基于适配体的核酸外切酶保护和酶循环切割扩增的多功能检测平台高灵敏性检验凝血酶

Aptamer-Based Exonuclease Protection and Enzymatic Recycling Cleavage A mplification Homogeneous Assa y for Highly sensitive Detection of thrombinQingwang Xue,‡ Lei Wang,*,†and Wei Jiang*,‡†Key Lab of Chemical Biology (MOE), School of Pharmaceutical Sciences, Shandong University,Jinan 250012, PR Chinaffi Key Laboratory for Colloid & Interface Chemistry of Education Ministry,School of Chemistry and Chemical Engineering, Shandong University, Jinan, Shandong, 250100, P.R . China.Corresponding author:Tel: +86 531 88363888; fax: +86 531 88564464. Email: wjiang@;Tel: +86 531 88382330. Email: wangl-sdu@ABSTRACTA critical challenge in homogeneous solution-based biomolecular detection is the separation and sensitivity compared to biomolecular detection in heterogeneous solution. In the work, a novel, separation-free and sensitive homogeneous protein detection assay based on combining aptameric exonuclease protection with nicking enzyme assisted fluorescence signal amplification(NEFSA)is developed for highly sensitive protein detection. We applied a special oligonucleotide probe containing the protein aptamer sequence at the 3’-terminus, which has the capacity to recognize protein target with high affinity and specificity. Specifically, the aptamer probe can be protected from exonuclease-catalyzed digestion upon binding to the protein target. The protected aptamer probe may hybridize with the molecular beacon (MB) probe, a reporter signal oligo-DNA. Consequently, NEFSA process is triggered in the presence of nicking enzyme, resulting in continuous enzyme cleavage of many MBs, providing a fluorescent cascadic amplification detection signal for the target. Thrombin is used as model analyte in the current proof-of-concept experiments. This method can permits detection of human thrombin specifically with a detection limit as low as 1.0 pM without using washes or separations. Our method exhibited excellent sensitivity. In addition, this new method is simple and avoids specifically conformational design of aptasensor probe for the elimination of the washing and separation steps. The mechanism, moreover, may be generalized and used for other forms of protein analysis by changing the corresponding aptamer without other conditions. So our news strategy may provide a homogeneous fluorescence detection platform for manyproteins.IntroductionAptamers are single-stranded nucleic acids isolated from random-sequence DNA or RNA libraries by an in vitro selection process termed the systematic evolution of ligands by exponential enrichment (SELEX).1, 2As nucleic acid ligands, aptamers have many advantages over antibodies as recognition elements such as simple synthesis, good stability, high-affinity, design flexibility and wide applicability, making them suitable candidates for biological application.3,4,5 In recent years, several methods using aptamers as the molecular recognition component have been developed to achieve the reliable detection of proteins by combining with electrochemistry,6 fluorescence,7colorimetry,8 surface plasmon resonance and chemiluminescent technique.9,10Among them, the homogeneous fluorescence aptasensors is one of the most attractive and interesting areas due to the stability and robustness of fluorescence and the simplicity and rapidness of homogeneous reaction as opposed to heterogeneous(the binding kinetics).11,12The disperse features of homogeneous reaction facilitate the binding kinetics. However, homogeneous assays generally need separation and being less as sensitive as their heterogeneous counterparts because of their high background. Although the sensitivity of the heterogeneous assays can be improved, all these methods need to perform the assay on the heterogeneous formats with multiplex binding and washing steps, which may decrease analytical accuracy and reproducibility. Therefore, a critical challenge in homogeneous solution-basedbiomolecular detection is the sensitivity and the separation.13Considering aptamers can be easily incorporated in oligonucleotide-based signal amplification processes, aptamer based amplification assays have been paid more and more attention for improving the sensitivity, such as the polymerase chain reaction (PCR),14rolling circle amplification(RCA),15 and nanoparticle assisted amplification.16,17 However, there are some limitations that hampers their widespread use for routine analysis such as the highly precise temperature cycling for PCR,18 the design of an effective aptamer probe that can undergo a complex conformational change to effectively initiate RCA,19the particles’ strong tendency to aggregate over time. In addition, the variability of the nanoparticles often affects the reproducibility and quant ification, especially for the real samples.20,21Recently, DNA nicking endonucleases, a special family of restriction endonucleases which cut one strand of a double stranded DNA at the specific sequence site,22 has been applied to DNA sensors and homogeneous aptasensors due to their high sensitivity.23 Xing et al developed an isothermal and sensitive fluorescence assay for protein detection using aptamer-protein-aptamer conjugates based on combining nicking enzyme amplification with magnetic separation. 24 The method achieved high sensitivity and showed good specificity. However, utilizing the magnetic beads, the assay needs separation procedures for reducing the background. In addition, the steric hindrance from magnetic beads also effect on following reactions. To address this problem, the homogeneous approaches for protein detection were developed based on target-triggered aptamer hairpin switch and nicking enzyme assisted fluorescencesignal amplification.25-27These reported approaches are all based the specifically conformational designed aptamer hairpin switch. Although the specifically conformational designed hairpin switch aptamer probe eliminate the washing and separation steps, the hairpin structure probe need to be specifically conformational designed. The designed aptamer probe is difficultly guaranteed to completely form the structure of desired hairpin switch. And, the complex handling procedures of specifically conformational designed aptamer probes frequently result tedious processing. Consequently, the development of a novel, simple and signal-amplified approach for further improving sensitivity and selectivity is highly desirable.To address aforementioned these problems, in the study, we developed a novel, separation-free and sensitive homogeneous protein detection assay based on aptameric exonuclease protection and nicking enzyme assisted fluorescence signal amplification (NEFSA). In the assay, only one aptamer was applied as recognition element, overcoming the limitations of sandwich assays. And, the aptamer probe need n’t to be specifically designed for hairpin switch. What’s more, the developed approach eliminates the washing and separation steps by utilizing exonuclease I. It avoided the specifically conformational design of aptamer probe, and completes a high sensitive protein analysis in a homogeneous format by NEFSA. Moreover, the developed technique may be generalized and used for other forms of protein analysis with high sensitivity and excellent specificity by changing the corresponding aptamer without other conditions. So it was believed that this developed technique possesses great potential for the simple, easy, and convenient detection of protein.ExperimentalChemicalsHuman α-thrombin, Human immunoglobulin G (IgG), and bovine serum albumin were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). The oligonucleotides with the following sequences were obtained from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (China): primer-aptamer (5’-GCT GCT GAG GAA GTT TT TT G GTT GGT GTG GTT GG-3’) (this sequence is consists of thrombin Apt15 sequences in green and ex-DNA signal transduction sequence in violet), molecular beacon (MB), (5’-FAM-CCAGCCAACCTT C C▽T CAGC AGCTGG-DabcyL-3’). Nicking endonuclease, Nt.BbvCΙ, and NEBuffer 4 were purchased from New England BioLabs (Ipswich, MA, USA). Other chemicals (analytical grade) were obtained from standard reagent suppliers. Water (≥18.2M) was used and sterilized throughout the experiments.ApparatusAll the fluorescence measurements were performed on a Hitachi F-4500 spectrofluorimeter (Hitachi, Japan). The excitation wavelength was 494 nm, and the spectra are recorded between 500 and 605 nm. The fluorescence emission intensity was measured at 518 nm.Protein DetectionThe model protein thrombin was dissolved by 50% glycerin and diluted by water. 10μL200 nM aptamer probe in 50 μL of 1 ×NEB4 buffer (50 mM KAc, 10 mM Mg(Ac)2, 20 mM Tris-Ac, 1 mM dithiothreitol (DTT), pH = 7.9) was firstly heated to 95 °C for 5 min, and slowly cooled to room temperature. To aforementioned composite aptamer probe was added 3 µL of different concentration thrombin, followed by incubation at 37 °C for 1 h to allow complete binding. Then, 40 units of exonuclease I in 7 µL 10 × reaction buffer ( 670 mM glycine-KOH, 67 mM MgCl2, 10 mM DTT) was added to the mixture in order to degrade the unbound probe and the resultant mixture incubated at 37 °C for 2 h, followed by a heating step at 80 °C for 15 min to inactivate the exonuclease I. After this step, 10μL 750 nM MB probes were added, and the mixture was incubated at 37 °C for 30 min. Subsequently, the aforementioned product was mixed with 4 units of Nt.BbvCI in 4 µL 10 ×NEB4 buffer (500 mM KAc, 200 mM Tris-Ac, 10 mM Mg(Ac)2, 10 mM DTT, pH = 7.9). The reaction mixture was incubated at 37 °C for 1 h. Then, the solution was moved to a fluorescence cuvette forfluorescence measurement.Results and discussionDesign of the assayThe designed assay is conceptually depicted in Scheme 1. Thrombin is used as model analyte in the current proof-of-concept experiments. In the assay, a specialoligonucleotide probe containing the thrombin aptamer sequence at the 3’ -terminus and signal transduction at the 5’-terminus was applied. The aptamer probe has the capacity to recognize protein target with high affinity and specificity. In the present of thrombin, thrombin binds the aptamer probe and thereby protects it from degradation by Exonuclease I, whereas any unbound probe is degraded by exonuclease I. Subsequently, the probes that were protected from exonuclease I by thrombin act as catalysator to trigger hybridizing with the MB probe. The MB probe is a reporter signal oligo-DNA l abeled with the fluorophore and its quencher at the 5’- and 3’-ends, respectively. And the MB probe carried the cleavage site for the nicking endonuclease Nb.BbvcI. A fluorescence signal appears only when the MB probe was hybridized with the signal transduction portion of the probe and cleaved by Nb.BbvCI. After nicking, the hybrid MB probe became less stable, and the cleaved strand dissociated from the complex, resulting in the complete disconnection of the fluorophore from the quencher. The released aptamer-target complex could then hybridize to another MB probe and initiated the second cycle of cleavage. NEFSA process is triggered, resulting in continuous enzyme cleavage of many MBs. Finally each target strand could go through many cycles, resulting in the fluorescent cascadic amplification detection signal. T he fluorescence signal s are detected, which can be used as a quantitative measure for the protein concentration. Therefore, the aptamer probes protected from exonuclease I by target reflect the identity and amount of target protein and can be quantified by NEFSA.Optimization of the aptamer oligonucleotide probe concentrationThe concentration of aptamer probe plays an important role in the sensing process. To guarantee to all protein molecules be recognized and detected, an appropriate concentration of aptamer oligonucleotide probe is expected to yield the optimization signal. The effect of the concentration of aptamer probe on the fluorescent signal was shown in Fig. 1A. It is clear that the fluorescent intensity response was very slow when the concentration of aptamer oligonucleotide probe is at the initial 50 nM, indicating a relatively low bond. The fluorescent intensity response exhibits a rapid increase with a further increase in the concentration of aptamer oligonucleotide probe after 50 nM and trends to a constant value at 200 nM, indicating the saturation of the bond between aptamer and thrombin. Thus, 200 nM was selected as the optimum concentration in the experiment due to its best signal-to-noise level.Optimization of the exonuclease IIn the assay, to realize the separation-free procedure, we introduce exonuclease I to degrade all unbound protein aptamer probes. Exonuclease I catalyzes the removal of nucleotides from single-stranded DNA in the 3’ to 5’ direction. Without a protein target, the aptamer probe would be degraded to 5’-terminal dinucleotides with the release of deoxyribonucleoside 5’-monophosphates from the 3’-terminal of the single-stranded DNA chains. In the assay described here, the oligonucleotides thatbind thrombin are protected, whereas free single-strands aptamer probes are degraded by exonuclease I. To guarantee to completely degrade all free single-strands aptamer probes, we investigate the concentration of exonuclease I. The effect of the exonuclease I on the fluorescent signal was shown in Fig. 1B. It shows that 40 units exonuclease I was selected as the optimum concentration for degrading the free protein aptamer probes.Optimization of the concentration of MBIn the assay, the MB probe is a reporter signal oligo-DNA, which carried the recognition sequences for the signal transduction portion of the aptamer probe and the cleavage site for the nicking endonuclease Nb.BbvcI, and was labeled with the fluorophore and its quencher at the 5’- and 3’- ends, respectively. To achieve desirable analytical characteristics, the effect of the concentration of MB probe was investigated. As shown in Fig. 1C, the fluorescence intensity of the aptasensor and the background increased with increasing the concentration of MB probe. The results indicated that the optimum concentration of MB probe used in this aptasensor was 750 nM due to its best signal-to-noise level (the positive signal deduct the negative signal).Effect of the Basic Components of the Amplification Method .To provide convincing proofs of the detection mechanism of Exonuclease protection and the nicking enzyme reaction, spectra and/or relative fluorescence intensities of the various phases of the MB probe were recorded, respectively. Fig. 2 shows that the fluorescence intensities of the MB probe in the absence of protected protein detection probes or hybridized to its complementary sequence in protein detection probes are rather low. In the absence of nicking enzyme and target, a 59% increase in the signal was observed. When the MB probe hybridized to the ex-DNA recognition probe sequence of protected protein detection probes and in the presence of nicking enzyme, significant fluorescence responses were achieved. As shown in Fig. S4, we observed a 314% signal increase b y introducing endonuclease amplification. The a mplification strategy based on the nicking endonuclease led to a dramatic increase in the final fluorescence intensity, assuring a higher sensitivity for detection of target. Therefore, the signal amplification of the nicking enzyme cascadic catalytic beacon is obvious.Detection of proteinIn view of the outstanding ability of the developed assay, the dynamic range of the designed assay for detection of human thrombin analyte was examined. As shown in Fig. 3, a dramatic increase in the fluorescence intensity was observed as the concentration of human thrombin from 0 pM to 100 nM. Meanwhile the fluorescence intensity was linear to the concentration of human thrombin in the range from 0 pM to1 nM. We estimate the limit of detection is 1.0 pM in a 3σ rule. The sensitivity of this method is about three orders of magnitude higher than that of traditional homogeneous aptasensors.1-3The lower detection limit represents that this nicking enzyme amplification method for protein detection is sensitive. These results clearly demonstrate that the endonuclease does indeed play an important role in the signal amplification. The method for protein detection is sensitive. The sensitivity mainly depended on the nicking enzyme reaction and the low background fluorescence. Additiona lly, the largest value of coefficients of variation for triplicate measurements was 4.6%, indicating that this proposed method exhibited excellent reproducibility.Selectivity of the bioassayT he detection specificity was basically determined by the aptame ric recognition function. The selectivity of this assay has been tested with some common proteins, BSA, human IgG and serine protease each at concentration of 10 nM. The resulting signal was negligible compared with that obtained using 10 nM thrombin, as shown in Fig. 4.High signal was obtained only when the pecific protein (thrombin) was tested. The measured data demonstrated that this aptasensor assay could be used as a sensitive and selective platform for target protein detection, which might support its potential in clinical applications.Detection of Human Thrombin in Human Blood SerumA big challenge for an excellent protein assay is its ability to be applied in complex biological matrixes. To demonstrate the feasibility of the approach in complex biological matrixes, we applied this aptasensor assay to detect the human thrombin spiked in serum. Serum is what remains from whole blood after coagulation; its chemical composition is similar to plasma but does not contain coagulation proteins such as thrombin or other factors. Standard human serum was isolated from the blood of healthly individuals, which was first diluted in 1:10 ratio with NEB buffer and then spiked with thrombin at concentration of 10 nM. As shown in Fig. 5, comparable responses were obtained for thrombin in both buffer and serum. We noted that the signal obtained in serum was slightly higher than the signal measured in buffer. Probably, this increase was due to the nonspecific binding of the aptamer to other proteins in human blood serum, which protected it from degradation by Exonuclease I and caused the binding of the MB probe and ex-DNA signal transduction sequence of the aptamer probe, and the serum contained some DNA nucleases not present in the standard solution which also caused the increase of fluorescence intensity. Overall, the current assay shows potential for application in protein detection in a biological system.ConclusionsIn conclusion, a novel, separation-free and sensitive homogeneous proteindetection assay based on aptameric exonuclease protection and nicking enzyme assisted fluorescence signal amplification (NEFSA) was proposed. It was demonstrated that the proposed strategy offers several advantages such as isothermal experimental condition, simple preparation, convenient operation, and low-cost devices. First, only one aptamer is used to trigger the NEFSA, which avoids the limitations of the sandwich assay, circumventing the requisite of two binding sites per target protein. Additionally, this assay has high sensitivity and selectivity. It permits detection of as low as 1.0 pM human thrombin. Third, the aptamer probe without any modification and specifically conformational design or label is involved, which is important for retaining the high target binding activity. Fourth, it could be expanded easily to other proteins by changing the corresponding aptamer. Moreover, this proposed assay could be applied in a complex biological system, which is important for its application in biological or clinical target detection. Thus, the proposed assay can be expected to provide a sensitive and selective platform for the detection of proteins.AcknowledgmentsThis project was supported by the National Natural Science Foundation of China (Grant No., 21175081, 21175082).References1 A. D. Ellington, J. W. Szostak, Nature, 1990, 346, 818-22.2 C. Tuerk, and L. Gold, Science, 1990, 249, 505-10.3 L. Xue, X. Zhou, D Xing, Chem. Commun.,2012. 46, 7373-5.4 T. Hermann, D. J. Patel, Science, 2000, 287, 820-5.5 Z. Zhou,, Y. Du, S Dong, Biosens. Bioelectron., 2011, 28, 33-7.6 Z. S. Wu, M. M. Guo, S. B. Zhang, C. R. Chen, J. H. Jiang, G. L. Shen, and R. Q. Yu, Anal. Chem., 2007, 79, 2933-9.7 V. Pavlov, Y. Xiao, B. Shlyahovsky, I. Willner, J. Am. Chem. Soc.,126, 11768-9.8 W. 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Higgins, C. Besnier, H. Kong, Nucleic Acids Res., 2001, 29, 2492-501.20 A. R. Connolly, M. Trau, Angew. Chem., Int. Ed., 2010 49, 2720-3.21 L. Xue, X. Zhou, D. Xing, Anal. Chem., 2012, 84, 3507-13.22 A. X. Zheng, J. R. Wang, J. Li, X. R. Song, G. N. Chen, H. H. Yang, Chem. Commun., 2011, 48, 374-6.23 A. X. Zheng, J. R. Wang, J. Li, X. R. Song, G. N. Chen, H. H. Yang, Biosens.Bioelectron., 2012 36, 217-21.Scheme 1 Illustration of the proposed assay for amplification detection of protein. (1) Thrombin aptamers adequately bind thrombin in the proper buffer. (2) Exonuclease I is added to hydrolyze free aptamers. (3) The aptamer-target complex may hybridize with the molecular beacon (MB) probe. (4) After nicking, the hybrid MB probe became less stable, and the cleaved strand dissociated from the complex, resulting in the complete disconnection of the fluorophore from the quencher. The released aptamer-target complex could then hybridize to another MB probe and initiated the second cycle of cleavage.Fig. 1.Effect of the different concentrations on the fluorescent signal intensity：(A) Aptamer concentration (nM). (B) Exonuclease concentration (Units). (C) MB concentration (nM). These reactions have been carried out with 100 nM of thrombin.Fig. 2.Fluorescence emission spectra and fluorescence responses of the sensing system under different conditions: (1) Aptamer probe + exonuclease I + MB (blue line); (2) Aptamer probe + thrombin (100 nM )+ exonuclease I + MB (black line); (3) Aptamer probe + thrombin (100 nM)+ exonuclease I + MB + Nt.BbvCΙ (red line).Fig. 3. (A) Fluorescence emission spectra obtained in the developed homogeneous protein detection assay of human thrombin with varying concentrations from 0 to 100 nM. (B) The relationship between the fluorescent intensity and the concentration of thrombin (n = 3). Each data point represents an average of 3 measurements. The insetshows the linear region from 0 pM to 1.0 nM of thrombin, yielding the detection limit of 1.0 pM.Fig. 4.Fluorescence intensity responses of the assay to (1) NEB buffer, (2) 10 nM BSA in NEB buffer, (3) 10 nM human IgG in NEB buffer, (4) 10 nM serine protease in NEB buffer, (5) 10 nM thrombin in NEB bufferFig. 5.Fluorescence intensity responses of the assay to (1) NEB buffer, (2) 10% human serum, (3) 10 nM thrombin in NEB buffer, (4) 10 nM thrombin in 10% human serum. 10% human serum (human serum was diluted in 1:10 ratio with NEB buffer)。