Analytical MethodsUltra-sensitive and absolute quantitative detection of Cu2+ based on DNAzyme and digital PCR in water and drink samples
Introduction
Native DNA performed the genetic function for most organisms. However, the latest research has shown that single-stranded DNA in a complex hairpin structure can perform molecular recognition and catalysis. The catalysis functions of single-strand DNA have broken the common idea that all catalysis functions should be performed by proteins and has raised concern in researchers. Among different kinds of biosensors, deoxyribozyme sensors for the detection of metal ions have improved outcomes compared with normal detection methods with simpler protocols (Li et al., 2009, Liu and Lu, 2007a, Liu and Lu, 2007b, Wang et al., 2008). Most of them are based on complex structures caused by nuclear strand folding, similar to the tertiary structure of common enzymes. Heavy metal ions act as the co-effectors to heavy metal biosensors, while the existing ions induce a reaction between the DNAzymes and substrates. Copper, as a type of widely used heavy metal in industry, can be leaked into the environment through different methods. High copper exposure for a prolonged period may cause permanent liver and kidney damage. Although the U.S. Environmental Protection Agency (EPA) has recommended the highest concentration of copper in drinking water to be 1.3 ppm, research has shown that trace amounts of copper can induce neurotoxicity by interacting with cholesterol in the diet after a treatment with 0.06 ppm copper for 16 weeks (Lu et al., 2006). Thus, the sensitive and selective detection of copper is important for human nutrition. In the 2000s, Liu found that a specific nucleic acid sequence could lead to the cleavage of a DNA strand in the presence of Cu2+ (Liu & Lu, 2007a). That study showed a sensitive and selective detection method using a biosensor that increased fluorescence. However, signal amplification steps were not included for the original biosensor assay; thus, the sensitivity might not be sufficient for practical detection. On the other hand, by measuring fluorescence emission through a spectrophotometer, only qualitative detection could be achieved. This method could not determine whether the concentration of copper is above a certain value. Therefore, to promote the detection of copper by a biosensor method, signal amplification steps and quantitative detection are essential.
The most attractive aspect of heavy metal ion biosensors is that the ion signals can be transferred to nucleic acids, so that PCR methods could be introduced to improve the sensitivity and specificity. PCR is a popular tool to amplify nucleic acids. PCR can amplify nucleic acids sensitively and selectively in an exponential manner within 2 h (Bustin, Benes, & Garson, 2009). Digital PCR, a newly developed quantitative PCR-based detection method, has been regarded as the third generation of PCR technology for its better sensitivity and directly absolute quantitative detection (Miotto et al., 2014, Pinheiro et al., 2012, Sanders et al., 2011, Vogelstein and Kinzler, 1999). After separating the original reaction volume in a microfluidic or oil-in-water manner (Pinheiro et al., 2012, Sanders et al., 2011), a large number of separating reaction cells are generated so that the original target is also divided into each cell. Thus, the target copy number for each partition results from the Poisson distribution based on the ratio of positive partitions to total partitions. After first being reported in the 1990s (Vogelstein & Kinzler, 1999), much progress has been achieved in the field of copy number determination (Hindson, Ness, Masquelier, & Colston, 2011), single nucleotide polymorphisms and absolute quantitative detection of low-abundant target samples (Burns et al., 2010, Corbisier et al., 2010, Demeke et al., 2014). Compared with real-time PCR, digital PCR can determine the copy number of a target template in the original solution without the generation of standard curves (Morisset, Štebih, Milavec, Gruden, & Žel, 2013). This feature mostly avoids the uncertainties caused by standard curves and their procedures. Thus, digital PCR is regarded as an improved quantitative detection compared with real-time PCR. In the 2000s, Bio-Rad commercialized digital PCR with the production of the Bio-Rad QX100 digital PCR system. After development for nearly a decade, this system has been regarded as the digital PCR system with better accuracy, stability and cost performance compared with other platforms by meeting the requirements.
Based on this original biosensor, different detecting methods have been combined to increase the sensitivity or the feasibility in latter researches, including the paper-based stripes (Shing et al., 2013, Zhao et al., 2008), the G-quadruplex structure (Zhang et al., 2012, Zhang, Fan, et al., 2015) or the isothermal amplification (Li et al., 2014, Torres-Chavolla and Alocilja, 2011, Zhang et al., 2014). However, all the above researches could only achieve the quantitative detection of target ions by the analysis of standard curves through relative quantitation. This relative quantitation could be easily effected by the operation or the data analysis. Therefore, the absolute quantitation of target ion was essential to the risk assessment of heavy metal ions. For absolute quantitative detection of copper using the nucleic biosensor, understanding the binding relationship between the DNAzymes and cofactors is essential. Previous research on different types of biosensors has focused on the standard curves between the fluorescence emission and concentration of heavy metal ions (Hao et al., 2014, Li et al., 2014, Liu and Lu, 2007a). However, this method of data analysis cannot measure the absolute concentration of ions without serial dilutions of ion solutions. Therefore, the binding relationship of heavy metal ions with DNAzymes is essential for the absolute quantitation of heavy metal ions using the biosensor approach.
Considering the demand for quantitative detection with good sensitivity and specificity, we developed a detection system based on the deoxyribozyme biosensor and digital PCR. This detection system takes advantage of the nucleic acid signal translation of the biosensor and absolute quantitative detection of digital PCR. In our study, the binding model between the DNAzyme and ion was identified and validated. This knowledge is the key for achieving the absolute quantitative detection of ions by biosensor-based methods. According to the final detection results, the limit of quantitation (LOQ) was determined to be 0.5 pmol evaluating different replicates, while the sensitivity was 50 fmol as determined by the obvious difference from the negative control group.
Section snippets
Oligonucleotides and reagents
All the oligonucleotides are listed in Supplementary Table 1. All the primers and probes were synthesized by Invitrogen (Life Technologies, California, US). All chemical reagents used in our assays including copper (II) chloride dihydrate, Tris base and other chemicals in the buffer for the cleavage assay were purchased from Sigma-Aldrich (St. Louis, MO). The reagents used in our experiment are all Analytically Pure. All the reagents related to the droplet-digital PCR were purchased from
Principle of our detection system
The scheme of sensing system was described in Fig. 1. The whole system mainly consisted two parts, the biosensor step (Fig. 1a and b) and the digital PCR step (Fig. 1c and d). The designation of the biosensor sequences used in our study was shown in Fig. 1a. The sequence of the substrate contained both the locations of primers and the cleavage site of DNAzyme. For the first step (Fig. 1b), the single strand of the substrate could be hydrolyzed by the DNAzyme with the existence of Cu2+ during the
Discussion
In our study, we have described a quantitative detection method for copper ions based on a biosensor and digital PCR. The binding model between DNAzyme and Cu2+ has been revealed. According to the binding model and evaluation results, the quantitative detection system has both good quantitative and qualitative sensitivity by taking advantage of digital PCR.
To achieve absolute quantitative detection for copper by a biosensor-based method, the binding model between DNAzyme and the copper ion is
Conclusion
Here, a flexible and stable quantitative detection method with ultra-sensitivity was developed by combining a biosensor and digital PCR. By revealing the binding model between DNAzyme and Cu2+, we solved the relationship of the signal generation and Cu2+ content. Taking advantage of the absolute quantitative character of digital PCR, an absolute quantitative detection system of Cu2+ was established. Thus, using the ultra-sensitivity of digital PCR, we have achieved the LOQ and LOD values as low
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