A Novel Strategy For Biological Molecule Detection Based On Isothermal Amplification And DNA Nanotechnology

Biosensor is analytical equipment consisting of the bioactive materials (such as peptides, nucleic acids, antibodies, cell, tissue, et al.), the transducer and the signal analysis device. In recent years, with the development of the molecular biology, materials science, bioanalytical chemistry, clinical laboratory diagnostics, the biosensor has been developed rapidly based on isothermal amplification and DNA nanotechnology, which can, sensitively detect a large number of substances, including cells, bacteria, nucleic acids, inorganic ions, polypeptide, small molecules, et al. In our work, rapid, accurate, and sensitive methods for detection of biology molecules cocaine and miRNAs based on isothermal amplification and DNA nanotechnology have been established, which might become a powerful tool for clinical diagnostics, and forensic toxicological analysis. The study has two parts as follow:1. Electrochemical aptasensor for highly sensitive determination of cocaine using a supramolecular aptamer and rolling circle amplificationCocaine, a small molecule, is one of the most illegally abused drugs available all over the world because of its instantaneous and overwhelming effects on the central nervous system. Quantification of cocaine plays an important role in forensic science, pharmacy, therapy, and metabolomics. We report on a novel strategy for the electrochemical detection of cocaine. It is based on the use of a supramolecular aptamer, rolling circle amplification (RCA), and multiplex binding of a biotin-strepavidin system. The aptamer fragments were assembled to a supramolecular aptamer which, in the presence of cocaine, conjugates to streptavidin for anchoring of biotinylated circular DNA. This initiates RCA and enables sensitive electrochemical-enzymatic readout. A significant signal amplification was obtained by using streptavidin linked to alkaline phosphatase that binds to the remaining biotinylated detection probes and catalyzes the hydrolysis of the synthetic enzyme substrate α-naphthylphosphate. This dual amplification strategy tremendously increases the detection limit of the aptasensor. Under optimal conditions and using differential pulse voltammetry, cocaine can be detected in the concentration range between 2 and 500 nM with a detection limit as low as 1.3 nM (S/N=3). The method is specific and acceptably reproducible. It was successfully applied to the detection of cocaine in (spiked) urine samples. The data were in good agreement with those obtained by the GC-MS reference method.2. A novel and versatile nanomachine for ultrasensitive and specific detection of microRNAs based on strand displacement amplification and catalytic hairpin assembly with DNAzyme formationMiRNAs are a class of small (21-24 nt), endogenous, and non-coding RNA molecules. The unnatural expression of miRNAs may lead to serious diseases. Thus, quantitative analysis of miRNAs is crucial for better understanding their roles in cancer cells and further validating their function in biomedical research and clinical diagnosis. In this study, a simple and versatile colorimetric biosensor has been developed for ultrasensitive and specific determination of microRNAs (miRNAs) based on strand displacement amplification (SDA) and catalytic hairpin assembly (CHA) with DNAzyme formation. The amplification nanomachine primarily consists of the versatile molecular beacon (MB), hairpin H1, hairpin H2, polymerase, and nicking enzyme. The presence of target miRNAs triggers strand displacement amplification to release nicking DNA triggers, which initiate CHA to produce large amount of CHA products. Meanwhile, the numerous CHA products can combine with hemin to form G-quadruplex/hemin DNAzyme, a well-known horseradish peroxidase (HRP) mimic, catalyzing a colorimetric reaction. Under the optimal conditions, the established colorimetric biosensor shows high sensitivity and selectivity in a dynamic response range from 5 fM to 5 nM with a detection limit as low as 1.7 fM (S/N=3). This simple and highly efficient signal amplification machine can provide a versatile platform for different miRNAs determination. Thus, this colorimetric biosensor may become a potential alternative tool for clinical molecular diagnostics.