Gold nanoparticles decorated reduced graphene oxide nanolabel for voltammetric immunosensing

Abstract

This study describes the development and testing of a simple and novel enzyme‐free nanolabel for the detection and signal amplification in a sandwich immunoassay. Gold nanoparticles decorated reduced graphene oxide (rGOAu) was used as the nanolabel for the quantitative detection of human immunoglobulin G (HIgG). The rGOAu nanolabel was synthesised by one pot chemical reduction of graphene oxide and chloroauric acid using sodium borohydride. The pseudo‐peroxidase behaviour of rGOAu makes the nanolabel unique from other existing labels. The immunosensing platform was fabricated using self‐assembled monolayers of 11‐mercaptoundecanoic acid (11‐MUDA) on a gold disc electrode. The covalent immobilisation of antibody was achieved through the bonding of the carboxyl group of 11‐MUDA and the amino group of the antibody using chemical linkers [1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide] and N ‐hydroxysuccinimide. The fabricated immunosensor exhibited a linear range that included HIgG concentrations of 62.5–500 ng ml⁻¹. The sensor was also used for the testing of HIgG in the blood sample.

1 Introduction

The routine monitoring of protein levels is highly essential to assess the normal condition of the human immune system. The determination of abnormal levels of proteins can be made possible by performing immunochemical reactions. Immunoassays are affinity‐based bioanalytical methods in which the quantification of the analyte depends on the specific interaction between antigen and antibody [1]. The importance and widespread use of these methods in clinical diagnostics are attributed to their high specificity and sensitivity. In general, immunoassays can be grouped into three main categories: direct [2], indirect [3] and sandwich [4] types. Sandwich immunoassay is more advantageous than direct and indirect methods due to its inherent specificity [5]. The assay involves two antibodies, primary and secondary, which bind to different sites on the antigen. Depending on the label of the secondary antibody there are different detection techniques in sandwich immunoassay that includes radioimmunoassay [6], chemiluminescent immunoassay [7], fluorescent immunoassay [8] and enzyme‐linked immunosorbent assay [9]. The principle behind all these techniques is that analyte concentration is proportional to the signal obtained from the label. However, these methods require expensive and sophisticated equipment, time‐consuming procedures and trained technicians. Therefore quantitative determination of low levels of antigen in a user friendly way is a big challenge in the early detection of diseases. Hence electrochemical immunosensors gained much attention for quantitative determination of the very minute concentration of biomarkers due to its high sensitivity, the capability for rapid recognition and ease of access [10].

Most of the electrochemical sandwich immunoassays make use of electroactive enzymes such as horseradish peroxidase (HRP) [11], alkaline phosphatase [12] and glucose oxidase [13] as labels. The major drawback of the enzyme‐based label system is slow electron transfer, pH and temperature dependent. Recently, enzyme‐free nanolabels have been used to overcome these problems. The application of non‐enzymatic redox active nanocomposites that includes doping of redox mediators such as thionin [14], ferrocene [15] and Prussian Blue [16] on graphene nanocomposites, reduced graphene oxide (rGO) decorated with metal nanoparticles such as silver [17], gold [18] and platinum [19]. The main disadvantage of all these methods is the leaching of redox mediators and oxidation of metal nanoparticles. The pseudo‐peroxidase activity of metal nanoparticles based graphene oxide (GO)‐like gold nanoparticle decorated on reduced GO (rGOAu) has been extensively studied [20].

The gold nanoparticles decorated on GO provide a favourable microenvironment for biomolecules immobilisation maintaining their biological activity and facilitate fast electron transfer to the electrode surface [21]. The oxygen‐containing functionalities on the edge and basal planes of rGO give more nucleation sites for the formation of gold nanoparticles and hence more antibodies are physically adsorbed on the label [22]. Multiple signal amplification can be achieved through the use of bioconjugated nanoparticles. Hence, in this work, the pseudo‐peroxidase property of rGOAu was used as an enzyme‐free label for highly sensitive detection of antigen in an electrochemical immunosensor.

The immobilisation of antibodies is one of the crucial steps in developing a reliable immunosensor. Electrode modification based on self‐assembled monolayers (SAMs) of organic thiols has attracted attention in the immunosensor fabrication due to its well‐ordered and closely packed structure [23]. The adsorption tendency of thiols on the gold surface is more when compared to other planar metal substrates such as silver and platinum [24]. Hence, the SAM‐modified gold substrate was taken as an effective and stable platform for antibody immobilisation. Reports show that in most of the electrochemical immunosensors long chain alkanethiol, 11‐mercaptoundecanoic acid (11‐MUDA) was used to covalently bind antibodies on the electrode surface [25].

In this work, we used human immunoglobulin G (HIgG) as a model analyte. Here, we report a simple approach to HIgG immunosensing using a non‐enzymatic nanolabel. The anti‐HIgG conjugated rGOAu was used as the nanolabel and its pseudo‐peroxidase property enabled the quantitative determination of HIgG over a wide range of concentration. The rGOAu was prepared by simple one pot synthesis using a common reducing agent, sodium borohydride (NaBH₄). The AuNP on the rGO offered a fast electron transfer rate of catalytic reaction of H₂ O₂ by the pseudo‐peroxidase activity of rGOAu in the presence of thionin. The MUDA‐based SAMs formed on the Au surface was used as a platform for the immobilisation of anti‐HIgG.

2 Experimental

2.1 Apparatus

All electrochemical measurements were carried out using a CHI 6088D electrochemical workstation (CH Instruments, Texas, USA) with a three‐electrode cell. A gold disc electrode was used as the working electrode, a platinum wire as a counter electrode and a saturated calomel electrode as a reference electrode. The surface morphology of the nanolabel was determined by high‐resolution transmission electron microscopy (HRTEM; JEOL, JEM‐2100). Ultraviolet (UV)–Visible spectra were recorded using a biospectrometer (Eppendorf, Germany), purification of the nanolabel was done using a Centrifuge 5810 R (Eppendorf, Germany) and ultrasonication was done using an ultrasonic cleaner DSA100 SK2 having a frequency of 400 kHz and power of 100 W (Fuzhou Desen Precision Instruments, China).

2.2 Reagents

Graphite powder, HIgG, polyclonal anti‐HIgG produced in rabbit, HRP‐labelled polyclonal anti‐HIgG produced in rabbit, bovine serum albumin (BSA), 11‐MUDA, 1‐ethyl‐3‐(3‐dimethyl aminopropyl)carbodiimide (EDC), N ‐hydroxysuccinimide (NHS), thionin acetate salt, and hexaammineruthenium(III) chloride (Ru(NH₃)₆ Cl₃) were purchased from Sigma Aldrich. All other reagents such as ethanol, sulphuric acid (H₂ SO₄), sodium borohydride, chloroauric acid (HAuCl₄), sodium borohydride (NaBH₄), potassium permanganate (KMnO₄), sodium hydroxide, hydrochloric acid, sodium chloride, and potassium chloride were purchased from FINAR chemicals, India and used without further purification. Disodium hydrogen phosphate and potassium dihydrogen phosphate were of analytical grade and supplied by LOBA chemicals, India. Hydrogen peroxide (30%) was obtained from Merck. All stock solutions were prepared with Millipore water (18.2 MΩ cm, Millipore, Germany). Antibody and antigen solutions were serially diluted in 0.01 M phosphate buffer saline (PBS) solution, pH 7.4.

2.3 Synthesis of rGOAu nanocomposite

For the synthesis of rGOAu, graphite oxide was prepared first by modified Hummer's method [26]. The method involves the chemical oxidation of graphite using KMnO₄ in 18 M H₂ SO₄ medium. The pure graphite oxide powder was obtained by several washing until the acidic environment was removed. GO was obtained by exfoliating graphite oxide by ultrasonication for 2 h.

To 5 mg GO, 40 ml of 1% HAuCl₄ was added and ultrasonicated in 50 ml milliQ water for 1 h to form a homogeneous solution. Freshly prepared 5 ml of 15 mM NaBH₄ was added slowly to the aqueous dispersion of the above solution and vigorously stirred for 12 h. The obtained solution was washed thrice using milliQ water and ethanol by centrifugation at 8000 rpm for 10 min. The obtained brown precipitate was dried at 60°C overnight [27].

2.4 Conjugation of anti‐HIgG on rGOAu nanocomposite

20 µl of 0.5 mg ml⁻¹ anti‐HIgG was added to 0.2 ml of 1% (w/w) rGOAu suspension. The mixture was continuously stirred for 8 h at 4°C. The reaction mixture was centrifuged three times at 4000 rpm, 15 min, 4°C, and the precipitate was resuspended in a 10% diluted ethanol–water mixture.

2.5 Fabrication of modified electrode

Fig. 1 presents the steps involved in the fabrication of the voltammetric immunosensor. Prior to the experiment, a gold disc electrode (2 mm diameter) was polished with 0.05 µm alumina powder, washed thoroughly and sonicated for 12 s in milliQ water. The electrode was pretreated with 0.1 M H₂ SO₄ by scanning the potential between −0.2 and 1.4 V for ten potential cycles at a scan rate of 100 mV s⁻¹. The SAMs were formed by dipping the Au electrode in an ethanolic solution of 10 mM 11‐MUDA for 1 h at room temperature. After washing with absolute ethanol and distilled water, the MUDA/Au electrode was incubated in 400 mM EDC–NHS solution for 1 h at 4°C. Then the electrode was washed thrice with 0.1 M PBS, pH 7.4, to remove unreacted EDC and NHS molecules. Subsequently, the activated electrode was incubated in 100 µg ml⁻¹ anti‐HIgG (Ab₁) solution for 2 h at 4°C. The antibody‐modified electrode was washed with PBS and blocked by incubating the electrode in 5 µl of 1% BSA solution for 15 min at 4°C. Then the electrode was washed and incubated for 2 h in 5 µl of various concentrations of antigen, HIgG (Ag) solution at 4°C. After the incubation, the modified electrode was washed with PBS and incubated in 5 µl of anti‐HIgG conjugated rGOAu solution (rGOAu‐Ab₁) for 2 h at 4°C. The electrode was washed with PBS. Similarly, another set of electrodes were fabricated without nanolabel and stored at 4°C before use.

Fig. 1.

Schematic representation of the fabricated electrochemical immunosensor for HIgG

2.6 Electrochemical measurements

After every step of fabrication, the electrode was characterised by cyclic voltammetry (CV) by scanning the potential from 0.2 to −0.5 V at a scan rate of 100 mV s⁻¹ and electrochemical impedance spectroscopy (EIS) with frequency ranges from 0.01 to 10⁶ Hz and amplitude 5 mV in 1.5 M KCl solution containing 5 mM Ru(NH₃)₆ Cl₃. The electrochemical measurements on the modified electrodes were carried out using differential pulse voltammetry (DPV) in the potential range from 0.2 to −0.6 V in 0.1 M PBS of pH 7.4 containing 50 µM thionin and 2 mM H₂ O₂. Before the experiment, PBS was thoroughly deaerated with high purity nitrogen for 2 h.

2.7 Real sample analysis

The fabricated electrode was used for the quantitative determination of IgG in human serum. The blood sample was collected from volunteers and centrifuged at 2500 rpm for 15 min at 4°C to separate the serum from blood. The serum was diluted 10⁵ times with 10 mM PBS (pH 7.4) before analysis.

3 Results and discussion

3.1 Spectroscopic characterisation of rGOAu‐anti‐HIgG nanolabel

Fig. 2 a depicts the UV–Visible absorption spectra of GO, rGOAu, and rGOAu‐anti‐HIgG. The peaks obtained at 228 and 304 nm (curve a) are the characteristic peaks of pi–pi excitation and n–pi excitation of carboxyl moieties in GO. Curve b shows the spectrum of rGOAu, the peak observed at 228 nm red shifted to 260 nm due to the extension of conjugation while reduction. The presence of a peak at 304 nm (curve b) indicates the incomplete reduction of GO and the appearance of the new absorption band at 546 nm showed the formation of gold nanoparticles. After conjugation with an antibody, the peak observed at 546 nm red shifted to 559 nm and a decrease in absorbance was observed. This is due to the change in the dielectric environment surrounding gold nanoparticle on the rGO sheet and thus confirms the effective binding of antibody on the gold nanoparticle.

Fig. 2.

UV–Visible spectra of (a) GO, (b) rGOAu, (c) rGOAu‐anti‐HIgG nanolabel

3.2 Morphological characterisation of rGOAu nanocomposite

Figs. 3 a and b show the HRTEM images of the single sheet GO with wrinkles at two different magnifications and Figs. 3 c and d represent reduced GO decorated with dense coverage of AuNPs at the folded edges and wrinkled sites. This clearly proves the formation AuNPs by the in situ reduction of gold ions adsorbed on the basal planes and edges of exfoliated GO dispersions. The oxygen elements on the GO act as a nucleation centre for nanoparticles and stabilise them after growth.

Fig. 3.

HRTEM images of

( a, b ) GO, ( c, d ) rGOAu at different magnifications

3.3 Electrochemical characterisation of the immunosensor

Cyclic voltammograms obtained after each step of the fabrication of the sensor electrode on the gold electrode are shown in Fig. 4 A. Well‐defined redox peaks of ruthenium hexaammine(III) ions were observed at −0.201 and −0.139 V with ΔE _p of 62 mV exhibiting a fast electron transfer on the unmodified Au electrode (curve a). After the MUDA SAM formation on the Au electrode the peak currents were decreased (curve b). This is due to the presence of thiol molecules that hindered the electron transfer of ruthenium ions from the solution to the electrode surface. The dense immobilisation of anti‐HIgG on the MUDA/Au electrode significantly decreased the peak current as shown in curve c. The anodic peak almost disappeared and the cathodic peak potential shifted considerably. The presence of a large number of biomolecules on the electrode surface further blocked the electron transfer of ruthenium ions to the electrode surface and thus there is a drastic reduction in the peak current. The blocking of BSA molecules on the electrode surface resulted in an increase in peak current (curve d), this may be due to the immobilisation of a maximum amount of anti‐HIgG and curve e showed the current response after the addition of HIgG, which was almost similar to that of the response of BSA. The fully fabricated immunosensor with anti‐HIgG conjugated rGOAu (rGOAu‐Ab₁) showed a slight increase in peak current (curve f) and this might be due to the presence of AuNP on the nanolabel that favoured the electron transfer of ruthenium ions.

Fig. 4.

Cyclic voltammograms obtained after each step of the fabrication of the sensor electrode on the gold electrode

(A) CVs at 100 mV s⁻¹ under a potential window of −0.5 to 0.2 V for the different fabrication steps, (a) bare Au electrode, (b) MUDA/Au, (c) anti‐HIgG/MUDA/Au, (d) BSA/anti‐HIgG/MUDA/Au, (e) HIgG/BSA/anti‐HIgG/MUDA/Au and (f) rGOAu‐anti‐HIgG/HIgG/BSA/anti‐HIgG/MUDA/Au, which were measured in 5 mM [Ru(NH₃)₆]Cl₃ and KCl mixture, (B) EIS of each modification step of the electrode in the frequency range of 0.01–10⁶ Hz with an amplitude of 5 mV. Table shows the values of charge transfer resistance obtained for each modification step

EIS has been a widely used technique to further confirm each modification step in the immunosensor (Fig. 4 B inset, The table shows the charge transfer resistance values (R _ct) obtained for each modification on the immunosensor). The R _ct values increased when the proteins bound on the electrode surface and decreased after the binding of the rGOAu‐Ab₁ nanolabel to the immunocomplex formed.

3.4 Studies on the influence of particle size on the catalytic activity of nanolabel

The particle size and surface area are inversely proportional and have a great influence on the catalytic activity of the nanocomposite. Hence, the effect of the particle size of nanoparticle on the catalytic activity of the nanolabel was studied by CV. The rGOAu‐anti‐HIgG/HIgG/BSA/anti‐HIgG/MUDA/Au electrode was fabricated using nanolabel synthesised by two different concentrations of a reducing agent. In Fig. 5, curve a represents the current response of complete immunosensor fabricated using the nanolabel synthesised by 0.5 M NaBH₄ in PBS containing 50 µM thionin. The high concentration of the reducing agent resulted in smaller‐sized nanoparticles as reported in our previous work [28] and hence more surface area obtained. After the addition of 2 mM H₂ O_2, there was only a slight increase in the current response (curve c). This may be due to the more binding of an antibody that decreased the catalytic activity of rGOAu. Curve b shows the current response of the modified electrode with nanolabel synthesised by 15 mM NaBH₄ in the testing medium without H₂ O₂ and curve d shows a significant current increase after the addition of H₂ O₂. The low concentration of the reducing agent resulted in bigger sized particles and therefore less surface area and less number of antibody binding, which retained the catalytic activity of the nanolabel. Therefore nanolabel synthesised using 15 mM NaBH₄ was used for further experiments.

Fig. 5.

Cyclic voltammograms of rGOAu‐anti‐HIgG/HIgG/BSA/anti‐HIgG/MUDA/Au using different concentrations of NaBH₄ 0.5 M (a and c), 15 mM NaBH₄ (b and d) in 0.1 M PBS, pH 7.4, containing 50 µM thionin in the absence (a and b), presence (c and d) of 2 mM H₂ O₂

3.5 Analysis of the pseudo‐peroxidase activity of rGOAu and rGOAu‐anti‐HIgG

Fig. 6 shows the CVs of (Fig. 6A) rGOAu/Au and (Fig. 6B) rGOAu‐anti‐HIgG/Au with increasing concentration of H₂ O₂. An increase in current response was observed on both the modified electrodes by the addition of 1 mM (a), 2 mM (b) and 3 mM (c) H₂ O₂. This clearly shows the pseudo‐peroxidase behaviour of both nanocomposite and nanolabel. The study also confirmed the catalytic activity of rGOAu towards H₂ O₂ still remained even after the conjugation of anti‐HIgG and showed an increased current response in comparison with rGOAu/Au. There was not much current increase between 2 and 3 mM H₂ O₂ on rGOAu‐anti‐HIgG/Au and hence the concentration of H₂ O₂ was fixed at 2 mM.

Fig. 6.

Cyclic voltammograms of

(A) rGOAu/Au, (B) rGOAu‐anti‐HIgG/Au in the absence (a) and presence of varying concentrations of H₂ O₂ – 1 mM (b), 2 mM (c) and 3 mM (d)

3.6 Studies on the selectivity of the nanolabel

This electrochemical study was conducted mainly to understand the selective nature of the nanolabel, rGOAu‐anti‐HIgG on the HIgG/BSA/anti‐HIgG/MUDA/Au modified electrode. The immunosensors were fabricated using 50 µg ml⁻¹ (a, b, d, f) and 100 ng ml⁻¹ (c, e) HIgG concentrations. Fig. 7 A shows the cyclic voltammograms of rGOAu/HIgG/BSA/anti‐HIgG/MUDA/Au in 0.1 M PBS pH 7.4 containing 50 µM thionin without (a) and with 2 mM H₂ O₂ (b, c). An increase in current was observed after the addition of 2 mM H₂ O₂ and this might be due to the presence of physically adsorbed rGOAu. However, there was no significant difference observed in the current responses between the two antigen concentrations. In Fig. 7 B the response of rGOAu‐anti‐HIgG on HIgG/BSA/anti‐HIgG/MUDA/Au in the absence (d) and presence of H₂ O₂ (e, f) was studied. Here also the current increased after the addition of H₂ O₂, but there was a distinguishable difference in the responses of two different antigen concentration modified electrodes. This clearly proved the highly selective behaviour of the antibody labelled nanolabel towards the antigen on the electrode surface. Thus, from this study, it is understood that rGOAu‐anti‐HIgG specifically binds to the immunocomplex formed on the electrode and responded to varying concentrations of HIgG.

Fig. 7.

Cyclic voltammograms on the modified electrodes in 0.1 M PBS pH 7.4 containing 50 µM thionin,

(A) rGOAu/HIgG/BSA/anti‐HIgG/MUDA/Au, (B) rGOAu‐anti‐HIgG/HIgG/BSA/anti‐HIgG/MUDA/Au in the absence (a, d) and presence (b, c, e, f) of 2 mM H₂ O₂. The resulting immunosensors were fabricated using 50 µg ml⁻¹ (a, b, d, f) and 100 ng ml⁻¹ (c, e) of HIgG

3.7 Examining the role of the nanolabel in signal amplification

The signal amplification performance of the fabricated immunosensor using rGOAu‐anti‐HIgG as the nanolabel was studied using CV in 0.1 M PBS, pH 7.4, containing 50 µM thionin and 2 mM H₂ O₂. In Fig. 8 A, the CV responses of the modified electrode with (b) and without (a) nanolabel in the presence of H₂ O₂ are shown. The immunosensor with nanolabel showed a higher current response when compared to the one with the enzyme label. This is because of the enhanced catalytic activity of the nanolabel with a large number of enzyme‐free catalytic centres instead of horseradish peroxide (HRP) enzyme. Fig. 8 B shows the CVs of rGOAu‐anti‐HIgG/HIgG/BSA/anti‐HIgG/MUDA/Au in the absence (a) and presence of H₂ O₂ (b and c). The reduction peak current was significantly enhanced for higher HIgG concentration, (50 µg ml⁻¹ (c)) and current response decreased for lower concentration (100 ng ml⁻¹ (b)). This proves that the synthesised nanolabel forms a sandwich immunocomplex and hence the fabricated immunosensor responds to different antigen concentrations. The synergistic effect of rGOAu‐anti‐HIgG, thionin, and H₂ O₂ resulted in an amplified current response and explained in (1)

$$\mathrm{rGOAu}+{\mathrm{H}}_2{\mathrm{O}}_2\to \mathrm{rGOAu}+{\mathrm{H}}_2\mathrm{O},$$ (1)

$$\mathrm{rGOAu}+\mathrm{Thioni}{\mathrm{n}}_{(\mathrm{Red})}\to \mathrm{rGOAu}+\mathrm{Thioni}{\mathrm{n}}_{(\mathrm{Oxi})},$$ (2)

$$\mathrm{Thioni}{\mathrm{n}}_{(\mathrm{Oxi})}+2{\mathrm{H}}^{+}+2{\mathrm{e}}^{-}\to \mathrm{Thioni}{\mathrm{n}}_{(\mathrm{Red})}.$$ (3)

Fig. 8.

Cyclic voltammograms of

(A) Modified electrodes (a) HRP‐labelled anti‐HIgG/HIgG/BSA/anti‐HIgG/MUDA/Au and (b) rGOAu‐anti‐HIgG/HIgG/BSA/anti‐HIgG/MUDA/Au in 0.1 M PBS, pH 7.4, containing 50 µM thionin and 2 mM H₂ O₂, (B) CVs of rGOAu‐anti‐HIgG/HIgG/BSA/anti‐HIgG/MUDA/Au in the absence (a) and presence of 2 mM H₂ O₂ (b and c). The resulting immunosensor was incubated with (b) 100 ng ml⁻¹ and (c) 50 µg ml⁻¹ of HIgG

3.8 Analytical performance of the immunosensor

DPV studies were carried out on the immunosensor with varying concentration of antigen and the results obtained are shown in Fig. 9. From the graph, it is observed that a linear relationship exists between peak current and HIgG concentrations (62.5–500 ng ml⁻¹). The calibration plot showed a linear segment for the HIgG concentrations with a correlation coefficient, R ²  = 0.9251 and the linear regression equation of y  = 0.0017x  + 0.1239 (inset).

Fig. 9.

DPV responses of the immunosensor to increasing concentrations of HIgG (62.5 ng ml⁻¹ (a), 125 ng ml⁻¹ (b), 250 ng ml⁻¹ (c) and 500 ng ml⁻¹ (d)), the inset shows the calibration plot

3.9 Studies on reproducibility and selectivity of the immunosensor

The reproducibility of the immunosensor was studied by comparing the electrochemical response of three sensors towards HIgG concentration of 500 ng ml⁻¹. The results obtained proved that the fabrication process is highly reproducible with a relative standard deviation of 2%.

The selectivity of the sensor was studied on four electrodes which were fabricated independently. The serum protein HER 2 (15 ng ml⁻¹) and blocking buffer BSA (1%) were taken as interfering molecules and phosphate buffer solution as a blank. From Fig. 10, it is clear that the percentage response obtained for target analyte is much higher in comparison with other proteins indicated high selectivity of the immunosensor towards HIgG concentration of 62.5 ng ml⁻¹.

Fig. 10.

Interference studies of the rGOAu‐anti‐HIgG/HIgG/BSA/anti‐HIgG/MUDA/Au towards other proteins

4 Clinical applications of the immunosensor

The current response obtained for the samples were compared with the calibration plot and the concentration of HIgG in the sample was determined at 15.4 mg ml⁻¹ and for another sample, the concentration obtained was 25 mg ml⁻¹. Clinical analysis of the same blood samples revealed the HIgG concentration to be 12.9 mg ml⁻¹ for sample 1 and 18.3 mg ml⁻¹ for sample 2. Even though the HIgG was used as a model analyte the real sample analysis shows that the fabricated immunosensing platform with non‐enzymatic nanolabel has great potential in clinical applications for the monitoring of cancer biomarkers.

5 Conclusion

A non‐enzymatic sandwich type of electrochemical immunosensing platform was fabricated for the quantitative determination of HIgG which was used as a model analyte. For signal amplification, rGOAu‐anti‐HIgG (nanolabel) was used for the immunosensing application. The sensor with non‐enzymatic nanolabel showed much superior performance compared to that of the HRP‐labelled anti‐HIgG. The developed sensor was capable of detecting HIgG concentrations from 62.5 to 500 ng ml⁻¹. The concentration of HIgG in the blood sample was determined using the fabricated sensor and which was in good agreement with that obtained from clinical laboratory.