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Electrochemical selective detection of carnitine enantiomers coupling copper ion dependent DNAzyme with DNA assistant hybridization chain reaction

https://doi.org/10.1016/j.jelechem.2019.02.020Get rights and content

Abstract

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 large-scale precision instruments.

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], [2], [3], [4]], it is still urgent to explore a simple, rapid, and signal-amplified 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], [6], [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 analytes [[21], [22], [23], [24], [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], [27], [28], [29], [30], [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], [33], [34], [35], [36], [37], [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 acid-assistant 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.

Section snippets

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 Milli-Q system (Millipore, USA). In this study, the DNA sequences were synthesized and purified by

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 d-carnitine. 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

Conclusions

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

Acknowledgements

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.

References (38)

  • R. Zeng et al.

    Ultrasensitive and label-free electrochemical aptasensor of kanamycin coupling with hybridization chain reaction and strand-displacement amplification

    Anal. Chim. Acta

    (2018)
  • G.A. Hembury et al.

    Chirality-sensing supramolecular systems

    Chem. Rev.

    (2008)
  • Y. Wang et al.

    Enantioselective discrimination of alcohols by hydrogen bonding: a SERS study

    Angew. Chem. Int. Ed.

    (2014)
  • P. Li et al.

    A homochiral microporous hydrogen-bonded organic framework for highly enantioselective separation of secondary alcohols

    J. Am. Chem. Soc.

    (2014)
  • S. Nieto et al.

    31P NMR spectroscopy and pattern-recognition techniques as tools for the identification and enantiodiscrimination of α-amino acids

    New J. Chem.

    (2012)
  • Y. Zhang et al.

    Self-assembled core–satellite gold nanoparticle networks for ultrasensitive detection of chiral molecules by recognition tunneling current

    ACS Nano

    (2016)
  • J. Liu et al.

    DNAzyme based nanomachine for in situ detection of microRNA in living cells

    ACS Sens.

    (2017)
  • N. Carmi et al.

    Cleaving DNA with DNA

    Proc. Natl. Acad. Sci. U. S. A.

    (1998)
  • P. Zhang et al.

    An “off–on” electrochemiluminescent biosensor based on DNAzyme-assisted target recycling and rolling circle amplifications for ultrasensitive detection of microRNA

    Anal. Chem.

    (2015)
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