Bovine serum albumin (BSA), trypsin, PBS (pH 7.2–7.4, 136.89 mM NaCl, 2.67 mM KCl, 8.24 mM Na₂HPO₄, 1.76 mM NaH₂PO₄), and sulfhydryl-modified CEA aptamer HS-C6-AAAAAAATACCAGCTTATTCAATT (Tang et al., 2020) were purchased from Shanghai Sangon Biotech Co., Ltd. (Shanghai, China). Human IgG was purchased from Beyotime Biotechnology. The CEA protein and alpha-fetoprotein (AFP) protein were purchased from Fitzgerald Inc. Chloroauric acid (HAuCl₄) was purchased from Macklin Biotech Co., Ltd. (Shanghai, China). Milli-Q ultrapure water (18.2 MΩ cm specific resistance) was used throughout. All the other chemicals were of analytical reagent grade.

A platinum wire with a diameter of 21.3 μm (Conghang Co., Ltd., Shanghai, China, 99.9%) was used to fabricate the platinum microelectrode, which is denoted as PtμE, and the procedure is similar to that in the previous report (Zhao et al., 2019). The prepared PtμE electrodes were left in 1.0 M HNO₃ for 15 min and then were cleaned ultrasonically in deionized water and ethanol for 5 min. As shown in Figure 1A, the voltammetric characteristic of the PtμE shows a sigmoid-shaped voltammogram, which is the typical characteristic of the microelectrode (Gyurcsányi et al., 1998).

The gold nanoparticles were electrodeposited onto the surface of PtμE through galvanostatic electrochemical polymerization in an aqueous solution of 1 μM HAuCl₄ under a constant current of 50 nA for 50, 100, 200, and 300 s to produce a total polymerization charges of 2.5, 5, 10, and 15 μC, respectively. The microelectrodes modified with gold nanoparticles were denoted as the PtμE/Au electrodes (Ihalainen et al., 2011; Hupa et al., 2015). The polymerization was carried out in a three-electrode cell using a Pt wire as the counter electrode, an Ag/AgCl/3 M KCl microelectrode as the reference electrode, and the above-prepared PtμE/Au electrodes as the working electrode. After electrodeposition, the PtμE/Au electrodes were rinsed with deionized water and allowed to dry in air for 1 day.

Proper folding of the CEA aptamer was obtained by heating at 95°C for 5 min and then annealing immediately on ice for 15 min. After being incubated with 20 μl CEA aptamer (1 μM) in a 0.2-ml centrifuge tube for 1 h at room temperature, the PtμE/Au electrodes were rinsed with PBS buffer to remove the nonspecific absorbed CEA aptamer. The PtμE/Au electrodes immobilized with CEA aptamer were denoted as PtμEs/Au aptasensor. The incubation conditions were also optimized to achieve a high signal.

SWV was used to characterize each step of the PtμEs/Au aptasensor fabrication using a CHI 660E electrochemical workstation (Shanghai Chenhua Apparatus Corporation, China). SWV was performed from −0.1 to 0.5 V in a 5.0 mM [Fe(CN)₆]^4-/3- solution containing 0.1 M KCl, the amplitude was 50 mV, step potential was 5 mV, and the frequency was 25 Hz. Cyclic voltammetry (CV) was carried out in 0.1 M KCl solution. The SWV and CV measurements were both performed using a three-electrode system, comprising the PtμE or PtμE/Au electrode as the working electrode, the Ag/AgCl/3 M KCl microelectrode as the reference electrode, and a Pt wire as the counter electrode.

In order to investigate the availability of the prepared PtμEs/Au aptasensors in clinical diagnosis, the blood samples were collected from in-patients of Yantai Affiliated Hospital of Binzhou Medical University for analysis. The fabricated PtμEs/Au aptasensors were dipped into 20 μl of each blood sample in a 0.2-ml centrifuge tube without pretreatment. After being incubated with each blood sample for 1 h at room temperature, the PtμEs/Au aptasensors were washed thoroughly with PBS solution for SWV measurements, and the values of the concentration of CEA [CEA] were calculated through the plotting linear curve of net current change (∆I) between the peak current of the PtμEs/Au aptasensors without CEA versus each [CEA] (Gui et al., 2018; Rizwan et al., 2018). For comparison, the [CEA] in blood samples was also measured in the Laboratory Center of the Yantai Affiliated Hospital of Binzhou Medical University through the electrochemiluminescence method.

The CV was used to investigate the redox capacitance of the microelectrodes before and after electrodeposition of the gold nanoparticles (Figure 1B). The interfacial capacitance of the PtμEs/Au could be calculated by summing the charge current in the positive and negative scan directions and dividing the sum by twice the scan rate. As shown in Fig. S1, the capacitance of the PtμEs/Au is calculated to be 78.3 nF cm⁻², which is much higher than that of the bare PtμE electrodes (41.3 nF cm⁻²) (Zheng et al., 2009) The capacitive current of the PtμE/Au electrode is much higher than that of the bare PtμE electrodes, which reveals that the redox capacitance of the microelectrodes is enhanced due to the presence of a gold nanoparticle film. Moreover, according to the Randles–Sevcik equation: i_p = 2.69 × 10⁵ n^3/2AD^1/2 V^1/2C₀, where i_p is the peak current (A), n is the number of electrons, A is the electrode area, D is the diffusion coefficient 6.7 × 10^–6 (cm² S⁻¹), V is the scan rate (V s⁻¹), and C₀ is the concentration (mol cm⁻³), and the surface area A of the PtμEs, PtμEs/Au, and PtμEs/Au aptasensor can be determined (Rizwan et al., 2018). It is found that the PtμEs/Au possessed about 116% more surface area than the bare PtμEs and about 183% higher than the PtμEs/Au aptasensor, and the electronic conductivity is decreased obviously due to the immobilization of the CEA aptamer. Therefore, the capacitive current of the PtμEs/Au decreases after the immobilization of the CEA aptamer, which results from the decrease of the surface area A of the electrodes.

The gold nanoparticles electrodeposited onto the surface of PtμE could not only act as solid contact which would improve the electrochemical property of the PtμE but could also make the CEA aptamer modified with sulfhydryl conjugated onto the surface of the PtμE directly. The SEM images revealed that the PtμE electrode has a smooth surface with a diameter of 21.3 μm (Figure 2A), while the PtμE/Au electrode has a rough and compact morphology (Figure 2B). The thickness of the gold nanoparticle layer could be reflected through the capacitive current of the cyclic voltammograms, which can be well-controlled by the amount of the polymerization charge from 2.5 to 15 μC. As shown in Supplementary Figure S1, the capacitive current of the bare microelectrode is less than 20 nA, while the capacitive current increases with the increase in the deposited polymerization charge, and the capacitive current is more than 40 nA when the polymerization charge reaches 10 μC. Therefore, the redox capacitance of the electrodes is enhanced obviously due to the modification of the gold nanoparticles, while the capacitive current of the electrodes no longer increases obviously even if the polymerization charge is up to 15 μC (Crespo et al., 2009).

As the recognition element, the CEA aptamer superstructure could be assembled onto the surface of the PtμEs/Au electrodes to form the electrochemical CEA aptasensor (Jiang et al., 2019). In order to obtain the optimal response of the experiment, the concentration of the CEA aptamer used for the preparation of the PtμE/Au aptasensor was optimized. As shown in Figure 3A the SWV response of the PtμE/Au was recorded after being incubated with various concentrations of the CEA aptamer from 10^–9 M to 10^–6 M. The SWV peak current decreases with the increase of the concentration of the CEA aptamer, while the peak current no longer decreases when the CEA aptamer concentration is up to 10^–7 M. Therefore, the 10^–7 M CEA aptamer was selected for further assay (Figure 3B).

The influence of the incubation time of the determined CEA aptamer and PtμE/Au electrodes was also investigated. The results show that the peak current of the SWV curve decreases with the increase of the incubation time of the PtμE/Au electrodes incubated with the 10^–7 M CEA aptamer from 0.25–2 h, and it would no longer decrease when the incubation time is up to 1 h (Figure 4). Therefore, the incubation time of 1 h was used for further study.

The influence of the deposited polymerization charge of the gold nanoparticles onto the surface of the PtμEs on the SWV performance of the PtμEs/Au aptasensor was investigated. The PtμEs modified with gold nanoparticles with different deposited polymerization charges from 2.5 to 15 μC were incubated with the 10^–7 M CEA aptamer for 1 h, and then the peak current of the SWV response was recorded. As shown in Supplementary Figure S2, the SWV peak current decreases with the increase of the polymerization charge of the gold nanoparticles, while the peak current almost stays the same when the polymerization charge ranges from 5 to 15 μC. Taking the redox capacitance of the PtμEs/Au and the SWV performance of the PtμEs/Au aptasensor into account, the polymerization charge of 10 μC was used for further assay.

Under the optimized conditions mentioned above, the sensitivity of the PtμE/Au aptasensor against CEA was investigated by measuring SWV starting at the concentration of 1.0 × 10^–7 g/ml and diluting the CEA solution by a factor of 10 each time through PBS solution until a limit of detection (LOD) could be detected, and SWVs were recorded in each concentration three times (Taylor et al., 2019; Hannah et al., 2020). A linear relationship between the ∆I and each [CEA] was observed (Caviglia et al., 2020). The PtμEs/Au aptasensor exhibits a linear response toward CEA in the concentration range of 10^–11-10^–7 g/ml (S = 5.5 nA/dec, R ² = 0.999), and the LOD is 7.7 × 10^–12 g/ml, which is calculated according to LOD = 3σ/b, where σ is the standard deviation of “n”, the number of SWV in blank solution, and b represents the slope of the calibration plot (Figure 5) (Shah et al., 2019; Gupta et al., 2020).

The selectivity of the PtμE/Au aptasensor was also investigated (Figure 6), and the PtμE/Au aptasensor can selectively distinguish between CEA and other interfering compounds with similar protein structures existing in the blood, such as AFP, BSA, pancreatin, and human IgG, even if the concentration of these proteins was ten times higher than that of the CEA (1.0 μg/ml vs. 0.1 μg/ml). As reproductivity is one of the major concerns of the sensing devices, five freshly prepared PtμEs/Au aptasensors were used for SWV measurement of CEA at the concentration of 0.1 μg/ml, and the standard deviation is 5.1% (Rizwan et al., 2018). Herein, the PtμEs/Au aptasensors have good reproductivity.

In order to investigate the feasibility of the designed detection assay in clinical applications, the prepared PtμE/Au aptasensor was used for the CEA measurement of the blood samples. As shown in Table 1, the results agree well with those obtained from the electrochemiluminescence measurements, which indicates that the PtμE/Au aptasensor is available for CEA detection in real blood samples.