(1 (3-dimethylamino-propyl) -3-ethylcarbon diimide hydrochloride/N-hydroxysuccinimide (EDC/NHS), 4-mercaptobenzoic acid (4-MBA) was purchased from Aladdin Industrial Co., Ltd. Chloroauric acid (HAuCl₄), silver nitrate (AgNO₃), hydrochloric acid (HCl), trisodium citrate (Tc), hydroquinone (Hq), potassium ferricyanide (potassium ferricyanide), potassium chloride (KCl), bovine serum albumin (BSA), glutathione (GSH) and glycine (Gly) were purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd. IL-6 antibody, IL-6 antigen and neuron specific enolase (NSE) were purchased from Sigma Aldrich (Shanghai) Trading Co., Ltd. Ultra-pure water was filtered through Milli-Q reagent water system in the experiment. Electrochemical workstation (CHI660E), platinum carbon electrode, reference electrode and counter electrode were purchased from Shanghai Chenhua instrument Co., Ltd. It was purchased from Sedorius Scientific Instruments (Beijing) Co., Ltd. with PB21 acidity meter.

Selected 50 mL beaker, washed detergent and ultra-pure water for three times in sequence. Added 30 mL ultra-pure water to the beaker, boil, added 300 μL 1% HAuCl₄ and 900 μL 1% Tc, stir, continued to heat until the solution turns wine red, and cool to room temperature to complete the preparation of gold seed solution. Selectd the 100 mL beaker, cleaned according to the above method, took 99 mL ultra-pure water to add the beaker, stir, added 1% HAuCl₄ of 1 mL, 100 μL dilute HCl, gold seed solution of 1 mL, AgNO₃ solution of 1 mL (10^–3 mol/L), Hq solution of 4 mL (10^–3 mol/L), stir until dark blue.

The spherical AuNPs were modified on the PC electrode, and then 4-MBA was modified on the surface of the electrode after the complete reaction. In order to activate the carboxyl group of 4-MBA to facilitate closer connection with IL-6 antibodies, we used EDC/NHS to do this. After the full action of EDC/NHS, the IL-6 antibody was modified on the 4-MBA modified electrode, and finally the electrochemical sensor was prepared. All the reaction steps were carried out in a 37°C incubator.

The standard solution of IL-6 antigen of known concentration was prepared with 0.1 mol/L PBS. Then it was added to the mixed solution of potassium ferricyanide (K₃[Fe(CN)₆]) and potassium chloride (KCl) (reaction system solution). The electrochemical sensor electrode was placed in the mixture, and the reference electrode and counter electrode were added at the same time. The correlation between CV and DPV was detected, and the linear relationship between concentration and peak current was obtained. The detection limit of the sensor was calculated. According to the linear relationship, the concentration of IL-6 in the sample solution to be tested was calculated.

The fabrication process of AuNPs was described above. In this part, we mainly introduced their characterization results. As shown in Figure 1A, the ultraviolet and visible light (UV-vis) absorption spectrum of the gold seed made of Venus was detected. The absorption peak of UV-vis of AuNPs was about 520 nm, which was consistent with the previous research. In addition, we used scanning electron microscope (SEM) to observe the nano-Venus particles, as shown in Figure 1B, the AuNPs was obviously angular, and its diameter was about 90 nm, which was in line with our experimental expectations.

According to the above methods, we prepared electrochemical sensors for specific recognition of IL-6. In order to verify the specific recognition function of the sensor, we used CV and DPV to detect the sensor. As shown in Figure 2A, we successively added BSA, Gly, GSH, NSE, and IL-6 to the reaction solution and detected it by electrochemical sensor. The experimental results showed that the clear redox peak was only seen in the cyclic voltammogram of the reaction solution with IL-6, which indicated that the sensor recognizes and specifically binds IL-6 in the solution. In addition, in the differential pulse voltammogram (Figure 2B), we also found that only the DPV curve of the solution added with IL-6 had a peak, which further proved the specific recognition and binding function of the sensor to IL-6. Through the atlas results of CV and DPV, we determined that the IL-6 electrochemical sensor had the ability to specifically recognize and bind IL-6, which indicated that our sensor was constructed successfully.

In order to evaluate the kinetic characteristics of the sensor, the cyclic voltammogram (Figure 3) of the sensor electrode in the reaction solution of standard concentration of IL-6 was recorded at different scanning rates. The oxidation peak current and reduction peak current increased with the change of scanning speed (0.1V/S-1V/S). Through the cyclic voltammogram, it was found that the peak potential did not change with the change of the scanning rate, which indicated that the redox state of the IL-6 electrochemical sensor is reversible, and the redox center was located on the surface of the electrode.

To explore the best working pH of the electrochemical sensor to allow it to successfully recognize IL-6 antigen in the pH environment of the real sample, we studied its performance in different pH reaction systems. The detection results of CV and DPV in different pH environments (Figure 4) showed that the sensor works better in neutral environment. As shown in Figure 4A, CV was detected in the reaction solution of pH 2.5–9.6. The results showed that the redox peak changed obviously in the reaction solution of pH 7.2. Compared with the test results of CV, the test results of DPV (Figure 4B) were more obvious. In the reaction solution of pH 7.0, the peak current reached the maximum, indicated that the sensor may be the best working environment in the neutral solution, which was close to the pH value of human blood and in line with the expectation of clinical transformation.

To evaluate the electrochemical analysis performance of IL-6 sensor, IL-6 detection was carried out under the above optimized conditions. Differential pulse voltammetry (DPV) had been recognized as an important tool for rapid electrochemical detection and analysis because of its high current sensitivity (Ou et al., 2007; He et al., 2008; Cevik et al., 2016; Zhang et al., 2017). Therefore, in order to analyze the sensitivity of the sensor and its relationship with concentration, we measured the DPV curves (Figure 5A) of many standard solutions with known IL-6 concentrations. As shown in Figure 5A, the oxidation peak current of the sensor increased gradually with the increase of IL-6 concentration, and the position of the oxidation peak was stable at about 0.12 V. There was a good linear relationship between oxidation peak current and concentration (I (μA) = 0.0019C_IL-6 (ng.mL⁻¹) + 0.75, R ² = 0.9626), which was obtained by IL-6 of 100 pg/mL-700 pg/mL concentration (Figure 5B). According to these relationships, we preliminarily calculated that the detection of the sensor was 3 pg/mL.

In order to study the reproducibility of IL-6 electrochemical sensors, we prepared six sensors to detect DPV in the same detection environment under the same conditions. As shown in Figure 6, a three-dimensional waterfall map was made based on the DPV data of six electrochemical sensors. From the graphic point of view, the waveforms of the six sensors had excellent consistency. At about 0.12 V, the relative standard deviation of the peak current was 2.35%. This showed that the sensor had excellent reproducibility and had a broad prospect in the detection of clinical samples.

To further evaluate the prospect of the sensor in the detection of clinical real samples, we collected serum samples from patients with spinal cord injury. In order to compare its accuracy, the test results of ELISA WEre used as reference data. As shown in Figure 7, the blood samples of the same sample were detected by ELISA and electrochemical sensor respectively, and the results showed high consistency. The experimental results showed that the prepared electrochemical sensor had good accuracy, and the operation was more simple and fast than ELISA. Moreover, we also compared other technical data for the detection of IL6 (Table1). The electrochemical sensor also showed better detection performance, which had a very broad prospect in clinical application.