Application of Graphene Oxide in Electrochemical Biomarker Detection

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1		KOYAPPAYIL ANEESH	--	2362	2023-01-31 10:21:50	\|
2	format correction	Dean Liu	-29 word(s)	2333	2023-02-01 02:47:34	\|

This entry is adapted from the peer-reviewed paper 10.3390/bios13010091

metal nanoparticles immunosensor MXene MoS2

1. Introduction

The definition of biomarkers has evolved over time, and a broader definition was suggested by the World Health Organization as “a biomarker is any substance, structure, or process that can be measured in the body or its products and influence or predict the incidence of outcome or disease” ^[1]^[2]. More specific definitions such as “a biological molecule found in blood, other body fluids, or tissues that is a sign of a normal or abnormal process, or of a condition or disease and can be tested to see how well the body responds to treatment for a disease or condition” ^[3], and “a characteristic that can be objectively measured and quantitatively evaluated as an indicator of a normal biological and pathological process, or pharmacological responses to a therapeutic intervention” ^[4] were coined by the US National Cancer Institute, and the US National Institutes of Health, respectively. Biomarkers can be biological, chemical, or physical, and are measurable parameters indicative of a specific biological state. The detection of biomarkers is crucial for the diagnosis and treatment of numerous diseases ^[5]. Biomarkers are classified broadly into imaging biomarkers and molecular biomarkers based on their characteristics. Imaging biomarkers are often used in combination with various imaging tools, whereas molecular biomarkers comprise RNA, DNA, and proteins ^[6]. Molecular biomarkers are easily quantifiable from biological samples and can complement clinical characteristics ^[7]^[8]. Another category, known as pharmacodynamic biomarkers, is applied in drug development during dose optimization studies ^[9]. Based on the application, biomarkers are classified into prognostic biomarkers, diagnostic biomarkers, predictive biomarkers, and monitoring biomarkers ^[10]. Prognostic biomarkers help to identify the risk of disease progression in the future ^[11]. Diagnostic biomarkers help physicians to identify a specific disease condition ^[12], and predictive biomarkers predict the responses related to therapeutic interventions ^[11], whereas a monitoring biomarker is usually measured for assessing the status of a medical condition or disease ^[13].

An ideal biomarker sensor must capture the biomarker selectively from the complex biological matrix of interfering molecules. Although nonspecific binding is still a concern, electrochemical detection methods, specifically electrochemical impedance spectroscopy (EIS), allow the selective analysis of biomarker detections by the resistive and/or capacitive changes due to physical and/or biomolecular interactions of the electrode surfaces coated with nanomaterials, DNA, proteins, etc. ^[14]^[15]^[16]. It is one of the basic and widely used approaches to determine the fundamental redox events at the electrode-electrolyte interface. However, evaluations are made by comparing the results of the EIS with cyclic voltammetry (CV) measurements. Also, differential pulse voltammetry (DPV) and square wave voltammetry (SWV) techniques are used in biomarker detection systems for both label and label-free approaches ^[17]^[18]. Among these techniques, CV-based detection sensing is widely reported due to its ability to explain the electrochemical events, such as oxidation-reduction reactions and electron-transfer kinetics occurring at the electrode-electrolyte interface, and the mass transport towards the electrode surface ^[19]^[20]^[21]. The search for advanced functional materials for electrochemical biomarker detection has sparked a research interest in layered 2D materials over the past few years and several novel approaches were reported for the synthesis of various 2D materials and their nanocomposites with exciting immunosensor applications. The interest and demand for 2D materials have increased significantly, and the global market for 2D materials is expected to grow rapidly with a CAGR of 3.9% between 2020 and 2027 and a corresponding increase in valuation from 2.27 billion to 2.86 billion USD ^[22].

2. Graphene Oxide Conjugated with Nanoparticles for Electrochemical Biomarker Detection

Graphene, a single layer (monolayer) of SP² 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.

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 ^[23]^[24]^[25].

Researchers discussed 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 ^[26] with minimal contributions from labeling methods.

Recently, Ranjan et al. ^[27] 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. The researchers 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). 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. ^[28] 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. 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. 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. ^[29] reported on the detection of prostate-specific antigen (PSA) by using a sandwich-type transducer composed of graphene oxide (GO) and gold nanoparticles (AuNPs). Researchers 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. 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. ^[30] 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. 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. ^[31] reported on the use of laser-induced graphene oxide ^[26] as a new-generation electrode in cancer research for the detection of human epidermal growth factor receptor 2 (HER-2). 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. 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. ^[32] 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). 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 ^[33]. 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 ^[34]. 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 ^[35]^[36]^[37]^[38]^[39]. 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 researchers analyze the analytical parameters of those sensors.

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

Sensing Platform	Biomarker	Technique	Linear Range	LOD	Real Sample	Ref.
RGO-NP/ITO	CRP	EIS	1–10,000 ng/mL	0.08 ng/mL	Human serum	^[28]
GO-CoPP	CPEB4	DPV	0.1 pg/mL–10 ng/mL	0.074 pg/mL	Human serum	^[40]
AuNP-RGO/ITO	TNF-α	EIS	1–1000 pg/mL	0.43 pg/mL	Human serum	^[41]
rGO@AgNPs	LA	CV	10–250 μM	0.726 μM	Human serum	^[42]
AgPdNPs/rGO	RAC	LSA	0.01–100 ng/mL	1.52 pg/mL	----	^[43]
	SAL			1.44 pg/mL
	CLB			1.38 pg/mL
MWCNTs-AuNPs/CS-AuNPs/rGO-AuNPs	OTC	DPV	1.00–540 nM	30 pM	----	^[44]
GO-Fe₃O₄-β-CD	MGMT	DPV	0.001–1000 nM	0.0825 pM	Human plasma	^[45]
AuNPs/GQDs/GO/SPCE	miRNA-21	SWV	0.001–1000 pM	0.04 fM	Human serum	^[46]
	miRNA-155			0.33 fM
	miRNA210			0.28 fM
rGO/RhNPs/GE	HER-2-ECD	DPV	10–500 ng/mL	0.667 ng/mL	Human serum	^[47]
AuNPs-rGO/ITO	IL8	DPV	500 fg/mL–4 ng/mL	72.73 pg/mL	----	^[48]
Pd@Au@Pt/rGO	CEA	DPV	12 pg/mL–85 ng/mL	8 pg/mL	Human serum	^[49]
Pd@Au@Pt/rGO	PSA	DPV	3 pg/mL–60 ng/mL	2 pg/mL	Human serum	^[49]
AgNPs/GO/SPCE	PSA	DPV	0.75–100 ng/mL	0.27 ng/mL	Human serum	^[50]
rGO-GNPs-Cr.6/GCE	L-Trp	SWV	0.1–2.5 μM	0.48 μM	Human serum	^[51]
GO/AgNPs/Au	PSA	LSV	5–20,000 pg/mL	0.33 pg/mL	Human serum	^[52]
AuNP/RGO/GCE	CA125	SWV	0.0001–300 U/mL	0.000042 U/mL	Human serum	^[53]
ErGO-SWCNT/AuNPs	HER2	EIS	0.1 pg/mL–1 ng/mL	50 fg/mL	Human serum	^[54]
Au-PtBNPs/CGO/FTO	MUC1	DPV	1 fM–100 nM	0.79 fM	Human serum	^[55]
BNPAu-Fe-rGO/GCE	Acetaminophen	DPV	50–800 nM	0.14 nM	Human urine	^[56]

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Aneesh Koyappayil

Ajay Kumar Yagati

Min-Ho Lee

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1. Introduction

2. Graphene Oxide Conjugated with Nanoparticles for Electrochemical Biomarker Detection

References