Nanobiosensing with graphene and carbon quantum dots: Recent advances

Abstract

Graphene and carbon quantum dots (GQDs and CQDs) are relatively new nanomaterials that have demonstrated impact in multiple different fields thanks to their unique quantum properties and excellent biocompatibility. Biosensing, analyte detection and monitoring wherein a key feature is coupled molecular recognition and signal transduction, is one such field that is being greatly advanced by the use of GQDs and CQDs. In this review, recent progress on the development of biotransducers and biosensors enabled by the creative use of GQDs and CQDs is reviewed, with special emphasis on how these materials specifically interface with biomolecules to improve overall analyte detection. This review also introduces nano-enabled biotransducers and different biosensing configurations and strategies, as well as highlights key properties of GQDs and CQDs that are pertinent to functional biotransducer design. Following relevant introductory material, the literature is surveyed with emphasis on work performed over the last 5 years. General comments and suggestions to advance the direction and potential of the field are included throughout the review. The strategic purpose is to inspire and guide future investigations into biosensor design for quality and safety, as well as serve as a primer for developing GQD- and CQD-based biosensors.

FIGURE 1

(a) Schematic outline of possible system configurations – with emphasis on graphene and carbon quantum dots interfacing with the biological recognition entity at the level of the biotransducer to form a nanobiosensor. Analyte detection is enabled by a biological recognition element co-joined with the physiochemical transducer. Depending on the engineered interface, physical detection method, and overall system integration, considerable signal processing may be required to achieve an actionable output. (b) An archetypical biosensor system showing the biotransducer which combines biomolecular specificity and the physiochemical transducer, the instrumentation subsystem and the readout device. (I) Matrix containing the analyte of interest and interfering substances, (II) the molecular recognition peptide loop with analyte capture at an engineered interface, (III) the electrochemical cell-on-a-chip comprising a microdisc array working electrode, a large area counter electrode and a reference electrode, (IV) a dual responsive biochip for multiplexed biotransduction, (V) a Bluetooth^® dual potentiostat, and (VI) a computer-enabled base station for data analysis and presentation of actionable results. Adapted from [15].

FIGURE 2

Electronic and band structure of graphene and carbon quantum dots. (a) Is a summary of key biosensor enabling properties of GQDs and CQDs, with the structures ordered by decreasing crystallinity (single graphene sheets being the highest and amorphous carbon dots being the lowest). (b) Illustrates examples of molecular orbitals which provide insight as to the band-like states and the surface states of CQDs. (c) and (d) Highlight the band gap of CQDs, with various contributing surface and band-like states, revealing a dependence on the size (all work from Margraf et al.). (e) Is a schematic reproduced from Li et al. which discusses how terminal groups participate in orbital hybridization as well as charge transfer processes, both of which alter the band gap of GQDs. (f) Highlights how each of the terminal groups alters GQD band gap (Li et al.). (g) and (h) Are work by which illustrate how π-orbital delocalization and various chemical groups influence the band gap of GQDs (Yan et al.). Current work highlights familiar patterns – such as increasing band gap with decreasing length scale – though the underlying etiology of such electronic states is considerably more complex than conventional quantum dots. (b), (c), and (d) Reproduced from Ref. [146], © 2015 American Chemical Society. (e) and (f) reproduced from Ref. [103], © 2015 American Chemical Society. (g) and (h) Reproduced from Ref. [105], © 2018 American Chemical Society.

FIGURE 3

Li et al. developed a general profiling method based on the photoluminescent properties of GQDs, which is detailed in (a). Based on the metabolite of interest, an oxidoreductase enzyme which catalyzed the oxidation of the analyte and formation of H₂O₂ was selected, some examples of which are listed. The response factor to this catalytic event is the oxidative cross-linking of tyramine-conjugated GQDs in the presence of H₂O₂, quenching the original photoluminescent tyramine-GQD conjugates. The generality of this system is its power, as any enzyme which produces H₂O₂ is potentially compatible. (b) Highlights time dependent cross-linking of the tyramine-GQDs in the presence of H₂O₂ (top) and the response to different concentrations (bottom). (c) Is a representative response which uses GOx, showing the luminescent response (top), the linear response range (middle), and the response in serum (bottom). Reproduced from reference [159], 2016 American Chemical Society.

FIGURE 4

Direct electron transfer of GOx as facilitated by GQDs. Razmi and Mohammad-Rezaei immobilized GOx onto a GQD modified electrode – which is characterized in (a), noting an increase in charge transfer resistance, R_CT, upon GQD functionalization and a dramatic increase upon enzyme immobilization. (b) Details characteristic cyclic voltammagrams (CVs) of the modified electrode. Note, on the red curve, (d), the appearance of a quasi-reversible set of peaks. (c) Highlights CV evolution in the presence of glucose, with (d) detailing mM addition of glucose and changes in the CVs. (e) Details amperometric response at constant potential of −0.42 V in 0.1 M PBS (pH 7.4). In this schematic (b) and (d) are the standard curves of (a) and (c) respectively, exploring the dynamic range and linearity of response. K_M was determined via a Lineweaver-Burk plot, which was reported to be 0.76 mM. Reproduced from Ref. [171], © 2013 Elsevier.

FIGURE 5

An electrochemical aptasensor for the detection of lysozyme developed by Rezaei et al. (a) and (b). In their system (a), a glassy carbon electrode modified with multiwalled carbon nanotubes, reduced GO, chitosan, and CQDs was used as the base substrate. This system was modified with an aptamer specific to lysozyme, and then blocked with bovine serum albumin to prevent non-specific binding. This biotransducer, in the absence of the target analyte, was electrochemically competent and could facilitate the redox reaction of ferri-/ferrocyanide. Upon a binding event, the electrode was passivated, which could be monitored electrochemically via the redox current or electrically with EIS. (a) Highlights the amperometric and impedimetric response of the system. A photoluminescent, intracellular aptasensor developed by Zhang et al. for the detection of ATP (c)–(e). In their system, detailed in (c), they developed a probe which consisted of the quenching pair gold nanocrosses and GQDs. The nanocrosses were conjugated to a strand of ssDNA, and the GQDs were conjugated to folic acid for tumor targeting and to the ATP aptamer. In the presence of ATP, the aptamer would preferentially bind to ATP, dissociating the complex, and restoring fluorescent capacity of the GQDs. (d) Shows the resulting standard curve in the presence of ATP, respectively. (e) Shows the intracellular sensing capacity of the system in various cell lines. (a) and (b) Reproduced from Ref. [193], © 2018 Elsevier. (c), (d), and (e) reproduced from Ref. [187], © 2019 Elsevier.

FIGURE 6

A photoelectrochemical (PEC) aptasensor developed by Wang et al. for zeatin detection. In their schematic, the PEC active material was a composite graphite-like carbon nitride doped with GQDs (a) and (b). Detailed in (a), this material was modified with ssDNA responsive to the probe aptameric DNA. In the absence of zeatin, the aptameric sequence would hybridize with the ssDNA on the surface of the electrode, preventing degradation from an ssDNA exonuclease. The aptamer probe was conjugated to biotin, which would hybridize with streptavidin and block PEC current. In the presence of zeatin, the aptamer would preferentially bind to the analyte, and hybridization would not occur. This left the surface-confined ssDNA susceptible to exonuclease activity, the electrode then became unblocked and PEC current spiked. (b) Is the response of the electrode to zeatin, showing both the PEC current and the standard curve. Lu et al. reported an electrochemiluminescent system to detect ATP. Their system (c) was a simple hybridization schematic wherein an electrode was modified with one ssDNA strand and the complementary strand was conjugated to an SiO₂/GQD particle, which served as the ECL source. In the presence of ATP, the two would hybridize, and the ECL intensity would increase. (d) and (e) Show, respectively, the ECL response to increasing concentrations of ATP and the linear response range after a logarithmic transform. (a) and (b) Reproduced from Ref. [201], © 2018 Elsevier. (c), (d), and (e) Reproduced from Ref. [81], © 2013 Elsevier.

FIGURE 7

A universal photoluminescent immunosensing regime using graphene and GQDs detailed by Zhao et al. (a) and (b). In (a), GQDs modified with a target-specific antibody (mouse anti-human IgG in this work) were free to interact with graphene in the absence of the analyte, resulting in the quenching of the GQDs. In the presence of the target epitope, the interaction between the GQDs and the graphene would be sterically inhibited, and the GQDs would again begin fluorescing. (b) Shows the increasing fluorescent intensity as a function of target concentration (human IgG in this work; left), and the dose response curve of intensity vs. analyte concentration (right). An electrochemiluminescent (ECL) immunosensor from work by Nie et al. with an application to cancer biosensing (c)–(e). They designed a sandwich immunosensor for the detection of carcinoembryonic antigen (CEA, c), where the dual sandwich binding event would result in an appreciable increase in ECL intensity. (d) Shows the characteristic cyclic voltammograms (CVs) of their system during stepwise synthesis and antigen presence (left) and their respective EIS responses. (e) Shows the increase in ECL intensity as the concentration of CEA increases and the resulting standard curve. (a) and (b) Reproduced from Ref. [204], © 2013 Royal Society of Chemistry. (c), (d), and (e) Reproduced from Ref. [217], © 2018 Elsevier.

FIGURE 8

(a) Details an electrochemical sandwich immunosensor developed by Serafín et al. for the detection of IL-13 receptor a2. One half of the sandwich was a GQD-modified CNT complex doped with HRP and a detector antibody. The other half was a carbon electrode modified with streptavidin/para-aminobenzoic acid, which in turn immobilized the capture antibody conjugated to biotin. On a dual binding event, the HRP was brought in close proximity to the electrode, and the H₂O₂ byproduct could be detected amperometrically. Various CVs of the different surface modified electrodes in the presence of the precursor (hydroquinone, b), and the precursor plus 100 mM H₂O₂ (c). (d) Is the electrochemical impedance spectroscopy (EIS) response of the system during the various surface modification steps, while (e) shows the EIS of the full system in the presence and absence of the analyte. Equivalent circuits that were used to fit the data are detailed above each of their respective EIS spectra (d) and (e). Reproduced from Ref. [213], © 2019 Elsevier.

FIGURE 9

A supramolecular biosensing regime developed by Huang et al. for the quantification of human telomeric DNA. They coupled the DNAzyme activity of the hemin/G-quadruplex complex (which forms in human telomeric DNA sequences) with the photoluminescent quenching of GQDs. Pictured in (a) is a rendition of a parallel quadruplex structure, though other configurations are possible. The DNAzyme was capable of converting ortho-phenylenediamine to 2,3-diaminophenazidine, which in turn quenched GQDs and formed a turn-off biosensor (a). (b) Shows the fluorescent spectra of varying compounds and system configurations, and (c) shows the fluorescent quenching of GQDs in the presence of hemin (plotted as a standard curve in d). (e) Shows the ratiometric response of the GQD system to varying concentrations of hemin/G-quadruplex DNAzyme, with the associated colorimetric response shown in (f). This response was visualized as a standard curve shown in (g). Reproduced from Ref. [231], © 2017 Elsevier.