Immunoelectrochemical Detection of Prostate-Specific Antigen on PEDOT/Cerium Phosphate Nanotubes Modified ITO Electrode

Herein, a label-free electrochemical immunosensor was developed to detect prostate-specific antigen (PSA), a biomarker for prostate cancer. The immunosensor was fabricated by modifying one-dimensional nanomaterial CePO4 nanotubes, electrodepositing poly-3,4-ethylenedioxythiophene layer, anchoring monoclonal antibodies to PSA, and blocking with bovine serum albumin on the surface of the indium tin oxide sheet. The morphologies and electrochemical performance of the electrochemical sensor were characterized by Fourier transform infrared spectrophotometry, field-emission scanning electron microscopy, energy dispersive X-ray spectroscopy, X-ray diffraction microscopy, cyclic voltammetry, and electrochemical impedance spectroscopy. For PSA, it exhibits a wide dynamic range from 0.1 ng ml−1 to 100 ng ml−1 and a detection limit of 0.012 ng ml−1 (at a signal-to-noise ratio of 3) with differential pulse voltammetry. Average recoveries from rat serum (a simulated human serum) are between 97.00%–103.17% with relative standard derivations less than 4.25% (n = 3) at three spiked levels. Moreover, it shows high reproducibility, well selectivity, and good stability. The immunosensor provides an effective tool in the clinical diagnosis of prostate cancer.

Prostate cancer is a kind of cancer with high incidence and high mortality.Nowadays, it ranks as the second most frequent cancer in men. 1 Prior research shows prostate cancer is curable in its early stages and can be treated when it becomes metastatic and castrate-resistant. 2 Therefore, developing an effective and precise method for the early diagnosis of prostate cancer is critical to increase the chances of cure and reduce rates of mortality.Prostate-specific antigen (PSA) is a serine protease that is only produced by the human prostate tissue. 3And its serum level in healthy adult males is generally less than 4 ng ml −1 . 4In clinical trials, PSA has been used as an identified biomarker of prostate cancer. 5,6By far, there have been developed various methods to monitor PSA in biofluid, including surface plasmon resonance, 7 fluorescent labeling, 8 enzyme-linked immunosorbent assay, 9 chemiluminescence, 10 and electrochemical immunoassay. 11,12And the electrochemical immunoassay receives much more attention due to its good selectivity, high sensitivity, simple operation, and easy miniaturization.In comparison, the labelfree immunoassay can be prepared much more simply and costeffectively than the labeled one.Especially, it does not need the use of expensive secondary antibodies. 13abricating a high-performance electrochemical immunoassay generally requires that the substrate material owns high conductivity, as well as a large specific surface area to immobilize more biomolecules for high-performance electrochemical immunosensor assembly. 14,157][18][19][20] As known, cerium phosphate (CePO 4 ) nanotubes are also a kind of 1D material with high stability, a large specific surface area, and good biocompatibility. 21It has been introduced to fabricate electrochemically modified electrodes (CMEs) for detecting dopamine, acetaminophen, hydroquinone, etc. [22][23][24] Those CMEs show high specificity, selectivity, and sensitivity towards targeted small molecules.Whether it can be applied to detect macromolecules with the same high performance is still a maze.Thus, a novel label-free immunosensor was designed and constructed for specifically monitoring PSA by successively modifying CePO 4 nanotubes, conducting polymer poly-3,4-ethylenedioxythiophene (PEDOT), and monoclonal antibody (Ab) on the surface of ITO sheet.The preparation procedure is schematically represented in Scheme 1. Apparatus and electrodes.-Field-emissionscanning electron microscopy (FE-SEM, Phenom Scientific Instruments Co., Ltd., Netherlands) was used to characterize the morphology of CePO 4 /ITO and PEDOT/CePO 4 /ITO.Local elemental composition was determined using ISIS-2000 energy-dispersive spectroscopy (EDS, Oxford Industries, Co., Ltd., Britain).CePO 4 nanotubes was characterized by Fourier transform infrared spectra (FT-IR, Thermo Fisher Scientific Co., Ltd., USA), X-ray diffraction (XRD, PANalytical B.V., Netherlands), and UV-vis spectrometer (UV-1800PC, AOE Instruments Co., Ltd., China).

Experimental
Preparation of CePO 4 nanotubes.-CePO4 nanotubes were synthesized based on our prior research. 24In brief, Na 3 PO 4 solution was added in 0.8 M Ce(NO 3 ) 3 solution with a ratio of 1:1 (v/v).Then, the pH of the mixed solution was adjusted to pH 1.3 with 6 M z E-mail: fengshunxd@hotmail.comECS Advances, 2023 2 040507 HCl.Subsequently, the solution was heated at 200 °C for 8 h in a sealed autoclave.Finally, the resulted CePO 4 nanotubes was washed with pure water triplicates, and dried at a temperature of 60 °C.
Fabrication of immunosensor.-Firstly,the ITO sheet was fixed with insulating tape to keep it having a naked area of about 1 cm × 1 cm.Then, it was immersed in the ethanol solution containing 0.5 mg of CePO 4 nanotube.After being shaken for 10 min, the sheet was removed and washed with pure water triplicates.The immersing and washing steps were repeated 8 times.In this way, CePO 4 nanotubes was anchored on the surface of ITO.Subsequently, the PEDOT layer was fabricated on the CePO 4 /ITO surface according to Wang's work 25 with few modifications.Briefly, the electrodeposition was performed by immersing CePO 4 /ITO in 30 ml of 0.01 M pH 7.4 phosphate buffer containing 0.02 M EDOT and 0.2604 g sodium citrate at a constant potential of 1.1 V for 200 s.After that, PEDOT/CePO 4 /ITO was immersed in 10 mM phosphate buffer (pH 7.4) containing 0.4 M EDC and 0.1 M NHS for 30 min to activate carboxylic groups of citrates.Afterward, 30 μl Ab solution (10 μg ml −1 ) was dropped on the surface of PEDOT/CePO 4 /ITO and stood to immobilize Ab.About 12 h later, the slide was removed out and washed with 0.01 M pH 7.4 phosphate buffer triplicates to elute  For PSA detection, BSA/Ab/PEDOT/CePO 4 /ITO was immersed in 0.01 M pH 7.4 phosphate buffer containing 1 ng ml −1 PSA for 50 min.After that, the electrode was removed and washed with 0.01 M pH 7.4 phosphate buffer triplicates.Then, differential pulse voltammetry (DPV) was carried out in 0.01 M pH 7.4 phosphate buffer containing 0.10 M KCl and 5.0 mM Fe(CN) 6 3−/4− in the potential range of −0.4-0.8V with a pulse amplitude of 50 mV and a pulse width of 0.2 s.

Results and Discussion
Characterization of CePO 4 nanotubes, CePO 4 /ITO, and PEDOT/CePO 4 /ITO.-InFig. 1a FT-IR spectra, the peak at 3475 cm −1 is attributed to the stretching vibration of -OH, 1043 cm −1 to the stretching of P-O, 623 cm −1 to the stretching of P-O, and 543 cm −1 to the bending of O-P-O, respectively. 22In Fig. 1b, the characteristic peaks of CePO 4 nanotubes well match those in the standard comparison card.In Fig. S1, characteristic adsorption maxima of CePO 4 nanotubes are observed at 258 nm and 275 nm. 26E-SEM images indicate that the topology of the ITO sheet changes from evenly distributing tubular (Fig. 1c) to tubular and granular materials (Fig. 1d).Meanwhile, the peaks of P, O, and Ce, and C, S, O, P, and Ce can be observed in Fig. 1e (CePO 4 /ITO) and 1f (PEDOT/CePO 4 /ITO).The above results demonstrates that CePO 4 nanotubes and PEDOT are successfully modified on ITO and CePO 4 /ITO sheets, respectively.
Electrochemical behavior of electrodes.-Ascan be seen in the cyclic voltammetry (CV) curves of Fig. 2a, compared with the bare ITO, the current response decreases after modified CePO 4 nanotubes, while increases with the formation of PEDOT, and then steadily weakens after separately immobilized Ab, BSA, and antigen PSA.It testifies that the immunosensor is constructed successfully as designed.From Fig. 2b EIS spectra, the same conclusion can also be drawn.
Effects of pH and incubation time.-Asshown in Fig. S2a, the response value of DPV increases at first and then levels down in the pH range of 5.8-8.2, and the maximum value is obtained at pH 7.4.It is attributed to the highly alkaline or acidic environment that will affect the activity and stability of the immune protein.Therefore, pH 7.4 is finally chosen as the optimal pH in the following electrochemical experiments.The optimum incubation time for PSA is 50 min based on Fig. S2b.Detection of PSA.-BSA/Ab/PEDOT/CePO 4 /ITO electrode was used to detect PSA as described in the section on Electrochemical measurements.
Under optimal experimental conditions, well-defined DPV responses are obtained from different concentrations of PSA with BSA/Ab/PEDOT/CePO 4 /ITO as the working electrode (Fig. 3a).Whereas, the peak current gradually decreases along with the increase of PSA concentration.In concentrations of 0.1 ng ml −1 -100 ng ml −1 , the linear regression equation is I = −0.083log C + 0.276 (linear regression factor R 2 = 0.992).And limits of detection (LOD) and quantitation (LOQ) are 0.012 ng ml −1 and 0.041 ng ml −1 (based on a signal-to-noise ratio of 3 and 10, respectively), respectively.Which is far lower than 4 ng ml −1 , the normal level of PSA in the serum of healthy adult males.It suggests that the immunosensor constructed without secondary antibodies also can have enough sensitivity and the high feasibility applied in clinical trials.
Table I summarizes the reported label-free immunosensors for PSA detection.As seen, their performances are equivalent to each other.Notably, the preparation of BSA/Ab/PEDOT/CePO 4 /ITO is much simpler, cheaper, and without noble metal needed, compared to the others.
Reproducibility, selectivity, and stability.-Fiveindependently immunosensors were constructed in parallels and used to detect PSA (1 ng ml −1 ).The relative standard deviation (RSD) of response current is 2.37% (S3a).In the presence of uric acid (UA), glucose (GLU), ascorbic acid (AA), dopamine (DA), BSA, or alpha-fetoprotein (AFP) (100 ng ml −1 each), there is few differences among DPV curves, indicating that either of the above interferences shows little influence on the detection of PSA (S3b).The stability was investigated by storing the immunosensor at 4 °C and the current response was measured every 3 d.After 21 d, it can still maintain 90.75% of the original current response (Fig. S3c).
Detection of PSA in stimulated human serum.-Inthis work, PSA-spiked rat serum was used to simulate human serum.After being collected, rat serum was diluted 10 times with 10 mM pH 7.4 phosphate buffer.Afterward, PSA was spiked in the diluted serum to final concentrations of 0.2, 0.4, and 0.8 ng•ml −1 , respectively.And then, DPV tests were performed under the optimized conditions.As seen in Table S1, average recoveries range from 97.00%-103.17%with RSDs between 1.79%-4.25%(n = 3).The above results testifies that BSA/Ab/PEDOT/CePO 4 /ITO shows great potential for the detection of PSA in clinical settings.

Conclusions
In this work, a novel label-free electrochemical immunosensor was designed and fabricated for the detection of PSA.In which, 1D nanomaterial CePO 4 and conducting polymer PEDOT were used cooperatively to improve the electro-chemical performance towards PSA. the results testified that CePO 4 also can be applied for the monitoring trace-level macromolecule.Moreover, the resulted BSA/Ab/PEDOT/CePO 4 /ITO showed good sensitivity and selectivity towards PSA. it was accomplished to monitor trace PSA in stimulated human serum, and match the clinical requirement for the detection of PSA.Furthermore, the fabrication procedure for preparing the immunosensor was relatively simple and cheap, making it suitable for biomedical sensing and clinical applications.

Figure 3 .
Figure 3. (a) DPV curves of the immunosensor detect different doses of PSA; (b) calibration curve between I and the logarithm of PSA concentration in 10 mM PBS solution containing 5 mM Fe(CN) 6 3−/4− and 0.1 M KCl.