Novel Piezoelectric Biosensors For Point Mutant Detection And The Fabrication Of Release-controlled Nano-vesicle Probes

Sequence-selective DNA detection has become increasingly important as scientists unravel the genetic basis of disease and use this new information to improve medical diagnosis and treatment. Many pathogenic and genetic diseases are associated with changes in the sequence of particular genes. Among these changes, the point mutation, i.e. single nucleotide polymorphisms (SNPs), are the most abundant form of genetic variation. Achieving early, accurate, simple and rapid identification of these single-base mutations is of particular importance for the pathogeny and early therapy of corresponding diseases. Up to now, many techniques have been developed for SNP detection. However, these conventional procedures are applied to date in very few laboratories. Being generally time-consuming, semi quantitative, complicated, and requiring expensive instrumentation and technical skills. Accordingly, exploring some simple, cost-effective, accurate and easy to be clinically popularized detection methods for SNPs is still of considerable interest. In additional, the DNA piezoelectric biosensors have been widely used for the assay of DNA analyte and detection of SNPs due to its ease in operation, high mass sensitivity, rapid response, low cost and simplicity. In this thesis, we proposed a series of novel piezoelectric methods for DNA point mutation detection based on DNA ligase reaction and nanoparticle amplified DNA probes which are described in following sections:A novel piezoelectric method for DNA point mutation detection based on DNA ligase reaction and nano-Au amplified DNA probes has been proposed in chapter 2. A capture probe was designed with the potential point mutation site located at the 3'-end and a thiol group at the 5'-end to be immobilized on the gold electrode surface of QCM. Successive hybridization with the target DNA and detection probe of nano-Au labeled DNA forms a ds-DNA. After the DNA ligase reaction and denaturing at an elevated temperature, the QCM frequency would revert to the original value for the target with single base mismatch, while a reduced frequency response would be obtained for the case of the perfect-match target, thereby the purpose of point mutation discrimination could be achieved. The present approach has been demonstrated with the identification of a single-base mutation in artificial codon CD17 ofβ-thalassemia gene and a detection limit of 2.6 pmol/L of oligonucleotides was achieved. Owing to its ease of operation and low detection limit, it was expected that the proposed procedure might hold great promise in both research-based and clinical genomic assays. This system was then exploited for the regeneration of the sensor's surface and the purification of the oligonucleotides through the Fe3O4/Au nanoparticle assembly and the ligase reaction (in Chapter 3). Briefly, after the DNA ligase reaction and denaturing at an elevated temperature, the biotin-modified probe reacts with avidin on the electrode surface for the perfect match target, causing a change of the crystal frequency, while actually no frequency change for mismatch target would be recorded. Point mutation discrimination could be achieved successfully. The present approach has been demonstrated with the identification of a single-base mutation in -28 site (AAA mutates to AGA) forα-thalassemia gene which is one of the four common point mutation types in Southwest of China, and the wild type and mutant type were successfully scored.Due to label-free, sensitivity and high selectivity, molecular beacons have proven to be a useful method for detection of oligonucleotides. Here in chapter 4, we described a new sensitive, selective, reagentless DNA biosensor for detection of hybridization based on the allosteric switched of molecular beacon, and the presented biosensor can also be used for indentication of SNPs. An electrode-attached, molecular beacon-like DNA stem–loop labeled with biotin was attached on the piezoelectric DNA sensor as a mass magnifier and use the self-assembly layer of alkanethiols as insulator to form the molecular print structure. When the hybridization taken place for the perfect match target, the biotin was moved away from the electrode surface and react with avidin, thus the mass on the sensor surface was changed and cause the frequency shift. The DNA target, associating with theα-thalassemia gene containing the codon 142, was determined in the range of 1.41×10-11-1.41×10-7M, and the detection limit can be reached 10-11M.Besides, a novel release-controlled probe by using phosphatidylcholine and cysteine has been prepared (in Chapter 5). In the immunoassay, signal amplification by means of cysteine marker-loaded, analyte-tagged liposomes provide high sensitivity. Immunoassay will become easy and sensitivity by using this kind of system. The proposed experimental procedure provides a novel convenient method for the immumoassay.