Homogeneous Bioluminescence Detection of Biomolecules Using

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Homogeneous Bioluminescence Detection of Biomolecules Using Target-triggered Hybridization Chain Reaction-Mediated Ligation without Luciferase label Qinfeng Xu, Guichi Zhu, and Chun-yang Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac401334r • Publication Date (Web): 14 Jun 2013 Downloaded from http://pubs.acs.org on June 22, 2013

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Analytical Chemistry

Homogeneous Bioluminescence Detection of Biomolecules Using Target-triggered Hybridization Chain Reaction-Mediated Ligation without Luciferase label

Qinfeng Xu, Guichi Zhu and Chun-yang Zhang* Single-molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China

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ABSTRACT: We develop a new homogeneous method for sensitive detection of various biomolecules on the basis of bioluminescence monitoring the released AMP from the target-triggered hybridization chain reaction-mediated ligation. The introduction of hybridization chain reaction not only improves the sensitivity of DNA assay, but also facilitates the sensitive detection of proteins by designing specific aptamer triggers, providing a universally amplified platform for simultaneous detection of different kinds of biomolecules. Importantly, this bioluminescence assay employs the target-dependent ATP from the ligation byproduct of AMP as the reporter without the requirement for the sophisticated luciferase manipulation, complicated immobilization, and separation steps. The proposed method has significant advantages of simplicity, high sensitivity, low-cost, and high throughput, and holds a great promise for practical point-of-care applications.

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Sensitive detection of biomolecules is essential to the biomedical research and early clinic diagnosis.1

Optical

detection

approaches such

as colorimetric,

fluorescence, and

bioluminescence assays are usually used to quantify the biomolecules.2 However, fluorescence assay requires the fluorescent-labeled probes and suffers from the autofluorescence of biomolecules, the scattered excitation light, and photobleaching;3 Colorimetric assay needs to avoid the optical absorption of matrix of many biological samples.4 In contrast, bioluminescence assay has the advantages of sensitive signal response, high throughput, and self-illumination without the requirement for excitation.5 Bioluminescence assay has been employed for the detection of nucleic acids and proteins,6 but most of the assays are heterogeneous, laborious, and time-consuming due to the involvement of sandwich assay format with the photoprotein (luciferase) as the labeled reporters,7 which requires immobilization, separation, and washing steps. In addition, the luciferase labeling requires either chemical or genetic methods to conjugate the biomolecules with luciferase, which involves the sophisticated luciferase manipulations such as bioconjugation, protein fusion, and expression,7 and suffers from the risk of activity loss.8 Recently, great efforts have been put into the development of homogenous bioluminescence assay without the requirement for luciferase manipulation.9 A luciferase manipulation-free method for DNA detection has been demonstrated on the basis of bioluminescence monitoring of DNA polymerase reaction with ATP as the reporter.10 However, this method requires thermostable firefly luciferase, and the template replication-based signal amplification might increase the risk of cross-contamination from amplicons.

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Ligase chain reaction is a simple and powerful technique for nucleic acid amplification.11 DNA ligase is capable of covalently ligating two oligonucleotide probes hybridized to one target DNA,12 and the ligation reaction can be exponentially amplified by repeated thermal cycling.11 However, ligase chain reaction is not an optimal biosensing platform due to the involvement of thermal-recycling process. An alternative approach is the hybridization chain reaction in an isothermal condition13 in which a cascade of hybridization events is triggered by one target molecule, yielding nicked alternating copolymers of hairpin probes in an enzyme-free way at room temperature.13a Herein, we develop a new homogeneous method for sensitive detection of various biomolecules on the basis of bioluminescence monitoring target-triggered hybridization chain reaction-mediated ligation without luciferase label.

EXPERIMENTAL SECTION Materials. Adenosine 5’-monophosphate sodium salt (AMP), nicotinamide adenine dinucleotide hydrate (NAD+), phospho(enol)pyruvic acid monosodium salt hydrate (PEP), pyruvate kinase from rabbit muscle (PK), adenylate kinase (AK) from chicken muscle, thrombin (lyophilized powder, 2000 units/mg), and horseradish peroxidase (HRP) were obtained from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Bovine serum albumin (BSA) was purchased from Shanghai Excell Biology, Inc. (Shanghai, China). Hemoglobin was obtained from Worthington Biochemical Corporation (Lakewood, NJ, USA). The dCTP was purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China). All oligonucleotides (Table 1) were synthesized and purified by Sangon Biotechnology Co. Ltd. (Shanghai, China). E.coli ligase

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and the ligation buffer were purchased from New England Biolabs (Ipswich, MA, USA). ATP determination kit containing D-luciferin, recombinant firefly luciferase, and dithiothreitol was obtained from Invitrogen (Carlsbad, CA, USA). Corning 96-well white microplates were purchased from Fisher Scientific (Pittsburgh, Pennsylvania). Milli-Q water (Millipore, Bedford, MA, USA) was used in all experiments. Table 1.

Gel Electrophoresis. All DNA probes were heated individually to 95 °C for 5 min followed by gradual cooling to room temperature for 1 h. DNA target (0.3 µM) was incubated overnight with 10 µL probe 1 (1 µM) and probe 2 (1 µM) in the absence or in the presence of E.coli ligase (0.5 µL, 10 U/µL) in 1× ligation buffer (30 nM Tris-HCl (pH 8.0), 4 mM MgCl2, 1 mM DTT, 26 µM NAD+, 50 µg/mL BSA). For nondenaturing polyacrylamide gel electrophoresis assay, the reaction products were prestained by SYBR Green II, and loaded on a 10% nondenaturing gel, followed by electrophoresis in 1× TBE buffer (45 mM Tris-boric acid, 10 mM EDTA, pH 8.0) at a 110 V constant voltage for 35 min. For denaturing polyacrylamide gel electrophoresis assay, the reaction products were mixed with an equivalent volume of formamide, respectively, followed by heating at 95°C for 5 min to disassociate the unligated DNA polymer. The above products were prestained by SYBR Green II, and loaded on a 10% denaturing gel (prepared with 8 M urea), followed by electrophoresis in 1× TBE buffer (45 mM Tris-boric acid, 10 mM EDTA, pH 8.0) at a 110 V constant voltage for 35 min. The electrophoresis images were obtained by Kodak 4000MM (Rochester, NY, USA).

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For the detection of thrombin, thrombin (2.6 µM) was incubated overnight with probe 3 (1 µM), probe 4 (1 µM), and the aptamer probe (1 µM) in 10 mM Tris-HCl buffer (pH 8.0) containing 100 mM NaCl. These reaction products were prestained by SYBR Green II, and loaded on a 2% agarose gel. The gel was run at 110 V for 35 min in 1× TAE buffer (40 mM Tris-acetate, 2 mM EDTA, pH 8.0). The electrophoresis images were acquired with the Image Station 4000MM (Rochester, NY, USA). Bioluminescence Spectra. Aliquots of 50 µL of hybridization chain reaction products, 10 µL of AK (2 U/µL), 10 µL of PK (2 U/µL), 10 µL of dCTP (10 mM), and 10 µL of PEP (4.8 mM) were added into 300 µL of ATP detection buffer containing 0.5 mM D-luciferin, 1.25 µg/mL firefly luciferase, 25 mM Tricine buffer, pH 7.8, 5 mM MgSO4, 100 µM EDTA, and 1 mM DTT. Bioluminescence spectra were recorded on a spectrofluorometer (F-4600, Hitachi, Japan) with the excitation light source turned off. Bioluminescence Detection. For nucleic acid detection, various concentration of DNA targets were mixed with 0.2 µM probe 1, 0.2 µM probe 2 in 65 µL of ATP detection buffer containing 0.5 mM D-luciferin, 1.25 µg/mL firefly luciferase, 25 mM Tricine buffer (pH 7.8), 5 mM MgSO4, 100 µM EDTA and 1 mM DTT, and were then added into the 96-well plate. With the addition of reaction buffer containing 0.5 µL of AK (2U/µL), 0.5 µL of PK (2U/µL), 1.0 µL of dCTP (10 mM), 1.0 µL of PEP (4.8 mM), 0.5 µL of NAD+ (30 µM), 0.5 µL of E.coli ligase (10 U/µL), the bioluminescence signals were recorded with a Glomax 96-well luminometer (Promega, Madison, Wisconsin), and the signals obtained at 60 min were used for data analysis.

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For the detection of thrombin, 1 µM aptamer probe, 1 µM probe 3 and 1 µM probe 4 were mixed with various concentration of thrombin in 10 mM Tris-HCl buffer (pH 8.0) containing 100 mM NaCl, and incubated at room temperature for 4 h. Then aliquots of 2.0 µL of reaction solution were added into the 96-well plate. After the addition of reaction buffer containing 0.5 µL of AK (2U/µL), 0.5 µL of PK (2U/µL), 1.0 µL of dCTP (10 mM), 1.0 µL of PEP (4.8 mM), 0.5 µL of NAD+ (30 µM), 0.5 µL of E.coli ligase (10 U/µL), 65 µL of ATP detection buffer, the bioluminescence signals were recorded with a Glomax 96-well luminometer (Promega, Madison, Wisconsin), and the signals obtained at 20 min were used for data analysis. For simultaneous detection of different kinds of DNA targets, fifteen samples with different combinations of four DNA targets (Table 1) were prepared. Four specific probe sets were designed (Table 1), respectively. Prior to the assay, four probe sets were added into the different wells, respectively. Each sample was split into four aliquots, and was put into the four different wells. After the addition of different samples, a series of reaction mixtures containing 0.5 µL of AK (2 U/µL), 0.5 µL of PK (2 U/µL), 1.0 µL of dCTP (10 mM), 1.0 µL of PEP (4.8 mM), 0.5 µL of NAD+ (30 µM), 0.5 µL of E.coli ligase (10 U/µL) were injected, respectively, and the bioluminescence signals were recorded after the addition of 65 µL of ATP detection buffer containing 0.5 mM D-luciferin, 1.25 µg/mL firefly luciferase, 25 mM Tricine buffer (pH 7.8), 5 mM MgSO4, 100 µM EDTA, and 1 mM DTT. The final concentration of each probe was 200 nM, and the concentration of targets a, b, c and d was 2.0 nM, 0.5 nM, 3.0 nM, and 1.0 nM, respectively.

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RESULTS AND DISCUSSION Principle of Target-Triggered Hybridization Chain Reaction-Mediated Ligation for DNA Detection. As shown in Scheme 1, in the absence of target DNA, both two probe monomers of probe 1 and probe 2 keep their stable hairpin conformation, and no ligatable nick is available. While in the presence of target DNA, a cascade of hybridization events is triggered by the target DNA,13a yielding a large number of ligatable nicks, which can be covalently ligated by the ligase.12 As a result, the target DNA can be quantified by monitoring the hybridization chain reaction-mediated ligation events. Notably, the bioluminescence monitoring is realized using the ligation byproduct of AMP rather than the ligation products of dsDNA polymers. AMP is easily converted to ATP which is the specific substrate of firefly luciferase-luciferin bioluminescence system.14 The bioluminescence response is dependent on ATP generated from the target-triggered ligation at room temperature, and is not dependent on the firefly luciferase, thus avoiding the complicated labelling of DNA probes with luciferase and making the proposed method much simple and cost-effective. Scheme 1.

Detection of DNA Targets. To verify the feasibility of the proposed method for DNA detection, nondenaturing and denaturing gel electrophoresis were performed to investigate the hybridization chain reaction-mediated ligation. As shown in the Figure 1A, in the absence of target DNA, a well-defined band of probe monomers (Figure 1A, part a, lanes 1 and 3) is observed no matter the ligase presents or not. While in the presence of target DNA, a series of DNA bands with high molecular weight are observed (Figure 1A, part a, lanes 2 and 4),

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suggesting the occurrence of target-triggered hybridization chain reaction. However, the ligation is not illustrated yet because there is not obvious difference between the bands of ligated DNA polymers (Figure 1A, part a, lane 4) in the presence of ligase and those of unligated polymers (Figure 1A, part a, lane 2) in the absence of ligase due to the similar migration rate between the ligated hybrids and the nicked hybrids in the nondenaturing gel electrophoresis.15 To further confirm the ligation of nicked polymers, the reaction products were further analyzed by the denaturing gel electrophoresis. As shown in Figure 1A (b), the bands of DNA polymers disappear in the absence of ligase (Figure 1A, part b, lane 2). In contrast, a series of DNA bands with high molecular weight are still observed in the presence of ligase (Figure 1A, part b, lane 4), suggesting that the ligation reaction can occur only in the presence of both target and ligase. To obtain the bioluminescence signal, the ligation byproduct of AMP is converted to ATP through coupling the enzymatic reactions of adenylate kinase and pyruvate kinase. ATP is the specific substrate of firefly luciferase-luciferin system.16 The combination of adenylate kinase with pyruvate kinase is chosen because of theirs high conversion efficiency and commercial availability.17 The converted ATP solution is finally mixed with luciferin and firefly luciferase to obtain the bioluminescence signal. The involved reactions are shown as follows: Nicked dsDNA + NAD+ → ligated dsDNA+AMP

(1)

AMP + dCTP → ADP + dCDP

(2)

ADP + PEP → ATP + pyruvate

(3)

ATP + luciferin → AMP + oxylucifrin + light

(4)

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Enzymatic coupled reactions are catalyzed sequentially by NAD+-dependent ligase (1), adenylate kinase (2), pyruvate kinase (3), and firefly luciferase (4). As shown in Figure 1B, strong bioluminescence signal with a characteristic emission peak of 556 nm18 is observed only in the presence of both target and ligase (Figure 1B, red line), indicating that the ligation byproduct of AMP is converted to ATP. However, in the absence of either target or ligase, no significant bioluminescence signal is observed. These results are consistent with the those of denaturing gel electrophoresis in which ligation occurs only in the presence of both ligase and target DNA (Figure 1A, part b, lane 4), demonstrating that the bioluminescence monitoring the released AMP from target-triggered hybridization chain reaction-mediated ligation can be applied for DNA detection. To examine the detection sensitivity of the proposed method for DNA detection, we measured the bioluminescence intensity in response to various target concentrations. The measurement was performed using a 96-well luminometer in a high-throughput and homogeneous way. Figure 1C shows the variation of bioluminescence intensity with the concentration of target DNA. The logarithm value of bioluminescence intensity is linearly dependent on the logarithm of target DNA concentration in the range from 3 pM to10 nM. The regression equation is log10 B = 0.89 log10 C + 12.78 with a correlation coefficient of 0.9893, where B is the bioluminescence intensity and C is the DNA concentration (M). The detection limit is calculated to be 3 pM based on the average signal of blank plus three times standard deviation. The sensitivity of the proposed method has improved by up to two orders of magnitude as compared with that of homogeneous bioluminescence assay using

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luciferase-labelled stem-loop probes (0.4 nM).19 The improved sensitivity can be attributed to both the signal amplification of target-triggered hybridization chain reaction and the recycling utilization of AMP. Moreover, the bioluminescence signal in response to the complementary target is much higher (ca. 3.3-fold) than that in response to single-base mismatched target (Figure 1D) due to the inhibition of hybridization chain reaction by the mismatched target.13b, 20

These results demonstrate that the proposed method has the capability to discriminate the

single-base mismatched target DNA. Figure 1.

Simultaneous Detection of Different Kinds of Biomarkers. Simultaneous detection of different kinds of biomarkers can increase the throughput, and shorten the analysis time.21 We further investigated the feasibility of the proposed method for simultaneous detection of different kinds of DNA targets. Fifteen samples with different combinations of four DNA targets were prepared, and four specific probes sets were designed with each set specific to one of four targets. As shown in Figure 2, the bioluminescence signals corresponding to the specific probe sets are observed only when theirs specific targets present in the samples. Both the compositions and the concentrations of target DNAs can be read directly from the bioluminescence signals in response to the four probes sets, and there is neither cross-reactivity nor false-positivity observed. These results demonstrate that the proposed method can be used for simultaneous detection of different kinds of biomolecules.

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It should be noted that the bioluminescence signals vary from the target to the target depending on the nucleotide sequence of both probes and targets (Figure 2). This can be ascribed to the different efficiency of hybridization chain reaction in response to the different DNA targets and the different sets of probes. 13a, 22 In the practical point of view, a linear correlation between the bioluminescence signal and the target concentration exists only between the specific target and its corresponding probe set. Figure 2.

Principle of Target-Triggered Hybridization Chain Reaction-Mediated Ligation for Protein Detection. The propose method can be further applied for the detection of proteins by using the aptamer probes.13a As a proof of concept, the thrombin was selected as a model protein. We designed a hairpin aptamer probe which consisted of an aptamer recognition domain and a DNA initiator domain for triggering hybridization chain reaction. As shown in Scheme 2, in the absence of thrombin, the DNA initiator in the aptamer probe hybridizes with the intramolecular complementary sequence and forms a stable hairpin structure, preventing the hybridization chain reaction from occurring. However, in the presence of thrombin, the conformation of aptamer probe changes from the hairpin structure to the open single-stranded form, exposing the DNA initiator which can trigger the hybridization chain reaction and the subsequent proximity ligation between probe 3 and probe 4. As a result, thrombin can be simply quantified through bioluminescence monitoring the ligation byproduct of AMP in a homogeneous way.

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Scheme 2.

Detection of Protein Targets. The feasibility of the proposed method for the detection of thrombin was validated by agarose gel electrophoresis (Figure 3). In the absence of either aptamer probe (Figure 3A, lane 3) or the mixture of probe 3 and probe 4 (Figure 3A, lane 4), no obvious band of DNA polymers is observed even in the presence of thrombin. Moreover, even though in the presence of aptamer probe and the mixture of probe 3 and probe 4, no obvious band of DNA polymers is observed in the absence of thrombin (Figure 3A, lane 5) or in the presence of BSA (Figure 3A, lane 7). In contrast, the bands of DNA polymers are observed only in the presence of thrombin and the mixture of aptamer probe, probe 3 and probe 4 (Figure 3A, lane 6), suggesting that thrombin-triggered hybridization chain reaction-mediated ligation occurs only in the presence of thrombin and the mixture of aptamer probe, probe 3 and probe 4. Only the binding of thrombin to the aptamer probe can change the hairpin conformation, exposing the DNA initiator which can trigger the hybridization chain reaction and the subsequent proximity ligation between probe 3 and probe 4. These results are further confirmed by two control experiments with the synthesized DNA initiators in place of thrombin. In the absence of synthesized DNA initiators, only a well-defined band of probe 3 and probe 4 (Figure 3A, lane 1) is observed due to the absence of DNA initiator-triggered hybridization chain reaction-mediated ligation. While in the presence of synthesized DNA initiators, a series of bands of DNA polymers with high molecular weight are observed (Figure 3A, lane 2) due to the occurrence of DNA initiator-triggered hybridization chain reaction-mediated ligation.

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To examine the detection sensitivity of the proposed method for protein detection, we measured the bioluminescence intensity in response to various target concentrations. As shown in Figure 3B, the bioluminescence intensity increases with the increasing concentrations of thrombin (Figure 3B). The regression equation is log10 B = 0.51 log10 C + 9.25 with a correlation coefficient of 0.9883, where B is the bioluminescence intensity and C is the thrombin concentration (M). The detection limit is calculated to be 10 pM based on the average signal of blank plus three times standard deviation. The sensitivity of the proposed method has improved by three orders of magnitude as compared with the bioluminescence assay using the luciferase fusion protein as the label (10 nM).23 To further verify the detection specificity, the proposed method was challenged with three control experiments of BSA, HRP and hemoglobin, respectively (Figure 3B, inset). High bioluminescence intensity is obtained only in the presence of 1.3 nM thrombin. In contrast, even though in the presence of 100 nM BSA, 100 nM HRP, and 100 nM hemoglobin, no significant bioluminescence signal is observed. Such high specificity can be ascribed to the specific binding of aptamer probe to thrombin.24 These results clearly demonstrate that the proposed method can be used to sensitively and selectively detect proteins. Because the proposed method employs ATP as the reporter which comes from the conversion of AMP and ADP, it might suffer from the background interference in the biological samples containing ATP, ADP or AMP. To solve this issue, we may use the adenosine phosphate deaminase to degrade ATP, ADP, AMP, and adenosine derivatives in the process of sample pretreatment.25 The adenosine phosphate deaminase can catalyze the

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deamination of adenosine derivatives to inosine monophosphate which is not active in the luciferin–luciferase system.26 Figure 3.

CONCLUSIONS In summary, we have developed a homogeneous bioluminescence method for sensitive detection

of

various

biomolecules

based

on

target-triggered

hybridization

chain

reaction-mediated ligation. The introduction of hybridization chain reaction not only improves the sensitivity of DNA assay, but also facilitates the sensitive detection of proteins by designing specific aptamer triggers, providing a universally amplified platform for sensitive detection of various biomolecules. Notably, this bioluminescence assay employs target-dependent ATP from the ligation byproduct of AMP as the reporter without the requirement for the sophisticated luciferase manipulation, complicated immobilization, and separation steps. Taking advantage of low-cost, simplicity, and inexpensive equipment for bioluminescence ‘self-illuminating’ detection, the proposed method holds a great promise for practical point-of-care applications. 2, 27

AUTHOR INFORMATION Corresponding Author *Tel.: +86 755 86392211. Fax: +86 755 86392299. E-mail: [email protected]

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Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Basic Research Program 973 (Grant Nos. 2011CB933600 and 2010CB732600), the Award for the Hundred Talent Program of the Chinese Academy of Science, and the Natural Science Foundation of China (Grant Nos. 21075129 and 21205128), and the Fund for Shenzhen Engineering Laboratory of Single-molecule Detection and Instrument Development (Grant No. (2012) 433).

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Figure Legends

Scheme 1. Bioluminescence assay for DNA detection based on target-triggered hybridization chain reaction-mediated ligation without luciferase label. A cascade of hybridization events is triggered by the target DNA, yielding a large number of ligatable nicks, which can be covalently ligated by the ligase. The ligation byproduct of AMP is easily converted to ATP which is the specific substrate of firefly luciferase-luciferin bioluminescence system. Consequently, the target DNA can be simply quantified by monitoring the ATP-dependent bioluminescence signals.

Figure 1. (A) Polyacrylamide gel electrophoresis and (B) bioluminescence analysis of the products from hybridization chain reaction. a and b represent nondenaturing and denaturing gel electrophoresis, respectively. P1 and P2 represent the probe 1 and probe 2, respectively. The concentration of each DNA probes is 1.0 µM, and the concentration of DNA targets is 0.3 µM. The bioluminescence spectra were recorded on a spectrofluorometer with the excitation light source turned off, and the bioluminescence is shown with arbitrary unit. (C) Linear relationships between the logarithm of bioluminescence intensity and the logarithm of target concentration. (D) Specificity of the proposed method for DNA detection: a, 1 nM complementary target; b, 1 nM target with single-base mismatched; c, 1 nM random DNA sequence; d, the buffer. Error bars show the standard deviation of three experiments.

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Figure 2. Simultaneous detection of four targets from 15 samples using four specific probe sets. Samples 1-15 contain targets a, b, c and d (1); target a (2); target b (3); target c (4); target d (5); targets a and b (6); targets a and c (7); targets a and d (8); targets b and c (9); targets b and d (10); targets c and d (11); targets a, b and c (12); targets a, b and d (13); targets a , c and d (14); targets b, c and d (15), respectively. The concentration of each probe is 200 nM; the concentration of targets a, b, c, d is 2.0 nM, 0.5 nM, 3.0 nM and 1.0 nM, respectively.

Scheme 2. Bioluminescence assay for thrombin detection based on target-triggered hybridization chain reaction-mediated ligation without luciferase label. A cascade of hybridization events is triggered by thrombin, yielding a large number of ligatable nicks, which can be covalently ligated by the ligase. The ligation byproduct of AMP is easily converted to ATP which is the specific substrate of firefly luciferase-luciferin bioluminescence system. Consequently, thrombin can be simply quantified by monitoring the ATP-dependent bioluminescence signals.

Figure 3. (A) Agarose gel electrophoresis analysis of the products from hybridization chain reaction. P3 and P4 represent the probe 3 and probe 4, respectively. The concentration of thrombin is 2.6 µM; the concentration of BSA is 2.6 µM; the concentration of synthesized DNA initiators is 0.3 µM; the concentration of each DNA probe is 1.0 µM. (B) Bioluminescence signal in response to various concentrations of thrombin. Inset shows the bioluminescence signal in response to 1.3 nM thrombin (a), 100 nM BSA (b), 100 nM HRP (c),

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and 100 nM hemoglobin (d), respectively. Error bars show the standard deviation of three experiments.

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Table

Table 1. Sequences of the Oligonucletides. note

sequence (5’-3’)

probe 1 of probe set a

5’-PO4-TTA ACC CAC GCC GAA TCC TAG ACT CAA AGT AGT CTA GGA TTC GGC GTG-3’

probe 2 of probe set a

5’-PO4-AGT CTA GGA TTC GGC GTG GGT TAA CAC GCC GAA TCC TAG ACT ACT TTG-3’

DNA target a DNA

target

5’-AGT CTA GGA TTC GGC GTG GGT TAA-3’ with 5’-AGT CTA GGA TTC AGC GTG GGT TAA-3’

single-base mismatched thrombin aptamer probe

5’-ACG GAC TAA CAA GAA AGC CAA ACC TCT TGT TAG TCC GTG GTA GGG CAG GTT GGG GTG ACT-3’

probe 3 of probe set b

5’-PO4-CAT CTC GGT TTG GCT TTC TTG TTA ACG GAC TAA CAA GAA AGC CAA ACC-3’

probe 4 of probe set b

5’-PO4-TAA CAA GAA AGC CAA ACC GAG ATG GGT TTG GCT TTC TTG TTA GTC CGT-3’

DNA target b

5’-ACG GAC TAA CAA GAA AGC CAA ACC-3’

probe 5 of probe set c

5’-PO4-ATT CAA GCG ACA CCG TGG ACG TGC ACC CAC GCA CGT CCA CGG TGT CGC-3’

probe 6 of probe set c

5’-PO4-GCA CGT CCA CGG TGT CGC TTG AAT GCG ACA CCG TGG

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ACG TGC GTG GGT-3’ DNA target c

5’-GCA CGT CCA CGG TGT CGC TTG AAT-3’

probe 7 of probe set d

5’-PO4-TAA GCG CGT GAT CAG ATG CCG ACG TGC TGG CGT CGG CAT CTG ATC ACG-3’

probe 8 of probe set d

5’-PO4-CGT CGG CAT CTG ATC ACG CGC TTA CGT GAT CAG ATG CCG ACG CCA GCA-3’

DNA target d

5’-CGT CGG CAT CTG AT CAC GCG CTT A-3’

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Scheme-1 82x52mm (300 x 300 DPI)



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Figure-1 177x133mm (300 x 300 DPI)


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Figure-2 82x79mm (300 x 300 DPI)



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Scheme-2 82x69mm (300 x 300 DPI)


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Figure-3 82x185mm (300 x 300 DPI)



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For TOC Only 34x13mm (300 x 300 DPI)


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Homogeneous Bioluminescence Detection of Biomolecules Using

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