Graphene-like two-dimensional layered nanomaterials: applications in biosensors and nanomedicine

Abstract

The development of nanotechnology provides promising opportunities for various important applications. The recent discovery of atomically-thick two-dimensional (2D) nanomaterials can offer manifold perspectives to construct versatile devices with high-performance to satisfy multiple requirements. Many studies directed at graphene have stimulated renewed interest on graphene-like 2D layered nanomaterials (GLNs). GLNs including boron nitride nanosheets, graphitic-carbon nitride nanosheets and transition metal dichalcogenides (e.g. MoS₂ and WS₂) have attracted significant interest in numerous research fields from physics and chemistry to biology and engineering, which has led to numerous interdisciplinary advances in nano science. Benefiting from the unique physical and chemical properties (e.g. strong mechanical strength, high surface area, unparalleled thermal conductivity, remarkable biocompatibility and ease of functionalization), these 2D layered nanomaterials have shown great potential in biochemistry and biomedicine. This review summarizes recent advances of GLNs in applications of biosensors and nanomedicine, including electrochemical biosensors, optical biosensors, bioimaging, drug delivery and cancer therapy. Current challenges and future perspectives in these rapidly developing areas are also outlined. It is expected that they will have great practical foundation in biomedical applications with future efforts.

1. Introduction

A key to develop nanodevices with high performance for biosensors and nanomedicine applications is to explore advanced functional nanomaterials. Two-dimensional (2D) nanomaterials, due to their potential applications, have been widely discussed in the last few decades;^1,2 however, these nanomaterials have attracted intensive global interests across academia and industry since the Nobel Prize award in 2010.³ Graphene, which is a novel 2D nanosheet of sp²-bonded carbon atoms arranged in a honeycomb lattice, has been realized as one of the most powerful nanomaterials for diversified applications in the field of materials science.⁴ Because of their extraordinary physicochemical and structural properties,^5–7 graphene and its derivatives have generated enormous excitement in the areas of nanocomposites, specific optoelectronics, energy storage and conversion, as well as bioscience/biotechnology.^8–13 Our group has also achieved great progress on graphene-based nanocomposites and their applications in bioassays.^14–19 In particular, we have reviewed recent important advances of graphene-related nanomaterials for biosensors and nanomedicine applications, with emphasis on their future and potential value.^20–23 Since then, the widespread investigation of graphene has also drawn a current surge of interest in other 2D nanomaterials for different fields of applications owing to their theoretical values and reliable practical perspective.^24–27 The atomically-thick 2D nanosheets with unprecedented excellent properties have opened up novel research branches from science to engineering.

Among them, graphene-like 2D layered nanomaterials (GLNs), such as boron nitride (BN) nanosheets, graphite-carbon nitride (g-C₃N₄) nanosheets, and various transition metal dichalcogenides (TMDs), have also been widely investigated because of their structural similarities to graphene.^28–36 For example, BN nanosheets were employed in polymer matrices, leading to a nanocomposite with superior thermal transport performance;²⁸ MoS₂ ultrathin nanosheets were reasonably engineered to act as a catalyst, showing excellent hydrogen evolution reaction activities;³⁴ g-C₃N₄ nanosheets also exhibited superior photocatalytic abilities under both UV-visible and visible light.³⁶ Exciting results and reviews have been reported, offering insights to achieve enhanced or even develop unprecedented properties.^37–45 Many techniques have been developed to obtain these GLNs often with heterostructures (called “van der Waals”³⁹) such as top-down exfoliation from bulk layered crystalline solids and bottom-up synthesis from small molecules.

GLNs, including BN nanosheets, g-C₃N₄ nanosheets and TMDs (e.g. MoS₂ and WS₂), with unique combinations of structural, electronic, catalytic, mechanical, and related properties can find superiority in applications of biosensors and nanomedicine to realize detection, diagnosis and therapy with high sensitivity, specificity and accuracy. Great achievements have been made in the related areas during the last few years, and a number of significant breakthroughs have taken place using graphene and GLNs-based platforms across academia and industry globally.^46,47 Herein, we comprehensively summarized recent studies on GLNs, including BN nanosheets, g-C₃N₄ nanosheets and TMDs (e.g. MoS₂ and WS₂), in biosensors and nanomedicine. Specific areas of interest include electrochemical biosensors, optical biosensors, bioimaging, drug delivery, and cancer therapy (as shown in Fig. 1). Finally, future research directions are proposed regarding this fast-growing field for experts in chemistry, materials science, medicine and biology, with promising application opportunities in a wide scope of technologies and markets. It is also anticipated that this review would assist related researchers to further develop this hot area.

Fig. 1.

Schematic illustration of graphene-like 2D layered nanomaterials for applications in biosensors and nanomedicine, including electrochemical biosensors, optical biosensors, bioimaging, drug delivery and cancer therapy.

2. Synthesis of GLNs

Over the last several years, many studies have been carried out utilizing GLNs, and they have shown their enormous potentials in biosensors and nanomedicine. Large-scale fabrication of GLNs and their derivatives with controlled shape, layers, size and defection is essential, which is the first step towards their high-performance applications. To date, many methods have been developed to prepare high-quality GLNs. The current synthesis methodologies of GLNs mainly include two categories: top-down and bottom-up methods.

For top-down methods, mechanical exfoliation and liquid-phase exfoliation are the two typical ways. Since the production of single and several layered graphene nanosheets from highly ordered pyrolytic graphite using “Scotch tape” was first reported by Novoselov and coworkers in 2004,³ the mechanical exfoliation process has proven to be an easy and fast way to prepare and study the properties of GLNs.^48,49 For example, Gorbachev et al.⁵⁰ showed a standard mechanical exfoliation technique to prepare BN mono- and bi-layers. The obtained high-quality nanomaterials were sufficiently large to allow the fabrication of proof-of-concept devices with a variety of new experiments. However, the yield with few-layer nanosheets from mechanical exfoliation is low and not scalable to mass production. As an alternative method, liquid-phase exfoliation has been demonstrated to effectively achieve large quantities of few layer nanosheets from bulk and stacked structures.² nanosheets with less defects could be directly obtained through this method, avoiding the severe destruction of samples. Coleman et al.⁵¹ reported that BN and TMDs nanosheets could be exfoliated under ultrasonic waves in certain solvents, including N-methyl-2-pyrrolidone and isopropanol. Ultrasonic radiation would create cavitation bubbles with the generation of high-energy jets, leading to the damage of the layered crystallites to some degree followed by the exfoliation of layers. Moreover, dispersants and intercalants can also be used to improve the exfoliation process as well as to functionalize the corresponding nanomaterials.^52,53

In the case of the bottom-up method, chemical vapor deposition (CVD) is a popular way to generate GLNs on substrates,⁴¹ which is beneficial for large-scale device fabrication. Using Mo and S as precursors, Zhan et al.⁵⁴ prepared single- and few-layered MoS₂ nanosheets on insulating SiO₂ substrates by CVD. During the process, the Mo thin layer was first deposited onto SiO₂ substrates. Then, the resultant film was reacted with the introduced S vapor to grow 2D MoS₂ nanosheets. Thermal decomposition of small molecules is another effective strategy. In a recent study, Duan et al.⁵⁵ reported the use of packed silica spheres as hard templates and mixed them with melted cyanamide; the mixture was then reacted at 550 °C, and finally the 2D porous C₃N₄ nanolayers was obtained after simple sonication. Thus, one can control the morphology of nanomaterials making use of the bottom-up method.

There are some other available synthetic routes we did not mention, and some disadvantages still exist for each method. It is also vital to functionalize and disperse GLNs to prevent them from agglomeration for their increased bioapplications. The more detailed information on GLNs preparations can be found in previous reviews.^37,40,41 The notable considerations in selecting suitable methods for bioassays include cost, modifications, convenience, and biocompatibility of related nanomaterials, as well as specific working environment. More cases will be discussed in the following sections.

3. BN nanosheets

BN nanosheet, a novel 2D nanomaterial with a wide band gap, has been used as an economically affordable advanced material with many breakthroughs in studies of multifunctional fillers, electronics, catalysis, and sensing. It can be isoelectric to carbon, with a structure analogous to that of graphene (so called “white graphene”⁵⁶). As shown in Fig. 2, 2D BN nanosheets consisting of alternating boron and nitrogen atoms have an atomically smooth surface. The unique honeycomb lattice structures possess several promising advantages, including high mechanical strength, high thermal conductivity, large specific surface area, and low fluorescence quenching.^57–59 These unique combinations of properties promote their usage in various applications.^60–62 It is worth noting that several bioapplications of BN nanosheets have already been proposed, although most of the studies are still at an early stage.

Fig. 2.

Structural basics of 2D BN nanostructures, reproduced with permission from ref. 57. Copyright 2012 Royal Society of Chemistry.

3.1. BN nanosheets-based bioassay

It was reported that BN nanotubes are noncytotoxic and can be functionalized through covalent and non-covalent interactions for ecological and biological applications.^63,64 BN nanotubes can accommodate molecules of various sizes such as proteins and cells, indicating their relatively flexible interlayer space. Recently researchers have also explored BN nanotubes as nanocarriers and nanotransducers, studying their possible biomedical applications.^65,66 Therefore, the similar advantages are likely applicable to the BN nanosheets, which is another allotrope of BN nanotubes.

For instance, with the properties of BN nanosheets mentioned above, we reported that graphene quantum dots (GQDs) with green fluorescence can be incorporated onto hexagonal BN nanosheets (HBN) to fabricate novel nanocomposites (HBN-GQDs). The HBN-GQDs nanocomposites exhibited strong green fluorescent property, high stability, water solubility, very low cytotoxicity towards HeLa cells, and were successfully applied for cell imaging.⁶⁷ Thus, BN nanosheet-based systems can offer appealing diagnostic and therapeutic opportunities, e.g. tracking the delivery of drugs and genes.

More recently, a sensitive immunosensor was designed to detect interleukin-6 with a fluorescent and electrochemical method (Fig. 3), using a multifunctional BN nanosheets-Au nanocluster nanocomposite as the label. In the process, because of the hydrophobic nature of BN, poly-diallyldimethyl-ammonium chloride was first used as both a stabilizer and a linker to fabricate the nanocomposite with outstanding fluorescent and electrochemical properties, as well as good stability and bioactivity. The excellent performance of the biosensor could be attributed to the as-formed architecture, which provided a favorable and large-surface-area microenvironment for antibody immobilization with enhanced signal amplification.⁵³ Xu et al. used chitosan to increase the solubility of BN nanosheets in an aqueous solution. Then, catalase was immobilized onto the well-dispersed BN nanosheets to construct an enzyme biosensor for the detection of forchlorfenuron (CPPU). A detection linear range of CPPU from 0.5 to 10.0 mM with a detection limit of 0.07 μM could be realized.⁶⁸ As a pioneer study, Uosaki et al. reported that BN nanosheets supported on Au could act as an electrocatalyst that had never been considered before. It has been witnessed by theory and experiment that it is possible to functionalize inert nanosheets for oxygen reduction reaction. This discovery opened new ways to design effective biocatalysts based on BN nanosheets, paving a novel route to electroanalysis.⁶⁹

Fig. 3.

(A) Schematic illustration of the fabrication process of PDDA-BN-GNC-Ab₂ bioconjugates. (B) Schematic representation of the fabrication and measurement process of the sandwich-type immunosensor, reproduced with permission from ref. 53. Copyright 2013 Royal Society of Chemistry.

H₂O₂ is also an essential compound involved in chemical and biological procedures; thus, we proposed a novel electrochemical biosensor based on gold nanoparticles (Au NPs)/BN nanosheets nanocomposites for the detection of H₂O₂, in which a facile sonochemical route was proposed to prepare the nanocomposites without using reducing or stabilizing agents. This study also demonstrated a universal approach for decorating BN nanosheets with nanoparticles, which holds great promise in biological sensing.⁷⁰ Despite the use of BN nanosheets being largely unexplored in this field, these reports suggested that the BN nanosheets-based nanomaterials were supposed to be promising biomaterials as a biological probe and are suitable for biomolecule detection and related applications.

4. g-C₃N₄ nanosheets

Ultrathin g-C₃N₄ nanosheets are becoming increasingly significant as an appealing class of nanomaterials from the point of view of their properties.⁷¹ g-C₃N₄ involve van der Waals interactions between adjacent C–N layers with strong covalent bonding within each layer. There are two structural isomers proposed to build a g-C₃N₄ network, in which the one composed of condensed tri-s-triazine units is energetically favoured and widely accepted. Tri-s-triazine units connected by planar amino groups construct the graphitic planes (Fig. 4).⁷² g-C₃N₄ have been prepared on a large scale through the pyrolysis of nitrogen-rich precursors, and then exfoliated to ultrathin nanosheets by a variety of methods.^44,73,74 It is stable upon thermal or chemical attack with a band gap of ca. 2.7 eV, possessing inherent photoluminenscence (PL) and other excellent characteristics for unusual structures.^75–77 g-C₃N₄ nanosheet-based nanomaterials have been widely utilized in photoelectronics and energy fields, especially in photocatalysis. However, it is also expected that they may couple with various functional materials and play key roles in biosensing and related applications.

Fig. 4.

Schematic diagram of a perfect g-C₃N₄ nanosheet constructed from melem units, reproduced with permission from ref. 72. Copyright 2009 Nature Publishing Group.

4.1. g-C₃N₄ nanosheet-based optical bioassay

The high-degree condensation of the tri-s-triazine unit in g-C₃N₄ endows it with a strong photoluminescent property, which renders it a potential for sensing, diagnosis and therapy in biological systems upon versatile functionalization.^71,72,78 The as-obtained g-C₃N₄ nanosheets possess high quantum yields and stability with good biocompatibility and low toxicity. Qiao’s group prepared ultrathin 2D g-C₃N₄ nanosheets by the exfoliation of protonated g-C₃N₄ powders in aqueous solutions, which exhibited strong interaction with heparin.⁷⁹ Considering the inherent fluorescence of g-C₃N₄ nanosheets at the same time, the quantification of heparin based on a metal-free and label-free sensing platform was realized. The best sensing efficiency that they reached can be attributed to the excellent biocompatibility and high surface area in the constructed system, and thus they might be used for detecting other biomolecules.⁷⁹ Sun and co-workers synthesized ultrathin g-C₃N₄ nanosheets using different methods, both the types of nanosheets can be employed as an efficient fluorosensor for Cu²⁺ due to the fluorescence quenching via photoinduced electron transfer.^80,81 Being favourable for manipulation and cyclic utilization, g-C₃N₄ nanohybrid films as a solid optical sensor was reported to detect Cu²⁺ and Ag⁺, both of which are important to the health of humans.⁸² It is clear that g-C₃N₄ nanosheets obtained through different methods would be widely used in the optical analysis of a variety of biological molecules.^83,84

In another report, a special fluorescence sensor based on a g-C₃N₄ nanosheets-MnO₂ sandwich nanocomposite was developed for the rapid and selective sensing of glutathione (GSH) in both aqueous solutions and living cell enviroments.⁸⁵ Because of the fluorescence resonance energy transfer (FRET) from a g-C₃N₄ nanosheet to the deposited MnO₂, the fluorescence of g-C₃N₄ nanosheets was quenched. Upon the addition of GSH, the nanoscaled MnO₂ was reduced to Mn²⁺, which led to the elimination of FRET. As a result, the fluorescence of g-C₃N₄ nanosheets was restored. Moreover, g-C₃N₄ nanosheets have been also used as quenchers in FRET-based systems for the detection of biomolecules. It was shown by Wang et al. that the strong interaction between g-C₃N₄ nanosheets and DNA could quench the fluorescence of a fluorophore labelled to DNA via a static quenching approach through photoinduced electron transfer.⁸⁶ On this basis, fluorescence sensing of DNA and extensive DNA related analytes, including metal cations, small molecules, and proteins, have been realized. Liao et al. assembled two dye-ssDNAs and folate onto dispersed g-C₃N₄ nanosheets to construct a multi-functionalized probe (mf-g-C₃N₄) for target cell specific monitoring of multiple intracellular microRNAs (miRNAs) (Fig. 5).⁸⁷ It was shown that g-C₃N₄ nanosheets exhibited strong fluorescence quenching abilities toward the dye labels. The fluorescence could be recovered upon the specific recognition of the dye-ssDNA to miRNA as a result of the release of the formed DNA-miRNA duplex helix from the g-C₃N₄ nanosheets. Finally, the simultaneous detection of multiple miRNAs in living cells was achieved.

Fig. 5.

Schematic illustration of (A) mf-g-C₃N₄ probe preparation and (B) simultaneous detection of intracellular miRNAs with an mf-g-C₃N₄ probe, reproduced with permission from ref. 87. Copyright 2013 Royal Society of Chemistry.

It was also found that g-C₃N₄ nanosheets possessed intrinsic peroxidase-like activity, and could catalytically oxidize 3,3′,5,5′-tetramethylbenzidine (TMB) to produce a colored solution upon reaction with H₂O₂. Thus, a colorimetric method was proposed to detect glucose in serum samples using g-C₃N₄ nanosheets combined with glucose oxidase by Lin et al.⁸⁸ It is suggested to synthesize g-C₃N₄ nanosheet-based nanocomposites to enhance the peroxidase-like activity, which will have potential for clinical diagnosis as peroxidase mimetics.

Unlike organic fluorophores, inorganic QDs exhibit improved fluorescent performance for bioimaging with the advantages of enhanced photostability and tunable wavelength. As shown in Fig. 6, Zhang et al. prepared ultrathin g-C₃N₄ nanosheets with high fluorescent quantum yield by a “green” route from bulk g-C₃N₄ according to a theoretical guidance, and found that water-soluble g-C₃N₄ nanosheets showed excellent biocompatibility and nontoxicity. The cell imaging results indicated that the as-prepared fluorescent g-C₃N₄ nanosheets could serve as bioimaging probes for further biomedical applications.⁸⁹ Moreover, several strategies were developed to synthesize C N QDs from g-C₃N₄ nanosheets, which showed unique fluorescent properties such as upconversion⁹⁰ and two-photon fluorescence properties.⁹¹ Among them, g-C₃N₄ QDs prepared by environmentally friendly synthetic routes could emit stable and strong two-photon fluorescence with a large two-photon absorption cross section. It is the first study about two-photon fluorescence imaging of a cellular nucleus based on g-C₃N₄ QDs. Compared to one-photon imaging, multi-photon imaging exhibited more advantages.⁹¹ In their report, it was shown that the two-photon fluorescence imaging using g-C₃N₄ QDs had advantages of a long observation time, large penetration depth, low photodamage and background signal, as well as nuclei details were obviously distinguishable, such as nucleolus positions could be clearly seen, while one-photon fluorescence imaging did not exhibit such properties. These improvements demonstrated that the light-up responses of two-photon fluorescence imaging based on g-C₃N₄ QDs were much more effective in visualizing and distinguishing of cellular nucleus imaging than one-photon fluorescence imaging. Moreover, the two-photon absorption (TPA) performance of g-C₃N₄ QDs was better than that of some organic dyes, which was evaluated by the TPA cross section. The cost of g-C₃N₄ QDs was also lower than commonly used nuclear dyes. These typical features make g-C₃N₄ QDs promising and economical probes in analytical and bioanalytical applications.

Fig. 6.

(a) Schematic illustration of liquid-exfoliation process from bulk g-C₃N₄ to ultrathin nanosheets (b) confocal fluorescence image and (c) overlay image of bright field and confocal fluorescence image of the HeLa cells incubated with ultrathin g-C₃N₄ nanosheets for about 1 h, reproduced with permission from ref. 89. Copyright 2013 American Chemical Society.

4.2. g-C₃N₄ nanosheet-based electrochemical biosensors

The study of g-C₃N₄ nanosheets has become popular with respect to electrochemical properties.⁹² To date, g-C₃N₄ nanosheet-based nanomaterials have been utilized to construct novel electrochemical biosensing platforms for the detection of various analytes, including small molecules, immunoassays, and DNA sensors.

In one report,⁹³ ultrathin g-C₃N₄ nanosheets were first prepared by conditional liquid exfoliation of bulk g-C₃N₄ under sonication. The authors studied its electrochemical properties, and found that g-C₃N₄ nanosheets had excellent electrocatalytic activity toward the reduction of H₂O₂. Furthermore, a biosensor based on a glucose oxidase-g-C₃N₄ modified electrode had also been constructed for the determination of glucose in both buffer solution and human serum medium, revealing g-C₃N₄ as a good candidate for immobilizing enzymes with high activity and loading efficiency. Gu et al.⁹⁴ also synthesized graphene supported g-C₃N₄ nanosheet-metal-free layered nanocomposites, which exhibited enhanced performance for the electrochemical biosensing of uric acid (UA), norepinephrine, tyrosine, tryptophan, acetaminophen and rutin on the modified glassy carbon electrode. It was said that graphene-g-C₃N₄ has the advantage of promoting electron transfer and improving the redox current.

Besides its ultraviolet-visible adsorption and PL properties, Cheng et al. recently investigated the cathodic and anodic electrochemiluminescence (ECL) behaviors of g-C₃N₄ nanosheets. A fairly intense ECL emission was observed, and corresponding ECL sensors based on g-C₃N₄ nanosheets have been designed to detect traces of Cu²⁺ and rutin, respectively, demonstrating that the g-C₃N₄ semiconductor might be an intriguing type of efficient candidate luminophore for ECL sensing.^95,96 Unlike graphene, pristine g-C₃N₄ can generate the ECL effect. However, electron-induced passivation of g-C₃N₄ nanosheets will occur at high energy. Chen and co-workers⁹⁷ developed a novel ECL immunosensor for the determination of carcinoembryonic antigen (CEA) based on the Au NP-modified g-C₃N₄ nanosheets nanohybrid, as shown in Fig. 7. Au NPs played critical roles in trapping and storing the electrons from the conduction band of g-C₃N₄ nanosheets, preventing electrode passivation. The obtained nanohybrids exhibited strong and stable cathodic ECL activity, which was better than that of the g-C₃N₄ nanosheets itself, resulting in high performance for the detection of CEA.

Fig. 7.

(A) Schematic representation of the stable ECL emission mechanism of the Au–g-C₃N₄ nanohybrid-co-reactant system. (B) Principle of ECL immunosensor based on Au-g-C₃N₄ nanohybrids, reproduced with permission from ref. 97. Copyright 2014 American Chemical Society.

Photoelectrochemical (PEC) sensors combine the advantages of optical methods and electrochemical sensors, and thus show great promise for analytical applications. Owing to its narrow band gap, g-C₃N₄ exhibits unique electronic and optical properties. For instance, a PEC sensor has been proposed and used to detect trace amounts of Cu²⁺ in an aqueous environment based on g-C₃N₄ nanosheets, thus showing great promise for bioanalysis.⁹⁸ Very recently, Li et al.⁹⁹ realized the specific detection of kanamycin with a novel PEC aptamer biosensor using water-dispersible g-C₃N₄ (w-g-C₃N₄) as a visible light-active material and the aptamer as the biorecognition element. However, graphene oxide was doped to improve the dispersion and film-forming ability of w-g-C₃N₄ as well as the PEC performance of the w-g-C₃N₄ modified electrode. The captured kanamycin molecules on the sensor surface were quickly oxidized by the photogenerated holes, and the recombination of photogenerated electrons and holes was inhibited, leading to the high photocurrent of the PEC sensor. Such study not only broadens the application of w-g-C₃N₄ to the field of aptamer sensors but also provides a novel strategy for the fabrication of various aptamer sensors for other target molecules.

5. TMDs nanosheets

TMDs are MX₂-type compounds, where M is a transition metal of groups IV, V, and VI, and X is a chalcogen such as S, Se, and Te.^39–41,100 For bulk layered TMDs, there are strong intralayer covalent M-X bonds, while weak van der Waals interactions connect the adjacent layers, which could be cleaved to few-layer nanosheets. As shown in Fig. 8, taking MoS₂ as an example, it consists of a sandwich structure of Mo atoms between two S atoms layers, forming a stable compound.² Many strategies used in the synthesis and characterization of graphene were employed with equal effectiveness in the case of the 2D TMDs nanosheets. Beyond classic crystal growth methods, such as CVD, other techniques specific to generating or altering 2D TMDs nanosheets, including mechanical exfoliation, heterostructure restructuring and topochemical deintercalation approaches, have also been used. Recently, 2D TMDs have attracted considerable attention of scientists and led to wide-ranging and diversified technological applications because of their intriguing properties.^{33–35,43,101–103} It was found that the properties of 2D TMDs are also favorable for other branches of science, such as biosensors and nanomedicine, taking advantage of large surface area, good biocompatibility, fluorescence, electrical conductivity and fast heterogeneous electron transfer.^104,105

Fig. 8.

Crystal structures of MoS₂, reproduced with permission from ref. 2. Copyright 2013 American Association for the Advancement of Science.

5.1. TMDs nanosheet-based electrochemical biosensors

Researchers have considered utilizing TMDs nanosheets for electrochemical detection, which is one type of important and effective sensing systems for various biological species because an increasing consensus has been achieved on their environmentally-friendly aspects.¹⁰⁶ Wang et al.¹⁰⁷ reported the application of layer-structured MoS₂ nanosheets as a matrix for horseradish peroxidase (HRP) immobilization and biosensor construction for the first time. The resultant HRP/MoS₂ modified glassy carbon electrode exhibited favourable electrocatalytic response toward the reduction of H₂O₂ when hydroquinone was used as mediator, indicating MoS₂ as an acceptable candidate for enzyme electrodes. Moreover, Wang et al.¹⁰⁸ reported a study on the electrochemical detection of nanomolar levels of H₂O₂ secreted by living cells using ultrasmall MoS₂ platelets, which showed high performance for sensing. This was possibly the result of the enhancement of planar electric transportation properties assisted by the 2D electron–electron correlations. In another report, the HRP-MoS₂-graphene nanocomposite was prepared and utilized as a sensing platform, which displayed electrocatalytic activity to H₂O₂ with high sensitivity and fast amperometric response. The linear detection range was wide (0.2 μM–1.103 mM) and the detection limit was considerably low (0.049 μM).¹⁰⁹

To improve the electrocatalytic activity toward small biomolecules, Sun et al.¹¹⁰ designed an Au NP functionalized MoS₂ nanosheet-based electrode to individually or simultaneously detect dopamine (DA), uric acid (UA) and ascorbic acid (AA) by differential pulse voltammetry. The peak potential separations were large enough to permit the corresponding analysis. The Au NPs@MoS₂ modified electrode exhibited linear responses toward AA, DA and UA in the range of 50–100 000, 0.05–30 and 50–40 000 μM, respectively. Moreover, the biosensor had been successfully employed to determine DA in biological fluids with satisfactory results. Xia and co-workers also synthesised a ternary nanocomposite made of Ag NPs, MoS₂ and chitosan for the electrochemical detection of tryptophan.¹¹¹ In comparison with MoS₂ and Ag NPs, Ag-MoS₂/chitosan exhibited better electron transfer properties. The nanomaterials introduced co-operated with each other and finally contributed synergistically to promote the analytical performance.

As we know, it is of significance to detect specific DNA sequences, which is important in the field of medical diagnostics, environmental monitoring, and food safety. Loo et al.¹¹² developed a facile approach of employing MoS₂ nanosheets as inherently electroactive labels for the voltammetric determination of DNA hybridization, which was correlating to the diagnosis of Alzheimer’s disease (Fig. 9). According to the proposed strategy, the range of detection was established to be from 0.03 to 300 nM with good reproducibility. The obtained phenomenon mainly originated from the detection principle that was the differential affinity of MoS₂ nanosheets towards ssDNA and dsDNA, as well as the oxidation of the nanosheets.

Fig. 9.

MoS₂ nanosheets as inherently electroactive labels for DNA hybridization detection, reproduced with permission from ref. 112. Copyright 2014 Royal Society of Chemistry.

Huang et al.¹¹³ synthesized a novel 2D graphene analogue MoS₂/multi-walled carbon nanotube (MoS₂/MWCNT), and subsequently constructed an ultrasensitive electrochemical DNA biosensor by assembling a thiol-tagged DNA probe on an MoS₂/MWCNT and Au NPs-modified electrode that had already been coupled with glucose oxidase (GOD). The proposed DNA sensor showed a relatively low detection limit (0.79 fM), wide linear range (10 M to 107 fM) and satisfactory selectivity. Another important contribution was made by Wang et al. They synthesized an MoS₂ nanosheet-thionin nanocomposite with the help of the ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate. Then, the nanocomposite was used for the construction of a dsDNA electrochemical biosensor using a stable electrochemical response. Moreover, there was a signal change because of the strong interaction between thionin and DNA.¹¹⁴ The as-prepared biosensor could be used for the determination of ssDNA and RNA with acceptable results. This study provided a facile one-step way to obtain functionalized MoS₂ nanosheets by surface modification; research efforts along these directions are expected to greatly expand the application of MoS₂ in sensing and other fields. Therefore, it is believed that TMDs nanosheet-based nanomaterials would be a notable platform for high-performance electrochemical biosensing.

5.2. TMDs nanosheet-based optical biosensors

Fluorescence quenching effects by graphene and graphene oxide have been widely explored. Very recently, TMDs nanosheets as universal highly efficient quenchers were also proposed, making them useful nanomaterials for various optical bioassays.^115,116 To the best of our knowledge, in 2013, Zhu and co-workers were first to successfully report the binding and quenching abilities of MoS₂ nanosheets against a fluorophore-labeled DNA.¹¹⁷ A dye-labeled ssDNA probe was first adsorbed and quenched by the basal plane of MoS₂ nanosheets, while dsDNA did not bind tightly to MoS₂. However, the probe desorbed from MoS₂ upon the hybridization of target and probe in the presence of a target, with the restoration of the fluorescence emission. Ge et al.¹¹⁸ developed a universal WS₂ nanosheet-based fluorescent biosensor, using T4 polynucleotide kinase (PNK) as a model target, to analyze the activity of PNK based on the phosphorylation-specific exonuclease reaction and efficient fluorescence quenching of ssDNA by WS₂ nanosheets (Fig. 10). The efficient cleavage capacity of λ exonuclease and the super quenching ability of WS₂ nanosheets both contributed to the sensitive screening of T4 PNK activity, suggesting the high performance of synergistic effects. They also found that shorter ssDNA oligonucleotides possessed weaker affinity to WS₂ nanosheets as compared to longer ssDNA oligonucleotides.¹¹⁹ With the fluorescence quenching ability of WS₂ nanosheets and the specific signal amplification, it was possible to realize the ultrasensitive detection of miRNAs. Obviously, except for fluorescent dyes, QDs nanocrystal quenching by TMDs nanosheets will also soon be exploited for optical biosensing applications.

Fig. 10.

Schematic representation of WS₂ nanosheet-based platform for T4 PNK activity and inhibition analysis, reproduced with permission from ref. 118. Copyright 2014 Royal Chemical Society.

TMDs can also fluoresce over a wide range of wavelengths; however, the reported synthetic methods that generate photoluminescent TMDs nanosheets in organic solvents have a few drawbacks, such as low dispersity and considerable cytotoxicity, limiting their universal applicability for biological applications.^120,121 To address this issue, strongly luminescent monolayered MoS₂ ¹²² and MoS₂ QDs^123,124 have been rationally designed. Among them, MoS₂ QDs were proved as an efficient photoluminescent probe to construct a PL quenching sensor for the detection of 2,4,6-trinitrophenol with high sensitivity.¹²⁵ Ha et al.¹²⁴ successfully synthesized homogenous MoS₂ QDs through a Li intercalation method, which exhibited a distinct blue photoluminescence at 415 nm regardless of the excitation wavelength. The capability of the MoS₂ QDs as a fluorescence tag was systematically investigated using the Alexa Fluor 430-dsDNA-MoS₂ FRET system. The MoS₂ QDs could play dual roles in the FRET system with high efficiency, serving as a donor at a certain distance, as well as working for a strong fluorescent quencher acceptor, expanding the types of the donor and acceptor molecule. Spontaneously, researchers have suggested TMD QDs as effective fluorescent probes for biomedical imaging due to their unprecedented characteristics under physiological conditions. Like graphene and graphene oxide, a TMDs-based analytical technique integrated with another platform would be on the way.

Intriguingly, Lin et al. made the surprising discovery that MoS₂ and WS₂ nanosheets could serve as the effective peroxidase mimics, with a fluorescent signaling change in the bioassay.^125,126 Based on this finding, they developed a simple, selective and sensitive colorimetric method for the visual detection of blood glucose. Furthermore, the glucose levels in human serum samples was successfully evaluated by a portable test kit using agarose hydrogel based platforms as a proof of concept. These studies would facilitate the applications of TMDs from immunoassays to stem cell growth. However, comparing with natural enzymes, there are still many disadvantages. Combining it with other nanozymes may provide new paradigms.

5.3. TMDs nanosheet-based bioelectronics

The integration of electronics with biological components is one of the current challenges on the path towards bioelectronics, which is the foundation for many human healthcare and medical technologies. It is hard to realize direct sensing with high-performance using graphene because it is a nanomaterial without an energy gap. For having a considerable band gap, the use of TMDs nanosheets in biosensors not only allows the development of flexible sensors, but also improves the impedance and biocompatibility to high degrees. TMDs nanosheets with 2D layered structures are considered promising platforms for next-generation wearable, flexible, stretchable, and transparent electronics because of their superior electrical, optical, and mechanical properties.^127–129 They have provided opportunities for constructing ultrasensitive biosensors because they are compatible with commercial planar processes for the large-scale circuits.¹³⁰ Therefore, applications are expected for the detection of proteins, nucleic acids, and cells.

For example, Sarker et al.¹³¹ demonstrated an MoS₂ nanosheet-based label-free, real-time field-effect transistor (FET) biosensor. They first showed that the biosensor could exhibit sensitivity as high as 713 for a pH change by 1 unit along with efficient operation over a wide pH range ( pH of 3 to 9). Finally, ultrasensitive and specific protein detection was achieved for the sensing of streptavidin, providing high sensitivity, easy patternability and device fabrication. The performance of the MoS₂-based FET biosensor was higher than that of the reduced graphene oxide-based sensor. The reason might be that the uncontrollable band gap together with the low purity of the reduced graphene oxide led to low sensitivity.

Graphene and its derivatives have been applied as a BioFET, which is still a relatively young research area. The water or cell growth media can easily affect the Fermi level of graphene such that it is away from the Dirac point, and the practical biomolecule detection performed in the real-world would be hindered. However, by combining graphene with 2D nanomaterials having proper structural and morphological designs, the limitation of graphene with a zero band gap may be overcome. Loan et al.¹³² reported an excellent platform based on a graphene/MoS₂ hetero-structural stacking film to detect DNA hybridization with high selectivity and sensitivity. Graphene served not only as a protection layer to prevent the reaction of MoS₂ with the ambient environment but also as a biocompatible interface layer to host DNA molecules on its surfaces. Other probe-target system biosensors could be further developed using the reported principle.

Moreover, Liu et al. presented a facile method to produce nanopore membranes using the exfoliated MoS₂ nanosheets with subnanometer thickness.¹³³ Compared to the conventional silicon nitride nanopores, the device translocated various types of dsDNA through such a novel architecture, offering considerably higher sensitivity. No other process treatment was needed to avoid the hydrophobic interaction between DNA and the surface, which are the advantageous features of the MoS₂ membranes with nanopores in comparison with graphene nanopores. Furthermore, atomistic and quantum simulations indicated that single-layer MoS₂ nanosheets showed a distinct ionic current signal for single-nucleobase detection with the improved signal and reduced noise.¹³⁴ These results were consistent with the experimental results mentioned above, holding great promise in DNA base detection using the proposed 2D nanomaterials. We can see that the needs of medical sensing in this field are vast.

5.4. TMDs nanosheet-based bioimaging

To date, various nano-devices or nano-agents have been developed using graphene-based nanomaterials for practical optical and non-optical imaging studies in the area of nanomedicine. Their success would promote the exploration of TMDs nanosheets as a new class of fluorescent probes. Owing to the excellent biocompatibility, ready cellular uptake, flexible chemical modifications and unique optical properties, TMDs have been explored for biological imaging.¹³⁵ In this section, we highlight some noticeable applications in fluorescence-based cell imaging. As an example, Lin et al.¹³⁶ created WS₂ QDs from bulk WS₂ flakes. In their contribution, the majority of the QDs were monolayered with a lateral size of around 8–15 nm. It was demonstrated that WS₂ QDs exhibited a direct semiconductor nature and activated strong luminescence observed at the region of green-blue light. The quantum yield was low (~4%), but much higher than that of raw nanosheets. The authors then used as-prepared nanomaterials directly for intracellular imaging without any additional process (Fig. 11). In another study, MoS₂ and WS₂ quantum dots were successfully prepared with the combination of sonication and solvothermal techniques.¹³⁷ The quantum yield was highly improved, and in vitro imaging witnessed them as biocompatible probes. It should be noted that most of the present studies have concentrated on the synthesis strategies of the fluorescent TMDs; thus, more attention needs to be paid to the functionalization of nanomateials for the targeted bioimaging with high-throughout.

Fig. 11.

(a) Agglomerated WS₂ QDs surrounding each nucleus (cells are stained by WS₂ QDs only). (b) Individual nucleus stained with DAPI. (c) Cell nucleus and cytoplasm were stained with DAPI and WS₂ QDs, respectively. (d) The overlay image of panels b and c, reproduced with permission from ref. 136. Copyright 2013 American Chemical Society.

5.5. TMDs nanosheet-based drug delivery and cancer therapy

TMDs have been proposed as a good material for attachment and delivery of drugs due to their large surface area and versatile chemistry, such as anticancer agents. They have also received close attention from the cancer therapy field because of their strong optical adsorption in the near-infrared reflectance region with multimodal CT/photoacoustic imaging guided biomedical applications. Photodynamic therapy, chemotherapy and gene therapy with TMDs nanosheets-based nanomaterials are also on the way. Chou et al.¹³⁸ reported that hydrophilic MoS₂ nanosheets exhibited strong optical absorption in the near-infrared (NIR) region, and then described its effectiveness as a NIR photothermal agent. MoS₂ nanosheets had large biomolecules loading capacity, which was comparable to graphene oxide with good biocompatibility as well. The results also displayed approximately 7.8 times greater absorbance in the NIR relative to graphene oxide with a good extinction coefficient, which was higher than that of gold nanorods and was comparable to that of reduced graphene oxide.

In 2014, MoS₂ nanosheets functionalized with lipoic acid modified poly-ethylene glycol (MoS₂-PEG) were synthesized and then employed as a multi-functional drug carrier for combined cancer therapy.¹³⁹ The authors demonstrated combined photothermal and chemotherapy in a mouse tumor model, using the as-obtained atomically-thin MoS₂-PEG nanosheets, which enabled highly efficient loading of therapeutic molecules. In vitro and in vivo experiments proved the outstanding synergistic anti-cancer effect in inhibiting tumor growth of functionalized MoS₂ nanosheets. It was observed that the doses of MoS₂-PEG were considerably lower than those of nano-graphene used in previous reports, indicating the great potential of TMDs nanosheets. Photothermally enhanced photodynamic therapy using chlorin e6 loaded MoS₂-PEG for improved synergistic cancer killing was also reported by the same group both for in vitro cellular and in vivo animal experiments.¹⁴⁰ Combining with photothermal therapy, this new type of multifunctional nano-carrier for the delivery of photodynamic therapy appeared to be an effective therapeutic approach for cancer treatment. In another study, Yin et al.¹⁴¹ prepared chitosan modified MoS₂ nanosheets and used them as chemotherapeutic drug nanocarriers for NIR photothermal-triggered drug delivery, facilitating the combination of chemotherapy and photothermal therapy into one system (Fig. 12). Loaded doxorubicin could be controllably released upon the photothermal effect induced by 808 nm NIR laser irradiation. Furthermore, MoS₂ nanosheets can also be used as the contrast agent for X-ray computed tomography bioimaging due to the obvious X-ray absorption ability of Mo. With the major advantage of high surface area, it is expected to investigate the co-operative delivery of drug and gene using TMDs as the vehicles.

Fig. 12.

Comparative investigation of inhibiting tumor effectiveness in vivo, as indicated, reproduced with permission from ref. 141. Copyright 2014 American Chemical Society.

On the other hand, multimodal bio-imaging offers better accuracy for cancer diagnosis and therapy. Cheng et al. found that WS₂-PEG nanosheets showed strong absorbance in the NIR region with excellent compatibility in physiological environments, making it a highly effective photothermal theranostic agent (Fig. 13).¹⁴² In their report, PEGylated WS₂ nanosheets could serve as a bimodal contrast agent for X-ray computed tomography (CT) and photoacoustic tomography (PAT) imaging, respectively. In vivo experiments uncovered that the as-prepared WS₂ nanosheets enabled highly effective photothermal ablation of tumors. Multimodal imaging-guided cancer therapy making use of PAT, positron emission tomography imaging and magnetic resonance imaging together was proposed as well with iron oxide/MoS₂-based multifunctional probes.¹⁴³ Definitely, toxicity of nanomaterials is a critical issue in biomedicine, which is associated with different biological phenomena. Most studies agree on the low cytotoxicity of TMDs nanosheets towards living cells.^141–144 Recently, Teo et al. demonstrated that MoS₂ and WS₂ nanosheets were less hazardous than graphene oxide by cytotoxicity test assays against human lung carcinoma epithelial cells A549,¹⁴⁵ revealing a better payoff. Although some mechanism of interactions between nanomaterials and biomolecules are not clear, these studies have given the promise of using such TMDs nanosheets for diagnosis and therapy, and would later encourage in-depth studies of 2D nanomaterials for other biomedical applications.

Fig. 13.

In vivo dual-model imaging in 4T1-tumor bearing mice. (a) CT images of WS₂-PEG solutions with different concentrations. (b) Hounsfield units values of WS₂-PEG as the function of its concentrations. (c) CT images of mice before and after i.t. injection with WS₂-PEG. (d) CT images of mice before and after i.v. injection with WS₂-PEG. The CT contrast was obviously enhanced in the mouse liver (green dashed circle) and tumor (red dashed circle). (e) PAT images of tumors on mice before and after i.t. or i.v. injection with WS₂-PEG. (f) Photoacoustic signals in the tumors from mice before and after i.t. or i.v. injections of WS₂-PEG solution. (i.t.: intratumorally; i.v.: intravenously), reproduced with permission from ref. 142. Copyright 2014 Wiley-VCH.

6. Conclusions

Development of a variety of inorganic/organic nanosystems for the biosensors and nanomedicine research is of great significance. GLNs, including BN nanosheets, g-C₃N₄ nanosheets and TMDs (e.g. MoS₂ and WS₂), have been extended to a wide range of applications due to their unique structure and properties, and also have achieved great success in the fields of biosensing and biomedical systems within a short time. In this review, we described state-of-the-art GLNs-based biosensors according to their distinct transduction approaches, particularly fluorescence and electrochemistry, as well as the progress based on GLNs in bioimaging, drug delivery, and cancer therapy. It continues to be a research focus due to the outstanding merits of the related nanomaterials and the fast development of nanotechnology.

On the other hand, research toward the wider applications of GLNs for biosensors and biomedicine are still in its infancy. More efforts are needed and will appear in the coming years. First of all, significant efforts should be devoted to develop facile strategies for controllable, reproducible and scalable synthesis, and functionalization of GLNs with defined structure and properties. The size, surface state and wettability of nanomaterials are indeed of great importance. Exploiting biofunctionalized products with various materials through covalent or non-covalent interactions should be of high interests for the biosensing and nanomedicine fields. To broaden the scope of their bioapplications in the future and open new probabilities for fundamental research, constructing GLNs based nanocomposites with ideal building blocks such as functional nanoparticles or biomolecules is also highly desirable. Second, it is critical to have a better understanding of GLNs based on theoretical and experimental facts, as well as detection mechanisms and interactions between nanomaterials and varieties of interfaces, molecules, cells. On these factors, it is anticipated that GLNs can be employed to design reliable and portable devices that would exhibit high performance such as the simultaneous detection of multiple targets with high selectivity and sensitivity. Third, understanding the impact of GLNs on various pharmacological parameters is imperative, which will facilitate the control over in vitro/in vivo behaviours. Moreover, sufficient attention should be paid to the safety of GLNs by studying their long term toxicity, cellular-uptake mechanism and metabolic pathway, which is critical and inevitable for applications of bioimaging, drug delivery and cancer therapy. How to diminish the toxicity and accelerate the biodegradability of GLNs-based nanomaterials in the biological system is a task that needs to be urgently solved. Last but not least, it is necessary to strengthen collaborations between different disciplines and technologies to make further progress.

In summary, GLNs emerge as the novel nanomaterial platforms for biosensors and nanomedicine. It is clear that GLNs offer considerable opportunities for promising bioapplications despite the large challenges. Thus, the future of GLNs in bioscience and technology will be bright with the efforts of researchers in these areas. Finally, we hope that this review will provide critical insights for understanding the biosensors and nanomedicine related fields and hence help to develop new nanomaterials in nanobiotechnology.

Biographies

Guohai Yang

Guohai Yang received his B.S. degree from Lanzhou University in China in 2008. He earned his Ph.D. degree in chemistry under the supervision of Prof Junjie Zhu at Nanjing University in 2013. Currently, Dr Yang is working as a joint postdoctoral fellow with Prof Junjie Zhu in the School of Chemistry and Chemical Engineering at Nanjing University and Prof Yuehe Lin in the School of Mechanical and Materials Engineering at Washington State University. His research interests mainly focus on the research in biochemical analysis and nanomedicine based on functional materials.

Chengzhou Zhu

Chengzhou Zhu received his Ph.D. in Analytical Chemistry at the Changchun Institute of Applied Chemistry under the supervision of Prof Shaojun Dong in January 2013. Since then, he is involved in the postdoctoral work with Prof Alexander Eychmüller supported by the Alexander von Humboldt Foundation at the Dresden University of Technology. Currently, he is a postdoctoral research associate in the School of Mechanical and Materials Engineering at Washington State University under the supervision of Prof Yuehe Lin. He has co-authored over 55 peer-reviewed publications. His scientific interests focus on carbon and metal nanomaterials for electrochemical and analytical applications.

Dan Du

Dan Du received her Ph.D. in Analytical Chemistry from Nanjing University in 2005. She joined Central China Normal University in 2005 and was promoted to Full Professor in 2011. Currently, she is a Research Professor in Washington State University, Pullman, USA. Her research interests include functional nanomaterials for immuno/biosensing and drug delivery. Dr Du has published more than 110 papers, with citations of ~3300, h-index 36.

Junjie Zhu

Junjie Zhu received his BS (1984) and PhD (1993) degrees from the Department of Chemistry, Nanjing University, China. Then, he began his academic career at School of Chemistry and Chemical Engineering, Nanjing University. He entered Bar-Ilan University, Israel, as a postdoctoral researcher from 1998 to 1999. Since 2001, he has been a full professor at Nanjing University. His main research activities focus on the preparation and bioapplication of functional nanomaterials.

Yuehe Lin

Yuehe Lin is a professor in the School of Mechanical and Materials Engineering at Washington State University, and a Laboratory Fellow at the Pacific Northwest National Laboratory, USA. His research is focused on the nanotechnology area, particularly in the development of new nanobioelectronic devices and nanomaterials for biosensors, biomedical diagnosis and drug delivery, as well as for energy and environmental applications. Dr Lin is a fellow of American Association for the Advancement of Science, Royal Society of Chemistry, and American Institute of Medical and Biological Engineering. He has published 350 papers, with >26 000 total citations and an h-index of 85.