Fluorescence and Sensing Applications of Graphene Oxide and Graphene Quantum Dots: A Review

Abstract

Graphene oxide and graphene quantum dots are attractive fluorophores, which are inexpensive, non-toxic, photo-stable, water-soluble, biocompatible and environmentally friendly. They find extensive applications in fluorescent biosensors and chemi-sensors, in which they either serve as fluorophores or quenchers. As fluorophores, they display the tunable photoluminescence emission and the “Giant Red-Edge Effect”. As quenchers, they exhibit a remarkable quenching efficiency via either electron transfer or Förster resonance energy transfer (FRET) process. In this review article, the origin of fluorescence and the mechanism of excitation wavelength-dependent fluorescence of graphene oxide and graphene quantum dots are discussed. The sensor design strategies based on graphene oxide and graphene quantum dots are presented. The applications of such sensors in health care, environment, agriculture and food safety are highlighted.

1. Introduction

Graphene-based nanomaterials are the two-dimensional (2D) atomic crystals, which are built on the sp² hybridized carbon atoms.^[1] Its family includes graphene, graphene oxide (GO), reduced graphene oxide (rGO) and graphene quantum dots (GQDs). Ideally graphene is completely composed of the sp² hybridized carbon atoms, which exhibits no fluorescence due to the zero optical band gap. GO, rGO and GQDs consist of the mixed sp² and sp³ carbons due to the introduction of functional groups, which opens the optical band gap, rendering fluorescence.^[2] GO is rich in oxygen-containing functional groups. Chemical reduction of partial oxygen-containing functional groups in GO leads to the formation of rGO.^[3] GQDs are cut from the single to several atomic layers of graphene sheets to several nanometers in lateral size.^[4] Typically GQDs contains both sp² and sp³ carbon atoms, which are inherent from synthesis processes. GQDs have a size-dependent optical band gap due to the quantum confinement.^[5] GO and GQDs are inexpensive, non-toxic, photo-stable, water-soluble, biocompatible, and environmentally friendly. And they exhibit excitation wavelength-dependent fluorescence.^[6] In addition, their 2D surface shows strong non-covalent interaction with adsorbed biomolecules via π-π interaction, electrostatic force or hydrogen bonding, which provides a chemically tunable platform for bio-conjugation.^[6–7] Hence both GO and GQDs have found extensive applications in fluorescent sensors in recent years. They can be employed as not only a tunable fluorophore but also an effective fluorescence quencher.

This review article starts with discussion of the physics principle of fluorescence of GO and GQDs. It will then show how to design fluorescent sensors by utilizing unique physicochemical properties of GO and GQDs. Also, this paper highlights the applications of GO- and GQD-based fluorescent sensors in a variety of fields.

2. Fluorescence Principles of Graphene Oxide

2.1. Fluorescence Origin

Electronic energy transition is responsible for fluorescence of GO.^[5] As shown in Figure 1, each “fingerprint” fluorescence band of functionalized GO comes from specific electronic transitions between the antibonding and the bonding molecular orbitals such as σ*→n, π*→n, and π*→π.^[5] GO generally contains various oxyen-containing functional groups such as hydroxyl groups (C-OH), carboxyl groups (COOH), carbonyl (C<C=>O), epoxy (C-O-C) and aromatic rings (C<C=>C). As a result, multiple fluorescence peaks, which correspond to different electronic transitions, are usually excited simultaneously. When these fluorescence peaks are overlapped with each other, a single broad peak displays as shown in Figure 1.^[8]

Figure 1.

(a) UV/Visible absorption of as-synthesized GO and GO treated with KOH and HNO₃; (b) Fluorescence spectra corresponding to (a) with an inset showing different electronic transitions. Reproduced from Ref. 8 with permission from The Royal Society of Chemistry.

Functional groups, dopants, lateral size, localized domains and strain in GO can alter the electronic energy transition in GO, leading to change in fluorescence peak position, width and shape. For example, after GO is incubated in the aqeuous KOH and HNO₃ solution, GO is enriched with -OH and-COOH, respectively. Consequently, the fluorescence peak position and intensity are tuned as shown in Figure 1.^[8] Keeping in mind, the band gap of GO is created as a result of a mixture of sp² and sp³ carbon atoms after introducing functional groups. The electronic band structure can be tuned by varying the ratio of sp²/sp³ carbon atoms, which renders fluorescence to be tunable from the visible light to near-infrared (NIR) wavelength range. The fluorescence of GQDs has the same origin as GO. Doping was found to modulate the electronic structure of GQDs and thus affect the fluorescence emission. For example, in nitrogen-doped GQDs, the fluorescence is dominated by the n→π* transition between the aromatic ring containing N and the conjugate structure of graphene.^[9] Increasing the doping percentage of nitrogen in GQDs could red-shift the emission and improve the photostability.^[10] Due to quantum confinement, changing the size of GQDs can modulate the electronic structure and thus can shift the fluorescence from green, red to NIR. In addition, change in the localized domains, where π-electrons are confined in localized sp² regions, can also alter fluorescence of GO.^[10–11]

2.2. Excitation Wavelength-Dependent Fluorescence

2.2.1 Breakdown of Kasha’s Rule

According to the Kasha’s Rule, the peak fluorescence wavelength for organic dyes and inorganic quantum dots is independent of the excitation wavelength because fluorescence always starts at the band edge after a rapid internal relaxation. However, Kasha’s Rule does not hold for GO. Excitation wavelength-dependent fluorescence occurs in GO suspended in polar solvents as shown in Figure 2.^[8] When the excitation wavelength increases from 325 nm to 650 nm, the fluorescence band red-shifts to longer wavelengths.

Figure 2.

Excitation wavelength-dependent fluorescence of GO. Reproduced from Ref. 8 with permission from The Royal Society of Chemistry.

2.2.2 Giant Red-Edge Effect

Excitation wavelength-dependent fluorescence of GO is due to the “Giant Red-Edge Effect”.^[12] As shown in Figure 3, the Giant Red-Edge Effect occurs in a polar solvent such as water where the solvation introduces an additional relaxation step. For an excited fluorophore in a polar solvent, the dipole is not in equilibrium with the solvent dipole. During solvation, these dipoles are realigned with one another, and reduce the interaction energy in order to achieve equilibrium. For a common fluorophore in a polar solvent, the solvation dynamics is orders of magnitude faster than fluorescence. The solvation is usually completed prior to fluorescence. Therefore, the final fluorescence only undergoes a small red-shift. However, if the solvation dynamics is slowed down to the time scale of fluorescence, fluorescence occurs simultaneously as the energy of the excited fluorophore is reduced, leading to strong dependence of fluorescence on the excitation. The Giant Red-Edge Effect takes place not only in GO but also in GQDs and carbon dots.

Figure 3.

Fluorescence properties of GO. GO exhibits an excitation wavelength dependent fluorescence (or Giant Red-Edge Effect) due to solvation. It also displays an excited state protonation from COOH groups. The measured fluorescence of GO is a superposition of contributions from C-OH and COOH groups. Reprinted with permission from Ref. 12. Copyright (2014) American Chemical Society.

2.2.3 Excitation Wavelength-Dependent Fluorescence due to Strain

Given the fast solvation dynamics (≈10⁻¹² s⁻¹) of the polar solvents used, there must be some important factors relevant to material itself, which drastically slow down the dipole realignment process to the timescale of fluorescence. In this regard, efforts have been made to study the effects of oxygen percentage, doping percentage, disorder, and strain in GO, which have been commonly hypothesized to be the underlying reasons for Giant Red-Edge Effect.^{[11a, 13]} Of the aforementioned factors, the strain has been found to correlate with the Giant Red-Edge Effect.^[14] When there is an out-of-plane strain, the plane of the GO sheet gets curved, generating a strong local reorganization potential. The local potential prevents a fast re-orientation of the excited dipoles with the solvated dipoles in the oxygen-containing functional groups nearby, and drastically slows down the dipole realignment process to the timescale of fluorescence, generating the Giant Red-Edge Effect.^[14] The red-shift of the peak fluorescence wavelength in GO is also found to scale linearly with the increase of the excitation wavelength.^{[8, 12]} The peak fluorescence can be tuned from visible to the NIR region by simply changing the excitation wavelength without changing any other factor such as chemical composition and size.

2.2.4 Influence of pH on GO Fluorescence

In addition, the fluorescence of GO is dependent on the pH value of the solvents, as shown in Figure 4.^[11a] Under acidic conditions, GO exhibits a broad fluorescence peak centered at around 680 nm. When the pH gradually increases, the 680 nm peak reduces its intensity, and finally disappears with the emergence of two new peaks at around 500 nm under basic conditions. This pH-dependent fluorescence is believed to be caused by the excited state proton transfer, as shown in Figure 3. Under an acidic condition, the −COOH group is largely deprotonated and exists in the form of ionic −COO⁻ in the ground state. Upon excitation, the excited state protonation from ionic −COO⁻ to −COOH group contributes to the broad fluorescence peak at 668 nm. In comparison, the fluorescence emission under basic conditions is associated with the excited −COO⁻ moieties.

Figure 4.

pH-dependent photoluminescence of GO excited at 440 nm. (a) The pH effect on photoluminescence; (b) Plot of the photoluminescence intensities at 683 nm, 506 nm, and 479 nm with respect to the pH of GO solutions. Reprinted by permission from Macmillan Publishers Ltd: [Scientific Reports] (Ref. 11a), copyright (2011).

3. Sensing Applications of Graphene Oxide and Graphene Quantum dots

GO can be used as a fluorophore with tunable fluorescence emission by varying the chemical composition, size and other factors. GO can also be used as a highly efficient quencher via either the charge transfer or the resonance energy transfer processes given its super electrical conductivity and 2 D planar structure. The dual roles of GO as both a fluorophore and a quencher open up new opportunities for sensor design, as shown in Figure 5. Hence graphene-based nanomaterials are finding increasing applications in environment, agriculture, biomedicine, food safety and homeland security. This section outlines different design strategies for fluorescent biosensors and also highlights some important applications.

Figure 5.

Schemastic illutration of sensor design based on GO. (a) GO as a fluorescent label; (b) GO as a FRET donor and its fluorscence can be quenched by a quencher, such as a gold nanoparticle, through FRET; (c) Charge transfer occurs between GO and a fluorophore in its close proximity; (d) GO as a quencher and can quench the fluorescence of a nearby fluorophore through FRET.

3.1. Graphene Oxide as a Fluorescent Label

GO has been widely used as a fluorescent label, just like organic dyes and inorganic quantum dots.^[15] However, the broad emission peak limits the sensing performance when the fluorescence spectral intensity is transduced as the signal. Instead, GO is remarkably suitable for NIR biological imaging using two-photon excitation spectroscopy thanks to the Giant Red-Edge Effect.^[12] Two-photon excitation is based on the simultaneous absorption of two photons which have only half the energy of a single photon traditionally used to excite the same event.^[16] The selection of light source with a longer wavelength, especially NIR excitation, greatly helps reduce photo-toxicity, lower the background signal, and increase the light penetration depth in a biological sample. GO with an excitation wavelength dependent fluorescence is well coupled with two-photon excitation spectroscopy, as its excitation wavelength can be deliberately chosen to be fallen into one of the biological windows, allowing selective biological imaging.^[17] The first and second NIR biological windows are located at 650≈950 nm and 1000≈1350 nm, where the biological sample is almost transparent to the incident light.^[18] As an example, GO exhibits different fluorescence when excited in the first and second biological windows using two-photon excitation fluorescence spectroscopy, as shown in Figure 6 (a) and (b).^[17] The advantages of the use of GO in biological imaging under two-photon excitation are further manifested by applications in cancer treatment as shown in Figure 6 (c) to (f).^[19] Graphene oxide nanoparticles (GON) are functionalized with transferrin (Trf) molecules to target the cancer cells. The Trf-GO only needs a much smaller laser power to generate sharp and well-resolved GON images. As Trf can specifically target cancer cells, so does the Trf-GON complex. The process of killing cancer cells using Trf-GON complex can thus be directly reflected on the GON fluorescence images. Liu et al. have also reported the cellular and deep-tissue imaging using two-photon fluorescence spectroscopy based on graphene quantum dots.^[20] Wang et al. took advantage of two-photon spectroscopy to image endogenous biological cyanide in plant tissues based on GQDs-gold nanoparticles complex.^[21] Two-photon imaging with GO as a fluorescent label has been employed in biomedical fields to target liver tumor cells,^[22] breast tumor cells,^[23] melanoma cells,^[24] and also for food safety.^[21]

Figure 6.

Two-photon fluorescence imaging (a) tunable excitation wavelength dependent two-photon imaging in the first and second biological transparency windows; (b) Two-photon fluorescence imaging of methicillin-resistant Staphylococcus aureus (MRSA) using aptamer modified GO, where the excitation wavelength for (b1) to (b4) are 1160 nm, 880 nm, 980 nm, and 760 nm, respectively. Reprinted by permission from Macmillan Publishers Ltd: [Scientific Reports] (Ref. 17), copyright (2014). (c)–(f) how a comparison of in vitro two-photon luminescence imaging of graphene oxide nanoparticles and molecular dye FITC. Reprinted from Ref. 19 with permissions from Wiley.

3.2. Graphene Oxide as a Donor in Charge Transfer

The fluorescence of GO can be quenched via a charge transfer process, in which GO acts as a charge donor.^[25] Compared to FRET, which is a long-range energy transfer mechanism, charge transfer is a short-range energy transfer process. It requires the charge donor and the acceptor to be separated within 10 Å.^[26] Such a short distance is often realized through chemical bonding or physical adsorption. As an example, dopamine molecules have been detected in a GO fluorescent sensor based on charge transfer (Figure 7 (a).^[15c] The dopamine molecule is chemically bonded to GO via the π-π interaction between GO and the aromatic rings in the molecules. In the excited GO-dopamine complex, the excited electrons in GO are transferred to the dopamine molecule, which results in the quenching of the GO fluorescence. In addition, Wu’s research group has utilized the charge transfer mechanism to detection metal cations,^[25] in which GO serves as a fluorophore and an electron donor while the metal ions act as the electron acceptor (Figure 7 (b)). GO is initially functionalized with a single-stranded DNA (ssDNA) aptamer, which can capture Hg²⁺ and form a hairpin structure on GO due to the formation of thymine-Hg²⁺-thymine complex. Once the GO-aptamer-Hg²⁺ complex is formed, fluorescence of GO is quenched due to charge transfer from GO to Hg²⁺. A similar sensor platform has been used for I⁻ detection, as shown in Figure 7 (c).^[27] As the binding constant^[28] for Hg²⁺ and I⁻ is >10²⁹, much larger than that for Hg²⁺ and thymine which is ≈10⁶, HgI₂ is preferentially formed and pulls the Hg²⁺ ions away from the GO surface, leading to recovery of the quenched fluorescence and allowing I⁻ to be detected. In addition, the GO sensor platform based on charge transfer has been successfully applied for various other environmental and agricultural pollutants detection such as Au³⁺, Fe³⁺, Fe²⁺, Co²⁺, Ni²⁺, Cd²⁺, HCl, et al.^{[15d,e, 29]}

Figure 7.

Fluorescent sensors based on charge transfer. (a) Dopamine detection based on charge transfer between GO and dopamaine through π–π stacking. Rerpinted with permission from Ref. 15c. Copyright (2011) American Chemical Society. (b) Hg²⁺ detection where Hg²⁺ is captured through thymine-Hg²⁺-thrymine interaction and quenches the fluorescence of GO. Reprinted from Ref. 25, Copyright (2013), with permission from Elsevier. (c) Hg²⁺ and I⁻ can both be detected on the same GO fluorescent biosensor platform. Reprinted with permission from Ref. 27. Copyright (2015) American Chemical Society.

3.3. Graphene Oxide as a Donor in FRET

In a FRET process, GO can serve as an energy donor where its fluorescence gets quenched by an energy acceptor such as gold nanoparticles and organic dyes.^[30] FRET is a non-radiative energy transfer process based on the dipole-dipole interaction.^[26] It requires the donor’s emission spectrum to be overlapped with the acceptor’s absorption spectrum. The FRET efficiency is inversely proportional to the six power of the separation distance of the donor and acceptor. Therefore, in order for FRET to be efficient, the energy donor and the acceptor need to be brought close to each other, usually within ≈6 nm (the Förster distance) if organic dyes serve both the energy donors and acceptors. This can be realized through antigen-antibody interaction, DNA hybridization, et al.^{[30b, 31]} Since GO has a broad fluorescence emission peak, the condition of spectrum overlap can be easily met with noble metal nanoparticles as a quencher which have a large absorption cross section with absorption band tunable from the ultraviolet light to the NIR region. As an example, GO has been used as an energy donor in an immuno-biosensor system for rotavius detection (Figure 8).^[30b] Initially, gold nanoparticles are functionalized with the detection antibody of the rotavirus; and the capture antibody is immobilized the GO surface. The gold nanoparticles and GO can be brought in a proximity through the antigen-antibody interaction after capturing the rotavirus cell. Consequentially, the fluorescence of GO is quenched by gold nanoparticles via the FRET process. GO has also been exploited as an energy donor in FRET sensors for drug detection, disease diagnosis, pharmaceutical screening, and so on.^{[30a, 32]}

Figure 8.

GO as a FRET donor in a sensor for rotavirus detection where the fluorescence of GO gets quenched by gold nanoparticles. Reprinted from Ref. 30b with permissions from Wiley.

3.4. Graphene Oxide as an Acceptor in FRET

In a FRET process, GO can also act as an energy acceptor where it quenches the fluorescence of an energy donor such as quantum dots and organic dyes.^{[29a, 33]} Use of GO as a quencher has several advantages: (i) The 2 D planer surface area renders a large number of bonding sites; (ii) The oxygen-containing functional groups enables further bioconjugation; (iii) It is water-soluble, non-toxic, biocompatible and intercellular penetrable. Also, the effective quenching distance could be extended to ≈30 nm if GO as an energy acceptor. Figure 9 highlights several FRET detection schemes.^{[29a, 33]} For example, (i) The fluorophore-labelled single-stranded aptamers are initially captured on GO via the π–π stacking force between the aromatic nucleobases of aptamer and the aromatic rings in GO, as shown in Figure 9 (a). The fluorescence of fluorophore such as organic dyes and quantum dots are quenched by GO in a proximity via the FRET process. After the presence of analytes, the aptamers form hairpin or G-quadruplex structures, which have a weak interacting force with GO. They go away from GO, turning on fluorescence. (ii) In Figure 9 (b), the fluorophore-labelled double-stranded DNA molecules are initially captured on the GO surface, in which one strand of DNA is covalently bounded to GO, and the other strand of DNA is labeled with a fluorophore. Fluorescence of the fluorophore is quenched if the fluorophore is controlled to be close to the GO surface (typically less than 6 nm). The addition of analytes causes DNA dehybridization. The fluorophore-labeled DNA strand form a hairpin structure, and is detached from the GO surface. (iii) In Figure 9 (c), the free-standing fluorophore-labelled detection antibodies initially display fluorescence. When analytes are present, the analytes are sandwiched between the detection antibody and the capture antibody, which brings the fluorophore close to the GO surface, leading to quenching of fluorescence. (iv) In Figure 9 (d), the fluorophore-labelled antigens are initially attached to the capture antibody on the GO surface with the fluorescence quenched. After addition of analytes, the fluorophore-labelled antigens are displaced away from GO due to the stronger affinity of analyte with the capture antibody, which leads to recovery of fluorescence.

Figure 9.

Schematic illustration of sensor design strategies with GO as a quencher. (a) An aptamer-based sensor; (b) A DNA-based sensor; (c) A sandwich-structured immunoassay; (d) A competitive immunoassay.

For example, microRNA (miRNA) is a type of important biomarkers as it regulates the expression of diverse genes and can be related to many diseases such as cancers and diabetes. GO with its capability of penetration through intracellular membranes has been used to build FRET sensors for in vitro detection of miRNA, as shown in Figure 10 (a).^[33a] In this sensor, a dye-labelled peptide nucleic acid (PNA) is initially quenched after being adsorbed to GO. The GO-PNA-dye complex is then delivered into the cytoplasm, where the target miRNA hybridizes with dye-labelled PNA and detaches from GO, leading to the fluorescence recovery. In addition, GO as a FRET quencher has also been used for in vitro and in vivo molecular probing based on two-photon excitation spectroscopy.^[34] By replacing the capture and detection reagents, the GO sensor platform has been extended for the detection of proteins, DNA, drugs, heavy metals, et al.^{[33b, 35]}

Figure 10.

GO as a quencher in sensors. (a) miRNA detection based on nano-sized graphene oxide (NGO) and peptide nucleic acid (PNA) where the fluorescence of the dye-labelled on the PNA probe is initially quenched by GO but later recovered after the probe detaches from NGO and hybridizes with a target miRNA. Reprinted with permission from Ref. 33a. Copyright (2013) American Chemical Society. (b) Ovarian cancer biomarker CA-125 detection based on chemiluminescence resonance energy transfer (CRET). Reproduced from Ref. 36 with permission of The Royal Society of Chemistry.

In addition to fluorescence, chemiluminescence provides an alternative for optical biosensing. Chemiluminescence occurs as a result of chemical reactions rather than direct photo-excitation, which could lead to a low signal-to-noise ratio. It has been reported that strong quenching capability of GO and GQDs can be used to quench chemiluminescence via chemiluminescence resonance energy transfer (CRET), as shown in Figure 10 (b), where the cancer biomarker CA-125 is detected based on CRET.^[36] GQDs are used as the quenchers (energy acceptors) and assembled on a glass substrate, which are further functionalized with the capture antibody for CA-125. The detection antibody for CA-125 is labelled with horseradish peroxidase (HRP) enzyme. In the presence of CA-125 antigen, the antibody-antigen-antibody complex is formed with HRP, and brought in a proximity to GQDs. As a result, the HRP enzyme catalyzes H₂O₂ to produce reactive oxygen species, which further oxidizes luminol to generate chemiluminescence. In case that HRP is brought close to GQDs in the presence of CA-125 antigen, the produced chemiluminescence gets quenched by GQDs nearby. This “turn-off” sensor enables the cancer biomarker CA-125 to be detected sensitively. In addition, CRET sensors with GO or GQDs as quenchers has also been used for detection of other protein biomarkers, DNA, environmental pollutants.^[36–37]

3.5. Graphene Quantum Dots in Fluorescent Biosensors

While GQDs shares many common excellent properties with GO such as easy functionalization, photo-stability, water-solubility, biocompatibility, and non-toxicity, GQDs has its own unique properties. For example, GQDs has a much smaller steric effect due to its small size; it can easily penetrate through intracellular membranes. This unique property makes GQDs a candidate material in drug delivery, in vivo and in vitro bioimaging.^[38] For example, Lannazzo et al. have employed GQDs for cancer targeted drug delivery.^[38c] Since GQDs also have an excitation wavelength-dependent emission, it can display different colors in bioimaging under different excitations, meeting the needs of various types of biosensing, as shown in Figure 11 (a).^[38e] The penetrating ability of GQDs in intracellular membranes also allows in-depth bioimaging using near-infrared light, enormously extending the scope of fluorescent bioimaging, as shown in Figure 11 (b).^[38d]

Figure 11.

bio-imaging with GQDs utilizing the Giant Red-Edge Effect. (a) Fluorescence images of A549 cells cultured with GQDs under different excitations (shown on the upper right of each image). Reprinted by permission from Macmillan Publishers Ltd: [Light: Science & Applications ] (Ref. 38e), copyright (2015). (b) Fluorescence images with GQDs at different depths in tissue. Reprinted with permission from Ref. Ref. 38d. Copyright (2017) American Chemical Society.

4. Conclusions and Perspectives

There are many superior properties for GO and GQDs. Compared to organic dyes, GO and GQDs are photo-stable. Compared to most inorganic semiconductor quantum dots, GO and GQDs are non-toxic and biocompatible. The strong excitation wavelength-dependent fluorescence of GO and GQDs are unique to fluorescent sensing and imaging; and it can be extended to other photoluminescent devices. However, GO and GQDs exhibit broad fluorescence peak in the presence of a large variety of oxygen-containing moieties, as compared to the sharp fluorescence peak of organic dyes and inorganic quantum dots. If a narrow fluorescence peak is needed for some applications, it is necessary to purify and functionalize GO.

Interestingly, GO is suitable for NIR biological imaging using two-photon excitation fluorescence spectroscopy due to their large two-photon absorption cross section. On the other hand, GO can be purified to eliminate various oxygen-containing moieties, and further functionalized to tailor the fluorescence peak position and width. In the future, further studies needs to tune the fluorescence of the purified and functionalized GO.

GO and GQDs can be employed not only as fluorophores but also as quenchers, which provide a plenty of room for development of new fluorescent biosensors and bio-imaging systems. It is envisioned that the applications of GO and GQDs in in vitro and in vivo sensing and imaging will continue to be expanded.

Multiplex detection is significant to practical applications of biosensors because it will enormously improve the specificity and lowers the cost of detection.^[39] The dual roles GO and GQDs as a fluorophore as well as a quencher make them highly promising. They can transduce an excitation wavelength dependent fluorescence signal and simultaneously quench the fluorescence from external fluorophores, which have strong implications in the detection of multiple events on a single biosensor.

Furthermore, the 2 D planar structure of GO makes it ideally coupled with lab-on-chip devices.^[40] The integration of all these functionalities into a single chip would greatly promote point-of-care and field-deployable applications of biosensing devices in the future. It is expected that GO and GQDs along with other materials in the family of graphene-based materials will find increasing applications in healthcare, environmental protection, agriculture, food safety, homeland security, and et al.