Biosensors and Bioelectronics 63 (2015) 14–20
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Highly selective and sensitive electrochemical biosensor for ATP based on the dual strategy integrating the cofactor-dependent enzymatic ligation reaction with self-cleaving DNAzyme-ampliﬁed electrochemical detection Lu Lu a, Jing Cao Si a, Zhong Feng Gao a, Yu Zhang a, Jing Lei Lei b, Hong Qun Luo a,n, Nian Bing Li a,n a Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China b School of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044, China
art ic l e i nf o
a b s t r a c t
Article history: Received 3 April 2014 Received in revised form 4 July 2014 Accepted 4 July 2014 Available online 9 July 2014
A dual strategy that combines the adenosine triphosphate (ATP)-dependent enzymatic ligation reaction with self-cleaving DNAzyme-ampliﬁed electrochemical detection is employed to construct the biosensor. In this design, the methylene blue-labeled hairpin-structured DNA was self-assembled onto a gold electrode surface to prepare the modiﬁed electrode through the interaction of Au–S bond. In the procedure of ATP-dependent ligation reaction, when the speciﬁc cofactor ATP was added, the two split oligonucleotide fragments of 8-17 DNAzyme were linked by T4 DNA ligase and then released to hybridize with the labeled hairpin-structured DNA substrate. The linked 8-17 DNAzyme catalyzes the cleavage of the hairpin-structured substrate by the addition of Zn2 þ , causing the methylene blue which contains high electrochemical activity to leave the surface of the gold electrode, therefore generating a dramatic decrease of electrochemical signal. The decrease of peak current was readily measured by square wave voltammetry and a relatively low detection limit (0.05 nM) was obtained with a linear response range from 0.1 to 1000 nM. By taking advantage of the highly speciﬁc cofactor dependence of the DNA ligation reaction, the proposed ligation-induced DNAzyme cascades demonstrate ultrahigh selectivity toward the target cofactor ATP. A catalytic and molecular beacons strategy is further adopted to amplify the electrochemical signal detection achieved by cycling and regenerating the 8-17 DNAzyme to realize enzymatic multiple turnover, thus one DNAzyme can catalyze the cleavage of several hairpin-structured substrates, which improves the sensitivity of the newly designed electrochemical sensing system. & 2014 Elsevier B.V. All rights reserved.
Keywords: T4 DNA ligase 8-17 DNAzyme Signal ampliﬁcation Electrochemical biosensor ATP detection
1. Introduction Adenosine triphosphate (ATP), serving as the ubiquitous energy currency for all living organisms (Imamuraa et al., 2009) through breaking the phosphoanhydride bond, plays a vital role in the regulation and integration of cellular metabolism. The multifunctional nucleotide is involved in a series of biological processes, including muscle contraction, cells functioning, synthesis and degradation of important cellular compounds, and membrane transport (Abraham et al., 1997; Gruenhagen et al., 2004; Patriarca et al., 2009), thus it has been treated as a critical indicator
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(H.Q. Luo), [email protected]
http://dx.doi.org/10.1016/j.bios.2014.07.007 0956-5663/& 2014 Elsevier B.V. All rights reserved.
for cell viability and cell injury. ATP is also identiﬁed to act as an extracellular and intracellular signal molecule, which plays a very important role in the central and peripheral nervous system (Bush et al., 2000; Przedborski and Vila, 2001). Therefore, the detection of ATP is of great signiﬁcance in biochemical study, clinic diagnosis, food quality control, and environmental analysis (Lee et al., 2008b; Yu et al., 2008). To date, there have been many previous reports on the bioanalytical detection of ATP, including ATP bioluminescence technology (Park et al., 2014). ATP measurement was developed to estimate the number of somatic cell (Gallez et al., 2000) or bacterial cell including Escherichia coli (Hunter et al., 2011; Lee and Deininger, 2004; Park et al., 2014), Staphylococcus epidermidis (Park et al., 2014), Salmonella (Urata et al., 2009), Shigella bacteria (Nhieu et al., 2003) in biological samples, to ensure microbiological quality in water and food, and to detect
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bacterial contamination in speciﬁc areas of medicine or the food industry (Ratphitagsanti et al., 2012). DNA ligases seal 5′-PO4 and 3′-OH polynucleotide ends via three nucleotidyl transfer steps involving ligase-adenylate and DNA-adenylate intermediates. According to the substrate required for ligase-adenylate formation, DNA ligases are grouped into two families, ATP-dependent ligases and NAD þ -dependent ligases (Shuman, 2009). For the ATP-dependent ligase, it is inactive until it binds a molecule of ATP, which leads to the loss of the pyrophosphate moiety from ATP and the formation of a covalent enzyme–adenosine monophosphate (AMP) intermediate linked to a lysine side-chain in the enzyme (Banin et al., 2007). Thus the enzymatic ligation reaction shows speciﬁc dependence on its cofactor ATP, which, in turn, provides an efﬁcient platform for constructing ultrahighly selective biosensing systems for the target biological molecule ATP (Ma et al., 2013, 2008; Wang et al., 2010). However, most of these reported methods lack the process of signal ampliﬁcation, so their sensitivities are relatively lower than those of the biosensors with the enzyme-ampliﬁcation. DNAzymes are a series of synthetic DNA oligonucleotides that exhibit catalytic activities toward their speciﬁc substrates when certain cofactors are introduced (Robertson and Ellington, 1999; Santoro and Joyce, 1997). Furthermore, DNAzymes exhibit many excellent characteristics, such as simple synthesis, good stability, easy labeling, and design ﬂexibility, which enable DNAzymes to be particularly attractive as sensing platforms and signal ampliﬁcation elements for the design of biosensors that are highly speciﬁc for a number of targets such as metal ions and small biological molecules (Liu et al., 2009; Zhang et al., 2011). Due to their remarkable speciﬁcity, several DNAzymes that can speciﬁcally recognize target molecules like Pb2 þ (Breaker and Joyce, 1994), Zn2 þ (Santoro et al., 2000), Co2 þ (Mei et al., 2003), Cu2 þ (Liu and Lu, 2007), UO22 þ (Liu et al., 2007), and L-histidine (Roth and Breaker, 1998) have been selected in vitro, and transformed into biosensors with ultrahigh selectivity (Lee et al., 2008a; Liu and Lu, 2004; Shen et al., 2007; Wang et al., 2009; Xiang et al., 2009). Taking advantage of the unique self-cleavage activity of DNAzymes, several researchers also employ DNAzymes as the signal ampliﬁcation units because the catalytic reaction can undergo many cycles to trigger the cleavage of several substrates when the speciﬁc cofactor is added; by the DNAzyme-based cyclic ampliﬁcation process, the sensitivity of DNAzyme-based biosensors can be improved signiﬁcantly (Kong et al., 2013; Lu et al., 2011; Zhang et al., 2010; Zhao et al., 2011, 2013). Recently, detection methods based on peptides, polymers, host–guest receptors, and DNA/RNA aptamers have been investigated for the quantitative determination of ATP (Wang et al., 2008), among which the analysis method based on ATP aptamers has already been developed to be one of the most frequently reported strategies. Nevertheless, a major disadvantage of aptamers was their relatively low association constant with the ATP, which led to a rather poor detection limit (Du et al., 2008). As a consequence, for most of the aptamer-based biosensors, the detection limits of ATP are usually in the micromolar range, demonstrating only moderate sensitivity. Furthermore, most of these approaches have the limitation to distinguish ATP from its analogues like adenosine, AMP, and adenosine diphosphate (ADP), though the aptamers obtain the advantage to speciﬁcally identify ATP from other small biological molecules and its analogues such as uridine triphosphate (UTP), guanosine triphosphate (GTP) and cytidine triphosphate (CTP) (Lu et al., 2011). In order to solve these defects, Tan group has proposed a design which combines DNA ligation reaction-based process with catalytic and molecular beacon-based ampliﬁed detection to constitute an enhanced ﬂuorescence biosensor for the sensitive and selective detection of ATP (Lu et al., 2011). The methods for ATP determination based
on the ATP-dependent enzymatic ligation reaction are relatively more selective than the above proposed ones because the biochemical reaction cannot occur in the absence of ATP (Lu et al., 2011; Wang et al., 2010). Compared to the existing optical methods, electrochemical techniques have attracted increasing attention because they possess excellent characteristics such as high sensitivity, promising response speed, simple instrumentation, and less expensive components and electrochemical measurements could be made in turbid samples (Hansen et al., 2006; Sadik et al., 2009). Electronic devices can be routinely miniaturized in terms of size and power consumption. Unlike ﬂuorescencebased detection, the instrumentation cost associated with electrochemical detection is much lower. These outstanding characteristics make such biosensors attractive in terms of practical applications, thus electrochemical-based detection methods have also been adopted to design nucleic acid enzyme biosensors (Liu et al., 2009). Although many electrochemical biosensing platforms which select DNAzyme as their recognition and signal ampliﬁcation elements have been well developed for various target molecules (Shen et al., 2008; Xiao et al., 2007; Yang et al., 2010), the ligationtriggered electrochemical DNAzyme biosensor has not been proposed previously. The novel electrochemical biosensor based on this strategy can not only distinguish ATP from its analogues including adenosine, AMP, and ADP but also obtain the potential to detect ATP in the nanomolar range without employing expensive and cumbersome optical instruments. Thus, the highly selective and sensitive electrochemical biosensing platform based on the dual strategy integrating the ATP-dependent enzymatic ligation reaction with self-cleaving DNAzyme-ampliﬁed electrochemical signal detection is very signiﬁcant for constructing a quantitative analysis method of ATP.
2. Material and methods 2.1. Materials and reagents T4 DNA ligase and all synthetic oligonucleotides were synthesized and puriﬁed by Takara Biotechnology Co., Ltd. (Dalian, China). The lot number and enzyme units of the T4 DNA ligase were CK5721B and 25,000 units, respectively. The sequences of oligonucleotides (Lu et al., 2011) used in this work are listed as follows: Hairpin-structured DNA (HP DNA): 5′-HS-(CH2)6-CCACCAC ATTCAAATTCACCAACTATrAGGAAGAGATGTTACGAGGCGGTG GTGG-Methylene Blue-3′ DNA 1: 5′-CATCTCTTCTCCGAGCCGGTCG-3′ DNA 2: 5′-P-AAATAGTGGGTG-3′ C-DNA: 5′-CCCCCCCCCCACCCACTATTTCGACCGGCTCGG CCCCCCC-3′ Invasive DNA: 5′-GGGGGGGCCGAGCCGGTCGAAATAGTG GGTGGGGGGGGGG-3′ 8-17 DNAzyme: 5′-CATCTCTTCTCCGAGCCGGTCG-AAATAGTG GGTG-3′ The underlined sequences in HP DNA represent the stem of the hairpin-structured DNA, and rA denotes adenosine ribonucleotide at that position, while all others are deoxyribonucleotides. The accurate graph about the exact base pairing between hairpinstructured DNA and 8-17 DNAzyme is demonstrated in Scheme S1 (see Supplementary material). ATP, uridine triphosphate (UTP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and 6-mercapto-1-hexanol (MCH) were obtained from Sigma-Aldrich. Adenosine, AMP, and ADP were obtained from Shanghai Sangon
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Biotechnology Co., Ltd. (Shanghai, China). All other chemicals, purchased from Aladdin Chemistry Co. Ltd., were of analytical reagent grade and used without further puriﬁcation. All solutions were prepared in ultrapure water (resistance 418 MΩ cm). 2.2. Apparatus The pH measurements were performed on a pHs-3B pH meter (Dazhong, Shanghai, China). All electrochemical measurements were performed on a CHI 660D electrochemical workstation (Shanghai CH Instruments Co., China). A conventional threeelectrode system was involved, which was consisted of an Ag/AgCl reference electrode (saturated KCl), a modiﬁed gold electrode as working electrode, and a platinum wire counter-electrode. The square-wave voltammetric (SWV) measurements were performed in 5 mL of 100 mM phosphate buffer (PB) solution (pH 7.40) and the SWV curves were scanned from 0 to 0.50 V. The parameters were set as follows: Increase E ¼0.001 V, Amplitude ¼0.05 V, Frequency ¼ 25 Hz. 2.3. Preparation of modiﬁed electrode and ATP detection The gold electrode ( 2 mm diameter) was polished with 0.05 μm alumina powder, followed by ultrasonic cleaning with ultrapure water, ethanol, and ultrapure water for 3 min each, followed by electrochemically cleaning in 0.5 M H2SO4 by potential scanning between 0.2 and 1.6 V until a characteristic cyclic voltammogram of a clean Au electrode was obtained. Finally, the electrode was washed with ultrapure water and dried under a mild nitrogen stream. To fabricate the biosensor, the pretreated gold electrode was held upside down, and 25 μL of thiolated and methylene blue-labeled DNA solution (2.0 μM) was dropped onto the electrode surface to prepare hairpin-structured DNA modiﬁed electrode through the interaction of Au–S bond. In order to avoid the evaporation of solution drops, the electrode immersed with hairpin-structured DNA solution was further covered with a 0.5-mL tapered plastic centrifuge tube. After being kept for 12 h at room temperature, the electrode was immersed in 25 μL of 2 mM 6-mercaptohexanol (MCH) for 2 h to avoid non-speciﬁc adsorption and block the remaining bare area. Subsequently, the electrode was rinsed thoroughly with a copious amount of ultrapure water. For the ligation procedure, a mixture containing ATP with various concentrations, 10 U T4 DNA ligase, the two oligonucleotide fragments (300 nM DNA 1 and 300 nM DNA 2), 200 nM template C-DNA, 20 mM Tris–HCl, and 5 mM MgCl2 was freshly prepared in the volume of 50 μL. After incubation at 37 °C for 1 h, the invasive DNA at a ﬁnal concentration of 0.5 μM was added to the produced DNA ligation solution to inhibit the ligation reaction by forming a more stable invasive C-DNA hybrid and releasing the active 8-17 DNAzyme to perform its catalytic function. For the detection of ATP, the hairpin-structured DNA/MCH modiﬁed electrode was held upside down, and 25 μL of resulting mixture that contains the released intact 8-17 DNAzyme was pipetted onto the electrodes surface. After hybridization with the methylene blue labeled hairpin-structured substrate at room temperature for 2 h, the ds-DNA surfaces were then allowed to react with 200 μM Zn2 þ in buffer (25 mM Tris–HCl and 100 mM NaCl, pH 7.60) for 2 h at 37 °C to obtain the maximum cleavage of the substrate strand on the modiﬁed gold surface. Before electrochemical detection, the resulting electrode was thoroughly washed with ultrapure water. All the SWV measurements were performed in 100 mM PB (pH 7.40) at room temperature, using a CHI 660D electrochemical workstation (Shanghai CH Instruments Co., China).
3. Results and discussion 3.1. Design of the ligation-triggered electrochemical DNAzyme biosensing system Since the hairpin-structured DNA is dually labeled with 5′-SH and 3′-methylene blue, it is self-assembled on gold electrode through Au–S bond. If the hairpin-structured DNA is self-assembled on gold electrode successfully, SH-labeled 5′-terminal of the hairpin-structured DNA will be immobilized on the surface of the gold electrode, and the 3′-terminal which is labeled with methylene blue will also be immobilized on the surface of the gold electrode because the two terminals on the stem of the hairpinstructured DNA is aligned. During the electrochemical measurement in PB buffer (pH 7.4), the electro-active substance (methylene blue) immobilized on the surface of the gold electrode would be reduced at its reduction potential when the scanning potential scanned from 0 to 0.6 V. Therefore, the modiﬁed gold electrode can be characterized through the square wave voltammograms. We have investigated the electrochemical signals gained from the different modiﬁed stages of the gold electrode. The square wave voltammograms of the gold electrode in different modiﬁed stages in 100 mM PB solution (pH 7.40) are illustrated in Fig. 1. As illustrated in Fig. 1, the peak current obtained from MB/MCH modiﬁed gold electrode (curve a) was the highest. After treated with the released 8-17 DNAzyme, the peak current decreased slightly (curve b) because the hybridization reaction between the labeled hairpin-structured DNA and 8-17 DNAzyme made a certain amount of methylene blue probe away from the electrode surface due to the unwinding of the stem part of the hairpin-structured DNA. When the cofactor Zn2 þ was added, the peak current decreased dramatically (curve c) due to the cyclic self-cleaving catalytic reaction which made a large amount of cleaved DNA fragments labeled with methylene blue probe leave the electrode surface. According to the experimental results mentioned above, we intended to employ the 8-17 DNAzyme generated by ATP-dependent enzymatic ligation reaction to establish a novel electrochemical biosensing platform for ATP detection. The principle of this novel electrochemical biosensor based on a cascade of ligationinduced DNAzyme is displayed in Scheme 1. To amplify the electrochemical signal, 8-17 DNAzyme was selected as the catalytic unit because it indicates high self-cleavage activity when adopting either Pb2 þ or Zn2 þ as cofactors. The two separate oligonucleotide fragments, DNA 1 and DNA 2, which demonstrate
Fig. 1. Square wave voltammograms of the different electrodes in 100 mM PBS (pH 7.4): (a) MB/MCH modiﬁed gold electrode; (b) MB/MCH modiﬁed gold electrode incubated with released 8-17 DNAzyme; (c) MB/MCH modiﬁed gold electrode incubated with released 8-17 DNAzyme and subsequently incubated with Zn2 þ .
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Scheme 1. Schematic illustration of the electrochemical sensing system based on the dual strategy of ATP-dependent enzymatic ligation reaction and self-cleaving DNAzyme-based cyclic ampliﬁcation.
no catalytic activity to cleave the methylene blue-labeled hairpinstructured DNA substrate containing an adenosine ribonucleotide (rA) cleavage site, can be ligated to constitute the intact DNA sequence of active 8-17 DNAzyme in the presence of T4 DNA ligase and the speciﬁc cofactor ATP. In order to form a ligatable nick, both the two fragments were hybridized with the template C-DNA, though the two spare terminals of C-DNA were left unhybridized. After the ligation procedure, the invasive DNA is introduced to release the activated 8-17 DNAzyme from C-DNA by the formation of a completely complementary duplex of invasive DNA and C-DNA. The released 8-17 DNAzyme can then be hybridized with the hairpin-structured substrate to form the catalytic and molecular beacon system (Hausler et al., 2000). The hairpin-structured DNA is dually labeled with 5′-SH and 3′-methylene blue and it is self-assembled on gold electrode through Au–S bond. The 8-17 DNAzyme catalyzes the cleavage of the hairpin-structured substrate by the addition of Zn2 þ , causing the methylene blue which contains high electrochemical activity to leave the surface of the gold electrode, therefore generating a dramatic decrease of electrochemical signal. Due to the cleavage, the 8-17 DNAzyme can be liberated from the hairpin-structured substrate and then hybridize with another intact hairpin-structured substrate self-assembled on gold electrode, so this procedure can undergo many cycles to trigger the cleavage of several hairpin-structured substrates (Kong et al., 2013; Lu et al., 2011; Zhao et al., 2013), providing an ampliﬁed detection signal for the target ATP. Possessing the ability to speciﬁcally recognize its cofactor ATP, T4 DNA ligase was chosen to establish the sensing system. As depicted in Scheme 1, in the absence of ATP, relatively high peak current for methylene blue was obtained, which demonstrated that T4 DNA ligase did not have catalytic activity without the speciﬁc cofactor, and could not catalyze the ligation reaction of the two separated DNA 1 and DNA 2 fragments. As a consequence, the two separated DNA fragments could not hybridize with the hairpin-structured substrate, and they could not construct a complete strand of 8-17 DNAzyme to catalyze the cleavage of the hairpin-structured substrate though the cofactor Zn2 þ was added (Lu et al., 2011). However, when the speciﬁc cofactor ATP was introduced, T4 DNA ligase could be activated so that the two separated DNA segments could be ligated to generate a complete sequence of 8-17 DNAzyme which contains the catalytic activity to cleave the hairpin structured substrate at the rA cleavage site. After the ligation procedure, the invasive DNA was added to release the ligated 8-17 DNAzyme. After hybridized with the hairpin structured substrate, combined with the cofactor Zn2 þ , 8-17 DNAzyme could cleave the substrate strand into
two pieces at the cleavage site and then made the fragment labeled with methylene blue at the 3′-terminal released from the gold electrode surface, thus the peak current generated by the reduction of electroactive substance on the electrode surface decreased dramatically. 3.2. Optimization of the electrochemical DNAzyme biosensing system In order to achieve the best detection performance for the ligation-triggered electrochemical biosensing system, the concentration of thiolated and methylene blue-labeled DNA solution used in this work, the self-assembly time of thiolated and methylene blue-labeled hairpin-structured DNA to the surface of gold electrode, the hybridization time of released 8-17 DNAzyme hybridized with immobilized hairpin-structured DNA, the molar ratio of DNA 1 and DNA 2 to the template C-DNA, the concentration of T4 DNA ligase, the ligation time, the concentration of Zn2 þ , and the concentration of Mg2 þ were optimized. Experimental results indicated that the following conditions could offer the best sensing performance for the proposed electrochemical biosensor: 2.0 μM thiolated and methylene blue-labeled DNA solution, a self-assembly time of 12 h, a hybridization time of 2 h, a molar ratio of 1.5:1 for DNA 1 and DNA 2 to the template C-DNA (ﬁxed at 200 nM) with incubation time of 60 min for the ligation reaction, 0.2 U/μL T4 DNA ligase, 200 μM Zn2 þ and 5 mM Mg2 þ (Figs. S1–S8, in Supplementary material). Because the two separate oligonucleotide fragments (DNA 1 and DNA 2) were linked to form the whole sequence of 8-17 DNAzyme in the process of enzymatic ligation reaction, thus except for the nick between the two oligonucleotide fragments, the base sequence of DNA 1 and DNA 2 was the same as the intact 8-17 DNAzyme. After invasive DNA was added, the generated 8-17 DNAzyme as well as unreacted DNA 1 and DNA 2 were released from C-DNA and all of them would hybridize with the hairpinstructured DNA substrate because they contained the same base sequence which was complementary to the loop part of the hairpin-structured DNA. The two separate oligonucleotide fragments (DNA 1 and DNA 2) could not hybridize with the substrate efﬁciently (Lu et al., 2011) because the number of the complementary pairing bases between the two separate DNA fragments (DNA 1 and DNA 2) and hairpin-structured DNA substrate was relatively few compared to that of the whole sequence of 8-17 DNAzyme. However, a small quantity of DNA 1 and DNA 2 would still hybridize with the substrate because of the hydrogen bond existing between the complementary pairing bases. If the quantity
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3.3. Sensitivity of the ligation-triggered electrochemical DNAzyme biosensor Under optimum conditions, the hairpin-structured substrate was self-assembled on the polished gold electrode at room temperature, and the ligation solution which contains DNA 1, DNA 2, template C-DNA, and T4 DNA ligase was prepared. Then a series of different concentrations of ATP were introduced in the ligation solution to trigger the ligation reaction. After incubation for 60 min at 37 °C the invasive DNA was added to the ligation solution to release the 8-17 DNAzyme. Subsequently, the gold electrode modiﬁed by hairpin-structured DNA was immersed in the ligation solution that contains the released 8-17 DNAzyme. After hybridization for 2 h, the electrode was thoroughly washed with ultrapure water and then immersed in 200 μM Zn2 þ for 2 h to induce the self-cleaving reaction. After the process of DNAzyme-based cyclic ampliﬁcation, the electrode was thoroughly washed and then put into 100 mM PB buffer to conduct
A 0.9 0 nM 0.1 nM 1 nM 10 nM 100 nM 1 µM 10 µM 100 µM
Current / µA
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0
Potential / V
B 0.7 0.6
Current / µA
of unligated DNA 1 and DNA 2 was too large, the amount of the two oligonucleotide fragments which would hybridize with the substrate would increase. As a consequence, the amount of 8-17 DNAzyme which hybridized with the substrate would decrease to a certain extent, which resulted in negative effects to the following self-cleaving reaction. In addition, the hybridization efﬁciency of the two separate DNA fragments and template C-DNA, which had a signiﬁcant impact on the process of ligation reaction, should also be taken into consideration. When the molar ratio of DNA 1 and DNA 2 to the template C-DNA was 1:1, the hybridization efﬁciency of the two separate DNA fragments and template C-DNA was relatively too low for the process of ligation reaction. As a consequence, the quantity of the intact 8-17 DNAzyme that was generated through the ligation reaction would be very less. Thus, there was less generated 8-17 DNAzyme hybridizing with the methylene blue-labeled hairpin-structured DNA. When the cofactor Zn2 þ was added, the amount of the labeled hairpin-structured DNA substrates that were cleaved in the self-cleaving catalytic reaction would decrease. Therefore, the electrochemical signal decreased slightly because there were less labeled DNA fragments leaving the electrode surface. Thus, the peak current was relatively high when the molar ratio is 1:1 as shown in Fig. S4 in Supplementary material. When the molar ratio was 1.5:1, the hybridization efﬁciency of the two separate DNA fragments and template C-DNA was sufﬁcient and the amount of the unreacted DNA 1 and DNA 2 that had the potential to hybridize with the hairpin-structured DNA instead of the generated 8-17 DNAzyme was not so large. As a result, the electrochemical signal was the lowest as shown in Fig. S4 in Supplementary material. When the molar ratio was bigger than 1.5:1, the amount of the unreacted DNA 1 and DNA 2 would be too large, which made the quantity of generated 8-17 DNAzyme that would hybridize with the hairpinstructured DNA decrease. Therefore, a molar ratio of 1.5:1 for DNA 1 and DNA 2 to the template C-DNA could offer the best sensing performance for the proposed electrochemical biosensor. The quantity of hairpin-structured DNA substrates self-assembled on the gold electrode was constant. The DNAzyme can undergo many cycles to trigger the cleavage of several hairpinstructured substrates. With an increasing number of hairpinstructured substrates cleaved during the DNAzyme based selfcleaving catalytic process, the quantity of intact hairpin-structured DNA substrates immobilized on the gold electrode decreased due to the self-cleaving catalytic reaction. When most of the hairpinstructured DNA substrates were cleaved through the catalytic and molecular beacons system, the rate of the catalytic cleavage reaction slowed down due to the remarkable reduction of the substrate quantity.
Current / µ A
0.5 0.4 0.3 0.2 -10
Log C / M
C / µM
Fig. 2. (A) Square wave voltammograms of modiﬁed gold electrodes incubated with the resulting mixture containing different concentrations of ATP (from top to bottom: 0 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, respectively). The LOD based on S/N ¼ 3 is 0.053 nM. (B) Relationship between SWV peak current and ATP concentration. Inset depicts the responses of the biosensing system toward ATP at low concentration. The error bars demonstrate the standard deviations of three independent experiments.
electrochemical measurement. The SWV responses of the sensor to target ATP at different concentrations under optimal experimental conditions are illustrated in Fig. 2A, from which it can be seen that the reduction peak current decreased signiﬁcantly with the increase in target concentration. Fig. 2B depicts the relationship between the response current and the increasing ATP concentration ranging from 0 nM to 100 μM. And a linear relationship between peak current and the concentration of ATP in the range of 0.1–1000 nM was obtained with a correlation factor of 0.998. The SWV peak currents were systematically corrected from the background current by simply subtracting a tangent line under the peak (Deféver et al., 2011). The detection limit was 0.053 nM, obtained according to the rule of three times standard deviation over the background signal (S/N ¼3). Compared to the previously reported aptamer-based ATP assay, the sensitivity of this novel DNAzyme-based cyclic ampliﬁed electrochemical biosensor built on ATP-dependent enzymatic ligation reaction was improved remarkably, and the linear range and detection limit of this sensing system are also superior to other ligase-based biosensors (Table S1, in Supplementary material). 3.4. Selectivity of the electrochemical DNAzyme biosensing system Besides sensitivity, selectivity is another important issue to evaluate the performance of a sensor. A highly selective response to the analyte over other potentially interfering materials is very
Current Change / nA
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300 250 200 150 100 50 0
Fig. 3. Selectivity of the DNAzyme cascade for ATP compared to its analogues. The concentration of ATP is 0.1 μM, while the concentrations of adenosine (A), AMP, ADP, CTP, UTP, and GTP are 1 μM, respectively. The error bars represent the standard deviation of three measurements.
conducted under the same experimental conditions. The square wave voltammograms for ATP detection at three different concentrations in human serum sample are illustrated in Fig. S9 (see Supplementary material). According to the illustration of Fig. S9, the peak currents generated by the labeled probe (methylene blue) were not affected obviously by the interference substances (the endogenous Zn2 þ and bio-macromolecules including HSA, DNA long chains) in serum, compared to the detection signal in buffer (not including serum). The analytical results are shown in Table S2 (see Supplementary material). By comparing our work with other reported references (Chen et al., 2013; Song et al., 2014), we found that both the recovery and relative standard deviation (RSD) values were sufﬁcient for the detection of ATP in human serum sample, which proved that the electrochemical biosensing system was able to provide the potential for the detection of target biological molecule ATP in real samples.
4. Conclusions essential for constructing a practical sensor platform, especially for a biosensor with potential application in bio-samples. Though several anti-ATP DNA aptamer-based sensing platforms have been reported with satisfactory sensitivity, almost none of them can discriminate ATP from its analogues which contain adenosine. Our sensing system can achieve improved selectivity because the ATPdependent enzymatic ligation reaction cannot occur unless the speciﬁc target cofactor is added. Thus, only the cofactor ATP can trigger the T4 DNA ligase-based ligation reaction, so the interferences are easily excluded through enzyme speciﬁcity. In this work, the selectivity experiments for the proposed ligase-based electrochemical biosensor were extended to various ATP analogues including adenosine (A), AMP, ADP, CTP, UTP, and GTP by comparing the peak current changes of samples containing ATP and the other six analogues, respectively. Changes in the electrochemical response were investigated individually when ATP and its competing species with the concentration of 0.1 μM and 1 μM were introduced, respectively. As depicted in Fig. 3, the reduction peak current did not show much change when adenosine, AMP, ADP, UTP, CTP, or GTP was added, though the concentrations of these ATP analogues are 10 times higher than those of target ATP. However, it decreased signiﬁcantly in the presence of ATP. These results obviously demonstrated that the proposed ligase-based ATP electrochemical biosensing platform exhibited ultrahigh selectivity in the detection of ATP. It was proved that this newly designed electrochemical biosensor did have the ability to distinguish ATP from its analogues including adenosine, AMP, and ADP, so the selectivity of the sensing platform is obviously superior to the previously reported anti-ATP DNA apatmer-based biosensors. 3.5. Application of the ligation-triggered electrochemical DNAzyme biosensor for ATP detection In order to further testify the potential of the newly developed electrochemical biosensor for the detection of ATP in real samples, we employed the proposed electrochemical biosensor to determine ATP in human serum sample by the standard addition method. The serum sample offered by a healthy human was gained from Southwest University Hospital in Chongqing City, China, and was subsequently diluted 20 times with buffer solution (20 mM Tris–HCl, 5 mM MgCl2, pH 7.6). At last, a series of ATP solutions with various concentrations were prepared by adding ATP standards into the diluted human serum. As mentioned in the experimental section, the same experimental steps and processes were implemented for the three replicates of each concentration. The detection of ATP at three different concentrations was
In conclusion, a new enzymatic ligation reaction based electrochemical ATP biosensor that combines the strategy of DNAzymebased cyclic ampliﬁcation was presented. In this method, owing to the highly speciﬁc cofactor-dependence of T4 DNA ligase, the proposed biosensing platform is provided with the advantage of speciﬁcally distinguishing the target biological molecule ATP from its six analogues, which results in ultrahigh selectivity. In the procedure of ATP-dependent ligation reaction, the two separate oligonucleotide fragments are ligated to form an intact strand of 8-17 DNAzyme which possesses the activity to catalyze the cleavage of hairpin-structured substrate with the cofactor Zn2 þ . Due to the self-cleavage, the DNAzyme was released from the split hairpin-structured substrate and subsequently hybridizes with another intact hairpin-structured substrate. This self-cleaving process is cyclic, so it can amplify the signal response by multiple turnover of catalytic beacons. Thus, the design that combines the ATP-dependent ligation procedure with the method of self-cleaving DNAzyme-based cyclic ampliﬁcation in one sensing system provides a novel sensing platform for the detection of cofactor ATP with highly improved selectivity and sensitivity. And it is expected that the proposed ligase-based and self-cleaving DNAzyme-ampliﬁed ATP electrochemical biosensor would ﬁnd wide applications in clinical diagnosis of ATP-related diseases, environmental monitoring as well as food quality control.
Acknowledgments This work was ﬁnancially supported by the National Natural Science Foundation of China (No. 21273174) and the Municipal Science Foundation of Chongqing City (No. CSTC-2013jjB00002).
Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.07.007.
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