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An Ultrasensitive Fluorescent Assay Based on Rolling Circle Amplification-Assisted Multi-Sites Strand Displacement Reaction Signal Amplification Strategy Xin Peng, Wen-Bin Liang, Zhi-Bin Wen, Cheng-Yi Xiong, Ying-Ning Zheng, Yaqin Chai, and Ruo Yuan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01015 • Publication Date (Web): 28 May 2018 Downloaded from http://pubs.acs.org on May 28, 2018

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Analytical Chemistry

An Ultrasensitive Fluorescent Assay Based on Rolling Circle Amplification-Assisted Multi-Sites Strand Displacement Reaction Signal Amplification Strategy Xin Peng, Wen-Bin Liang, Zhi-Bin Wen, Cheng-Yi Xiong, Ying-Ning Zheng, Ya-Qin Chai∗, Ruo Yuan∗ Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China

∗

Corresponding authors at: Tel.: +86 23 68252277, fax: +86 23 68253172. E-mail addresses: [email protected] (Y. Q. Chai), [email protected] (R. Yuan).

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ABSTRACT: Heavy metals are persistent environmental contaminant and pose great threat to human health, which have prompted demand for new method to selectively identify and detect these metal ions. Herein, a novel fluorescent assay was proposed based on rolling circle amplification (RCA)-assisted multi-sites strand displacement reaction (SDR) signal amplification strategy for ultrasensitive detection of heavy metal ions with lead ions (Pb2+) as a model. The proposed strategy not only achieved the target recycling, but also introduced the RCA induced by the released DNAzyme. Most importantly, the RCA product was adapted as the initiator to provide multi-sites for SDR, which could displace signal duplex down from RCA product to effectively avoid the self-quenching of signal probes assembling on RCA product. Therefore, the amplification efficiency and the detection sensitivity could be improved significantly. As expected, the proposed strategy demonstrated good performances for the determination of Pb2+ with a liner range from 0.1 nM to 50 nM and a detection limit down to 0.03 nM. Taking this strategy into the intracellular Pb2+ detection, a favorable property was obtained. Furthermore, the proposed strategy could be also expanded for determination of microRNA, proteins, and other biomolecules, offering a novel avenue for environmental assays and clinical diagnostics. Keywords: Ultrasensitive Fluorescent Assay, Signal Amplification, DNAzyme, Intracellular Pb2+

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INTRODUCTION Heavy metal ions in environment posed a serious threat to human health, especially lead ions (Pb2+), which as the major environmental pollutant have been widely concerned due to its neurotoxicity, nephrotoxicity and cardiovascular toxication to human.1-4 Therefore, establishing a fast, sensitive and high efficient detection method for Pb2+ has always been an active area of research, which is significantly important not only for the human health but also for environmental protection. Currently, detection methods such as high performance liquid chromatography,5 atomic absorption spectrometry6 and atomic emission spectrometry7 have widely been applied in the Pb2+ determination. However, those techniques are still limited by low sensitivity, complicated operation, and time-consuming procedures. Significantly, even a small amount of Pb2+ could also induce severe damages on human health due to the long-term accumulation in body. Over the past years, although great efforts have been made to improve the detection performance of Pb2+,8-10 it is still urgent for an ultrasensitive, efficient and convenient detection method for the low concentration of Pb2+ detection. Fortunately, fluorescent assay as a mature detection technique with simple equipment and facile operation has been proved to be a promising strategy for detection of heavy metal ions.11-14 Recently, DNAzyme with special DNA molecular structures and specific enzymatic activities have been applied to fluorescent assays for the Pb2+ determination.15,16 Yu’s group has proposed a simple and efficient DNAzyme-based fluorescent assay for the Pb2+ detection, by incorporating the specific Pb2+-dependent DNAzyme as signal probe and graphene oxide (GO) as 2



fluorescence quencher17. However, such a one-step detection method without signal amplification strategy is still limited by low detection sensitivity. To overcome the deficiency, several amplification methods have been proposed, such as rolling circle amplification (RCA),18,19 strand displacement reaction (SDR),20,21 and hybridization chain reaction (HCR).22,23 Especially, RCA as a representative enzymatic isothermal nucleic acid amplification method involving a circular template to produce a lot of reduplicated oligonucleotide sequences has been widely used in signal amplification strategy. However, RCA product usually worked as substrate for signal probes assembling,24-26 the self-quenching of signal probes assembling on RCA product could be unavoidable.27 Therefore, displacing signal probes down from RCA product and improving the utilization efficiency of RCA product are favorable for signal amplification strategy. Notably, SDR as an effective amplification tool which can be modulated by toeholds to co-localize reactant DNA molecules has been widely used in signal conversion.28 Therefore, an ultrasensitive signal amplification strategy could be expected by introducing SDR on RCA product. Herein, an ultrasensitive fluorescent assay was proposed for Pb2+ determination based on RCA-assisted multi-sites SDR signal amplification strategy (Scheme 1). Typically, the target Pb2+ specifically identified the duplex 1 to initiate catalytic cleavage activity of DNAzyme, resulting in ribonucleotide (rA) cleavage on the substrate to release the Pb2+ and DNAzyme. The released Pb2+ then identified the next duplex 1. In the meantime, the released DNAzyme opened the hairpin1 (H1) and further provided the binding sites of circular template to generate the RCA reaction,

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so a lot of duplicate DNA sequences on RCA product were produced, which provided multi-sites

for

initiating

SDR.

Next,

the

hairpin

2

(H2)

labeled

with

6-Carboxyfluorescein N-succinimidyl ester (FAM) at 5’ end hybridized with the RCA product to expose the 5’- sticky end of H2. Immediately, the hairpin 3 (H3) labeled with 5-Carboxy-tetramethylrhodamine N- succinimidyl ester (TAMRA) at 3’ end displaced H2 down from the RCA product to form signal duplex based on the toehold-mediated SDR of the 5’- sticky end of H2 and the 3’- sticky end of H3. The signal duplex brought FAM and TAMRA into close proximity to generate the fluorescence resonance energy transfer (FRET) signal, which avoided the false positive signals and minimized effects of system fluctuations for the low concentration of Pb2+ detection. Furthermore, the proposed ultrasensitive fluorescent assay for Pb2+ determination based on RCA-assisted multi-sites SDR signal amplification strategy could be readily expanded for the detection of microRNA, proteins and other biomolecules, offering a promising tool for environmental assays and clinical diagnostics.29-31

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Scheme 1. Schematic diagram of fluorescent assay based on RCA-assisted multi-sites SDR signal amplification strategy. EXPERIMENTAL SECTION Chemicals and Materials. T4 DNA ligase (3U/µL), and Phi29 polymerase (10U/µL) were purchased from Vazyme Biotech Co., Ltd. (Nanjing, China). Exconuclease I and Exconuclease III were obtained from Thermo Fisher Scientific Inc. (Shanghai, China). Deoxynucleotides (dNTPs) was purchased from Genview Scientific Inc. (El Monte, CA, USA). All oligonucleotides (Table S1) were custom-synthesized by Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China). Phosphate buffer solutions (PBS) were composed of 0.1 M K2HPHO4, 0.1 M NaH2PO4, and 0.1 M KCl and the pH was adjusted to 8.0. TM buffer solutions (pH 8.0) were prepared by 20 mM Tris and 12.5 mM MgCl2 standard stock solutions.

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Pb(NO3)2 were purchased from Sigma Chemical Co.(St. Louis, MO, USA). MgCl2·6H2O, AgCl, CuCl2, ZnCl2, CdCl3, NiCl2·6H2O, FeCl3, CoCl2, HgCl2, CaCl2, and MnCl2 were obtained from Chengdu Kelong Co. Ltd. (Chengdu, China). The deionized water was purified by a water purification system with the electrical resistance of 18.2 MΩ/cm. Apparatus. FL-7000 fluorescence spectrophotometer (Hitachi, Tokyo, Japan) was used to detect the fluorescent responses. The pH-3C digital pH-meter (Shanghai LeiCi Device Works, Shanghai, China) was adopted to test the pH of all buffers in the experiment. Gel Doc XR+ System (Bio-Rad, California, USA) was used to take gel-imaging. Preparation of Circular Template. First, 1 µM of the padlock and assistant were mixed at the ratio with 1:3. Next, the solution was heated to 65 °C for 10 min and cooled down to room temperature for 20 min. When 10 × T4 DNA ligase reaction buffer and T4 DNA ligase have been added, the reaction system was incubated at 16 °C for 5 h to finish the intramolecular ligation of padlock. Finally, Exconuclease I and Exconuclease III were added to cut off the extra nucleotides in order to form circular templates. The Cleavage of Duplex 1 with Assistance of Pb2+. 1 µM of the DNAzyme and substrate were mixed at the ratio with 1:3 in the volume of 10 µL hybridized at 37 °C for 2 h. Afterwards, a series of Pb2+ at different concentration (0.1, 0.2, 0.5, 1, 2.5, 5, 10, 25 and 50 nM) were added and incubated at 37 °C for 30 min to initiate the catalytic activity of DNAzyme.

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RCA Reaction. H1 has been added to the reaction system and opened by the released DNAzyme. Immediately, the RCA reaction was generated by the exposed sequences of H1 on the basis of circular template, 2 U Phi29 DNA polymerases, 1 x Phi29 DNA Polymerase buffer and 600 µM dNTPs. And then, the reaction system was incubated at 37 °C for 3 h. The Preparation of Cell lysate. Human breast cancer cells (MDA-MB-231 cells) and human pulmonary adenocarcinoma cells (A-549 cells) were provided by the cell bank of the Committee on Type Culture Collection of Chinese Academy of Science (Shanghai, China). They were respectively cultured in DMEM medium containing 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C with a humidified atmosphere (5% CO2) and treated with different concentration of Pb2+ for 24 h. After that, 106 cells were obtained and washed with sterile PBS. Then, the cell lysate has been prepared by repeatedly freezing and thawing cells in ice-cold water. Fluorescent Experiments. After RCA reaction, signal probes H2 and H3 were added to keep the solution finally contained 200 µM H2 and H3 in the totally volume of 50

µL and incubated at 37 °C for 2 h. The fluorescence was recorded on the F-7000 fluorescence spectrophotometer. The excitation wavelength was 497 nm and the emission wavelength ranged from 510 nm to 650 nm. RESULTS AND DISCUSSION Feasibility of the Proposed Strategy. To demonstrate the feasibility of the proposed strategy, a series of in vitro tests were performed as shown in Figure 1. In absence of Pb2+, the test solution exhibited a low FRET signal (curve a in Figure 1). However, in

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presence of Pb2+ but without RCA product for SDR, a little of SDR was generated by the sticky end of H1. Therefore, an obvious FRET peak could be observed at 575 nm (curve b in Figure 1). When the target Pb2+ was added and RCA was triggered by the addition of circular template, dNTPs and Phi29 DNA polymerase, a lot of duplicate DNA sequences on RCA product were produced, which provided multi-sites for initiating SDR. Therefore, abundant signal duplexes were generated to induce a strong FRET signal (curve c in Figure 1). The results demonstrated that the proposed strategy was feasible for Pb2+ detection.

Figure 1. The FRET signal spectra of the detection solutions without target Pb2+ (curve a), with target Pb2+ but without RCA product for SDR (curve b), and with target Pb2+ and RCA product for SDR (curve c). Native Polyacrylamide Gel Electrophoresis (PAGE). PAGE was adapted to approve the practicability of the proposed fluorescent assay based on RCA-assisted multi-sites SDR signal amplification strategy. As shown in Figure S3, lane 1 and lane 2 represented DNAzyme and substrate, respectively. Lane 3 was the hybrid product of DNAzyme and substrate at the ratio with 1: 3. Lane 4 was the cleavage of duplex 1 8



with the concentration of 1 µM Pb2+. Lane 5, lane 6 and lane 7 represented H1, H2 and H3, respectively. Lane 8 was the hybrid product of DNAzyme and H1. Lane 9 was circular template of RCA reaction. Lane 10 was the RCA reaction. Lane 11 and lane 12 were the detection system without and with target Pb2+. The RCA product and the signal duplex in lane 12 could be clearly observed compared to lane 11. Optimization of Experimental Conditions. In order to confirm the optimum conditions for the determination of Pb2+, the concentration of dNTPs, the dosage of Phi29 DNA polymerase, reaction time and temperature of RCA, and reaction time of SDR have been investigated, respectively. Firstly, 200 µM, 400 µM, 500 µM, 600 µM, 800 µM, 1000µMdNTPs have been studied. As shown in Figure 2A, the FRET signal ratio (peak intensity value of TAMRA at 575 nm / peak intensity value of FAM at 497 nm) increased gradually with the concentration of dNTPs increased until 600 µM was added. Secondly, as shown in Figure 2B, the FRET ratio increased with the increasing dosage of Phi29 DNA polymerase until the dosage reached 2 U. Thirdly, the reaction time and temperature of RCA could also affect the detection performances of the proposed strategy. Samples have been incubated from 1 h to 6 h at 30 °C and 37 °C, respectively. When samples incubated at 37 °C for 3 h, the FRET ratio exhibited a favorable performance (Figure 2C). Therefore, the optimum conditions for RCA reaction were 600 µM dNTPs, 2 U Phi29 DNA polymerases, and samples incubated at 37 °C for 3 h. Finally, the reaction time of SDR has also been investigated. According to Figure 2D, the FRET ratio increased with the reaction time from 0.5 h to 2 h and then the growth trend was flatten out, so the optimum reaction time of SDR was 2 h.

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Figure 2. (A) The relationship between the FRET signal ratio and the concentration of dNTPs, (B) the dosage of Phi29 DNA polymerase, (C) the reaction time and temperature of RCA, and (D) the reaction time of SDR. Sensitivity of the Proposed Strategy. Under the optimal conditions, the correlation between the FRET signal ratio and the concentration of Pb2+ had been investigated. As shown in Figure 3A, the FRET signal gradually increased with the Pb2+ concentration increasing from 0.1 nM to 50 nM. As shown in Figure 3B, it suggested a favorable liner relationship between the FRET signal ratio and the logarithmic concentration of Pb2+, there was a regression equation of I = 0.410 lgcpb2+ + 0.662 and a correlation coefficient of r = 0.994, where I represents the FRET ratio of TAMRA to FAM. Furthermore, the limit of detection (LOD) and limit of quantitation (LOQ) were calculated to be 0.03 nM and 0.10 nM, respectively, according to IUPAC recommendation.32,33 10



Figure 3. (A) The FRET signal spectra of the detection solutions with different Pb2+ concentration (0.1, 0.2, 0.5, 1, 2.5, 5, 10, 25 and 50 nM). (B) Calibration curve of the FRET signal ratio and the logarithmic concentration of Pb2+. In order to evaluate the performance of the proposed strategy, several other detection methods were selected for comparison, such as colorimetry, electrochemical, and other fluorescent assay (Table 1). Obviously, higher detection sensitivity was obtained with the proposed fluorescent assay, indicating a potential application for the low concentration of Pb2+ detection. Furthermore, the applicability of the proposed fluorescent assay was superior to electrochemical approaches because the complicated and time-consuming preparation of electrode was omitted. In terms of the proposed fluorescent assay, all the oligonucleotides were mixed in buffer with detection samples so that the concentration of Pb2+ could be determined simply on account of the response from fluorescence spectrophotometer. Meanwhile, all procedures were completed in a homogeneous solution, which could avoid the interface differences from the two phase of electrode and solution. Table 1. Comparison of the proposed fluorescent assay with other methods from the 11


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literatures. Detection method

LOD

Linear range

Ref.

Colorimetry

13 nM

0.03 - 2 µM

34

Colorimetry

0.048 µM

0.24 - 5 µM

35

Electrochemical Assay

21 nM

25 - 1000 nM

36

Electrochemical Assay

0.6 nM

1.51 nM - 302 nM

9

Fluorescent Assay

0.64 nM

1 - 1000 µM

6

Fluorescent Assay

20 nM

100 - 4000 nM

12

Fluorescent Assay

10nM

10nM - 10 µM

37

Fluorescent Assay

0.03 nM

0.1 - 50 nM

This work

Application of the Proposed Strategy. In order to demonstrate practical application of the proposed strategy for intracellular Pb2+ determination, MDA-MB-231 cells and A-549 cells were selected as the models incubated with different concentration of Pb2+. Firstly, the cell lysate was detected by the ICP-MS, which has been widely used and approved in the analysis of metal ions, and then, the cell lysate was measured by the proposed strategy. The Figure 4 was the correlation comparison between ICP-MS and the proposed strategy for intracellular Pb2+ detection, where the x-axis represented the logarithm of the intracellular Pb2+ concentration detected by ICP-MS and the y-axis was the logarithm of the intracellular Pb2+ concentration detected by the proposed strategy. The two methods exhibited good correlations with slope value to 0.971 and intercept value to 0.011 for MDA-MB-231 cells (A), and slope value to 0.953 and intercept value to 0.028 for A-549 cells (B), which indicated the practical application of the proposed fluorescent assay based on RCA -assisted multi-sites SDR signal amplification strategy for the detection of intracellular Pb2+. 12



Figure 4. Correlation comparison between ICP-MS and the proposed strategy for intracellular Pb2+ detection, where Pb2+ came from MDA-MB-231 cells (A) and A-549 cells (B). Specificity of the Proposed Strategy. To evaluate the specificity of the proposed strategy for the detection of Pb2+, other metal ions including Cd3+, Zn2+, Ni2+, Ag+, Fe3+, Cu2+, Ca2+, Hg2+ and Co2+ with the concentration at 250 nM were measured as controls. According to the experimental results exhibited in Figure 5A, there was no obvious FRET signal for these interfering ions. In contrast, in the presence of Pb2+, even at a much lower concentration at 25 nM, the FRET signal had also been significantly enhanced. After normalized the data as shown in Figure 5B, which was illustrated in formula with Normalized FRET Ratio = (Iinterfering ion–I0) / (IPb2+–I0) × 100%, the mixture (Mix) of the interfering metal ions and Pb2+ has a little negative influence on the FRET signal intensity. These results approved that the proposed fluorescent assay based on RCA-assisted multi-sites SDR signal amplification strategy has an excellent selectivity to Pb2+ and other interfering metal ions, which can be ascribed to the specific Pb2+-dependent DNAzyme.38-41 Generally speaking, DNAzyme have been regarded as novel and ideal platforms in sensing due to their 13


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catalytic ability and binding activity toward specific metal ions, which are isolated through in vitro selection.42 The interfering metal ions could not been identified by the specific Pb2+-dependent DNAzyme due to the high selectivity of DNAzyme, thus it could not generate the following amplified reactions with FRET signal.

Figure 5. (A) The FRET signal spectra with different metal ions. (B) Selectivity of the proposed strategy towards Pb2+. The excitation wavelength was 497 nm. CONCLUSION In conclusion, an ultrasensitive fluorescent assay was proposed based on RCA-assisted multi-sites SDR signal amplification strategy. The proposed strategy not only achieved the target recycling, but also introduced the RCA induced by the released DNAzyme. Most importantly, the RCA product was adapted as the initiator to provide multi-sites for SDR, which could displace signal duplex down from RCA product to effectively avoid the self-quenching of signal probes assembling on RCA product. Therefore, the amplification efficiency and the detection sensitivity could be improved significantly. Taking this strategy into the detection of Pb2+, good performances were achieved for the detection of intracellular Pb2+, which suggested the successful establishment of the proposed fluorescent assay based on RCA-assisted 14



multi-sites SDR signal amplification strategy. Importantly, the proposed fluorescent assay could be also expanded for the determination of microRNA, proteins, and other biomolecules, offering a new avenue for environmental assays and clinical diagnostics. AUTHOR INFORMATION *Corresponding authors Tel.: +86 23 68252277, fax: +86 23 68253172. E-mail addresses: [email protected] (Y. Q. Chai), [email protected] (R. Yuan). Notes The authors declare no competing financial interest. ACKNOWLEDGEMENT This work was financially supported by the National Natural Science Foundation of China (NFSC) (21775124 and 51473136). ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: XXXXXX. Model of DNA secondary structures analysis, figure of native polyacrylamide gel electrophoresis and table of nucleic acid sequences. REFERENCES (1)

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An Ultrasensitive Fluorescent Assay Based on ... - ACS Publications

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