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Electrochemical selective detection of carnitine enantiomers coupling copper ion dependent DNAzyme with DNA assistant hybridization chain reaction
Journal of Electroanalytical Chemistry 837 (2019) 137–142
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Short communication
Electrochemical selective detection of carnitine enantiomers coupling copper ion dependent DNAzyme with DNA assistant hybridization chain reaction
T
Fenfen Zhaia,b, Qiao Yub,d, Hong Zhoub, , Jing Liub,c, , Wenrong Yangb,c, Jinmao Youa, ⁎
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a
Shandong Province Key Laboratory of Life-Organic Analysis, College of Chemistry and Chemical Engineering, Qufu Normal University, Qufu 273165, PR China Shandong Province Key Laboratory of Detection Technology for Tumor Markers, College of Chemistry and Chemical Engineering, Linyi University, Linyi 276005, PR China c Centre for Chemistry and Biotechnology, School of Life and Environmental Sciences, Deakin University, Geelong, Victoria 3216, Australia d Shandong Sino-Japanese Center for Collaborative Research of Carbon Nanomaterials, College of Chemistry and Chemical Engineering, Qingdao University, Qingdao, Shandong 266071, PR China b
ARTICLE INFO
ABSTRACT
Keywords: Electrochemical detection DNAzyme Hybridization chain reaction Chiral molecules recognition Signal amplification
An electrochemical method for carnitine enantiomers recognition is proposed employing Cu (II)-L-cysteine complexes and signal amplification strategies based on DNAzyme and DNA assistant hybridization chain reaction (HCR). The reported method combined Cu (II)-amino acids complexes with DNAzyme and proposed a smart and efficient electrochemical method to recognize chiral molecules through the difference between homochiral interaction and heterochiral interaction. Moreover, further employment of hairpin DNA assistant HCR greatly enhanced the detecting signal, realizing the selective recognition of carnitine enantiomers in low concentrations. This study creatively provides a general sensing method for sensitive chiral molecules recognition without largescale precision instruments.
1. Introduction Due to several drawbacks of current detecting method for chiral molecules, including the requirement of high-cost and large-scale instruments, additional laser radiation and unsatisfied sensitivity and selectivity [1–4], it is still urgent to explore a simple, rapid, and signalamplified sensing method for selective analysis of chiral molecules enantiomers. Recently, Cu (II) complexes formed from interactions between Cu (II) ions and enantiomeric amino acid have been studied and applied for chiral-sensing systems [5–7]. Recent work from Yang's group employed Cu (II)-amino acids complexes to find out molecular recognition mechanism between amino acid L (levorotary)-cysteine and L/D(dextrorotary)-carnitine using Surface-enhanced Raman spectrum (SERS) technology and then in their following studies proposed an electrochemical method to recognize carnitine enantiomers [8,9]. Carnitine, with two enantioselective L/D configurations due to the asymmetric secondary carbon, is a safe food supplement in its L-form and could support the amino acid development in human body. While, D-carnitine demonstrates some unwanted side effects and thus is limited
in pharmaceutical and nutritional formulations. Therefore, recognition of chiral carnitine is very important in biological and medicinal studies. However, more improvement in sensitivity and selectivity of the sensing method for carnitine enantiomers is urgently needed in order to realize highly enantioselective recognition of carnitine in low concentrations. DNAzymes, based on DNA molecules with a catalytic activity have attracted much attention with good stability, such as Hemin/G-quadruplex DNAzymes [10,11] and metal ions dependent DNAzyme [12]. Metal ions dependent DNAzyme are DNA molecules generally containing a substrate strand containing a specific cleavage site by specific metal ions and an enzyme strand. Thus in the presence of metal ions, such as Cu2+ [13,14], Pb2+ [15,16], Mg2+ [17], Zn2+ [18,19] or Ca2+ [20], as catalytic cofactors, the substrate strand will be cleaved into two parts. Benefit from this high recognition function and catalytic activity, DNAzyme showed many advantages of easy operation, low cost, and ability to be renatured many times without losing activity, DNAzyme showed significantly improved selectivity and simplicity in the construction of functional molecular probes for the detection of various
Corresponding authors. Correspondence to: J. Liu, Centre for Chemistry and Biotechnology, School of Life and Environmental Sciences, Deakin University, Geelong, Victoria 3216, Australia. E-mail addresses: [email protected] (H. Zhou), [email protected] (J. Liu), [email protected] (J. You). ⁎
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https://doi.org/10.1016/j.jelechem.2019.02.020 Received 11 January 2019; Received in revised form 12 February 2019; Accepted 13 February 2019 Available online 14 February 2019 1572-6657/ © 2019 Elsevier B.V. All rights reserved.
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analytes [21–25]. Besides DNAzyme, DNA assistant isothermal amplification strategies have been widely employed in recent biosensing platforms, providing efficiently amplified detecting signal, excellent stability, sensitivity, and designability [26–31]. For example, hybridization chain reaction (HCR) has been widely studied and developed for amplification strategy due to no needed additional DNA tool enzyme [32–38]. Here, we combined Cu2+-dependent DNAzyme, Cu (II)-amino acids complexes and proposed a smart and efficient electrochemical method to recognize chiral molecules through the difference between homochiral interaction and heterochiral interaction and different amount of replaced Cu2+ which can be involved in DNAzyme cleavage and then resulting in a measurable electrochemical signal. Benefit from the excellent stability, recycling abilities from DNAzyme and nucleic acidassistant HCR amplification technology, greatly enhanced signals were obtained for selective recognition of L/D carnitine molecules in low concentrations. Moreover, using simple electrochemical method, this study may provide a novel sensing method for efficient chiral molecules recognition in low concentration without the corresponding large-scale precision instruments.
2 h. In this way, the MCH/probe modified electrodes were formed. Following this, electrodes were washed with PBS buffer (pH = 7.4) and dried for future experiments. 2.4. Preparation of Cu2+-L-cysteine self-assembled Au electrode The bare gold electrode was immersed in 0.5 M of the L-cysteine for 2 h at RT in order to form a self-assembled cysteine monolayer on the electrode surface. After that, the L-cysteine modified electrode was dipped in 0.5 M CuSO4 (pH = 5.5) for 30 min at RT. Cu2+-L-cysteine self-assembled Au electrode was formed by this way. Then, they were washed with 0.01 M PBS (pH = 5.5) and dried for future experiment. 2.5. The recycling amplification detection electrochemical ferrocene signal of DNA3/4/Cu2+/MCH/DNA1/2/Au electrode Cu2+-L-cysteine self-assembled Au electrode was dipped in centrifugal tube with 300 nM D/L-Carnitine (pH = 5.5) for 25 min at RT. After that, Cu2+-L-cysteine self-assembled Au electrode was taken out and MCH/DNA1/2/Au electrode was immersed in the above centrifugal tube for 1 h at RT to form Cu2+/MCH/DNA1/2/Au electrode. Following this Cu2+/MCH/DNA1/2/Au electrode was washed there times with purified water. Finally, 10 μL of electrochemical signal probe (Fc-DNA3 and Fc-DNA4) solution with a concentration of 10 μM were dropped to this Cu2+/MCH/DNA1/2/Au electrode for 2 h at RT. DNA3/4/Cu2+/ MCH/DNA1/2/Au electrode was formed by this way. After washed three times with PBS (pH = 7.4), electrochemical impedance spectroscopy (EIS) and electrochemical signal of ferrocene tag on modified gold electrode was tested through differential pulse voltammetry (DPV) by CHI660B electrochemical work station.
2. Experimental 2.1. Reagents L-Cysteine, copper (II) sulfate (CuSO4) and L/D-carnitine were obtained from Aladin (Shanghai China). 6-Mercapto-1-hexanol (MCH) was obtained from Kaivo (Zhuhai, China). Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was purchased from Sigma-Aldrich. All other reagents were of analytical grade and used without purification. All aqueous solutions were prepared using ultrapure water from a MilliQ system (Millipore, USA). In this study, the DNA sequences were synthesized and purified by Takara Bio Inc. (Dalian). The sequences were as follows:
DNA
Sequence
DNA1
5′-GTCGACAATTAATCTCTTCTTTCTAATACGGCTTACC TAGCATCTCTACAAGTGTCGACTTTTTT-(SH)-3′ 5′-GGTAAGCCTGGGCCTCTTTCTTTTTAAGAAAGAAC-3′ 5′-(Ferrocene)-TTTGTCGACACTTGTAGAGATGCTAGCATAA GACTTCTAGCA TCTCTACAAG-3′ 5′-(Ferrocene)-TTTCTTCTAGCATCTCTACAAGTGTCGACGTAG AGATGCTAGAAGTCTTATG-3′
DNA2 DNA3 DNA4
2.6. Electrochemical detection All electrochemical measurements were carried out with a CHI660B electrochemical work station (Shanghai, CHI instruments Inc., China). A conventional three-electrode system was applied with the Au electrode, a platinum wire and an Ag/AgCl electrode as the working electrode, the auxiliary electrode and the reference electrode respectively. Electrochemical impedance spectroscopy (EIS) experiments were tested in a 5 mM of K4Fe(CN)6/K3Fe(CN)6 (1:1) mixture containing KCl (0.1 M) with the frequency range from 0.1 Hz to 100 kHz. Cyclic voltammetry (CV) experiments were performed in 10 mM PBS solution before and after immersion in copper ions solution and target carnitine solution. The scan rate in our experiment is 0.1 V/s and curves scanned from −0.1 V to 0.6 V. The differential pulse voltammetry (DPV) experiments were measured in 10 mM PBS (pH 7.4) and curves scanned from 0 to 0.6 V with pulse amplitude 50 mV, pulse period 0.2 s.
2.2. Pretreatment of the gold electrode The gold electrode (2 mm diameter) was firstly polished with 0.05 mm Al2O3 powder slurry on a microcloth pad to obtain a mirror surface. Then, it was washed with purified water and ethanol for 10 min respectively. After that, the electrode was refreshed in 1 M H2SO4 solution from −0.2 V to +1.5 V versus Ag/AgCl for 30 cycles at a scan rate of 100 mV s−1 to clear away adsorbents. Finally, these electrodes were slightly washed with purified water and dried with nitrogen.
3. Results and discussion 3.1. Principle of electrochemical detection of L/D-carnitine with signal amplification strategies The principle of the strategy is shown in Fig. 1 with two parts. Part (A) is the formation of Cu (II)-L-cysteine complexes on the electrode I and the following Cu2+ replacement in the presence of L-carnitine or Dcarnitine. Part (B) is the formation and cleavage reaction of DNAzyme and further HCR process occurred on the surface of electrode II, generating significantly amplified electrochemical signal, which relies on the amount of replaced Cu2+ from part (A). Due to different combining capacity between homochiral interaction (L-carnitine to L-cysteine) and heterochiral interaction (D-carnitine to L-cysteine), different amount of Cu2+ was replaced from the Cu (II)-L-cysteine complexes in the presence of L-carnitine and D-carnitine. The binding of Cu2+ in DNAzyme structure on the electrode will activate DNAzyme, thus results in cleavage of the substrate strand at specific site and many DNA fragments on
2.3. Preparation of MCH/DNA1/2/Au electrode 20 μL of the template probe DNA1 and assistant probe DNA2 (10 μM) with the molar concentration ratio of 2:1 were added to a solution of 10 mM PBS buffer (0.5 M NaCl, 1 mM MgCl2, pH = 7.4). Then, the mixture was heated at 95 °C for 5 min, then allowed to cool to room temperature (RT) for at least 60 min before use. There with, 1.0 mM TCEP was added in above probes mixture for 2 h to open the disulfide bond. Afterwards, 10 μL of the above mixture were loaded onto treated bare gold electrodes overnight at room temperature (RT). Then, 1 mM freshly MCH was dropped onto above gold electrodes for 138
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Fig. 1. Schematic illustration of selective detection of L/D carnitine assisted with DNAzyme and DNA based hybridization chain reaction.
the electrode. And the amount of Cu2+ could influence the amount of DNA fragments cleaved by Cu2+-activated DNAzyme on the electrode. During further HCR process, two partially complementary ssDNA are employed with a long-stem and short-loop hairpin structure (DNA3 and DNA4), both of which are labeled with ferrocene as electrochemical tag. The mixture of two DNA hairpins is stable and concomitant at room temperature in the solution. While facing at the remaining ssDNA (production of DNAzyme cleavage reaction) served as an initiator, the hairpin structure of the employed two DNA hairpins tend to open and thus trigger an alternating hybridization chain reaction to form a long nicked dsDNA. The formed nicked dsDNA thus labeled a large amount of ferrocene, obtaining significantly amplified electrochemical detection signal for L/D-carnitine. In order to find how carnitine enantiomers influence the electrochemical results, differential pulse voltammetry (DPV) was then employed to test the electrical responses from ferrocene tag under different experiment conditions. First, different culture time of carnitine enantiomers with previously formed Cu (II)-L-cysteine complexes on the electrode I were studied and the results were showed in Fig. 2. An obvious DPV peak current was found after 5 min treatment of L/D-carnitine (300 nM in PBS, pH = 5.5) which was due to the Fc tag on the formed nicked dsDNA (HCR products). This peak current gradually increased along with the increase of culture time of L-carnitine or Dcarnitine (Fig. 2a and b, separately) and tend to be stable after 25 min. It suggested that Cu2+ has been successfully replaced by carnitine and be involved in DNAzyme cleavage, thus generating long dsDNA with a large amount of Fc on the gold electrode surface. As reported in previous studies [8,9], homochiral interaction (L-cysteine/L-carnitine a two-point contact model) demonstrated weaker interaction compared with that from heterochiral interaction (L-cysteine/D-carnitine with a three-point contact model), thus affects copper ion replacement rate and the following different DPV peak current changes. Our results (Fig. 2c) were consistent with the previous report and further found that D-carnitine treatment showed more energetically favorable interaction and thus larger DPV current obtained from long HCR products within 35 min in comparison with that of L-carnitine treatment. To confirm the above obvious current changes were exact from
carnitine as well as to evaluate the sensitivity of this sensing method, different concentrations of L-carnitine and D-carnitine (from 1 nM to 500 nM) were employed in the following experiment, respectively. From Fig. 2d–e, we could find that the current value from Fc tag gradually increased with the increased concentration of L-carnitine or Dcarnitine. It means more copper ions in Cu (II)-L-cysteine complexes could be replaced at higher carnitine concentration. Moreover, as showed in Fig. 2f, the current response of electrode with the treatment of solution after the Cu2+ replacement reaction in the presence of Dcarnitine is larger than that in the presence of L-carnitine at different concentrations. This can be well assigned that less copper ions were replaced in the presence of L-carnitine compared with that of its D-form due to the weaker interactions between L-carnitine and L-cysteine, which is consistent with former data of DPV performances of Fig. 2a–c. Cyclic voltammogram signals from Cu2+ were further employed to verify the replacement of Cu2+ on the electrode. As shown in Fig. 3a, Lcysteine modified electrode without Cu2+ did not show obvious cyclic voltammogram signals in PBS solution, while after immersion in copper ions solution, the electrode demonstrated significant electrochemical signal which is from the oxidation and reduction process of copper ions. After further treatment of L/D-carnitine solution, the current peak current from copper ions significantly decreased, indicating that the Cu2+ on the electrode were replaced by carnitine in the solutions. Meanwhile, the lower current found from the electrode treated with D-carnitine compared with that of L-carnitine suggested the favorite binding trend between L-cysteine and D-carnitine. Electrochemical impedance spectroscopy (EIS) was employed to monitor the step-by-step modification and assembly of molecules on the surface of electrode I and electrode II during the preparation of the sensing interface (Fig. 3b,c). The largely increased Ret during the preparation and sensing process on electrode I indicated the successful construction of sensing interfaces and capture of target carnitine molecules. From Fig. 3c, it can be observed that the introduction of hairpin DNA1 and DNAzyme strand DNA2 and following assembly of MCH on electrode surface largely increased Ret due to the repelled diffusion of [Fe(CN)63−/4−] toward the electrode surface by negatively charged DNA molecules and MCH molecules. However, the further treatment of Cu2+ solution led to a 139
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Fig. 2. DPV for the modified electrode GE-II in 10 mM PBS (pH 7.4) with treatment of the solution after the reaction between L-carnitine (a,d) or D-carnitine (b,e) and Cu (II)-L-cysteine complexes modified electrode I; (c) and (f) are the relationship between the DPV peak current and the reaction time (or concentration) of L- or Dcarnitine reaction. Error bars are the standard deviation of three repetitive experiments.
current. The ratio of replaced Cu2+ by L/D-carnitine was estimated from the ratio of DPV current intensity (R = A − A0/Amax − A0). A is the intensity of the DPV current after treatment of carnitine induced Cu2+ replacement solution (or without carnitine A0). Current intensity from reaction in the presence of 300 nM D-carnitine for 35 min is employed as Amax. Higher Cu2+ replacement rate of D-carnitine under the same conditions indicated higher binding opportunities between L-cysteine and D-carnitine. This can be interpreted from Fig. 3e that the interaction between L-cysteine and D-carnitine is relatively stronger with threepoint binding (NH3+/COO−, NH3+/OH and COO−/N(CH3)3+) compared with that of L-cysteine-L-carnitine with two-point interaction (NH3+/COO− and NH3+/OH). And the difference of Cu2+ replacement
decreased Ret. This could be attributed to the activated DNAzyme in the presence of Cu2+, which cleaved DNA1 into two parts and destroyed the hybrid structures of DNA1/DNA2, resulting in a short DNA fragment remained on the electrode surface. However, under conditions of two Fc labeled hairpin DNA3 and DNA4, a long nicked dsDNA formed via an alternating HCR, resulting in a further significantly increased Ret. And when using L-carnitine or D-carnitine, we could find a different Ret which may be assigned to the different amount of replaced Cu2+ and thus different amount of HCR products on the electrode (Fig. 3d). The comparison of Cu2+ replacement rate by L/D-carnitine was shown in Fig. 3f. The replaced copper ions activated DNAzyme and triggered the following HCR reaction, generating the significant DPV 140
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Fig. 3. (a) Cyclic voltammograms in PBS solution of a) GE-I/L-cysteine, b) GE-I/L-cysteine/Cu2+, c) GE-I/L-cysteine/Cu2+/L-carnitine and d) GE-I/L-cysteine/Cu2+/Dcarnitine modified electrodes; (b) Nyquist plots obtained from bare GE-I, GE-I/L-cysteine, GE-I/L-cysteine/Cu2+, GE-I/L-cysteine/Cu2+/L-(or D) carnitine modified electrodes; (c) Nyquist plots obtained from a) bare gold electrode GE-II, b) GE-II/DNA1/DNA2, c) GE-II/DNA1/DNA2/MCH, d) GE-II/DNA1/DNA2/MCH/Cu2+ (replaced by D-carnitine) and e) GE-II/DNA1/DNA2/MCH/Cu2+ (replaced by D-carnitine)/HCR(DNA3/DNA4 modified electrodes in 5 mM [Fe(CN)63−/4−] and 0.1 M KCl solution; (d) Nyquist plots obtained from a) GE-II/DNA1/DNA2/MCH, b) GE-II/DNA1/DNA2/MCH/Cu2+ (replaced by L-carnitine)-/HCR(DNA3/DNA4) and c) GE-II/DNA1/DNA2/MCH/Cu2+ (replaced by D-carnitine)-/HCR(DNA3/DNA4) modified; (e) binding structures of L-cysteine/L-(or D) carnitine; (f) estimated Cu2+ replacement ratio in the presence of L-(or D) carnitine.
rate of L/D-carnitine has been transferred to amplified measureable DPV current response of formed DNA products with a large amount of Fc tags benefit from DNAzyme and HCR amplification strategies.
[5]
4. Conclusions [6]
In summary, we have designed a novel electrochemical strategy to recognize carnitine enantiomers employing Cu (II)-L-cysteine complexes and signal amplification strategies based on DNAzyme and DNA assistant HCR reaction. The proposed method has several combined advantages. First, Cu2+-dependent DNAzyme was employed to monitor the information of carnitine enantiomers with high recognition capability, low cost and recycling ability without losing activity. Moreover, DNA assistant HCR amplification strategy further amplified the signal response. We expect that this study may offer a new direction in design of high performance sensing methods for chiral molecules.
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Acknowledgements
[10]
This work was supported by the National Natural Science Foundation of China (Grant Nos.: 21675074, 21675075), the “Innovation Team Development Plan” of the Ministry of Education Rolling Support (IRT_15R31) and the Taishan Scholar Project of Shandong Province.
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Short communication
Electrochemical selective detection of carnitine enantiomers coupling copper ion dependent DNAzyme with DNA assistant hybridization chain reaction
T
Fenfen Zhaia,b, Qiao Yub,d, Hong Zhoub, , Jing Liub,c, , Wenrong Yangb,c, Jinmao Youa, ⁎
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a
Shandong Province Key Laboratory of Life-Organic Analysis, College of Chemistry and Chemical Engineering, Qufu Normal University, Qufu 273165, PR China Shandong Province Key Laboratory of Detection Technology for Tumor Markers, College of Chemistry and Chemical Engineering, Linyi University, Linyi 276005, PR China c Centre for Chemistry and Biotechnology, School of Life and Environmental Sciences, Deakin University, Geelong, Victoria 3216, Australia d Shandong Sino-Japanese Center for Collaborative Research of Carbon Nanomaterials, College of Chemistry and Chemical Engineering, Qingdao University, Qingdao, Shandong 266071, PR China b
ARTICLE INFO
ABSTRACT
Keywords: Electrochemical detection DNAzyme Hybridization chain reaction Chiral molecules recognition Signal amplification
An electrochemical method for carnitine enantiomers recognition is proposed employing Cu (II)-L-cysteine complexes and signal amplification strategies based on DNAzyme and DNA assistant hybridization chain reaction (HCR). The reported method combined Cu (II)-amino acids complexes with DNAzyme and proposed a smart and efficient electrochemical method to recognize chiral molecules through the difference between homochiral interaction and heterochiral interaction. Moreover, further employment of hairpin DNA assistant HCR greatly enhanced the detecting signal, realizing the selective recognition of carnitine enantiomers in low concentrations. This study creatively provides a general sensing method for sensitive chiral molecules recognition without largescale precision instruments.
1. Introduction Due to several drawbacks of current detecting method for chiral molecules, including the requirement of high-cost and large-scale instruments, additional laser radiation and unsatisfied sensitivity and selectivity [1–4], it is still urgent to explore a simple, rapid, and signalamplified sensing method for selective analysis of chiral molecules enantiomers. Recently, Cu (II) complexes formed from interactions between Cu (II) ions and enantiomeric amino acid have been studied and applied for chiral-sensing systems [5–7]. Recent work from Yang's group employed Cu (II)-amino acids complexes to find out molecular recognition mechanism between amino acid L (levorotary)-cysteine and L/D(dextrorotary)-carnitine using Surface-enhanced Raman spectrum (SERS) technology and then in their following studies proposed an electrochemical method to recognize carnitine enantiomers [8,9]. Carnitine, with two enantioselective L/D configurations due to the asymmetric secondary carbon, is a safe food supplement in its L-form and could support the amino acid development in human body. While, D-carnitine demonstrates some unwanted side effects and thus is limited
in pharmaceutical and nutritional formulations. Therefore, recognition of chiral carnitine is very important in biological and medicinal studies. However, more improvement in sensitivity and selectivity of the sensing method for carnitine enantiomers is urgently needed in order to realize highly enantioselective recognition of carnitine in low concentrations. DNAzymes, based on DNA molecules with a catalytic activity have attracted much attention with good stability, such as Hemin/G-quadruplex DNAzymes [10,11] and metal ions dependent DNAzyme [12]. Metal ions dependent DNAzyme are DNA molecules generally containing a substrate strand containing a specific cleavage site by specific metal ions and an enzyme strand. Thus in the presence of metal ions, such as Cu2+ [13,14], Pb2+ [15,16], Mg2+ [17], Zn2+ [18,19] or Ca2+ [20], as catalytic cofactors, the substrate strand will be cleaved into two parts. Benefit from this high recognition function and catalytic activity, DNAzyme showed many advantages of easy operation, low cost, and ability to be renatured many times without losing activity, DNAzyme showed significantly improved selectivity and simplicity in the construction of functional molecular probes for the detection of various
Corresponding authors. Correspondence to: J. Liu, Centre for Chemistry and Biotechnology, School of Life and Environmental Sciences, Deakin University, Geelong, Victoria 3216, Australia. E-mail addresses: [email protected] (H. Zhou), [email protected] (J. Liu), [email protected] (J. You). ⁎
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https://doi.org/10.1016/j.jelechem.2019.02.020 Received 11 January 2019; Received in revised form 12 February 2019; Accepted 13 February 2019 Available online 14 February 2019 1572-6657/ © 2019 Elsevier B.V. All rights reserved.
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analytes [21–25]. Besides DNAzyme, DNA assistant isothermal amplification strategies have been widely employed in recent biosensing platforms, providing efficiently amplified detecting signal, excellent stability, sensitivity, and designability [26–31]. For example, hybridization chain reaction (HCR) has been widely studied and developed for amplification strategy due to no needed additional DNA tool enzyme [32–38]. Here, we combined Cu2+-dependent DNAzyme, Cu (II)-amino acids complexes and proposed a smart and efficient electrochemical method to recognize chiral molecules through the difference between homochiral interaction and heterochiral interaction and different amount of replaced Cu2+ which can be involved in DNAzyme cleavage and then resulting in a measurable electrochemical signal. Benefit from the excellent stability, recycling abilities from DNAzyme and nucleic acidassistant HCR amplification technology, greatly enhanced signals were obtained for selective recognition of L/D carnitine molecules in low concentrations. Moreover, using simple electrochemical method, this study may provide a novel sensing method for efficient chiral molecules recognition in low concentration without the corresponding large-scale precision instruments.
2 h. In this way, the MCH/probe modified electrodes were formed. Following this, electrodes were washed with PBS buffer (pH = 7.4) and dried for future experiments. 2.4. Preparation of Cu2+-L-cysteine self-assembled Au electrode The bare gold electrode was immersed in 0.5 M of the L-cysteine for 2 h at RT in order to form a self-assembled cysteine monolayer on the electrode surface. After that, the L-cysteine modified electrode was dipped in 0.5 M CuSO4 (pH = 5.5) for 30 min at RT. Cu2+-L-cysteine self-assembled Au electrode was formed by this way. Then, they were washed with 0.01 M PBS (pH = 5.5) and dried for future experiment. 2.5. The recycling amplification detection electrochemical ferrocene signal of DNA3/4/Cu2+/MCH/DNA1/2/Au electrode Cu2+-L-cysteine self-assembled Au electrode was dipped in centrifugal tube with 300 nM D/L-Carnitine (pH = 5.5) for 25 min at RT. After that, Cu2+-L-cysteine self-assembled Au electrode was taken out and MCH/DNA1/2/Au electrode was immersed in the above centrifugal tube for 1 h at RT to form Cu2+/MCH/DNA1/2/Au electrode. Following this Cu2+/MCH/DNA1/2/Au electrode was washed there times with purified water. Finally, 10 μL of electrochemical signal probe (Fc-DNA3 and Fc-DNA4) solution with a concentration of 10 μM were dropped to this Cu2+/MCH/DNA1/2/Au electrode for 2 h at RT. DNA3/4/Cu2+/ MCH/DNA1/2/Au electrode was formed by this way. After washed three times with PBS (pH = 7.4), electrochemical impedance spectroscopy (EIS) and electrochemical signal of ferrocene tag on modified gold electrode was tested through differential pulse voltammetry (DPV) by CHI660B electrochemical work station.
2. Experimental 2.1. Reagents L-Cysteine, copper (II) sulfate (CuSO4) and L/D-carnitine were obtained from Aladin (Shanghai China). 6-Mercapto-1-hexanol (MCH) was obtained from Kaivo (Zhuhai, China). Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was purchased from Sigma-Aldrich. All other reagents were of analytical grade and used without purification. All aqueous solutions were prepared using ultrapure water from a MilliQ system (Millipore, USA). In this study, the DNA sequences were synthesized and purified by Takara Bio Inc. (Dalian). The sequences were as follows:
DNA
Sequence
DNA1
5′-GTCGACAATTAATCTCTTCTTTCTAATACGGCTTACC TAGCATCTCTACAAGTGTCGACTTTTTT-(SH)-3′ 5′-GGTAAGCCTGGGCCTCTTTCTTTTTAAGAAAGAAC-3′ 5′-(Ferrocene)-TTTGTCGACACTTGTAGAGATGCTAGCATAA GACTTCTAGCA TCTCTACAAG-3′ 5′-(Ferrocene)-TTTCTTCTAGCATCTCTACAAGTGTCGACGTAG AGATGCTAGAAGTCTTATG-3′
DNA2 DNA3 DNA4
2.6. Electrochemical detection All electrochemical measurements were carried out with a CHI660B electrochemical work station (Shanghai, CHI instruments Inc., China). A conventional three-electrode system was applied with the Au electrode, a platinum wire and an Ag/AgCl electrode as the working electrode, the auxiliary electrode and the reference electrode respectively. Electrochemical impedance spectroscopy (EIS) experiments were tested in a 5 mM of K4Fe(CN)6/K3Fe(CN)6 (1:1) mixture containing KCl (0.1 M) with the frequency range from 0.1 Hz to 100 kHz. Cyclic voltammetry (CV) experiments were performed in 10 mM PBS solution before and after immersion in copper ions solution and target carnitine solution. The scan rate in our experiment is 0.1 V/s and curves scanned from −0.1 V to 0.6 V. The differential pulse voltammetry (DPV) experiments were measured in 10 mM PBS (pH 7.4) and curves scanned from 0 to 0.6 V with pulse amplitude 50 mV, pulse period 0.2 s.
2.2. Pretreatment of the gold electrode The gold electrode (2 mm diameter) was firstly polished with 0.05 mm Al2O3 powder slurry on a microcloth pad to obtain a mirror surface. Then, it was washed with purified water and ethanol for 10 min respectively. After that, the electrode was refreshed in 1 M H2SO4 solution from −0.2 V to +1.5 V versus Ag/AgCl for 30 cycles at a scan rate of 100 mV s−1 to clear away adsorbents. Finally, these electrodes were slightly washed with purified water and dried with nitrogen.
3. Results and discussion 3.1. Principle of electrochemical detection of L/D-carnitine with signal amplification strategies The principle of the strategy is shown in Fig. 1 with two parts. Part (A) is the formation of Cu (II)-L-cysteine complexes on the electrode I and the following Cu2+ replacement in the presence of L-carnitine or Dcarnitine. Part (B) is the formation and cleavage reaction of DNAzyme and further HCR process occurred on the surface of electrode II, generating significantly amplified electrochemical signal, which relies on the amount of replaced Cu2+ from part (A). Due to different combining capacity between homochiral interaction (L-carnitine to L-cysteine) and heterochiral interaction (D-carnitine to L-cysteine), different amount of Cu2+ was replaced from the Cu (II)-L-cysteine complexes in the presence of L-carnitine and D-carnitine. The binding of Cu2+ in DNAzyme structure on the electrode will activate DNAzyme, thus results in cleavage of the substrate strand at specific site and many DNA fragments on
2.3. Preparation of MCH/DNA1/2/Au electrode 20 μL of the template probe DNA1 and assistant probe DNA2 (10 μM) with the molar concentration ratio of 2:1 were added to a solution of 10 mM PBS buffer (0.5 M NaCl, 1 mM MgCl2, pH = 7.4). Then, the mixture was heated at 95 °C for 5 min, then allowed to cool to room temperature (RT) for at least 60 min before use. There with, 1.0 mM TCEP was added in above probes mixture for 2 h to open the disulfide bond. Afterwards, 10 μL of the above mixture were loaded onto treated bare gold electrodes overnight at room temperature (RT). Then, 1 mM freshly MCH was dropped onto above gold electrodes for 138
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Fig. 1. Schematic illustration of selective detection of L/D carnitine assisted with DNAzyme and DNA based hybridization chain reaction.
the electrode. And the amount of Cu2+ could influence the amount of DNA fragments cleaved by Cu2+-activated DNAzyme on the electrode. During further HCR process, two partially complementary ssDNA are employed with a long-stem and short-loop hairpin structure (DNA3 and DNA4), both of which are labeled with ferrocene as electrochemical tag. The mixture of two DNA hairpins is stable and concomitant at room temperature in the solution. While facing at the remaining ssDNA (production of DNAzyme cleavage reaction) served as an initiator, the hairpin structure of the employed two DNA hairpins tend to open and thus trigger an alternating hybridization chain reaction to form a long nicked dsDNA. The formed nicked dsDNA thus labeled a large amount of ferrocene, obtaining significantly amplified electrochemical detection signal for L/D-carnitine. In order to find how carnitine enantiomers influence the electrochemical results, differential pulse voltammetry (DPV) was then employed to test the electrical responses from ferrocene tag under different experiment conditions. First, different culture time of carnitine enantiomers with previously formed Cu (II)-L-cysteine complexes on the electrode I were studied and the results were showed in Fig. 2. An obvious DPV peak current was found after 5 min treatment of L/D-carnitine (300 nM in PBS, pH = 5.5) which was due to the Fc tag on the formed nicked dsDNA (HCR products). This peak current gradually increased along with the increase of culture time of L-carnitine or Dcarnitine (Fig. 2a and b, separately) and tend to be stable after 25 min. It suggested that Cu2+ has been successfully replaced by carnitine and be involved in DNAzyme cleavage, thus generating long dsDNA with a large amount of Fc on the gold electrode surface. As reported in previous studies [8,9], homochiral interaction (L-cysteine/L-carnitine a two-point contact model) demonstrated weaker interaction compared with that from heterochiral interaction (L-cysteine/D-carnitine with a three-point contact model), thus affects copper ion replacement rate and the following different DPV peak current changes. Our results (Fig. 2c) were consistent with the previous report and further found that D-carnitine treatment showed more energetically favorable interaction and thus larger DPV current obtained from long HCR products within 35 min in comparison with that of L-carnitine treatment. To confirm the above obvious current changes were exact from
carnitine as well as to evaluate the sensitivity of this sensing method, different concentrations of L-carnitine and D-carnitine (from 1 nM to 500 nM) were employed in the following experiment, respectively. From Fig. 2d–e, we could find that the current value from Fc tag gradually increased with the increased concentration of L-carnitine or Dcarnitine. It means more copper ions in Cu (II)-L-cysteine complexes could be replaced at higher carnitine concentration. Moreover, as showed in Fig. 2f, the current response of electrode with the treatment of solution after the Cu2+ replacement reaction in the presence of Dcarnitine is larger than that in the presence of L-carnitine at different concentrations. This can be well assigned that less copper ions were replaced in the presence of L-carnitine compared with that of its D-form due to the weaker interactions between L-carnitine and L-cysteine, which is consistent with former data of DPV performances of Fig. 2a–c. Cyclic voltammogram signals from Cu2+ were further employed to verify the replacement of Cu2+ on the electrode. As shown in Fig. 3a, Lcysteine modified electrode without Cu2+ did not show obvious cyclic voltammogram signals in PBS solution, while after immersion in copper ions solution, the electrode demonstrated significant electrochemical signal which is from the oxidation and reduction process of copper ions. After further treatment of L/D-carnitine solution, the current peak current from copper ions significantly decreased, indicating that the Cu2+ on the electrode were replaced by carnitine in the solutions. Meanwhile, the lower current found from the electrode treated with D-carnitine compared with that of L-carnitine suggested the favorite binding trend between L-cysteine and D-carnitine. Electrochemical impedance spectroscopy (EIS) was employed to monitor the step-by-step modification and assembly of molecules on the surface of electrode I and electrode II during the preparation of the sensing interface (Fig. 3b,c). The largely increased Ret during the preparation and sensing process on electrode I indicated the successful construction of sensing interfaces and capture of target carnitine molecules. From Fig. 3c, it can be observed that the introduction of hairpin DNA1 and DNAzyme strand DNA2 and following assembly of MCH on electrode surface largely increased Ret due to the repelled diffusion of [Fe(CN)63−/4−] toward the electrode surface by negatively charged DNA molecules and MCH molecules. However, the further treatment of Cu2+ solution led to a 139
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Fig. 2. DPV for the modified electrode GE-II in 10 mM PBS (pH 7.4) with treatment of the solution after the reaction between L-carnitine (a,d) or D-carnitine (b,e) and Cu (II)-L-cysteine complexes modified electrode I; (c) and (f) are the relationship between the DPV peak current and the reaction time (or concentration) of L- or Dcarnitine reaction. Error bars are the standard deviation of three repetitive experiments.
current. The ratio of replaced Cu2+ by L/D-carnitine was estimated from the ratio of DPV current intensity (R = A − A0/Amax − A0). A is the intensity of the DPV current after treatment of carnitine induced Cu2+ replacement solution (or without carnitine A0). Current intensity from reaction in the presence of 300 nM D-carnitine for 35 min is employed as Amax. Higher Cu2+ replacement rate of D-carnitine under the same conditions indicated higher binding opportunities between L-cysteine and D-carnitine. This can be interpreted from Fig. 3e that the interaction between L-cysteine and D-carnitine is relatively stronger with threepoint binding (NH3+/COO−, NH3+/OH and COO−/N(CH3)3+) compared with that of L-cysteine-L-carnitine with two-point interaction (NH3+/COO− and NH3+/OH). And the difference of Cu2+ replacement
decreased Ret. This could be attributed to the activated DNAzyme in the presence of Cu2+, which cleaved DNA1 into two parts and destroyed the hybrid structures of DNA1/DNA2, resulting in a short DNA fragment remained on the electrode surface. However, under conditions of two Fc labeled hairpin DNA3 and DNA4, a long nicked dsDNA formed via an alternating HCR, resulting in a further significantly increased Ret. And when using L-carnitine or D-carnitine, we could find a different Ret which may be assigned to the different amount of replaced Cu2+ and thus different amount of HCR products on the electrode (Fig. 3d). The comparison of Cu2+ replacement rate by L/D-carnitine was shown in Fig. 3f. The replaced copper ions activated DNAzyme and triggered the following HCR reaction, generating the significant DPV 140
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Fig. 3. (a) Cyclic voltammograms in PBS solution of a) GE-I/L-cysteine, b) GE-I/L-cysteine/Cu2+, c) GE-I/L-cysteine/Cu2+/L-carnitine and d) GE-I/L-cysteine/Cu2+/Dcarnitine modified electrodes; (b) Nyquist plots obtained from bare GE-I, GE-I/L-cysteine, GE-I/L-cysteine/Cu2+, GE-I/L-cysteine/Cu2+/L-(or D) carnitine modified electrodes; (c) Nyquist plots obtained from a) bare gold electrode GE-II, b) GE-II/DNA1/DNA2, c) GE-II/DNA1/DNA2/MCH, d) GE-II/DNA1/DNA2/MCH/Cu2+ (replaced by D-carnitine) and e) GE-II/DNA1/DNA2/MCH/Cu2+ (replaced by D-carnitine)/HCR(DNA3/DNA4 modified electrodes in 5 mM [Fe(CN)63−/4−] and 0.1 M KCl solution; (d) Nyquist plots obtained from a) GE-II/DNA1/DNA2/MCH, b) GE-II/DNA1/DNA2/MCH/Cu2+ (replaced by L-carnitine)-/HCR(DNA3/DNA4) and c) GE-II/DNA1/DNA2/MCH/Cu2+ (replaced by D-carnitine)-/HCR(DNA3/DNA4) modified; (e) binding structures of L-cysteine/L-(or D) carnitine; (f) estimated Cu2+ replacement ratio in the presence of L-(or D) carnitine.
rate of L/D-carnitine has been transferred to amplified measureable DPV current response of formed DNA products with a large amount of Fc tags benefit from DNAzyme and HCR amplification strategies.
[5]
4. Conclusions [6]
In summary, we have designed a novel electrochemical strategy to recognize carnitine enantiomers employing Cu (II)-L-cysteine complexes and signal amplification strategies based on DNAzyme and DNA assistant HCR reaction. The proposed method has several combined advantages. First, Cu2+-dependent DNAzyme was employed to monitor the information of carnitine enantiomers with high recognition capability, low cost and recycling ability without losing activity. Moreover, DNA assistant HCR amplification strategy further amplified the signal response. We expect that this study may offer a new direction in design of high performance sensing methods for chiral molecules.
[7]
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Acknowledgements
[10]
This work was supported by the National Natural Science Foundation of China (Grant Nos.: 21675074, 21675075), the “Innovation Team Development Plan” of the Ministry of Education Rolling Support (IRT_15R31) and the Taishan Scholar Project of Shandong Province.
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