*2.2. MoS2 Conjugated Nanoparticles for Electrochemical Biomarker Detection*

Recently, transition metal dichalcogenides (TMDCs) found their applications in various biosensors due to their large surface-to-volume ratio, tunable electronic and optical properties, low toxicity, and unique van der Waals layered structure [65]. In TMDCs, one layer of transition metal atoms (M) lies between two layers of chalcogen atoms (X) resulting in a formula MX2. Various kinds of TMDCs can be realized by altering the chalcogen atoms such as Sulphur (S), Selenium (Se), and Tellurium (Te), and metal atoms like Molybdenum (Mo) and Tungsten (W). Among these, MoS2 is commonly used because its fundamental constituents are surplus and innoxious [66]. MoS2 molybdenum (Mo) atoms lie between the two sulfide atoms layers (S-Mo-S) and atoms in the crystal are associated by strong

covalent bonding and adjacent layers of MoS2 are held by weak van der Waals forces. MoS2 possesses a mobility of 200 cm2/Vs at room temperature, high on/off current ratio of 108, and a direct band gap of 1.8 eV. Based on these properties, MoS2 becomes a promising alternative to graphene and is applied in various electrochemical and optical sensors [67–69]. MoS2 can be synthesized in both top-down and bottom-up approaches (Figure 8). The top-down approach includes the exfoliation of MoS2 [70], while the bottom-up approaches include (i) chemical vapor deposition [71]; (ii) physical vapor deposition [72]; (iii) solutionbased processing [73]. For a more detailed synthesis of MoS2, readers are encouraged to go through the literature survey of the desired synthesis approach. Thus, like graphene, MoS2 offers a large surface area that enhances its biosensing performance.

**Figure 8.** Various synthetic methods for MoS2 preparation. Reprinted with permission for Ref. [74]. Copyright 2022 MDPI.

MoS2 possesses a direct band gap of 1.8 eV in the monolayer, lattice defects of zero dimensionality, grain boundary defects, and an enhanced surface-to-volume ratio. Also, the feasibility of surface modification and chemical functionalization makes these characteristics of MoS2 to adopt and study in scientific and industrial fields [75] (Figure 9). Furthermore, to increase the electroactivity/conductivity of graphene and/or other 2D materials, mostly nanoparticles were incorporated to achieve the synergistic effects from both nanomaterials, which ultimately resulted in an improvement in the overall analytical performance of the biosensor. In this section, we review various types of biosensors that incorporate metal nanoparticles on MoS2 for the detection of various biomarkers.

**Figure 9.** MoS2 nanostructures-based electrochemical sensing application in various fields. Reprinted with permission from Ref. [76]. Copyright 2018, Elsevier.

In a recent report that mentions the usage of MoS2-Au nanoparticles, Yagati et al. [77] reported on the applications of MoS2 conjugated Au nanoparticles on indium tin oxide (ITO) electrodes for the detection of the thyroid-stimulating hormone biomarker, triiodothyronine (T3), as shown in Figure 10. Electrodeposition procedures allowed the formation of MoS2 and Au nanostructures on the ITO electrode. Subsequently, T3 antibodies were immobilized on the MoS2-Au/ITO surface by forming a self-assembled monolayer of dithiobis (succinimidyl propionate) (DSP). For any nonspecific bindings, the surface is coated with casein and then subjected to different concentrations of the T3 biomarker diluted in both PBS and serum samples. Electrochemical impedance spectroscopy was used to analyze the bindings of T3 to its antibodies and a linear correlation was observed for different concentrations. Based on the quantifications made by this sensor for the detection of T3, a linear range of 0.01–100 ng/mL with a detection limit of 2.5 pg/mL was observed. The sensor also showed a good correlation with data observed by the conventional method (Roche Cobas) and possessed high sensitivity and selectivity in discriminating the healthy and cancer samples. Based on the findings, the developed sensor could apply to cancer-related biomolecule analysis.

Su et al. [78] developed dual target sensing (adenosine triphosphate (ATP) and thrombin) detection electrochemical biosensors based on gold nanoparticles-decorated MoS2 (AuNPs–MoS2) nanocomposites which feature both "signal-on" and "signal-off" elements in the detection system, and thrombin and ATP could act as inputs to activate an AND logic gate (Figure 11). In this approach, two different aptamer probes labeled with redox tags (ferrocene (Fc) and methylene blue (MB)) were simultaneously immobilized on an AuNPs-MoS2 modified glassy carbon electrode (GCE) through Au-S bond formations. Subsequently, the electrode was immersed in 6-mercaptohexanol to block the uncovered spots of AuNPs–MoS2/GCE. Square wave voltammetry (SWV) was used to determine the

various concentrations of ATP and thrombin applied to the GCE. From concentration vs. change in the current results, it was evaluated that the sensor had a linear range for the determination of ATP, which was 1 nM to 10 mM with a detection limit of 0.32 nM, while for the thrombin determination, the linear range was 0.01 nM to 10 μM with a detection limit of 0.0014 nM.

**Figure 10.** Schematic illustration of the total triiodothyronine (T3) receptive interface fabrication through the immobilization of the antibody on a step-by-step modification process of MoS2–Au formation and subsequent functionalization with a dithiobis (succinimidyl propionate) monolayer on an indium tin oxide electrode surface. With increasing concentration of the T3 analyte in serum, the EIS (Nyquist plot) shows increased semi-circle (Rct) for quantification. Reprinted with permission from Ref. [77]. Copyright 2020, Elsevier.

**Figure 11.** Schematic representation for the development of the aptasensor for the determination of ATP and thrombin. Reprinted with permission from Ref. [78]. Copyright 2016, ACS.

The authors also suggested that this mechanism can be acted as an AND logic gate by using ATP and thrombin as inputs and the electrochemical signals of Fc and MB as outputs (Figure 12). The logic gate works on the structural conversion of the aptamer probe triggered by ATP and thrombin. The working mechanism was the individual peak current enhancement of Fc or the suppression of MB as electron transfer OFF (eTOFF) or "zero" output, and the simultaneous peak current enhancement of Fc and suppression of MB as electron transfer ON (ON) or "one" output. From the inset table, a "one" output was achieved only when both inputs were "one". When there were no inputs (0, 0) or only one input (0, 1 or 1, 0), the result was "zero" output. Thus, the MoS2-based multiplexed aptasensor could also serve as an "AND" gate.

**Figure 12.** Schematic description of the MoS2-Based AND logic gate for determination of ATP and thrombin. Reprinted with permission from Ref. [78]. Copyright 2016, ACS.

In another work, Chen et al. [79] reported on the development of a growth differentiation factor-15 (GDF-15) expression sensor which is a potential biomarker for the diagnosis, risk stratification, and prognosis of various cardiovascular diseases (Figure 13). Here, a sandwich-type immunosensor was constructed using amine-modified graphenesupported gold nanorods (NG/AuNPs) as a substrate platform, and the durian-shaped MoS2/AuPtPd nanodendrite (NDs) as a label for secondary antibodies (Ab2) for the quantification of growth differentiation factor-15 (GDF-15). NG/AuNPs are used to enhance the surface area and for the immobilization of primary antibodies through the binding of amino or sulfhydryl groups. Subsequently, the electrodes were blocked with 1wt% BSA. Finally, the signal probe MoS2/AuPtPd-Ab2 was added to the sample.

The developed sensor was also applied to evaluate the efficacy towards the clinical sample analysis and compared with traditional sensing methods, such as ELISA, to evaluate the accuracy of the results. The sensor showed a linear range of 1.5 pg/mL to 1.5 μg/mL with a detection limit of 0.9 pg/mL. Due to its high sensitivity, rapid response, and feasibility to miniaturization, the proposed sensor could be applied to a point-of-care diagnostic tool for cardiovascular diseases and paves the path toward "liquid biopsies".

**Figure 13.** Schematic illustration for the development of a sandwich-type electrochemical sensor for GDF-15 detection sensor. Reprinted with permission from Ref. [79]. Copyright 2022, Elsevier.

Nong et al. [80] reported on the detection of cortisol which is a glucocorticoid hormone that adrenal glands produce and release, and this hormone regulates stress, inflammation, blood pressure, sugar, and overall metabolism. In this work, copper tungstate-molybdenum sulfide (CuWO4@MoS2) and chitosan-gold (Chit-Au) nanocomposite were synthesized and applied to GCE (Figure 14). Subsequently, the cortisol antibody (C-Mab) was immobilized using the EDC/NHS reaction and subsequent blocking with BSA. Once the transducer surface was fabricated, SWV was performed to analyze the bindings of various concentrations of cortisol and a linear relationship was observed concerning different concentrations. The sensor showed a linear range of 0.1 fg/mL to 1 μg/mL with a detection limit of 0.014 fg/mL (S/N = 3). The sensor showed excellent storage stability and reproducibility and it can detect the content of cortisol in saliva.

Su et al. [81] reported on the use of a MoS2-Au nanocomposite for the detection of a carcinoembryonic antigen (CEA). In this work, CEA antibodies labeled with horseradish peroxidase resulted in an amplified electrochemical signal by catalyzing o-phenylenediamine (o-PD) in the presence of hydrogen peroxide (H2O2). As can be seen in Figure 15, the MoS2-Au conjugated HRP labeled antibodies enhance the overall sensitivity when the different concentrations of CEA were measured using cyclic voltammetry. From the analytical performance, the sensor displayed a linear range of 10 fg/mL to 1 ng/mL with a detection limit of 1.2 fg/mL. The sensor also exhibited good stability, and high selectivity suggesting that the proposed immunosensor could detect CEA in real samples.

**Figure 14.** Schematic representation for (**A**) Synthesis of MoS2, CuWO4@MoS2, AuNPs, and Chit-Au nanocomposites; (**B**) Preparation process of the immune electrode. Reprinted with permission for Ref. [80]. Copyright 2022, Elsevier.

Also, Ma et al. [82] reported similar works using MoS2@Cu2O-Au nanoparticles for the detection of alpha-fetoprotein (AFP), a tumor marker to identify adult primary liver cancer (Figure 16). In this work, AuNPs were electrodeposited on GCE which acted as antibody carriers and sensing platforms. Further, MoS2@Cu2O was combined with the AuNPs as a strategy to obtain the signal amplification resulting in a composite MoS2- Cu2O-Au as a triamplification electrochemical signal. A sandwich immunosensor was developed by immobilizing primary antibodies on Au-deposited GCE and blocked with a surface with BSA for nonspecific bindings. Then, the electrodes were dipped with different concentrations of AFP. Subsequently, the HRP-labeled secondary antibodies coupled with MoS2@Cu2O were then allowed to conjugate with the electrode. Amperometric response, under suitable experimental conditions, exhibited that the sensor possessed a linear range of 0.1 pg/mL to 50 ng/mL and a detection limit of 0.037 pg/mL (S/N = 3). The sensor showed satisfactory recoveries when tested in human serum samples, and the proposed approach could extend the potential application of electrochemical immunosensors to medical applications.

**Figure 15.** The schematic diagram for the stepwise modification of the GCE with MoS2 and Au nanoparticles for anti-CEA antibody immobilization for developing a CEA detection sensor. Reprinted with permission from Ref. [81]. Copyright 2019, Elsevier.

**Figure 16.** The schematic diagram for the preparation procedure for the sandwich-type electrochemical immunosensor. Reprinted with permission from Ref. [82]. Copyright 2019, Elsevier.

Likewise, several reports demonstrated the usage of a MoS2-Au nanocomposite for the detection of electrochemical biosensors for various types of biomarker detection in clinical applications. However, very few reports show the possibility of point-of-care applications. Here, we analyzed the analytical parameters of the reports that adopt the MoS2-Au nanocomposite used for electrochemical sensors and presented them in the following Table 2.


**Table 2.** Literature reports on the analytical parameters of MoS2 conjugated nanoparticles for various biomarker detections.

#### *2.3. Biomarker Detection on MXenes Conjugated with Metal Nanoparticles*

MXenes are transition-metal carbides/nitrides/carbonitrides with a 2D structure and general formula Mn+1XnTx (n = 1–3), where M is an early transition metal, X can be carbon or nitrogen, and Tx corresponds to the surface terminations (Figure 17A,B). The ideal electronic structure [95], structural stability [96], high surface-to-volume ratios [97], outstanding mechanical [98] and optical properties [99], versatile surface chemistries [100], tunable bandgap [101], and high thermal and chemical stability [102,103] make them promising materials for biomarker detection (Table 3). The initial synthesis approach for MXenes was realized based on the etching of Ti3AlC2 with 50% HF for 2 h at room temperature [104]. Later many environmentally friendly approaches were formulated [105] (Figure 17C). However, similar to any other pristine 2D materials, MXenes suffer from poor selectivity, low sensitivity, and slow response [106]. These disadvantages were usually overcome by synthesizing MXene-metal nanoparticle nanocomposites. MXene-metal nanoparticle nanocomposites possess a large specific surface area, superior electron conductivity, and enhanced electron transfer properties for biosensing applications [107]. To expand beyond the limitations of MXenes, Liu et al. [108] reported the covalent grafting of PAMAM onto MXene (MXene@PAMAM) (Figure 18A). Here, the PAMAM acted as an efficient stabilizer and spacer, thereby preventing the restacking and oxidation of the MXene. Moreover, the aminoterminals of PAMAM acted as adsorption sites for AuNPs. The AuNPs@MXene@PAMAM nanobiosensing platform was applied for the detection of the cardiovascular disease biomarker cTnT. The sensor performance was remarkable with a wide detection range (0.1–1000 ng/mL) and a very low detection limit (0.069 ng/mL). Medetalibeyoglu et al. [109] fabricated a d-Ti3C2TX MXene@AuNPs/Ab2 bioconjugate-based sandwich-type electrochemical immunosensor for the detection of PSA. Here, AuNPs at the bioconjugate were used to label PSA secondary antibody-2 for signal amplification (Figure 18B). In one study, Laochai et al. [110] fabricated thread-based L-Cys/AuNPs/MXene working electrodes for the noninvasive electrochemical detection of sweat cortisol, which is an important biomarker for identifying adrenal gland disorders (Figure 18C). Here, MXene served as a 2D platform to anchor the monoclonal anticortisol antibodies, whereas AuNPs increased the specific surface area, and thereby the sensitivity of the detection system. Mesoporous nanoparticles (MNPs), comprising metallic and nonmetallic counterparts, show better catalytic performance compared to their bulk nanoparticles [111]. Liu et al. [112] reported sandwich-type PdPtBP MNPs/MXene-based immunosensor for the ultrasensitive detection of urine kidney injury molecule-1(KIM-1) (Figure 18D). Yang et al. [113] reported an interesting cascaded signal amplification strategy on in situ reduced gold nanoparticle deposited Ti3C2 MXene (Figure 18E), where MXene

acted as a stabilizer and reductant. Here, AuNPs with the predominant (111) facet on MXene provided high electrocatalytic activity and were also used as a carrier of the C-DNA and to make DNA hybridization. Mohsen et al. [114] reported Au nanoparticles on Ti3C2 MXene for synergistic signal amplification (Figure 18F). Here, the perfectly distributed Au nanoparticles on the flaky architecture of MXene contributed to the enhanced electrochemical performance and the attomolar detection of multiple micro-RNAs (miRNAs) achieved on an AuNP@MXene/Au electrode. Wang et al. [115] proposed a competitive electrochemical aptasensor for the breast cancer biomarker Mucin1 based on Au nanoparticles decorated Ti3C2 MXene. Here, aptamer binding to the electrode surface was achieved through Au-S bonds by the electrodeposited gold nanoparticles. The electrochemical aptasensor reported a wide linear range (1.0 pM–10 μM) and a low detection limit (0.33 pM) with promising clinical applications. Cheng et al. [116] demonstrated a gold nanoparticle-modified MXenebased sandwich-type immunosensor platform for squamous cell lung cancer cytokeratin fragment antigen 21-1 (CYFRA 21-1).

**Figure 17.** (**A**) Structure of various MXenes with surface terminations. (**B**) Periodic table elements experimentally used for the synthesis of MXenes, and (**C**) Timeline of the various synthesis routes to MXenes. Reproduced with permission from Ref. [117]. Copyright 2021, Wiley.

**Table 3.** Recent literature reports on metal nanoparticles incorporated MXenes for electrochemical biomarker detection.


**Figure 18.** (**A**) Schematic illustration of the fabrication of AuNPs/MXene@PAMAM for the electrochemical detection of cTnT. Reproduced with permission from Ref. [108]. Copyright 2022, Nature. (**B**) Preparation of d-Ti3C2 MXene@AuNPs/Ab2 for the detection of PSA. Reproduced with permission from Ref. [109]. Copyright 2020, Elsevier. (**C**) Fabrication of L-cys/AuNPs/MXene on a thread-based electrochemical biosensor for noninvasive sweat cortisol detection. Reproduced with permission from Ref. [110]. Copyright 2022, Elsevier. (**D**) Fabrication of PdPtBP nanoparticles/MXene-based enzyme-free electrochemical biosensor for the detection of kidney injury molecule-1 (KIM-1). Reproduced with permission from Ref. [111]. Copyright 2021, Elsevier. (**E**) Schematics of the AuNPs-based cascaded signal amplification process for the detection of miRNA-21. Reproduced with permission from Ref. [113]. Copyright 2022, ECS, and (**F**) Schematic diagram based on AuNPs decorated MXene for the multiplex and concurrent detection of miR-21 and miR-141. Reproduced with permission from Ref. [114]. Copyright 2020, Elsevier.

#### *2.4. MOFs Conjugated Metal Nanoparticles for Electrochemical Biomarker Detection*

As an emerging material with exceptional properties, metal-organic frameworks (MOFs) have been studied exceptionally during the past decades. MOFs are porous materials comprising a framework of metal ions or metal-containing clusters and organic ligands [122]. MOFs have been reported to have excellent properties such as a tunable structure [123], large surface area [124], abundant functional groups [125], high porosity [126], good conductivity [127], and thermal stability [128]. MOFs have been traditionally synthesized by hydrothermal/solvothermal methods [129]. The solvothermal method is a general concept where a solvent other than water is used, and the synthesis is usually performed at a temperature above the boiling temperature of the solvent in closed chemical reactors at higher pressures. Moreover, the greater pressure inside the closed reactor results in enhanced salt solubility. The benefits of the solvothermal process allowed researchers to develop reproducible protocols with total control of the long-term synthesis

processes. The solvothermal method has the advantage of higher product yield with improved crystallinity [130]. The hydrothermal/solvothermal method has been optimized for the synthesis of MOFs such as Ni-MOF [131], Co-MOF [131], Fe-MOF [132], Cu-MOF [133], Zn-MOF [134], and mixed-ligand metal-organic frameworks [135]. In recent years, electrochemical synthesis gained attention, and several MOFs such as Cu3(HHTP)2 [136], Mn-DABDC(ES) [137], 2D/3D Zn(II)-MOF hybrid [138], Fe-MIL-101 and Fe-MIL-101-NH2 [139], etc. have been reported for various MOFs' electrocatalytic applications. Electrochemical synthesis has the advantages of mild synthesis conditions, shorter synthesis times, and controllability of morphology and thickness by the applied current/voltage [140]. During electrochemical synthesis, the metal ions enter the solution through the dissolution of the anode and the process is usually continuous with the availability of dissolved linker molecules [141]. Researchers have also developed a variety of other synthesis approaches such as ultrasound and microwave-assisted [142], mechanochemical [143], and sonochemical [144] methods for the synthesis of MOFs with different morphology and applications (Figure 19). As shown in Table 4, modified MOF nanocomposites often outperform unmodified MOF and are often exploited for diverse biosensor applications [145]. MOFs are often decorated with metal nanoparticles in immunosensor applications for anchoring antibodies and enhancing the electrochemical signal. Nanoparticles decorated MOFs with versatile ligands and metal clusters, low cost, and simple operation provide researchers with an adequate 2D platform for biosensing applications. Li et al. [146] fabricated such an interesting immunosensor platform with core-shell Cu2O@Cu-MOF@AuNPs nanostructures for the sensitive detection of CEA (Figure 20A). Here, the sandwich-type electrochemical immunosensor achieved a tripled electrical signal amplification due to the synergistic effect of Cu-MOF, Cu2O, and AuNPs. Nanowires had more surface area to accommodate proteins and were used to fabricate label-free sensors with exceptional performance [147,148]. Li et al. [149] constructed such an ultrasensitive label-free platform for the detection of NMP-22 based on CuAu nanowires decorated Co-MOFs (Figure 20B). The outstanding catalytic capabilities of Co-MOFs/CuAu NWs achieved a highly sensitive immunosensor with a good linear response (0.1 pg/mL–1 ng/mL), with a lower detection limit (33 fg/mL) suitable for the detection of NMP-22 from human urine samples. An immunoprobe based on AuNPs decorated Fe-MOF for the detection of PSA was reported by Feng et al. [150]. In this study, the labeling antibody was immobilized on AuNPs/Fe-MOF, and methylene blue (MB) covered by a thin layer of AuNPs-rGO served to covalently attach the coating antibodies. An amperometric signal at 0.18 V was measured to quantitatively measure PSA from urine samples (Figure 20C). Zhang et al. [27] reported a similar MB-based strategy for the detection of PSA (Figure 20D). Here, the MOF-325 adsorbed and stabilized MB, thereby solving the problem of MB leakage. A similar nanocomposite comprising MOF, rGO, and AuNPs was reported by Mehmandoust et al. [151] for the detection of a GFAP biomarker (Figure 20F). Here, AuNPs were anchored onto zeolitic imidazolate MOFs and were deployed as a recognition element for the detection of GFAP in urine samples. The intrinsic properties of unique nanomaterials are advantageous for specific immunosensor applications. Zhao et al. [152] fabricated an immunosensor for the detection of NMP-22 based on AuNPs and PtNPs decorated MOFs. The nanoparticles decorated MOF sowed an increased surface area to anchor antibodies through Pt-S and Au-N bonding (Figure 20E), and the immunosensor reported a sensitive response towards NMP-22.

**Figure 19.** (**A**) Various literature reported conditions and approaches for the synthesis of MOFs. Reprinted with permission from Ref. [153]. Copyright 2021, Elsevier. (**B**) Structures of porous MOFs reported by several research groups. Reprinted with permission from Ref. [154]. Copyright 2015, Royal Society of Chemistry. (**C**) Various biomedical applications of 2D MOFs. Reprinted with permission from Ref. [155]. Copyright 2022, BMC (Springer).

**Table 4.** Recent literature reports on metal nanoparticles incorporated MOFs for electrochemical biomarker detection.


**Figure 20.** Schematic illustrations of (**A**) Fabrication of core-shell Cu2O@Cu-MOF@AuNPs-based electrochemical immunosensor for CEA detection. Reproduced with permission from Ref. [146]. Copyright 2020 Springer, (**B**) Preparation of Co-MOFs/CuAu NWs based label-free immunosensor for the detection of NMP-22. Reproduced with permission from Ref. [149]. Copyright 2019 Royal society of chemistry, (**C**) Fabrication of Au-MOF-based amperometric immunosensor for the detection of PSA. Reproduced with permission from Ref. [150]. Copyright 2020 Springer, (**D**) Preparation steps of AuNPs decorated MOF235/MB based electrochemical immunosensor for PSA detection. Reproduced with permission from Ref. [28]. Copyright 2021 Elsevier, (**E**) Stepwise assembly of AuNPs-PtNPs-MOFs based electrochemical immunosensor for the detection of NMP-22 in urine samples. Reproduced with permission from Ref. [152]. Copyright 2019 Elsevier, and (**F**) Preparation of GFAP-BSA-Anti-GFAP-Au@ZIF-8@rGO/SPE based electrochemical immunosensor for the detection of GFAP. Reproduced with permission from Ref. [151]. Copyright 2022 ACS.
