A Simple Ratiometric Electrochemical Aptasensor Based on Exonuclease III-Assisted Target Recycling for Ultrasensitive Detection of Prostate Specific Antigen

Wang, Ping; Xie, Yaoyao; Guan, Zhimin; Ma, Huikai; Xi, Shoumin

doi:https://doi.org/10.1155/2023/8897036

Journal of Chemistry

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Abstract Introduction Results and Discussion Conclusions Data Availability Conflicts of Interest Authors’ Contributions Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2023 | Article ID 8897036 | https://doi.org/10.1155/2023/8897036

A Simple Ratiometric Electrochemical Aptasensor Based on Exonuclease III-Assisted Target Recycling for Ultrasensitive Detection of Prostate Specific Antigen

Ping Wang,¹Yaoyao Xie,¹Zhimin Guan,¹Huikai Ma,¹and Shoumin Xi¹

Academic Editor: Takeshi Kondo

Received08 May 2023

Revised03 Aug 2023

Accepted04 Aug 2023

Published11 Aug 2023

Abstract

Prostate specific antigen (PSA) is considered as an important biomarker in prostate cancer diagnosis. Herein, an ultrasensitive ratiometric electrochemical aptasensor was constructed for analyzing PSA. The ferrocene (Fc)-labeled thiolated hairpin probe 2 (Fc-HP2) was conjugated to the gold nanoparticles (AuNPs)-modified gold electrode (AuE) surface by Au-S bonds. With the introduction of PSA, the aptamer region of hairpin probe 1 (HP1) preferred to bind with PSA, and the rest of HP1 could hybridize with Fc-HP2 to form Fc-HP2/HP1/PSA complex with a blunt 3′ terminus, which triggered the exonuclease III (Exo III) cleavage process accompanied by Fc leaving from the electrode and recycling of HP1/PSA. The remaining Fc-HP2 fragments left on the electrode surface were then hybridized with methylene blue-labeled DNA (MB-DNA). This resulted in an enhancement of MB signal (I_MB) and a decrement of Fc signal (I_Fc). Under the optimal conditions, the proposed aptasensor showed good analytical performance for sensitive detection of PSA with a linear range from 100 fg·mL⁻¹ to 10 ng·mL⁻¹ and a detection limit of 34.7 fg·mL⁻¹. The fabricated aptasensor had been applied to detect PSA in diluted human serum samples and obtained satisfied results, suggesting it had great potential in clinical diagnosis.

1. Introduction

Prostate cancer is one of the most common types of cancer affecting men worldwide, and in early stages, the symptoms are not obvious. When found in late stages, the mortality rate is high [1, 2]. Therefore, detection in its early stage is critical for the successful treatment of prostate cancer and it improves the survival rates. Prostate specific antigen (PSA), a single-chain serine protease produced by the epithelial cells of the prostate gland, is a commonly used biomarker in prostate cancer diagnosis [3]. In healthy male serum, the concentration of PSA is lower than 4.0 ng·mL⁻¹, and serum PSA levels above 10 ng·mL⁻¹ are likely to develop prostate cancer [1, 4]. Therefore, establishment of an ultrasensitive and simple method for the analysis of low levels of PSA is of great significance for the early diagnosis of prostate cancer.

Currently, a series of detection methods have been used for the detection of PSA, such as enzyme-linked immunosorbent assay (ELISA) [5], surface-enhanced Raman scattering (SERS) [6], colorimetric method [7], chemiluminescence immunoassay [8], and electrochemical methods [9, 10]. Among them, electrochemical methods have attracted the attention of researchers due to their high sensitivity, simple operation, low cost, and potential for miniaturization.

Early PSA electrochemical biosensors usually used antibody-based approaches, and other recognition elements, such as aptamers, are gaining the attention today. Aptamers are single-stranded DNA or RNA oligonucleotides isolated using systematic evolution of ligands by exponential enrichment (SELEX) [11]. They are capable of binding to a number of targets including proteins, small molecules, metal ions, viruses, bacteria, and even the whole cell with high affinity and specificity [12–14]. Compared with conventional antibodies, aptamers possess unique advantages such as high selectivity, low cost, better stability, and ease of chemical modification [15]. Different aptasensing platforms for PSA detection have been recently developed [16–19].

To improve the detection sensitivity, various signal amplification strategies are used to amplify the electrochemical signals. Among them, enzyme digestion amplification method [20] has been widely used recently. For example, exonuclease III (Exo III)-assisted target recycling amplification has emerged as a powerful amplification technique and attracted great attention due to its ease of operations and the simplicity of equipment [21–25]. Besides detection sensitivity, the reproducibility, stability, and reliability are also important indicators to investigate the performance of the sensors. Most of the reported electrochemical sensors used a single signal output, which are easily affected by internal or external factors such as electrode and environment. However, the ratiometric strategy [26–28] which uses two independent electrochemical signals of probes has been introduced to overcome the limitations. Methylene blue (MB) and ferrocene (Fc) are mainly selected as electrochemical species to fabricate ratiometric electrochemical aptasensors [29–32]. The analyte concentration can be determined by using the ratio of the peak current intensity of two electroactive probes. Therefore, the accuracy and reproducibility of detection can be greatly improved by eliminating the interference of the possible external and internal influence [33].

Herein, a simple ratiometric electrochemical aptasensor based on Exo III-assisted target recycling amplification was constructed for the detection of PSA (Scheme 1). Firstly, the gold nanoparticles (AuNPs)-modified gold electrode (AuE) was obtained through in situ electro deposition (AuNPs/AuE electrode). Subsequently, Fc-labeled hairpin probe 2 (Fc-HP2) was self-assembled onto the AuNPs/AuE surface via Au-S bond, and Fc was close to the electrode surface. MCH was used to the block the unbound sites. In the presence of target PSA, the aptamer sequence (red region) of hairpin probe 1 (HP1) bound specifically with PSA, and the hairpin structure of HP1 was opened to form HP1/PSA complex. Then, the rest fragment of HP1 (black region) hybridized with Fc-HP2 to form Fc-HP2/HP1/PSA complex with a blunt 3′ terminus. In the presence of Exo III, it could recognize the Fc-HP2/HP1/PSA complex and catalyze stepwise removal of mononucleotides from the blunt 3′ terminus, which led to the releasing of Fc molecules and liberating HP1/PSA complex to achieve Exo III-assisted signal amplification. The Fc-HP2 fragments left on the sensor surface were then hybridized with MB-labeled DNA (MB-DNA), making the MB confined near the electrode. As a result, the ratiometric assay for PSA detection based on the decrease of oxidation peak current of Fc (I_Fc) and accompanied by the increase of that of I_MB was achieved. By measuring the ratio of I_MB/I_Fc, PSA could be quantitatively analyzed. By combining the Exo III-assisted target recycling and ratiometric dual-signal strategy, the aptasensor has the advantage of good specificity and reproducibility and high sensitivity and low detection limit.

2. Experimental Section

2.1. Reagents and Materials

Hydrogen tetrachloroaurate trihydrate (HAuCl₄·3H₂O, ≥99.9%), tris (2-carboxyethyl) phosphine (TCEP, 98%), and 6-mercapto-1-hexanol (MCH, 98%) were all acquired from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Exo III (100 U·μL⁻¹) was obtained from New England Biolabs Ltd. (Beijing, China). PSA, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP) antigen, and human serum albumin (HSA) were purchased from Shanghai Linc-Bio Science Co., Ltd. (Shanghai, China). Ultrapure water (18.2 MΩ·cm) was used for preparing the solutions. Tris-ethylenediaminetetraacetic acid (EDTA) (TE) buffer (10 mM Tris-HCl, 1 mM EDTA, and pH 8.0) was purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China) and used to dilute DNA oligonucleotides. DNA immobilization buffer (I-buffer) was 20 mM Tris-HCl (50 mM NaCl, 10 mM MgCl₂, 10 mM TCEP, and pH 8.0); the electrode washing buffer (W-buffer) was 10 mM Tris-HCl (100 mM NaCl and pH 7.4); DNA hybridization buffer (H-buffer) was 10 mM Tris-HCl (1 mM EDTA, 500 mM NaCl, 1 mM MgCl₂, and pH 7.4). The solution for cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) was 0.1 M KCl solution containing 5 mM [Fe(CN)₆]^3−/4− (1 : 1). The oligonucleotides used in the experiment were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China), and the sequences were as follows: hairpin probe 1 (HP1): 5′-TTT TTA ATT AAA GCT CGC CAT CAA ATA GCT TTG GCG AGC TTT AAC GTC GAC AC-3′; Fc-labeled hairpin probe 2 (Fc-HP2): 5′-SH-SH-(CH₂)₆-CCA TAG CTT TAA GTG CGA CGT TAA AGC TCG CC-Fc-3′; and MB-DNA: 5′-TTA AAG CTA TGG-MB-3′.

2.2. Apparatus and Measurements

The Autolab PGSTAT 128N station (Metrohm Autolab, The Netherlands) was used for CV and EIS tests, and other electrochemical measurements were performed on a CHI840D electrochemical workstation (CH Instrument, China). A traditional three-electrode system was used with a modified gold electrode (AuE, 2 mm) as the working electrode, a platinum wire as the counter electrode, and an Ag/AgCl electrode as reference electrode, respectively.

2.3. Fabrication of the Aptasensor and PSA Detection

The gold electrode (AuE) was polished following the reported method [34]. Then, gold nanoparticles (AuNPs) were electrodeposited on AuE in 10 mM HAuCl₄ solution at −0.2 V for 200 s to obtain AuNPs/AuE. Prior to attachment to the AuNPs/AuE surface, thiolated Fc-HP2 probe was mixed with TCEP (1 mM) for 1 h at room temperature and diluted to 1 μM with I-buffer for use. 5 μL of Fc-HP2 was applied to the AuNPs/AuE surface and incubated for 12 h at 4°C. After thoroughly rinsed with W-buffer, MCH (1 mM) was used to block the unoccupied binding sites for 1 h to obtain the sensing interface (MCH/Fc-HP2/AuNPs/AuE).

Firstly, different concentrations of PSA were mixed with HP1 (1 μM) and Exo III (2 U) in 50 mM Tris-HCl (10 mM MgCl₂ and pH 7.4). Then, 5 μL of the above solution was dropped on the MCH/Fc-HP2/AuNPs/AuE surface and incubated for 80 min at 37°C to perform the Exo III-assisted recycling process. After washing with W-buffer, the modified electrode was incubated with MB-DNA (1 μM) in H-buffer at 37°C for 1 h and rinsed with ultrapure water. Finally, the electrode was subjected to the square-wave voltammetry (SWV) (step potential, 4 mV; frequency, 25 Hz; amplitude, 25 mV) test in 10 mM Tris-HCl (pH 7.4) from −0.4 to 0.6 V.

3. Results and Discussion

3.1. Feasibility of the Aptasensor

To illustrate the feasibility of the constructed aptasensor for PSA detection, a set of SWV experiments were conducted. As depicted in Figure 1, a low peak current of MB at −0.240 V and a high peak current of Fc at 0.368 V were observed when in the absence of PSA and Exo III (curve a). In the presence of only PSA (curve b) or Exo III (curve c), the SWV intensity almost changed compared to that of curve a, indicating that the PSA or Exo III alone could not trigger the amplification reaction. However, an obvious reduced Fc signal and increased MB signal were obtained in the presence of both PSA and Exo III (curve d), suggesting the occurrence of the Exo III-assisted signal amplification process and Fc was released from the electrode surface. Then, MB-DNA hybridized with the Fc-HP2 fragment was left on the electrode, and MB was near to the electrode surface. These results clearly demonstrated the feasibility of the ratiometric aptasensor for PSA detection.

3.2. Verification of Exo III-Assisted Target Recycling Process

15% polyacrylamide gel electrophoresis (PAGE) was employed to verify the Exo III-assisted target recycling process. As shown in Figure 2, lane 1 and 2 represented the bands of HP1 and Fc-HP2, respectively. A new band with slower migration shift was observed (lane 3) after the hybridization of HP1 and Fc-HP2 with the presence of PSA. When Exo III was added, a new band with faster migration shift was displayed in lane 4, which was attributed to the generation of short output DNAs digested from the duplex by Exo III. After the addition of MB-DNA, a band migrated slower than lane 4 was appeared due to the success of DNA hybridization (lane 5).

3.3. Characterization of the Modified Electrodes

The stepwise fabrication of the aptasensor was monitored by EIS and CV, and the results are displayed in Figures 3(a) and 3(b), respectively. Compared with the bare AuE (Figures 3(a) and 3(b), curve (a)), the value of electron transfer resistance (R_et) was significantly reduced and the corresponding redox peak current was obviously increased after being modified with AuNPs (Figures 3(a) and 3(b), curve (b)), implying the excellent electrical conductivity of AuNPs. The assembly of Fc-HP2 on the electrode surface led to an increase in R_et and a decrease of the redox peak current (Figures 3(a) and 3(b), curve (c)) due to the electrostatic repulsion between the negatively charged probes and [Fe(CN)₆]^3−/4−. Blocking with MCH led to a further enhancement in R_et and reduced redox peak current (Figures 3(a) and 3(b), curve (d)). Subsequently, HP1 recognized PSA and paired Fc-HP2 on the electrode to form a Fc-HP2/HP1/PSA complex. With the addition of Exo III, the R_et decreased obviously and the redox peak current increased (Figures 3(a) and 3(b), curve (e)), which was ascribed to the fact that Fc-HP2 probes were digested by Exo III. The introduction of MB-DNA on the electrode surface led to the increase of R_et and decrease of the redox peak current again (Figures 3(a) and 3(b), curve (f)), indicating the success of DNA hybridization. The CV results were in line with the EIS results, demonstrating the successful fabrication of the aptasensor.

(a)

(b)

3.4. Optimization of Experimental Conditions

The experimental conditions including the Fc-HP2 probe concentration, Exo III dosage, and incubation time of the Exo III were optimized, and the results are presented in Figure 4. As shown in Figure 4(a), the I_MB/I_Fc values increased when the concentration of Fc-HP2 increased from 0.25 to 1 μM and then began to decrease with the increasing of concentration, which may be due to the steric hindrance at high concentrations. Hence, 1 μM was the optimized concentration for Fc-HP2. The dosage of Exo III was also optimized. As displayed in Figure 4(b), increment in the concentration of Exo III from 0.5 to 2 U led to the increased in I_MB/I_Fc values and reached the maximum at 2 U. Therefore, 2 U was selected as the optimal dosage for Exo III. The effect of incubation time of Exo III was also investigated. In the range from 20 to 80 min, the I_MB/I_Fc values increased with the increase of time and reached the maximum at 80 min, indicating that the Fc-HP2 probes on the electrode surface were almost completely digested. Thus, the best incubation time of Exo III was 80 min (Figure 4(c)).

(a)

(b)

(c)

3.5. Analytical Performance of the Ratiometric Electrochemical Aptasensor

Under optimal experimental conditions, the proposed aptasensor was employed to detect PSA at different concentrations. Figure 5(a) demonstrates the relationship between the SWV responses and PSA concentration. With the increment of PSA concentration, more HP2 probes were digested by Exo III and many MB-DNA probes were captured on the electrode surface, so that I_Fc gradually decreased, while I_MB increased. Figure 5(b) showes the relationship between the peak currents and the logarithm of PSA concentration, and from the figure, we could see that the peak current of MB was positively related to logarithm of PSA concentration, while the peak current of Fc was negatively related to logarithm of PSA concentration. A good linear relationship between I_MB/I_Fc and the logarithm of PSA concentration in the range of 100 fg·mL⁻¹ to 10 ng·mL⁻¹ could be seen in Figure 5(c). The obtained linear equation was I_MB/I_Fc = 0.102 lgC + 1.160 (R² = 0.9979) with a limit of detection (LOD) of 34.7 fg·mL⁻¹ (S/N = 3). In addition, our proposed aptasensor was compared with the previously reported PSA detection methods [35–41]. As shown in Table 1, the analytical performance of the aptasensor developed in this study was comparable or better than those of the reported methods.

(a)

(b)

(c)

3.6. Specificity, Reproducibility, and Stability Test

CEA, AFP, and HSA were employed as interferences to demonstrate the specificity of the proposed aptasensor. Although the concentration of the interferences (100 ng·mL−1) was 10-fold higher than that of PSA (10 ng·mL−1), their response signals were obviously lower than that of PSA (Figure 6), indicating acceptable specificity. The reproducibility of the aptasensor was investigated by using five aptasensors prepared under the same conditions to detect 1 ng·mL⁻¹ PSA. The relative standard deviation (RSD) obtained was 3.3%, suggesting its good reproducibility. Furthermore, when the aptasensor was stored for 4°C for one and two weeks, it still maintained 90.5% and 85.1% of the original signal, respectively, indicating its good stability.

3.7. Analysis of Serum Samples

To assess the clinical application of the aptasensor in biological samples, three healthy human serum samples (obtained from Henan University of Science and Technology Hospital) were diluted 10-fold with 20 mM phosphate buffered saline (pH 7.4) and spiked with PSA. The recoveries were from 89.0% to 102.3% with a RSD of 2.1%–4.6% (Table 2), indicating the proposed aptasensor had good accuracy in clinical analysis.

4. Conclusions

In summary, we constructed a simple ratiometric electrochemical aptasensor for ultrasensitive detection of PSA. Compared with conventional electrochemical aptasensors, this method only used two hairpin DNA probes and a MB-labeled DNA, thus reducing the measurement cost. Secondly, the ratiometric signal of I_MB/I_Fc was used to detect the target, which could improve the assay reproducibility and accuracy. Thirdly, the AuNPs-modified electrode provided a large surface area for Fc-HP2 immobilization and improved electron conductivity. The utilization of Exo III-assisted target recycling could further enhance the detection sensitivity. In addition, the proposed strategy had been applied to detect PSA in diluted human serum samples. Although good performances have been achieved, the present method required multiple hybridization and washing steps, which was the main limitation of the study. Further design of the simple and new aptamer-based assays for PSA detection might be the future scope.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

Ping Wang was involved in methodology and investigation and wrote the original draft. Yaoyao Xie conducted experiments and assisted in the writing of the article. Zhimin Guan and Huikai Ma constructed figures and tables. Shoumin Xi conceptualized and supervised the study.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (61701171).

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Copyright © 2023 Ping Wang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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