Immobilized-free miniaturized electrochemical sensing system for Pb2+ detection based on dual Pb2+-DNAzyme assistant feedback amplification strategy
Introduction
Lead ion (Pb2+), which is one of the most toxic and wildly distributed heavy metal ions in the environment, has caused significant concerns of the scientific community (Sun et al., 2018, Cai et al., 2017, Rudd et al., 2016). In the past years, the detection of Pb2+ was mainly dependent on instrument analysis such as mass spectrometry, spectrometry and inductive coupled plasma (ICP), but the inherent shortcomings (e.g., complicated pretreatment, bulky apparatus and expensive testing costs) limited their application to the laboratory (Zhou et al., 2016, Li et al., 2017). Therefore, the development of high sensitive and low-cost technologies for routine monitoring of Pb2+ hold great significance (Liang et al., 2017, Zang et al., 2014). Electrochemical methods with distinct virtues of low-cost, simple instrumentation and excellent analytical performance, have achieved great progress in the past few years (Yu et al., 2018, Shin et al., 2017). Nevertheless, most of current existing electrochemical biosensors still suffer from redundant immobilization procedures, tedious clean steps and non-renewable electrode surface (Shin et al., 2017, Hou et al., 2015, Tan et al., 2016). To overcome these disadvantages, researchers focus their attention on the development of immobilized-free and reusable miniaturization electrochemical biosensors for applying to complex biological and environmental samples.
With the breakthrough of microfabrication and printing technologies, miniaturized electrochemical biosensors have became increasingly popular in the area of point-of-care testing (Sempionatto et al., 2017, Hrdy et al., 2017, Hsieh et al., 2012), environmental protection (Huang et al., 2017, Chalupniak and Merkoci, 2017, Zhao et al., 2017), as well as biological analysis (Liu et al., 2017a, Zhang et al., 2017a, Zhang et al., 2017b, Xu et al., 2018). Among them, carbon fiber microelectrode (CFME) attracts considerable interests in biomedical detection and environmental monitoring due to its virtue of inexpensive, biocompatibility, high mechanical strength and easy miniaturization. Although CFME exhibits excellent analytical performance and obvious advantages than typical bulk electrode, the small size also poses great difficulty for its manufacturing. Ohsaka's group reported a third-generation biosensor for superoxide anion detection based on a CFME (Tian et al., 2005). In this method, the CFME is sealed by epoxy resin at 100 °C for 2 h, the harsh seal conditions inevitably lead to poor sealing and increase the difficulty in the fabrication process. Subsequently, Cheng's group have developed a facile method to fabricate a low-noise CFME by using flame-fuse seal procedure to replace the traditional epoxy sealing, achieving effective insulation without any pollution (Huang et al., 2001). However, it is difficult to guarantee the carbon fiber survival from the flame of gas lamp in sealing process. Recently, Huang's group has provided a facile way to solve this problem by using wax to seal the SiC nanowire in glass pipette (Zhang et al., 2017). While the good electrical contact of the SiC nanowire is achieved by filling liquid metal in glass pipette which inevitably cause unnecessary heavy metal pollution. In this context, we constructed a facile, high production and environmentally friendly CFME that covered the aforementioned drawbacks and successfully demonstrated its application to Pb2+ detection in river water samples.
At present, Pb2+-dependent DNAzyme-based methodologies have gradually emerged as dominant sensing strategies for Pb2+ detection because of their versatility and high catalytic activity and good compatibility (Li et al., 2010, Shi et al., 2016). These fascinating virtues make them easily integrate with various kinds of DNA amplification techniques, such as polymerase chain reaction (PCR) (Wang et al., 2010, Zhu et al., 2015), hybridization chain reaction (HCR) (Zhuang et al., 2013) and rolling circle amplification (RCA) (Kim and Lee, 2016, Tang et al., 2013). As far as we know, there are two types of DNAzyme, GR-5 DNAzyme and 8–17 DNAzyme, have been extensively used to develop Pb2+ specific recognition biosensors. Interestingly, we found that 8–17 DNAzyme and GR-5 DNAzyme could recognize the same substrate at the same conditions (Zhao et al., 2011, Elbaz et al., 2008). Therefore, we speculated that if we could integrate the two categories of Pb2+-DNAzymes into one amplification system to develop a novel signal amplification strategy for high sensitivity detection of Pb2+. Recently, Li's group reported a feedback amplification strategy that incorporates DNAzyme into RCA process to create a feedback circuit for DNA amplification sensing (Liu et al., 2017b). Inspire by the feedback design, we attempt to incorporate 8–17 DNAzyme and GR-5 DNAzyme into RCA process to create a novel feedback strategy, which could obviously enhance the sensitivity than the method that only has one Pb2+-DNAzyme.
In this work, we integrated dual Pb2+-DNAzyme into RCA process to create a novel feedback amplification strategy and demonstrated its high potential for amplifying Pb2+ sensing in river water samples. The use of dual Pb2+-DNAzyme as recognition elements for triggering RCA process endow this amplification technology with excellent selectivity and sensitivity toward Pb2+. Furthermore, we also presented an extremely facile yet efficient method for fabricating CFME by using wax as a unique sealant to replace traditional epoxy-seal or flame-fuse methods, which make the fabrication process more simple and robust. As the dimension of CFME perfectly matched with commercial micropipette tip (10 µL), it allow us to develop a miniaturized electrochemical device to achieve sample volume down to 10 µL. Therefore, combining with the proposed DDFA strategy with the CFME-based miniaturized electrochemical device, this sensing system may hold a promising prospect for Pb2+ detection in real samples at the microliter level.
Section snippets
Procedures of the DDFA
Phosphorylated circular DNA template C1 (100 nM), C2 (100 nM) and L-DNA (200 nM) was mixed in 40 µL H2O. Then, the mixture was denatured at 90 °C for 5 min, followed slowly cooled down to room temperature. After that, 5 µL of 10 × T4 DNA ligase buffer (400 mM Tris-HCl, 100 mM MgCl2, 100 mM DTT, 5 mM ATP, pH 7.8) and 1 µL T4 DNA ligase (5 U) was added and watered total volume to 50 µL. The obtained mixture was incubated at 4 °C for overnight to ensure the formation of DNA complexes L-DNA/Cir1
Design and detection principle
The design and sensing principle of the DDFA is illustrated in Scheme 1. In this system, two circular DNA templates (C1 and C2) and linear DNA (L-DNA) have been subtly designed: (I) C1 consists of a sequence of 8–17 DNAzyme and an antisense sequence of G-quadruplex (anti-G-quadruplex), (II) C2 contains an antisense sequence of GR-5 DNAzyme (anti-GR-5 DNAzyme) and the same sequence of anti-G-quadruplex, and (III) L-DNA comprises of three parts, a linear oligonucleotide sequence for hybridizing
Conclusions
In summary, we have designed a novel feedback amplification strategy for highly sensitive detection of Pb2+ based on a dual Pb2+-DNAzyme assistant RCA process and an immobilized-free homogeneous miniaturized electrochemical device. Using DNAzyme as the recognition element to trigger two categories of RCA process ensures the excellent selectivity of the sensing system. As Pb2+ was able to induce two paths of RCA process to create feedback amplification, a small amount of target Pb2+ could
Acknowledgements
This work was financially supported by the National Natural Science Foundation of China (21705013), the Chongqing Science and Technology Commission of China (cstc2017jcyjAX0282, cstc2015jcyjBX0126, cstc2016shmsZX20001), the Science and Technology Research Program of Chongqing Municipal Education Commission China (KJ1711265, KJ1711285), the Foundation for High-level Talents of Chongqing University of Arts and Sciences, China (R2016CH08), Chongqing Yongchuan District Water Authority (2016-07),
Notes
The authors declare no competing financial interest.
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