Introduction

Much attention has been paid to research on brain science, among which neurotransmitters are widely studied and involve a lot of neuropathies. Dopamine is a member of the catecholamine neurotransmitter family with a variety of functions [1–3]. It has a strong influence on the brain’s control of learning, feeding and neurocognition [4–6]. The temporal fluctuation of the dopamine concentrations in the human brain has a critical effect on several neurological disorders such as Harrington’s disease and Parkinson’s disease [7, 8]. Thus, dopamine detection at the nanometric scale is one of the main objectives to keep in mind since it would be of great help for monitoring patients with impaired release of this neurotransmitter in vivo.

Methods for dopamine detection include liquid chromatography [9], chemiluminescence [10], capillary electrophoresis [11], fluorescence [12], colorimetry [13, 14] and electrochemical detection [15]. However, almost all these methods require sophisticated equipment and/or time-consuming procedures, which bring significant limitations on its application. Electrochemical determination of dopamine is mainly compromised by the interferents ascorbic acid and uric acid which are present in most samples that contain dopamine. Dopamine, ascorbic acid and uric acid possess similar oxidation/reduction potentials. Aptamer technology can resolve problems of limited selectivity and therefore has been widely used because of its excellent recognition and binding ability with the target molecular. Thus, dopamine aptamer would only interact with dopamine and thereby will eliminate the interferences caused by ascorbic acid and uric acid [16, 14].

Signal amplification is an efficient way to improve the sensitivity of a biosensor. So far, signal amplification techniques such as Polymerase Chain Reaction (PCR) [17]. Rolling Circle Amplification (RCA) [18, 19]. Loop-mediated Isothermal Amplification of DNA (LAMP) [20], Nicking Endonuclease signal amplification [21–23] have been reported. But, it is obviously that these methods listed above require enzymes and its complicated manipulation also limited the application. AuNPs has been used as substrate to immobilize electrochemical marker to amplify the electrochemical signal in recent years. It’s obviously that there needs no enzymes in this procedure [24].

In this article, AuNPs was used to immobilize electrochemical marker thionine, and the complementary strand of dopamine aptamer was linked onto the surface of AuNPs through Au-S binding to fabricate the electrochemical probe. Carbon nanoparticles (CNPs) were used to modify the gold electrode [25, 26]. CNPs were used to interact with ssDNA to adsorb electrochemical probe on the surface of the electrode. In this method, dopamine aptamer was used to improve the selectivity and electrochemical probe was applied to enhance the sensitivity. Therefore, it is believed that this detection method possess great potential for highly sensitive and selective detection of dopamine.

Experimental

Chemicals

HAuCl4, thionine, and sodium citrate (C6H5O7) were obtained from Shanghai Chemical Reagent Co. (Shanghai, China); Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and tris(hydroxymethyl)methyl aminomethane (Tris) were purchased from Guoyao Chemical Company. Dopamine, ascorbic acid and uric acid were obtained from Tianjin Bodi Chemical Reagent Co. (Tianjin, China).

The following oligonucleotides were purchased from SBS Genetech Co. Ltd.

  • DNA1: 5′-GGG CCT CAT TCT GTG CTG GCC CAT ATC TGC CCC AGT GTT CTC TGG CGC ACA CAG AGA C-SH-3′

  • DNA2 (aptamer): 5′-GTC TCT GTG TGC GCC AGA GAA CAC TGG GGC AGA TAT GGG CCA GCA CAG AAT GAG GCC C-3′

Apparatus

All the electrochemical experiments were carried out with CHI 660D Electrochemical Analyzer (CH Instruments, Shanghai, China), using a conventional three-electrode system with the gold electrode or carbon nanoparticles (CNPs) modified gold electrode as the working electrode, a platinum wire as the counter electrode, a Ag/AgCl electrode as the reference electrode. The change of electrochemical characters of the electrode surface when modified with DNA was examined using CHI 660D. The shape and character of CNPs was examined using a JSM-6700 F scanning electron microscope (Hitachi High-Technology Co., Japan).

The synthesis of carbon nanoparticles

All glassware was washed with aqua regia, rinsed with distillated water then dried before used. At first, 0.3 g active carbon was added into 60 mL, 5 mol L−1 HNO3 solution, then the mixture was heated until boiling and continue heating for 12 h. During this time, the active carbon gradually dissolved in the solution and the color of the solution turned to dark brown. The final CNPs prepared by this method had an average diameter of approximately 3 nm as measured by SEM. The prepared 3 nm CNPs was stored in brown glass bottles at 4 °C in a refrigerator.

The preparation of electrochemical probe

Firstly, thionine was directly immobilized onto the surface of AuNPs according to methods reported previously with slightly modification [27]. A 100 μL of 1 × 10−4 mol L−1 thionine solution was added into 1 mL AuNPs solution for 30 min to immobilize thionine on its surface. Then 10 μL of 1.0 × 10−5 mol L−1 DNA1 activated by TCEP was added and incubated at 37 °C for 16 h with shaking. The DNA1 is immobilized on the AuNPs through Au-S binding. The product was washed with 1.0 mL of 0.1 mol L−1 phosphate buffer three times, resuspended in 1.0 mL phosphate buffer and stored at 4 °C for further use.

The preparation of carbon nanoparticle-modified gold electrode

The gold electrode was polished with Al2O3 powders for a certain time. Then gold electrode was rinsed by ultrasonic apparatus with ethanol and distilled water, respectively. The electrode was allowed to dry at room temperature. The gold electrode was modified with AuNPs by dropping 10 μL CNPs on its surface to enlarge the ratio-surface areas and improve the immobilizing efficiency of probe DNA1.

The detection of dopamine

A 10 μM solution (20 μL) of the aptamer DNA2 was added to a solution of the electrochemical probe and incubated for 2 h. Then the solution was centrifuged for 30 min at 13,050 r.c.f. at room temperature and washed by 10 mmolL−1 phosphate buffer, then the product was resuspended in 1.0 mL phosphate buffer and stored at 4 °C for use.

A 20 μL of the above solution was added into 2 mL tube, then 10 μL of dopamine was added and incubated for 30 min. The CNPs modified electrode (CNPs/GE) was put in the tube to make sure that the surface of the electrode was immersed in the solution. After adsorption for 30 min the electrode was washed by 10 mmol L−1 phosphate buffer three times and used as working electrode in the follow experiment.

A conventional three-electrode cell with an Ag/AgCl reference electrode and a platinum (Pt) wire counter electrode was used. The CV experiments were scanned from −0.1 to −0.6 V, with a scan rate of 100 mV s−1, unless otherwise stated. For establishing the analytical method, the DPV experiments were employed and I-E curve was recorded in the potential window between −0.1 to −0.6 V. The optimum parameters for DPV were: Amplitude 0.05 V, pulse width 0.05 s, Incr E 0.001 V, pulse period 0.2 s.

Result and discussion

Fabrication of biosensor and detection process

Lots of works were reported about the DNA adsorption on CNPs. And detection methods for DNA and protein were developed based on the π-π stacking and electrostatic repulsion between CNPs and DNA [26]. So inspiring by these works, we use the CNPs to modify the gold electrode to fabricate the electrochemical method for the dopamine detection. In this method, the AuNPs was used to immobilize Thionine and DNA1 to fabricate the electrochemical probe. After then dopamine aptamer being added into electrochemical probe solution, aptamer and its complementary strand would hybridize to form a double-strand DNA (dsDNA). When dopamine was added, the interaction between aptamer and dopamine would take precedence over hybridization and the electrochemical probe was released as a result. Then CNPs/GE was dipped to adsorb the electrochemical probe and the electrochemical signal was produced. AuNPs with a large ratio-surface area can immobilize a large amount of thionine and the signal was amplified. Scheme 1 illustrated the procedure of the fabrication of dopamine biosensor.

Scheme 1
scheme 1

Schematic illustration of dopamine detection based on thionine modified AuNPs and carbon nanoparticle-modified gold electrode

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Investigation the effect of carbon nanoparticles

The CNPs was firstly characterized by TEM which is shown in Fig. 1a. The CNPs were approximately 3 nm diameter. The prepared CNPs were homogenous and high dispersive. To examine the successful adsorption of electrochemical probe to CNPs modified gold electrode, we tested the electrochemical intensities of electrochemical probe with or without dopamine adding to investigate experimentally if the electrochemical probe was adsorbed to CNPs modified gold electrode. Three kinds of experiments were done. DNA 2 was first added into the solution of electrochemical probe. After incubated for 30 min, dopamine was added. Then the bare gold electrode or CNPs/GE was dipped into the solution and taken for electrochemical detection. As shown in Fig. 1b, the current of DPV at bare gold electrode is very low shown as cure b. When the electrode was modified with the CNPs, the current was increased greatly shown as curve c. Without adding dopamine the electrochemical probe hybridized with DNA2 formed the ds-DNA. The ds-DNA is not adsorbed at CNPs/GE and therefore there was nearly no current response. It is suggested that the CNPs increase the adsorption ability.

Fig. 1
figure 1

The TEM image of CNPs (a). DPV at a CNPs/GE; b Th/AuNPs/DNA1/GE; c Th/AuNPs/DNA1/CNPs/GE (b). The concentration of dopamine was 0 mol L−1 in a, 1.0 × 10−7 mol L−1 in b and c

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The calibration curve of dopamine detection

The analytical method based on AuNPs signal amplification coupled with the aptamer technique can be applied to sensitive and selective detection of dopamine. Differential pulse voltammetry (DPV) was used to get higher sensitivity. As shown in Fig. S2 (A), the peak current increased with increasing concentration of dopamine. Under the optimized test conditions (see the supporting information, Fig. S1), the linear range and detection limit of this bioassay to dopamine were investigated. The results showed that the peak current linearly increased with the concentration of dopamine in the range from 3.0 × 10−8 ~ 3.0 × 10−6 mol L−1 and the detection limit is 1.0 × 10−8 mol L−1. The regression equation is Ip = 2.4096 C + 6.3 × 10−8 with a regression coefficient of 0.9988; the RSD is 3.1 % at a 3.0 × 10−7 mol L−1 level (for n = 11). To better demonstrate the advantage of the developed dopamine biosensor, the analytical characteristic have been compared with other similar strategies and listed in Table 1.

Table 1 Comparison between the current method and other reported techniques for the detection of dopamine
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Selectivity of the biosensor

Ascorbic acid and uric acid were investigated with respect to their potential interference. The concentration of Ascorbic acid and Uric acid were both 100 fold of the concentration of dopamine and DPV was used to record the electrochemical signal. The electrochemical signals of them were shown in Fig. 2, the peak currents of Ascorbic acid and Uric acid were much lower the electrochemical signal of dopamine. Thus the bioassay proved to be a selective method for the detection of dopamine.

Fig. 2
figure 2

Electrochemical responses of different relative substances, Ascorbic acid (a) and Uric acid (b), Dopamine (c). The concentrations were 3 × 10−7 for dopamine and 3.0 × 10−5 mol L−1 for Ascorbic acid and Uric acid, respectively

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Determination of dopamine in the human urine samples

The feasibility of applying the developed method to measure dopamine in a complex matrix was studied. The accuracy of this method for dopamine detection in human urine sample was also evaluated by determining the recovery of dopamine by addition of a known quantity of standard dopamine solution into the corresponding urine samples. The results of recovery test were shown in Table 2. The recoveries were between 96.7 and 102.7 % (n = 9). The results indicate that the developed method is highly reliable.

Table 2 Results of determination of dopamine in human serum samples
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Conclusions

A strategy for the simple and sensitive detection of dopamine based on CNPs modified gold electrode and aptamer recognition action coupling AuNPs signal amplified strategy was developed. The detection limit of 1.0 × 10−8 mol L−1 of dopamine was obtained. The resulting dopamin biosensor exhibited ultrasensitivity, high selectivity, and good reusability as a promise alternative technique for other bioassays. It holds great promise for small molecule diagnostics, microarrays, and microchips, as well as for bioanalysis in general.