNPs-GMCr/ GCE MPOs 0.1 pg/ mL 1 to 300 pg/ mL [146]AuNPs-3DCNSst/ GCE A549 cells 14 cells/ mL 4.2 × 10−1 to 4.2 × 10−6 cells/ mL [147]

a: toluidine blue, b: Cu2O nanowires, c: alpha–fetoprotein, d: carbohydrate antigen 15–3, e: poly–dopamine, f: polydiacetylenes, g: iridium, h: nitrogen–doped graphene, i: nitrogen–doped graphene nanoribbons, j: metal–organic frameworks, k: nitrogen–doped graphene quantum dots, l: carboxylatedmulti-walled carbon nanotube, m: whiskered nanofibers, n: cardiac troponin I, o: Nitrogen/ Sulfur, p: nanocubes, q: pyrrole−3–carboxylic acid, r:graphitized mesoporous carbons, s: myeloperoxidase, t: three dimensional carbon nanospheres

Fig. 6 (a) different types of electrochemical affinity-based biosensors(considering the publications since 2017 to August 2019 according toScopus data) (b) Using nitrogen–doped graphene (NG), streptavidin,AuNPs and HRP (horseradish peroxidase) label for preparing anelectrochemical immunosensor in order to detect CEA. (c) Using three-

dimensional graphene–modified GCE as an electrochemical biosensor forhybridization of DNA. MB was used as a redox element. (d) Usingaptamer in combination with Gr, MoSe2 and AuNPs for detection ofCEA (Reproduced from [63, 151, 152] respectively)


element in various biosensors due to their unique ability forfast and specific hybridization to the complementary se-quences [118, 119, 153–155]. These unique characteristicsof DNA, which results in the specific detection of varioustarget molecules, including nucleic acid-based targets, pro-teins, and antibodies, for disease diagnostics [3, 49, 71, 153,156, 157]. DNA-based biosensors, also sometimes known asgenosensors, are composed of a single-strand synthetic DNAimmobilized on the surface of the electrode, which then inter-acts with its complementary DNA strand in the solution in aprocess called “hybridization” [158, 159]. Table 4 representsthe recent advancements in this field based on carbonaceousnanomaterials. Herein, the role of carbonaceous nanomaterialswas investigated for the detection of DNA, microRNA andsome other biological analytes. Accordingly, GO has beenwidely used in the DNA based biosensors to provide a suitablesubstrate for DNA capture probe. This process occurs withinteractions between functional groups of GO and DNA cap-ture probe, therefore, precise development of GO to achievehigh surface area, electrical conductivity and electrical contactwith its substrate are of important challenges.

Chen et al. [151] prospered an electrochemical biosensorplatform based on three-dimensional nitrogen-doped grapheneand Fe3O4 for DNA detection. The sensing electrode was madeby mixing FeCl3·6H2O and GO solutions followed by addingof urea to facilitate the reaction of Fe3+ to Fe3O4, and then byaltering the basicity and reduction of GO to nitrogen-dopedreduced graphene oxide simultaneously. Methylene blue wasused as the redox element to generate the electrochemical sig-nal in the biosensing process (Fig. 6c). The results showed high

sensitivity because of the presence of nitrogen-doped grapheneand a wide linear range (0.01 pM to 1 μM), which is related tothe interconnected structure of this electrode and its potential inadsorbing ions and transferring electrons. The three-dimensional structures of graphene have recently attracted alot of attention in the field of biosensors.

Gong et al. [174] used Gr and nafion film modified GCEas an impedimetric nucleic acid biosensor for detection ofHIV. The biosensor offered a very low LOD (10 fM) and awide range of detection from 0.1 nM to 0.1 pM. Thesingle-strand DNA (ssDNA) was immobilized by non-covalent interactions on the nafion surface. They observedthat nafion can stabilize Gr and increase Gr dispersity onthe electrode. In another study, Wang et al. [160] producedgold nanoclusters and Gr nano-hybrids by one step ultra-sonication. The modified electrode offered a DNA detec-tion range from 0.02 fM to 20 pM with a detection limit aslow as 0.057 fM owing to the simultaneous presence of Grand gold nanoclusters, and their synergistic effects.

In addition to detection of DNA and viruses, fast and reli-able detection of bacteria such asMycobacterium tuberculosis(MTB) [165] is another important application of DNA-basedbiosensors. According to the world health organization(WHO), tuberculosis infection is one of the top 10 causes ofdeath worldwide. Chen et al. [165] reported a nanohybridstructure of rGO, polyaniline (PANI) and gold nanoparticles(AuNPs) for detection of sequence-specific DNA of MBT inthe range of 0.1 pM to 10 nM. The nanohybrid structure wasfunctionalized with a signaling probe, and the hybridizationbetween target DNA and the signaling probe was recorded by

Table 4 Electrochemical DNA-based biosensors usingcarbonaceous nanomaterials


AuNCsa-Gr/ AuE Detection of DNA 0.057 fM 0.02 fM to 20 pM [160]

GO-AgNPs/ AuE Detection of DNA 7.6 fM 10 fM to 10 nM [161]

3DNGb-Fe3O4/ GCE Detection of DNA 36.3 fM 0.01 pM to 1 μM [151]

AuNPs-rGO/ GCE Detection of DNA 0. 428 aM 0.01 fM to 1 pM [162]

MWCNTs/ PGEc Escherichia coli 17 nM 7 to 12 μg/ mL [163]

GO/ PGE Hepatitis C virus 0.43 nM 0.1 nM to 0.5 mM [164]

PANI-rGO-AuNPs/ AuE Mycobacterium tuberculosis 50 fM 0.1 pM to 10 nM [165]

AuNPs- rGONRd/ ITO Mycobacterium tuberculosis 0.1 fM 0.1 fM to 1 μM [166]

AgNPs/ 3DGFe CYFRA21-1 10 fM 10 fM to 0.1 μM [167]

AuNPs- GQDs/ AuE VEGFf165 0.3 fM 1.0 fM to 120 pM [168]

C3N4-PCNSg/ GCE VEGF165 3 fM 10 fM to 102 nM [169]

Au-PtNPs-CGOh/ FTOi microRNA-21 1 fM 1 fM to 1 μM [170]

GO-Dox-CdTe QDj/ GCE microRNA-21 1 fM 1 fM to 0.1 nM [171]

GO microRNA-141 100 aM 1 fM to 1 nM [172]

GO/ AuE microRNA-122 1.5 fM 10 fM to 10 nM [173]

a: gold nanoclusters, b: three-dimensional nitrogen–doped graphene, c: pencil-graphite electrode, d: reducedgraphene oxide nanoribbons, e: three-dimensional graphene foam, f: vascular endothelial growth factor, g: porouscarbon nanosphere, h: carboxylated graphene oxide, i: fluorine tin oxide, j: doxorubicin-modified cadmiumtelluride.


differential pulse voltammetry (DPV). Noticeably, the rGOincreased the immobilization of PANI and enhanced electro-chemical signals to achieve an ultra-low LOD (50 fM).

Another application of DNA based biosensors is detec-tion of micro-RNA. microRNAs are single strandRibonucleic acids (RNA) molecules that have a shortlength (usually 19–25 nucleotides) and have an importantrole in gene adjustments [172, 175]. These nucleotides arealso known as specific tissue biomarkers. Therefore, theconcentration of these biomarkers can be measured in or-der to monitor diagnosis and prognosis of deadly diseasessuch as cancer and inflammations [175].

Considering the numerous functional groups on thesurface of GO, Wang et al. [173] used GO to make anelectroactive Prussian blue (PB)/ GO to monitor micro-RNA 122 detection. In fact, GO can interact with captureDNA probe through π interaction between its functionalgroups and DNA probe. On the other hand, GO can pro-vide sufficient active cites for deposition of PB on thesurface. After the interaction between micro-RNA andDNA probe, the current on the surface of the electrodedecrease. Therefore, in this research the change in thecurrent was monitored by electrochemical techniques,confirmed the ability of the sensor to detect micro-RNAin low volumes.

It is worth noting that, an ultra-low LOD (usually in therange of fM), particularly towards target DNA biomarkers, isone of the remarkable properties of DNA-based biosensors.

Aptasensors / aptamer-based biosensors

Aptamers are single-strand DNAs or RNAs that can bind withdifferent kinds of molecules with high specificity and highaffinity. DNA aptamers usually have more stability thanRNA aptamers, while RNA aptamers have higher flexibility[176]. Aptamers have advantages over antibodies due to theirsmaller size, controllable synthesis procedure, and the ease ofmodification by a variety of molecules [176]. These makeaptamers exciting candidates for biosensing, featuring out-standing specifications for applications in diagnostics. Theaptamer-based biosensors also known as aptasensors [177].Table 5 reveals the recent advancement in the aptasensorsbased on carbonaceous nanomaterials.

Table 5 shows that a diversity of carbonaceousnanomaterials is used for aptasensors, where, inimmunosensor and DNA based biosensors the focus waspointed to rGO and GO. This can be related to the more novelfield of aptasensors compared to immunosensors and DNAbased biosensors. The aptasensors are known as syntheticbioreceptors which their properties and interactions withnanomaterials are still unknown for most of the researchers.

They [152] suggested an aptasensor based on MoSe2-rGOhybrids (Fig. 6d) for detection of CEA. The MoSe2-rGO

hybrids were prepared via a hydrothermal method. AuNPswere also added to the system by chronoamperometry tech-nique to increase the electrocatalytic behavior of the biosen-sor. Overall, the synergistic interaction of AuNPs and MoSe2-rGO hybrid improved the electrochemical behavior of the bio-sensor. Zhang et al. [178] used three-dimensional reducedgraphene oxide (3D-rGO) and silver nanoclusters (AgNCs),graphene quantum dots (GQDs) and aptamer for detection ofplatelet-derived growth factor (PDGF). PDGF is a well-known biomarker for detection of diseases such as hepaticfibrosis, liver cancer and colorectal cancer [187, 188]. In thisresearch 3D-rGO/AgNCs/Apt-based showed a low LOD as0.82 pg mL−1. It is believed that the extended surface area of3D-rGO improved the affinity of PDGF binding to the surfaceand thus enhanced the sensitivity of the aptasensor. Low LOD,as well as controllable procedure of producing aptasensors,made them an interesting candidate in biosensors develop-ment. In addition to detection of different kinds of proteins,the thrombin detection is also known as one of the specificapplications of aptasensor, which can be used for fast andaccurate detection of myocardial infarction [180].

Carbonaceous screen-printed electrodesin electrochemical biosensing

Screen-printed wearable electrochemical biosensors based oncarbon have drawn tremendous attention towards non-invasive glucose sensing in both sweat and interstitial fluid(ISF) [51, 189, 190]. There have been advances towards car-bonaceous screen-printed biosensors by introducing body-compliant wearable electrochemical devices that combinehigh elasticity with an attractive electrochemical performance[191, 192]. For instance, Chaiyo et al. [193] used a screen-printed configuration as a non-enzymatic sensor for glucosedetection (Fig. 7). They fabricated PADs by wax-printingtechnique, in which paper-based sensor was patterned by adesigner software (Adobe Illustrator) and printed onto filterpaper using a wax printer. In this system, a working electrode(area = 0.196 cm2) a counter electrode and a pseudo-referenceelectrode were screen-printed using carbon ink, followed bydrying at 55 °C for 1 h.

In another study, Bandodkar et al. [189] reported a tattoo-based electrochemical device, using carbon electrodes andaimed to sense glucose in the ISF (Fig. 8a). The perfor-mance of this electrochemical device was optimizedin vitro and later tested for on-body glucose monitoring.The tattoo-based sensor was designed to monitor glucosebased through screen-printing two compartments of reverseiontophoresis electrodes: carbon and prussian blue ink asthe working electrode and a pseudo counter/ reference Ag/AgCl electrode. In the reverse iontophoretic reaction, glu-cose was attracted to the cathodic contingent, where glucose


oxidase (GOx) was immobilized on the carbon-based work-ing electrode. In order to prevent skin irritation caused byelectrodes, an agarose hydrogel was used to cover the elec-trodes. Chronoamperometry was used to test the perfor-mance of the sensor. In-vitro testing of a tattoo-based elec-trochemical biosensor demonstrates that it enables the se-lective sensing of micromolar levels of glucose in buffersolution [189]. Abellán-Llobregat et al. [51] reported ontwo sets of tattoo-based all-printable flexible glucose sen-sors in physiological fluids based on non-enzymatic andenzymatic platinum (Pt) decorated graphite electrode(Fig. 8b). Chronoamperometry of H2O2 reduction was usedto identify the performance of these glucose monitoringdevices. A distinct LOD of 10 μM for glucose monitoringwas recorded for the Pt-decorated graphite electrodeimmobilized with GOx. This is while the LOD achievedby non-enzymatic Pt-decorated graphite electrode reportedas 6.6 mM. The GOx immobilized Pt-decorated graphite

revealed competency for human perspiration glucose mon-itoring enabling the non-invasive wearable use of this elec-trochemical device to detect spikes in the glucose level. Thestretchability of these devices was reported 75% with ac-ceptable sensing performance.

Kim et al. [190] demonstrated the use of carbon-basedwearable screen-printed biosensors for non-invasive alco-hol monitoring in induced sweat. In this work, the authorsreported an effective noninvasive ethanol monitoring basedon coupling amperometric enzymatic electrode and sweat-inducing iontophoresis. The performance of the reportedbiosensor was tested both in-vitro and in-vivo. The LODof ethanol detection achieved for this biosensor was 3 mM,demonstrated by the Chronoamperometric response of thedevice.

By further technology advancements in carbon-based biosen-sors to detect biological analytes, Wang’s group recentlyattempted to engage these screen-printed electrochemical devices

Table 5 Some electrochemical aptasensors based on carbonaceous nanomaterials


MoSe2-rGO-AuNPs/ GCE CEA 0.03 pg/ mL 0.1 pg/ mL to 100 ng/ mL [152]

GR-3DAu-AuNPs-HRPa/ GCE Oxytetracycline 0.498 pg/ mL 0.5 pg/ mL to 2 μg/ mL [62]

3DrGOb-AgNCsc/ AuE Platelet derived growth factor 26.5 fM 32.3 fM to 1.61 pM [178]

PCNRd-Gr-Fe3O4-AuNPs/ GCE Streptomycin 28 pg/ mL 50 pg/ mL to 0.2 μg/ mL [179]

PANI-MWCNTs/ GCE Thrombin 80 fM 0.2 to 4 nM [180]

GO/ GCE AFP 3 pg/ mL 10 pg/ mL to 100 ng/ mL [109]

THe- rGO- AuNPs/ CEf AFP 0.05 μg/ mL 0.1–100 μg/ mL [181]

AuNPs- CNPsg-CNFsh/ GCE Staphylococcus aureus 1 CFU/ mL 12 to 1.2 × 108 CFU/ mL [182]

Gr-QDsi-ILj-Nfk/ GCE CEA 0.34 fg/ mL 0.5 fg/ mL to 0.5 ng/ mL [183]

GO- PEDOTl/ GCE Adenosine triphosphate 0.03 pM 0.1 pM to 1 μM [184]

AuNPs- CIPNPs-NG/ CE Troponin I 20 fM 0.1 pM to 10 nM [185]

AuNPs-GQDs/ GE VEGFn165 0.3 fM 1 fM to 120 pM [168]

ERGO-SWCNTs/AuNPs/ GCE HER2o 50 fg/ mL 0.1 pg/ mL to 1 ng/ mL [186]

a: Horseradish peroxidase, b: three-dimensional reduced graphene oxide, c: silver nanoclusters, d: porous carbon nanorods, e: thionin, f: carbon electrode,g: carbon nanoparticles, h: cellulose nanofibers, i: quantum dots, j: ionic liquids, k: nafion, l: Poly(3,4 ethylenedioxythiophene), m: cyclometalatediridium(III)-polyvinylpyridine polymer nanoparticles, n: vascular endothelial growth factor o: Human epidermal growth factor receptor 2

Fig. 7 A screen-printed electrode modified with Gr for electrochemical detection of glucose (Reproduced from [193])


with other substrates to sense explosives and organophosphates[194–196]. Mishra et al. [194] reported a carbon-based electro-chemical enzymatic biosensor, screen-printed on a glove withhigh stretchability, which can be used to detect organophosphates(Fig. 9a). This “lab-on-a-glove” biosensor is coupled with anelectronic interface for the electrochemical detection andwirelessdata transmission to enable on-site detection of organophosphatenerve-agent compounds by a smartphone.A carbon inkmodifiedwith an elastomeric styrene-isoprene copolymer, a silver layer ofAg/AgCl particles and an elastomeric styrene-isoprene copoly-mer carbon ink was used as a counter electrode, a referenceelectrode and a working electrode, respectively. Square WaveVoltammetry (SWV) technique was used to successfully detectthe peak for p-nitrophenol hydrolysis product. Sempionatto et al.[195] demonstrated a ring-based wearable chemical sensor forrapid electrochemical detection of organophosphate nerve agentsand explosives in vapor and liquid phases (Fig. 9b). In this work,they used a screen-printed carbon counter electrode, an Ag/AgClreference electrode and two working electrodes made of carbonand prussian blue conductive carbon as working electrodes 1 and2, respectively. The substrate that hosts the electrochemicalscreen-printed electrodes is a 3D-printed compact ring structurecoupled with a coin battery and electronic kit. Agarose in phos-phate buffer conductive hydrogel was used to detect explosivesand organophosphates. The bare carbon working electrode wasused to detect dinitrotoluene (DNT) using squarewave voltamm-etry technique. On the other hand, the bare carbon prussian bluewas used to detect H2O2 using chronoamperometry technique.However, in order to detect methyl paraoxon (MPOx) as amodelof the organophosphate nerve agents, the carbon working

electrode was immobilized with OPH prior to themeasurements.The LOD achieved using this set up was 4 ppm, 1 mM and200 μM for DNT, H2O2 and MPOx vapors, respectively.

Later, this group reported a “Lab-on-a-Glass”, wherethey introduced a sweat metabolite/electrolyte eyeglassessensor platform with wireless signal transmission basedon carbon electrodes (Fig. 9c) [196]. The electrodes werefabricated on the pads placed on two sides of the nosewhich reduces the potential crosstalk between correspond-ing sensors. The whole electrochemical device wascoupled with a wireless electronic backbone placed onthe arm of the glasses. This device was designed basedon two major electrochemical techniques; amperometricand potentiometric to detect various analytes forhealthcare and fitness. The performance of this biosensorwas tested for Lactate and potassium, both in-vitro and in-vivo. Ag/AgCl ink was screen-printed as the referenceelectrode for both sensors. For the potassium sensor side,valinomycin immobilized carbon ink along with polyvinylchloride (PVC) and bis(2-ethylhexyl) sebacate (DOS)were screen-printed as the working electrode. In Lactatesensor, prussian blue/graphite film was screen-printed forthe counter electrode and prussian blue/graphiteimmobilized with lactate oxidase (LOx) along with bo-vine serum albumin (BSA) and chitosan was used asworking electrode. The advantage of eyeglasses sensingplatform over the other screen-printed platforms is theability to replace the pad sensor stickers for lactate andpotassium with a variety of different sensor stickers. Forinstance, one can replace the lactate pad sensor with a

Fig. 8 Screen-printed electrode based on carbon ink for electrochemicaldetection of glucose (a) tattoo-based iontophoretic electrochemicaldevice based on carbon electrodes, aimed to sense glucose in the ISF,

(b) flexible glucose sensors based on enzymatic and non-enzymaticplatinum (Pt) decorated graphite electrode (Reproduced from [51, 189]respectively)


glucose one to conveniently monitor the sweat glucose.Table 6 summarizes the recent advances achieved on car-bonaceous screen-printed electrochemical biosensors.

General considerations, challenges and futureprospects

Development of fast and reliable biosensors for detection ofbiological markers of various diseases is extremely challengingdue to complications in sensitivity, specificity, rapidness, and

cost. The more aiming for earlier stage diagnostics, the morecomplexity will appear for the design of a rapid sensitive sensorplatform. Recent advancement in the development of advancedmaterials and nanostructures has pushed the boundaries in thefield of biosensing towards rapid diagnostics. In this review,recent applications of highly conductive carbonaceousnanomaterials (particularly Gr and CNTs) in electrochemicalbiosensing and their interface with biological recognition ele-ments (DNA, antibodies, aptamers) were investigated.

There is a wide range of applications for carbonaceous elec-trochemical biosensors including catalytic and affinity

Fig. 9 Screen–printed electrode based on carbon ink for electrochemicaldetection of explosives and organophosphates (a) flexible carbon-basedelectrochemical device implemented on a glove, aimed to senseorganophosphates on–site, (b) ring-shaped electrochemical sensor forrapid electrochemical detection of organophosphate nerve agents and

explosives in vapor and liquid phases based on carbon electrodes, (c) asweat metabolite/electrolyte eyeglasses sensor platform with wirelesssignal transmission based on carbon electrodes (Reproduced from[194–196] respectively)

Table 6 Summary of recent advances achieved on carbonaceous screen-printed electrochemical biosensors

Platform Recognition Element Target Biofluid Technique Reference

Temporary Tattoo GOx–Prussian blue/Carbon Glucose ISFa Reverse IontophoreticChronoamperometry

[189]

Temporary Tattoo GOx–Pt–Prussian blue/Carbon Glucose Human Perspiration Chronoamperometry [51]

Temporary Tattoo AOx–Prussian blue/Carbon Alcohol Induced Sweat Iontophoretic Chronoamperometry [190]

Glove OPHb/ Elastomeric styrene-IsopreneCopolymer Modified Carbon Ink

MPOxc Agarose Hydrogel SWV [194]

3D Printed Ring Carbon DNTd Agarose Hydrogel SWV [195]Prussian Blue/Carbon H2O2 Chronoamperometry

OPH/Carbon MPOx SWV

Glasses Frame Loxe–Prussian blue/Carbon Lactic Induced Sweat Amperometry [196]Valinomycin /Carbon Ink Potassium Potentiometry

Flexible paper Gr–AuNPs/Carbon C-reactive protein Human serum Impedimetric detection [197]

Flexible cellulosepaper

Silver/ Gr AFPe Human serum Impedimetric detection [198]

a: interstitial fluid, b: organophosphorus hydrolase, c: methyl paraoxon, d: dinitrotoluene, e: lactate oxidase, e: Alpha- fetoprotein


biosensors, as they potentially provide elevated sensitivity due totheir high conductivity. Gr and its derivatives, including GO,rGO, Gr with p-blocked elements, and 3DGr can be recognizedas a growing field in electrochemical biosensing, while the num-ber of published papers in the field of CNTs is relatively constantafter 2012. This can be evident by the huge potential of Grcompared to CNTs including its higher surface area and electricaland thermal conductivity, as well as feasible controllable produc-tion procedures to achieve different types of Gr platforms.

On the other hand, immunosensors are an important fieldof research in electrochemical biosensors for the detectionof various diseases including cancers and infections.Synthetic nucleic acid-based recognition molecules, partic-ularly aptamers in combination with nanomaterials are anamazing and growing field. They can be used asbioreceptors with high affinity, selectivity and highly con-trollable synthesis parameters, high availability and lowcost compared to antibodies and proteins receptors. In ad-dition, in catalytic-based biosensors, non-enzymatic elec-trochemical sensors are replacing the enzymatic biosensors.This is because of their higher stability, lower cost of pro-duction and also acceptable sensitivity and selectivity.

Finally, the use of carbonaceous nanomaterials as the elec-trode platform in electrochemical sensors can guaranty highsensitivity. In addition, the presence of organic groups such ascarboxyl on the surface of carbonaceous nanomaterials such asGO can facilitate their attachment and connection to the posi-tively charged biological recognition elements such as antibod-ies, DNAs, and enzymes. Therefore, GO, rGO, ERGO andfunctionalized Gr have been investigated vastly as potentialsubstitutes for graphene. However, functionalizing the surfaceand increasing the active surface area together with improvingthe reduction process, conductivity and interaction with bio-chemical specious, are considered the main challenges and per-spectives towards their future research.

Acknowledgements The authors thank Faculty of Engineering at McGillUniversity, Natural Science and Engineering Research Council of Canada(NSERC, G247765 and G248584) and Canada Foundation forInnovation (CFI, G248924) for financial support. M.J. is grateful forMEDA award by the Faculty of Engineering at McGill University. Theauthors acknowledge Nanotools-Microfab and the Facility for ElectronMicroscopy Research at McGill University and the research facilities ofNanoQAM at the Université du Québec à Montréal.

Compliance with ethical standards

Conflict of interest The authors declare no conflict of interest.

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