**2. Metal Nanoparticles on 2D Materials for Biomarker Detection**

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<sup>2</sup> 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 cm2/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 × 103–5.30 × 103 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.

**Figure 1.** The schematic diagram for the synthesis of graphene. Reprinted with permission from Ref. [30]. Copyright 2018, Elsevier.

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 [31–33] 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, electrochemicalbased 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.

**Figure 2.** (**A**) Schematic diagram shows the synthesis of GO-IL-AuNPs hybrid nanocomposite and (**B**) Stepwise fabrication shows the surface modification procedures for the fabrication of BSA/anti-CD44/GO-IL-AuNPs/GCE Immunosensor. Reprinted with permission from Ref. [35] Copyright 2022, ACS.

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.

**Figure 3.** (**A**) Fabrication of 8-channel Indium-tin oxide electrodeposited with reduced graphene oxide-nanoparticle microdisk electrode array as working electrodes with a shared counter electrode. (**B**) Chemical functionalization of modified ITO electrode with EDC/NHS to couple antibodies for CRP detection in real samples. Reprinted with permission from Ref. [36]. Copyright 2016, Elsevier.

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.

**Figure 4.** (**A**) Procedures for the fabrication of Go/GNP/Ab. (**B**) Procedure for preparing the electrochemical sensor. (**C**) Schematic illustration of the novel electrochemical sensor for PSA marker detection. Reprinted with permission from Ref. [37]. Copyright 2019, Wiley.

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.

**Figure 5.** Schematic representation of the (**A**) Synthesis of rGO/Au nanocomposite, (**B**) Fabrication of rGO/Au nanocomposite-based miRNA-122 electrochemical detection platform. Reprinted with permission from Ref. [38]. Copyright 2021, Elsevier.

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 oxidechitosan/pencil graphite electrode (GO-chit/PGE). Later, the electrodeposition of Au-Pt bimetallic nanoparticles (Au-Pt BNPs) was accomplished on the surface of the GOchit/PGE-modified electrode. Subsequently, DNA was immobilized on the Au-Pt BNPs/GOchit/PGE, applying a constant potential of 0.5 V.

**Figure 6.** The schematic diagram for the formation of laser-induced graphene (LIG) electrode sensor. (**A**) LIG electrode on polyimide sheet, (**B**) Formation of Au nanostructures on working electrode area with electrodeposition, inset shows the SEM images of the tree-like structure of Au. (**C**) Bindings of DNA aptamer on the electrode through self-assembly of mecaptohexanol (MCH), (**D**) Surface blocking procedures with BSA and measurement of electrochemical signal with [Fe(CN)6] <sup>3</sup>−/4<sup>−</sup> redox probe, (**E**) Incubation with the HER-2 antigen and measurement of EC signal, and (**F**) Quantification of HER-2 by evaluating the electrochemical signal. Reprinted with permission from Ref. [39]. Copyright 2021, Elsevier.

**Figure 7.** Schematic route for the fabrication of DNA/Au−Pt BNPs/GO−chit/PGE transducer surface for the development of an electrochemical biosensor for the detection of ZDV. Reprinted with permission from Ref. [40]. Copyright 2021, Elsevier.

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 [43–47]. 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.

**Table 1.** Literature reports on the analytical parameters of graphene oxide conjugated nanoparticles for various biomarker detection.

