Sensitive and Homogeneous Protein Detection Based on Target

Mar 28, 2012 - A short Black Hole Quencher. (BHQ)-quenching fluorescence DNA probe (BQF probe) carrying the recognition sequence and cleavage site for...
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Sensitive and Homogeneous Protein Detection Based on TargetTriggered Aptamer Hairpin Switch and Nicking Enzyme Assisted Fluorescence Signal Amplification Liyun Xue, Xiaoming Zhou, and Da Xing* MOE key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China ABSTRACT: Specific and sensitive detection of proteins in biotechnological applications and medical diagnostics is one of the most important goals for the scientific community. In this study, a new protein assay is developed on the basis of hairpin probe and nicking enzyme assisted signal amplification strategy. The metastable state hairpin probe with short loop and long stem is designed to contain a protein aptamer for target recognition. A short Black Hole Quencher (BHQ)-quenching fluorescence DNA probe (BQF probe) carrying the recognition sequence and cleavage site for the nicking enzyme is employed for fluorescence detection. Introduction of target protein into the assay leads to the formation change of hairpin probe from hairpin shape to open form, thus faciliating the hybridization between the hairpin probe and BQF probe. The fluorescence signal is amplified through continuous enzyme cleavage. Thrombin is used as model analyte in the current proof-of-concept experiments. This method can detect thrombin specifically with a detection limit as low as 100 pM. Additionally, the proposed protein detection strategy can achieve separation-free measurement, thus eliminating the washing steps. Moreover, it is potentially universal because hairpin probe can be easily designed for other proteins by changing the corresponding aptamer sequence.

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which hamper its widespread use for routine analysis.25,26 Some aptamer based RCA assays need to design an aptamer probe to undergo a complex conformational change for initiating the RCA effect, which is considerably difficult.16 The DNAzymes used for protein detection are partly G-quadruplex−hemin based, aiming at more convenient operation and directly displaying results.27,28 Protein detection methods based on DNAzymes can achieve high sensitivity when combined with other amplification elements, such as nanoparticles.29,30 The use of nanoparticles in many assays is relatively difficult because of the particles’ strong tendency to aggregate over time, such as the most used gold nanoparticles.6 Therefore, there is a wide need for fast, convenient, and easy-to-use analytical methods for protein detection from diagnostics to basic research. DNA nicking endonucleases are a special family of restriction endonucleases which cut one strand of a double stranded DNA.31−33 Recently, many nicking enzyme based assays for sensitive DNA detection had been demonstrated.34−38 As a new nucleic acid detection technique, nicking enzyme signaling amplification (NESA) has been reported to have distinct specificity for the detection of a single-base mismatch and offers high sensitivity.39,40 Nicking enzyme is employed as an assistance element for colorimetric and high sensitive DNA

roteins are of great importance in medical and biological fields. Methods for specific and sensitive detection of proteins are necessary in many biotechnological applications and medical diagnostics. Antibody-linked immunological assays (ELISA) are commonly used as classical methods to detect proteins.1−3 Aptamers (DNA or RNA) are selected from random sequence nucleic acid libraries by an in vitro evolution process which is named systematic evolution of ligands by exponential enrichment (SELEX).4,5 Numerous high-affinity and highly specific aptamers have been selected against a wide variety of target molecules including small organics, peptides, proteins, and even supramolecular complexes such as viruses or cells.6 Aptamers offer several advantages over antibodies, such as being easy-to-stock, having stability and reusability, and having general availability for almost any given protein.6−9 Nowadays, the development of the aptameric biosensors recognition is known as highly useful tools for many applications. Several techniques using aptamers as the molecular recognition component have been developed to achieve the reliable detection of proteins. In order to obtain high sensitivity, aptamer based amplification assays have been paid more and more attention, such as the polymerase chain reaction (PCR),10−13 rolling circle amplification (RCA),14−16 DNAzyme based isothermal amplification,17−19 and nanoparticle assisted amplification.20−24 However, PCR suffers from the highly precise temperature cycling and complex sample preparation, © 2012 American Chemical Society

Received: October 8, 2011 Accepted: March 28, 2012 Published: March 28, 2012 3507

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detection.41 Additionally, taking advantage of the nicking enzyme, reactions can be performed in an isothermal condition without specialized instrumentation and have the potential to be widespread for routine analysis.42 Moreover, the product can be easily detected by common detection modalities, such as fluorescence36 and electrochemistry.43 Some techniques based on nicking enzyme are also demonstrated for protein detection.44,45 However, part of the currently available protein detection methods assisted by nicking enzyme suffer from some disadvantages, such as time-consuming, complex handling procedures, and modification needed for aptamer probes. We chose human α-thrombin as the model analyte of interest. Thrombin, the last enzyme protease involved in the coagulant cascade, plays important role in the thrombosis, hemostasis, and many other coagulation-related reactions.46 Changes in thrombin concentration levels in the blood are known to be associated with some diseases, including thromboembolic disease and Alzheimer’s disease.47−49 In our last work, we developed an isothermal and sensitive fluorescence assay for protein detection using aptamer− protein−aptamer conjugates based on nicking enzyme amplification, which was combined with magnetic microparticles separation.45 To the best of our knowledge, it was the first time that aptamer and nicking enzyme based fluorescence signal amplification were simultaneously used for protein detection. The method achieved high sensitivity and showed good specificity. The magnetic beads serving as solid support made it easy to collect target from the complex sample matrix by a magnetic field, making this assay suitable for protein detection in biological samples. However, there are also some disadvantages, such as time-consuming and separation procedures. Utilizing the magnetic beads, the assay needed a separation procedure. Moreover, two aptamers are needed to form the sandwich complex, which also limit the universal application because not all the target protein has two aptamers. To address these problems, we developed a novel, simple, and homogeneous approach for protein detection based on targettriggered aptamer hairpin switch and nicking enzyme assisted fluorescence signal amplification (NEFSA). In this assay, only one aptamer was required as recognition element, thus overcoming the limitations of sandwich assays. The aptamer based approach could complete protein analysis in a homogeneous format, resulting in the elimination of the washing and separation steps. Hairpin probe used for the scheme can be easily designed for other proteins by changing the corresponding aptamer without other conditions, making this method potentially universal. Therefore, it was believed that this homogeneous NEFSA technique possesses great potential for the simple, easy, and convenient detection of protein and biological molecules.

Table 1. Oligonucletides Used in the Present Study

the hairpin probe could match perfectly with the underlined sequence in the 3′ terminal. Nicking endonuclease, Nb.BbvC, and buffer were purchased from New England BioLabs (Ipswich, MA, USA). Pure human thrombin was supplied by Ding Guo Biotech Co., Ltd. (Beijing, China). Lysozyme was bought from TianGen Biotech Co, Ltd. (Beijing, China). Bovine serum albumin was produced by ZhanChen biologic-tech Co., Ltd. (GuangZhou, China). Other reagents were bought from Sangon Biotech Co., Ltd. (Shanghai, China). The buffer used in the present work: NEBuffer 4 containing 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM dithiothreitol, pH 7.9. Water (⩾18.2MΩ) used throughout the experiments was generated by a Milli-Q water purification system (Millipore, Bedford, MA, USA). Apparatus and Fluorescence Measurements. Fluorescence spectra were measured using a Perkin-Elmer LS-55 fluorometer equipped with a xenon lamp excitation source and a personal computer data processing unit. Excitation and emission slits were set for 10.0 and 5.0 nm band-pass, respectively. The fluorophore of FAM was excited at 488 nm, and the emission spectra from 510 to 580 nm were collected. The fluorescence intensity at 518 nm was used to evaluate the performance of the proposed assay strategy. All measurements were carried out at room temperature unless stated otherwise. Protein Detection. The model analyte of protein thrombin was dissolved by 50% glycerin and diluted by water. HP needed a preparation process before the experiments. HP was diluted in 1 μM, then heated to 95 °C for 2 min, and then slowly cooled to 27 °C to ensure a hairpin structure. The preparation process needed for HP is about 20 min. After the preparation procedure, 5 μL of NEbuffer 4 (10× ), 5 μL of HP, 25 μL of BQF probe, and 5 μL of thrombin were mixed together, followed by incubation at 27 °C for 40 min. Then, 0.5 U/μL of Nb.BbvC I was added; fluorescence signal amplification reaction was carried out at 37 °C for 45 min. The same reaction mixtures without thrombin were used as negative controls. The last solution contained 100 nM HP, 3 μM BQF probe, 1× NEbuffer 4 (50 mM potassium acetate, 20 mM Trisacetate, 10 mM magnesium acetate, 1 mM dithiothreitol), and different concentrations of thrombin in a volume of 50 μL. Then, the solution was moved to a fluorescence cuvette for fluorescence measurement.

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EXPERIMENTAL SECTION Chemicals. Oligonucleotides designed in this study were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China), and the sequences of all oligonucleotides were listed in Table 1. A Black Hole Quencher (BHQ)-quenching fluorescence DNA probe50 (BQF probe) was used as the detection probe, whose 5′ and 3′ ends were modified with the fluorescent dye 6carboxyfluorescein (6-FAM) and its quencher Black Hole Quencher I (BHQ I), respectively. The hairpin probes (HP, shown in Table 1) were made up with three parts, thrombin aptamer (the blue), target DNA (the red), and extend DNA (the black). The underlined sequence near the 5′ terminal of

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RESULTS AND DISCUSSION Design for NEFSA. The scheme of the approach was separation-free protein detection based on target-responsive aptamer hairpin switch and nicking enzyme assisted fluorescence signal amplification (NEFSA). For some assays, each detected protein generally required two aptamers in order to form a sandwich structure.20,51−54 Herein, we proposed a new strategy using one aptamer based on structure switching3508

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triggered fluorescence signal amplification, circumventing the limitations of sandwich assays. The construction of the assay scheme was relatively simple, where the hairpin probe without any chemical modification or immobilization was involved. As shown in Scheme 1, the

Additionally, retaining the high binding activity of aptamer/ protein is important for an outstanding strategy; compared with other aptamer based assays,59−61 aptamer probe needed in this study did not require any modification or immobilization. Two DNA probes were required for the proposed work, which was simpler than most of the other signal amplification protein detection methods.16,21,62 Moreover, the assay has separationfree measurements without utilizing magnetic beads, thus eliminating the washing steps used to separate unbound from bound ligands. A minimal number of handling steps is needed for sample preparation and reaction, making this approach simple and easy. Furthermore, the proposed strategy is potentially universal since the sequences of aptamer probe could be easily designed for detection of other proteins by changing the corresponding aptamer. However, one of the great challenges in fluorescence amplification sensors is that the downstream amplifier can pick up the signal generated by the upstream amplifier in the absence of the target, resulting in a high background. In order to obtain low background, BHQ 1 was employed as the quencher for the quenching of fluorophore FAM in the design of detection probe. BHQ 1-FAM pairs in dual-labeled oligonucleotides can have a strong enough affinity for each other so that they form an intramolecular, nonfluorescent complex.63 In this assay, as shown in Figure 1a, the background fluorescence induced by 100 nM of the HP (group 2) was almost equivalent to the fluorescence intensity of water (group 1). After adding 3 μM of BQF probe (group 3), the fluorescence intensity had a slight increase. Then, after 0.5 U/μL of Nb.BbvC I was added, the fluorescence intensity displayed by group 4 was almost as low as the fluorescence without nicking enzyme (group 3). However, in the presence of 10 nM thrombin, significant fluorescence responses were achieved (shown in group 5). This phenomenon showed a low background fluorescence signal, which indicated that this assay was suitable for analytical applications. Further support that confirms the activation of the aptamer machine was obtained by gel-electrophoresis experiments. As shown in Figure 1b, it was observed that no cleaved BQF probe was obtained in the absence of target protein (shown in lane 2, 3, 4). However, a new product (cleaved BQF probe) appeared in lane 5, which is only generated in the presence of the thrombin. The experimental data strongly demonstrated that the design of our proposed strategy is successful. Sequence Optimization of Hairpin Probe. The key point of our proposed strategy was that, only when the hairpin probe was opened, the BQF probe could hybridize with TDNA. Therefore, the length of E-DNA was optimized in order to make sure that the aptamer bound preferentially to the target protein while the T-DNA hybridized with the BQF probe only in the presence of the target protein to specifically trigger the reaction. We compared fluorescence characteristics of three different HPs by changing the length of the stem for selecting a proper probe to initiate the homogeneous NEFSA effect (shown in table 1). As shown in Figure 2, the thrombin samples represented the fluorescence signals upon addition of 100 nM thrombin. The control group contained all the components in the sample group except thrombin. The signal-to-background ratio was used to evaluate the assay performance; the highest signal-to-background ratio was observed for HP 2. For this group, E-DNA of HP 2 perfectly matched thrombin aptamer with 9 base-pairs, while HP 3 matched with 6 base-pairs and HP 1 matched with 12 base-pairs. When the matched base-pairs

Scheme 1. Schematic Representation of Homogeneous Aptamer and Nicking Enzyme Assisted Fluorescence Signal Amplification (NEFSA) Assay for Protein

hairpin probe (HP) contained three sections: thrombin aptamer (blue), target-DNA (T-DNA, red) and extend-DNA (E-DNA, black). E-DNA was designed to be complementary to a part of the aptamer, in order to stabilize the hairpin-sharp form of the HP in the absence of protein. BQF probe carrying a specific enzyme cleavage site served as detection probe, the sequence of which could completely hybridize with the T-DNA to form a double strand. A BQF probe of the double strand can be recognized and cleaved by nicking enzyme. The design of the complementary domain of HP was to prevent the hybridization of T-DNA to BQF probe in the absence of target protein. The loop was complementary to the BQF probe, which might help T-DNA completely and quickly hybrid to BQF probe in the presence of protein. The binding of aptamer to protein unzipped the double-stranded aptamer/E-DNA of the HP. HP was energetically more stable when aptamer binding to the target protein formed the aptamer/protein complex, causing the HP to adopt the open form. BQF probe could easily hybrid to T-DNA to form the double-stranded structure because T-DNA was single stranded when HP was opened. The double stranded probe is a substrate of Nb.BbvC I. Then, the nicking enzyme bound to and cleaved only the BQF probe. The cleaved BQF probe was too short to maintain the double-stranded conformation, resulting in the complete disconnection of the fluorophore from the quencher, and then a fluorescence signal appeared. The T-DNA could be reused to hybridize to the next uncleaved BQF probe. Finally, each target strand could go through many cycles, leading to cleavage of many BQF probes. The traditional assays had been usually based on recognition interaction between protein and detection probe in a 1:1 stoichiometric ratio.55−57This stoichiometric recognition interaction limited the detection sensitivity, because one target could only cause one quenched fluorescent probe to fluoresce for the detection. Therefore, the current method will have a lower detection limit than the traditional ones.58 3509

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Signal Generation and Optimization. In our study, we developed an assay in homogeneous solution, where the cleaved BQF probes were used as the signal reporters and the reactions involved were simple and fast. Detection signal was amplified through protein−aptamer recognition and nicking enzyme cleavage. As a result, one protein produced many signal probes. Only when the nicking reaction took place could the BQF probes be cleaved, creating the amplified fluorescence signal. To achieve desirable analytical characteristics, the nicking enzyme reaction time was optimized. As shown in Figure 3, when the cleavage time was prolonged, the

Figure 3. Time course of the fluorescence intensity in the presence of 10 nM thrombin or without thrombin (the controls). The fluorescence intensity was obtained every 5 min, and the results showed a linear increase in fluorescence between 0 and 50 min. The error bar was calculated from three independent experiments.

Figure 1. (a) Fluorescence intensities of various phases of the assay. The components of the five groups are 1, water; 2, HP; 3, HP and BQF probe; 4, HP, BQF probe, and Nb.BbvC I; 5, HP, BQF probe, Nb.BbvC I, and thrombin (10 nM). The error bar was calculated from three independent experiments. (b) Nondenaturing PAGE analysis of the products for the mechanism. Lane M, marker; lane 2, HP; land 3, HP and BQF probe; lane 4, HP, BQF probe, and Nb.BbvC I; lane 5, HP, BQF probe, Nb.BbvC I, and thrombin (100 nM). Samples were separated on a nondenaturing 15% polyacrylamide gel in 1× TBE at 100 V constant voltage for about 90 min and then stained with silver. M represents 20-bp DNA ladder.

fluorescence of the final solution increased as well, which indirectly displayed the procedure of cleavage by nicking enzyme. The results showed a linear increase in fluorescence between 0 and 50 min, and then, the intensity continued to increase slowly. Control groups also had a slight intensity increase which might be caused by the contamination of the Nb.BbvCI with DNA nucleases. Taking both the effectiveness and the speed into consideration, we chose 45 min as the nicking enzyme reaction time for our reactions. Analytical Performance of Protein Detection. To confirm the ability of the described strategy to sensitively detect target protein, a series of different concentrations of thrombin were measured. As shown in Figure 4, the results reveal a concentration dependent response. Figure 4 displayed the relationship between the fluorescence intensity and thrombin concentration between 0 pM (control) and 100 nM. The phenomenon that the fluorescence intensity increased gradually with the concentrations of thrombin was related to the accumulation of the fluorescence, and it implied that more and more BQF probes were cleaved by nicking enzyme in solution. Additionally, the fluorescence of the control sample was relatively low because the T-DNA can be hybridized only in the presence of target protein. Inset in Figure 4 is the amplification of the linear range from 0 pM to 1 nM for thrombin determination. The detect limit was estimated to be 100 pM from three times the standard deviation corresponding to the blank sample detection. Such detection sensitivity was comparable to many exisiting homogeneous assay techniques for protein detection.64−66

Figure 2. Sequence optimization of hairpin probe. The thrombin in the control groups was absent, but all the other compositions and reaction steps were the same as in the detection of thrombin sample. The concentration of the thrombin sample is 100 nM. The error bar was calculated from three independent experiments.

were too long, the binding efficiency between aptamer and thrombin were not good enough for the opening of the hairpin probe, and when the matched base-pairs between thrombin aptamer and E-DNA were only 6, the hairpin-probe was unstable. Thus, we chose HP 2 for the experiments. 3510

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Detection of Human Thrombin in Human Blood Serum. A 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, the amount of thrombin spiked in serum was evaluated with the aptasensor. 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 NEBbuffer and then spiked with thrombin at three concentrations (0.1, 1, and 10 nM thrombin). As shown in Figure 6, comparable responses were

Figure 4. Fluorescence intensities obtained in the presence of control or different concentrations of thrombin. Concentrations of thrombin are 0 pM (control), 30 pM, 60 pM, 120 pM, 240 pM, 500 pM, 1 nM, 10 nM, and 100 nM. The inset shows the linear region from 0 pM to 1 nM of thrombin, yielding the detection limit of 100 pM. The error bar was calculated from three independent experiments.

Detection Specificity. Sensitivity and specificity were the two key factors for a successful assay system for protein detection. In the proposed strategy, the sensitivity mainly depended on the nicking enzyme reaction and the low background fluorescence associated with the specific hybridization of BQF probe with T-DNA. The detection specificity was basically determined by the aptameric recognition function. Two proteins, BSA and lysozyme, at concentration of 10 μM were used to test the specificity of this assay. The resulting signal was compared with that obtained using 10 nM thrombin, as shown in Figure 5. High signal was obtained only when the

Figure 6. Results obtained from the testing of serum samples spiked with thrombin (diluted in 1:10 ratio) and thrombin in buffer (thrombin concentration is 0.1, 1, and 10 nM). The same reaction mixtures without thrombin were used as controls. The error bar was calculated from three independent experiments.

found for thrombin in both buffer and serum. However, the sensitivity for the detection in blood serum cannot be as high as that for the reaction in buffer. 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 caused the binding of the BQF probe and TDNA, 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.

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DISCUSSION AND CONCLUSIONS In summary, we have successfully demonstrated a homogeneous approach for the development of an aptameric detection method for protein using nicking enzyme amplification technology based on the function of aptameric recognition. First, this homogeneous assay system has several advantages, such as isothermal experimental condition, simple preparation, convenient operation, and low-cost devices. The entire time needed for the preparation and reaction processes was less than 2 h, which was faster than some of the other signal amplification assays.44,54,67 Additionally, the detection of protein with picomolar sensitivity is achieved by this study. The detection limit is down to 100 pM. Third, the aptamer probe without any modification or label is involved, which is important for retaining the high target binding activity. Moreover, the aptamer/target binding indirectly initiates

Figure 5. Test of human thrombin and other proteins, showing the specificity of the assay. The concentration of the thrombin was 10 nM. However, the concentration of BSA and lysozyme is 10 μM. There is no thrombin or test proteins contained in control group. The error bar was calculated from three independent experiments.

specific protein (thrombin) was tested, whereas the fluorescence signals were negligible in the presence of BSA and lysozyme. The measured data demonstrated that the fluorescence signal was specifically triggered by the aptamer/ target binding for parallel samples, which indicated that the proposed sensing system could offer a high specificity. This revealed excellent selectivity of fluorescence protection assay, which might support its potential in clinical applications. 3511

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homogeneous NEFSA reaction where T-DNA is used to mediate the signaling process. In other words, the homogeneous NEFSA do not directly depend on the sequence of aptamer probe. Thus, it can be applied to the detection of other target analytes by changing the corresponding aptamer, which means the design of the screening scheme is potentially universal. Furthermore, only one aptamer is used to trigger the NEFSA, which overcomes the limitations of the sandwich assay, circumventing the requisite of two binding sites per target protein. Moreover, this proposed assay could be applied in a complex biological system, which is important for its application in biological or clinical target detection. In view of these advantages, this one-step homogeneous NEFSA strategy has promising application in the monitoring of many other proteins with high sensitivity and excellent specificity.

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AUTHOR INFORMATION

Corresponding Author

*Tel: (+86-20) 8521-0089. Fax: (+86-20) 8521-6052. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The first two authors (L.X. and X.Z.) contributed equally to this work. This research is supported by the National Basic Research Program of China (2010CB732602), the National Natural Science Foundation of China (81101121), the Key Program of NSFC Guangdong Joint Funds of China (U0931005), the Program for Changjiang Scholars and Innovative Research Team in University (IRT0829), and the Natural Science Foundation of Guangdong Province (S2011040005386).

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Analytical Chemistry

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dx.doi.org/10.1021/ac2026783 | Anal. Chem. 2012, 84, 3507−3513