Nanoprobe-Initiated Enzymatic Polymerization for Highly Sensitive Electrochemical DNA Detection

Abstract

Electrochemical DNA (E-DNA) sensors have been greatly developed and play an important role in early diagnosis of different diseases. To determine the extremely low abundance of DNA biomarkers in clinical samples, scientists are making unremitting efforts toward achieving highly sensitive and selective E-DNA sensors. Here, a novel E-DNA sensor was developed taking advantage of the signal amplification efficiency of nanoprobe-initiated enzymatic polymerization (NIEP). In the NIEP based E-DNA sensor, the capture probe DNA was thiolated at its 3'-terminal to be immobilized onto gold electrode, and the nanoprobe was fabricated by 5'-thiol-terminated signal probe DNA conjugated gold nanoparticles (AuNPs). Both of the probes could simultaneously hybridize with the target DNA to form a "sandwich" structure followed by the terminal deoxynucleotidyl transferase (TdT)-catalyzed elongation of the free 3'-terminal of DNA on the nanoprobe. During the DNA elongation, biotin labels were incorporated into the NIEP-generated long single-stranded DNA (ssDNA) tentacles, leading to specific binding of avidin modified horseradish peroxidase (Av-HRP). Since there are hundreds of DNA probes on the nanoprobe, one hybridization event would generate hundreds of long ssDNA tentacles, resulting in tens of thousands of HRP catalyzed reduction of hydrogen peroxide and sharply increasing electrochemical signals. By employing nanoprobe and TdT, it is demonstrated that the NIEP amplified E-DNA sensor has a detection limit of 10 fM and excellent differentiation ability for even single-base mismatch.

Synthesis of enzymatic hydrogels and their application in biosensors

“…图 17 基于OTA适配体和TDT介导超长聚胸腺嘧啶转换的 Cu NPs的OTA荧光测定示意图 [49] (网络版彩图) 图 19 基于近端双向酶修复扩增的ATP荧光检测原理图 [51] (网络版彩图) 图 18 末端脱氧核苷酸转移酶(TdT)辅助扩增策略在黄瓜 绿斑驳花叶病毒视觉检测中的应用原理图 [50] (网络版彩图) [46] (网 络版彩图) 图 21 基于NIEP的E-DNA生物传感原理 [52] (网络版彩图)…”

Biosensing and Delivery of Nucleic Acids Involving Selected Well-Known and Rising Star Functional Nanomaterials

“…Furthermore, due to AuNPs’ wide specific surface area, they can be filled with a high number of labels and used for amplified detection of nucleic acids. Indeed, AuNPs have been functionalized with unlabeled [13] or redox-labeled [14] nucleic acid probes, redox molecules [15,16], and enzymes or DNAzymes.…”

“…Attractive strategies for the sensitive electrochemical detection of nucleic acids have also been proposed using AuNPs as nanocarriers of redox-labeled DNA probes [14,15], DNA probes and enzymes [13], and redox reporters [15,16]. Wang et al [14] reported a sandwich hybridization assay at an Au electrode modified with a thiolated capture probe and the use of AuNPs modified with two types of signaling reporter DNAs labeled with methylene blue (where just one was complementary to the target DNA).…”

“…Wan et al [13] described a detection scheme where the target DNA helped the setup of sandwich complexes between electrode-immobilized capture probes and reporter probe-linked AuNPs. The terminal desoxynucleotidyl transferase (TdT)-catalyzed elongation of the free 3´-terminal of DNA on the AuNPs led to the incorporation of multiple biotin moieties into the generated long DNA strands, which were further used for conjugating avidin-modified HRP molecules (Figure 5a).…”

“…Electrochemical biosensing of nucleic acids based on the use of AuNPs as nanocarriers of probes further elongated with TdT to generate long DNA strands bearing multiple biotin moieties, further conjugated with avidin–HRP complex ( a ), and electron transfer regulators through their non-covalent interaction with ssDNA ( b ). Reprinted from [13] ( a ) and [17] ( b ) with permission. Copyrights ACS, 2015 ( a ) and ACS, 2014 ( b ).…”