Label-Free Fluorescence Strategy for Sensitive Detection of

Jul 1, 2014 - A simple, rapid, label-free, and ultrasensitive fluorescence strategy for adenosine triphosphate (ATP) detection was developed using a l...
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Label-Free Fluorescence Strategy for Sensitive Detection of Adenosine Triphosphate Using a Loop DNA Probe with Low Background Noise Chunshui Lin,† Zhixiong Cai,† Yiru Wang,† Zhi Zhu,† Chaoyong James Yang,*,† and Xi Chen*,†,‡ †

Department of Chemistry and the MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005, China ‡ State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen, Fujian 361005, China S Supporting Information *

ABSTRACT: A simple, rapid, label-free, and ultrasensitive fluorescence strategy for adenosine triphosphate (ATP) detection was developed using a loop DNA probe with low background noise. In this strategy, a loop DNA probe, which is the substrate for both ligation and digestion enzyme reaction, was designed. SYBR green I (SG I), a double-stranded specific dye, was applied for the readout fluorescence signal. Exonuclease I (Exo I) and exonuclease III (Exo III), sequence-independent nucleases, were selected to digest the loop DNA probe in order to minimize the background fluorescence signal. As a result, in the absence of ATP, the loop DNA was completely digested by Exo I and Exo III, leading to low background fluorescence owing to the weak electrostatic interaction between SG I and mononucleotides. On the other hand, ATP induced the ligation of the nicking site, and the sealed loop DNA resisted the digestion of Exo I and ExoIII, resulting in a remarkable increase of fluorescence response. Upon background noise reduction, the sensitivity of the ATP determination was improved significantly, and the detection limitation was found to be 1.2 pM, which is much lower than that in almost all the previously reported methods. This strategy has promise for wide application in the determination of ATP.

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Recently, several novel strategies for ATP detection based on the ATP-dependent ligation reaction are reported.12−14 Owing to the high sensitivity of the molecular beacon and T4 DNA ligase’s high substrate dependence, these methods have shown very good analytical performances with a detection limit at the pM level. However, most of these strategies involve fluorophore labels causing higher cost and complexity to the assay. Although label free detection strategies can provide a fast and costeffective assay, most label-free strategies suffer from high background noise which results in a poor determination limit and even causes a false positive signal. Recently, graphene oxide (GO) was used15 to reduce the fluorescence background of SYBR green I (SG I) due to the preferential binding of GO to single-stranded DNA over a double-stranded one, and this reduces the fluorescence signal of the analytes. Furthermore, the addition of GO makes the sensing process complicated. We developed a simple, fast, highly sensitive, and label-free fluorescence strategy for the ultrasensitive and selective detection of ATP with a low background noise. In this strategy, we designed a loop DNA probe as the substrate for ATPdependent ligation and a double-stranded specific dye SG I as a

denosine-5′-triphosphate (ATP) is a multifunctional nucleoside triphosphate, which is generally used as a universal energy storage molecule and plays a critical role in the regulation of cellular metabolism and biochemical pathways in cell physiology.1,2 In addition, ATP has been widely used as an indicator for cell viability, cell injury, food quality control, and environmental analyses.3,4 The highly sensitive and selective detection of ATP is essential for biochemical study as well as clinical diagnosis. Luciferase-mediated bioluminescence5 is a traditional method in the ATP assay. However, this method involves the use of costly and unstable bioluminescence agents. Several strategies have been developed for the determination of ATP level based on synthetic host−guest receptors,6,7 peptides,8 and conjugated polymers.9 However, most of these methods suffer from poor selectivity,multistep reactions, or the synthesis of complex compounds. The aptamer-based strategy for the ATP assay has attracted much attention.10,11 Usually, the signaling aptamer is labeled with a fluorophore which undergoes a conformational change upon the recognition of ATP. However, the aptamer shows a low association constant with ATP, and the detection limit for ATP is in the micromolar range. In order to improve the sensitivity, many amplification techniques have been reported including the use of nanoparticles, enzymes, and graphene oxide, but these methods are time-consuming and increase the complexity of the detection method. © 2014 American Chemical Society

Received: May 8, 2014 Accepted: July 1, 2014 Published: July 1, 2014 6758

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readout signal. Exonuclease III (Exo III) catalyzes the stepwise removal of mononucleotides from the 3-hydroxyl ends of DNA duplexes, but its activity on single-stranded DNA and 3′protruding termini of double-stranded DNA is limited. Exonuclease I (Exo I) is another sequence-independent nuclease that catalyzes the stepwise removal of 3′ mononucleotides from the three termini of single-stranded DNA and leaves the double-stranded DNA intact. Both Exo I and Exo III were applied to digest the loop DNA probe to minimize the background fluorescence to an extremely low level. Since the strategy obviously reduced the background noise, the signal-tonoise ratio and sensitivity were improved significantly, and a detection limit of 1.2 pM ATP could be achieved.

the emission spectra were collected from 516 to 600 nm with both excitation and emission slits of 5 nm. Polyacrylamide Gel Electrophoresis (PAGE). A 10% native polyacrylamide gel was prepared using 1× TBE (89 mM Tris-HCl, pH 8.0, 89 mM boric acid, 2.0 mM Na2EDTA). 50 μL mixtures of (1) 4 μM P1 + P2; (2) 4 μM P1 + P2 +Exo I + Exo III + T4 DNA ligase; (3) 4 μM P1 + P2 +Exo I + Exo III + T4 DNA ligase + 1 μM ATP; (4) 4 μM P1; (5) 4 μM P2 were used for PAGE. The gel was run at 100 V for 60 min in 1× TBE buffer. Then, it was stained in the stains-all solution (0.1 g of stains-all, 450 mL of formamide, 550 mL of H2O) for 30 min. After this process, the gel was illuminated under sunlight for 5 to 10 min to obtain the stained bands. Finally, the PAGE results were photographed using a digital camera.

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EXPERIMENTAL SECTION Materials. T4 DNA ligase and Exo I and Exo III were purchased from the Takara Biotechnology Co., Ltd. (Dalian, China). SG I, ATP, adenosine (A), adenosine diphosphate (ADP), adenosine 5′-monophosphate (AMP), uridine triphosphate (UTP), cytidine triphosphate (CTP), and guanosine triphosphate (GTP) were obtained from the Sangon Biotechnology Co., Ltd. (Shanghai, China). The original solution of SG I was 10 000-fold concentrated. SG I was first diluted to 100-fold with ultrapure water before usage. All other reagents were of analytical grade and used without additional purification. All solutions were prepared with ultrapure water (≥18 MΩ, Milli-Q, Millipore, USA). The oligonucleotide probes P1 and P2 were “synthesized” and purified by the Sangon Biotechnology Co., Ltd. (Shanghai, China), and their detailed base sequences are listed as follows: P1: 5′-PO4-GGAAGGTTGCGGAGTTTTTTTTTTCTCCGCAACCTTCCTGTATGTGAACTGA-3′ P2: 5′-PO4-GGAAGGTTGCGGAGTTTTTTTTTTCTCCGCAACCTTCCTCAGTTCACATACA-3′ Fluorescence Measurement. Twenty-five mM Tris-HCl (pH 7.6) buffer solution, 100 mM NaCl, 5 mM MgCl2, and 1 mM dithiothreitol (DTT) were used for Exo I- and Exo IIIbased digestion and the ATP-based ligation reaction, as well as the fluorescence assay. The DNA absorbance at 260 nm was measured on a Nanodrop ND-1000 (Thermo Scientific, USA) for calculation of the DNA concentration. In a typical procedure, first, a 20 μL loop DNA probe solution (final concentration, 4 μM) could be achieved by mixing equal volumes of P1 and P2 solution; then, the solution was denatured at 90 °C for 5 min and cooled to room temperature for further experiments. Exo I and Exo III (final concentration, 1 U/μL) were added to the 20 μL, 4 μM loop DNA solution to induce the digestion for 60 min. After this procedure, SG I (final concentration, 10-fold) was added for a 5 min incubation and its fluorescence intensity was measured as the background noise. In the second step, T4 DNA ligase (final concentration, 0.1 U/μL) with varying concentrations of ATP was introduced to the 20 μL, 4 μM loop DNA solution to induce ligation at room temperature (25 °C) for 30 min. Then, ExoI and Exo III were introduced to the ligated solution to digest the unligated loop DNA. After this step, SG I was added for a 5 min incubation. Finally, the mixture was diluted to 200 μL, and its fluorescence intensity with different concentrations of ATP was measured. All fluorescence measurements of samples were performed on an F-4500 spectrophotometer (Hitachi, Japan) at room temperature. The excitation wavelength was set at 497 nm, and

RESULTS AND DISCUSSION Design Principle of the Strategy. The method design is shown in Scheme 1. A loop DNA probe was prepared using

Scheme 1. Schematic Illustration of the Mechanism for the Label-Free Fluorescence Assay of ATP Using Loop DNA with a Low Background Signal

two hairpin-like DNA probes: P1 and P2. Both P1 and P2 were 5′-end PO4 modified. In the absence of cofactor ATP, the ligation reaction could not occur. The loop DNA probe was completely digested by Exo I and Exo III, resulting in a low background signal. Whereas, when ATP was added to the system, the loop DNA probe was sealed by the T4 DNA ligase, and the sealed loop resisted digestion by Exo I and Exo III. When stained with SG I, a strong fluorescence signal could be obtained. The background signal reduction led to a dramatic enhancement in the signal-to-noise ratio and a significant improvement in sensitivity. Feasibility of the Strategy. To demonstrate the feasibility of the present strategy, the fluorescence emission spectra under different conditions were investigated. As shown in Figure 1, a highly reduced background fluorescence intensity (curve b) was obtained which was almost the same as the fluorescence response of SG I in the blank solution (curve a). This result indicated that SG I bound weakly to the mononucleotides. The lower background signal was caused mainly by the efficient 6759

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control experiment in which no ligase or nucleases were included (lane 1). The above results confirmed that the fluorescence background signal was weak on addition of Exo I and Exo III, and the strategy would give fluorescence enhancement only by the addition of ATP. The gel-electrophoresis experiment results further verified the feasibility of this strategy. To achieve optimal analytical performance, the experimental parameters including the concentration and the intercalation time of SG I as well as the amount of T4 DNA ligase, Exo I, and Exo III were optimized. As shown in Figure S1, Supporting Information, after optimizing, the following conditions were selected for further experiments: 10-fold SG I, 5 min intercalation time of SG I, 0.1 U/μL of T4 DNA ligase and 30 min ligation time, and 1.00 U/μL of Exo I and Exo III and 60 min digestion time. Detection Performance. Since a too large amount of the loop DNA probe could cause a slight increase in the background signal, a suitable concentration of the loop DNA was helpful to increase the signal. In the experiments, 1 μM loop DNA probe was applied for the determination of ATP at the nM level. At lower concentrations of ATP (such as the pM level), a higher concentration of the loop DNA probe would ensure the completeness and stability of the reaction, and in our experiments, a 4 μM loop DNA probe was selected. As shown in Figure 3, with the addition of ATP, the system fluorescence intensity increased obviously. We found that the fluorescence signal for 100 nM of ATP (∼9000) was ∼50 times larger than that in the absence of ATP (∼180). As indicated in Figure 3b, the fluorescence intensity shows a good linear correlation with the logarithmic concentrations of ATP from 10 to 1000 pM with a correlation coefficient of 0.9854. The detection limit was estimated to be 1.2 pM (3σ/slope), which provided superior detection sensitivity compared with the aptamer-based fluorescence strategy16−18 and previously reported T4 DNA ligase-based strategies12−15 (Table S1, Supporting Information). This sensitive analysis of ATP could be achieved due to the great signal-to-noise ratio, which made it applicable to detect ATP even in a complex biological environment (Figure S2, Supporting Information). The high sensitivity of the strategy was caused mainly by the following factors: the Exo I and Exo III could efficiently digest the loop DNA probe with only a very weak background signal, and T4 DNA ligase together with its cofactor ATP could easily recognize the ligation site and seal the loop DNA, which could resist digestion by the nucleases. In addition, the fluorescence intensity of SG I increased 800−1000-fold when it was intercalated into the double-stranded DNA grooves. The high selectivity of the ligation reaction-based strategy has an inherent advantage among these methods. To demonstrate the selectivity of the analytical strategy toward ATP, the fluorescence responses of several ATP analogs were tested under the same experimental conditions (Figure 4). When the system contained 1 μM A, ADP, AMP, UTP, CTP, or GTP, no obvious increase in fluorescence intensity could be observed, and their fluorescence intensities were almost the same as that of the blank solution without ATP. However, when 100 nM ATP was added to the system, a very strong fluorescence intensity could be observed. The excellent specificity was attributed to the specific dependence of the T4 DNA ligase on the cofactor ATP.

Figure 1. Fluorescence emission spectra under different conditions: (a) 10-fold SG I; (b) 10-fold SG I + 1 μM loop DNA + Exo I + Exo III + T4 ligase; (c) 10-fold SG I + 1 μM loop DNA + Exo I + Exo III + T4 ligase + 100 nM ATP.

digestion of the loop DNA, which reduced remarkably the accumulation of SG I on the mononucleotides. In contrast, the fluorescence of the SG I dyed sealed loop DNA probe showed a sharp increase in the presence of 100 nM ATP. The signal to background ratio was superior to the previous ATP-dependent ligation strategy.12−15 The feasibility of the proposed strategy was also verified using PAGE (Figure 2). In the presence of ligase, the Exo I, and

Figure 2. Gel electrophoresis image for the label-free assay of ATP compared with the control experiment. Lane 1: unsealed loop DNA with no enzyme treatment; Lane 2: unsealed loop DNA digested by Exo I and Exo III without ATP; Lane 3: sealed loop DNA with ATP digested by Exo I and Exo III; Lane 4: P1 only; Lane 5: P2 only, control experiments.

Exo III, but without ATP, the ligation reaction could not occur and the nucleases cleaved the loop DNA probe to mononucleotides, resulting in the failure to dye with stainsall. Thus, it is difficult to visualize the band in lane 2. However, in the presence of ATP, the sealed loop DNA could resist the digestion of Exo I and Exo III. As a result, a band in lane 3 could be easily identified, indicating the same mobility with the 6760

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Figure 3. Fluorescence spectra of the label free fluorescence assay for ATP at various concentrations. (A) 1 μM loop DNA for ATP at nM concentration levels; (B) 4 μM loop DNA for ATP at pM concentration levels; (C) dependence of fluorescence intensity on ATP concentration (nM); (D) dependence of fluorescence intensity on the logarithm of ATP concentration (pM). The error bars represent the standard deviations based on three independent measurements.

reported methods for ATP detection, the proposed strategy revealed several advantages including: (1) the ease of preparation of the fluorescence signal readout, a doublestranded specific dye, SG I, and a convenient to operate process; (2) the label-free strategy avoided laborious and timeconsuming labeling, modification, and separation steps; (3) the novel design employing Exo I and Exo III reduced background noise resulting in improvement in the sensitivity. The detection limit of 1.2 pM could be found, and it was much lower than that obtained from previously reported methods; and (4) because of the unique role of cofactor in the ligation reaction, this strategy showed an outstanding selectivity for ATP from its analogues. In view of these advantages, the developed method is expected to provide a new strategy in designing a loop DNAbased biosensor for simple, fast, and sensitive ATP detection.

Figure 4. A comparison of fluorescence intensity between the blank and solutions containing ATP (0.1 μM) and its analogs (1 μM). Error bars indicate the standard deviation of three independent experiments.

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CONCLUSIONS In summary, a label-free, fast, and simple fluorescence sensing strategy for the ultrasensitive detection of ATP was reported. A loop DNA probe was selected as the substrate for both the ligase and nuclease system. The strategy relied on the Exo Iand Exo III-based digestion reaction to give only a weak fluorescence background. ATP-dependent ligation of the loop DNA blocked the digestion site of Exo I and Exo III, resulting in a strong fluorescence enhancement. Compared with the

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ASSOCIATED CONTENT

S Supporting Information *

Additional information including optimization of the assay conditions, comparison of ATP detection strategies, and evaluation of ATP in human blood serum. This material is available free of charge via the Internet at http://pubs.acs.org/. 6761

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +86 592 2184530. Fax: +86 592 2184530. E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by the National Basic Research Program of China (2010CB732402), the National Nature Scientific Foundation of China (No. 21175112 and No. 21375112), and the NFFTBS (No. J1030415), which are gratefully acknowledged. We thank Professor John Hodgkiss of The University of Hong Kong for assistance with the English in the paper.

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