Nucleic Acid Amplification by Template-Dominated Click Chemistry for Ultrasensitive DNA/RNA Detection on an Electrochemical Readout Platform.

As an enzyme-free exponential nucleic acid amplification method, the click chemistry-mediated ligation chain reaction (ccLCR) has shown great prospects in the molecular diagnosis. However, the current optics-based ccLCR is challenged by remarkable nonspecific amplification, severely hindering its future application. This study demonstrated that the severe nonspecific amplification was generated probably due to high random collision in the high DNA probe concentration (µM level). To solve this hurdle, a nucleic acid template-dominated ccLCR was constructed using nM-level DNA probes and read on an electrochemical platform (cc-eLCR). Under the optimal conditions, the proposed cc-eLCR detected a low-level nucleic acid target (1 fM) with a single-base resolution. Furthermore, this assay was applied to detect the target of interest in cell extracts with a satisfactory result. The proposed cc-eLCR offers huge possibility for click chemistry-mediated enzyme-free exponential nucleic acid amplification in the application of medical diagnosis and biomedical research.

Ultrasensitive Detection of RNA with Single-Base Resolution by Coupling Electrochemical Sensing Strategy with Chimeric DNA Probe-Aided Ligase Chain Reaction.

Accurate and sensitive detection of single-base mutations in RNAs is of great value in basic studies of life science and medical diagnostics. However, the current available RNA detection methods are challenged by heterogeneous clinical samples in which trace RNA mutants usually existed in a large pool of normal wild sequences. Thus, there is still great need for developing the highly sensitive and highly specific methods in detecting single-base mutations of RNAs in heterogeneous clinical samples. In the present study, a new chimeric DNA probe-aided ligase chain reaction-based electrochemical method (cmDNA-eLCR) was developed for RNA mutation detection through the BSA-based carrier platform and the horseradish peroxidase-hydrogen peroxide-tetramethylbenzidine (HRP-H2O2-TMB) system. The denaturing polyacrylamide gel electrophoresis and a fluorophore-labeled probe was ingeniously designed to demonstrate the advantage of cmDNA in ligation to normal DNA templated by RNA with the catalysis of T4 RNA ligase 2 as well as its higher selectivity than DNA ligase system. Finally, the proposed cmDNA-eLCR, compared with the traditional eLCR, showed excellent performance in discriminating single base-mismatched sequences, where the signal response for mismatched targets at a high concentration could overlap completely with that for the blank control. Besides, this cmDNA-eLCR assay had a wide linear range crossing six orders of magnitude from 1.0 × 10-15 to1.0 × 10-10 M with a limit of detection as low as 0.6 fM. Furthermore, this assay was applied to detect RNA in real sample with a satisfactory result, thereby demonstrating its great potential in diagnosis of RNA-related diseases.

A novel ligase chain reaction-based electrochemical biosensing strategy for highly sensitive point mutation detection from human whole blood.

Challenged by the detection of trace amounts of mutants and disturbance from endogenous substances in clinical samples, herein, we present a novel electrochemical biosensor based on ligase chain reaction (eLCR) via the thermostable ligase with high mutation recognizing ability. The lengthened double-stranded DNAs exponentially generated via LCR were uniformly distributed on a bovine serum albumin-modified gold electrode, in which the phosphate buffer was tactfully added to remove adsorbed uninterested-probes, and thereafter the amperometry current was collected for the specific binding of streptavidin-poly-HRP and subsequent catalysis in the 3, 3', 5, 5'-tetramethylbenzidine substrate that contained hydrogen peroxide. It found that, under optimized conditions, the proposed biosensor exhibited a high selectivity of mutant targets from the 104-fold excess of co-existent wild targets within a detection limit of 0.5 fM. Impressively, without the involvement of pre-PCR, the homozygous mutants were specifically distinguished from the wild genotype of CYP2C19*2 allele in human whole blood samples. Therefore, the proposed eLCR, due to its advantages in simple primer design, operational ease and ease of miniaturization, has demonstrated its considerable potential for point-of-care testing in the diagnosis of point mutation-related diseases and personalized medicine.

Ultrasensitive Electrochemical Biosensor Developed by Probe Lengthening for Detection of Genomic DNA in Human Serum.

As an alternative to most of the reported nucleic acid amplification-based electrochemical DNA biosensors used for detection of trace levels of genomic DNA, we herein present a novel detection concept. The proposed system involves the conversion of two short double-stranded DNAs (dsDNAs), labeled with a thiol-tag or biotin-tag, into a single integrated dsDNA containing thiol and biotin at both terminals in the presence of target DNA through ligase chain reaction (LCR) and followed by the immobilization of these integrated dsDNAs on a bovine serum albumin (BSA)-modified gold electrode surface. Owing to rapid depletion of the two short dsDNAs via LCR, the integrated dsDNAs were generated in an exponential manner so that this sensoring approach offered a limit of detection of 25 yoctomoles (15 copies in 50 µL sample volumes), a high discrimination of single-base mismatch and a wide linear concentration range (across 6 orders of magnitude) for target DNA. Significantly, the proposed sensor, which has simplicity in operation and ease of miniaturization, detected the target of interest in total nucleic acid extracts derived from clinical serum samples with excellent results, thereby demonstrating its considerable diagnostic potential in fields ranging from virus detection to the diagnosis of genetic diseases.

Facilely prepared low-density DNA monolayer-based electrochemical biosensor with high detection performance in human serum.

Presently, most reported electrochemical biosensors, for highly sensitive and selective detection of nucleic acid, still require multiple, time-consuming assembly steps and high-consumption DNA probes as well as lack good performance in human serum, which greatly limit their applicability. Herein, an easy-to-fabricate electrochemical DNA biosensor constructed by assembly of bovine serum albumin (BSA) followed with direct incubation of amplified products has been proposed. This method combined terminal deoxynucleoside transferase (TdTase)-mediated isothermal amplification and polyHRP catalysis to achieve dual-signal enhancement, and was featured with low-density DNA monolayer for its employment of only 2 nM capture probes. Surprisingly, based on the low-density DNA monolayer, the steric hindrance effect of polyHRP could effectively restrain the background compared with HRP, which further pushes the signal-to-noise (S/N) ratio to 70 than that of most currently available methods. Additionally, this strategy also showed favorable specificity and powerful anti-interference in human serum, and thus potentially attractive for diagnosis of diseases.