Graphene and its derivatives for cell biotechnology

Abstract

Every few years, a novel material with salient and often unique properties emerges and attracts both academic and industrial interest from the scientific community. The latest blockbuster is graphene, an increasingly important nanomaterial with atomically thin sheets of carbon, which has become a shining star and has shown great promise in the field of material science and nanotechnology. In recent years, it has changed from being the exclusive domain of physicists to the new passion of chemists and biologists. Graphene and its derivatives are now at the forefront of nearly every rapidly developing field of science and engineering, including biochemistry, biomedicine and certain cutting-edge interdisciplines that have intense popularity. The aim of this review is, firstly, to provide readers with a comprehensive, systematic and in-depth prospective of graphene's band structure and properties, and secondly, to concentrate on the recent progress in producing graphene-based nanomaterials, including mechanical exfoliation, chemical vapor deposition, plasma enhanced chemical vapor deposition, chemical reduction of graphene oxide, total organic synthesis, electrochemical synthesis and other fabrication strategies widely accepted by research scientists. At the same time, important definitions related to graphene are also introduced. The focus of this Tutorial Review is to emphasize the current situation and significance of using this new kind of two-dimensional material in the hot and emerging fields that are closely related to human life quality, for instance, cell biochemistry, bioimaging along with other frontier areas. Finally, the latest developments and possible impact that affect the heart of the whole scientific community have been discussed. In addition, the future trends along with potential challenges of this rapidly rising layered carbon have been pointed out in this paper.

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Mei Yang

Mei Yang was born in Sichuan, China, and studied chemistry at China West Normal University. As an undergraduate, she conducted research on the development of new biosensors and their applications in biomolecules analysis such as amino acid, protein and nucleic acid, etc. After receiving her B.S. in chemistry in 2010, she joined the lab of Professor Yixiang Duan in the Research Center of Analytical Instrumentation at Sichuan University, China. Her research interests are in the area of bioanalytical chemistry. Current work focuses on the green and morphology controlled nanomaterials synthesis, the functionalization of inorganic nanoparticles (mainly graphene and quantum dots) with inorganic or organic ligands and integration of these novel materials with various photoelectric analysis techniques. Her other scientific interests include the applications of GC, HPLC and LC-MS on medical diagnostics and pharmaceutical analysis.

Jun Yao

Jun Yao studied Chemistry at China West Normal University, where he obtained his B.S. degree in Chemistry in 2006. He worked in the field of bioanalytical chemistry, receiving his M.S. degree from the same university in 2009. Following graduate studies, he became a university teacher of chemistry and pharmacy at Chongqing Three Gorges Medical College, China. Subsequently, he joined the Analytical and Testing Center of Sichuan University as a PhD candidate to research biochemical or medical analysis based on nanomaterials as well as spectroscopy and electrics related technology, especially carbon nanomaterials and fluorescence technique. His research interests revolve around graphene-based nanomaterials, quantum dots and multifunctional nanostructures, spanning from synthetic methodology to device fabrication, with the desire of pursuing nanostructures for useful photoelectric applications, mainly focusing on a variety of biochemical and biomedical applications of graphene and other inorganic nanomaterials such as biosensor, bioimaging, drug delivery, medical diagnostics and biotherapy.

Yixiang Duan

Yixiang Duan received his B.S. degree from Fudan University and M.S. degree in analytical chemistry from Changchun Institute of Applied Chemistry, Chinese Academy of Science in 1988 and PhD degree in analytical chemistry jointly from Jilin University, China, and Indiana University, USA in 1994. Then he did his postdoctoral research at Los Alamos National Laboratory. From 1997 to 2010, he was a Principal Investigator and Staff Scientist with the Chemical Diagnostics and Engineering Group, Los Alamos National Laboratory. He is currently a National Special Professor and Director of Research Centre of Analytical Instrumentation, Sichuan University, China. His current research interests include nanomaterial-based photoelectric biosensors, molecular spectrometry, non-invasive medical diagnostics, novel analytical instrumentation, as well as various applications of chemistry to nanomaterials, nanomedicine, and biological science, especially fluorescence instrumentation, fluorescence imaging and development of novel strategies and methods for utilizing graphene in bioanalysis and biomedicine. His other research interests are in the areas of spectrometry, chromatography, mass spectrometry and analytical instrumentation.

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1. Introduction

Nanoscience is always inspiring the imagination of biologists and biotechnologists.¹ Recent decades have witnessed the magical glamour of nano- and bio-technology, two important hot topics of interdisciplinary research. They have undergone a rapid growth all over the world and have turned out to be the source of new research directions.^1–5 In these cutting-edge fields, the emergence of a broad array of new-found nanomaterials will gradually affect our life. Because of their unique structures, components and properties, these materials have merged in many daily-used products and have provided appealing opportunities for biotechnological development.^6–11

Graphene is one of the most fascinating nanomaterials with important applications.^12–18 Its discovery has spawned huge interest in the fields of chemistry, physics, biology, medicine, nanoscience and material science.^19–27 In the meantime, a lot of papers are published every day according to the bibliometrics predictions.²⁸ There is no doubt that researches on graphene have gone forward at a truly relentless pace and this gathered momentum will keep rapidly increasing over the next few years.^29,30 Till now, the blue ribbon for graphene is the Nobel Prize in physics in 2010 that was awarded to Geim and Novoselov, the first group to isolate single-layer graphene from graphite.^19,31–33 Since the discovery of graphene, considerable efforts have been dedicated to its structure, fabrication, functionalization and application.^13,34–42 A number of researches have suggested that graphene is a single-atom-thick sheet with carbon atoms of sp² bonding and forming a hexagonal 2D lattice.^43–45 It is the thinnest material ever measured,^34,46–49 yet is harder than diamond, and possesses a large specific surface area, outstanding thermal conductivity, and favorable electric conductivity, along with eminent quantum Hall effect.^13,50–54 Due to these unique physicochemical properties,^55–60 graphene has been considered as a promising candidate in many applications and technological aspects such as nanoelectronics,^34,61,62 polymer composites,^{51,53,63–67} sensors,^68–86 batteries,^87–94 fuel cells and super capacitors.^95,96 In this paper, after a brief presentation of its structures, unique properties and preparations, together with the content what is discussed in the vast majority of research articles and reviews, we put most of our energy on its applications for biotechnology that are seldom involved in other peer publications.

2. Basic structure and properties of graphene

Most of the amazing properties of graphene derive from its peculiar lattice structure. Thus, it is imperative to understand the structure of graphene before introducing its properties.⁹⁷ Graphene comprises a single layer of sp²-hybridized carbon atoms arranged densely in a planar hexagonal honeycomb crystal structure (Fig. 1).^98–106 In this configuration, one s-orbital and two p-orbitals of each carbon atom hybridized to form three in-plane σ bonds and have a strong wave function overlap with their three nearest neighbor atoms to give rise to a very strong covalent bond, which results in the mechanical stability of the carbon sheet. While, the unaffected p_z orbital, oriented perpendicular to the plane of the 2D sheet, can bind covalently with the adjacent carbon atoms, leading to the formation of a delocalized π bond.

Fig. 1 Photograph of planar hexagonal honeycomb crystal structure of graphene. Reproduced with permission from ref. 106. Copyright 2006, Nature Publishing Group.

Unsurprisingly, the essential 2D structure is responsible for the prominent properties and other excellent characteristics of graphene.^{13,56,107–109} Here, we will only introduce graphene's characters that are most relevant to cell biotechnology. Because of the largely dislocated π-electrons, graphene possesses remarkably high electron mobility and size-dependent electrical properties, which make it a promising material in high-performance biosensors as well as an efficient fluorescence nanoquencher in the optical-based detection of biomolecules.^110–112 Its large specific surface area with a value up to 2600 m² g⁻¹ can provide an ultrahigh loading capacity for biomolecules and drugs, leading to increased sensitivity.^{79,97,113,114} Experimental results reveal that graphene has a Young's modulus of 1060 GPa and a breaking strength of ∼40 N m⁻¹.¹¹⁵ Therefore, it has been recognized as an ideal scaffold for cell culture growth.

3. Fabrication and functionalization of graphene

So far, inspired by pioneering work, tremendous efforts have been devoted to exploring synthetic approaches for graphene and its derivatives.^116–121 Broadly speaking, the preparations of graphene can be arranged into four main methods as follows. The first is a physical approach, known as ‘mechanical exfoliation’, where mechanical or chemical energy are essentially used to break the weak van der Waals forces and separate out individual graphene sheets (Fig. 2).^122–125 It is promising to produce graphene on a large scale and some of the recent modifications have been employed to produce large-size pieces. But there is still a long way to apply this process into an industrial scale production level due to its low yield. The second is chemical vapor deposition (CVD),^126–131 first reported for graphene in 2006, a typical method that eradicates residual metallic impurities and has been used to grow large areas of single- or few-layer graphene on a wide range of metal substrates, e.g. Ni, Ru, and Cu, without changing its properties.^132–146 The third strategy is plasma enhanced CVD (PECVD). It has demonstrated the versatility of synthesizing graphene on any substrate and is capable of growing single-layer graphene with high throughput at shorter reaction times and lower deposition temperatures compared to CVD.¹⁴⁷ The fourth is the chemical reduction of graphene oxide (GO), both an economical and practical method to acquire graphene.^{118,148–151} Graphene synthesized by this approach possesses many structural defects, which is advantageous for biosensing applications. Additionally, there are other innovative categories including total organic synthesis, electrochemical synthesis, thermal decomposition of SiC, and unzipping of carbon nanotubes (CNTs).^{22,55,118,122,125,152–161}Fig. 3 shows the different existing ways to unzip CNTs to yield graphene nanoribbons.¹⁶² The above-mentioned details revealed that remarkable progress has been made on manufacturing graphene; however, opportunities and challenges still co-exist in developing new synthetic routes for preparing graphene with controlled size, shape, tailored surfaces and defined mesoscopic morphology. For better utilization of graphene in biology and medicine, appropriate functionalization of pristine graphene and the immobilization of biomaterials on it are necessary and play important roles in biocompatibility because the functional groups can create defects on the graphene surface and reduce the strong hydrophobic interaction of graphene with cells and tissues.¹⁵⁶ Biomolecules such as protein, DNA and small molecules have been reported to functionalize graphene. The biological activity of graphene-based materials can be associated with their capacities to interfere with molecular processes and functions.¹⁶³

Fig. 2 Mechanical exfoliation of graphene using scotch tape from highly oriented pyrolytic graphite (HOPG). Reproduced with permission from ref. 124. Copyright 2011, Elsevier.

Fig. 3 Schematics and representative images of several methods for unzipping CNTs into graphene nanoribbons. Reproduced with permission from ref. 162. Copyright 2010, Elsevier.

4. Applications in cell biotechnology

4.1 Graphene-based scaffold for cell culture

As we know, the backbone of biomolecules and biological structures is carbon, so it is no doubt to integrate biological systems with nanocarbons. Plenty of evidence has demonstrated that CNTs are able to support the growth and adhesion of live cells;^164,165 however, there are also researches certified that this one-dimensional nanocarbon has certain toxic effects on living cells.^166–173 Compared with CNTs, the 2D carbon nanomaterials, graphene and its derivatives, being only one atom thick, have a larger surface area and better biocompatibility, introducing the least amount of artificial material possible. Therefore, they have recently captured great interests from scientists as cell culture substrates.^174–178 The highest Young's modulus of graphene allows it to be converted to the desired shape.^179–181

Graphene and its derivatives have been used as coating materials of substrates to culture human mesenchymal stem cells (hMSCs), a kind of significant stem cell for a myriad of ground-breaking therapies in regenerative medicine.^182–190 The survival, proliferation and differentiation of hMSCs are extremely susceptible to numerous environmental factors such as impact from nearby cells, interaction with soluble growth factors and the influence of extracellular matrices. So it is of the utmost urgency to control these factors.^191–193 Using graphene-based materials in this field is an elegant solution to these questions as well as a new ray of hope for groups of researches. Firstly, graphene is covered onto the substrates, for example, glass slides, Si/SiO₂, polyethylene terephthalate (PET) and polydimethylsiloxane (PDMS), and then hMSCs are seeded on the test chips followed by culturing in normal stem cell medium. Examining the viability and morphology of the hMSCs showed that there was no significant difference between graphene-coated and uncoated substrates. However, these substrates without or with a graphene covering had a different influence on the conversion of hMSCs into specific cell types. In the absence of graphene, hMSCs on the soft substrates, PDMS and PET, could convert into neuronal cells and muscle cells respectively, whereas on stiff substrates such as glass slides and Si/SiO₂, these stem cells did not differentiate. Once these substrates had been covered with graphene, the hMSCs were able to convert into osteoblasts. These results proved that graphene played an important role in the differentiation of hMSCs into the bone cells. Besides, the study of cultivating induced pluripotent stem cells (iPSCs) has further proved that graphene and its derivatives could support the cell culture and that their surfaces led to distinct cell proliferation and differentiation characteristics. Other cells like NIH-3T3 fibroblasts,¹⁹⁴ L929 cells,¹⁹⁵ MC3T3-E1 cells,¹⁹⁶ A549¹⁹⁷ and mouse hippocampal cells¹⁷⁶ can also grow on the graphene-based substrates. A focal adhesion study, proliferation assay, and cell shape analysis together with many other investigations revealed that the cells grow well on the graphene-coated substrates with improved cellular adhesion, proliferation and differentiation, which suggested that these nanomaterials held great potential as surface-coating materials for cell culture. Fig. 4 shows the cell adhesion patterns on different substrates.¹⁹⁴ From Fig. 4 we can see that there are large dot-shaped plaques of vinculin in the peripheral regions of the cells cultured on glass, while cells grown on the coated substrates exhibit less vinculin-containing focal contacts at the periphery, which indicate the superior biocompatibility of graphene and its derivatives-coated substrates.

Fig. 4 Illustration of NIH-3T3 cells incubated on different substrates for 24 and 48 h. Actin, vinculin and the DAPI-stained nucleus are represent in red, green and blue respectively. Reproduced with permission from ref. 194. Copyright 2010, American Chemical Society.

Cellular adhesion is a vital prerequisite to subsequent cell functions, such as proliferation, synthesis of proteins and the formation of mineral deposits. It usually depends on the properties of substrates including surface topography, chemical composition, dimensions and wettability.^198–203 Cell proliferation is regulated by the induction and inhibition of positive and negative cell cycle regulators. Most reports have shown that graphene-based materials were biocompatible by facilitating the cell adhesion and proliferation of L-929 cells,²⁰⁴ osteoblasts,²⁰⁵ kidney cells,²⁰⁶ and embryonic cells.²⁰⁶ Graphene has a different effect on the differentiation of stem cells into specific tissues. Lee et al. demonstrated that graphene was likely to accelerate the differentiation of hMSCs into the osteoblast lineage; however, they inhibit their differentiation into adipocytes. This was mainly because of different binding interactions between graphene and the growth factors.¹⁷⁷ In addition to the literature reporting the good biocompatibility of graphene, there are papers revealing that graphene exhibits concentration-dependent toxicity to cells.^207–210 For example, Fan et al. demonstrated that GO nanosheets with a lower dose of 20 μg mL⁻¹ did not show toxicity to A549, and the dose of 85 μg mL⁻¹ had increased cytotoxicity.⁶⁹ Similar results were also obtained by Cui and colleagues reported that a GO concentration of less than 20 μg mL⁻¹ did not cause appreciable toxicity and GO at a higher concentration (>50 μg mL⁻¹) led to obvious cytotoxicity.²¹¹

4.2 Graphene for cell labeling and real-time monitoring

Cellular labeling is a technology that connects special reagents, such as organic dyes or inorganic NPs, to the target cells to trace their functions and behaviours.^212–216 It is the foundation of cell imaging, drug delivery and many other cell-related applications. Aptamers are single-stranded nucleic acids selected by a molecular selection process called Systematic Evolution of Ligands by Exponential Enrichment (SELEX).^217,218 They can specifically bind to given target molecules with numerous merits, such as high affinity, specificity, recognition capability, small size and little immunogenicity,^219–221 and thus have often been used for labeling and differentiating between different cell types.²²² However, when delivered into cells the oligonucleotides are prone to be cleaved by enzymes. This problem can be solved smoothly by attaching aptamers to graphene-based structures like graphene oxide nanosheets (GO-nS) because the DNA is able to be effectively constrained on the surface of the graphene via non-covalent binding and the strong interactions between them will generate a steric hindrance effect.^223,224 It is this effect that prevents DNAse from attaching to the DNA to initiate enzymatic digestion. Moreover, compared with adsorbing onto CNT where DNA is hypothesized to wrap around it,²²⁵ DNA linked to graphene can be more readily released from the surface in the presence of its complementary sequence. Therefore, graphene-based structures can figure as an efficient DNA protector as well as delivering oligonucleotides into cells for real-time monitoring.^226,227 In the above example, graphene is just playing a role of vehicle and preserver for the fluorescent probe. Actually, graphene can be applied as a robust biological tag for cell labeling as well because of its distinctive qualities. Not long ago, Zhang and colleagues²²⁸ successfully synthesized a kind of graphene-based material, graphene quantum dots (GQDs), with bright yellow photoluminescence by an electrochemical method. After incubation with target cells, these uniform sized GQDs were effortlessly internalized in the stem cells for their unusual penetrating power (Fig. 5). Evidence also demonstrated that the presence of the GQDs did not affect the special characteristics of the cells, which open up a significant opportunity for their biomedical purposes.

Fig. 5 Living neurosphere cells (a and b) and pancreas progenitor cells (c and d) labeled by GQDs. Reproduced with permission from ref. 228. Copyright 2012, Royal Society of Chemistry.

4.3 Cell analysis based on microfluidic device

Mounting evidence indicates that some cells (e.g., cancer stem cells and circulating cancer cells) in blood or tissues indicate the onset of human diseases and effect of treatment.^229,230 But there usually are a small proportion of these cells. As a result, it is a complicated operation procedure needing rigorous conditions to survey these cells for disease diagnosis at an early stage that is ultrasensitive yet simple. Several kinds of promising tricks for cell testing include microfluidic cell separation,²³¹ flow cytometry analysis,²³² histological examination,^233–236 nanoparticle-based biosensing²³⁷ and endogenous nucleic acid analysis.²³⁸ Here we emphatically introduce two of them: microfluidic cell separation and flow cytometry analysis. Owing to their large specific surface area and need for less time to separate the target molecule and cell, microfluidic devices have been in widespread use.²³⁹ Recently, a microfluidic device with GO for the selective and sensitive detection of circulating tumor cells (CTCs) aimed at the early diagnosis of cancer has been reported (Fig. 6).²⁴⁰ The overall size of the microfluidic device is just 35 mm × 10 mm × 3 mm. It comprises a PDMS layer with a microchannel and a silicon substrate with gold posts. GO functionalized with PEG was attached to the gold nanoposts and then the epithelial cell adhesion molecule (EpCAM) was immobilized on GO. When CTCs go through the microchannel, it would be captured by the nano microfluidic device, consequently, sensitive and specific detection of CTCs was achieved. Flow cytometry is a clinical diagnosis technique for determining thousands of blood-borne cells in a few seconds.²⁴¹ For its distinct electronic nature that would be easily affected by electrostatic forces from long-range charge scatters, graphene has been integrated with microfluidic flow cytometry for the ‘flow-catch-release’ assay malaria-infected red blood cells at the single-cell level. According to the conductance change and dwell times, specific microscopic information related to the development of disease is capable to be achieved and a dynamic disease diagnostic patterns formed with the help of graphene. In addition, several other features of graphene, for instance optical transparency, easiness of integrating into microfluidic flow cytometry, ability to interface with cell-recognition proteins, endow it immense potential in clinical medicine.

Fig. 6 Picture of the integrated nano microfluidic device for capturing CTCs on the functionalized GO/gold posts (left) and photograph of the microfluidic device (right). Reproduced with permission from ref. 240. Copyright 2011, Chemical and Biological Microsystems Society.

4.4 Cell imaging

Although numerous experimental data employed to structure the mathematical models of molecular networks come from routine analysis, an increasing amount of evidence demonstrates that the determination if unable to provide both the complex and the individual information in living cells. The emergence of cell imaging is capable of resolving this dilemma. Cell imaging provides an opportunity to easily glimpse into the world of biology in a natural setting, which is often difficult or even impossible with other strategies. Among numerous imaging techniques, fluorescence imaging is the most popular and important method for cell biological research. The graphene-based materials, for instance GO and reduced graphene oxide (RGO), have turned out to be fluorescent and thus have been utilized for fluorescence imaging.^26,242–245 In a recent study, Wang and co-workers prepared graphene oxide nanoparticles (GONs) approximately sized at around 30 nm. They then functionalized them with transferrin (Trf) molecules and poly(ethylene glycol) (PEG) for targeting cancer cells and prolonging the blood circulation of nanoparticles respectively. After labeling these composites on cancer cells and irradiating them with an ultrafast pulsed laser, strong photoluminescence of GONs can be observed. As shown in Fig. 7, signal disturbance from cell auto-fluorescence and a molecular dye, fluorescein isothiocyanate (FITC), is possible to be avoided effectively with a low laser power of 7 mW.²⁴⁶ When the laser power was reduced to 4 mW, intensive microbubble and instant cell damage occurred, which showed that this system was not only suitable for target cell imaging but also for possible cell therapy. In addition, graphene and its derivatives can also be applied to cellular magnetic resonance imaging (MRI). Due to its capability of producing excellent quality and high-resolution images, allowing a fast scan time as well as no need of radiochemicals, MRI has been proved as the most important modality in both clinical and research settings.^247–257 Therefore, integrating this advanced technique into cell (especially tumor cell) imaging will greatly boost the development of diagnostics. In MRI, superparamagnetic Fe₃O₄ NPs are widely used. They are usually decorated with suitable functionalities to obtain better biocompatibility and physiological stability along with an enhanced MRI contrast effect.²⁵⁸ Thanks to the specific structure and characters of GO, Fe₃O₄ NPs can anchor onto the surface of the GO sheet to prevent them from aggregating, which will result in a considerable relaxation rate.^259–263 Compared with isolated Fe₃O₄ NPs, the Fe₃O₄–GO composites show significantly enhanced cellular MRI.

Fig. 7 Comparison of the in vitro two-photon luminescence of GONs with cell auto-fluorescence and the molecular dye FITC. Reproduced with permission from ref. 246. Copyright 2012, Wiley.

4.5 Gene delivery

Gene therapy is a novel and promising approach for the treatment of many acquired and inherited life-threatening diseases, including AIDS, cystic fibrosis, cancer, Parkinson's disease, etc.^264–266 An efficacious treatment result requires that a nucleic acid drug can be successfully delivered to the interior of the target cell.²⁶⁷ So the development of a safe gene delivery vector with the ability to protect DNA from enzymatic cleavage and facilitate cellular uptake of DNA with high efficiency is a significant factor.²⁶⁸ It proved that graphene could bind to single-stranded DNA effectively through the π–π interaction and was able to protect oligonucleotides from nuclease degradation. Zhang et al. reported a gene delivery system based on GO functionalized with branched polyethylenimine (PEI-GO).²⁶⁹ The complexation of PEI-GO with plasmid DNA (pDNA) was allowed via an electrostatic interaction arising from the cationic PEI. Intracellular tracking of Cy3-labelled pGL-3 demonstrated that PEI-GO was able to effectively deliver pDNA into cells (Fig. 8). In addition, Kim and colleagues fabricated a hybrid gene carrier by conjugating low-molecular weight branched polyethylenimine (BPEI) to GO. Results confirmed that BPEI−GO possessed high gene delivery efficiency and exhibited high cell viability.²⁷⁰ In another investigation, GO was covalently functionalized with chitosan (CS) by an amide linkage.²⁷¹ At the same time, pDNA could be condensed on the GO-CS sheet to form stable nanoparticle, which exhibited reasonable transfection efficiency in HeLa cells at certain nitrogen/phosphate (N/P) ratios.

Fig. 8 Intracellular tracking of Cy3-labelled pGL-3 (red) in HeLa cells. A and B are for 4 h and 24 h post-transfection respectively. Reproduced with permission from ref. 269. Copyright 2011, Royal Society of Chemistry.

4.6 Determining dynamic secretion of living cells

Cell secretion is a fundamental and ubiquitous cell function involved in the regulation of various physiological processes, such as the digestion of food, neurotransmission, hormonal control of cell- and reproductive-cycles, and many other life processes.²⁷² Investigation of cell secretions is of great value in diagnosing, monitoring, assessing and treating related disease. Routine assays using ensemble biochemical measurements from a large cell population with a low temporal resolution are unable to resolve the fast kinetics of cell secretion and detect trace amounts of released molecules.²⁷³ Fortunately, the emergence and utilization of nanomaterials provide new opportunities for the analysis of this cellular event and provide spatiotemporal information on fundamental researches and drug discovery. Nanomaterials such as nanoparticles, carbon nanotubes and more recently graphene have been reported to determine cell secretions. For example, Chen and co-workers employed single-walled carbon nanotube (SWNT) network-based field-effect transistors (SWNT-net FET) for the label-free detection of ATP release from living astrocytes.²⁷⁴ Subsequently, they coupled SWNT-net FET with neuroendocrine PC12 cells to detect their secretion of hormone catecholamines upon triggering.²⁷⁵ Compared to CNTs, graphene is more advantageous for detecting cell secretions because of its easy functionalization, large sensing area and suitability for interfacing with cell membranes. Zhang et al. synthesized large-area ultrathin RGO films to fabricate nanoelectronic FETs and then applied these RGO-based devices to the label-free monitored dynamic secretion of catecholamine molecules from PC12 cells.¹⁴⁹ As illustrated in Fig. 9, PC12 cells were directly cultured on top of the FET devices and membrane depolarization resulting from a physiological stimulation opened voltage-gated Ca²⁺ ion channels. Once catecholamines were released from the cells, they quickly diffused onto the RGO films and interacted with them by π–π stacking, which would lead to current spikes. As a result, cell secretions were detected by monitoring the change in current. Although it is at an early stage, graphene for analysing the dynamic secretion of biomolecules is promising.

Fig. 9 (A) Schematic of the interface between a PC12 cell and an RGO FET. (B) Real-time response of RGO to the vesicular secretion of catecholamines from PC12 cells stimulated by high K⁺ solution. Reproduced with permission from ref. 149. Copyright 2010, American Chemical Society.

4.7 Probing the activity of enzymes

Enzymes are a kind of biocatalyst generated from the living cells of the biosome and, in fact, a majority of enzymes are proteins.^276–281 They can catalyze all kinds of biochemical reactions, promoting metabolic progress with high efficiency. Accordingly, it is of vital significance for diagnosis and treatment to monitor the activity of enzymes. As its synthesis is facile, can hold surface functionalities, has a large surface area to mass ratio and a fluctuant surface, graphene can act as a potential artificial receptor and inhibit the activity of α-Chymotrypsin (ChT), a proteolytic enzyme capable of decomposing the denatured protein quickly.^282–284 Results showed that graphene was strongly and compatibly attached by ChT through reversible interactions between the carboxylate groups of functionalized GO and the cationic surface residues of ChT (Fig. 10),^285,286 hence there were no changes of the native conformation of ChT. Another strategy of the fluorescence resonance energy transfer (FRET)-based platform to assess enzymatic activity is a very popular means too. In this method, a pair of energy transfer acceptor and donor are obligatory. Under ordinary circumstances, the acceptor is graphene whereas the donor may be a quantum dot (QD) or fluorescent dye. According to the quenching and recovery of fluorescence, enzymatic activity is able to be obtained.^287–291 Taking the assay of protease activity for example, in this measurement, as demonstrated in Fig. 11, a protease substrate peptide with one end attached to biotin and another end bonded with cysteine was employed as the link bridge between GO and QDs.^292,293 Due to the ultra-efficient quenching capability of GO, the fluorescence of the QDs could be completely quenched through the FRET procedure. In the presence of target protease, the designated peptide was cleaved into two parts followed by the release of QDs from the surface of GO and the protease activity was detected based on the recovered fluorescence initially quenched. This system is useful for diagnosing protease-related disorders and screening potential drugs.

Fig. 10 The structures of (a) functionalized GO, and (b) α-chymotrypsin. (c) Schematic of reversible interactions between carboxylate groups of functionalized GO and the cationic surface residues of ChT. Reproduced with permission from ref. 286. Copyright 2011, American Chemical Society.

Fig. 11 (A) Schematic representation of constructing the peptide conjugated QDs. (B) Detecting protease activity through energy transfer by using graphene oxide. Reproduced with permission from ref. 292. Copyright 2011, Elsevier.

5. Conclusions and outlook

In summary, the recent advances of graphene applied for biotechnology, in particular, cell culture, real-time monitoring, living cell detection, imaging, gene delivery, investigation of cell secretions and surveying enzymatic activity, are revealed to readers. It is well known that there have been just 8 years in total since the discovery of graphene in 2004 and successful transfer from its original application field, physics, to the realm with better practical significance including biomedicine, biochemistry, materiology and leading-edge disciplines rising in recent times, has been realized with great achievements.²⁹⁴ Certainly, it is no exaggeration to say that the introduction of graphene into these areas brings infinite vitality to scientific researches and enormously enlivens the scientist's enthusiasm for study. Nevertheless, merging graphene into the aforementioned domains is still in its infancy and is conducted only at the laboratory level; consequently, a long journey with numerous challenges lies ahead to summon scientists to apply graphene in reality (e.g. hospitals).^{53,69,156,206,224,295–298} Therefore, future works are mainly concentrated on the following aspects, for example, tailoring the synthesis of nanohybrid materials for meeting specific applications and needs;^{224,295,296,299–305} investigating and clarifying the long-term exposure effects of this nanomaterial; combining graphene with other nanomaterials to form multifunctional materials accompanied by better performance; and integrating it into various advanced instruments and techniques such as MS, LC, MRI, photodynamic therapy (PDT), etc. Although these goals cannot be easily obtained, there is every reason to believe that with the joint efforts of biologists, chemists, physicists and medics, all of the difficulties and obstacles will be triumphed over as a better tomorrow follows.

Acknowledgements

The authors are grateful to the financial support from National Major Scientific Instruments and Equipments Development Special Funds (no. 2011YQ030113), National Recruitment Program of Global Experts (NRPGE), the Hundred Talents Program of Sichuan Province (HTPSP), and the Startup Funding of Sichuan University for setting up the Research Center of Analytical Instrumentation.

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