Bin-Cheng Yin

One-Step, Multiplexed Fluorescence Detection of microRNAs Based on Duplex-Specific Nuclease Signal Amplification

Traditional molecular beacons, widely applied for detection of nucleic acids, have an intrinsic limitation on sensitivity, as one target molecule converts only one beacon molecule to its fluorescent form. Herein, we take advantage of the duplex-specific nuclease (DSN) to create a new signal-amplifying mechanism, duplex-specific nuclease signal amplification (DSNSA), to increase the detection sensitivity of molecular beacons (Taqman probes). DSN nuclease is employed to recycle the process of target-assisted digestion of Taqman probes, thus, resulting in a significant fluorescence signal amplification through which one target molecule cleaves thousands of probe molecules. We further demonstrate the efficiency of this DSNSA strategy for rapid direct quantification of multiple miRNAs in biological samples. Our experimental results showed a quantitative measurement of sequence-specific miRNAs with the detection limit in the femtomolar range, nearly 5 orders of magnitude lower than that of conventional molecular beacons. This amplification strategy also demonstrated a high selectivity for discriminating differences between miRNA family members. Considering the superior sensitivity and specificity, as well as the multiplex and simple-to-implement features, this method promises a great potential of becoming a routine tool for simultaneously quantitative analysis of multiple miRNAs in tissues or cells, and supplies valuable information for biomedical research and clinical early diagnosis.

An allosteric dual-DNAzyme unimolecular probe for colorimetric detection of copper(II).

An effective dual-DNAzyme-based unimolecular probe design employing intramolecular signal transduction is demonstrated. The probe is composed of three domains: a DNA-cleaving DNAzyme, a substrate, and an HRP-mimicking DNAzyme. When the probe meets its target, cleavage of the substrate by the DNA-cleaving DNAzyme activates the HRP-mimicking DNAzyme, producing a colorimetric signal. The Cu(2+)-dependent DNAzyme engineered to demonstrate this design revealed a sensitivity corresponding to 65 ppb, which is sufficient to detect Cu(2+) in drinking water. The new probe has excellent selectivity toward Cu(2+). This three-component design is simple and easy to engineer. It may provide the basis for future development of other nucleic acid-based probes for toxicological and environmental monitoring.

Attomolar ultrasensitive microRNA detection by DNA-scaffolded silver-nanocluster probe based on isothermal amplification.

MicroRNAs (miRNAs) play vital roles in a plethora of biological and cellular processes. The levels of miRNAs can be useful biomarkers for cellular events or disease diagnosis, thus the method for sensitive and selective detection of miRNAs is imperative to miRNA discovery, study, and clinical diagnosis. Here we develop a novel method to quantify miRNA expression levels as low as attomolar sensitivity by target-assisted isothermal exponential amplification coupled with fluorescent DNA-scaffolded AgNCs and demonstrated its feasibility in the application of detecting miRNA in real samples. The method reveals superior sensitivity with a detection limit of miRNA of 2 aM synthetic spike-in target miRNA under pure conditions (approximately 15 copies of a miRNA molecule in a volume of 10 μL) and can detect at least a 10 aM spike-in target miRNA in cell lysates. The method also shows the high selectivity for discriminating differences between miRNA family members, thus providing a promising alternative to standard approaches for quantitative detection of miRNA. This simple and cost-effective strategy has a potential of becoming the major tool for simultaneous quantitative analysis of multiple miRNAs (biomarkers) in tissues or cells and supplies valuable information for biomedical research and clinical early diagnosis.

An ultrasensitive electrochemical DNA sensor based on the ssDNA-assisted cascade of hybridization reaction

We have developed a simple and ultrasensitive E-DNA sensor based on the ssDNA-assisted cascade of a hybridization reaction mechanism to form a long concatamers structure to improve its sensitivity, significantly. The proposed sensor was applied to sequence-specific DNA and ATP detection. Experimental results showed a quantitative measurement with the detection limit as low as 1 aM for specific DNA and 10 fM for ATP.

Copper-Mediated DNA-Scaffolded Silver Nanocluster On–Off Switch for Detection of Pyrophosphate and Alkaline Phosphatase

We present a new copper-mediated on-off switch for detecting either pyrophosphate (PPi) or alkaline phosphatase (ALP) based on DNA-scaffolded silver nanoclusters (DNA/AgNCs) templated by a single-stranded sequence containing a 15-nt polythymine spacer between two different emitters. The switch is based on three favorable properties: the quenching ability of Cu(2+) for DNA/AgNCs with excitation at 550 nm; the strong binding capacity of Cu(2+) and PPi; and the ability of ALP to transform PPi into orthophosphate (Pi). The change in fluorescence of DNA/AgNCs depends on the concentrations of Cu(2+), PPi, and ALP. Copper(II) acts as a mediator to interact specifically with the Probe, while PPi and ALP convert the signal of the Probe by removing and recovering Cu(2+), operating as an on-off switch. In the presence of Cu(2+) only, DNA/AgNCs exhibit low fluorescence because the combination of Cu(2+) and DNA template disturbs the precise formation of DNA/AgNCs. When PPi is added to the system containing Cu(2+), free DNA template is obtained due to the stronger interaction of PPi and Cu(2+), leading to a significant fluorescence increase (ON state) which depends on the concentration of PPi. Further addition of ALP results in the release of free Cu(2+) via ALP-catalysis of hydrolysis of PPi into Pi, thereby returning the system to the low fluorescence OFF state. The switch allows the analysis of either PPi or ALP by observation of the fluorescence status, with the detection limit of 112.69 nM and 0.005 U/mL for PPi and ALP, respectively. The AgNCs on-off switch provides the advantages of simple design, convenient operation, and low experimental cost without need of chemical modification, organic dyes, or separation procedures.

DNAzyme-based microarray for highly sensitive determination of metal ions.

A novel microarray for the multiplex determination of heavy metal ions in aqueous solution based on DNAzymes has been developed with good sensitivity and selectivity. In the present work, metal ion dependent DNAzymes of copper (Cu-Enz) and lead (Pb-Enz) were first associated with their corresponding DNA substrates (Cu-Sub and Pb-Sub) immobilized on the surface of aldehyde-coated slides. After introducing the corresponding metal ions, the DNA cleavage of the substrates caused by the DNAzymes took place, resulting in a dramatic change in fluorescent signal intensity. The proposed microarray method, which can be used as a multi-component assay with high efficiency, combines the high sensitivity and selectivity of DNAzymes with the high throughput and parallel analysis of microarray technology. The method reveals a sensitivity corresponding to 0.6ppb and 2ppb for Cu(2+) and Pb(2+), respectively, which is sufficient to detect them in drinking water. This approach may find potential applications in environmental monitoring, food safety monitoring, clinical toxicology, waste treatment, the cosmetic industry and industrial process monitoring.

Sensitive DNA-Based Electrochemical Strategy for Trace Bleomycin Detection

Bleomycins (BLMs) are widely used in combination with chemotherapy for the treatment of a variety of cancers. The clinical application of BLMs is featured by the occurrence of sometimes fatal side effects, such as renal and lung toxicity, and the potential dose-limiting side effect of pulmonary fibrosis. Therefore, it is highly desirable to develop a sensitive method to quantitatively determine the BLM content in both pharmaceutical analysis and clinical samples, to make full use of therapeutic efficacy and to weaken its toxicity. Here, we proposed a simple, rapid, and convenient electrochemical assay for trace BLM detection. A reported DNA motif, as substrate for BLMs, is prepared to self-assemble onto the gold electrode to fabricate an electrochemical DNA (E-DNA) sensor, with a terminus tethered on the electrode surface and the other terminus labeled with ferrocenyl moiety as a signal reporter to form a stem-loop structure, giving an arise of remarkable faradaic current. In the presence of Fe(II)·BLM, the E-DNA sensor undergoes the irreversible cleavage event, which can be transduced into a significant decrease in current peak. This proposed sensor reveals an impressive sensitivity as low as 100 pM BLMs and exhibits a good performance as well as in serum sample. Considering the high sensitivity and specificity of this proposed sensor, as well as the cost-effective and simple-to-implement features of the electrochemical technique, we believe that this method shows distinct advantages over conventional methods and it is a promising alternative for the determination of trace amounts of BLMs in clinical samples.

A novel polydopamine-based chemiluminescence resonance energy transfer method for microRNA detection coupling duplex-specific nuclease-aided target recycling strategy

MicroRNAs (miRNAs), functioning as oncogenes or tumor suppressors, play significant regulatory roles in regulating gene expression and become as biomarkers for disease diagnostics and therapeutics. In this work, we have coupled a polydopamine (PDA) nanosphere-assisted chemiluminescence resonance energy transfer (CRET) platform and a duplex-specific nuclease (DSN)-assisted signal amplification strategy to develop a novel method for specific miRNA detection. With the assistance of hemin, luminol, and H2O2, the horseradish peroxidase (HRP)-mimicking G-rich sequence in the sensing probe produces chemiluminescence, which is quickly quenched by the CRET effect between PDA as energy acceptor and excited luminol as energy donor. The target miRNA triggers DSN to partially degrade the sensing probe in the DNA-miRNA heteroduplex to repeatedly release G-quadruplex formed by G-rich sequence from PDA for the production of chemiluminescence. The method allows quantitative detection of target miRNA in the range of 80 pM-50 nM with a detection limit of 49.6 pM. The method also shows excellent specificity to discriminate single-base differences, and can accurately quantify miRNA in biological samples, with good agreement with the result from a commercial miRNA detection kit. The procedure requires no organic dyes or labels, and is a simple and cost-effective method for miRNA detection for early clinical diagnosis.

Simultaneous Surface-Enhanced Raman Spectroscopy Detection of Multiplexed MicroRNA Biomarkers

Simultaneous detection of cancer biomarkers holds great promise for the early diagnosis of cancer. In the present work, an ultrasensitive and reliable surface-enhanced Raman scattering (SERS) sensor has been developed for simultaneous detection of multiple liver cancer related microRNA (miRNA) biomarkers. We first proposed a novel strategy for the synthesis of nanogap-based SERS nanotags by modifying gold nanoparticles (AuNPs) with thiolated DNA and nonfluorescent small encoding molecules. We also explored a simple approach to a green synthesis of hollow silver microspheres (Ag-HMSs) with bacteria as templates. On the basis of the sandwich hybridization assay, probe DNA-conjugated SERS nanotags used as SERS nanoprobes and capture DNA-conjugated Ag-HMSs used as capture substrates were developed for the detection of target miRNA with a detection limit of 10 fM. Multiplexing capability for simultaneous detection of the three liver cancer related miRNAs with the high sensitivity and specificity was demonstrated using the proposed SERS sensor. Furthermore, the practicability of the SERS sensor was supported by the successful determination of target miRNA in cancer cells. The experimental results indicated that the proposed strategy holds significant potential for multiplex detection of cancer biomarkers and offers the opportunity for future applications in clinical diagnosis.

A universal real-time PCR assay for rapid quantification of microRNAs via the enhancement of base-stacking hybridization

Via the base-stacking hybridization strategy, we have developed a universal, one-step real-time quantitative PCR assay for sensitive and selective detection of microRNAs. This proposed assay has several intrinsic features including rapid response, low cost, simple handling procedures, etc.