Impedimetric thrombin aptasensor based on chemically modified graphenes

Abstract

Highly sensitive biosensors are of high importance to the biomedical field. Graphene represents a promising transducing platform for construction of biosensors. Here for the first time we compare the biosensing performance of a wide set of graphenes prepared by different methods. In this work, we present a simple and label-free electrochemical impedimetric aptasensor for thrombin based on chemically modified graphene (CMG) platforms such as graphite oxide (GPO), graphene oxide (GO), thermally reduced graphene oxide (TR-GO) and electrochemically reduced graphene oxide (ER-GO). Disposable screen-printed electrodes were first modified with chemically modified graphene (CMG) materials and used to immobilize a DNA aptamer which is specific to thrombin. The basis of detection relies on the changes in impedance spectra of redox probe after the binding of thrombin to the aptamer. It was discovered that graphene oxide (GO) is the most suitable material to be used as compared to the other three CMG materials. Furthermore, the optimum concentration of aptamer to be immobilized onto the modified electrode surface was determined to be 10 μM and the linear detection range of thrombin was 10–50 nM. Lastly, the aptasensor was found to demonstrate selectivity for thrombin. Such simply fabricated graphene oxide aptasensor shows high promise for clinical diagnosis of biomarkers and point-of-care analysis.

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Introduction

In recent years, biosensors have attracted much attention due to their potential roles in clinical diagnosis, genetic screening, food quality analysis and environmental monitoring. Currently, the potential application of biosensors in clinical diagnosis for the detection and quantification of proteins is one of the most popular research areas.^1–3

Thrombin is a specific serine protease involved in the coagulation cascade and it catalyses the conversion of soluble fibrinogen to insoluble fibrin.⁴ In addition, it also regulates many processes in inflammation and tissue repair at the blood vessel wall.⁵ It is usually produced in blood and tissues when physiological and pathological blood coagulation happen.⁶ Therefore, thrombin plays a vital role in a number of pathological conditions. Under ordinary health conditions, the concentration of thrombin present in blood during coagulation process ranges from nM to low μM levels.⁷ Hence, it is of utmost relevance to develop a biosensor for the detection of thrombin with high sensitivity, selectivity and that would be easy to use.

Aptamers are synthetic single-stranded oligonucleotides which are in vitro selected through SELEX (systematic evolution of ligands by exponential enrichment) to bind to specific target biomolecules.^8,9 They have many advantages such as high specificity, low molecular weight, simple structure, easy synthesis and long-term stability. As such, aptamers have great potential for the applications in clinical diagnosis, genetic screening, food quality analysis and environmental monitoring^10,11 and concerted efforts have been put into developing aptasensors for thrombin based on several electrochemical techniques such as electrochemiluminescence (ECL),¹²differential pulse voltammetry (DPV),¹³electrochemical impedance spectroscopy (EIS)^14–16 and ultraviolet-visible spectroscopy.¹⁷

In recent years, electrochemical impedance spectroscopy (EIS) has been perceived to be one of the most promising tools for interfacial investigation.^18–21EIS is capable of measuring the response of an electrochemical system to an applied oscillating potential as a function of the frequency.^22,23 In addition, impedance is also a useful method for the detection of protein binding process on the surface of an electrode. In fact, when the high molecular weight/charge density protein binds, the impact on the electron transfer kinetics between the redox probe and the electrode surface is significant.^15,24 Furthermore, EIS is also simple, economical and requires no additional modification on the biomolecules involved in the sensing protocol. In view of all these advantages, it is of no wonder that several impedimetric aptasensors for thrombin have been reported in recent years.^15,24,25 However, few reports have been made on aptasensors which are able to fulfil the desired requirements of high sensitivity, selectivity and simplicity.

Graphene is considered a highly promising material for biosensing.²⁶ Research on graphene materials has underwent tremendous growth over the past few years.²⁷ This is attributed to the expectations that the remarkable electronic, mechanical and thermal properties of graphene will lead to technological breakthroughs. For application in electrochemical devices, such as aptasensors, graphene possesses advantageous properties such as high electron conductivity,²⁸ fast heterogeneous electron transfer (HET) rate at the edges and basal plane defects of its sheets in the order of ∼0.01 cm s⁻¹, high surface area,²⁹ low cost and simple synthetic routes.³⁰

On that note, we wish to evaluate here for the first time the performance of various chemically modified graphene materials (CMG; according to Ruoff et al.³¹ this class of graphene materials include graphite oxide and various reduced graphene oxides) for impedimetric aptasensing of thrombin. The aim of this work is to develop an impedimetric thrombin aptasensor which is capable of fulfilling the desired requirements. We employ CMGs-based screen printed disposable electrodes which hold promise for point-of-care testing.

Experimental section

Materials

Thrombin from human plasma (THR), thrombin aptamer (THR-APT) (5′-TTT TTT TTT TTT TTT GGT TGG TGT GGT TGG-3′), immunoglobulin G (IgG) from rabbit serum, albumin from bovine serum (BSA), avidin, graphite microparticles (<20 μm), sodium borohydride, fuming nitric acid (conc. > 90%), sulphuric acid (conc. 95–98%), potassium chlorate, hydrochloric acid (conc. 37%), sodium phosphate dibasic, sodium chloride, tris(hydroxymethyl)aminomethane, potassium hexacyanoferrate (II) trihydrate, potassium hexacyanoferrate (III) were purchased from Sigma-Aldrich, (Singapore). DEP-chips (disposable electrical printed chip) were obtained from BioDevice Technology (Nomi, Japan). Chemically modified graphenes were prepared with detailed characterization according to our previous report.³²

Apparatus

Electrochemical experiments were performed with an Autolab potentiostat PGSTAT302 (Eco Chemie, Utrecht, The Netherlands) connected to a personal computer and controlled by GPES and FRA software version 4.9.

Impedance measurements were recorded between 0.1 MHz and 0.1 Hz at a sinusoidal voltage perturbation of 10 mV amplitude. The experiments were carried out at room temperature using DEP-chip electrodes and 10 mM K₄[Fe(CN)₆]/K₃[Fe(CN)₆] (1 : 1 molar ratio) as a redox probe in Tris buffer solution (0.025 M NH₂C(CH₂OH)₃ + 0.3 M NaCl, pH = 8.2). Randles equivalent circuit was used to fit the obtained impedance spectra, represented as Nyquist plots in the complex plane.

Protocol

Graphite oxide (GPO), graphene oxide (GO) and thermally reduced graphene oxide (TR-GO), were immobilized onto each DEP-chip surface by physical absorption. 3 μL of chemically modified graphene (CMG) at a concentration of 1 mg mL⁻¹ in milli-Q water was deposited on the electrode surface and left to dry at room temperature overnight. Excess of chemically modified graphene (CMG) material that was not well adsorbed on the electrode surface was then removed by gentle washing with milli-Q water. Prior to depositing the chemically modified graphene (CMG) materials onto electrode surfaces, graphene oxide (GO) and thermally reduced graphene oxide (TR-GO) were sonicated for 15 min in order to ensure maximum dispersion of the materials. To obtain electrochemically reduced graphene oxide (ER-GO) modified electrode, graphene oxide (GO) modified electrode was reduced at −1.2 V for 900 s with 20 μL of PBS buffer solution (pH = 7) at room temperature.

Thrombin aptamer (THR-APT) was next immobilized onto the modified electrode surfaces by dry physical absorption. 3 μL of thrombin aptamer (THR-APT) in PBS buffer solution at a concentration of 10 μM was deposited onto the modified electrode surface for 10 min at a temperature of 60 °C, after which, the electrode was washed in PBS buffer solution with gentle stirring at room temperature to remove the excess aptamer which was not well adsorbed on the modified electrode surface.

DEP-chips modified with thrombin aptamer (THR-APT) were then incubated in Eppendorf tubes with Tris buffer solution containing the desired concentrations of protein target (total volume: 100 μL). The incubation was performed at 37 °C for 60 min with gentle stirring. Two washing steps were then carried out in Tris buffer solution at 37 °C. Negative controls were performed using the above sensing protocol with IgG, BSA and avidin proteins.

Results and discussion

We investigated in this work the suitability of different chemically modified graphene (CMG) surfaces for impedimetric aptasensing of thrombin. We employed chemically modified graphene (CMG) materials consisting of graphite oxide (GPO), graphene oxide (GO), thermally reduced graphene oxide (TR-GO) and electrochemically reduced graphene oxide (ER-GO). Scheme 1 illustrates the ways of preparation of various graphene oxides, also known as CMGs.

Scheme 1 Illustration for the preparation of chemically modified graphene (CMG) materials with graphite as the starting material. (A) Graphite oxide was first generated viaoxidation from graphite. This was followed by: (B) thermal reduction/exfoliation of graphite oxide to yield TR-GO; (C) exfoliation by ultrasonication to produce graphene oxide; (D) electrochemical reduction of graphene oxide to generate ER-GO.

We employed following analytical protocol (see Scheme 2). Briefly, the DEP-chip was modified by CMG material. Such an electrode exhibits relatively low impedance as it is fully accessible by redox probe, [Fe(CN)₆]^3−/4−. Consequently, the aptamer was immobilized³³ and impedance increases due to the electrostatic repulsion between the negatively charged backbone of the aptamer and the redox probe. Moreover, the accessibility to the electrode also decreases. Finally, the electrode was exposed to thrombin which binds specifically to the immobilized aptamer. This results in a decrease in impedance due to the partial removal of the immobilized aptamer from the electrode surface.³⁴ The conformational changes of the aptamer from a coil-like structure to quadruplex structure¹⁶ when binding to the protein can also partially influence the impedance change. Impedance spectra were recorded after each stage for all four chemically modified graphene (CMG) materials; they are shown in Fig. 1.

Scheme 2 Illustration of the protocol of thrombin detection based on EIS method. (A) Bare DEP-chip modified with CMG material; (B) CMG material modified DEP-chip after the immobilization with THR-APT; (C) THR-APT modified DEP-chip after the incubation with THR.

Fig. 1 Nyquist plots (−Z′′ vs. Z′) of the various CMG surfaces: (A) Graphite oxide; (B) graphene oxide; (C) TR-GO; (D) ER-GO (black), THR-APT (red), THR (blue). Concentration of the THR-APT used is 10 μM, concentration of THR target is 40.5 nM. All measurements were performed with 10 mM K₄[Fe(CN)₆]/K₃[Fe(CN)₆] in Tris buffer solution (pH = 8.2) at room temperature.

From Fig. 1, it can be observed that for all four chemically modified graphene (CMG) materials, charge transfer resistance (R_ct), which corresponds to the diameter of the semi-circle, increases with immobilization of thrombin aptamer (THR-APT) and decreases after incubation with thrombin. In addition, it should also be noted that the CMG surface which exhibited the greatest change in impedimetric signal after incubating with thrombin is graphene oxide (GO).

Impedance spectra recorded were then analyzed and represented in terms of average R_ct ratio ((R_ct protein − R_ct blank)/(R_ct aptamer − R_ct blank)) and plotted in a histogram, as seen in Fig. 2. From Fig. 2, it can be concluded that the graphene oxide (GO) surface exhibits the largest change in impedimetric signal (lowest R_ct ratio value) if compared to other materials. This suggests that the graphene oxide (GO) surface is the most sensitive surface for impedimetric aptasensing of thrombin and graphene oxide (GO) was therefore the chosen material to be used for subsequent experiments performed.

Fig. 2 Histogram representing a comparison of impedimetric signals on GPO, GO, TR-GO, ER-GO and bare DEP electrode. Signal is represented as average R_ct ratio ((R_ct protein − R_ct blank)/(R_ct aptamer − R_ct blank)). Error bars correspond to triplicate experiments. All measurements were performed with 10 mM K₄[Fe(CN)₆]/K₃[Fe(CN)₆] in Tris buffer solution (pH = 8.2) at room temperature.

Thrombin aptamer (THR-APT) optimization was then carried out to determine the optimum concentration of aptamer to be immobilized onto the graphene oxide (GO) modified electrode surface. Impedance spectra recorded in the experiment were analyzed and plotted respectively in a histogram as shown in Fig. 3.

Fig. 3 Impedimetric response towards the concentration of THR-APT deposited on graphene oxide modified DEP electrode surface. Signal is represented as average (R_ct aptamer/R_ct blank). Error bars correspond to triplicate experiments. All measurements were performed with 10 mM K₄[Fe(CN)₆]/K₃[Fe(CN)₆] in Tris buffer solution (pH = 8.2) at room temperature.

From Fig. 3, it can be seen that the impedimetric signal is the highest when 10 μM of thrombin aptamer (THR-APT) was immobilized on the modified electrode surface. Therefore, it can be concluded that the optimum concentration of aptamer to be immobilized onto the graphene oxide (GO) modified electrode surface in order to ensure maximum coverage of the surface is 10 μM. As such, 10 μM of aptamer was used for subsequent experiments conducted.

We then studied the impedimetric response towards the thrombin target concentrations in order to determine the range of detection on graphene oxide (GO) platform. Impedimetric response was recorded for thrombin concentrations from 10 nM to 50 nM. The concentration of thrombin aptamer (THR-APT) was kept constant at the optimized value of 10 μM.

As shown in Fig. 4, increasing thrombin target concentration led to a lower R_ct ratio, corresponding to a higher analytical signal variation. In addition, it is also noted that there is a linear relationship between the impedimetric signal and thrombin target concentration in the range of 10 nM to 50 nM.

Fig. 4 Calibration plot corresponding to the changes in the charge transfer resistance (R_ct) after the incubation with different concentrations of THR. Signal is represented as average R_ct ratio ((R_ct protein − R_ct blank)/(R_ct aptamer − R_ct blank)). Error bars correspond to triplicate experiments. All measurements were performed with 10 mM K₄[Fe(CN)₆]/K₃[Fe(CN)₆] in Tris buffer solution (pH = 8.2) at room temperature.

We compared our findings with some references and found out that the linear detection range obtained by adopting differential pulse voltammetry (DPV)¹³ was reported to be from 5 pM to 7 nM. This range is wider than that obtained by our proposed method. However, it should be noted that our obtained detection range lies in the range of the actual concentration of thrombin present in blood during the coagulation process, which ranges from nM to low μM levels.⁷

To illustrate the selectivity of the aptasensor, the graphene oxide (GO) modified electrodes after the immobilization of THR-APT were incubated with IgG, BSA, and avidin as negative controls. From Fig. 5, it can be seen that the R_ct ratio values for IgG, BSA and avidin are much greater than that for thrombin. This indicates that IgG, BSA and avidin do not interact with the thrombin aptamer (THR-APT) and the effects of IgG, BSA and avidin on thrombin detection are negligible. Hence, the impedimetric aptasensor based on graphene oxide platform is considered to be selective for thrombin in the presence of IgG, BSA and avidin.

Fig. 5 Comparison of impedimetric response after the incubation with different protein targets. IgG, BSA and avidin were used as negative controls. The concentrations of IgG, BSA, avidin and THR used were 1.62 μM, 0.25 μM, 0.25 μM, and 50 nM, respectively. The signal is represented as the average R_ct ratio ((R_ct protein − R_ct blank)/(R_ct aptamer − R_ct blank)). Error bars correspond to triplicate experiments. All measurements were performed with 10 mM K₄[Fe(CN)₆]/K₃[Fe(CN)₆] in Tris buffer solution (pH = 8.2) at room temperature.

Conclusions

In conclusion, we have for the first time investigated the suitability of different chemically modified graphene (CMG) surfaces, namely graphite oxide (GPO), graphene oxide (GO), thermally reduced graphene oxide (TR-GO) and electrochemically reduced graphene oxide (ER-GO), for impedimetric aptasensing of thrombin. We found out that graphene oxide is the most sensitive surface for the detection of thrombin with the adopted protocol. Furthermore, we also discovered that the optimized concentration of thrombin aptamer (THR-APT) to be immobilized is 10 μM. The impedimetric aptasensor also exhibits a linear relationship between the impedimetric signal and thrombin target concentration in the range of 10 nM to 50 nM. Lastly, it was also observed that the impedimetric aptasensor is highly selective for thrombin in the presence of IgG, BSA and avidin.

Acknowledgements

This work was supported by Nanyang Technological University (NTU) via Nanyang Assistant Professorship fund.

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