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Highly Sensitive and Selective Bifunctional Oligonucleotide Probe for Homogeneous Parallel Fluorescence Detection of Protein and Nucleotide Sequence Li-Ping Qiu, Zai-Sheng Wu,* Guo-Li Shen, and Ru-Qin Yu* State Key Laboratory of Chemo/Biosensing and Chemometrics, Chemistry and Chemical Engineering College, Hunan University, Changsha 410082, P. R. China
bS Supporting Information ABSTRACT: The existing isothermal polymerization-based signal amplification assays are usually accomplished via two strategies: rolling circle amplification (RCA) and circular strand-displacement polymerization. In essence, the two techniques are based on cyclical nucleic acid strand-displacement polymerization (CNDP), limiting the application of isothermal polymerization in medical diagnosis and bioanalysis. In the present study, circular common target molecule (non-nucleic acid strand)-displacement polymerization (CCDP) is developed to amplify the fluorescence signal for biomolecule assays, extending isothermal polymerization to an aptameric system without any medium. Via combining an aptamer with a common hairpin DNA probe, we designed a self-blocked fluorescent bifunctional oligonucleotide probe (signaling probe) for the homogeneous parallel detection of two disease markers, PDGF-BB and the p53 gene. On the basis of CNDP and CCDP signal amplification, highly sensitive (e.g., detecting PDGF down to the concentration level of 1.8 1010 M) and selective detection (no interference even in the presence of a significantly higher concentration (7200 times) of nontarget proteins) was accomplished, and the linear response range was considerably widened. Furthermore, the bifunctional signaling probe exhibits impressive simplicity, convenience, and short detection time. Herein, the design of the signaling probe was described, factors influencing fluorescence signal were investigated, analytical properties were characterized in detail, and the assay application in a complex medium was validated. The proposed biosensing scheme as a proof-ofconcept is expected to promote the application of oligonucleotide probes in basic research and medical diagnosis.
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roteins are ubiquitous and essential in all living organisms. The recognition and quantification of disease-marker proteins, especially those associated with cancers, are of particular importance in fundamental research and corresponding applications such as medical diagnosis, prevention, and treatment. Nowadays, methods for protein analysis have become indispensable tools in new disease-marker discovery and their function studies.1 On the other hand, single nucleotide polymorphisms (SNPs) are abundant genetic variations in the human genome sequence, and point mutations as a deleterious type of SNPs are the hallmarks of several diseases, such as cancers. Reliable methods for screening point mutations are beneficial for human health.2,3 Despite proteins and SNPs having been both investigated using separate sensing probes, it remains a challenge and is of significance to design a multifunctional recognition probe for the detection of different target analytes. Aptamers are artificial, short, single-stranded DNA and RNA oligonucleotides screened by an in vitro evolution process called SELEX (systematic evolution of ligands by exponential enrichment) on the basis of their high affinity in binding specifically to proteins or other targets.46 Although those aptamers r 2011 American Chemical Society
composed of nonmodified RNAs tend to be digested by ribonucleases in serum,7,8 the DNA aptamers can be used under a wide range of conditions without losing binding activity to their target molecules.911 In addition to the desirable stability compared with protein bodies that are only stable in a physiological environment, other intrinsic advantages of nucleic acid components in synthesis,6 labeling, signaling, and amplification,12,13 make aptamers appear as promising alternative candidates to traditional antibodies in analytical devices for biodetection applications.1,6,14 For a sensitive aptameric assay, the conformational change of the aptamer upon target binding usually becomes the starting point for the design of a novel probe and signaling scheme regardless of whether optical1519 or electrochemical2026 analysis systems are developed. To date, aptamer-based bioassays have received tremendous attention in scientific and industrial communities.2731 However, up to now, only a few bifunctional aptamer probes20,3235 Received: December 16, 2010 Accepted: February 28, 2011 Published: March 29, 2011 3050
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Table 1. Oligonucleotides Designed in the Present Study
have been developed for parallel analysis of different analytes despite their potential applications. For example, Elbaz and coworkers32 developed a bifunctional aptamer for the analysis of cocaine and adenosine 50 -monophosphate (AMP). In that work, while the analytical significance of bifunctional aptamers was demonstrated, the bioelectronic logic gate system was fabricated along this line. However, the target detection is limited to the micromolar range. Du et al.20 and Zhang et al.33 separately reported a multifunctional electrochemical biosensor based on a multifunctional integrated aptamer for the detection of two different targets. Deng et al.34 recently designed a bifunctional electrochemical biosensor based on two separate aptamers rather than an integrated aptamer for the analysis of small molecules (adenosine) or proteins (lysozyme). Despite good performance, these sensing schemes20,33,34 were still confronted with some limitations from interface detection, such as the labor-intensive and time-consuming interface fabrication, poor reproducibility, and low-efficiency biomolecule activity. To simplify testing procedures, improve the sensitivity, and circumvent potential problems associated with interface-related assay systems, developing new bifunctional oligonucleotide probes and proposing efficient homogeneous signaling schemes36 are currently a subject of concern. Because of the relatively low association constants of aptamers to their target molecules, it is difficult to develop a highly sensitive aptamer probe by a conventional transducing scheme. It is essential to design a signal amplification protocol when proposing aptamer-based sensing systems regardless of the fluorescent,3740 chemiluminescent,41 electrochemical,42,43 or microgravimetric44 measurements. Among those amplification protocols, rolling circle amplification (RCA), an isothermal nucleic acid circular amplification technique, has been attracting much attention in aptameric systems.13,37,38,4549 However, the circular single-stranded DNA templates make the operation complicated, limiting the applications in many cases. When a linear DNA sequence is used as the template and a trigger is designed to repeatedly trigger the polymerization reaction, this biochemical technique is called isothermal circular strand-displacement polymerization (ICSDP).50 In this case, the former products can be displaced by polymerases during the polymerization, promoting the generation of the latter ones. This results in repeating sequences and in turn amplifies the target analyterecognition binding event. Although the ICSDP technique has proved to possess attractive amplification and multiplexing properties for the development of sensitive biosensing systems,51,52 very few ICSDP-based aptamer probes39,53 are proposed. In principle, two protocols might be adopted in ICSDP-based aptasensing system: circular nucleic acid strand-displacement
polymerization (CNDP) and circular common target moleculedisplacement polymerization (CCDP). Just as its name implies, in the former, nucleic acid strands are circularly displaced during the polymerization reaction, while common target molecules (non-nucleic acid strand) are circularly displaced in the latter. For the CCDP, to introduce the isothermal polymerization reaction into aptasensing systems, transferring the targetaptamer binding into the circular nucleic acid stranddisplacement polymerization is the primary prerequisite. Platelet-derived growth factor BB (PDGF-BB), a cancerrelated protein, has been directly implicated in many cell transformation processes and in tumor growth and progression.54 The p53 tumor suppressor gene is a transcription factor of cell regulation. Because the mutation of the p53 gene is the most common phenomenon in carcinogenesis and tumor progression, this gene was usually chosen as a model to confirm a new sensing tool.55 Sensitive and rapid detection of such proteins and mutations could help early diagnosis, treatment, and prognosis of cancers. On the basis of the above considerations, in this study, we developed a sensitive and selective fluorescent self-blocked bifunctional oligonucleotide probe (consisting of an aptamer sequence and common DNA strand) with the combination of CCDP and CNDP for the homogeneous parallel detection of two analytes, PDGF-BB and the p53 gene. As a proof-of-concept, the CCDP technique was for the first time introduced into an aptasensing system for the analysis of the target protein. In the presence of PDGF or the p53 gene, the target binding can induce the conformational change of the designed bifunctional probe that in turn triggers an isothermal circular polymerization reaction, contributing to the signal amplification. Utilizing this signaling scheme, the aptasensing system developed possesses a simple experimental procedure, needs a short detection time, is suitable for detecting a broader range of analytes, and exhibits improved analytical properties. Herein, the design and signaling principle of bifunctional oligonucleotide probes were elucidated, and the working conditions were optimized. The detection performance (e.g., the sensitivity, selectivity, and linear dynamic range) and the capability to amplify the response signal were evaluated by horizontal and longitudinal comparison between different measured data. The preliminary application of this signaling probe was also investigated.
’ EXPERIMENTAL SECTION Chemicals. Oligonucleotides designed in this study were synthesized by Invitrogen Bio Inc. (Shanghai, China), and their sequences are listed in Table 1. The bifunctional oligonucleotide probe (signaling probe) consists of two recognition units: one in 3051
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Analytical Chemistry bold italics for PDGF-BB and the other underlined for the p53 target DNA. Lowercase letters, “t” in the middle of signaling probe and “a” at the 30 terminus, are labeled with FAM (fluorescein) and DABCYL (4-(40 -dimethylaminophenylazo)benzoic acid), respectively. The two regions in the boxes are designed to be complementary to each other. All oligonucleotides were dissolved in water prior to use. Unless otherwise stated, the primer3 was used throughout. Platelet-derived growth factor (PDGF)-BB, human IgG, and human serum albumin (HSA) were obtained from Dingguo Biochemical Reagents Company (Beijing, China). Immunoglobulin E (IgE) purified from human plasma was purchased from Meridian Life Science, Inc. (Saco, ME). The deoxynucleotide solution mixture (dNTPs) and polymerase Klenow fragment exo (5 u/μL) accompanied by 10Klenow Fragment exo buffer (500 mM Tris-HCl pH 8.0, 50 mM MgCl2, 10 mM DTT) were purchased from Shanghai Sangon Biological Engineering Technology and Services Co. Ltd. (Shanghai, China). The polymerase used was prepared by diluting stock solution with water to l u/μL. All other chemicals involved were of analytical-reagent grade. Deionized and sterilized water (resistance > 18 MΩ 3 cm) was used throughout. Apparatus. Fluorescence spectra were determined using a Hitachi F-7000 fluorescence spectrometer (Hitachi Ltd., Japan) controlled by FL Solution software. The optical path length of a quartz fluorescence cell was 1.0 cm. Excitation and emission slits were all set for a 5.0 nm band-pass. The mixtures in square quartz cuvettes were excited at 491 nm, and the emission spectra were collected from 500 to 600 nm. The fluorescence intensity at 518 nm was used to evaluate the performances of the proposed assay strategy. All optical measurements were performed under room temperature unless otherwise indicated. Trigger-Displacement Polymerization Reaction and Amplified Target Detection. The protein determination could be briefly described as follows: the signaling probe (5 μL, 1 μM) was added to the mixture of PDGF-BB sample at specific concentration (5 μL) and 2.5Klenow Fragment exo buffer (2.5 μL) (KF buffer); then, 5 μL of 2 μM primer3 and 2.5 μL of 2 mM dNTPs were successively injected into the resulting solution; subsequently, 5 μL of Klenow Fragment exo (1 u/μL) was added. The resulting solution (called reaction buffer) was 0.25Klenow Fragment exo buffer, in which the targetprobe binding and subsequent polymerization occurred. After being incubated at 37 °C for 30 min, the polymerization reaction was stopped by addition of 0.5 M EDTA (1 μL).56,57 To obtain information on the amount of target in the sample, 174 μL of 0.25Klenow Fragment exo buffer was added to the resulting solution and the fluorescence spectrum was collected. The difference in the fluorescence peak between samples with and without target was used to quantify target molecules in sample. During evaluation of the detection specificity and the ability to determine the p53 target DNA, selected biomolecules (e.g., nontarget molecules and p53 target DNA) were used as the analytes, and the experiments were conducted according to the method described above. During characterization of the signaling probe and optimization of the experimental conditions, the polymerase, dNTPs, and primer might be replaced by an equal amount of corresponding solutions, and the KF buffer concentration as well as the reaction time could be also regulated according to the experimental requirements.
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’ RESULTS AND DISCUSSION Design of the Bifunctional Oligonucleotide Probe and Principle of Target Amplification Detection. The isothermal
circular polymerization-based bifunctional oligonucleotide probe facilitates the integration of multiple functional elements into a cooperative biosensing system: homogeneous assay format, isothermal signal amplification, aptameric target recognition, and hairpin DNA probe.58,59 In a traditional circular strand-displacement polymerization reaction, a nucleic acid strand acts as both the trigger of the polymerization reaction and a displaced sequence,51,52 and the design of the screening DNA probe is straightforward to a certain extent. By contrast, for an aptasensing probe, the trigger is the target species of interest while the displaced element is a common DNA sequence. This needs an ingenious strategy (called converting strategy) to convert the targetaptamer binding into the isothermal circular polymerization, making the design of the aptasensing probe very complicated. Although we have developed a fluorescent aptameric probe for the sensitive detection of cocaine using the standard strand displacement amplification technique,53 only the aptamer with a unique secondary structure that contains a long stretch of paired nucleotides can maintain its recognition activity after being split into two molecules.60 Up to now, the applications of circular strand-displacement polymerization amplification to aptasensing systems are few and far between despite the attractive properties of this biochemical technique.39 Presumably, this is due to the lack of converting strategies. Furthermore, incorporating isothermal circular polymerization amplification into the aptameric probe, especially into a bifunctional oligonucleotide probe composed of an aptamer and a common oligonucleotide probe for different targets, remains a challenge. To demonstrate a proof-of-concept of a novel strategy for designing biosensing systems, we developed a new isothermal polymerization reaction protocol, CCDP. If the signaling manner adopted in a CNDP-based aptameric sensing system39 is indirect, the CCDPbased aptasensing format represented in the present work is direct, in which the converting strategy is circumvented. The design of bifunctional oligonucleotide probe and principle of target amplification detection are elucidated in Scheme 1. The sequences of the signaling probes were designed with the help of DNA folding software (mfold) based on previous works.61,62 The original sequence of anti-PDGF aptamer63,64 can fold into a three-way helix junction, a consensus secondary structure motif (shown in Scheme 1A) with a conserved single-stranded loop at the branch point. Because the segments at both ends complementary to each other are not important for the high-affinity binding to target PDGF, the outer sequence of aptamer could be regulated while reserving a good binding affinity.63,64 In this study, the bifunctional oligonucleotide probe was designed by slightly changing the outer bases of original anti-PDGF aptamer and extending by the common recognition probe for detecting p53 target DNA at the 30 -end. To introduce the isothermal polymerization signal amplification and to make the signal amplification occur only in the presence of target molecules, a segment (antiprimer) is added to the outermost part at the 30 -end. The antiprimer is designed to be complementary to the primer and to compete with the 50 -terminal segment for the region in the box of the middle region (shown in Table 1). The basic design idea of the bifunctional oligonucleotide probe is demonstrated in Scheme 1A, while its secondary structure in the absence of target molecules is represented in Scheme 1B. Although the aptamer 3052
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Analytical Chemistry Scheme 1. (A) Design of Bifunctional Oligonucleotide Probe Consisting of anti-PDGF-BB Aptamer and Recognition Sequence for the p53 Gene. (B) Secondary Structure of the Bifunctional Oligonucleotide Probe Predicted by the Program “mfold”.a (C) Schematic Illustration of Homogeneous Parallel Fluorescent Detection of PDGF-BB and p53 Target DNA Based on the Structure-Switching-Triggered Isothermal Circular Target-Displacement Polymerization Signal Amplificationb
a
In the absence of target molecules, the isothermal polymerization can be inhibited by the formation of stem4-loop structure due to the intramolecular hybridization. In this case, the fluorescence is quenched because the fluorophore is brought into the proximity to the terminal quencher. b Upon addition of PDGF-BB, the well-known secondary structure motif, a three-way helix junction, is formed, and the segment close to the 30 -end that is complementary to the primer is released, triggering the CCDP in the presence of primer, dNTPs, and polymerase and enhancing considerably the fluorescence signal. Similarly, the hybridization of p53 target DNA to loop sequence can trigger CNDP reaction, and the signal amplification can be achieved. The impracticable process corresponding to the nontarget analytes is marked with a cross.
part shaded in Table 1 can form a three-way helix junction (predicted by the program “mfold”), stem4 rather than stem3 is obtained in the free bifunctional oligonucleotide probe. In this case, the primer is incapable of hybridizing to antiprimer, inhibiting (namely, locking) the subsequent isothermal polymerization process. Taking this and the common nonaptamer sequence involved into account, the designed probe is also called self-blocked bifunctional oligonucleotide probe. To generate fluorescence signal upon addition of target analyte, FAM and DABCYL are attached to the bifunctional signaling probe at the positions indicated in Table 1. For the free designed fluorescent
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probe, the fluorescence quenching occurs because the formation of stem4 can bring the quencher close to the fluorophore. The parallel analysis of PDGF and p53 target DNA is elucidated in Scheme 1C. When introducing PDGF into the developed sensing system, the binding of the target molecule makes the aptamer region of the bifunctional oligonucleotide probe fold into an expected three-way helix junction, facilitating the formation of stem3 and releasing the antiprimer at the 30 -end. As a result, the fluorophore and quencher are moved from each other, leading to the partial restoration of the FAM fluorescence and generating a small fluorescence signal. In this case, the primer can hybridize to the antiprimer and is subsequently extended by the polymerase from the 30 -end in the presence of dNTPs over the designed signaling probe as the template. The formation of rigid extension product/bifunctional oligonucleotide probe hybrid causes the complete stretching of bifunctional probe and resulting in a full fluorescence restoration. More importantly, the target protein, PDGF-BB, is expected to be displaced during the isothermal polymerization process and binds to another bifunctional probe, triggering a new isothermal polymerization reaction. This results in the protein target binding/displacement cycle-based polymerization, CCDP. Throughout this cyclical process, the designed fluorescent bifunctional probe serves as the recognition element, template for polymerization reaction, and fluorescence signal reporter, while the target protein acts as a trigger of circular polymerization reaction. Because the target can be displaced and trigger the polymerization reaction circularly, the amplification detection of target analytes can be accomplished. Under these conditions, even minute amounts of targets present in samples are able to induce obvious fluorescence signal. Therefore, by monitoring the change in fluorescence intensities, we could detect the target with high sensitivity. Similarly, upon the p53 target DNA, the hybridization between target and bifunctional probe can also open stem4 and promote the formation of primer/antiprimer complex, triggering the circular strand-displacement polymerization, CNDP. This produces an amplified fluorescence signal and improves the analytical performance of the biosensing system. Feasibility of Proposed Signaling Principle and Signal Amplification Capability of the Bifunctional Oligonucleotide Probe. According to a literature report,65 the target protein/ aptamer binding can protect the DNA strand against the exonuclease degradation. This result is also supported by other observations (e.g., PCR-based aptameric protein assay40). Furthermore, a recent work66 reported that binding of the target protein to the aptamer can affect the DNA replication by Klenow fragment exo, and the polymerase activity is inhibited by target protein in a concentration-dependent manner. Presumably, the binding of the target protein to the aptamer could increase the interaction between DNA segments and bring additional steric hindrance, generating a barrier to the accessibilities of polymerases. This, possibly as well as other factors, would influence the interaction between polymerase and DNA sequence. In our work, the same polymerase was involved, and the target species was also protein (PDGF-BB). However, CCDP was required to effectively proceed even in the presence of high concentrations of target protein. It seems that the designed bifunctional oligonucleotide probe is faced with a problem associated with proteinaptamer binding-induced inhibition of polymerization. Clearly, although the CNDP technique has been used to develop the DNA-based biosensing systems with desirable analytical characteristics,51,52 the CCDP protocol for interrogating PDGF is still challenging. 3053
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Figure 1. (A) Utility of CCDP technique for the amplification detection of PDGF-BB utilizing the designed bifunctional oligonucleotide probe. The fluorescence spectrum of the developed sensing system is collected in the absence (a) and presence (b) of PDGF where CCDP is not involved; c and d are the same as a and b, respectively, but the CCDP is introduced into the signaling process. The concentration of PDGF involved is 5.0 106 g/mL. (B) Utility of CNDP technique for the amplification detection of p53 target DNA. The fluorescence spectrum of the designed screening system is collected in the absence (a) and presence (b) of p53 target DNA without CNDP amplification; c and d are the same as a and b, respectively, but with the CCDP amplification process. In this section, 5.0 107 M p53 target DNA is used. The sample in the absence of target molecule mentioned might be also expressed as the blank.
To verify the feasibility of the designed fluorescent bifunctional oligonucleotide probe, the polymerization-based amplification detection of target and control experiments were conducted under identical conditions. The measured data are presented in Figure 1. As shown in Figure 1A, when CCDP is introduced into the biosensing system, a dramatic increase of the fluorescence peak (line d) is caused by addition of target PDGF
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compared with the value obtained for the blank (line c). In contrast, as demonstrated in the direct comparison of lines a and b, addition of target PDGF only induces a slight fluorescence change when the CCDP is not adopted. To accurately quantify the utility of CCDP, the fluorescence difference between the fluorescence peaks in the presence and absence of PDGF calculated from the fluorescence spectra of Figure 1A was used to evaluate the capability of CCDP to amplify the fluorescence signal. The fluorescence differences with and without the polymerization amplification are 330.2 and 23.9 au, respectively. This indicates that CCDP can substantially amplify the PDGF aptamer binding event, increasing the fluorescence signal by about 13.8 times. The experimental results demonstrate that PDGF bound to the bifunctional probe can be displaced by polymerase during the polymerization process, triggering a circular polymerization reaction and ensuring the working of CCDP. Probably, this should be attributed to the following reasons: (1) polymerase Klenow fragment exo exhibits desirable strand-displacement activity that has proved to be suitable for isothermal circular strand-displacement polymerization amplification;51,52 (2) the folded conformation with the threeway helix junction resulting from the PDGF binding essentially is the hybridized structure via the intramolecular base-pairing of the bifunctional oligonucleotide probe rather than the G-quadruplex structure that can serve as the barrier to polymerization.66 As a result, the degree of CCDP reaction is determined by the amount of free antiprimer that is closely related to the concentration of PDGF-BB in sample. Figure 1B illustrates the utility of the developed bifunctional probe for the detection of the p53 gene. Introduction of the CNDP reaction can lead to a considerable enhancement in the fluorescence response to the target, making the fluorescence difference increase from 183.2 to 441.0 au. More importantly, other assay performances (e.g., assay selectivity) are dramatically improved. These measured data indicate that the design of the fluorescent bifunctional signaling probe is successful and that the circular trigger-displacement polymerization-based sensing scheme is suitable for the parallel amplification detection of PDGF and the p53 gene. To characterize the designed biosensing system and to achieve versatile assay performance, the elements affecting the fluorescence response were investigated and the experimental conditions were optimized. The detailed results are shown in Figures S13 of Supporting Information. Analytical Performance of Protein Detection. To confirm the capability of the described bifunctional signaling probe to quantify target protein, a series of different concentrations of PDGF-BB were analyzed, and the blank fluorescence-subtracted fluorescence peak intensity was employed to evaluate the intensity of fluorescence response. As shown in Figure 2, the fluorescence peak increases with an increase in the concentration of target PDGF, indicating a signal-on sensing mechanism that possesses preferable sensing properties.67,68 The inset of Figure 2 clearly represents the linear relationship between fluorescence signal and the logarithm of PDGF concentration in the range from 2.5 108 to 1.0 105g/mL. When the target concentration is further increased or decreased, no considerable change in the fluorescence peak intensity is generated. The regression equation is Y = 124.8 log X 122.2, where Y and X denote the fluorescence peak intensity and target concentration, respectively. The corresponding correlation coefficient of calibration curve is 0.9989. The detection limit, which is defined as three 3054
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Figure 2. The blank-subtracted fluorescence spectra of the bifunctional oligonucleotide probe in the presence of different concentrations of PDGF-BB. Inset: the linear relationship between the fluorescence peak intensity at 518 nm and the logarithm of target concentration. The error bars indicate the standard deviation of repeated measurements. The detailed descriptions are presented in the corresponding section of text.
times the standard deviation of the blank measurements, is estimated to be 1.2 108 g/mL (4.2 1010 M, assuming that the molecular weight of PDGF-BB is 28 000). The detection capability achieved represents a 2.5- to 100-fold improvement in the sensitivity over the previously reported aptameric interrogating systems based on colorimetric,63 luminescent,69 fluorescent37 (close to an impressive value reported previously by fluorescence anisotropy, 70), and electrochemical71 techniques accompanied by a significantly widened linear dynamic range. In fact, a lower concentration (e.g., 5.0 109g/mL, namely, 1.8 1010 M) of target protein than the detection limit can still be detected, giving a fluorescence peak that is apparently higher than the value of the blank although the corresponding doseresponse point is outside the linear response range. Presumably, besides the versatile properties of targetaptamer binding, such excellent analytical performance should be attributed to several factors. First, the changes in the outer helix of the three-way junction including the modification of bases by functional groups, which are involved in the present study to design the bifunctional signaling probe, does not compromise the intrinsic bioactivity of original aptamer;1 second, binding of the target PDGF does not inhibit the isothermal polymerization over the developed probe as the template, and the extension of primer generates a complementary DNA, fully restoring the fluorescence of fluorophore; third, because the displaced PDGF triggers a circular polymerization, the designed CCDP can operate well, amplifying the target binding event. It is expected that the analytic performance could be further improved if the fluorescence peak of the blank is inhibited as mentioned in the subsequent section, Significance in Diagnostic Genomics and Proteomics and Further Improvement. To assess the reproducibility of the present biosensing system, target samples at different concentrations in the linear dynamic range (2.5 108 g/mL, 5.0 108 g/mL, 2.5 107 g/mL, 5.0 107 g/mL, 2.5 106 g/mL, 5.0 106 g/mL, and 1.0 105 g/mL) were repeatedly analyzed. The maximum value of the relative standard deviations obtained was only 5.6% for triplicate measurements, indicating that a desirable reproducibility for protein assay.
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Figure 3. The detection specificity of the proposed bifunctional oligonucleotide probe. IgE (36 μg/mL IgE), IgG (1.0 mg/mL), and HSA (1.0 mg/mL) were used to evaluate the detection specificity of the present biosensing system. The concentration of PDGF-BB is 5.0 μg/mL, and the corresponding fluorescence response is defined as 100%.
Detection Specificity. In addition to the detection sensitivity, the specificity is another critical factor for a potential assay system. For an aptasensing system, the detection specificity is essentially determined by the intrinsic properties of aptamer. Although the original PDGF aptamer has been proved to specifically recognize its target molecule,64 the aptamer in the proposed strategy was adapted by changing the sequence and modifying by optical reporter units. This possibly led to the change (deterioration or improvement) of target recognition ability of the aptamer region.72,73 In order to evaluate the detection specificity of the present detection system, the degree of nonspecific binding of other proteins possibly coexisting in target samples was investigated under the same experimental conditions as those involved for the PDGF assay. Figure 3 depicts the direct comparison between the fluorescent response intensities of target PDGF and nontarget proteins. Even though substantially higher concentration of nontarget proteins are involved, their relative fluorescence responses are not more than 7.0% based on the assumption that the fluorescence signal triggered by target PDGF is 100%, indicating insignificant nonspecific bindings. The measured data indicate that the fluorescence signal observed was generated by the specific interaction between the designed bifunctional oligonucleotide probe and the target protein. The applications of the proposed bifunctional oligonucleotide probe for the PDGF assay in serum samples and for p53 detection are validated in Supporting Information (Figures S47). Significance in Diagnostic Genomics and Proteomics and Further Improvement. During the design of the signaling probe, the novel signal amplification concept, CCDP, is introduced, extending the polymerization technique to aptasensing systems without any mediating elements (e.g., nicking enzyme39). When CCDP is combined with CNDP, the amplification detection of nonnucleic acid target and nucleic acid target can be accomplished in a parallel manner, achieving impressive assay characteristics and broadening the scope of target molecules. In 3055
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Analytical Chemistry addition to progress in the signal amplification technique, an advance in the design of nucleic acid assay probes has been achieved. The hairpin structure, stem4-loop shown in Scheme 1B, not only selectively recognizes the p53 target DNA but also prevents the circular polymerization reaction in the absence of target (self-polymerization), forming a self-blocked bifunctional oligonucleotide probe. In this case, the polymerization signal amplification process is capable of being specifically induced by target binding. This innovative probing design not only eliminates additional blockers of the polymerization amplification process39 but also circumvents the difficulties associated with the splitting of an aptamer sequence. Besides, the interactions, including the PDGF/signaling probe binding, p53 target DNA/signaling probe hybridization, and primer/antiprimer hybridization, occur between only two molecules. Compared with the probetargetprobe triplex structure,53 the effective collision probability can be increased. Moreover, in contrast to the literature sensing scheme where the target binding and polymerization amplification are separated,39 in the proposed sensing scheme, the target (namely PDGF and the p53 gene) binding and the replication of the signaling probe in essence can alternately proceed during the circular polymerization reaction. The two elements not only simplify the detection procedure but also reduce the assay time. For example, the total assay time required is about 40 min, being considerably shortened when compared to previous aptameric systems for the homogeneous optical detection of the same target protein.37,63 Even though, prior to the addition of polymerase, target binding was preincubated for a specific period (e.g., 30 min) after mixing the target with the designed signaling probe to form the targetprobe duplex, no detectable increase in the fluorescence response was observed, demonstrating the rationality of the employed assay procedure. Unfortunately, a blank fluorescence peak is detected for the present sensing system, compromising the assay capability. The possible reason is that there is a small amount of free antiprimer sequences. As indicated in the literature,51,25 the hairpin probe for the fluorescence detection of target nucleic acids based on isothermal circular strand-displacement polymerization has no detectable blank fluorescence peak. This probably offers a promising prospect for the further improvement in the analytical performance of bifunctional oligonucleotide probes. There is the competition (hybridization competition) between stem3 and stem4 as shown in Scheme 1. If stem4 is exceedingly stable, the structure-switching could not occur even in the presence of targets of interest. If the reverse happens, the antiprimer is easily released even in the absence of target molecules, inevitably leading to a blank peak. Thus, the hybridization competition should be further optimized via controlling the length of stem3 and stem4. Under optimized conditions, the binding of target analytes to the signaling probe is expected to induce structureswitching from single strand containing stem4 to targetprobe duplex with stem3, specifically triggering the subsequent polymerization amplification reaction. In this case, the assay characteristics should be further improved. Additionally, both targets create the same type of optical signal, and the bifunctional probe is unable to determine whether the signal is induced by the presence of PDGF or p53, or a mixture of both. Namely, this oligonucleotide probe in the present design cannot discriminate PDGF from the p53 target. However, it should be noted that the introduction of a common oligonucleotide sequence corresponding to p53 that folds into a
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hairpin structure can inhibit the self-polymerization of DNA, generating a self-blocked bifunctional oligonucleotide probe. This makes the CCDP signal amplification technique an analytical tool for PDGF, though the CCDP concept could be discovered without preintroduced hairpin structure. Furthermore, the common sequence and aptamer designed in the same probe not only cooperate to complete the parallel analysis of two disease markers but also share the fluorophore and quencher. Thus, in addition to the improved assay performance, the assay cost is substantially decreased compared with two separate monofunctional probes, making the proposed biosensing scheme affordable for an ordinary laboratory.
’ CONCLUSIONS This contribution describes the first instance of the use of CCDP amplification for proteinaptamer binding that is suitable for integration into a classical polymerization amplification technique, CNDP, accomplishing the development of a bifunctional signaling probe for the parallel analysis of important disease markers. The target binding-induced molecular structure-switching of the designed oligonucleotide probe allows transduction of the interaction between targets and signaling probes into the fluorescent signal, accomplishing the analyses of PDGF and the p53 gene in homogeneous solution. This method takes advantage of the dramatic increase in the intensity of the turn-on fluorescence signal upon target molecule binding that can trigger isothermal circular polymerization amplification. The developed biosensing system allowed the accurate quantification of PDGF-BB and p53 and achieved considerable improvement in assay characteristics compared with the common screening scheme. We have also validated that the self-blocked bifunctional signaling probe is an effective tool for recognizing target elements in a complicated environment, the human serum-containing sample. With its advantages of rapidity, sensitivity, and specificity, this described screening scheme is expected to hold great potential for use in protein analysis and cancer diagnosis. ’ ASSOCIATED CONTENT
bS
Supporting Information. Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Phone: 86-731-88821916. Fax: (þ86) 731-88821916. E-mail:
[email protected] (Z.-S.W.);
[email protected] (R.-Q. Y.).
’ ACKNOWLEDGMENT Financial assistance is gratefully acknowledged from the National Natural Science Foundation of China (Grants Nos. 90817101, 20905022, and 20775023) and “973” National Basic Research Program of China (No. 2007CB310500). ’ REFERENCES (1) Yang, C. J.; Jockusch, S.; Vicens, M.; Turro, N. J.; Tan, W. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 17278–17283. (2) Nie, B.; Shortreed, M. R.; Smith, L. M. Anal. Chem. 2006, 78, 1528–1534. 3056
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