Immobilized-free miniaturized electrochemical sensing system for Pb²⁺ detection based on dual Pb²⁺-DNAzyme assistant feedback amplification strategy

Highlights

• Integrating dual Pb²⁺-DNAzyme into one RCA-mediated feedback amplification for Pb²⁺ detection.

• Presenting a facile and efficient method for carbon fiber microelectrode fabrication.

• Reducing analyte sample volume down to microliter level.

• Analytical performance of the sensing system is down to picomole level.

• Displaying excellent applicability for evaluating Pb²⁺ in real samples.

Abstract

We presented a novel dual-DNAzyme feedback amplification (DDFA) strategy for Pb²⁺ detection based on a micropipette tip-based miniaturized homogeneous electrochemical device. The DDFA system involves two rolling circle amplification (RCA) processes in which two circular DNA templates (C1 and C2) have been designed with a Pb²⁺-DNAzyme sequence (8–17 DNAzyme, anti-GR-5 DNAzyme) and an antisense sequence of G-quadruplex. And a linear DNA (L-DNA), which consists of a primer sequence and a Pb²⁺-DNAzyme substrate sequence, could hybridize with C1 and C2 to form two DNA complexes. In presence of Pb²⁺, the Pb²⁺-DNAzyme exhibited excellent cleavage specificity toward the substrate sequence in L-DNA, leaving primer sequence to trigger two paths of RCA process and finally resulting in massive long nanosolo DNA strands with reduplicated G-quadruplex sequences. And then, methylene blue (MB) could selectively intercalate into G-quadruplex to reduce the free MB concentration in the solution. Thereafter, a carbon fiber microelectrode-based miniaturized electrochemical device was constructed to record the decrease of electrochemical signal due to the much lower diffusion rate of MB/G-quadruplex complex than that of free MB. Therefore, the concentration of Pb²⁺ could be correctively and sensitively determined in a homogeneous solution by combining DDFA with miniaturized electrochemical device. This protocol not only exhibited high selectivity and sensitivity toward Pb²⁺ with a detection limit of 0.048 pM, but also reduced sample volume to 10 µL. In addition, this sensing system has been successfully applied to Pb²⁺ detection in Yangtze River with desirable quantitative manners, which matched well with the atomic absorption spectrometry (AAS).

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.