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Mia, A.K.; Meyyappan, M.; Giri, P.K. Biosensing with 2D Transition Metal Dichalcogenides Materials. Encyclopedia. Available online: https://encyclopedia.pub/entry/41554 (accessed on 16 June 2024).
Mia AK, Meyyappan M, Giri PK. Biosensing with 2D Transition Metal Dichalcogenides Materials. Encyclopedia. Available at: https://encyclopedia.pub/entry/41554. Accessed June 16, 2024.
Mia, Abdul Kaium, M. Meyyappan, P K Giri. "Biosensing with 2D Transition Metal Dichalcogenides Materials" Encyclopedia, https://encyclopedia.pub/entry/41554 (accessed June 16, 2024).
Mia, A.K., Meyyappan, M., & Giri, P.K. (2023, February 22). Biosensing with 2D Transition Metal Dichalcogenides Materials. In Encyclopedia. https://encyclopedia.pub/entry/41554
Mia, Abdul Kaium, et al. "Biosensing with 2D Transition Metal Dichalcogenides Materials." Encyclopedia. Web. 22 February, 2023.
Biosensing with 2D Transition Metal Dichalcogenides Materials
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There has been an exponential surge in reports on two-dimensional (2D) materials ever since the discovery of graphene in 2004. Transition metal dichalcogenides (TMDs) are a class of 2D materials where weak van der Waals force binds individual covalently bonded X–M–X layers (where M is the transition metal and X is the chalcogen), making layer-controlled synthesis possible. These individual building blocks (single-layer TMDs) transition from indirect to direct band gaps and have fascinating optical and electronic properties. Layer-dependent opto-electrical properties, along with the existence of finite band gaps, make single-layer TMDs superior to the well-known graphene that paves the way for their applications in many areas. Ultra-fast response, high on/off ratio, planar structure, low operational voltage, wafer scale synthesis capabilities, high surface-to-volume ratio, and compatibility with standard fabrication processes makes TMDs ideal candidates to replace conventional semiconductors, such as silicon, etc., in the new-age electrical, electronic, and opto-electronic devices. Besides, TMDs can be potentially utilized in single molecular sensing for early detection of different biomarkers, gas sensors, photodetector, and catalytic applications. The impact of COVID-19 has given rise to an upsurge in demand for biosensors with real-time detection capabilities. TMDs as active or supporting biosensing elements exhibit potential for real-time detection of single biomarkers and, hence, show promise in the development of point-of-care healthcare devices. 

biosensors 2D materials transition metal dichalcogenides

1. Electrical Biosensors

Electrical sensors transform the information about the analytes into useful user-readable electrical signals, such as current or resistance change. This will discuss the historical progress of TMD-based FET biosensors and the current challenges. The basic working principle of FETs is that the current between the source and drain terminals is controlled by a third terminal called the gate. FETs are essential components of any modern-day electronic circuit and have well-established technologies for fabrication. Thus, electrical detection has the potential for integration with various modern-day electronic devices for real-time detection of bioanalytes. The electrical biosensors consist of a semiconductor transducer element along with a bioreceptor connected to the transducer. The bio-analytes interact with the semiconducting transducer element through the receptor and change the transducer’s electrical properties by the charge transfer mechanism. The attachment of bio-analyte to the semiconductor surface is equivalent to a potential bias at the gate terminal and hence, leads to a change in the drain current. The main advantage of FET biosensors is the enhancement of sensitivity and limit of detection (LoD).

1.1. Biomarker Detection

Biomarkers are biological markers for a specific medical state that can be observed or measured externally. They are direct outcomes of specific biological, pathogenic, or pharmacological processes [1]. Detection of biomarkers is crucial in disease diagnosis, state of progress, and treatments. Detection at ultralow concentration identifies the disease onset early, leading to a higher chance of treatment and recovery. Two-D TMDs, with their high density of active sites, give ultrasensitive responses and ultralow LoD [2]. For example, cancer is one of the deadliest diseases, and a large number of cancer patients can be treated successfully if detected at an early stage of the disease. According to estimates by the International Agency for Research on Cancer, there will be 18.1 million new cancer cases and 9.6 million cancer-related deaths. Lung cancer is the deadliest, followed by breast and prostate cancer [3].
Prostate cancer is one of the most common cancer diseases for males, and prostate-specific antigen (PSA) is a well-studied biomarker for prostate cancer [4]. Detection of PSA at low concentrations is highly desirable, and Wang et al. demonstrated biomodifications of multilayer MoS2-based microfluidic FETs for specific detection of PSA. The MoS2 flakes were mechanically exfoliated on a SiO2 substrate and passivated by HfO2 before functionalizing with anti-PSA to have specific interactions. The concentration-dependent analysis shows a sub-pM sensitivity with a detection limit of 375 fM [5]. The specific binding of PSA on the functionalized surface shows no response with bovine serum albumin (BSA), implying the highly selective detection of PSA. An ultra-thin layer of HfO2 helps biofunctionalization and protects the MoS2 layer from direct interaction with the aqueous medium.
MicroRNAs are common breast cancer biomarkers used for diagnosis [6]. Majd et al. used chemically synthesized MoS2 in a FET to detect miRNA-155 in human serum and breast cancer cell line samples toward PoC devices. The surface was functionalized with amino-modified probe RNAs through physical adsorption, which reduced the channel conductivity. In the presence of targeted RNA, the probe hybridized and detached from the surface, increasing the channel conductivity. A linear response over 0.1 fM to 10 nM was achieved with a detection limit of 0.03 fM at the optimized conditions. The linear response with concentrations of miRNA-21 in human serum is a significant step toward PoC systems [7]. Circulating protein (CP) is essential for diagnosing and treating cancer patients. Park et al. employed scotch-tape-assisted transfer of MoS2 onto an Al2O3-deposited substrate for the FET-based matrix metalloproteinase-9 (MMP-9) sensor and achieved a detection limit of 1 pM [8]. The same group has also used multilayer MoS2 functionalized with aptamer to detect cortisol in human serum and artificial saliva [9]. Sulfur vacancies aid in the functionalization of the bioreceptor and hence, the sensitivity. Additional sulfur defects by creating the nanopore on the MoS2 surface enhanced the sensitivity exceptionally. The nanopore-modified MoS2 has the lowest detection limit of 1 ag/mL with a wide range of sensitivity from ag/mL to µg/mL.

1.2. Detection of Bacteria and Virus

Bacteria and viruses are microorganisms living everywhere, including air, contaminated food, water, body fluids, etc. Some are beneficial to human health, while others are detrimental [10]. Bacterial and viral infections are mostly transferred via physical contact and non-contact spreading. The rapid spreading of these infections causes millions of deaths worldwide [11]. Thus, rapid detection of bacterial and viral infections is vital in human health and environmental monitoring. Detection of bacteria and viruses using TMDs is still in the developing stage. Graphene and reduced graphene oxide (rGO) have been studied in the FET-based detection of bacteria and viruses [12][13][14][15][16]. TMD-based FET biosensors for detecting bacteria and viruses are limited. Moudgil et al. have shown highly selective and sensitive detection of Gram-positive bacteria based on MoS2/TiO2 hybrid nanostructure FET [17]. The TiO2 surface functionalized with vancomycin was able to differentiate between Gram-positive and negative bacteria. A sensitivity of 49% was observed toward S. Aurus with a dynamic response range between 50–106 cfu/mL with a detection limit of 50 cfu/mL. The first WSe2-based FET biosensor for virus detection was reported by Fathi-Hafshejani et al. [18], wherein monolayer WSe2 was functionalized by SARS-CoV-2 antibody 11-mercaptoundecanoic acid (MUA) for the real-time detection of SARS-CoV-2. Selenium vacancies present in WSe2 help in covalently bonding with MUA. The ability of functionalization of monolayer WSe2 also opened avenues for the usage of other TMDs for different pathogenic bacteria and viruses. The MUA functionalized WSe2 FET was able to detect as low as 25fg/µL in real-time.

3.1.3. Detection of DNAs

Detection of DNA is crucial for disease diagnosis, drug delivery, food quality monitoring, environment monitoring, etc. The polymerase chain reaction (PCR) is the most trusted and standard DNA amplification and identification method, but its high cost and time remain a drawback. There is a need for a fast and cost-effective way of DNA sequencing and identification. Lee et al. used chemically synthesized MoS2-based FET sensors for the selective detection of DNA molecules. The van der Walls interaction allows direct functionalization of the MoS2 surface by probe ssDNA. Adsorption of negatively charged ssDNA on the basal plane of MoS2 reduces the effective positive gate voltage, reducing drain current significantly. On adding the targeted DNA, the probe DNA hybridized to dsDNA and detached from the MoS2 surface, increasing its conductivity. At optimum conditions, the sensing device has a highly selective response over 10 fM to 10 nM of complementary DNA [19]. The absence of dangling bonds ensures stability over different pH environments. Mei et al. reported MoS2 based FET sensor for the detection of targeted DNA by using phosphorodiamidate morpholino oligos (PMO) as a probe. The strong interaction between the PMO and targeted DNA improves the detection limit to 6 fM, compared to previous reports. Owing to its low detection limit and ability to detect different concentrations of targeted DNA in human serum, it is a step forward for the PoC diagnostics [20]. Liu et al. used monolayer MoS2 grown by CVD in bio-FETs for the detection of specific DNA targets. Thiol-modified ssDNA probes functionalize the MoS2 surface by using AuNPs through strong Au–SH bonding that enhances the sensitivity by many folds. The monolayer MoS2 can selectively detect 100 aM complementary targeted DNA [21]. Bahri. et al. used WS2 for the first time for the detection of DNA hybridization. The CVD monolayer WS2 was functionalized with an ssDNA probe by van der Walls interaction. The unreacted WS2 surface was blocked by blocking agent poly-C (C-15) to eliminate the non-specific binding for better selectivity. This DNA sensor has an excellent linear response over 0.1 fM to 1 nM DNA concentration with a detection limit of 3 aM [22].
Sarkar et al. fabricated pH and streptavidin sensors based on functionalized MoS2 FET. Lowering the solution pH increases positive ion concentration, which is equivalent to applying positive potential at the gate terminal. The biosensor has a linear response over 3 to 9 pH values and hence serves as a reliable pH sensor. For specific detection of streptavidin, the MoS2 surface was functionalized with biotin. The functionalized MoS2 sensor is highly selective to streptavidin with a detection limit of 100 fM [23]. The semiconducting MoS2 has enhanced the sensitivity by almost 74-fold over graphene. Man et al. fabricated a few layers of MoS2 FET by SF6-assisted plasma etching from bulk MoS2 and passivated it by HfO2. They functionalized the HfO2 by anti-human tumor necrosis factor–alpha (TNF-α) antibody corresponding to the TNF-α biomarker. At the optimum sensing environment, the device showed a linear response over 60 fM to 6 pM concentrations and a detection limit of 60 fM in linear and subthreshold regimes. The statistics over several devices showed an excellent repeatability [24]. Nam et al. compared electrical sensing of TNF-α and Streptavidin using MoS2 and WSe2 FET biosensors [25], and both TMDs exhibited similar detection limits. Chen et al. detected kanamycin (KAN) using an aptamer functionalized MoS2 FET biosensor. The highly specific interaction between the probe aptamer and KAN shows a selective response toward KAN. Surface modification by AuNPs enhances probe attachment and sensitivity [26].
Reactive oxygen species, such as H2O2, play a vital role in the functioning of cells and neuro systems. Detection of such species is essential for the continuous monitoring of cell functioning. Zheng et al. demonstrated the real-time detection capability of H2O2 in HeLa cells using MoS2/RGO heterostructure FET. The sensitivity toward H2O2 increased significantly compared to pristine RGO. Hela cells generate H2O2 when reacted by phorbol 12-myristate 13-acetate (PMA) and detected by the FETs, proving the capability to detect H2O2 in complex biofluids [27].
TMD-based FET biosensors are still new in the research domain compared to well-established silicon technologies. Generally, TMDs grown at high temperatures lead to degradation of the growth substrate. One major disadvantage of this for electrical biosensors is that the 2D TMD films need to be transferred from the growth substrate to the device substrate. The transfer process is complex and chemical residue is present on the transfer samples, leading to lower quality and performance issues. There is a need for a low-temperature growth mechanism or improved transfer methods to mitigate this problem in electrical biosensors.
The charge screening effect in FET devices, defined by the Debye length, remains a big challenge. This is the maximum distance from the sensing channel, where analytes can modulate the channel conductivity. As the ionic strength increases, the Debye length decreases. The complex and high ionic strength biofluids limit the Debye length to a few nm. Although TMDs have been successfully demonstrated as selective biosensors, most studies have been done in controlled environments, and further development is required in terms of rigorous testing in complex media. Thus, applying TMD-based FETs as state-of-the-art PoC devices has a long way to go.

2. Electrochemical Biosensors

Electrochemical sensors are three electrode-based sensing platforms where the electrochemical reaction reduction–oxidation (redox) creates ions and charges, changing the electrochemical response. The three electrodes are the working electrode (WE), the reference electrode (RE), and the counter electrode (CE). All the potentials are measured with respect to RE, while CE completed the electrochemical cell connections. The WE is modified by nanomaterials for better electrochemical activity. The basic operating principle is the detection of Faraday current using voltammetry or amperometry and the modulation of interfacial impedance by electrochemical impedance spectroscopy (EIS). The WE is modified with suitable biorecognition elements for detecting specific analytes via redox reactions, which generate or suppress electrons or ions and change the current across the WE. In the case of EIS, the analytes adsorbed on the modified WE surface modulate the electrochemical current or interfacial impedance. The quantitative correlations between the change in the interfacial impedance or electrochemical current with the number of analytes give the sensing response.

2.1. Detection of Biomarkers

Dopamine (DA) is a neuro biomarker and plays an important role in the functioning of the neurological system. Sakthivel et al. decorated cobalt oxide polyhedrons on an MWCNT/MoS2 hybrid system by conventional hydrothermal method for electrochemical detection of DA [28]. The modified electrode showed great stability, high sensitivity and selectivity, and a low detection limit of 13 nM. The electrode retained 97% of its initial current even after 30 weeks of use, indicating its robustness. The feasibility of real-time application was tested with physiological samples, such as rat and human serum, in an optimized lab environment. As discussed earlier, miRNAs are cancer biomarkers. Su et al. used gold nanoparticle (Au NPs)-modified MoS2 to detect miRNA-21 by EIS and differential pulse voltammetry (DPV) [29]. The results showed selective detection of miRNA-21 in the fM range for both methods. The miRNA-21 was added to human serum for accurate sample detection and studied with the same device. The results indicated that the Au NP decorated MoS2 was an excellent real-time detector for miRNA-21. Zhu et al. demonstrated thionine-reduced AuNP functionalized MoS2 sheets for electrochemical detection of miRNA-21 with a detection range of 1 pM to 10 nM and a detection limit of 0.26 pM [30]. Chand et al. synthesized copper ferrite-decorated MoS2 nanosheets functionalized with thiol-modified biotin for microfluidic-based electrochemical sensors to detect paratuberculosis-specific miRNAs [31]. Paratuberculosis (pTb), or Johne’s disease, is a deadly disease in dairy cattle as it is highly contagious and asymptomatic [32]. The microfluidic assisted sensing allows the screening of multiple samples simultaneously. The optimized conditions showed real-time detection of miRNA at the lowest concentration of 0.48 pM with excellent selectivity. The complex biological fluid analysis of infected blood and fecal samples helps in the detection of miRNA. This sensor also successfully detected actual samples from infected cows. Carcinoembryonic antigen (CEA) is a cancer biomarker produced by colorectal cancer and found at very low concentrations [33]. Thus, sensitive and low-level detection of CEA is vital for the early detection and treatment of colorectal cancer. Wang et. al. synthesized a nanocomposite of flower-like MoS2 with rGO and Ag NPs for ultrasensitive detection of CEA [34]. Incorporating Ag NPs in MoS2-GO composite enhances the electrochemical activity manyfold because the synergistic effect between MoS2 and Ag NPs improves the sensitivity. The wide range of detection is from 0.01 pg/mL to 100 ng/mL with a detection limit of 1.6 fg/mL, which is much lower than the clinically safe limit.
Liu et al. reported an AuNP-decorated MoS2/T3C2 hybrid structure for ultrasensitive electrochemical sensing of miRNA-182, a well-known lung cancer biomarker [35]. The thiol-modified probe ssRNA was mobilized by the hybrid structure of the well-known Au–SH solid bond. Because of its negative charge, the electrochemical activity of the hybrid decreased after the ssRNA modification. The binding of the targeted miRNA-182 with the probe RNA resulted in dsRNA hybrids, which, when released from the Au NPs, increased the electrochemical activity. At optimum conditions, quantitative analysis of the RNA hybridization gave a linear detection range of 1 fM to 0.1 nM with a detection limit of 0.43 fM. MicroRNA-155 is a cancer biomarker found in ultra-low concentrations in body fluids, such as plasma, at the early stage of cancer patients [36]. Liu et al. studied the electrochemical sensing of miRNA-155 using MoS2 thin films deposited by ALD and modified by AuNPs [37]. For the selective detection of miRNA-155, thiol-modified probe RNA was attached to the MoS2 surface via SH-Au chemistry. To avoid a non-specific response, unbounded Au NPs were blocked by blocking agent 6-mercaptohexanol (MCH). Cyclic voltammetry (CV) and EIS measurements of the modified electrode at different stages of the fabrication show that the Au NPs enhanced the electrical conductivity by synergistic effect and enhanced electron transfer process. After functionalization and blocking of the unreacted sites of Au NPs, the electrical conductivity decreased as the charge transfer was hindered. For the concentration-dependent study, toluidine blue (TB), a phenothiazine dye, was used as the hybridization monitor owing to the presence of π-π conjugate electron. The sensors could detect miRNA-155 ranging from 1 fM to 10 nM with a detection limit of 0.32 fM.

2.2. Detection of Bacteria and Virus

Hepatitis B virus (HBV) infection is one of the serious public health concerns and can cause some deadly diseases, such as cirrhosis and hepatocellular carcinoma (HCC) [38]. Hepatitis B e antigen (HBeAg) is one of the most reliable tumor biomarkers for identifying HBV infections. Gao et. al. developed an electrochemical immunosensor based on gold@palladium (Au@Pd) NP decorated MoS2 functionalized multiwall carbon nanotubes (Au@Pd/MoS2@MWCNTs) [39]. The MoS2/MWCNTs hybrid composite enhanced electrochemical activity, and the incorporated Au@Pd NPs amplified the sensitivity towards HBeAg detection by synergistic effect. The sensor showed a systematic current increase with the addition of HBeAg ranging from 0.1 pg/mL to 500 pg/mL and had a detection limit of 26 fg/mL.

2.3. Detection of DNAs and Other Bio-Analytes

Wang et al. demonstrated electrochemical sensing of dsDNA using thionin-functionalized MoS2 sheets as a working electrode. The electrostatic attraction of thionin to the MoS2 defect sites was confirmed by X-ray photoelectron spectroscopy (XPS) analysis and the redox peak at −0.27 V at square wave voltammetric measurements [40]. The redox peak current at −0.27 V decreased consistently with increasing concentration of dsDNA in the solution. The linear response was obtained from 0.09 to 1.9 ng per ml dsDNA with a detection limit of 0.09 pg/mL. Redox current decreased after the addition of complex biofluid, circulating DNA extracted from human serum, demonstrating its capability as a PoC device. Yang et al. synthesized ZnO/MoS2 hybrid nanocomposite for the electrochemical sensing of DNA with great sensitivity [41]. The hybrid composite not only helps in direct charge transfer but also in probe immobilization. The probe ssDNA is attached to the nanocomposite surface by electrostatic interaction between positively charged ZnO and negatively charged ssDNA. At an optimum environment, the DPV measurement of the modified electrode with different concentrations of promyelocytic leukemia (PML) and retinoic acid receptor alpha (RARA) exhibits a linear response over a wide range. The low detection limit of 0.66 fM demonstrates its potential for ultralow detection of DNA by the heterostructure.
Zhang et al. fabricated poly-xanthurenic acid (PXA) functionalized MoS2 electrochemical biosensors to detect circulating tumor DNA in blood samples [42]. The novel polymer XA has low toxicity, acceptable redox activity and good electrochemical performance. The probe ssDNA was immobilized on hybrid PXA/MoS2 nanostructure by π-π conjugate interactions. The immobilization of ssDNA on the surface decreased the current drastically due to charge transfer and blocking. With the addition of the targeted circulating tumor DNA, the ssDNA hybridized to form dsDNA. The weak interaction between PXA and dsDNA releases the DNA from the surface, restoring electrochemical activity. The quantitative analysis of the current restoration with DNA concentration shows a linear response range of 0.1 fM/l to 100 pM/l with a detection limit of 0.018 fM/L. The sensor shows excellent reproducibility. The van der Waals interaction between the ssDNA with MoS2 nanosheets was utilized by Zhou et al. for electrochemical sensing of Kanamycin, the widely used antibiotic for bacterial infections and tuberculosis [43]. In the presence of biotin-modified assist DNA, probe DNA and aptamer DNA formed a Y shape dsDNA structure, which has a very low adsorption probability with MoS2 sheets. The addition of Kanamycin aided in the binding with the aptamer DNA and breaking the Y shape of dsDNA to ssDNAs. The MoS2-modified glassy carbon electrode (GCE) adsorbed the ssDNA. The interactions between the biotin and streptavidin enhanced the mobilization of the biotin-modified assist DNA and probe DNA hybrid on MoS2-modified GCE. The catalytic effect between alkaline phosphatase and p-nitrophenol phosphate produced p-nitrophenol (PNP), an electrochemically active molecule. The quantitative analysis between the Kanamycin concentration and peak oxidation current showed a linear response from 0.1 nM to 100 nM. The threshold limit of detection was 0.03 nM. The Kanamycin in Kanamycin Sulfate Eye Drops was successfully detected with the sensor with less than 9% variation and remarkable selectivity. Zhang et al. synthesized WS2/graphite microfiber hybrid for the electrochemical detection of adenine and guanine. WS2 synthesis directly on graphite microfiber exhibited excellent charge transfer characteristics and electrocatalytic oxidation response. The adsorption of adenine and guanine on the WS2 surface changed its electronic and charge transfer properties. The CV analysis showed two oxidation peaks at +0.73 V and +1.03 V, corresponding to the oxidation of adenine and guanine, respectively. The concentration-dependent CV response showed a systematic increase in the reduction current due to charge transfer resulting from the adsorption on the WS2 surface. The electrochemical sensor had a linear response ranging from 0.5 µM to 20 µM concentration of adenine and guanine [44].
The partial reduction of oxygen produces H2O2 during various biological processes, which plays an essential role in signal processing and transduction of cells [45]. The imbalanced ions affect the stress in cells and eventually lead to several diseases, including cancer [46]. Wang et al. used MoS2 nanoparticles to detect H2O2 at the nM level without using any enzymes [47]. The high density of electroactive sites of MoS2 NP increases the electrochemical reduction of H2O2 and hence, improves the sensitivity along with a low detection limit. Ma et al. demonstrated H2O2 and cholesterol sensing by oxidized glutathione-modified MoS2 (MoS2-GSSG) NSs. The high affinity of MoS2-GSSG towards the 3,3′,5,5′-tetramethylbenzidine (TMB) substrate ensures uniform distribution. Peroxidase-like catalytic activities of MoS2-GSSG NSs convert the H2O2 into *OH, which oxidizes the TMB [48]. With this catalysis-based reaction, the detection limit was as low as 0.5 µM. Shu et al. synthesized nanoflower-like interlayer expanded MoS2 (IE-MoS2) and nonexpanded MoS2 (NE-MoS2) by thiourea-assisted hydrothermal route for electrochemical sensing of the H2O2 [49]. The GCE was modified by IE-MoS2 and NE-MoS2 to check the electrochemical performance for H2O2 detection. The IE-MoS2 modified electrode showed an enhancement in current with good sensitivity as it promoted the reduction of H2O2 to OH. Linear response with H2O2 concentration ranging from 2.3 × 10−1 to 14.2 × 103 μM was reported with remarkable selectivity in the presence of ascorbic acid (AA), glucose, sucrose, uric acid (UA), dopamine (DA), NaCl and KCl. Real-time detection of H2O2 was carried out in complex biological fluids, including living cancer cells, e.g., human breast cancer cells (MCF-7), for PoC applications.
Diabetes is one of the most common medical issues worldwide, causing many serious health problems. Blood glucose is the critical parameter for monitoring diabetes. Hence, continuous blood glucose monitoring is crucial [50]. Currently, the commercially available PoC system can detect glucose in human blood drawn by an end user from 1 to 27 mM [51]. Instead of using a blood sample, the glucose level can also be detected by the patient’s sweat and saliva, which is a non-invasive technique. However, the glucose level in sweat and saliva is in the order of µM. Thus, researchers need ultrasensitive sensing devices to detect glucose in sweat and saliva, which is much lower than the sensing capabilities of enzyme-based commercial systems [50][51][52][53]. TMDs have the potential for lower detection limits and can detect glucose even in the nM concentration [54]. Su et al. decorated MoS2 NSs with Au@Pt core-shell nanoparticles for the electrochemical detection of glucose in the human serum [55]. The synergistic effect of the nanoparticles enhanced the electrocatalytic activity of the modified electrode towards glucose reduction, improving the sensitivity. Glucose detection in human serum with excellent recovery and accuracy implies its application in real-time in biofluids. The low detection limit of 1.08 µM shows the capabilities for ultralow glucose detection.

3. Optical Biosensors

Optical biosensors utilize the advanced and superior optical properties of 2D TMDs to detect bioanalytes. They are some of the most sensitive biosensors and can detect even a single bioanalyte in real-time. The typical mechanisms for detection in optical biosensors include surface-enhanced Raman spectroscopy (SERS), surface plasmon resonance (SPR), fluorescence imaging, and Forster resonance energy transfer (FRET). SERS measures the enhancement of the Raman signal of the samples after and before analyte adsorption on a SERS substrate. SPR strongly depends on the refractive index. Upon adsorption of the bio-analyte, there is a local change in the material’s refractive index. FRET is a nonradiative energy transfer mechanism between two fluorophores through dipole–dipole coupling. Due to its inverse sixth power law dependence, it is extremely sensitive to changes in the distance between fluorophores. Hence, modulation in the fluorescence intensity in the presence of a target analyte has been extensively used for the purpose of detection.

3.1. Biomarker Detection

Biomarkers, as mentioned earlier, are direct outcomes of specific biological, pathogenic, or pharmacological processes [1]. 2D TMDs, with their high density of active sites, give ultrasensitive responses as biosensors used for biomarker detection [2]. Kong et al. carried out real-time detection of a prostate cancer biomarker in human serum for the first time using aptamer-functionalized MoS2 nanosheets. When added to the dye-labeled ssDNA aptamer, there is high fluorescence (FL) quenching of MoS2 NSs. This phenomenon was utilized for the purpose of sensing [56]. The adsorption of the target-specific ssDNA on the MoS2 surface almost entirely quenches its FL spectra due to charge transfer by van der Waals interaction. The addition of the target DNA leads to the binding with probe aptamers, and thus, desorption from the MoS2 surface restores the FL intensity. The quantitative study between the FL restoration and the targeted aptamer addition shows a linear response from 0.5 to 300 ng/mL with a detection limit of 0.2 ng/mL. With its high stability and specificity to aptamers, the sensor had a very high selectivity toward targeted DNA. The ability to detect PSA in complex biofluid human serum in real-time ensures its capability to be used as a PoC device. Dhenadhayalan et al. studied the molybdenum (Mo) series of 2D materials from MoO3, MoS2 to MoSe2 NSs for real-time detection of PSA by FL quenching [57]. Among those NSs, MoO3 had the lowest detection limit of 13 pM, whereas MoS2 and MoSe2 yielded 72 and 157 pM, respectively. The low detection limit of MoO3 NSs was attributed to the relatively electronegative element O as compared to S and Se. These ultrasensitive biosensors have the potential for practical use at preliminary testing facilities. Similar to prostate cancer in males, breast cancer is the most common cancer in women and shares a high percentage among all cancer-related deaths [3]. MicroRNAs are common biomarkers for breast cancer and are used for disease diagnosis [6]. Xi et al. first showed the detection of microRNA-21 (miRNA-21) using WS2 NSs through duplex-specific nuclease signal amplification (DSNSA) [58]. The binding of the dye-labeled ssDNA onto the basal plane of WS2 NSs quenches its FL intensity by almost 97%, indicating strong charge transfer between aptamers and WS2 NSs. Binding with the targeted miRNA-21, the probe DNA formed a DNA/RNA heteroduplex which acts as the substrate for duplex-specific nuclease (DSN) cleavage. The DSN selectively cleaves the ssDNA from the heteroduplex, allowing the miR-21 to hybridize with another ssDNA. This stimulated process improved the detection limit to 300 fM with extremely high selectivity and even allowed to differentiate a single mismatch RNA.
MiRNA extracted from various cancer cell detection shows excellent agreement with the quantitative real-time polymerase chain reaction (qRT-PCR) results, hence having the capability to use in PoC detection systems. Chi et al. demonstrated miRNA-21 sensing by ssDNA aptamer functionalized MoS2 NSs via an excellent FL quenching [59]. As reported previously, the FL of MoS2 NSs quenched on the adsorption of ssDNA on its surface. The sensing platform had a detection limit of 500 pM and distinguished even a single mismatch miRNA-21. The detection of miRNA-21 with human serum in different concentrations was demonstrated in a real-time analysis as a PoC system. Gómez et al. showed a red shift in the photoluminescence (PL) spectra of monolayer MoS2 when used for miRNA-21 detection. The thiol-modified probe ssDNA binds to the S vacancy sites and enhances the PL intensity because of charge transfer between them [60]. When the probe DNA captures complementary targeted DNA, it forms dsDNA, the PL peak shifts towards the lower energy region, and PL is quenched. Further study is needed for the concentration dependence of PL peak shift to find the sensitivity and detection limit. Lung cancer, as mentioned earlier, does not show symptoms and the biomarkers are at ultralow concentration. Cytokeratin 19 fragment (CYFRA21-1) is a well-studied biomarker for lung cancer [61]. Chiu and Yang used the principle of SPR with carboxyl-functionalized MoS2 (carboxyl-MoS2) to detect CYFRA21-1 in the human serum [2]. The sulfur vacancies in MoS2 act as attachment sites for the carboxyl group, making them more sensitive to incident light. The study showed that carboxyl-functionalized MoS2 SPR biosensor could detect breast cancer biomarker CYFRA21-1 from 0.05 pg/mL to 100 ng/mL with a detection limit of 0.05 pg/mL. The concentration-dependent measurement of CYFRA21-1 in human serum showed a detection limit corresponding to 3.125% CYFRA21-1 in human serum. Zhao et al. detected the tumor biomarker CEA by FL quenching of MoS2 NSs, on adsorption of ssDNA on its surface. The sensor had a linear response over 0.1 to 100 ng/mL with a detection limit of 300 pg/mL and an excellent selectivity [62].
Malaria is a health concern in many parts of the world and is responsible for a considerable loss of lives. The protozoan Plasmodium parasite causes malaria, and its early detection remains a big challenge. Kenry et al. used single-layer MoS2 sheets for the detection of Plasmodium lactate dehydrogenase (pLDH) protein [63]. FL intensity of high-affinity malaria biomarker aptamer quenches almost 90% after adding MoS2 because of van der Waals interaction. The quantitative analysis shows a linear restoration of FL intensity upon serial addition of 0 to 62.5 mM pLDH protein with a detection limit of 550 pM, much lower than that of the clinically accepted safe limit of a few nM. To practically use the biosensor, several other bioanalytes ranging from insulin to globulin, were also tested for FL restoration. Results showed high selectivity towards pLDH protein and low detection limit.

3.2. Detection of Bacteria and Virus

Zhang et al. studied the FL quenching of 6-carboxyfluorescein (FAM) dye-labeled ssDNA using MoS2, TaS2, and TiS2. The adsorption of FAM dye-labeled ssDNA probe aptamer corresponding to the Influenza A virus onto the TMD surface quenches its FL intensity. Among the three TMD NSs, TaS2 has the highest quenching capability of 99% [64]. The systematic FL intensity restoration shows a linear response in the 0 to 20 nM concentration range. The TaS2-based sensor has the lowest detection limit of 0.05 nM compared to 0.1 nM for MoS2 and 0.2 nM for TiS2.

3.3. Detection of DNAs and Other Biomolecules

Zhu et al. first demonstrated the FL quenching of single-layer MoS2 by the FRET mechanism in the vicinity of a biomolecule. The probe ssDNA has a higher affinity toward single-layer MoS2 than dsDNA; hence, the FL intensity almost quenches completely on the adsorption of ssDNA due to nonradiative energy transfer among them. Upon binding with the targeted DNA, the probe DNA hybridizes to dsDNA and detaches from the MoS2 surface, restoring the FL intensity. The quantitative study between the addition of the targeted DNA and the FL intensity restoration showed a linear relationship from 0 to 50 nM concentration range with a detection limit of 500 pM [65]. Ge et al. designed FL based aptasensor for specific detection of adenosine triphosphate (ATP) and human α-thrombin in the human serum [66]. FL intensity of the probe aptamers diminished once MoS2 NSs were added because of the charge transfer. With the attachment of the targeted ATP and α-thrombin, the aptamers formed a dsDNA hybrid and detached from the MoS2 surface, resulting in FL restoration. However, even after the addition of a high concentration of targeted DNA, the FL intensity did not restore to its original value as some of the aptamers bound with MoS2 in other configurations and remained bound to the MoS2 NSs. The concentration-dependent study shows a linear response over a wide range of 0 to 2 mM for ATP. The system successfully detected ATP in human serum and from the extraction of lung adenocarcinoma A549 cells, proving its application in real-time PoC devices. A detection limit of 4 µM ATP and 300 pM of thrombin was reported using a similar mechanism.
Loan et al. made a graphene-encapsulated MoS2 heterostructure-based DNA sensor using a PL enhancement study. The ssDNA attachment acts as a positive voltage gating to graphene, changing the optoelectronic properties. The biocompatible top graphene layer protects the MoS2 surface from the aqueous medium. The functional groups on the graphene surface also serve as biolinkers between the aptamers and the graphene surface. The systematic enhancement of the PL intensity with the DNA concentration shows a linear behavior from 1 aM to 1 fM concentration. A similar study with single-sequence mismatch DNA shows that the enhancement is only due to targeted binding with the probe DNA, and the sensor has excellent selectivity toward the targeted DNA [67].
Huang et al. developed a microfluidic-based DNA sensor for rapid and multiple DNA screening using single-layer MoS2 NSs [68]. Polydimethylsiloxane (PDMS)-assisted zigzag-shaped microchannel helped the screening of multiple samples simultaneously and ensured uniform mixing of ssDNA and MoS2 NSs. Using the microchannel, rapid screening of DNA can detect targeted DNA with fM concentration within a few minutes. Wang and his co-workers used Peptide nucleic acid (PNA) as a probe for DNA sensing. PNAs are similar to ssDNA, with a higher binding affinity toward targeted DNA. Similar to ssDNA, the FL intensity of probe PNA quenches entirely after adsorbing on the WS2 surface because of charge transfer among them. The restoration of FL correlated with the binding of specified targeted DNA, which was confirmed by adding a single base mismatch DNA. The result showed almost negligible restoration of FL intensity, which indicated the precise binding nature of the PNAs [69]. The linear detection capability of the sensing device was in the range of 1 nM to 20 nM with a detection limit of 500 pM. Jin et al. functionalized a single-layer MoS2 surface with thiol-modified aptamer through SH–Au bond [70]. The thiol-Au bond enhanced the adsorption of ssDNA on the surface of a single-layer MoS2. The adsorption of ssDNA modulates the local dielectric constant and affects the band energy. There is a continuous blueshift with the addition of modified aptamers. The study reveals that one can detect targeted DNA at an nM concentration level by employing PL spectroscopy measurement on a single-layer MoS2. Xi et al. synthesized thioglycolic acid (TGA) functionalized single-layer MoS2 NSs to detect dopamine. Upon the addition of DA, the FL intensity of TGA functionalized MoS2 NSs quenches. In the presence of DA, the C–O⋯H–O hydrogen bonding starts stacking single-layer MoS2 NSs and results in FL quenching as a result of the charge transfers. Dopamine interacts with the TGA functionalized MoS2 by hydrogen bonding [71]. Gao et al. demonstrated thrombin detection using Au NPs modified MoS2 NSs. The FL-based biosensor had a linear responsivity from 50 nM to 20 µM concentration of DA with a detection limit of 2.7 nM [72].

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