A Sensitive Aptamer-Based Biosensor for Electrochemical Quantification of PSA as a Specific Diagnostic Marker of Prostate Cancer

- Purpose: The current project aimed to design a simple, highly sensitive, and economical label-free electrochemical aptasensor for determination of prostate-specific antigen (PSA), as the gold standard biomarker for prostate cancer diagnosis. The aptasensor was set up using a screen-printed carbon electrode (SPCE) modified by gold nanoparticles (Au NPs) conjugated to thiolated aptamers. Methods: Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were implemented for electrochemical (EC) characterization of the aptasensor. The determination of PSA was also performed through differential pulse voltammetry (DPV) in [Fe (CN) 6 ] 3-/4- electrolyte solution. Results: The present aptasensor was shown an outstanding linear response in the concentration range of 1 pg/mL - 200 ng/mL with a remarkably lower limit of detection of 0.077 pg/mL. The optimum concentration for PSA separation and the optimum incubation time for antigen-aptamer binding were determined by observing and electing the highest electrochemical responses in a specified time or concentration. Conclusion: According to the results of the specificity tests, the designed aptasensor did not show any significant interactions with other analytes in real samples. Clinical functionality of the aptasensor was appraised in serum samples of healthy individuals and patients examining the PSA level through the fabricated aptasensor and the reference methods. Both methods are comparable in sensitivity. The present fabricated PSA aptasensor with substantial characteristics of ultra-sensitivity and cost-effectiveness can be conventionally built and used for the routine check-up of the men for prostate problems.


INTRODUCTION
Prostate cancer is the second most predominant cancer in men and the fifth leading cause of death worldwide (1). Despite its highest 5-survival rate in all stages among cancers, its later stages are powerfully lethal and usually severe to be cured (2,3). According to the evidence, prostate cancer is bound to cause no signs or symptoms at early stages, and thus early diagnosis seems necessary for treatment. Currently, prostatespecific antigen (PSA), a 34 kDa single-chain glycoprotein, is regarded as the gold standard biomarker for the diagnosis of prostate cancer (4). It is produced by both healthy and malfunctioning prostate cells; however, the rising serum levels above four ng/mL could be associated with prostate malignancies (5).
Therefore, the implementation of alternative strategies with such high sensitivity and specificity, like aptamers, can be of utmost importance. ___________________________________________ With their exclusive features such as unique stability in extreme conditions of pH and temperature and userfriendly pretreatment, aptamers have become superior to antibody-based assays (20,21). Aptamers are single-stranded DNAs or RNAs selected by a technology called SELEX (Systematic Evolution of Ligands by Exponential Enrichment). They are readily accessible but can be manufactured straightforwardly (22,23). Besides their high specificity and dynamic binding towards corresponding targets, aptamers can be effortlessly modified through the incorporation of a wide range of functional substitutes, making them suitable for a variety of applications (20,24). Generally, biosensorbased determination methods with their substantial selectivity and cost-effectiveness can offer reliable alternatives to the conventional analytical assays (25). In Table 1, the characteristics of the routine assays (ELISA) and biosensing techniques for the determination of PSA have been compared. A biosensor is typically composed of the biological sensing element and a transducer (26,27), which is divided into three categories: electrochemical, physical, and optical (28). The electrochemical (EC) biosensor was chosen and applied in the present study because of relatively low cost, miniaturization capacity, exceptional sensitivity, and less timeconsuming determination process (25). As for the electrical conductor, a screen-printed carbon electrode (SPCE) is selected due to its reproducible results with a small volume of sample needed for each measurement and feasibility for large scales of production (29,30). On account of the advantages of aptamers compared to antibodies, which was mentioned earlier, and the fact that specific aptamers for PSA have been sequenced previously, specific thiol functionalized aptamers are employed as receptors (19,20,31). The surface modification is carried out using gold nanoparticles (Au NP) to provide a larger electroactive surface area and achieve quicker and more selective analytical responses (32,33). Herein, we report on the development of a novel label-free electrochemical aptasensor based on aptamer conjugated Au NP-modified SPCE, which can offer accurate, reproducible, and highly sensitive determination of minimum levels of PSA in clinical serum samples.

Apparatus
To carry out the electrochemical analysis, an AUTOLAB PGSTAT 101 potentiostat/galvanostat (Methrohm Autolab BV, Utrecht, Netherlands), compatible with NOVA 2.1 software was employed. Characterization of modified-SPCEs was performed in 0.1 M PBS (pH 7.4) containing [Fe(CN)6] 3-/4-/ KCl (5 mM /0.1 M) electrolyte solution as a redox probe using three distinct EC techniques cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV). EIS was carried out through an AC (alternating current) voltage amplitude modulation of +10 mV with frequency ranging between 100 mHz and 100 kHz. CV technique was conducted to assess the procedure of fabrication of the aptasensor. The potential was calculated in the range between -0.4 and +0.7 V at a scan rate of 0.1 V/s. DPV method was used under experimental conditions of potential ranging from +0.7 V to -0.4 V, and amplitude modulation of 0.05 V. Following electrodeposition procedure, the morphology of Au NPs was thoroughly assessed by FESEM (Hitachi S-4160, Japan). SPCEs (DropSens, Spain) measuring 3.4 × 1.0 × 0.05 cm (length × width × height) were made up of carbon working, carbon counter, and silverbased reference electrodes.
Au NP-modified SPCEs assembly Surface modification of the working electrode interface was carried out by Au NP electrodeposition, using a solution of 10 mM HAuCl4.3H2O in 0.5M H2SO4. Firstly, the electrode surface was activated by a 0.5 M H2SO4 solution. Prior to the activation, the SPCE was characterized via CV measurements in the range between -0.4 and + 0.7 V in 0.1 M PBS (pH 7.4) containing [Fe(CN)6] 3-/4-/ KCl (5 mM /0.1 M) electrolyte solution as the electrochemical redox probe with scan rate of 0.1 V/s. After activation of the electrode, the Au NPs were electrodeposited under optimal conditions of the potential of -0.2 V in a medium of HAuCl4/ H2SO4 (10 mM/ 0.5 M) for 80 s. The efficiency of electrodeposition was assessed through CV measurements, and then the voltammograms of Au NP-modified SPCE and bareactivated SPCE were compared. Finally, the deposition of Au NPs on SPCE was confirmed by using FESEM.
Aptamer immobilization onto Au NPs /SPCEs surface To modify the electrode surface by immobilized bioreceptor, the stock solution of PSA aptamer (20 μM) was reduced with a 10 mM TCEP solution for one h at 25°C to break disulfide bonds. Subsequently, the stock solution of PSA aptamer was diluted with a ratio of 1:20 with 0.1 M PBS (pH= 7.4). Afterward, 10 μL of aptamer solution (1 μM) was dropped onto the Au NPs-modified SPCE following a 12 h dark incubation at 4°C, which resulted in the formation of selfassembled monolayers (SAM). At the end of the incubation period, the SPCE was thoroughly rinsed with ultrapure water to ensure the removal of any free aptamers. Remaining active binding sites were blocked by adding the MCH solution (1 mM). After 30 min incubation, modified SPCE was again rinsed with ultrapure water, and the electrode was dried under a stream of N2 gas.   Figures 1A and 1B depict the FESEM images of untreated and Au NPs-modified SPCEs. Signal transduction was substantially enhanced by exploiting Au NPs, which successfully expanded the SPCE's electroactive surface area and increased the number of immobilized thiolated aptamers (35). Activation by CV was performed in H2SO4 solution (0.5 M) with potential ranging between -0.1 and 1.3 V and a scan rate of 0.1 V/s to achieve steady and repetitive voltammograms. Recent studies suggest that the elimination of the oxide layer and the introduction of the highly active sites on the electrode surface after activation may lead to a significant improvement in electrochemical performance (36). Label-free aptasensor (PSA/MCH/ thiolated aptamer/ Au NPs /SPCEs) characterization Characterization of the PSA/thiolated aptamer/Au NPs/SPCEs was performed separately through EIS and CV to monitor each step involved in assembly processes. The whole process was carried out in 0.1 M PBS (pH 7.4) containing [Fe(CN)6] 3-/4-/KCl (5 mM/ 0.1 M) electrolyte solution (39). CV measurements were recorded over a potential range between -0.4 and + 0.7 V with a scan rate of 0.1 V/s. Figure 2 shows the redox probe CV scans of the bare SPCE (curve a), Au NPs/SPCE (curve b), aptamer/Au NPs/SPCE (curve c), MCH/aptamer/Au NPs /SPCE (curve d) and 1 ng/mL PSA/aptamer/Au NPs/SPCE (curve e). To demonstrate the process of EC aptasensor fabrication, CV and EIS techniques were applied. The functional properties of the redox probe were investigated in various stages with different current response values and peak-to-peak separations (ΔEp= Epa-Epc).

Characterization of the Au NPs
Regarding its fast heterogeneous electron transfer features, bare carbon SPE showed appropriate reversible peaks. The characteristics of [Fe (CN)6] 3-/4were evaluated, measuring redox peak separation and calculating ΔEp of the cathodic and anodic waves and current responses. According to Figure 2 curve a, the CV records of the redox probe at the SPCE indicated a pair of reversible reduction/oxidation peak (ΔEP 215.78 mV). After modifying the electrode with Au NPs, a decrease of ΔEP to (173.34 mV) and an increase of the oxidation peak current was observed ( Figure 2, curve b), which can be ascribed to the electrocatalytic activity, good conductivity and larger exposed area of Au NPs. Self-assembly of the thiolated aptamer onto the Au NP SPCE led to a significant fall in the peak current of redox probe [Fe(CN)6] 3-/4and a corresponding rise in ΔEP (202.87 mV) (Figure 2, curve c). This phenomenon could be associated with the repulsive steric hindrance of the negatively charged phosphate groups of the aptamer hampering the electrolyte transfer. Figure 2, curve d represents the MCH added onto the electrode surface for the blockade of non-specific sites, which caused a marked decline in the redox peak current and a similar rise in the peak separation (ΔEp 250.40 mV). Lastly, the peak current value underwent a noticeable decrease after the reaction of the aptasensor with PSA solution (1 ng/mL), and the peak potential separation was raised correspondingly (ΔEp 278.32 mV) (Figure 2, curve e). This may be ascribed to the steric hindrance resulting from the conjunction of DNA aptamer and PSA, which closely correlated with the concentration of PSA solution; the higher the extent of PSA, the more significant the decrease in the peak current would be (23).
Optimization of the analytical parameters for PSA determination Various parameters such as Au NPs deposition time, aptamer concentration, aptamer self-assembly time, and incubation time were optimized to boost the performance of the aptasensor. The CV measurements of the EC aptasensor were carried out at different deposition time of Au NPs treatment ( Figure 3).   The redox currents increases were directly proportional to the deposition time. Given the obtained CV records in a span of 20-120 s ( Figure 4A), the optimal aptasensor state was found at the electrodeposition time of Au NPs SPCE of 80 s.
The aptamer concentration is another contributing factor in the aptasensor responses. Thus different amounts (10 µL of 0.1-5 µM) of the thiolated aptamer were assessed to reach the maximum level of aptamer-PSA interaction. According to the results, the current absolute value experienced a considerable rise before the aptamer concentration of 1 µM and then remained approximately constant ( Figure 4B). This may be because aptamers could be immobilized onto the SPCE surface with saturating binding attained at a concentration of 1 µM. The impact of incubation time on the self-assembly process was evaluated, testing multiple samples with different incubation periods ranging between 4 and 20 h ( Figure 4C). According to the DPV measurements, the maximum peak current was recorded in a sample incubated for 12 h. Similar to the effect of aptamer concentration. It seemed that exceeding the time to more than 12 h resulted in the saturation of the active binding sites.
Moreover, a more extended incubation period led to the aggregation of aptamers, and the current absolute value gradually declined after this period. In addition to the self-assembly process, the effect of incubation time was evaluated on PSA/aptamer response following the addition of PSA solution (1 ng/mL) to the fabricated aptasensor ( Figure 4D). In a period of 30 -60 min, the maximum peak current was observed at 45 min PSA incubation; after that, it reached a steady state, presumably caused by the saturation of aptamers. EIS spectra of the modified electrode The EIS, as one of the vital electrochemical approaches, was implemented to assess the aptasensor condition in each step of the fabrication procedure. Impedance spectrum (Nyquist diagram) consists of two major compartments: a semicircle segment at high-frequency regions correlating to the chargetransfer resistance and a linear segment in the lowfrequency range corresponding to the diffusion process. The diameter of the semicircle stands for the charge-transfer resistance (Rct) at the electrode surface and the straight-line accounting for the diffusion resistance (40). The Randles equivalent circuit was employed to model the electrochemical impedance data ( Figure 5, inset).
EIS data was recorded at a frequency ranging between 100 mHz and 100 kHz and an AC voltage amplitude of 0.01 V, superimposed on a DC (direct current) of +0.13 V.  Figure 5 curve a, corresponding to the impedance of the bare SPCE, indicates the Rct value of about 3167 Ω at the electrode surface. The electrodeposition of Au NPs on the working electrodes of bare SPCE resulted in a sharp reduction of the Rct level to 64 Ω ( Figure 5, curve b). As previously confirmed by CV, conductivity was considerably enhanced due to the expansion of the active surface area.
Subsequent immobilization of thiolated aptamer and MCH onto the Au NPs /SPCE caused the semicircle's diameter of the Nyquist plot to increase significantly. As a result of the blockade of charge transfer between SPCE and the redox probe, the Rct level rose from 2887 Ω ( Figure 5, curve c) to 3646 Ω ( Figure 5, curve d).
Finally, the interaction between PSA and thiolated aptamer led to a noticeable rise in the amount of the Rct to 3976 Ω, indicating that the fabricated aptasensor interacted successfully with PSA ( Figure 5, curve e). PSA determination with the aptasensor Due to the higher sensitivity of the DPV technique compared to CV, determination by DPV technique is recommended; besides, DPV can lower the background current (41). The quantitative determination of PSA was achieved by DPV signals in the same solution used for CV and under the optimal experimental conditions (potential range of + 0. According to the results, a linear response was detected between cathodic peak current and logarithmic concentration of PSA ranging between 1 pg/mL and 200 ng/mL. Figure 6 B shows the performance of the aptasensor at different PSA concentrations by DPV. The limit of detection (LOD) was calculated to be 0.077 pg/mL (LOD= 3Sb/m, Sb: standard deviation of the blank, m: slope of the calibration curve) using the linear regression equation ΔI (μA)= 14.383 log CPSA (pg/mL)+22.695 and correlation coefficient of 0.992. Table 2 represents a brief overview of the analytical properties, LOD, and linear range of our novel aptasensor and the available assays for PSA determination. In summary, the LOD and linear range of our technique are comparable and even better than available published assays. Regarding its dominant characteristics, such as user-friendliness and inexpensiveness, our novel method introduces itself as a compelling alternative to the available traditional techniques.
When examined on three distinct modified SPCEs, the DPV method demonstrated that PSA (1 ng/mL) was measured with an acceptable relative standard deviation (RSD) value of 3.2%, which certified that the test was reproducible under the experimental condition. Also, intra-and inter-day accuracy and precision (low, middle, and high standard concentrations) data for the determination of PSA are summarized in Table 3. The stability of the fabricated aptasensor was investigated using three SPCEs kept at 4°C for 30 days. According to the results acquired from conventional assays (every five days) in this period, the response value experienced less than a 10% decline compared with the initial response, which was not statistically significant. This indicates that no considerable decomposition occurred, and fabricated biosensor remained perfectly stable in long-term storage.

Selectivity
The selectivity of aptasensors plays a decisive role in the acquisition of the quality to detect and quantify analyte of interest in real samples. To evaluate the selectivity of the aptasensor, the determination of both types of PSA concentration (including 100 pg mL −1 PSA1 and 10 pg mL −1 PSA2) was examined in the presence of different proteins. The selected proteins to do so were BSA, IgG, and hemoglobin, and the reason behind selecting them was their abundance in plasma. For this purpose, the aptasensor was firstly incubated for 45 min at 25°C with 200 pg/mL of each antigen (except for the PSA concentrations of 10 and 100 pg/mL) and DPV values were measured subsequently.
Next, each stage was tested in two types of samples, one with all the antigens and only PSA1 and the other containing all the antigens and PSA 2. According to the results, the signals obtained from samples 1 and 2 did not show any significant differences with PSA1 and PSA2 sample's data and unsubstantial responses of the interfering compounds could be related to the negligible affinity of the fabricated aptasensor and its strong specificity for PSA (Figure 7).

Quantitative assay of clinical serum samples
To further investigate the functionality of our aptasensor for clinical applications, human serum samples obtained from Firoozgar Hospital were analyzed without any specific preparations or dilutions.
The results corroborated our electrochemical analyses regarding the selectivity and specificity of the fabricated biosensor. Aptamer based-Au NPs/SPCE records were strictly comparable to the reference values obtained by the ELISA method (Sigma-Aldrich, Germany), which was performed in the hospital as a standard method. As indicated in  Table 4 shows the results of two analytical methods and their relative error which was calculated at 6.86%.

CONCLUSION
In summary, a novel aptasensor was fabricated for precise determination of PSA biomarker based on the immobilization of thiol terminated PSA binding DNA aptamers onto Au NPs/ SPCEs. Surface modification performed by gold nanoparticles offered a highly efficient electrode for aptamer immobilization. The superb conductivity of Au NPs modified SPCEs ensured an effective charge transfer at the interface between the electrodes and the aptamer receptor.
Regarding the substantial features of electrochemical transducers such as adaptability, simplicity, and swiftness, the biosensing technique can be much efficient. The fabricated biosensor in this study benefiting from distinguishing features of aptamerbased techniques (instead of antibodies) and nanoparticle-based signal amplification offers a viable option for prostate cancer screening. Under the optimum condition, our EC aptasensor is capable of detecting PSA levels as small as 0.077 pg/mL in a linear response range from 1 pg/mL to 200 ng/mL. Serum sample analyses demonstrated that our aptasensor could determine the amount of PSA as accurately as the reference method (ELISA). Therefore, it would be a credible and economical alternative to the conventional analyzing method for point-of-care testing (POCT) of prostate cancer. However, biosensor designing is one of the innovative methods used in the field of biotechnology to detect sensitively various types of biomarkers. It is noteworthy to say that this low limit of detection is a research goal, and it is not necessarily for clinical applications.
Additionally, further modifications could be applied to the SPCE to boost its performance and enable the aptasensor to detect multiple biomarkers simultaneously. This would be a promising strategy for an accurate determination that could be exploited in various types of diseases. Preparation of a conventional sensitive aptamer-based biosensor kit for electrochemical quantification of PSA in routine check-up and screening of prostate problems in the men is suggested.

HIGHLIGHTS
A novel strategy for electrochemical determination of PSA based on the aptamer/Au NPs modified SPCE was introduced.
The fabricated aptasensor was validated by comparison to the reference method (ELISA) for PSA determination in clinical serum samples.
A Label-free aptamer-based biosensor offers a straightforward, rapid, ultrasensitive, and selective method for the determination of PSA.

ACKNOWLEDGMENT
The work was supported by a grant from Tehran University of Medical Sciences (Code Number: 98-02-222-42563). Iran National Science Foundation is acknowledged for the Seat Award directed to the corresponding author.

AUTHOR CONTRIBUTION
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for relevant intellectual content; gave final approval of the version to be published; and agreed to be accountable for all aspects of the work.