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A highly sensitive homogeneous electrochemical assay for alkaline phosphatase activity based on single molecular beacon-initiated T7 exonucleasemediated signal amplification Lianfang Zhang, Ting Hou, Haiyin Li and Feng Li* Alkaline phosphatase (ALP), a class of enzymes that catalyzes the dephosphorylation of a variety of substrates, is one of the most commonly assayed enzymes in routine clinical practice, and an important biomarker related to many human diseases. Herein, a facile and highly sensitive homogeneous electrochemical biosensing strategy was proposed for the ALP activity detection based on single molecular beacon-initiated T7 exonuclease-assisted signal amplification. One 3’-phosphorylated and 5’methylene blue (MB) labeled hairpin probe (HP) is ingeniously designed. In the presence of ALP, the dephosphorylation of HP, the subsequent Klenow fragment (KF) polymerase-catalyzed elongation and T7 exonuclease-catalyzed digestion of the duplex stem of HP take place, releasing MB-labeled mononucleotides and the trigger DNA (tDNA). tDNA then hybridizes with another HP and initiates the subsequent cycling cleavage process. As a result, a large amount of MB-labeled mononucleotides are released, generating a significantly amplified electrochemical signal toward the ALP activity assay. A directly measured detection limit as low as 0.1 U L−1 is obtained, which is comparable to that of the fluorescence method and up to three orders of magnitude lower than that of the immobilization-based electrochemical strategy previously reported. In addition to high sensitivity and good selectivity, the as-proposed strategy also
Received 16th March 2015, Accepted 17th April 2015
exhibits the advantages of simplicity and convenience, because the assay is carried out in the homo-
DOI: 10.1039/c5an00516g
geneous solution phase and sophisticated electrode modification processes are avoided. Therefore, the homogeneous electrochemical method we proposed here is an ideal candidate for ALP activity detection
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in biochemical research and clinical practices.
1.
Introduction
Alkaline phosphatase (ALP) is a class of enzymes that catalyzes the hydrolysis of a phosphomonoester into an inorganic phosphate with the specificity on a variety of substrates including nucleic acids, proteins, and small molecules.1 ALP, which exists in almost all living organisms and can be found in various tissues such as bone, liver, kidney, intestine and placenta, regulates the phosphorylation-related biochemical behaviors, and is one of the most commonly assayed enzymes in routine clinical tests.1,2 The abnormal level of ALP has been proven to be closely related to many diseases such as breast and prostatic cancer, liver dysfunction, bone disease, and diabetes, so ALP can be used as an important biomarker in the diagnosis of such diseases.3–6 Therefore, it is crucial to
College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao 266109, China. E-mail: [email protected]; Fax: +(86) 53286080855; Tel: +(86) 53286080855
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develop facile, convenient and sensitive strategies for ALP activity detection. In recent years, numerous strategies have been developed to assay the activity of ALP, including fluorescence,7–16 electrochemiluminescence,17 colorimetry,18–20 and surface enhanced resonance Raman scattering (SERRS).21 For instance, Yu and co-workers developed a label-free fluorescence turn-on strategy for the ALP activity assay based on the polycation-induced noncovalent perylene probe self-assembly.13 Zhu et al. designed a label-free hairpin fluorescent biosensor for the detection of ALP activity utilizing a graphene oxide platform.14 Zheng et al. reported the first ratiometric fluorescent sensing system for ALP.16 Jiang and Wang developed an anodic electrochemiluminescence system based on the co-reaction of CdSe nanoparticles and triethylamine to assay ALP activity.17 Recently, Jiao et al. developed a colorimetric assay for ALP activity based on nucleic acid-regulated perylene probe-induced gold nanoparticle aggregation.20 Although the aforementioned detection methods have their own advantages, sophisticated optical instruments are needed,
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which limit their applications in practical ALP activity assay. As compared to these optical methods, electrochemical methods exhibit fascinating advantages of fast response, low cost and miniaturizability, and have been adopted to detect ALP activity.22–25 For example, Serra et al. realized rapid monitoring of ALP using an amperometric graphite-Teflon composite tyrosinase biosensor.22 Li’s group developed a novel electrochemical biosensor for the detection of ALP activity based on two complementary DNA probes coupled with λ exonuclease.24 However, such electrochemical approaches need the immobilization of the DNA probes or the dephosphorylation substrates on the electrode surface and other tedious processes for the construction of electrochemical biosensors. As a consequence, appropriate immobilization strategies and steps are needed to overcome any steric hindrance of the electrode surfaces. Thus, it is highly desirable to develop simpler and faster immobilization-free electrochemical methods to assay ALP activity. Recently, various immobilization-free electrochemical strategies have been developed to detect a variety of targets, including DNA, small biological molecules, metal ions and enzyme activity.26–34 For example, Xuan et al. demonstrated a solutionphase electrochemical molecular beacon-based strategy and realized the sensitive detection of DNA and mercury ions with signal amplification by exonuclease III-assisted target recycling.27,29 Tang and co-workers reported an ultrasensitive homogeneous electrochemical DNA biosensing platform for the target DNA and protein detection based on the exonuclease III-aided autocatalytic target recycling strategy.30 Our group developed a homogeneous electrochemical aptamerbased adenosine triphosphate assay,28 and highly sensitive homogeneous electrochemical assays of DNA methyltransferase activity,32 and telomerase activity at the single-cell level.33 As compared to the immobilization-based electrochemical means, homogeneous electrochemical strategies avoid the tedious and time-consuming steps of electrode modification, making the experimental processes much simpler and more convenient. Herein, we present a homogeneous electrochemical strategy for highly sensitive assays of ALP activity based on single molecular beacon-initiated T7 exonuclease-assisted signal amplification. The as-proposed strategy employs a 3′-phosphorylated and 5′-methylene blue (MB) labeled hairpin probe (HP) as the molecular beacon, Klenow fragment (KF) polymerase and T7 exonuclease as the tool enzymes, and a negatively charged indium tin oxide (ITO) electrode as the working electrode. HP acts as the substrate for the ALP dephosphorylation, and also initiates the cycling cleavage process. By making full use of the molecular beacon HP and taking advantage of the unique features of KF polymerase and T7 exonuclease, a large amount of MB-labeled mononucleotides are released via the T7 exonuclease-assisted cycling cleavage process, resulting in significantly amplified electrochemical signals of MB toward the highly sensitive ALP activity assay. The as-proposed homogeneous electrochemical strategy provides a facile and convenient biosensing platform for the ALP activity detection with high sensitivity and good selectivity. To the best of our knowl-
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edge, this is the first time to realize the homogeneous electrochemical assay of ALP activity based on single molecular beacon-initiated T7 exonuclease-assisted signal amplification. Due to its advantages of high sensitivity, good selectivity, as well as simple operation, the as-proposed homogeneous electrochemical strategy for ALP activity detection has great potential in the applications in biochemical research and clinical practices.
2. Experimental 2.1.
Reagents
T7 exonuclease, 10 × NEBuffer 4, and shrimp alkaline phosphatase were purchased from New England Biolabs, Ltd (Beijing, China). Klenow fragment (KF) polymerase (without 3′ to 5′ exonuclease activity), and deoxyribonucleoside triphosphates (dNTPs) were purchased from Shanghai Sangon Biotechnology Co., Ltd (Shanghai, China). Tris(hydroxymethyl)aminomethane (Tris), hydrochloric acid (HCl), acetic acid (HAc), dithiothreitol (DTT), Mg(CH3COO)2, CH3COOK, MgCl2, and NaCl were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China), which were of analytical grade and used without further purification. Lysozyme, bovine serum albumin (BSA), hemoglobin (Hb) and streptavidin (SA) were purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). The 3′-phosphorylated and 5′-MB labeled oligonucleotide was synthesized and HPLC-purified by Shanghai Sangon Biotechnology Co., Ltd (Shanghai, China), and the sequence of this hairpin probe (HP) is as follows:
in which the loop sequence is in italics, and the underlined letters represent the sequences complementary to each other. Ultrapure water (resistivity >18.2 MΩ cm at 25 °C) obtained from the Milli-Q water purification system (Millipore Corp., Bedford, MA, USA) was used throughout the experiments. 2.2.
Electrode pretreatment & electrochemical measurement
The ITO electrode was pretreated by being sequentially sonicated in an Alconox solution (8 g of Alconox per liter of water), propan-2-ol, and ultrapure water for 10 min each. Then, the electrode was immersed in 1 mM NaOH solution for 5 h at room temperature and sonicated in ultrapure water for 10 min. After these procedures, a negatively charged working electrode surface was obtained and the ITO working electrode with an active surface area of ca. 0.07 cm2 was ready to use. Differential pulse voltammetric (DPV) measurements were conducted on a CHI 660E electrochemical analyzer (Shanghai, China). A conventional three-electrode system was adopted: an ITO electrode as the working electrode, an Ag/AgCl as the reference electrode, and a platinum wire as the auxiliary electrode. Differential pulse voltammograms (DPV) were recorded with the potential window ranging from −0.6 to 0.1 V.
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2.3.
Alkaline phosphatase activity assay
Dephosphorylation experimentation was performed in 10 µL of dephosphorylation buffer (10 mM Tris-HCl, 10 mM MgCl2, 50 mM NaCl, and 1 mM DTT, pH 7.9) containing 2 µM HP and alkaline phosphatase (ALP) of various concentrations. The aforementioned solution was incubated at 37 °C for 60 min and then heated and kept at 90 °C for 5 min to inactivate ALP. Then the solution was slowly cooled to room temperature followed by addition of 5 µL of 0.5 U µL−1 KF polymerase and 5 µL of 400 µM dNTPs to obtain a reaction volume of 20 µL, and then this reaction solution was incubated at 37 °C for 80 min. After the above operation, 10 µL of 1 U µL−1 T7 exonuclease and 20 µL of T7 exonuclease reaction buffer (20 mM Tris-HAc, 10 mM Mg(CH3COO)2, 50 mM CH3COOK, and 1 mM DTT, pH 7.9) were added to obtain a final volume of 50 µL and the resulting solution was incubated at 37 °C for 60 min before electrochemical measurements. All experiments were repeated three times.
3. Results and discussion 3.1.
Principle of alkaline phosphatase activity assay
The principle of the homogeneous electrochemical assay for ALP activity is illustrated in Scheme 1. A hairpin DNA probe (HP) containing a recessed 3′-phosphoryl end and an overhanging 5′-MB labeled end is ingeniously designed and employed as the molecular beacon in this strategy. In the absence of ALP, the 3′-phosphorylated HP is an inactive substrate for KF polymerase, which is a 5′ to 3′ polymerase and catalyzes the elongation of the duplex DNA region with a 3′-hydroxyl end.35 Therefore, the 5′ terminus of HP remains the protruding configuration, and the digestion of HP by T7 exonuclease, a sequence-independent nuclease that catalyzes the removal of mononucleotides from the blunt or recessed 5′ termini of double-stranded DNA, does not proceed.36 Due to the electrostatic repulsion between the negatively charged ITO electrode and the 5′-MB labeled HP, negligible electrochemical response of MB was observed on the sensing electrode.27,28,31 However, in the presence of ALP, the phosphoryl group at the 3′ terminus of HP is removed, resulting in a 3′-hydroxyl
Scheme 1 The principle of a homogeneous electrochemical assay for alkaline phosphatase activity.
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hairpin probe. Then the KF polymerase recognizes the recessed 3′-hydroxyl terminus of the hairpin DNA and initiates the elongation of the duplex stem until the 3′ terminus becomes blunt. Next, the resulting hairpin DNA is specifically hydrolyzed by T7 exonuclease in the direction from 5′ to 3′, resulting in the release of a MB-labeled mononucleotide and the liberation of the single-stranded trigger DNA (tDNA). The released MB-labeled mononucleotide, with less negative charge and smaller size, possesses much higher diffusivity toward the negatively charged ITO electrode than that of HP,27,31 leading to a distinct increase of the electrochemical signal. Subsequently, the released tDNA partially hybridizes with another HP to form a duplex DNA, in which, the strand with the blunt 5′ terminus is selectively digested by T7 exonuclease to release the MB-labeled mononucleotide, but the tDNA with an overhanging 5′ terminus remains intact. Thus, tDNA is released once again to initiate the subsequent cycling cleavage process, resulting in a large amount of MB-labeled mononucleotides and thereby generating an amplified electrochemical signal. As a result, a highly sensitive alkaline phosphatase activity assay can be realized by monitoring the electrochemical signal change. It is worth noting that in this strategy, the molecular beacon, i.e. the 3′-phosphorylated and 5′-MB labeled HP, has dual functions: (1) acting as the substrate for the ALP dephosphorylation and (2) partially hybridizing with the released tDNA to initiate the cleavage process. Such a design makes full use of the molecular beacon and results in significantly amplified electrochemical signals. 3.2.
Feasibility study of alkaline phosphatase activity assay
DPV experiments were carried out to investigate the feasibility of the proposed homogeneous electrochemical strategy for ALP activity detection. As shown in Fig. 1, in the presence of
Fig. 1 Differential pulse voltammograms under different conditions: (a) HP only, (b) HP + KF polymerase + dNTPs, (c) HP + T7 exonuclease, (d) HP + KF polymerase + T7 exonuclease + dNTPs, (e) HP + ALP + KF polymerase + dNTPs, and (f ) HP + ALP + KF polymerase + T7 exonuclease + dNTPs. The concentrations of HP, ALP, KF polymerase, T7 exonuclease and dNTPs were 2 μM, 5 U L−1, 0.125 U µL−1, 0.2 U µL−1 and 100 μM, respectively.
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HP only, negligible DPV signal was detected (curve a), because the electrostatic repulsion between the negatively charged ITO electrode and HP prevented methylene blue from reaching the surface of the electrode. With the addition of KF polymerase (curve b), T7 exonuclease (curve c), or both (curve d), the electrochemical signals barely changed due to the lack of the dephosphorylation reaction, so the subsequent KF polymerization and T7 exonuclease cleavage reactions couldn’t proceed, and the electrochemical indicator MB still attached to HP. In the presence of HP, ALP, KF polymerase and dNTPs, a noticeable decrease of the DPV signal was observed (curve e), which could result from the greater repulsion between the hairpin probe with the elongated double-stranded stem and the ITO electrode. It also demonstrated clearly that, in the absence of T7 exonuclease, the digestion of the hairpin probe couldn’t occur, resulting in no MB-labeled mononucleotides and thus no electrochemical signal amplification. However, under the same conditions, with the addition of T7 exonuclease to the reaction system, a significant increase of the electrochemical signal of MB was observed (curve f ), indicating the occurrence of the elongation and the cleavage of HP, as well as the subsequent cycling cleavage process illustrated in Scheme 1. The aforementioned results clearly demonstrated the feasibility of the proposed homogeneous electrochemical assay for ALP activity. 3.3.
Optimization of experimental conditions
As shown in Scheme 1, besides being the substrate for ALP dephosphorylation, HP is also utilized in the cycling cleavage process to generate a large amount of MB-labeled mononucleotides. In order to achieve high sensitivity, sufficient amount of HP needs to be added into the reaction system to carry out enough rounds of cycles, so 2 μM HP was used in the experiments. In addition, the reaction time of ALP dephosphorylation, KF polymerization, and T7 exonuclease cleavage was optimized, respectively. As shown in Fig. 2A, the DPV signal (for simplicity, the absolute value of the peak current adopted here and afterwards) increased progressively with the increase of ALP reaction time up to 60 min, but with a longer reaction time the DPV current did not change much and reached a maximum value. Similarly, DPV currents were found to reach the maximum after 80 min for KF polymerization reaction (Fig. 2B) and after 60 min for T7 exonuclease reaction (Fig. 2C), respectively. Therefore, 60 min, 80 min and 60 min were chosen as the optimal reaction times for ALP, KF polymerase and T7 exonuclease throughout the subsequent experiments, respectively. In addition, the continuous increase of the electrochemical signal at the initial stage indicated that the as-proposed T7 exonuclease-assisted signal amplification did take place, while the eventual signal saturation suggested that the MB-labeled HP was completely consumed at the end of the reaction. 3.4.
Alkaline phosphatase activity detection
Under the optimal experimental conditions, the analytical performance of the proposed homogeneous electrochemical bio-
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Fig. 2 DPV peak currents versus the incubation time of (A) ALP, (B) KF polymerase, and (C) T7 exonuclease, respectively. The concentrations of ALP, KF polymerase and T7 exonuclease were 5 U L−1, 0.125 U µL−1 and 0.2 U µL−1, respectively, and the concentrations of HP and dNTP were 2 µM and 100 µM, respectively. The error bars represent the standard deviation of three measurements.
sensing platform was investigated by varying the concentration of ALP. As shown in Fig. 3A, the electrochemical response was highly dependent on ALP concentration, and the DPV signal of MB increased as the concentration of ALP increased. Fig. 3B depicts the relationship between the DPV peak current and the concentration of ALP. The DPV peak current increased gradually as the ALP concentration increased from 0 to 50 U L−1, which demonstrated that the T7 exonuclease-catalyzed release of MB from HP was highly dependent on the concentration of ALP. The DPV peak current was proportional to the ALP concentration ranging from 0.1 to 10 U L−1, and the calibration plot for the quantification of ALP activity is shown in the inset of Fig. 3B. As the result demonstrated, there was a good linear correlation between the DPV peak current change and the ALP
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than that of the colorimetric approach, and comparable to or one order of magnitude lower than that of the fluorescence strategies reported in literature (Table 1). The low detection limit makes it possible to dilute real samples more when ALP activity is detected, which will effectively reduce the interference of the matrix substances, making this assay more applicable for the detection of ALP activity in complex samples. The results demonstrated that the as-proposed single molecular beacon-initiated T7 exonuclease-assisted signal amplification strategy for a highly sensitive ALP activity assay was indeed realized. In addition, the reproducibility of the ALP activity assay was investigated through 5 successive assays in the presence of 5 U L−1 ALP. The relative standard deviation (RSD) was determined to be 3.65%, indicating an acceptable reproducibility of the asproposed strategy. Therefore, the homogeneous electrochemical method we proposed here could be used for detecting the ALP activity sensitively and conveniently. 3.5.
Fig. 3 (A) Differential pulse voltammograms of the biosensing system upon the addition of ALP with different concentrations: (a) 0, (b) 0.1, (c) 0.5, (d) 1, (e) 5, (f) 10, (g) 20 and (h) 50 U L−1. (B) DPV peak currents plotted against the concentration of ALP. Inset: the linear relationship between the DPV peak current change and the ALP concentration ranging from 0.1 to 10 U L−1. The error bars represent the standard deviation of three independent measurements.
concentration, and the calibration equation was determined to be Δip = 5.6560C + 8.5264 (Δip, nA; C, U L−1) with a correlation coefficient of R2 = 0.9977, where Δip is the DPV peak current change and C is the ALP concentration. The directly measured limit of detection for ALP activity was as low as 0.1 U L−1, three orders of magnitude lower than that of the immobilizationbased electrochemical method, two orders of magnitude lower
Table 1
Selectivity of alkaline phosphatase activity assay
To evaluate the selectivity of the as-proposed ALP activity assay, one interfering enzyme (lysozyme) and three interfering proteins, namely bovine serum albumin (BSA), hemoglobin (Hb), and streptavidin (SA), were investigated. In the reaction system, ALP was substituted by lysozyme, BSA, Hb and SA, respectively. As shown in Fig. 4, a high DPV peak current was obtained only when the target (ALP, 5 U L−1) was present, whereas in the presence of lysozyme, BSA, Hb or SA (all with the same concentration of 100 U L−1), the DPV peak current was fairly small and comparable to that in the control experiment. Therefore, the as-proposed homogeneous electrochemical strategy exhibited good performance for discriminating ALP against other interfering enzymes and proteins.
4.
Conclusions
In summary, we proposed here a facile and highly sensitive homogeneous electrochemical assay for ALP activity detection based on single molecular beacon-initiated T7 exonucleaseassisted signal amplification. This strategy makes full use of the molecular beacon HP and takes advantage of the unique
Comparison of analytical performance for the detection of ALP activity on DNA substrates by our strategy and those reported in literature
Method
Detection limit (U L−1)
Homogeneous electrochemistry Immobilization-based electrochemistry Colorimetry
0.1 100 32
Fluorescence
3
Fluorescence
0.3
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Strategy
Ref.
T7 exonuclease-assisted signal amplification λ Exonuclease-mediated signal amplification Nucleic acid-regulated perylene probe-induced gold nanoparticle aggregation A graphene oxide platform based on hairpin primer and polymerase elongation Polycation-induced noncovalent perylene probe self-assembly
This work 24 19 13 14
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Fig. 4 Comparison of the DPV peak current of the biosensing platform in the presence of lysozyme, BSA, Hb, SA and ALP, respectively, in which Blank indicates the conditions in the absence of ALP as well as the interfering enzyme and proteins. The concentration of ALP is 5 U L−1, whereas the concentrations of lysozyme, BSA, Hb and SA are all 100 U L−1. The error bars represent the standard deviation of three measurements.
features of KF polymerase and T7 exonuclease, and a large amount of MB-labeled mononucleotides are released via the cycling cleavage process, resulting in significantly amplified electrochemical signals. This biosensing system exhibits good selectivity and very high sensitivity for ALP activity assays, and the limit of detection down to 0.1 U L−1 is obtained, comparable to that of the fluorescence method and up to three orders of magnitude lower than that of the immobilization-based electrochemical strategy reported in literature. Moreover, compared to the immobilization-based electrochemical assays for ALP activity, this method is carried out in a homogeneous solution, thus exhibiting additional advantages of simplicity and convenience. Therefore, the homogeneous electrochemical ALP activity assay we proposed here has great potential in the applications in biochemical research and clinical practices.
Acknowledgements This work was funded by the National Natural Science Foundation of China (no. 21175076, 21375072 and 21445002), the Open Foundation of State Key Laboratory of Electroanalytical Chemistry (no. SKLEAC201402), and the Research Foundation for Distinguished Scholars of Qingdao Agricultural University (no. 6631113311 and 6631113320).
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A highly sensitive homogeneous electrochemical assay for alkaline phosphatase activity based on single molecular beacon-initiated T7 exonucleasemediated signal amplification Lianfang Zhang, Ting Hou, Haiyin Li and Feng Li* Alkaline phosphatase (ALP), a class of enzymes that catalyzes the dephosphorylation of a variety of substrates, is one of the most commonly assayed enzymes in routine clinical practice, and an important biomarker related to many human diseases. Herein, a facile and highly sensitive homogeneous electrochemical biosensing strategy was proposed for the ALP activity detection based on single molecular beacon-initiated T7 exonuclease-assisted signal amplification. One 3’-phosphorylated and 5’methylene blue (MB) labeled hairpin probe (HP) is ingeniously designed. In the presence of ALP, the dephosphorylation of HP, the subsequent Klenow fragment (KF) polymerase-catalyzed elongation and T7 exonuclease-catalyzed digestion of the duplex stem of HP take place, releasing MB-labeled mononucleotides and the trigger DNA (tDNA). tDNA then hybridizes with another HP and initiates the subsequent cycling cleavage process. As a result, a large amount of MB-labeled mononucleotides are released, generating a significantly amplified electrochemical signal toward the ALP activity assay. A directly measured detection limit as low as 0.1 U L−1 is obtained, which is comparable to that of the fluorescence method and up to three orders of magnitude lower than that of the immobilization-based electrochemical strategy previously reported. In addition to high sensitivity and good selectivity, the as-proposed strategy also
Received 16th March 2015, Accepted 17th April 2015
exhibits the advantages of simplicity and convenience, because the assay is carried out in the homo-
DOI: 10.1039/c5an00516g
geneous solution phase and sophisticated electrode modification processes are avoided. Therefore, the homogeneous electrochemical method we proposed here is an ideal candidate for ALP activity detection
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in biochemical research and clinical practices.
1.
Introduction
Alkaline phosphatase (ALP) is a class of enzymes that catalyzes the hydrolysis of a phosphomonoester into an inorganic phosphate with the specificity on a variety of substrates including nucleic acids, proteins, and small molecules.1 ALP, which exists in almost all living organisms and can be found in various tissues such as bone, liver, kidney, intestine and placenta, regulates the phosphorylation-related biochemical behaviors, and is one of the most commonly assayed enzymes in routine clinical tests.1,2 The abnormal level of ALP has been proven to be closely related to many diseases such as breast and prostatic cancer, liver dysfunction, bone disease, and diabetes, so ALP can be used as an important biomarker in the diagnosis of such diseases.3–6 Therefore, it is crucial to
College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao 266109, China. E-mail: [email protected]; Fax: +(86) 53286080855; Tel: +(86) 53286080855
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develop facile, convenient and sensitive strategies for ALP activity detection. In recent years, numerous strategies have been developed to assay the activity of ALP, including fluorescence,7–16 electrochemiluminescence,17 colorimetry,18–20 and surface enhanced resonance Raman scattering (SERRS).21 For instance, Yu and co-workers developed a label-free fluorescence turn-on strategy for the ALP activity assay based on the polycation-induced noncovalent perylene probe self-assembly.13 Zhu et al. designed a label-free hairpin fluorescent biosensor for the detection of ALP activity utilizing a graphene oxide platform.14 Zheng et al. reported the first ratiometric fluorescent sensing system for ALP.16 Jiang and Wang developed an anodic electrochemiluminescence system based on the co-reaction of CdSe nanoparticles and triethylamine to assay ALP activity.17 Recently, Jiao et al. developed a colorimetric assay for ALP activity based on nucleic acid-regulated perylene probe-induced gold nanoparticle aggregation.20 Although the aforementioned detection methods have their own advantages, sophisticated optical instruments are needed,
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which limit their applications in practical ALP activity assay. As compared to these optical methods, electrochemical methods exhibit fascinating advantages of fast response, low cost and miniaturizability, and have been adopted to detect ALP activity.22–25 For example, Serra et al. realized rapid monitoring of ALP using an amperometric graphite-Teflon composite tyrosinase biosensor.22 Li’s group developed a novel electrochemical biosensor for the detection of ALP activity based on two complementary DNA probes coupled with λ exonuclease.24 However, such electrochemical approaches need the immobilization of the DNA probes or the dephosphorylation substrates on the electrode surface and other tedious processes for the construction of electrochemical biosensors. As a consequence, appropriate immobilization strategies and steps are needed to overcome any steric hindrance of the electrode surfaces. Thus, it is highly desirable to develop simpler and faster immobilization-free electrochemical methods to assay ALP activity. Recently, various immobilization-free electrochemical strategies have been developed to detect a variety of targets, including DNA, small biological molecules, metal ions and enzyme activity.26–34 For example, Xuan et al. demonstrated a solutionphase electrochemical molecular beacon-based strategy and realized the sensitive detection of DNA and mercury ions with signal amplification by exonuclease III-assisted target recycling.27,29 Tang and co-workers reported an ultrasensitive homogeneous electrochemical DNA biosensing platform for the target DNA and protein detection based on the exonuclease III-aided autocatalytic target recycling strategy.30 Our group developed a homogeneous electrochemical aptamerbased adenosine triphosphate assay,28 and highly sensitive homogeneous electrochemical assays of DNA methyltransferase activity,32 and telomerase activity at the single-cell level.33 As compared to the immobilization-based electrochemical means, homogeneous electrochemical strategies avoid the tedious and time-consuming steps of electrode modification, making the experimental processes much simpler and more convenient. Herein, we present a homogeneous electrochemical strategy for highly sensitive assays of ALP activity based on single molecular beacon-initiated T7 exonuclease-assisted signal amplification. The as-proposed strategy employs a 3′-phosphorylated and 5′-methylene blue (MB) labeled hairpin probe (HP) as the molecular beacon, Klenow fragment (KF) polymerase and T7 exonuclease as the tool enzymes, and a negatively charged indium tin oxide (ITO) electrode as the working electrode. HP acts as the substrate for the ALP dephosphorylation, and also initiates the cycling cleavage process. By making full use of the molecular beacon HP and taking advantage of the unique features of KF polymerase and T7 exonuclease, a large amount of MB-labeled mononucleotides are released via the T7 exonuclease-assisted cycling cleavage process, resulting in significantly amplified electrochemical signals of MB toward the highly sensitive ALP activity assay. The as-proposed homogeneous electrochemical strategy provides a facile and convenient biosensing platform for the ALP activity detection with high sensitivity and good selectivity. To the best of our knowl-
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edge, this is the first time to realize the homogeneous electrochemical assay of ALP activity based on single molecular beacon-initiated T7 exonuclease-assisted signal amplification. Due to its advantages of high sensitivity, good selectivity, as well as simple operation, the as-proposed homogeneous electrochemical strategy for ALP activity detection has great potential in the applications in biochemical research and clinical practices.
2. Experimental 2.1.
Reagents
T7 exonuclease, 10 × NEBuffer 4, and shrimp alkaline phosphatase were purchased from New England Biolabs, Ltd (Beijing, China). Klenow fragment (KF) polymerase (without 3′ to 5′ exonuclease activity), and deoxyribonucleoside triphosphates (dNTPs) were purchased from Shanghai Sangon Biotechnology Co., Ltd (Shanghai, China). Tris(hydroxymethyl)aminomethane (Tris), hydrochloric acid (HCl), acetic acid (HAc), dithiothreitol (DTT), Mg(CH3COO)2, CH3COOK, MgCl2, and NaCl were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China), which were of analytical grade and used without further purification. Lysozyme, bovine serum albumin (BSA), hemoglobin (Hb) and streptavidin (SA) were purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). The 3′-phosphorylated and 5′-MB labeled oligonucleotide was synthesized and HPLC-purified by Shanghai Sangon Biotechnology Co., Ltd (Shanghai, China), and the sequence of this hairpin probe (HP) is as follows:
in which the loop sequence is in italics, and the underlined letters represent the sequences complementary to each other. Ultrapure water (resistivity >18.2 MΩ cm at 25 °C) obtained from the Milli-Q water purification system (Millipore Corp., Bedford, MA, USA) was used throughout the experiments. 2.2.
Electrode pretreatment & electrochemical measurement
The ITO electrode was pretreated by being sequentially sonicated in an Alconox solution (8 g of Alconox per liter of water), propan-2-ol, and ultrapure water for 10 min each. Then, the electrode was immersed in 1 mM NaOH solution for 5 h at room temperature and sonicated in ultrapure water for 10 min. After these procedures, a negatively charged working electrode surface was obtained and the ITO working electrode with an active surface area of ca. 0.07 cm2 was ready to use. Differential pulse voltammetric (DPV) measurements were conducted on a CHI 660E electrochemical analyzer (Shanghai, China). A conventional three-electrode system was adopted: an ITO electrode as the working electrode, an Ag/AgCl as the reference electrode, and a platinum wire as the auxiliary electrode. Differential pulse voltammograms (DPV) were recorded with the potential window ranging from −0.6 to 0.1 V.
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2.3.
Alkaline phosphatase activity assay
Dephosphorylation experimentation was performed in 10 µL of dephosphorylation buffer (10 mM Tris-HCl, 10 mM MgCl2, 50 mM NaCl, and 1 mM DTT, pH 7.9) containing 2 µM HP and alkaline phosphatase (ALP) of various concentrations. The aforementioned solution was incubated at 37 °C for 60 min and then heated and kept at 90 °C for 5 min to inactivate ALP. Then the solution was slowly cooled to room temperature followed by addition of 5 µL of 0.5 U µL−1 KF polymerase and 5 µL of 400 µM dNTPs to obtain a reaction volume of 20 µL, and then this reaction solution was incubated at 37 °C for 80 min. After the above operation, 10 µL of 1 U µL−1 T7 exonuclease and 20 µL of T7 exonuclease reaction buffer (20 mM Tris-HAc, 10 mM Mg(CH3COO)2, 50 mM CH3COOK, and 1 mM DTT, pH 7.9) were added to obtain a final volume of 50 µL and the resulting solution was incubated at 37 °C for 60 min before electrochemical measurements. All experiments were repeated three times.
3. Results and discussion 3.1.
Principle of alkaline phosphatase activity assay
The principle of the homogeneous electrochemical assay for ALP activity is illustrated in Scheme 1. A hairpin DNA probe (HP) containing a recessed 3′-phosphoryl end and an overhanging 5′-MB labeled end is ingeniously designed and employed as the molecular beacon in this strategy. In the absence of ALP, the 3′-phosphorylated HP is an inactive substrate for KF polymerase, which is a 5′ to 3′ polymerase and catalyzes the elongation of the duplex DNA region with a 3′-hydroxyl end.35 Therefore, the 5′ terminus of HP remains the protruding configuration, and the digestion of HP by T7 exonuclease, a sequence-independent nuclease that catalyzes the removal of mononucleotides from the blunt or recessed 5′ termini of double-stranded DNA, does not proceed.36 Due to the electrostatic repulsion between the negatively charged ITO electrode and the 5′-MB labeled HP, negligible electrochemical response of MB was observed on the sensing electrode.27,28,31 However, in the presence of ALP, the phosphoryl group at the 3′ terminus of HP is removed, resulting in a 3′-hydroxyl
Scheme 1 The principle of a homogeneous electrochemical assay for alkaline phosphatase activity.
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hairpin probe. Then the KF polymerase recognizes the recessed 3′-hydroxyl terminus of the hairpin DNA and initiates the elongation of the duplex stem until the 3′ terminus becomes blunt. Next, the resulting hairpin DNA is specifically hydrolyzed by T7 exonuclease in the direction from 5′ to 3′, resulting in the release of a MB-labeled mononucleotide and the liberation of the single-stranded trigger DNA (tDNA). The released MB-labeled mononucleotide, with less negative charge and smaller size, possesses much higher diffusivity toward the negatively charged ITO electrode than that of HP,27,31 leading to a distinct increase of the electrochemical signal. Subsequently, the released tDNA partially hybridizes with another HP to form a duplex DNA, in which, the strand with the blunt 5′ terminus is selectively digested by T7 exonuclease to release the MB-labeled mononucleotide, but the tDNA with an overhanging 5′ terminus remains intact. Thus, tDNA is released once again to initiate the subsequent cycling cleavage process, resulting in a large amount of MB-labeled mononucleotides and thereby generating an amplified electrochemical signal. As a result, a highly sensitive alkaline phosphatase activity assay can be realized by monitoring the electrochemical signal change. It is worth noting that in this strategy, the molecular beacon, i.e. the 3′-phosphorylated and 5′-MB labeled HP, has dual functions: (1) acting as the substrate for the ALP dephosphorylation and (2) partially hybridizing with the released tDNA to initiate the cleavage process. Such a design makes full use of the molecular beacon and results in significantly amplified electrochemical signals. 3.2.
Feasibility study of alkaline phosphatase activity assay
DPV experiments were carried out to investigate the feasibility of the proposed homogeneous electrochemical strategy for ALP activity detection. As shown in Fig. 1, in the presence of
Fig. 1 Differential pulse voltammograms under different conditions: (a) HP only, (b) HP + KF polymerase + dNTPs, (c) HP + T7 exonuclease, (d) HP + KF polymerase + T7 exonuclease + dNTPs, (e) HP + ALP + KF polymerase + dNTPs, and (f ) HP + ALP + KF polymerase + T7 exonuclease + dNTPs. The concentrations of HP, ALP, KF polymerase, T7 exonuclease and dNTPs were 2 μM, 5 U L−1, 0.125 U µL−1, 0.2 U µL−1 and 100 μM, respectively.
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HP only, negligible DPV signal was detected (curve a), because the electrostatic repulsion between the negatively charged ITO electrode and HP prevented methylene blue from reaching the surface of the electrode. With the addition of KF polymerase (curve b), T7 exonuclease (curve c), or both (curve d), the electrochemical signals barely changed due to the lack of the dephosphorylation reaction, so the subsequent KF polymerization and T7 exonuclease cleavage reactions couldn’t proceed, and the electrochemical indicator MB still attached to HP. In the presence of HP, ALP, KF polymerase and dNTPs, a noticeable decrease of the DPV signal was observed (curve e), which could result from the greater repulsion between the hairpin probe with the elongated double-stranded stem and the ITO electrode. It also demonstrated clearly that, in the absence of T7 exonuclease, the digestion of the hairpin probe couldn’t occur, resulting in no MB-labeled mononucleotides and thus no electrochemical signal amplification. However, under the same conditions, with the addition of T7 exonuclease to the reaction system, a significant increase of the electrochemical signal of MB was observed (curve f ), indicating the occurrence of the elongation and the cleavage of HP, as well as the subsequent cycling cleavage process illustrated in Scheme 1. The aforementioned results clearly demonstrated the feasibility of the proposed homogeneous electrochemical assay for ALP activity. 3.3.
Optimization of experimental conditions
As shown in Scheme 1, besides being the substrate for ALP dephosphorylation, HP is also utilized in the cycling cleavage process to generate a large amount of MB-labeled mononucleotides. In order to achieve high sensitivity, sufficient amount of HP needs to be added into the reaction system to carry out enough rounds of cycles, so 2 μM HP was used in the experiments. In addition, the reaction time of ALP dephosphorylation, KF polymerization, and T7 exonuclease cleavage was optimized, respectively. As shown in Fig. 2A, the DPV signal (for simplicity, the absolute value of the peak current adopted here and afterwards) increased progressively with the increase of ALP reaction time up to 60 min, but with a longer reaction time the DPV current did not change much and reached a maximum value. Similarly, DPV currents were found to reach the maximum after 80 min for KF polymerization reaction (Fig. 2B) and after 60 min for T7 exonuclease reaction (Fig. 2C), respectively. Therefore, 60 min, 80 min and 60 min were chosen as the optimal reaction times for ALP, KF polymerase and T7 exonuclease throughout the subsequent experiments, respectively. In addition, the continuous increase of the electrochemical signal at the initial stage indicated that the as-proposed T7 exonuclease-assisted signal amplification did take place, while the eventual signal saturation suggested that the MB-labeled HP was completely consumed at the end of the reaction. 3.4.
Alkaline phosphatase activity detection
Under the optimal experimental conditions, the analytical performance of the proposed homogeneous electrochemical bio-
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Fig. 2 DPV peak currents versus the incubation time of (A) ALP, (B) KF polymerase, and (C) T7 exonuclease, respectively. The concentrations of ALP, KF polymerase and T7 exonuclease were 5 U L−1, 0.125 U µL−1 and 0.2 U µL−1, respectively, and the concentrations of HP and dNTP were 2 µM and 100 µM, respectively. The error bars represent the standard deviation of three measurements.
sensing platform was investigated by varying the concentration of ALP. As shown in Fig. 3A, the electrochemical response was highly dependent on ALP concentration, and the DPV signal of MB increased as the concentration of ALP increased. Fig. 3B depicts the relationship between the DPV peak current and the concentration of ALP. The DPV peak current increased gradually as the ALP concentration increased from 0 to 50 U L−1, which demonstrated that the T7 exonuclease-catalyzed release of MB from HP was highly dependent on the concentration of ALP. The DPV peak current was proportional to the ALP concentration ranging from 0.1 to 10 U L−1, and the calibration plot for the quantification of ALP activity is shown in the inset of Fig. 3B. As the result demonstrated, there was a good linear correlation between the DPV peak current change and the ALP
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than that of the colorimetric approach, and comparable to or one order of magnitude lower than that of the fluorescence strategies reported in literature (Table 1). The low detection limit makes it possible to dilute real samples more when ALP activity is detected, which will effectively reduce the interference of the matrix substances, making this assay more applicable for the detection of ALP activity in complex samples. The results demonstrated that the as-proposed single molecular beacon-initiated T7 exonuclease-assisted signal amplification strategy for a highly sensitive ALP activity assay was indeed realized. In addition, the reproducibility of the ALP activity assay was investigated through 5 successive assays in the presence of 5 U L−1 ALP. The relative standard deviation (RSD) was determined to be 3.65%, indicating an acceptable reproducibility of the asproposed strategy. Therefore, the homogeneous electrochemical method we proposed here could be used for detecting the ALP activity sensitively and conveniently. 3.5.
Fig. 3 (A) Differential pulse voltammograms of the biosensing system upon the addition of ALP with different concentrations: (a) 0, (b) 0.1, (c) 0.5, (d) 1, (e) 5, (f) 10, (g) 20 and (h) 50 U L−1. (B) DPV peak currents plotted against the concentration of ALP. Inset: the linear relationship between the DPV peak current change and the ALP concentration ranging from 0.1 to 10 U L−1. The error bars represent the standard deviation of three independent measurements.
concentration, and the calibration equation was determined to be Δip = 5.6560C + 8.5264 (Δip, nA; C, U L−1) with a correlation coefficient of R2 = 0.9977, where Δip is the DPV peak current change and C is the ALP concentration. The directly measured limit of detection for ALP activity was as low as 0.1 U L−1, three orders of magnitude lower than that of the immobilizationbased electrochemical method, two orders of magnitude lower
Table 1
Selectivity of alkaline phosphatase activity assay
To evaluate the selectivity of the as-proposed ALP activity assay, one interfering enzyme (lysozyme) and three interfering proteins, namely bovine serum albumin (BSA), hemoglobin (Hb), and streptavidin (SA), were investigated. In the reaction system, ALP was substituted by lysozyme, BSA, Hb and SA, respectively. As shown in Fig. 4, a high DPV peak current was obtained only when the target (ALP, 5 U L−1) was present, whereas in the presence of lysozyme, BSA, Hb or SA (all with the same concentration of 100 U L−1), the DPV peak current was fairly small and comparable to that in the control experiment. Therefore, the as-proposed homogeneous electrochemical strategy exhibited good performance for discriminating ALP against other interfering enzymes and proteins.
4.
Conclusions
In summary, we proposed here a facile and highly sensitive homogeneous electrochemical assay for ALP activity detection based on single molecular beacon-initiated T7 exonucleaseassisted signal amplification. This strategy makes full use of the molecular beacon HP and takes advantage of the unique
Comparison of analytical performance for the detection of ALP activity on DNA substrates by our strategy and those reported in literature
Method
Detection limit (U L−1)
Homogeneous electrochemistry Immobilization-based electrochemistry Colorimetry
0.1 100 32
Fluorescence
3
Fluorescence
0.3
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Strategy
Ref.
T7 exonuclease-assisted signal amplification λ Exonuclease-mediated signal amplification Nucleic acid-regulated perylene probe-induced gold nanoparticle aggregation A graphene oxide platform based on hairpin primer and polymerase elongation Polycation-induced noncovalent perylene probe self-assembly
This work 24 19 13 14
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Fig. 4 Comparison of the DPV peak current of the biosensing platform in the presence of lysozyme, BSA, Hb, SA and ALP, respectively, in which Blank indicates the conditions in the absence of ALP as well as the interfering enzyme and proteins. The concentration of ALP is 5 U L−1, whereas the concentrations of lysozyme, BSA, Hb and SA are all 100 U L−1. The error bars represent the standard deviation of three measurements.
features of KF polymerase and T7 exonuclease, and a large amount of MB-labeled mononucleotides are released via the cycling cleavage process, resulting in significantly amplified electrochemical signals. This biosensing system exhibits good selectivity and very high sensitivity for ALP activity assays, and the limit of detection down to 0.1 U L−1 is obtained, comparable to that of the fluorescence method and up to three orders of magnitude lower than that of the immobilization-based electrochemical strategy reported in literature. Moreover, compared to the immobilization-based electrochemical assays for ALP activity, this method is carried out in a homogeneous solution, thus exhibiting additional advantages of simplicity and convenience. Therefore, the homogeneous electrochemical ALP activity assay we proposed here has great potential in the applications in biochemical research and clinical practices.
Acknowledgements This work was funded by the National Natural Science Foundation of China (no. 21175076, 21375072 and 21445002), the Open Foundation of State Key Laboratory of Electroanalytical Chemistry (no. SKLEAC201402), and the Research Foundation for Distinguished Scholars of Qingdao Agricultural University (no. 6631113311 and 6631113320).
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