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Cascade Signal Amplification Based on Copper NanoparticleReported Rolling Circle Amplification for Ultrasensitive Electrochemical Detection of the Prostate Cancer Biomarker Ye Zhu, Huijuan Wang, Lin Wang, Jing Zhu, and Wei Jiang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b10285 • Publication Date (Web): 14 Jan 2016 Downloaded from http://pubs.acs.org on January 19, 2016

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ACS Applied Materials & Interfaces

Cascade Signal Amplification Based on Copper Nanoparticle-Reported Rolling Circle Amplification for Ultrasensitive Electrochemical Detection of the Prostate Cancer Biomarker Ye Zhu a, Huijuan Wang b, Lin Wang c, Jing Zhu a, Wei Jiang a* a

Key Laboratory of Colloid and Interface Chemistry of Education Ministry, School of Chemistry

and Chemical Engineering, Shandong University, Jinan 250100, China. b

School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China.

c

Department of Radiation Oncology, Qilu Hospital, Shandong University, Jinan 250012, China.

KEYWORDS: rolling circle amplification, poly(thymine)-templated copper nanoparticle, signal amplification, electrochemical detection, prostate specific antigen

ACS Paragon Plus Environment

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ABSTRACT: An ultrasensitive and highly selective electrochemical assay was first attempted by combining the rolling circle amplification (RCA) reaction with poly(thymine)-templated copper nanoparticles (CuNPs) for cascade signal amplification. As proof of concept, prostate specific antigen (PSA) was selected as a model target. Using a gold nanoparticle (AuNP) as a carrier, the primer-AuNP-aptamer bioconjugate was synthesized for signal amplification by increasing

the

primer/aptamer

ratio.

The

specific

construction

of

primer-AuNP-

aptamer/PSA/anti-PSA sandwich structure triggered the effective RCA reaction, in which thousands of tandem poly-thymine repeats were generated and directly served as the specific templates for the subsequent CuNP formation. The signal readout was easily achieved by dissolving the RCA product-templated CuNPs and detecting the released copper ions with differential pulse stripping voltammetry. Because of the designed cascade signal amplification strategy, the newly developed method achieved a linear range of 0.05 fg/mL - 500 fg/mL, with a remarkable detection limit of 0.020 ± 0.001 fg/mL PSA. Finally, the feasibility of the developed method for practical application was investigated by analyzing PSA in the real clinical human serum samples. The ultrasensitivity, specificity, convenience, and capability for analyzing the clinical samples demonstrate that this method has great potential for practical disease diagnosis applications.


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INTRODUCTION A type of newly emerged signal reporters, DNA-templated metal nanoparticles, have attracted increasing interest in bioanalysis by virtue of their low toxicity, good biocompatibility, excellent optical properties, as well as facile integration with DNA-based recognition and signal amplification strategies.1-3 For example, cytosine-rich DNA can serve as an efficient template for the formation of silver nanoclusters, which has been widely utilized for the detection of nucleic acids, small molecules, proteins, and cancer cells.4 In particular, He and coworkers synthesized DNA-stabilized silver nanoclusters wires using the DNA amplification product as the scaffold, which exhibited great potential in sensing applications.5 In addition to DNA-templated silver nanoclusters, DNA-templated copper nanoparticles (CuNPs), including random double stranded DNA-templated CuNPs (dsDNA-templated CuNPs) and poly(thymine)-templated CuNPs,6,7 have been attracting growing attention for biosensing applications. The synthesis of DNAtemplated CuNPs can be completed within several minutes after the reaction beginning under ambient conditions, which is much faster and more convenient than the synthesis of other DNAtemplated metal nanoparticles.8 Highly efficient, simple, and rapid synthesis process enables DNA-templated CuNPs to be a promising signal reporter candidate for various bioanalytical applications. For example, dsDNA-templated CuNPs have been employed for the detection of small molecules,9 enzyme activity,10 nucleic acids,11 and metal ions.12 Furthermore, dsDNAtemplated CuNPs have been combined with DNA amplification techniques to achieve high sensitivity.1,13,14 Compared with dsDNA-templated CuNPs, the exploration of poly(thymine)-templated CuNPs for bioanalytical applications is still at a very early stage.8,15 The formation of poly(thymine)templated CuNPs is deemed to result from binding interactions between thymine and Cu2+ ions,


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which facilitate the formation of the thymine-Cu2+ complex. Next, the thymine-complexed Cu2+ ions are reduced to Cu0 by ascorbic acid, thereby leading to the formation of CuNPs along the contour of the poly(thymine) template.7,8 Note that poly(thymine)-templated CuNPs can be synthesized without hybridization of a secondary strand, that is, just a single stranded sequence containing more than 15 continuous thymine bases can induce obvious CuNP formation,7 which is simple and highly specific. Moreover, the productivity of CuNPs is highly dependent on the length and amount of poly(thymine) sequences,7 which holds an immense potential of being combined with programmable DNA amplification techniques for highly selective and sensitive biosensing applications. However, as summarized in Table S1 in the supporting information, most reported poly(thymine) templates used for CuNP formation are ready-made short sequences containing continuous 25-40 thymine bases.8,16-24 Compared with short poly(thymine) templates, poly(thymine) sequences that were synthesized through terminal deoxynucleotidyl transferaseassisted polymerization provide controllable templates for CuNP formation and superior sensitivity for analytical applications.15,25 In view of DNA’s high programmability in terms of length and sequence composition, the exploration of highly effective and flexible DNA templates for CuNP synthesis in ultrasensitive and versatile biochemical applications is still highly desirable.26,27 As a DNA amplification technique, rolling circle amplification (RCA) is a simple and powerful isothermal enzymatic process, in which a short DNA or RNA primer is elongated to form a long single stranded DNA or RNA with the assistance of a circular DNA template and unique DNA or RNA polymerases.28,29 Due to its speed, high efficiency, and specificity, RCA has been widely employed for signal amplification in numerous analytical applications, such as detection of nucleic acids, small molecules, proteins, and diseased cells, allowing detection of


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targets even at the aM concentration level.30-33 The RCA product contains thousands of tandem repeats that are complementary to the circular template. Effective signal readout approaches that can transduce these tandem repeats into detectable signals are crucial to achieve various analytical purposes.34-39 With the elaborative design of the circular DNA template sequence, RCA can be programmed to generate thousands of single stranded tandem repeats, which can directly serve as the template for CuNP formation. It can be imagined that RCA producttemplated CuNPs would be a perfect signal amplification strategy for an extremely sensitive bioanalysis applications. To date, almost all the reported analytical methods based on poly(thymine)-templated CuNPs are fluorescent (Table S1). Although these fluorescent methods provide considerable sensitivity, the fluorescence emission of monomeric CuNPs can last for only 20 min and thereafter decreases quickly, which might greatly impede its long-time monitoring application.1 It would be interesting to overcome this disadvantage by reading the signal through the electrochemical method, which shows superiority in the determination of metal ions.14,40 In the present work, a cascade signal amplification strategy based on RCA product-templated CuNPs was developed in electrochemical readout format for the detection of prostate specific antigen (PSA) as a model target molecule. PSA is secreted by prostatic epithelial cells and is present in the serum of healthy males at a low level (0 - 4 ng/mL), but it is elevated in males with prostate cancer and other prostate disorders. PSA is the most common serum marker for diagnosing prostate cancer and monitoring the recurrence of the disease after treatment.41,42 As illustrated in Scheme 1, using a gold nanoparticle (AuNP) as a carrier, the primer-AuNP-aptamer bioconjugate was first synthesized for signal amplification by increasing the primer/aptamer ratio. The formation of the sandwich construction of primer-AuNP-aptamer/PSA/anti-PSA led to RCA reaction and


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subsequent formation of CuNPs. The RCA process was demonstrated using gel electrophoresis. The formation of RCA product-templated CuNPs was characterized using a transmission electron microscope (TEM) and an atomic force microscope (AFM). Large amounts of copper ions released from the dissolution of the RCA product-templated CuNPs were detected by differential pulse stripping voltammetry (DPSV), which was utilized to evaluate the amount of PSA. The experimental conditions that might affect the analytical performance were optimized. The calibration curve and the detection limit for PSA analysis were determined as well. Finally, the amount of PSA in real clinical human serum samples was determined by using the developed method.

Scheme 1. Schematic Representation of Cascade Signal Amplification for the Detection of a Prostate Cancer Biomarker.

EXPERIMENTAL SECTION


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Chemicals and Materials. Human prostate specific antigen (PSA), monoclonal anti-human PSA (anti-PSA) from mouse, alpha fetoprotein (AFP), and carcino-embryonic antigen (CEA) were purchased from Shanghai Linc-Bio Science Co., Ltd. (Shanghai, China). 5'–thiolated PSAspecific DNA aptamer (5’-SH-TTA TTA TTA AAT TAA AGC TCG CCA TCA AAT AGC TTT-3’),41 5'–thiolated primer (5’-SH-TTA TTA TTA TGT CCG TGC TAG AAG GAA ACA GTT AC-3’), circular template (5′-p-TAG CAC GGA CAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AGT AAC TGT TTC CTT C-3′), bovine serum albumin (BSA), thrombin, and human immunoglobulin G (H-IgG) were obtained from Shanghai Sangon Biotech Co., Ltd. (China). Phi29 DNA polymerase, T4 DNA ligase, and dNTPs were purchased from Thermol Fermentas (Lithuania). Human serum samples were collected from Qilu Hospital. Phosphate-buffered saline (PBS) was prepared with 0.01 M disodium hydrogen phosphate, 0.01 M sodium dihydrogen phosphate, and 0.9% sodium chloride. Carbonate buffer solution (0.05 M, pH 9.6) prepared with sodium carbonate and sodium bicarbonate was used for antibody immobilization. TE buffer (pH 8.0) prepared with 10 mM tris(hydroxymethyl)aminomethane and 1 mM ethylenediaminetetraacetic acid was used to dilute DNA sequences. All other chemicals were of extra pure analytical grade and were used without further purification. Ultra-pure water that was purified with a resistance of 18.25 MΩ cm was used to prepare all aqueous solutions. Apparatus. The electrochemical measurements were performed on a CHI 660E electrochemistry workstation (Shanghai Chenhua, China) with an electrochemical cell containing a gold disk electrode (area 0.07 cm2), a Ag/AgCl electrode (in saturated KCl), and a platinum wire as the working, reference, and counter electrode, respectively. The formation of CuNPs was


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demonstrated from images taken using a transmission electron microscope (JEOL JEM-2100) and an atomic force microscope (Veeco Instruments, USA). Preparation

of

Primer-AuNP-Aptamer

Bioconjugate.

The

primer-AuNP-aptamer

bioconjugate was prepared via the formation of the Au-S bond between AuNPs and DNA sequences. First, AuNPs with diameters of approximately 10 nm were prepared according to the previously reported procedure.43 Briefly, 50 mL of 0.01 wt% HAuCl4 in H2O was mixed with 1 mL of 38.8 mM trisodium citrate. After 1 min, 0.5 mL of a freshly prepared NaBH4 solution was slowly added into the mixture. During the addition of NaBH4, the color of the resulting solution turned from yellow to pink, indicating the formation of AuNPs. Next, the obtained AuNPs were mixed with the thiolated aptamer (200 nM) and the thiolated primer (1 µM), followed by incubation at 4 ℃ for 12 h to form the Au-S bond. Subsequently, the unreacted DNA sequences were removed by centrifugation (13 000 rpm, 15 min). Afterward, the supernatant was removed, and the precipitate was washed three times with TE buffer. The resulting primer-AuNP-aptamer bioconjugate was dispersed in a 0.1 M TE buffer for further use. Construction of the Primer-AuNP-Aptamer/PSA/Anti-PSA Sandwich Structure. The principle and procedure for detection of PSA is illustrated in Scheme 1. In total, 50 µg/mL of anti-PSA (25 µL) in carbonate buffer solution was added into one well of a blank polystyrene plate and was incubated at 4 ℃ overnight to immobilize anti-PSA onto the surface of the well. The excess anti-PSA was thoroughly washed with carbonate buffer solution. To reduce unspecific adsorption, 1% BSA was incubated in the well at 37 ℃ for 1 h to block the unreacted active sites. After washing, PSA in 10 mM PBS (pH 7.4) was added into the anti-PSAimmobilized well, and this solution was incubated at 37 ℃ for 1 h. Next, the well was washed with PBS to remove the unbound PSA. Afterward, the prepared primer-AuNP-aptamer


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bioconjugate was added into the well and then incubated at 37 ℃ for 1 h, resulting in the immobilization of the bioconjugate through the recognition and binding between the aptamer and PSA. As a result, the sandwich structure of the primer-AuNP-aptamer/PSA/anti-PSA was constructed through the dual recognition of PSA by anti-PSA and PSA-specific aptamer. RCA Reaction and Formation of CuNPs. In total, 1 µM circular template was annealed at 90 ℃ for 5 min and then was added into the well to incubate with the primer in the primer-AuNPaptamer/PSA/anti-PSA construction at 37 ℃ for 30 min.36,38 Subsequently, 0.125 µL of T4 DNA ligase and 2.5 µL of 10× ligase buffer (400 mM Tris-HCl, 100 mM MgCl2, 100 mM DTT, 5 mM ATP, pH 7.8) were added into the well and incubated at 37 ℃ for 1 h. After ligation, the solution in the well was removed. To conduct the RCA reaction, 2.5 µL 10× reaction buffer (330 mM Tris–acetate, 100 mM Mg(Ac)2, 660 mM potassium acetate (KAc), 1% Tween 20 and 10 mM DTT, pH 7.9), 0.125 µL (10 u/µL) phi29 DNA polymerase, 5 µL 10 mM dNTPs, and 17.5 µL H2O were added into the well and incubated at 37 ℃ for 80 min. Next, the well was washed with TE buffer three times. According to the previous report,7 formation of CuNPs on the RCA product was conducted by adding sodium ascorbate and copper sulfate into the well. The CuNP formation reaction proceeded at room temperature for 30 min and then the well was gently washed three times. Electrochemical Measurements. Firstly, a gold disk electrode was polished to a mirror finish using alumina slurry (0.05 µm) on chamois leather, followed by sonication for 15 s to remove the alumina particles and other impurities. Then, the electrode was rinsed with ultrapure water and ethanol successively.44 To further clean and activate the electrode surface, the gold disk electrode was placed into a 0.5 M H2SO4 solution and then treated with cyclic voltammetry (CV) between −0.2 V and 1.6 V at 100 mV/s until a steady voltammogram was obtained. Next, the


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electrode was thoroughly cleaned with ultrapure water for further use. The CuNPs formed on the RCA product were converted into Cu2+ by the dissolution of concentrated nitric acid for 30 min. Subsequently, the resulting solution was diluted to 0.5 M HNO3. The obtained Cu2+ ions were detected by using the activated gold disk electrode. CV was performed between -0.2 V and 0.6 V at a scan rate of 50 mV/s. DPSV was performed from -0.2 V to 0.6 V under the following conditions: increased potential, 0.004 V; amplitude, 0.05 V; pulse width, 0.05 s; pulse period, 0.5 s; deposition potential, -0.5 V; deposition time, 300 s. Agarose Electrophoresis Analysis. Agarose gel electrophoresis was performed to demonstrate the RCA reaction. Briefly, 1 µM free primer and 1 µM circular template were mixed and annealed. Next, the RCA reaction was conducted with the assistance of T4 DNA ligase and phi29 DNA polymerase, as described in the preceding section. Meanwhile, the control experiment without primer was performed in the same procedure. The obtained reaction product was characterized with agarose gel electrophoresis, which was performed with 0.7% agarose gel in 1× TAE buffer (40 mM tris(hydroxymethyl)aminomethane, 20 mM acetic acid, and 2.0 mM ethylenediaminetetraacetic acid, pH 8.3) at a constant voltage of 120 V for 1.5 h. After staining with ethidium bromide for 5 min, the gel was imaged on a Tanon-2500R gel imaging system (Shanghai, China).

RESULTS AND DISCUSSION Principle of the Design. The principle of the developed method is illustrated in Scheme 1. The anti-PSA immobilized in the plate well and the PSA-specific aptamer on the primer-AuNPaptamer bioconjugate could specifically recognize PSA and form the sandwich construction of primer-AuNP-aptamer/PSA/anti-PSA. Herein, a AuNP served as a carrier to increase the


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primer/aptamer ratio, thereby amplifying the signal reflecting the aptamer/PSA/anti-PSA binding events. In the presence of T4 ligase and the circular template, the primer on the primer-AuNPaptamer bioconjugate could hybridize with the circular template and further trigger the RCA reaction with the assistance of phi29 DNA polymerase. Because the circular template was designed containing a continuous segment of 50 adenine bases, the resulting RCA product ought to be a long single stranded DNA containing thousands of tandem repeats of corresponding continuous 50 thymine bases, which are the specific template for CuNP formation in the presence of Cu2+ and ascorbate. Upon the addition of nitric acid, the RCA product-templated CuNPs could be dissolved, thereby releasing large amounts of Cu2+ and leading to significant electrochemical response in the subsequent stripping voltammetric measurement. In contrast, in the absence of a PSA target, the following process would not proceed, thus, no electrochemical response could be detected. Therefore, the sensitive detection of target molecule could be achieved through the developed CuNP-reported RCA strategy. Characterizations of the Cascade Signal Amplification Strategy. The agarose gel electrophoresis experiment was performed to verify the RCA reaction. As shown in Figure 1A, the RCA product exhibited extremely low mobility (lane a), suggesting the high molecular weight of the product. According to the indication of the marker (lane c), the number of the bases contained in the RCA product was estimated to be much higher than 15000 bp, indicating that the RCA reaction successfully occurred. In contrast, there was no band observed for the control experiment where the RCA reaction could not be triggered without the primer (lane b). These results demonstrated that the RCA reaction can be utilized for enormous signal amplification in bioanalysis.


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The formation of CuNPs was characterized via TEM using the free primer triggered RCA product. As displayed in Figure 1B, gray salt precipitates were observed in the image of the RCA product, which were actually the salt residues surrounding individual DNA-coils. The individual RCA-coil revealed a diameter of approximately 2.5 µm, which was comparable with the previous report.45 Interestingly, after the reaction with Cu2+ and ascorbate, accumulated particles appeared in the RCA-coil (Figure 1C). Obviously, the diameters of these particles were much larger than the CuNPs formed on a short fifty thymine sequence (approximately 7 nm, Figure S1 in supporting information). The increase of the CuNP size in this work was mainly attributed to the aggregation of CuNPs that resulted from the vast tandem 50-thymine repeats in one individual DNA-coil.

Figure 1. (A) Agarose gel (0.7%) electrophoresis images for (a) RCA product, (b) control experiment (without primer), and (c) marker; the band marked with the dashed circle represents 15000 bp. (B) TEM image of an individual RCA-coil. (C) TEM image of the RCA producttemplated CuNPs.


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Figure 2. AFM images of (A) an individual RCA-coil and (C) RCA product-templated CuNPs; (B) and (D) show the high resolution AFM images of the marked areas in image A and image C; (E) phase image of image D; (F) and (G) show the height analysis of the marked positions in image B and image D.

Free primer triggered RCA product and RCA product-templated CuNPs were further characterized using AFM, as exhibited in Figure 2. An individual DNA-coil with a diameter approximately 2.5 µm was observed for the RCA product (Figure 2A), which was in good agreement with that observed using TEM. The marked area in Figure 2A was characterized at high resolution and analyzed (Figures 2B and 2F). The height of DNA sequence was determined to be about 2 nm. After formation of the CuNPs, agglomerated particles were observed on the RCA-coil (Figure 2C); the marked area in Figure 2C containing a thin chain was carefully characterized (Figures 2D and 2E). Completely different from the RCA sequence in image 2B, this thin chain was composed of particles, as is more obvious in its phase image (Figure 2E). The


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height of this thin chain was determined to be about 45 nm (Figure 2G). The center position of this stack of RCA product-templated CuNPs in image C exhibited the largest height, which was approximately 150 nm and was attributed to the accumulation of CuNPs. TEM and AFM characterization demonstrated the successful synthesis of RCA products and the formation of RCA product-templated CuNPs. The electrochemical characterization of the developed method was performed with CV and DPSV. Figure 3A shows the CV responses toward the blank experiment without PSA and the presence of 1.0 pg/mL PSA. The CV for the blank experiment did not show any redox peak (curve a), whereas the CV for 1.0 pg/mL PSA exhibited a pair of significant redox peaks at approximately 0.33 / 0.28 V (curve b), which corresponded to the redox reaction of the dissolved Cu2+,46 demonstrating the feasibility of the developed CuNP-reported RCA signal amplification strategy for PSA detection. In addition, the response toward the presence of 1.0 pg/mL PSA was further amplified by DPSV, where the copper ions were electrochemically pre-concentrated and reduced onto the electrode surface. Then, the deposited copper was electrochemically oxidized with an enhanced oxidation current response (the inset of Figure 3A), thereby improving the sensitivity of the detection method. Therefore, DPSV was performed in the following experiments.


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Figure 3. (A) CV responses of the developed method toward the blank experiment without PSA (a) and the presence of 1.0 pg/mL PSA (b); the arrow marks the initial scanning direction of CVs, and the inset shows the corresponding DPSV responses; (B) DPSV responses toward 1.0 pg/mL PSA by using primer-AuNP-aptamer bioconjugate (a) and primer-aptamer (b).

To verify the role of the AuNPs in signal amplification, a control experiment without the AuNP carrier was conducted, where a sequence consisting of primer segment and aptamer segment (denoted as primer-aptamer) was used instead of the primer-AuNP-aptamer bioconjugate. The concentration of 1.0 pg/mL PSA was detected based on the formed primeraptamer/PSA/anti-PSA sandwich construction and the subsequent RCA reaction and CuNP formation. Cu2+ dissolved from the formed CuNPs was measured by DPSV. The DPSV


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responses toward the 1.0 pg/mL PSA by using the primer-AuNP-aptamer bioconjugate and the primer-aptamer were compared in Figure 3B (curve a and curve b, respectively). Obviously, the peak current intensity using the primer-AuNP-aptamer bioconjugate was about 7-fold higher than that using the primer-aptamer, demonstrating the ability of the primer-AuNP-aptamer bioconjugate to amplify the signal reflecting the aptamer/PSA/anti-PSA binding events. Optimization of the Experimental Conditions. To achieve the best performance of the developed method, the experimental conditions that might significantly affect the analytical performance were optimized, including the primer/aptamer ratio in the primer-AuNP-aptamer bioconjugate, the RCA reaction time, and the deposition potential and deposition time parameters for DPSV. To develop the best ability of the primer-AuNP-aptamer bioconjugate for signal amplification, the effect of the primer/aptamer ratio on the assay response was investigated by using the primer-AuNP-aptamer bioconjugate that was synthesized with varying primer/aptamer ratios (2:1, 3:1, 4:1, 5:1, 6:1, and 7:1 with a fixed aptamer concentration of 200 nM). As shown in Figure 4A, the current response significantly increased with the increasing primer/aptamer ratio and reached the highest value at a primer/aptamer ratio of 5:1, and then decreased when the primer/aptamer ratio was higher than 5:1. The decrease of the current might be attributed to the excess primers occupying the binding sites on AuNPs belonging to the aptamer, which may even result in a bioconjugate without aptamer. Thus, the primer/aptamer ratio of 5:1 was adopted to synthesize the primer-AuNP-aptamer bioconjugate.


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Figure 4. The effects of the (A) primer/aptamer ratio in the primer-AuNP-aptamer bioconjugate, (B) RCA reaction time, (C) deposition potential, and (D) deposition time for DPSV measurement on the analytical performance of the developed method toward 1.0 pg/mL PSA in PBS.

To generate a long RCA product sequence for significant signal amplification, the effect of the RCA reaction time on the electrochemical response was studied, as shown in Figure 4B. At the beginning, the response significantly increased with the increasing RCA reaction time, indicating the effective extension of the RCA product. However, the increased rate of the response slowed after 60 min, and then the saturation of the RCA product was observed at 80 min, which might be caused by the exhaustion of the RCA substrates or the inactivation of the phi29 DNA


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polymerase.38 Thus, 80 min was selected as the adequate reaction time for RCA in the following experiments. Because the concentration of the dissolved Cu2+ reflected the amount of PSA, the sensitive detection of Cu2+ was of significant importance in the whole assay. Therefore, the important DPSV parameters in terms of deposition potential and deposition time were optimized. The effect of the deposition potential on the electrochemical signal was investigated by applying a range of potentials for copper deposition. As exhibited in Figure 4C, the current response gradually increased as the applied potential varied from -0.1 V to -0.5 V and then almost reached a constant above -0.5 V. Thus, -0.5 V was applied as the deposition potential for the DPSV analysis of Cu2+. The effect of the deposition time on the electrochemical signal is displayed in Figure 4D. The current response rapidly increased with the increasing deposition time. However, there was no significant increase in the current observed when the deposition time was longer than 300 s due to the saturation of the deposited copper. Therefore, the deposition time for Cu2+ analysis was fixed at 300 s in the following experiments. Detection of PSA. Under the above optimal experimental conditions, varying concentrations of PSA in 10 mM PBS was detected by using the developed method. The overall detection time for PSA determination was about 6 h. The obtained DPSVs are exhibited in Figure 5A, which showed a gradual increase in the current with the PSA concentration increasing from 0 to 1000 fg/mL (from a to m). The corresponding calibration curve in Figure 5B exhibited a linear relationship between -Ip (the negative value of the peak current of DPSV) and log (cPSA (fg/mL)) in the range of 0.05 fg/mL to 500 fg/mL PSA. The linear dependencies of PSA analysis yielded an equation, -Ip (µA) = (5.96 ± 0.09) + (3.48 ± 0.08) [log (cPSA (fg/mL))], with a correlation coefficient of 0.995. Based on five measurements for the standard deviation of the blank noise


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(95% confidence level, k = 3, n = 5), the detection limit was determined to be 0.020 ± 0.001 fg/mL PSA, which was more than four orders-of-magnitude lower than the previously reported electrochemical methods for PSA detection.42,47-49 The ultrasensitivity for bioanalysis was finally achieved by the developed cascade signal amplification strategy.

Figure 5. (A) DPSV responses of the developed method toward varying PSA concentrations (from a to m: 0, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 500, and 1000 fg/mL); (B) the corresponding calibration curve.

To prove the signal amplification was achieved by the primer-AuNP-aptamer bioconjugateinduced RCA products, a DNA sequence composed of PSA specific aptamer and continuous fifty thymine bases (denoted as aptamer-T50) was used as a substitute and served as the template


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for CuNP formation to detect various concentrations of PSA. The obtained DPSVs and corresponding calibration curve are shown in Figure S2 in the supporting information. The linear relationship between -Ip and log (cPSA (ng/mL)) could be expressed as -Ip (µA) = (0.98 ± 0.02) + (0.52 ± 0.02) [log (cPSA (ng/mL))] with a correlation coefficient of 0.992. The detection limit was determined to be 0.055 ± 0.004 ng/mL PSA, which was approximately six orders-of-magnitude higher than the developed method using the primer-AuNP-aptamer bioconjugate. This result demonstrated that the effective signal amplification was successfully achieved by the developed strategy. Selectivity and Repeatability. To study the selectivity of the developed method, several biomolecules that might coexist with PSA in the real serum samples were detected, including AFP, CEA, thrombin, and H-IgG. The electrochemical responses of the developed method toward different analytes are exhibited in Figure 6. Obviously, a significant current response was obtained for 100 fg/mL PSA, whereas a negligible response was observed for 10 pg/mL AFP, CEA, thrombin, and H-IgG, indicating the satisfying selectivity of the developed method toward PSA. Such excellent selectivity was attributed to the dual recognition of PSA via the anti-PSA and the PSA-specific aptamer.


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Figure 6. Electrochemical responses of the developed method toward 100 fg/ml PSA, 10 pg/ml AFP, 10 pg/ml CEA, 10 pg/ml thrombin, and 10 pg/ml human IgG.

To investigate the repeatability of the developed assay, a batch of five parallel experiments was conducted under the same condition, where the same concentration of PSA was detected. As a result, a relative standard deviation of 6.8% was achieved, indicating good repeatability of the developed method. Real Sample Analysis. The capability of the developed method for detecting spiked PSA in real serum samples was first studied. In consideration of the PSA level in serum (0 - 4 ng/mL for healthy people and an elevated level for prostate cancer and other prostate disorders) and the linear range of the developed method (0.05 - 500 fg/mL PSA in PBS), serum from healthy people was firstly diluted 106 times with PBS and then used as the real matrix sample. Next, a standard solution of PSA was spiked into the diluted serum sample to obtain final PSA concentrations ranging from 0 to 1000 fg/mL that were detected by the developed method. The obtained DPSVs and the corresponding calibration plot are shown in Figure S3 in the supporting information. The relationship of -Ip vs. log (cPSA (fg/mL)) exhibited a linear response in the range of 0.1 - 500 fg/mL PSA, which was expressed as -Ip (µA) = (4.96 ± 0.05) + (3.26 ± 0.04) [log (cPSA (fg/mL))] with a correlation coefficient of 0.998. The detection limit was determined to be 0.045 ± 0.003 fg/mL PSA. Compared with the linear equation and the detection limit in standard PBS solution, the analytical performance of the developed method was not significantly influenced by the matrix effects of serum. This positive result was attributed to the ultrasensitivity of the method, which allowed a high dilution ratio of the serum sample and thus effectively reduced the matrix effects of serum.


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Table 1. Comparison of the Detection Results for Clinical Human Serum Samples by Using the Developed Method and the ECLIA Method. PSA concentration Relative deviation (mean ± SD, n=3, (mean ± SD, n=3, (%) ng/mL) ng/mL) this method

ECLIA

1

0.74 ± 0.05

0.77 ± 0.03

-3.90

2

0.46 ± 0.02

0.47 ± 0.02

-2.13

3

3.50 ± 0.10

3.36 ± 0.12

4.17

4

80.41 ± 1.62

82.48 ± 1.85

-2.51

5

6.60 ± 0.41

6.72 ± 0.33

-1.79

6

15.41 ± 0.25

15.21 ± 0.36

1.32

7

0.84 ± 0.07

0.82 ± 0.02

2.44

8

1.30 ± 0.06

1.28 ± 0.07

1.51

9

32.86 ± 2.13

32.79 ± 1.18

0.22

10

9.14 ± 0.34

9.72 ± 0.16

-5.97

11

19.83 ± 0.80

19.03 ± 1.25

4.22

12

7.27 ± 0.73

7.85 ± 0.37

-7.35

Sample

The practical feasibility of the developed method was investigated by analyzing several clinical human serum samples, which were obtained from Qilu Hospital in accordance with the rules of the local ethical committee. Because the PSA concentration in these serum samples was beyond the linear range of the developed method, an appropriate dilution of these samples was conducted prior to detection. The PSA concentration in each sample determined by the developed method is listed in Table 1, exhibiting an acceptable consistency with the results provided by the hospital, which were obtained using an electrochemiluminescence immunoassay


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(ECLIA) via a Cobas 6000 analyzer (Roche, Switzerland), with a relative deviation less than 8%. The capability of the developed method for real sample analysis demonstrated its potential in clinical applications.

CONCLUSIONS This work demonstrated the first attempt to design and synthesize RCA products containing numerous poly(thymine) repeats for CuNP formation, which was applied in electrochemical bioanalysis for cascade signal amplification. Consequently, an ultrasensitive, selective, and convenient electrochemical assay was developed with a remarkable detection limit of 0.020 ± 0.001 fg/mL PSA, which was more than four orders-of-magnitude lower than the reported electrochemical methods. The ultrasensitivity and high selectivity of this strategy might be attributed to the following factors: (1) the primer-AuNP-aptamer bioconjugate offers a high primer/aptamer ratio, which is proportional to the response; (2) the RCA product-templated CuNPs facilitate cascade signal amplification by offering an enormous ratio of metal components to target molecules; and (3) the dual recognition of the target molecule via antibody and aptamer guarantees the selectivity of the strategy. Moreover, the electrochemical readout of CuNPs avoids the restriction of the unstable fluorescence emission of CuNPs, ensuring convenient and long-time monitoring application. The capability of the developed method for analyzing PSA in clinical samples suggests the feasibility of practical applications. This method can be conveniently extended to analyze a wide range of disease markers by using corresponding affinity ligands and thus possesses enormous potential for disease diagnosis. In addition to the superior analytical performance and potential versatility, this method can be conducted with simple instruments and basic operation in moderate conditions, suggesting its potential in


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commercialization for practical applications. More importantly, this design opens new horizons for integrating different disciplines, providing a reference for the development of new bioanalytical methods.

ASSOCIATED CONTENT The Supporting Information is available free of charge via the Internet at http://pubs.acs.org. Table summarizing the analytical applications of poly(thymine)-templated CuNPs, TEM image of T50-templated CuNPs, calibration curve of the control experiment using aptamer-T50, calibration curve in real samples.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. Tel: +86-531-88362588. Fax: +86-531-88564464. Author Contributions The manuscript was written via the contributions of all authors. All authors have approved the final version of the manuscript. Notes The authors declare no competing financial interests.


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ACKNOWLEDGMENTS This work was supported by the National Science Foundation of China (21305077), the Research Award Fund for Outstanding Middle-aged and Young Scientist of Shandong Province (BS2013SW031), the Special Funding of China Postdoctoral Science Foundation (2014T70629), and the Independent Innovation Foundation of Shandong University (2012HW004).

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