建立循環流動系統壓電式生物感測器用於及時檢測病原菌大腸桿菌 O157:H7及登革熱病毒

Abstract

Pathogens infections have been a serious problem on the public health aspect. There are many classes of pathogenic microorganisms, including pathogenic viruses, bacteria, fungi, parasites, marine phytoplankton, and cyanobacteria, etc. in our daily lives. The pathogen transmission mechanism use not only environment factors, ex. water, air or soil, but also the food contamination, blood transfusion or contract infection etc… At every country, the deadly pathogens are usually accompanying economy damage and life loss. Hence, how to prevent and early detect pathogen infection and outbreak is important tasks. Biosensor is a novel technology for rapid detection of biomolecules. In this study, we develop a DNA piezoelectric biosensing method for In-time detection of pathogens or viruses in a circulating flow system. First, the specific probes of a 30-mer oligonucleotide with additional 12 deoxythymidine 5'-monophosphate (12-dT) is designed to solve steric interference on the biosensor system. The addition of 12-dT to the probes as a spacer, significantly enhanced (P < 0.05) the hybridization efficiency (H%). The results indicate that the spacer enhanced the H% by 1.4- and 2-fold when the probes are hybridized with 30-mer and 104-mer targets, respectively. The spacer reduced steric interference of the support on the hybridization behavior of immobilized oligonucleotides, especially when the probes hybridized with relatively long oligonucleotide targets. The DNA piezoelectric biosensing system is also applied in the detection of PCR-amplified DNA from real samples of Escherichia coli O157:H7. The resultant H% of the PCR-amplified double-strand DNA is comparable to that of the synthetic target T-104AS, a single strand DNA. Further, a circulating-flow piezoelectric biosensor, based on an Au nanoparticle amplification and verification method, is used for real-time detection of a foodborne pathogen, E. coli O157:H7. Continuing the first part study, a second thiolated probe with 12-dT, complementary to the target sequence, is conjugated to the Au nanoparticles and used as a “mass enhancer” and “sequence verifier” to amplify the frequency change of the DNA piezoelectric biosensor. The PCR products amplifing from concentrations of 1.2 × 10^2 CFU/ml of E. coli O157:H7 are detectable by the DNA piezoelectric biosensor. A linear correlation is found when the E. coli O157:H7 detected from 102 to 106 CFU/ml. The piezoelectric biosensor is also able to detect targets from real food samples. Besides bacteria detection, virus detection is a challenge in the filed of piezoelectric biosensor. It needs a lot of time to detect viruses by traditional methods. This part of study combined the previously developed In-time and on-line work, with Au nanoparticles layer by layer method for rapid detection of virus. In this study, a pair of universal primers of dengue virus envelope gene conserve region was used to amplify cDNA fragment, and two specific probes for the identification of dengue virus type II common subtypes are developed in Taiwan. According to pervious process, first probe hybridizes with the target to identify dengue virus in DNA piezoelectric biosensor surface, then, second probe conjugates with Au nanoparticles and hybridizes with target to enhance signal and verification. In order to increase more hybridization efficiency, the layer by layer method is recommended for the first probe to conjugate to Au nanoparticles in different sizes and to hybridize with free targets. Further, the second probes are hybridized with targets at other terminals. The structure is like dendritic form via layer by layer hybridization. Following layer by layer method, as low as 2.1×10^1 plaque forming unit (PFU)/ml DENV type 2 can be detected by the DNA piezoelectric biosensor. Linear correlation between frequency change and logarithmic number of virus concentration is found for DENV from 2.1×10^6 to 2.1×10^1 PFU/ml.