Novel Biosensor Technique For Detection Of Nucleic Acids And Proteins

Protein and nucleic acid are the most two important macro-biomolecules, playing essential roles in the fields of genetic disease and metabolism. Therefore, it is highly significant that constructing simple, sensitive and efficient analytical methods for detecting protein and nucleic acid in the area of clinical diagnosis and drug screening. To satisfy and achieve the demand of the simple, high sensitivity, selectivity and affinity, and high throughout for the detection of proteins and DNA, we develop a series of novel electrochemical biosensor methods and provide a high performance platform. These results primarily proved that the proposed technology is reasonably comparable with the traditional detection methods, indicating the practicability of using the proposed method in clinical diagnosis. The detailed content described as follows:(1) We develop a novel electrochemical DNA (E-DNA) biosensor for simple, rapid, and specific detection of nucleic acids based on the proximity-dependent surface hybridization assay in order to widening the application area. This E-DNA biosensor was constructed by self-assembly of a 3'short thiolated capture probe on the gold electrode. DNA detection was realized by outputting a remarkable redox current of 5'ferrocene (Fc) tail labeled probe. When the target DNA was introduced into the system, it was complementary to the 5'Fc labeled probe at the one half-segment and complementary to the 5'short thiolated capture probe at the other half-segment, resulting in forming a stable duplex complex. As a result, the Fc probe was proximate to the electrode surface and the Faradaic current was observed. This E-DNA biosensor was proved to have a low detection limit (1 fM) and a wide dynamic range (from 1 fM to 1 nM) due to the stable hybridization mode. In addition, the sensing system could discriminate the complementary sequence from mismatch sequences, with high sensitivity, stability, and reusability. Compared with the conventional sandwich hybridization assay, this approach ensured the ferrocene marker to enough close the electrode surface and increased the charge transport efficiency as well as improved the sensitivity remarkably.(2) We develop a surface proximity-dependent hybridization electrochemical aptasensor method for the high sensitive detection of model analyte PDGF-BB based on the proximity effect, that is to say a pair of affinity aptamer probes simultaneously recognize the target molecules and form the proximity probes so that some complementary sequence hybridization enhanced the stability. When aptamer pairs simultaneously binding to the homodimer of PDGF-BB, the tail sequence are brought into close proximity with their local concentration increased substantially to allow the pair of tail sequences to hybridize together with the surface-tethered DNA strands. Then the ferrocene labels of the tail sequence are drawn close to the electrode surface and produce a detectable redox current. In conclusion, this method can be implemented to the high affinity detection of PDGF-BB, dynamically increased DPV current with increasing PDGF-BB concentration ranging from 1.0 pg/mL to 20 ng/mL, with the detection limit of 1.0 pg/mL.(3) A novel rolling circle amplification (RCA) immunoassay based on DNA-encapsulating liposomes, liposome-RCA immunoassay, was developed for ultrasensitive protein detection. This technique utilized antibody-modified liposomes with DNA prime probes encapsulated and a linear RCA reaction for the determination of prostate-specific antigen (PSA),. The results revealed that the technique exhibited a dynamic response to PSA over a six-decade concentration range from 0.1 fg mL-1 to 0.1 ng mL-1 with a limit of detection as low as 0.08 fg mL-1 and a high dose-response sensitivity.