Attomolar Detection of Proteins via Cascade Strand-Displacement

Publication Date (Web): July 13, 2015 ... Herein, we develop a new type of amplified fluorescence polarization (FP) ..... The Supporting Information i...
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Attomolar Detection of Proteins via Cascade Strand-Displacement Amplification and Polystyrene Nanoparticle Enhancement in Fluorescence Polarization Aptasensors Yong Huang,* Xiaoqian Liu, Huakui Huang, Jian Qin, Liangliang Zhang, Shulin Zhao,* Zhen-Feng Chen, and Hong Liang* Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Ministry of Education), College of Chemistry and Pharmacy, Guangxi Normal University, Guilin 541004, China S Supporting Information *

ABSTRACT: Extremely sensitive and accurate measurements of protein markers for early detection and monitoring of diseases pose a formidable challenge. Herein, we develop a new type of amplified fluorescence polarization (FP) aptasensor based on allostery-triggered cascade strand-displacement amplification (CSDA) and polystyrene nanoparticle (PS NP) enhancement for ultrasensitive detection of proteins. The assay system consists of a fluorescent dye-labeled aptamer hairpin probe and a PS NP-modified DNA duplex (assistant DNA/trigger DNA duplex) probe with a single-stranded part and DNA polymerase. Two probes coexist stably in the absence of target, and the dye exhibits relatively low FP background. Upon recognition and binding with a target protein, the stem of the aptamer hairpin probe is opened, after which the opened hairpin probe hybridizes with the single-stranded part in the PS NP-modified DNA duplex probe and triggers the CSDA reaction through the polymerase-catalyzed recycling of both target protein and trigger DNA. Throughout this CSDA process, numerous massive dyes are assembled onto PS NPs, which results in a substantial FP increase that provides a readout signal for the amplified sensing process. Our newly proposed amplified FP aptasensor enables the quantitative measurement of proteins with the detection limit in attomolar range, which is about 6 orders of magnitude lower than that of traditional homogeneous aptasensors. Moreover, this sensing method also exhibits high specificity for target proteins and can be performed in homogeneous solutions. In addition, the suitability of this method for the quantification of target protein in biological samples has also been shown. Considering these distinct advantages, the proposed sensing method can be expected to provide an ultrasensitive platform for the analysis of various types of target molecules.

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enhancers for amplified analysis of various target molecules. Also, nicking endonuclease24 or exonuclease25 was used as the biocatalytic amplifiers for the FP detection of small molecules and proteins. In addition, an alternative amplification strategy based on target-triggered cyclic assembly of DNA−protein hybrid nanowires by hybridization chain reaction was also reported for dual-amplified FP detection of small molecules.26 Despite many advances in this field, it is still the quest to find new schemes that can improve the detection sensitivity of FP aptasensors for clinical assays. Recently, different enzyme-assisted nucleic acid amplification techniques have been proven to be increasingly valuable for quantitative analysis of various target molecules. Besides polymerase chain reaction, these include rolling circle amplification (RCA),27 exonuclease signal amplification,28,29 nicking endonuclease signal amplification,30 exponential amplification reaction (EXPAR),31,32 isothermal circular

ptamers are in vitro selected artificial nucleic acid receptors that possess distinct recognition properties toward a wide array of targets, including ions, small molecular compounds, peptides, proteins, and even cells.1,2 Moreover, aptamers have many unique advantages over natural receptors (i.e., antibodies and enzymes), such as synthesis convenience, easy modification, good stability, and design flexibility. So aptamers have been extensively used as sensing elements for developing bioassays, especially for aptasensors.3−5 Due to the simplicity, rapidness, and versatility, fluorescence polarization (FP) aptasensors have received more and more research interests.6,7 In the past few years, many FP aptasensors based on fluorescent dye-labeled aptamers probes or target-induced aptamer displacement have been developed for quantitative analysis of proteins,6−10 small molecules,11−16 and cells.17 However, these FP aptasensors are not sensitive enough due to the lack of an amplification mechanism. Various amplification strategies have been developed to improve the detection sensitivities of FP aptasensors. For example, gold nanoparticles were used as as an effective FP enhancer for the specific amplified sensing of Hg2+.18 Similarly, silica nanoparticles,19 graphene oxide,20,21 and proteins22,23 were used as FP © XXXX American Chemical Society

Received: November 8, 2014 Accepted: July 13, 2015

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Analytical Chemistry strand-displacement polymerization (ICSDP),33,34 et al. Among these amplification protocols, ICSDP is one of the most popular methods. This technique is based on repeated triggering toward an isothermal polymerization reaction on a single-stranded DNA template with the assistance of DNA polymerase and primer. During the polymerization, the bound target can be displaced by DNA polymerase to promote the next round of polymerization reaction, thus resulting in the repeating sequences for in turn amplification of targetrecognition binding event.35 This amplification function of ICSDP has been employed to elaborately design amplified nucleic acid-based biosensors for diverse biomolecules. For example, the hybridization of target DNA or RNA with a molecular beacon36,37 or a single-stranded DNA probe38,39 led to the initiation of ICSDP for the amplified fluorescence detection of nucleic acids. Similarly, the ICSDP strategy was used for amplified electrochemical40 and chemiluminescent41 analysis of DNA. Also, the catalytic recycling of common target molecules (non-nucleic acid strands) by ICSDP was utilized to develop the bioassays for amplified fluorescence,42−45 surfaceenhanced Raman scattering (SERS),46,47 or colorimetric48 analysis of aptamer substrates. However, to the best of our knowledge, such ICSDP systems have not been investigated in any attempts for their use in an FP sensor. In the present work, we report a new type of amplified FP aptasensor based on allostery-triggered cascade strand-displacement amplification (CSDA) and polystyrene nanoparticle (PS NP) enhancement for ultrasensitive detection of proteins. Compared with traditional homogeneous aptasensors, the detection sensitivity of proposed FP aptasensor can be significantly improved in 5 orders of magnitude by using CSDA and PS NP enhancement. Moreover, this method exhibits high specificity for target proteins and can be performed in homogeneous solution without any separation/ washing steps, which is very simple and convenient. With use of thrombin (Tb) and vascular endothelial growth factor-165 (VEGF165) as model analytes, our newly proposed FP aptasensor enables the quantitative measurement of Tb and VEGF with the detection limits of 28 aM and 86 aM, respectively. Furthermore, the suitability of this method for the quantification of target protein in complex biological samples has also been demonstrated. The proposed sensing method promises an ultrasensitive detection platform for the analysis of various target molecules.

(Shanghai, China). Streptavidin-coated polystyrene nanoparticles (SA-PS NPs) of different diameter sizes (i.e., 60, 80, 90, and 110 nm) were purchased from Microspheres-Nanospheres, Inc. (NY, USA). Assay buffer was 20 mM Tris-HCl buffer solution (100 mM NaCl, 20 mM KCl, and 2 mM MgCl2, pH 8.0). All other reagents used in this work were of analytical grade and were used without further purification. Water was purified with a Milli-Q plus 185 equip from Millipore (Bedford, MA, USA) and used throughout the work. Apparatus. Fluorescence polarization (FP) measurements were carried out using an FL3-P-TCSPC system (Jobin Yvon, Inc., Edison, NJ, USA) with a 300 μL cuvette. The FP of the sample solution was monitored by exciting the sample at 490 nm and measuring the emission at 520 nm for FAM, and slits for both the excitation and the emission were at 10 nm. FP was measured by using the L-format configuration and FluorEssenceTM software with constant wavelength analysis to achieve a FP value. The G factor was initially set to zero, to let the system measure G automatically. The FP value was also calculated automatically by the instrument. The integration time was set to 3 s for the FP measurements. The gel electrophoresis assays were performed by using a DYCP-31A Electrophoresis Cell (Beijing LiuYi Instrument Factory, China) equipped with DYY5 Electrophoresis Power Supply (Beijing LiuYi Instrument Factory, China) and the Omega 16ic Gel imaging system (ULTRA-LUM, USA). Prepararion of PS NP-Functionalized DNA Duplex Probes. To prepare the PS NP-functionalized DNA duplex probes, 10 μL of 3.6 μM assistant DNA-2 was mixed with 10 μL of 3.6 μM trigger DNA-1 in assay buffer, or 10 μL of 3.6 μM assistant DNA-5 was mixed with 10 μL of 3.6 μM trigger DNA2 in assay buffer. Then, the mixtures were annealed by heating to 95 ◦C for 10 min and slowly cooled down to room temperature to obtain DNA duplexes. Finally, 10 μL of 7.2 mg/ mL SA-PS NPs was added to the resulting mixtures and incubated for 1 h at 37 °C to obtained the PS NPfunctionalized DNA duplex probes via the streptavidin−biotin binding. The prepared PS NP-functionalized assistant DNA-2/ trigger DNA-1 duplex probe and PS NP-functionalized assistant DNA-5/trigger DNA-2 duplex probe were used directly for FP assays. Gel Electrophoresis. The preparation of samples for gel electrophoresis assays were described as follows: (1) 2.0 μM trigger DNA-1 in assay buffer was used as sample one; (2) 2.0 μM assistant DNA-2 in assay buffer was used as sample two; (3) the assistant DNA-2 (2 μM) was mixed with trigger DNA-1 (2 μM) in assay buffer and incubated for 2 h at 37 °C to obtain the assistant DNA-2 (2 μM)/trigger DNA-1 (2 μM) duplex, and the resulting solution was used as sample three; (4) 2.0 μM H1 probe in assay buffer was used as sample four; (5) 2.0 μM H1 probe was mixed with the assistant DNA-2 (2 μM)/trigger DNA-1 (2 μM) duplex in assay buffer was used as sample five; (6) the mixture of 2.0 μM H1 probe, assistant DNA-2 (2 μM)/ trigger DNA-1 (2 μM) duplex and Klenow fragment exo- (15 U) in assay buffer was incubated for 1.5 h at 37 °C and used as sample six; (7) sample seven was prepared by incubating the mixture of 2.0 μM H1 probe, assistant DNA-2 (2 μM)/trigger DNA-1 (2 μM) duplex in assay buffer and Klenow fragment exo- (15 U) with Tb (200 nM) 1.5 h at 37 °C; (8) sample eight was prepared by the procedure above-mentioned for sample seven but with the change of Tb concentration to be 800 nM. Electrophoresis analysis was carried out on 15% nondenaturing polyacrylamide gels, cast, and run in 1 × TBE buffer.

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EXPERIMENTAL SECTION Materials and Reagents. Thrombin (Tb, 133 NIH units/ vial (NIH units obtained by direct comparison to a NIH Thrombin Reference Standard49), 2582 NIH units/mg), human serum albumin (HSA), immunoglobulin G (IgG), immunoglobulin E (IgE), alpha-fetoprotein (AFP), and ethidium bromide (EB) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human vascular endothelial growth factor-165 (VEGF165) were purchased from Invitrogen (Carlsbad, CA, USA). All oligonucleotides used in this work were synthesized and purified by Sangon Biotechnology Co. Ltd. (Shanghai, China), and their sequences were listed in Table S1. DNA maker, the deoxynucleotide solution mixture (dNTPs), polymerase Klenow fragment exo(50 U/μL), 10 × Klenow Fragment exo-buffer (KF buffer, 500 mM Tris-HCl, 50 mM MgCl2, 10 mM DTT, pH 8.0), and 5 × TBE buffer (225 mM Tris-Boric acid, 50 mM EDTA, pH 7.9) were also purchased from Sangon Biotechnology Co. Ltd. B

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Analytical Chemistry Scheme 1. Principle of the Proposed Amplified FP Aptasensor Based on CSDA and PS NP Enhancement

DNA-2/trigger DNA-1 duplex probe/H1 probe system or the assistant DNA-2/trigger DNA-1 duplex probe/H1 probe system were carried out under identical conditions. For VEGF165 detection, the H2 probe (5 μL, 1.2 μM) in assay buffer was mixed with the VEGF165 sample at specific concentration (5 μL) in assay buffer and 2.5 × KF buffer (2.5 μL). Then, 5 μL of the PS NP-functionalized assistant DNA-5/trigger DNA-2 duplex probe in assay buffer and 2.5 μL of 3 mM dNTPs were added into the resulting solution. Other assay procedures for VEGF165 assay were the same as that of Tb detection noted above. Control experiments using the PS NPfunctionalized assistant DNA-5/trigger DNA-2 duplex probe/ H2 probe system or the assistant DNA-5/trigger DNA-2 duplex probe/H2 probe system were carried out under identical conditions. All experiments were repeated seven times, and each sample solution was repeatedly measured seven times.

Electrophoresis was performed at a constant potential of 80 V for 1 h with loading of 10 μL of each sample into the lanes. The resulting gel was stained by EB and scanned using the Omega 16ic Gel imaging system. Treatment of Plasma Samples. Five normal human plasma samples collected in the citrate anticoagulated tubes were provided by No. 5 People’s Hospital (Guilin, China). Fibrinogen was removed via precipitation according to the method previous reported.50 Briefly, 10 μL of human plasma was quickly mixed with 200 μL of ammonium sulfate (2 M) and 100 μL of NaCl (0.1 M). Four minutes later, the mixture was centrifuged, and the upper supernatant solution was collected. Then, CaCl2 (0.03 M) with 8 nM human factor Xa was added to the plasma to promote the transformation from prothrombin to Tb. Finally, the resulting plasma solution was diluted 104-fold with assay buffer and immediately used for Tb detection by the proposed amplified FP aptasensor. The level of Tb in human plasma was calculated from the measured concentrations after correction for dilution. Procedure for FP Detection of Proteins. In a typical experiment for Tb detection, the H1 probe (5 μL, 1.2 μM) in assay buffer was mixed with the Tb sample at specific concentration (5 μL) in assay buffer and 2.5 × KF buffer (2.5 μL). Then, 5 μL of the PS NP-functionalized assistant DNA-2/trigger DNA-1 duplex probe in assay buffer and 2.5 μL of 3 mM dNTPs were injected into the resulting solution. Subsequently, 5 μL of Klenow fragment exo- (3 U/μL) in 2.5 × KF buffer was added and incubated the resulting solution at 37 ◦ C for 60 min. The obtained sample solution was diluted with 175 μL of 0.25 × KF buffer and used for FP measurements. Control experiments using the PS NP-functionalized assistant

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RESULTS AND DISCUSSION Assay Principle. Scheme 1 outlines the working principle of the proposed amplified FP aptasensor based on CSDA and PS NP enhancement. Briefly, this system mainly consists of an aptamer hairpin probe with fluorescein amidite (FAM) labeled on its stem region, a PS NP-modified DNA duplex probe, and polymerase. The aptamer sequence (pink) for target is integrated into the 5′-end of the hairpin probe and a mismatch at the 3′-end of the hairpin probe to prevent elongation by DNA polymerase in the absence of target. The DNA duplex on PS NPs is the assistant DNA (red)/trigger DNA (green) duplex with a single-stranded part that is designed to be complementary to the 3′-end of the hairpin probe, which is also part of the hairpin stem region. The trigger DNA is designed to C

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Analytical Chemistry be partly complementary to the aptamer sequence of the hairpin probe. In the absence of target, the single-stranded part of the PS NP-functionalized DNA duplex probe is unable to bind with the aptamer hairpin probe and cannot trigger the CSDA reaction. In this case, the FAM dyes exhibits relatively low background of the FP value (P) due to the relatively small size of the aptamer hairpin probe. However, upon recognition and binding with a specific target, the stem of the aptamer hairpin probe is opened, after which the 3′-end of the opened hairpin probe anneals with the single-stranded part of the DNA duplex probe linked to the PS NPs and triggers a polymerization reaction in the presence of dNTPs and polymerase. During the polymerization process, a long complementary DNA duplex at the PS NP surface is synthesized, and the target and a trigger DNA is displaced, resulting in an increase of the P value. The displaced target can then recognize and bind with another aptamer hairpin probe, triggering the next round of polymerization reaction. On the other hand, the displaced trigger DNA can also recognize and hybridize with an aptamer hairpin probe, which triggers another polymerization reaction, resulting in the generation of a long complementary DNA duplex at the PS NP surface and the displacement of this trigger DNA and a new trigger DNA. Thus, in the presence of target, autonomous cascade strand-displacement polymerization reactions occur through the polymerase-recycled both the target and the trigger DNA, which leads to the continuous assembly of the FAM dye onto the PS NPs. Consequently, a single target can initiate the assembly of many FAM dyes onto the PS NPs, generating a substantial increase in the P value. By monitoring the increase in the P value, we could detect the target with very high sensitivity. Of note, the use of both polymerase and PS NPs in our new assay provides a novel means of signal amplification, which would significantly improve the sensitivity of the aptamer-based FP assay. Feasibility Study. To verify the feasibility of the proposed assay strategy, Tb, an anti-Tb aptamer hairpin (H1), the assistant DNA-2, and trigger DNA-1 were used as our models, we then investigated the FP responses of different sensing systems for the detection of Tb (3 nM). The experimental results are shown in Figure 1. With the use of H1 probe only, the P value increased slightly upon the addition of Tb, indicating the formation of the Tb/H1 complex. With the use of the H1/assistant DNA-2-trigger DNA-1 duplex system, the P value increased obviously upon the addition of Tb, suggesting the formation of stable Tb-mediated complexes between H1 and assistant DNA-2-trigger DNA-1 duplex. When the H1/PS NP-functionalized assistant DNA-2-trigger DNA-1 duplex system was used for Tb detection, the P value increased significantly compared with that obtained from the H1/ assistant DNA-2-trigger DNA-1 duplex system, indicating the formation of stable PS NP-functionalized tripartite complexes between H1, assistant DNA-2-trigger DNA-1 duplex, and Tb. Also, when the H1/PS NP-functionalized assistant DNA-2/ polymerase system was used to detect Tb, the P value of this system increased significantly compared with that obtained from the H1/PS NP-functionalized assistant DNA-2-trigger DNA-1 duplex system, suggesting that the strand-displacement polymerization reaction had taken place and generated numerous PS NP-functionalized longer DNA duplexes carrying the FAM dye. Further introduction of trigger DNA-1 into the H1/PS NP-functionalized assistant DNA-2/polymerase system, the P value further increased, indicating the occurrence of cascade strand-displacement polymerization reactions and the

Figure 1. FP values of different sensing systems in the presence (red columns) and absence (black columns) of 3 nM Tb. (A) the H1 system; (B) the H1/assistant DNA-2-trigger DNA-1 duplex system; (C) the H1/PS NP-functionalized assistant DNA-2-trigger DNA-1 duplex system; (D) the H1/PS NP-functionalized assistant DNA-2/ polymerase system; (E) the H1/PS NP-functionalized assistant DNA2-trigger DNA-1 duplex/polymerase system. A blank (H1 and assistant DNA-2-trigger DNA-1 duplex without Tb) was used as a control reference. Error bars were derived from seven times repeated measurement of each sample solution.

generation of more PS NP-functionalized longer DNA duplexes carrying the FAM dye. To further confirm cascade stranddisplacement polymerization reactions, trigger DNA-1 was also used as the target to detect the FP value with the H1/PS NPfunctionalized DNA-2-trigger DNA-1 duplex/polymerase system. As shown in Figure S1, the P value of the system increased upon the addition of the target trigger DNA-1, indicating the occurrence of the cascade strand-displacement polymerization reactions. These results demonstrate that the introduction of both PS NPs and polymerase can significantly amplify the reporting signal for the aptamer-based FP assay of the Tb target. Gel Electrophoresis Characterization. To evaluate the viability of the proposed sensing strategy, the strand-displacement polymerization reaction induced by Tb binding was examined by gel electrophoresis using the H1 probe, unmodified assistant DNA-2, and trigger DNA-1, and the results are shown in Figure 2. The first five lanes showed the trigger DNA-1, the assistant DNA-2, the mixture of assistant DNA-2/trigger DNA-1, the H1 probe, and the mixture of the H1 probe/assistant DNA-2-trigger DNA-1 duplex, respectively. A relatively fast migration band appeared in lane 3 due to the formation of the double-stranded structure between assistant DNA-2 and trigger DNA-1, while no obvious bands were observed in both lane 1 and lane 2 due to the single-stranded structure of trigger DNA-1 and assistant DNA-2. Lane 4 displayed a band for the H1 probe due to the stem helix of the H1 probe. Lane 5 showed two bands for the H1 probe/ assistant DNA-2-trigger DNA-1 duplex mixture at almost the same migration position as that in lane 3 and lane 4, indicating no interaction between the H1 probe and the assistant DNA-2trigger DNA-1 duplex. When polymerase was added into the mixture of H1 probe/assistant DNA-2-trigger DNA-2 duplex, two bands at the same migration position as in lane 5 were still observed. This indicated a nonpolymerization reaction occurring initiated by polymerase in the absence of Tb. D

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Figure 2. Results of polyacrylamide gel electrophoresis analysis. (M) DNA marker; (1) trigger DNA-1 (2 μM); (2) assistant DNA-2 (2 μM); (3) trigger DNA-1 (2 μM)/assistant DNA-2 (2 μM); (4) H1 (2 μM); (5) H1 (2 μM)/trigger DNA-1 (2 μM)/assistant DNA-2 (2 μM); (6) H1 (2 μM)/trigger DNA-1 (2 μM)/assistant DNA-2 (2 μM)/polymerase (15 U); (7) H1 (2 μM)/trigger DNA-1 (2 μM)/ assistant DNA-2 (2 μM)/polymerase (15 U)/Tb (200 nM); (8) H1 (2 μM)/trigger DNA-1 (2 μM)/assistant DNA-2 (2 μM)/polymerase (15 U)/Tb (800 nM).

Figure 3. Plots of FP changes as a function of Tb concentrations by using different sensing systems. (A) the H1/PS NP-functionalized assistant DNA-2-trigger DNA-1 duplex/polymerase system; (B) the H1/PS NP-functionalized assistant DNA-2-trigger DNA-1 duplex system; (C) the H1/assistant DNA-2-trigger DNA-1 duplex system. Error bars were derived from seven times repeated measurement of each sample solution.

However, when both Tb and polymerase were added to the mixture of the H1 probe/assistant DNA-2-trigger DNA-1 duplex, two bands from the H1 probe/assistant DNA-2-trigger DNA-1 duplex mixture became vague and disappeared finally with the increase of Tb concentration, and a new bright band with lower mobility was observed (lane 7 and lane 8), suggesting that the strand-displacement polymerization reaction had taken place in the presence of the Tb target, and generated longer double-stranded DNA structures. These results demonstrate the successful proceeding of the stranddisplacement polymerization reaction in the presence of Tb. Optimization of Assay Conditions. In order to improve the sensitivity of the proposed FP biosensing system, the elements affecting FP response were investigated, and the experimental conditions were optimized. The details and the related results are shown in Figures S2−S4 of the Supporting Information. From the results in the Supporting Information, the optimal conditions for detection of Tb were obtained as follows: assistant DNA-2 with 8-nt complementary bases to the H1 probe, 110 nm SA-PS NPs, and 60 min reaction time. Analytical Performance of Tb detection. To evaluate the sensitivity of the proposed amplified FP aptasensor based on CSDA and PS NP enhancement for target protein detection, a series of different concentrations of Tb were analyzed under the optimal conditions. As shown in Figure 3A, the relative FP value increased with an increase in the concentration of target Tb, consistent with the generation of more PS NP/FAMfunctionalized longer DNA duplexes through the polymerasecatalyzed recycling of the target Tb and trigger DNA-1. Interestingly, as shown in Figure S5 in the Supporting Information, the plots of the relative FP value versus the logarithm of Tb concentrations in the range from 50 aM to 100 pM showed a good linear relationship (R2 = 0.9975). The regression equation was ΔI = 47.282logC + 212.15, where ΔI and C were the relative FP value and Tb concentration, respectively. Based on 3δ/S (in which δ is the standard deviation for the blank solution, n = 10, and S is the slope of the calibration curve), the detection limit for Tb was estimated to be 28 aM, which was about 6 orders of magnitude lower than that of traditional homogeneous aptasensors and about 2 orders of magnitude lower than that of recently reported amplified homogeneous aptasensors (see Table S2). Very high sensitivity

in this method might be attributed to the reasons as follows. First, the association of the target thrombin with the aptamer region led to the unfolding of the hairpin structure of the H1 probe, the open H1 probe annealed with the single-stranded part of the assistant DNA-2-trigger DNA-1 duplex probe, which triggered a circular target molecule-displacement polymerization reaction via catalytic recycling of thrombin and leads to the assembly of numerous FAM-labeled H1 at the PS NPs surface and release of many trigger DNA-1 strands. This achieved the target molecule recycling amplification and could increase the detection sensitivity. Second, each of the released trigger DNA-1 strands from the circular target moleculedisplacement polymerization reaction could hybridize with the H1 probe to unfold the hairpin structure of the H1 probe, after which the open H1 probe annealed with the assistant DNA-2trigger DNA-1 duplex probe and triggered a circular nucleic acid strand-displacement polymerization reaction via catalytic recycling of trigger DNA-1. This circular nucleic acid stranddisplacement polymerization reaction also resulted in the assembly of numerous FAM-labeled H1 at the PS NPs surface and release of many new trigger DNA-1 strands. These new released trigger DNA-1 strands could act as “new DNA triggers” to initiate new circular nucleic acid strand-displacement polymerization reactions, leading to the continuous release and recycling of trigger DNA-1 strands and the continuous assembly of FAM-labeled H1 at the PS NPs surface. Eventually, each target can go through many cycles, resulting in the assembly of many FAM-labeled H1 probes onto PS NPs and generation of numerous PS NP-functionalized complementary DNA duplexes carrying the FAM dye and thus achieving the cascaded strand-displacement signal amplification. This cascaded strand-displacement signal amplification could also increase the detection sensitivity. In addition, because of the extraordinarily larger volume of PS NPs, the generation of PS NP-functionalized complementary DNA duplexes carrying the FAM dye from the cascaded cycle reactions mentioned above led to the significant decrease of the rotation rate of the FAM dye, resulting in a higher increase in the P value compared E

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Analytical Chemistry with that without PS NPs and thus achieving PS NP enhancement. This PS NP enhancement could amplify the reporting FP signal and also increased the detection sensitivity. In contrast, when the FP aptasensor with PS NP enhancement only was employed, a detection limit of 0.26 pM Tb was obtained (Figure 3B), and when FP aptasensor without PS NP enhancement and CSDA was used for Tb detection, a detection limit of 1.6 nM was obtained (Figure 3C). The substantial sensitivity improvement of our newly proposed amplified FP aptasensor was attributed to the slower rotation of the fluorescent unit when many FAM-labeled H1 probes were assembled onto the PS NP surface after the cascade stranddisplacement polymerization reactions in the presence of the target Tb. To assess the reproducibility of the proposed amplified aptamer-based FP sensing system, Tb samples at five different concentrations (200 aM, 1 fM, 20 fM, 400 fM, and 50 pM) were repeatedly analyzed seven times. The results showed that the relative standard deviations (RSDs) were found to be less than 4.8% for intraday assays and less than 6.5% for interday assays, indicating a desirable reproducibility for Tb assay. Selectivity of Amplified FP Aptasensor for Tb Detection. The proposed amplified FP aptasensor for Tb detection was also specific. To evaluate this property, we challenged the system with the target Tb and several nonspecific proteins: HSA, IgG, IgE, and AFP. It was found that the specific target Tb led to an increase in the P value, and while no apparent P value change was observed in the assay for all nonspecific proteins as compared to the background (Figure 4). These results clearly demonstrate the high specificity of our amplified FP aptasensor.

Table 1. Determination of Thrombin in Five Human Plasma Samples sample

present method (nM)

ELISA (nM)

relative deviation (%)

1 2 3 4 5

126.2 78.6 94.8 112.0 81.4

129.4 74.8 101.3 104.5 85.1

2.5 −5.1 6.4 −7.2 4.3

standard addition experiments were also performed with these plasma samples to validate the determination. The experimental results revealed that the recoveries of Tb were from 94.6% to 104.5% with relative standard derivation around 5%. These results indicate that the proposed amplified FP aptasensor could be acceptable for quantitative assays of Tb in complex biological samples. Analytical Performance of VEGF165 Detection. To illustrate the generality of our proposed amplification strategy, we applied this strategy to develop another amplified FP aptasensor for VEGF165 detection by using an anti-VEGF165 aptamer hairpin (H2), assistant DNA-5, and trigger DNA-2. As shown in Figure 5A, as the concentration of VEGF165 was

Figure 5. Plots of FP changes as a function of VEGF165 concentrations by using different sensing systems. (A) the H2/PS NP-functionalized assistant DNA-5-trigger DNA-2 duplex/polymerase system; (B) the H1/PS NP-functionalized assistant DNA-5-trigger DNA-2 duplex system; (C) the H1/assistant DNA-5-trigger DNA-1 duplex system. Error bars were derived from seven times repeated measurement of each sample solution. Figure 4. Specificity of the proposed amplified FP aptasensor for Tb detection. The concentration of Tb is 10 fM, and other proteins were 1 nM each. The ΔFP values were obtained by the subtraction of the background FP value. Error bars were derived from seven times repeated measurement of each sample solution.

higher, the relative FP value increased gradually, consistent with the generation of more amounts of PS NP/FAM-functionalized longer DNA duplexes through the polymerase-catalyzed recycling of the target VEGF165 and trigger DNA-2. Importantly, the plots of the relative FP value versus the logarithm of VEGF165 concentrations ranged from 150 aM to 1000 pM showed a good linear relationship (R2 = 0.9948), (Supporting Information, Figure S6). The regression equation was ΔI = 43.253logC+173.69, where ΔI and C were the relative FP value and VEGF165 concentration, respectively. This amplified FP aptasensor based on CSDA and PS NP enhancement could detect VEGF165 with a detection limit of 86 aM, which was about 7 orders of magnitude lower than that of the FP aptasensor without signal amplification (Figure 5B)

Determination of Tb in Human Plasma. To evaluate the applicability of this amplified fluorescence aptasensor to real samples, the determination of Tb in human plasma from healthy volunteers was conducted by the proposed method and compared with those obtained from the traditional ELISA method. As shown in Table 1, the assay results for Tb in human plasma by the presented method were in an acceptable agreement with those of the ELISA method. In addition, the F

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and the project of Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, Ministry of Education of China (CMEMR2012-A19, CMEMR2013-C06).

and about 4 orders of magnitude lower than that of the FP aptasensor with PS NP enhancement only (Figure 5C). Meanwhile, the detection limit for VEGF165 in this work was about 6 orders of magnitude lower than that of traditional homogeneous aptasensors and about 4 orders of magnitude lower than that of recently reported amplified aptasensors (see Table S2). This amplified FP aptasensor also exhibited high specificity for the VEGF165 target over other nonspecific proteins, such as HSA, IgG, IgE, and AFP (Supporting Information, Figure S7). In addition, the present method also exhibited an acceptable reproducibility. RSDs are less than 5.2% and 6.8% for repeatedly detecting VEGF165 (at 400 aM, 4 fM, 40 fM, 4 pM, and 400 pM) seven times within the same day and within different days.

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CONCLUSIONS In summary, we have developed a simple, highly sensitive amplified FP aptasensor based on CSDA and PS NP enhancement for ultrasensitive and selective quantification of proteins. This assay technique has several excellent features. First, it is very simple and convenient, needing only mixing of an aptamer hairpin probe, PS NP-functionalized duplex DNA probe, polymerase, and the target molecule in homogeneous solution. Second, it has extremely high sensitivity. Our newly proposed FP aptasensor enables the quantitative measurements of target proteins with the detection limit in the attomolar range, nearly 6 orders of magnitude lower than that of traditional homogeneous aptasensors. Third, the assay exhibits high specificity, which is capable of detecting target protein in complex biological samples. Fourthly, it also has a wide detection range up to 6 orders of magnitude. Finally, the method is based on FP enhancement, thus, the assay can be easily carried out in the multiwell plates, rendering it suitable for routine high-throughput applications. Also, given that the hairpin structure may be opened by other analytes, for example, nucleic acids and small molecules, this sensing platform may be extended for the sensing and amplified analysis of other analytes, such as DNAs, RNAs, or small molecules. These qualities of our proposed assay protocol make it a potential platform for ultrasensitive analysis of various target molecules.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ac5041692.

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*Phone: 86 773 5845973. Fax: 86 773 5832294. E-mail: [email protected] (S.Z.). *E-mail: [email protected] (Y. H.). *E-mail: [email protected] (H. L.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundations of China (No. 21305021, No. 81460544, No. 21175030) and the Natural Science Foundations of Guangxi Province (No. 2012GXNSFDA385001, 2013GXNSFBA019038) as well as BAGUI Scholar Program G

DOI: 10.1021/ac5041692 Anal. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/ac5041692 Anal. Chem. XXXX, XXX, XXX−XXX