The single-stranded DNA sulfhydrylated aptamer probe sequence was ACGTTTTATCTGCCCCAGTGTTCTC-(CH2)-6-SH, Apt single-stranded as Fc-GTCTCTGTGTGTGCGCCAGAGAGAACACTGGGGCAGATATGGGGCCAGCACAGAATGAGGCCC, and the nucleic acid sequence was purchased from Shanghai Sangong Bioengineering Technology Co. DA, MgCl₂, KCl, NaOH, NaCl, and potassium ferricyanide was purchased from Shanghai Aladdin Biochemical Technology Co. Tris-HCl was purchased from Amresco. 0.3 µm, 0.05 µm Al₂O₃ was purchased from Shanghai Chenhua Instrument Co. The working rod gold electrode (Au), auxiliary platinum wire electrode (Pt), and reference electrode were Ag/AgCl. The electrochemical workstation (CHI660e) was used to perform cyclic voltammetry (CV) curves in 0.5 mm [Fe(CN)₆]^3−/4−. 0.5 mM [Fe(CN)₆]^3−/4− was configured with PBS solution. Ultrasonic oscillator (JYD-250) and water-bath constant temperature oscillator (SHA-C).

Bare gold electrodes were immersed in a tigerfish solution containing 30% H₂O₂ and 70% concentrated H₂SO₄ for 1 hour. The main objective was to completely remove the oxide layer and thus regenerate the surface of the gold electrode. After completing the treatment, the electrode was then thoroughly rinsed with a thorough rinse of deionized water to ensure that a flawless, oxide-free surface was obtained. Next, the surface of the gold electrodes was refined, starting with 0.3 µm and 0.05 µm aluminum oxide powder (Al₂O₃), with a precise polishing process lasting about 30 min to obtain a properly smooth surface. Subsequently, residual Al₂O₃ powder is removed by an ultrasonic cleaning process. This thorough cleaning procedure consisted of successive washes with deionized water, ethanol, and deionized water, each lasting 5 min, to ensure complete removal of all impurities. Upon completion of the cleaning procedure, the cleaned gold electrodes were subjected to another thorough washing with deionized water, followed by drying using nitrogen gas. Finally, the electrode was immersed in cDNA solution for 10 h to allow the cDNA to be completely modified on the electrode surface by Au-S bonding.

10 × PBS was diluted 10-fold with deionized water to obtain 1 × PBS, which was used as a buffer. 0.038 g of DA was added to 100 mL of 1 × PBS to prepare a 20 μM DA solution, and the 20 μM DA solution was diluted with 1 × PBS in an appropriate ratio to obtain a lower concentration of DA solution.

A bare gold electrode was used as the working electrode. Ag/AgCl and Pt electrodes were used as reference and auxiliary electrodes, respectively. The electrochemical behavior of different concentrations of DA on the modified electrode was analyzed using CV. The CV curves were recorded for 30 cycles using an electrochemical workstation (CHI660e) scanned in the voltage range of −0.2 - 0.4 V at a scan rate of 100 mV s.

As shown in Figure 1 , the cDNA was first loaded on the bare gold electrode, which was subsequently filled with 6-MCH, and the S-Au bond ensured the stability of the cDNA binding to the gold electrode, and finally the Fc-labeled dopamine Apt strand was bound to the cDNA, at which time the electrochemical signal was maintained at a higher level due to the presence of the Fc electrochemical signaling molecule on the electrode. When DA is contained in the solution to be measured, DA binds to Apt, resulting in a subsequent Fc-Apt movement away from the electrode, leading to a decrease in the electrochemical signal, thus realizing the detection of DA.

The electrochemical behavior of the gold electrode was observed using electrochemical methods in potassium ferricyanide solution; the more loadings on the electrode, the lower the redox peak. We used CV to characterize the biosensor build change process. From Figure 2 , it can be seen that the bare gold electrode has the largest redox peak (curve a), followed by a gradual decrease in the current of the redox peak when the cDNA signaling probe (curve b) and 6-MCH (curve c) are sequentially modified on the electrode, the electrode transfer of electrons is blocked. After continuing to load on Fc-Apt (curve d), the redox peak value continued to decrease, and when DA solution was added, Fc-Apt reacted with DA (curve e), Fc-Apt moved away from the electrode, and the redox peak value increased. This indicates that the electrochemical sensor was successfully constructed.

We carefully considered our options when choosing the experimental setup to ensure the electrochemical sensor operated at its peak efficiency. Included in this are the ideal DA concentration and reaction time. Since the concentration of the DA solution and the reaction time of the Apt chain with the DA solution are important, we set up various DA solution concentrations and calculate the time of the solution’s steady state as well as the ideal reaction concentration at steady state in order to determine the ideal reaction concentration and reaction time.

As shown in Figure 3(A) , the current tends to stabilize when the reaction time reaches 10 min, and the current is stable at 15 min, so the optimal time for the solution to react to a steady state is 15 min. Configure different concentrations of DA solution 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, respectively, take the optimal reaction time of 15 min, and measure the electrochemical signals, as shown in Figure 3(B) , the concentration of 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, in that order, the 5 μM electrochemical signal response value is the largest, so we set the optimal response concentration as 5 μM.

The biosensor platform was used to measure different concentrations of DA solutions and analyze their electrochemical signal values to evaluate the sensitivity of the developed electrochemical biosensing platform to detect them. Figure 4(A) , Figure 4(B) shows how different concentrations of DA solutions affect the responsiveness of electrochemical signals.

The initial DA concentration in this experiment was 5 μM/L. As the DA solution concentration continued to increase, the electrochemical signal was linearly related to the logarithm of the DA solution concentration (lgC, with C representing the DA solution concentration), and in this study, a linear regression equation was established, which showed that there was a linear relationship between the level of the electrochemical signal and the concentration of the DA solution, with Y = −1.45 × 10⁻⁹ LgC + 3.91 × 10⁻⁷, R² = 0.993, and the limit of detection was determined to be 4.67 μmol/L according to the general equation LOD = 3σ/k, where k is the slope of the linear regression equation and σ is the standard deviation. Therefore, this biosensor is highly sensitive to the detection of the target target, which suggests that our constructed electrochemical biosensor can be used to detect DA.

We chose various approaches for the measurement of sensor specificity in order to confirm, under ideal experimental conditions, that the biosensor is specific for the detection of DA. This study was divided into five groups: potassium chloride (KCL), dopamine solution (DA), uric acid solution (UA), 5% glucose solution (GLU), and blank control group (Blank). Figure 5 illustrates this: while the sensor responds to the electrochemical signals of the Glu, UA, and blank control group in some way, there is almost no difference, indicating that the sensor lacks specificity for any of these groups. In addition, the electrochemical signals of the DA group are much lower than those of other groups, which indicates that the experimental method has good selectivity for DA solution. This is enough to prove that the electrochemical sensor platform we developed has obvious specificity for the detection of DA solutions.

We simulated the samples in a serum environment, diluted GLU, UA, and KCL with PBS solution, and added various doses of DA to assess the recoveries in order to test the method’s applicability. Table 1 displays the method’s recoveries, which ranged from 83.93% to 96.26%. These results suggest that the electrochemical biosensor we created is useful for DA detection.