Yibing Yin

Cascade Signal Amplification Strategy for Subattomolar Protein Detection by Rolling Circle Amplification and Quantum Dots Tagging

A cascade signal amplification strategy was proposed for detection of protein target at ultralow concentration by combining the rolling circle amplification (RCA) technique with oligonucleotide functionalized quantum dots (QDs), multiplex binding of the biotin-strepavidin system, and anodic stripping voltammetric detection. The RCA product containing tandem-repeat sequences could serve as excellent template for periodic assembly of QDs, which presented per protein recognition event to numerous quantum dot tags for electrochemical readout. Both the RCA and the multiplex binding system showed remarkable amplification efficiency, very little nonspecific adsorption, and low background signal. Using human vascular endothelial growth factor as a model protein, the designed strategy could quantitatively detect protein down to 16 molecules in a 100 microL sample with a linear calibration range from 1 aM to 1 pM and was amenable to quantification of protein target in complex biological matrixes. The proposed cascade signal amplification strategy would become a powerful tool for proteomics research and clinical diagnostics.

Electrochemical immunosensor for competitive detection of neuron specific enolase using functional carbon nanotubes and gold nanoprobe.

An electrochemical immunosensor for detection of neuron specific enolase (NSE) was designed by immobilizing NSE covalently functionalized single-walled carbon nanotubes (NSE-SWNTs) on a glassy carbon electrode. The NSE-SWNTs not only enhanced electrochemical signal but also presented abundant antigen domains for competitive immunological recognition to anti-NSE primary antibody and then gold nanoprobes labeled with alkaline phosphatase conjugated secondary antibody (AP-anti-IgG/AuNPs). The AP-anti-IgG/AuNPs exhibited highly catalytic activity toward enzyme substrate and significantly amplified the amperometric signal for target molecule detection. Based on the dual signal amplification of SWNTs and gold nanoprobe, the immunosensor could response down to 0.033 ng mL(-1) NSE with a linear range from 0.1 ng mL(-1) to 2 μg mL(-1), and showed acceptable precision and reproducibility. The designed immunosensor was amenable to direct quantification of target protein with a wide range of concentration in complex clinical serum specimens. The assay results were in a good agreement with the reference values. The proposed electrochemical immunosensor provided a pragmatic platform for convenient detection of tumor markers in clinical diagnosis.

A colorimetric biosensor for detection of attomolar microRNA with a functional nucleic acid-based amplification machine

A functional nucleic acid-based amplification machine was designed for simple and label-free ultrasensitive colorimetric biosensing of microRNA (miRNA). The amplification machine was composed of a complex of trigger template and C-rich DNA modified molecular beacon (MB) and G-rich DNA (GDNA) as the probe, polymerase and nicking enzyme, and a dumbbell-shaped amplification template. The presence of target miRNA triggered MB mediated strand displacement to cyclically release nicking triggers, which led to a toehold initiated rolling circle amplification to produce large amounts of GDNAs. The formed GDNAs could stack with hemin to form G-quadruplex/hemin DNAzyme, a well-known horseradish peroxidase (HRP) mimic, for catalyzing a colorimetric reaction. The modified MB improved the stringent target recognition and reduced background signal. The proposed sensing strategy showed very high sensitivity and selectivity with a wide dynamic range from 10 aM to 1.0 nM, and enabled successful visual analysis of trace amount of miRNA in real sample by the naked eye. This rapid and highly efficient signal amplification strategy provided a simple and sensitive platform for miRNA detection. It would be a versatile and powerful tool for clinical molecular diagnostics.

Catalytic Hairpin Assembly Actuated DNA Nanotweezer for Logic Gate Building and Sensitive Enzyme-Free Biosensing of MicroRNAs

A target-switched DNA nanotweezer is designed for AND logic gate operation and enzyme-free detection of microRNAs (miRNAs) by catalytic hairpin assembly (CHA) and proximity-dependent DNAzyme formation. The double crossover motif-based nanotweezer consists of an arched structure as the set strand for target inputs and two split G-rich DNAs at the termini of two arms for signal output. Upon a CHA, a small amount of binary target inputs can switch numerous open nanotweezers to a closed state, which leads to the formation of proximity-dependent DNAzyme in the presence of hemin to produce a highly sensitive biosensing system. The binary target inputs can be used for successful building of AND logic gate, which is validated by polyacrylamide gel electrophoresis, surface plasmon resonance and the biosensing signal. The developed biosensing system shows a linear response of the output chemiluminescence signal to input binary miRNAs with a detection limit of 30 fM. It can be used for miRNAs analysis in complex sample matrix. This system provides a simple and reusable platform for logic gate operation and enzyme-free, highly sensitive, and specific multianalysis of miRNAs.

Bis-three-way junction nanostructure and DNA machineries for ultrasensitive and specific detection of BCR/ABL fusion gene by chemiluminescence imaging

A novel G-quadruplex DNAzyme-driven chemiluminescence (CL) imaging method has been developed for ultrasensitive and specific detection of BCR/ABL fusion gene based on bis-three-way junction (bis-3WJ) nanostructure and cascade DNA machineries. Bis-3WJ probes are designed logically to recognize BCR/ABL fusion gene, which forms the stable bis-3WJ nanostructure for the activation of polymerase/nicking enzyme machineries in cascade, resulting in synthesis of DNAzyme subunits. These DNAzyme subunits can form integrated DNAzyme by self-assembly to catalyze CL substrate, thus providing an amplified signal for the sensing events or outputs for AND logic operation. The imaging method achieved ultrasensitive detection of BCR/ABL fusion gene with a low detection limit down to 23 fM. And this method exhibited wide linear ranges over seven orders of magnitude and excellent discrimination ability toward target. In addition, an acceptable recovery was obtained in complex matrix. It is notable that this biosensing strategy possesses merits of homogenous, isothermal and label-free assay system. Therefore, these merits endow the developed imaging method with a potential tool for CML diagnosis.

DNA transducer-triggered signal switch for visual colorimetric bioanalysis

A simple and versatile colorimetric biosensor has been developed for sensitive and specific detection of a wide range of biomolecules, such as oligonucleotides and aptamer-recognized targets. Combining the signal transducer and catalyzed hairpin assembly (CHA)-based signal amplification, the target DNA binds with the hairpin DNA to form a new nucleic acid sequence and creates a toehold in the transducer for initiating the recycle amplification reaction of CHA. The catalyzed assembly process produces a large amount of G-rich DNA. In the presence of hemin, the G-rich DNA forms G-quadruplex/hemin complex and mimic horseradish peroxidase activity, which catalyzes a colorimetric reaction. Under optimal conditions, the calibration curve of synthetic target DNA has good linearity from 50 pM to 200 nM with a detection limit of 32 pM. This strategy has been successfully applied to detect S. pneumoniae as low as 156 CFU mL−1, and shows a good specificity against closely related streptococci and major pathogenic bacteria. In addition, the developed method enables successful visual analysis of S. pneumoniae in clinical samples by the naked eye. Importantly, this method demonstrates excellent assay versatility for sensitively detecting oligonucleotides or aptamer-recognized targets.