Ultrasensitive electrochemical DNA sensor based on the target induced structural switching and surface-initiated enzymatic polymerization
Introduction
As DNAs are important biomarkers for disease diagnosis, ultrasensitive and selective DNA sensors are essential for extremely low abundance of DNA biomarkers in clinical samples (Cosnier and Mailley, 2008, D'Orazio, 2003, Teles and Fonseca, 2008). Among different types of DNA biosensors, electrochemical DNA sensor is a promising one with the ability to produce a simple, accurate and inexpensive platform for DNA sensing. In recent years, numerous efforts have been made to improve the performance of electrochemical DNA sensors (e.g., sensitivity, selectivity) (Lao et al., 2005, Li et al., 2010b, Lucarelli et al., 2008, Luo et al., 2013, Pei et al., 2010, Sun et al., 2010, Wang, 2005, Zhang et al., 2006).
To achieve high sensitivity, different amplified strategies have attracted increasing attention from bioanalytical scientists. There are several types of nucleic acid amplification methods, such as polymerase chain reaction (PCR), (Erlich et al., 1991, MacKnight, 2003) rolling circle amplification (RCA), (Demidov, 2002, Mahmoudian et al., 2008, Murakami et al., 2009) and other isothermal amplification (Detter et al., 2002, He et al., 2010, He et al., 2012, Hellyer and Nadeau, 2004, Notomi et al., 2000, Parida et al., 2008). However, there are several drawbacks including relatively long assay time, high assay cost, and complicated preparation process, which restrict their applications. To overcome these obstacles, Tjong et al., (2011) reported a novel isothermal, on-chip, post-hybridization labeling and amplification strategy for DNA sensing, which was termed surface initiated enzymatic polymerization (SIEP). We introduced this SIEP strategy into an electrochemical DNA sensor and made significant progress towards sensitivity improvement (Wan et al., 2013).
Besides high sensitivity, excellent selectivity is also essential for identifying specific nucleic acid sequences of DNA biomarkers, with the goal of high discrimination of single-nucleotide polymorphisms (SNPs). To realize high selectivity, several elegant strategies have been proposed, including allele-specific oligonucleotide (ASO) hybridization (Østergaard et al., 2013), oligonucleotide ligation assays (OLA) (Wan et al., 2009, Wan et al., 2010), and primer extension assays (Kisaki et al., 2010). However, extra reactions are involved in these strategies. A convenient and validated way is to use stem-loop probe (SLP). Compared with linear probes, SLPs inherently possess high specificity due to their conformational constraints (Fan et al., 2003, He et al., 2012, Hsieh et al., 2010, Huang et al., 2012, Li et al., 2010a, Lubin et al., 2006, Xiao et al., 2009, Yang and Lai, 2011, Yu and Lai, 2012, Zhang et al., 2010). Based on the hybridization induced conformational change of the SLPs, electrochemical signals are associated with tags of these surface-confined probes. The tags could be redox moiety or small molecular which can further capture other electrochemical labels. Taking advantage of SLPs, numerous electrochemical DNA sensors have been developed towards sequence-specific DNA detection. We have reported a strategy of electrochemical DNA sensor by using redox enzyme and stem-loop probe, which can detect as low as 1 fM target DNA (Liu et al., 2008).
Herein we developed two electrochemical DNA sensors that combine the SIEP strategy with SLPs. By employing terminal deoxynucleotidyl transferase (TdT) as a labeling enzyme and SLP as capture probe, we aim to provide a promising approach for ultrahigh sensitive and selective detection of target DNA.
Section snippets
Materials
DNA oligonucleotides were purchased from Invitrogen Co. (Shanghai, China) with their sequences listed in Table 1. The capture probes (oligo 1 and oligo 2) were designed with the same sequences which formed a stem-loop structure. There were eight complementary bases at their 5' and 3' ends, which will form the stem at appropriate ionic strength. Thiol and a –(CH2)6– alkyl chain were modified at either its 5'-terminal (5-SLP) or 3'-terminal (3-SLP). The sequence of the target (oligo 3) perfectly
The strategy of the two sensors
A schematic representation of the two sensors is shown in Scheme 1. The first type of electrochemical sensor was prepared by the immobilization of 5-SLP on gold electrodes via gold-thiol chemistry (5-SLP–SENS) while the other one was prepared by using 3-SLP (3-SLP–SENS). In the initial state of the sensors, the probes adopted the stem-loop structure, which shielded the unlabeled terminal from being approached. Upon hybridization, the SLPs would be open and lead to a rigid double-strand
Conclusions
Implementation of low-cost and commercially available electrochemical sensors in clinical laboratories requires highly sensitive and selective devices. Here, we developed two electrochemical DNA sensors for sequence specific DNA analysis via SIEP and SLP. Uniquely, these DNA sensors employ a considerably simple structure and a valid signal amplification system. TdT is used to incorporate multiple biotin labels into the extended ssDNA chain. Due to the limited folding efficiency, the detection
Acknowledgments
This work was supported by the National Science Foundation (Nos. 21105048 and 61371039), China Postdoctoral Science Foundation Funded Project (No. 2012T50475).
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