Nanoparticles used separately or in conjugation with other nanomaterials on 2D materials fulfill various roles in the design and development of electrochemical immunosensors. Also, they improve the analytical characteristics of the developed sensors such as linear range, LOD, and sensitivity [
23]. For instance, nanoparticles deposited on the surface of the working electrode result in an enhancement of the surface area, thereby leading to an increased molecule loading capacity [
24,
25]. Additionally, the unique properties of nanoparticles could enhance the signal for the sensitive determination of biomarkers [
23]. Also, the high electrical conductivity of metal nanoparticles at the electrode surface accelerates the redox electron transfer process. In some cases, nanoparticles could act as platforms for anchoring antibodies [
26]. Metal nanoparticles were also used as a transport medium to capture the analyte from the sample, thereby concentrating the analyte molecules towards the electrode surface to improve the analytical signal [
27]. Among various metal nanoparticles, AuNPs were extensively used to immobilize antibodies on the electrode surface to effectively amplify the immunosensor signal, anchor antibodies, and improve electrocatalytic activity [
28,
29].
2.1. Graphene Oxide Conjugated with Nanoparticles for Electrochemical Biomarker Detection
Graphene, a single layer (monolayer) of SP
2 carbon atoms with a molecular bond length of 0.142 nm, is tightly bound in a hexagonal honeycomb lattice. It is basically extracted from graphite and is merely a sheet of graphite. Graphene possesses excellent electrical conductivity (200,000 cm
2/Vs) due to its bonding and antibonding of pi orbitals, with the strongest compound around 100–130 times stronger than steel with a tensile strength of 130 GPa and a Young’s Modulus of 1 TPa-150,000,000 psi. It is also one of the best conductors of heat at room temperature (at (4.84 × 10
3–5.30 × 10
3 W/mK). As graphene is a subunit of graphite it can be synthesized by direct extraction from bulk graphite. From the high-quality sample of graphite, graphene can be extracted by micromechanical cleavage or the scotch tape method of production. It is a straightforward method that doesn’t need any specialized equipment. A piece of adhesive tape is placed onto and then peeled off the surface of a sample of graphite, resulting in a single to few layers of graphene. Other methods include the dispersion of graphite, exfoliation of graphite oxide, epitaxial growth, and chemical vapor deposition (CVD) as shown in
Figure 1.
Graphene oxide is a form of graphene that includes oxygen functional groups and possesses interesting properties that are different from graphene. By reducing graphene oxide, these functional groups can be removed resulting in reduced graphene oxide. The production of reduced graphene oxide can be done in (i) chemical reduction, (ii) Thermal reduction; (iii) microwave and photoreduction; (iv) photocatalyst reduction; (v) solvothermal/hydrothermal reduction. The detailed information for various synthesis routes can be found elsewhere [
31,
32,
33] and is beyond the scope of this review.
In this section, we discuss the development of various types of electrochemical sensors based on graphene oxide conjugated with nanoparticles that have been reported recently for various types of biomarkers. The development of biosensors that accurately measure the desired biomarker at high sensitivity and selectivity is crucial. However, sensitivity and selectivity are the two main factors that limit accuracy when performing the detections at the point of care with meager volumes of biological test solutions. For cancer cell analysis, the sensors should be able to detect tumors within the range of 100–1000 cell counts. To overcome these difficulties, innovative biosensor approaches with the optical, electrochemical, and piezoelectric transducer occupy the place of benchtop protocols adopted by the classical detection methods. Among these biosensors, electrochemical-based approaches competed with optical sensors which are widely used for the analysis of cancer biomarkers due to the characteristics of high sensitivity, selectivity, fast response, ease of use, low cost, and minimal fabrication procedures. In electrochemical biosensors, the right choice of transducer material is crucial, since it is the transducer that mainly influences the overall sensitivity [
34] with minimal contributions from labeling methods.
Recently, Ranjan et al. [
35] reported on the detection of breast cancer CD44 biomarkers using a gold-graphene oxide nanocomposite with ionic liquid with differential pulse voltammetry and electrochemical impedance spectroscopy. In this work, the authors reported the synthesis of RGO, ionic liquid (IL), and Au nanoparticles (Au NPs) by the citrate reduction method and other chemical procedures to form a nanocomposite on a glassy carbon electrode (GCE), as shown in
Figure 2. In this work, the addition of 1-butyl-3-methylimidazolium tetrafluoroborate, an ionic liquid in conjugation with Au nanoparticles enabled the enhancement in the overall sensitivity of the developed sensor. Once the nanocomposite is deposited on GCE, the surface is activated with EDC/NHS to covalently bind the anti-CD44 antibodies. After the surface is blocked with BSA for nonspecific binding, then different concentrations of CD44 antigen were allowed for electrochemical investigation with CV, DPV, and EIS. The sensor possessed a linear range of 5 fg/mL to 50 µg/mL with a LOD of 2.7 fg/mL and 2.0 fg/mL in serum and PBS samples, respectively. This sensor is a promising candidate for the onsite detection of CD44 in breast cancer patients.
In another study, Yagati et al. [
36] proposed indium tin oxide (ITO)-based electrodes modified with reduced graphene oxide-gold nanoparticles that were used for the electrochemical impedance sensing of the C-reactive protein in serum samples. This biomarker detection is crucial in analyzing the inflammation due to an infection, and the risk of heart disease. In this study, graphene oxide-Au nanoparticles were electrodeposited on ITO microdisk electrodes fabricated using standard photolithography techniques. Subsequently, the modified electrodes were coated with a self-assembled monolayer of 3-MPA and activated with EDC/NHS. After the surface-blocking protocol was performed, then the selective antibodies were immobilized on the rGO-NP surface. Once the transducer surface is ready, a different concentration of CRP in human serum (1: 200) was detected with the help of impedance spectroscopy (
Figure 3). The key feature of this sensor is that by forming the nanohybrid materials (RGO-NP hybrid) on the electrode, it results in an enhanced sensitivity toward CRP detection. The linear range of the sensor is 1–1000 ng/mL with an LOD of 0.08 ng/mL in serum samples. Based on the findings, it has the feasibility to employ multiplexed assay detection of biomarkers for point-of-care applications.
Jonous et al. [
37] reported on the detection of prostate-specific antigen (PSA) by using a sandwich-type transducer composed of graphene oxide (GO) and gold nanoparticles (AuNPs). In this work the authors utilized an 11-mercaptoundecanoic acid for self-assembled monolayer formation on the GO-coated glassy carbon electrode (GCE) and a subsequent modification with EDC/NHS to convert -COOH to -NH for antibody bindings (
Figure 4). After blocking with 1% BSA, different concentrations of PSA were allowed to bind to the electrode and with square wave voltammetry, and the quantification was made. The sensor possessed a limit of detection estimated to be around 0.2 and 0.07 ng/mL for total and free PSA antigens, respectively. The incorporation of AuNPs on GO/GCE enabled double functionality, i.e., specific recognition and signal amplification, for sensitive determination of PSA.
Also, Kasturi et al. [
38] reported on the development of a biosensor for the detection of microRNA-122 (miRNA-122) with AuNPs-decorated reduced graphene oxide (rGO) on the Au electrode surface (
Figure 5). The thiol-labeled DNA probes were attached to the Au-rGO transducer surface by forming a SAM layer, with subsequent blocking with 1% BSA. Then, the target miRNA was allowed to bind to the transducer surface to quantify the biomarker for liver diseases.
The sensor possessed a linear range from 10 µM to 10 pM and had a detection limit of 1.73 pM. The sensor possessed good biocompatibility, superior electron transfer characteristics, large surface area, and selective conjugation with biomarkers. Also, the sensor design can be applied to construct other types of biomarker detection. Furthermore, it can be integrated with a lab on a chip platform. It is also applicable to the large-scale production of sensors with a focus on the early detection of diseases.
In another interesting work, Rauf et al. [
39] reported on the use of laser-induced graphene oxide [
34] as a new-generation electrode in cancer research for the detection of human epidermal growth factor receptor 2 (HER-2). In this study, with laser printing technology, the structures of working, counter, and reference electrodes were formed on a polyimide sheet, then the gold nanostructures (Christmas-tree-like structures) were formed by electrodeposition on the working electrode (
Figure 6). Subsequently, the sensor surface is modified with thiol labeled HER-2 aptamer and blocked with BSA for any nonspecific bindings. Then, the HER-2 protein was allowed, in different concentrations, to interact with the aptamer immobilized surface. The electrochemical signals were then recorded for the aptamer surface after bindings with different concentrations with [Fe(CN)
6]
3−/4− redox probe. The CV analysis showed a decrease in current upon bindings of various concentrations of HER-2, and from the calibration, the limit of detection was found to be 0.008 ng/mL. It is claimed that with the incorporation of 3D Au nanostructures the sensor possessed a high electron transfer rate, which resulted in achieving a lower LOD and possessing high sensitivity and accuracy in detecting HER-2 in human serum samples. Furthermore, special software was developed to make it a POC device, in which the laboratory aptasensor could be converted into a hand-held aptasensor.
Also, Hasanjani et al. [
40] reported on the development of Zidovudine (ZDV). A modified pencil graphite electrode (PGE) was made using deoxyribonucleic acid/Au-Pt bimetallic nanoparticles/graphene oxide-chitosan (DNA/Au-Pt BNPs/GO-chit/PGE) (
Figure 7). The PGE was immersed in the GO-chit solution to create the graphene oxide-chitosan/pencil graphite electrode (GO-chit/PGE). Later, the electrodeposition of Au-Pt bimetallic nanoparticles (Au-Pt BNPs) was accomplished on the surface of the GO-chit/PGE-modified electrode. Subsequently, DNA was immobilized on the Au-Pt BNPs/GO-chit/PGE, applying a constant potential of 0.5 V.
Using differential pulse voltammetry, the I−V response was recorded for different concentrations of ZDV. The sensor showed a linear dynamic range from 0.01 pM to 10.0 nM, with a detection limit of 0.003 pM in human serum samples.
Recently, Kangavalli and Veerapandian reported on the development of a dengue biomarker using ruthenium bipyridine complex on the surface of graphene oxide [
41]. They also reported on various EC-based techniques for the electrodeposition and electroless deposition procedures of graphene oxide as a nanoarchitecture for a label-free biosensor platform [
42]. Some more information on electrochemical biosensors developed for biomarker detection that contain graphene oxide and metal nanoparticles can be found in some valuable studies recently reported, and are available in the literature [
43,
44,
45,
46,
47]. Graphene oxide-based nanomaterials offer a wide range of possibilities for developing sensitive electrochemical biosensors for biomarker detection. In recent years, significant advances in graphene-nanoparticle-based electrochemical sensors are made for the detection of cancer biomarkers, and here we analyze the analytical parameters of those sensors, as shown in
Table 1.
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 MX
2. 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, MoS
2 is commonly used because its fundamental constituents are surplus and innoxious [
66]. MoS
2 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 MoS
2 are held by weak van der Waals forces. MoS
2 possesses a mobility of 200 cm
2/Vs at room temperature, high on/off current ratio of 10
8, and a direct band gap of 1.8 eV. Based on these properties, MoS
2 becomes a promising alternative to graphene and is applied in various electrochemical and optical sensors [
67,
68,
69]. MoS
2 can be synthesized in both top-down and bottom-up approaches (
Figure 8). The top-down approach includes the exfoliation of MoS
2 [
70], while the bottom-up approaches include (i) chemical vapor deposition [
71]; (ii) physical vapor deposition [
72]; (iii) solution-based processing [
73]. For a more detailed synthesis of MoS
2, readers are encouraged to go through the literature survey of the desired synthesis approach. Thus, like graphene, MoS
2 offers a large surface area that enhances its biosensing performance.
MoS
2 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 MoS
2 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 MoS
2 for the detection of various biomarkers.
In a recent report that mentions the usage of MoS
2-Au nanoparticles, Yagati et al. [
77] reported on the applications of MoS
2 conjugated Au nanoparticles on indium tin oxide (ITO) electrodes for the detection of the thyroid-stimulating hormone biomarker, triiodothyronine (T
3), as shown in
Figure 10. Electrodeposition procedures allowed the formation of MoS
2 and Au nanostructures on the ITO electrode. Subsequently, T
3 antibodies were immobilized on the MoS
2-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 T
3 biomarker diluted in both PBS and serum samples. Electrochemical impedance spectroscopy was used to analyze the bindings of T
3 to its antibodies and a linear correlation was observed for different concentrations. Based on the quantifications made by this sensor for the detection of T
3, 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 MoS
2 (AuNPs–MoS
2) 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-MoS
2 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–MoS
2/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.
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 MoS
2-based multiplexed aptasensor could also serve as an “AND” gate.
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 graphene-supported gold nanorods (NG/AuNPs) as a substrate platform, and the durian-shaped MoS
2/AuPtPd nanodendrite (NDs) as a label for secondary antibodies (Ab
2) 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 MoS
2/AuPtPd-Ab
2 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”.
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 (CuWO
4@MoS
2) 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 MoS
2-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 (H
2O
2). As can be seen in
Figure 15, the MoS
2-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.
Also, Ma et al. [
82] reported similar works using MoS
2@Cu
2O-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, MoS
2@Cu
2O was combined with the AuNPs as a strategy to obtain the signal amplification resulting in a composite MoS
2-Cu
2O-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 MoS
2@Cu
2O 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.
Likewise, several reports demonstrated the usage of a MoS
2-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 MoS
2-Au nanocomposite used for electrochemical sensors and presented them in the following
Table 2.
2.3. Biomarker Detection on MXenes Conjugated with Metal Nanoparticles
MXenes are transition-metal carbides/nitrides/carbonitrides with a 2D structure and general formula M
n + 1X
nT
x (n = 1–3), where M is an early transition metal, X can be carbon or nitrogen, and T
x 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 Ti
3AlC
2 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-Ti
3C
2T
X 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 Ti
3C
2 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 Ti
3C
2 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 Ti
3C
2 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 MXene-based sandwich-type immunosensor platform for squamous cell lung cancer cytokeratin fragment antigen 21-1 (CYFRA 21-1).
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 Cu
3(HHTP)
2 [
136], Mn-DABDC(ES) [
137], 2D/3D Zn(II)-MOF hybrid [
138], Fe-MIL-101 and Fe-MIL-101-NH
2 [
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 Cu
2O@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, Cu
2O, 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.
2.5. Biomarker Detection on Other 2D Materials Conjugated with Metal Nanoparticles
2D materials such as graphitic carbon nitride, black phosphorous, 2D layered double hydroxides (LDHs), boron nitrides, graphdiyne, etc. have also been explored in conjunction with metal nanoparticles for immunosensor applications with interesting biomarker targets (
Figure 21,
Table 5). Graphdiyne, the new 2D carbon allotrope with its unique sp-sp2 carbon network and highly π-conjugated structure has been receiving increased attention [
163]. A graphdiyne-based self-powered biosensor platform was constructed by Hou et al. [
164] for the determination of miRNA-21. Here, both the cathode and bioanode were fabricated by different modifications of AuNPs/GDY (
Figure 21A). The 2D hexagonal boron nitride nanosheets, due to their electronic conductivity and large surface area were explored for immunosensor applications [
165]. A label-free aptasensor for the detection of cardiac biomarker myoglobin on AuNPs decorated 2D-Boron nitride nanosheets was reported by Adeel et al. [
166]. Here, the boron nitride nanosheets modified electrode AuNPs/BNNSs/FTO acted as a transducer for the immobilization of thiol-functionalized DNA aptamer for the specific binding of myoglobin (
Figure 21B). Carbon nitrides are polymeric materials mainly consisting of carbon and nitrogen [
167,
168]. At ambient temperature, graphitic carbon nitride (g-C
3N
4) is the most stable allotrope of carbon nitrides. Due to the presence of basic surface groups and rich surface properties, g-C
3N
4 is attractive for many applications including catalysis [
169]. Neto et al. [
170] fabricated a miniaturized PEC system based on AuNPs decorated g-C
3N
4 for the detection of the breast cancer biomarker CA15-3 (
Figure 21C). In this work, AuNPs on the g-C
3N
4 platform acted as a linker to 11-mercaptoundecanoic acid for the effective adsorption of antibodies. The performance of the PEC sensor was remarkable with a long linear range (0.1 fg/mL–10 ng/mL) and a very low detection limit (0.04 fg/mL). One of the promising candidates for immunosensor applications is 2D-Black phosphorus (BP) with high carrier mobility and controllable bandgap [
171]. The unique properties of BP at atomic thickness are valuable for diverse applications [
172,
173,
174]. Li et al. [
175] reported a 2D-black phosphorous-supported Pt-Pd nanoelectrocatalyst for the determination of 4-AP, a potent biomarker for aniline exposure. Layered double hydroxides (LDHs) received attention because of their tunable chemistry and high charge density [
176]. In one study, an electrochemical immunosensor based on AuNPs decorated ferrocene carboxylic acid conjugated MgAl layered double hydroxides for the label-free detection of CA-125 was reported by Wu et al. [
177]. In this work, an LBL approach was used to increase the number of ferrocenes and antibodies, thereby amplifying the signal. The sensor reportedly displayed a wide linear range (0.01–1000 U/mL) and LOD (0.004 U/mL) and was tested for clinical cancer diagnostics (
Figure 21D).