Label-Free Electrochemical Immunosensor Based on a Functionalized Ionic Liquid and Helical Carbon Nanotubes for the Determination of Cardiac Troponin I.

A label-free electrochemical immunosensor for cardiac troponin I was prepared by using a helical carbon nanotube-supported aldehyde-functionalized ionic liquid. Because of the good conductivity of ionic liquid and helical carbon nanotubes, high sensitivity of the immunosensor was obtained. Functionalized ionic liquid provided binding sites for antibody, which simplified the process of sensor construction. Cardiac troponin I was detected by this immunosensor with a linear range of 0.05-30 ng/mL and a detection limit of 0.03 ng/mL. The electrochemical immunosensor had satisfactory reproducibility, high sensitivity, and acceptable specificity.

Introduction

Cardiac troponin I (cTnI), as a marker protein of myocardial damage, was recognized as the “gold standard” biomarker test for acute myocardial infarction.[1] Accordingly, sensitivity and accuracy were very important for cTnI detection. Many detection strategies had been put forward for the quantitative detection of cTnI, such as enzyme-linked immunosorbent assay[2] and photoelectrochemical immunoassay.[3] Recently, fluorescent immunosensor and electrochemiluminescent immunoassay for cTnI was also reported.[4,5] However, these methods had some drawbacks including the need for a tedious labeling process, bulky and expensive equipment, and highly skilled operators. The electrochemical immunosensor had aroused researchers’ interest because of its high sensitivity, handling, portability, miniaturization, and low operating cost.[6,7] In most previous studies, linear carbon nanotubes (LCNTs) had widely been applied in the field of electrochemical immunosensors.[8−11] However, only few electrochemical immunosensors based on helical carbon nanotubes (HCNTs) had been reported to date.[12,13] The surface of HCNTs was more susceptible to chemical modification than that of LCNTs because of the high energy state as a result of inherent tensile and compressive stresses. In addition, the difference of charge density caused by the lattice defect of carbon nanotubes (CNTs) improved the electronical properties.[14,15] This motivated us to fabricate an electrochemical immunosensor using HCNTs for the detection of cTnI.

Because ionic liquids (ILs) had the advantages such as high ionic conductivity, chemical stability, and good biocompatibility, they were widely used in the fabrication of electrochemical biosensors by being incorporated into other matrixes including graphene, metal nanoparticles, fullerene, and cellulose.[16−20] Because of π–cation interaction between CNTs and IL, they could closely combine together to form nanocomposites which provided a platform for the fabrication of electrochemical biosensors.[21,22] However, most reported papers focus on LCNTs with ILs without functionalization and a few based on HCNTs with dialdehyde-functionalized IL (DIL).

Antibody immobilization using a simple method, such as DIL,[23] attracted many researchers. In this article, the DIL–HCNTs (the composite of DIL and HCNTs) was prepared by the ultrasound method, where DIL noncovalently bonded together with HCNTs through π–π and π–cation interactions.[13] Subsequently, the DIL–HCNTs was used to fabricate a label-free electrochemical immunosensor for cTnI detection. As the conductivity of the sensing interface was enhanced by DIL and HCNTs, this immunosensor had a high sensitivity. Furthermore, the fabrication process was simplified because the antibody was immobilized directly by DIL.

Results and Discussion

SEM Characterization of DIL–HCNTs

The surface characteristics of the HCNT before and after being functionalized with DIL were investigated by scanning electron microscopy (SEM, Figure ). DIL–HCNTs (Figure B) maintained the helical structure of HCNTs (Figure A) and looked thicker because DIL covers on the surface of HCNTs.[24]

Figure 1

SEM images of HCNTs (A) and DIL–HCNTs (B).

CV Characterization of the Modified Electrode

In order to investigate the electrochemical characteristics of the electrochemical immunosensor, cyclic voltammetry (CV) results were measured in 5 mmol/L potassium ferricyanide/potassium ferrocyanide at a scan rate of 100 mV/s from −0.2 to 0.6 V. As shown in Figure , the CV curve of the bare Au electrode (Figure a) was a typical redox wave. Because of the obstruction electron and mass transfer of Nafion film, no peak could be observed on CV curve (Figure b) when the electrode was covered with Nafion. Contrarily, obvious redox peaks could be observed when the electrode was modified with Nf/DIL–HCNT film (Figure c), which was attributed to the high conductivity of DIL–HCNTs. After the modified electrode adsorbed antibodies and antigens, the current of redox peak decreased (Figure d–f) because the electron and mass transfer had been blocked.

Figure 2

Cyclic voltammograms of bare Au (a), Nf/Au (b), DIL–HCNT–Nf/Au (c), anti-cTnI/DIL–HCNT–Nf/Au (d), BSA/anti-cTnI/DIL–HCNT–Nf/Au (e), and cTnI/BSA/anti-cTnI/DIL–HCNT–Nf/Au (f) in 5 mmol/L Fe(CN)63–/Fe(CN)64–. Scan rate was 100 mV/s.

Optimization of the Experimental Conditions

Experimental conditions, such as concentration and immobilization time of antibody and immunoreaction time between antibody and antigen, were optimized in order to improve the sensitivity and accuracy of cTnI detection. The peak current of differential pulse voltammetry (DPV) was used to evaluate the influence of the experimental conditions on detection of cTnI, and the concentration of cTnI remained at 10 ng/mL in these optimization experiments. According to the results, the peak current of DPV decreased significantly with the increase of the anti-cTnI antibody concentration from 20 to 60 μg/mL but continued to increase the concentration of anti-cTnI antibody, resulting in a minor change of the peak current of DPV (Figure A). Therefore, 60 μg/mL was the optimal concentration of anti-cTnI antibody. Similarly, DPV decreased with the prolongation of the fixed time of the antibody at room temperature and remained stable after 60 min. Similarly, the peak current of DPV decreased with the immobilization time of the antibody at room temperature and remained stable after 60 min (Figure B). Thus, 60 min was the select immobilization time of the antibody. Finally, 30 min was the optimal time for the immunoreaction time between antibody and antigen at 37 °C (Figure C).

Figure 3

Effect of the concentration of antibody (A), immobilization time (B), and immunoreaction time (C) on the peak current of immunosensor. The concentration of cTnI was 10 ng/mL.

Detection of cTnI

After antibody reacted with antigen, the immunocomplex formed on the electrode surface would hinder electron transfer, resulting in the decreasing of the peak current of DPV. Under the optimal experimental conditions, the signals corresponding to different concentrations of cTnI were tested. Figure shows that the peak current decreased with the increasing concentration of cTnI. The inset indicates a linear calibration that was obtained in the range of 0.05–30 ng/mL. The limit of detection is 0.03 ng/mL determined by 3 σ rule (where σ is the standard deviation of a blank solution).

Figure 4

DPV curves at different concentrations of cTnI. (Inset: calibration curve of the peak currents of DPV to different concentrations of cTnI. Error bars represent standard deviation, n = 3.) Experiment conditions: potential range: −0.4 to 0.6 V, pulse amplitude: 0.05 V, pulse width: 0.05 s, sample width: 0.02 s.

Comparing the analytical characteristics including the detection limit and linear range of the proposed immunosensor with those of other cTnI immunosensors, Table demonstrated that the fabricated immunosensor had a satisfied linear range and a lower detection limit.

Table 1

Performance of Different cTnI Immunosensors

modifying material	linear range (ng/mL)	detection limit (ng/mL)	refs
carbon nanofiber	0.25–1	0.2	(25)
Au nanoparticle	0.2–12.5	0.2	(26)
Au/Ag nanoparticle	0.1–32	0.1	(27)
porous graphene	0.1–10	0.07	(28)
nanostructured ZrO2	0.1–100	0.1	(29)
DIL–HCNT	0.05–30	0.02	this work

Specificity and Reproducibility of the Immunosensor

In order to investigate specificity of the immunosensor, the peak current of DPV toward blank solution was measured (48 μA). Carcinoembryonic antigen, BSA, and α-fetoprotein were used to replace cTnI as interfering species for immunoreaction. As shown in Figure , the peak currents of DPV corresponding to these interfering species of 20 ng/mL were close to the peak currents of DPV obtained from blank solution, while the peak current of DPV corresponding to cTnI (20 ng/mL) was 18 μA. These results indicated that the DIL–HCNT-modified immunosensor had good specificity.

Figure 5

Specificity of the immunosensor.

The reproducibility of the proposed immunosensor was also studied. The inter-and intra-assay coefficients of variation of detected cTnI (10 ng/mL) were 7.3 and 6.7%, respectively. These results showed that the prepared immunosensor had good reproducibility.

The immunosensor retained more than about 95% of the initial values after storing at 4 °C for 15 days. The results demonstrated that the designed immunosensor has satisfied stability.

Conclusions

Here, an electrochemical immunosensor based on DIL and HCNTs as a platform for the determination of cTnI was developed. The nanocomposite of DIL–HCNTs improved the conductivity of the electrode surface, resulting in high sensitivity. In addition, the DIL provided two −CHO groups for antibody immobilization, which simplified the process of sensor construction. The immunosensor for cTnI exhibited some good analytical performance such as satisfactory reproducibility and good specificity. Furthermore, the DIL–HCNT nanocomposite is an attractive modifying material in the fabrication of other electrochemical biosensors.

Experimental Section

Materials

cTnI and anti-cTnI (anti-cTnI monoclonal antibodies) were purchased from Shanghai Linc-Bio Science Co. Ltd. IgG (human immunoglobulin G) and BSA (bovine serum albumin) were purchased from Beijing Dingguo Biotechnology Company. PBS (0.1 mol/L, phosphate buffer solution, pH 7.0) was prepared using Na2HPO4 and KH2PO4, which were purchased from Sinopharm Chemical Reagent Co. Ltd. HCNTs were purchased from Nanjing Xianfeng Nanomaterial Technology Ltd. Nafion (Nf, 5%, v/v) was obtained from Sigma Chemical. 4-(Bromomethyl)benzaldehyde, 4,4′-bipyridine, acetonitrile, and N,N′-dimethylformamide (DMF) were purchased from Sinopharm Chemical Reagent Co. Ltd.

CV and DPV were performed on a CHI 660E electrochemistry workstation (Shanghai CH Instruments, China). The three-electrode system included a Pt electrode (counter electrode), a saturated calomel electrode (reference electrode), and a gold electrode (Au)-modified composite film (working electrode). SEM images were obtained by using a TESCAN MIRA3 (LMU) microscope.

Preparation of the DIL–HCNT Composite

4-(Bromomethyl)benzaldehyde (0.498 g, 2.5 mmol) and 4,4′-bipyridine (0.156 g, 1 mmol) were added into 20 mL acetonitrile, and the mixture was refluxed for overnight and cooled to room temperature. Subsequently, DIL, a yellow solid (0.42 g, yield about 76%), was obtained by filtration.[23] The DIL was directly used for the preparation of DIL–HCNTs without further purification.[23]

The DIL–HCNTs was prepared according to ref (13) with minor modification. Typically, 1 mL of HCNTs (dissolved in DMF, 2 mg/mL) was mixed with 4 mL DIL (dissolved in ethanol, 10 mg/mL), and black DIL–HCNT suspensions were obtained after 1 h with ultrasonication. Subsequently, this mixture was centrifuged (12 000 rpm, 10 min) and washed three times with ultrapure water. Then, the DIL–HCNT product was redispersed into 1 mL ethanol solution of Nf (0.25%, v/v) under ultrasonication for 10 min. The preparation of the DIL–HCNT composite is shown in Figure A.

Figure 6

Preparation of the nanocomposite of DIL–HCNT (A) and the electrochemical immunosensor (B).

Fabrication of the Immunosensor

At first, a gold electrode (Au, 3 mm in diameter) was successively polished with 0.3 and 0.05 μm alumina slurries and ultrasonication cleaned in ultrapure water. Subsequently, 10 μL of Nf/DIL–HCNT solution was dropped on the cleaned electrode and dried in the air. Then, 10 μL of antibody (60 μg/mL) was dropped on the Nf/DIL–HCNT membrane-modified electrode and incubated at room temperature for 60 min. The unreacted antibodies were removed by washing with ultrapure water, and then, the electrode was incubated for 30 min at 37 °C with 10 μL BSA (2.0 wt %) in order to eliminate nonspecific binding. Ultimately, 10 μL of antigen solution with various concentrations was dropped on the surface of electrode and incubated for 40 min at 37 °C, followed by washing with ultrapure water and then measuring the electrochemical signals. The whole process of the immunosensor fabrication is shown in Figure B.