The Study Of The Biochips For Non-invasive Prenatal Diagnosis On Inherited Disorder

The ultimate goal of biochip study is to integrate the entire analytical processes (i.e. sample preparation, biochemical reaction and detection) to achieve the so-called micro total analysis system (μ TAS) or lab-on-a-chip, the advantages of which are to achieve automation and miniaturization of the whole analytical process decrease waste of resource and avoiding contamination of the results. The miniaturization of biochemical reaction and detection has been demonstrated by many research groups. However, microchips for non-invasive prenatal diagnosis on hereditary disease are still on the horizon.Objectives: The purpose of this study is to investigate and optimize three independent steps, i.e., sample preparation, biochemical reaction and outcome diagnosis on the basis of non-invasive prenatal diagnosis: (1) sample preparation: to increase the efficiency of enrichment of fetal cells from maternal blood, which is achieved by centrifugation traditionally and is a main obstacle in instrumental miniaturization, to facilitate the automatization and miniaturization of fetal cell enrichment process; (2) biochemical reaction: to construct the microarray PCR reaction chamber using a non-microprocessing method to decrease the cost and facilitate to integrate it with thermo-controlling system; (3) outcome detection: to construct the solid phase diagnostic PCR microarray and further to optimize the constructing conditions of which. In the end, a technical platform of non-invasive prenatal diagnotic microarray was to be preliminarily set up based on the hereditary disease of Duchenne muscular dystrophy (DMD).Methods:Sample preparation: First, the experimental model of maternal peripheral blood was established. Then the appropriate concentration of red cell agglutinative agent was determined and the difference between antibodies of CD50 and CD45 in removing maternal white blood cells and enriching NRBC was further assessed based on this model. Afterwards, maternal white blood cells and mature red blood cells were removed with CD50 antibody and red cell agglutinative agent on appropriate concentration. Fetalnucleated red blood cells were obtained by further removing mature red blood cells on the basis of cell diameter disparity using miniature silicon membrane filtration microarray made by microprocessing. In the end, mimicked and real maternal blood was used to compare the enrichment efficiencies of NRBC and the clearance rate of WBC/RBC of our method with the conventional one.Biochemical reaction: Seal rings of in situ PCR and commonly used glass slides in microarray laboratory were used to construct PCR microarray reaction chamber. The material compatibility of the constructed chamber with PCR was further tested. Then the minimum limit of amplification detection was determined with optimized concentration of surface treatment agent BSA and amount of enzyme. After the yield of whole genome amplification of single cell using the microarray was determined following gel purification, further amplifications of multiple genes were performed using the previous product as a template, whose homogeneity and usability were also examined.Outcome detection: The solid phase diagnostic PCR microarray was constructed and the principle verification was performed using artificially synthesized oligonucleotide. We also systematically optimized and screened the film base thermostability, spotting concentration and truss arm length of the probe, and intensity of ultraviolet cross-linking for probe fixation on the constructed microarray. Based on the model of deletion form of DMD, the solid phase diagnostic PCR microarray was constructed. Meanwhile, DMD oligonucleotide microarray was also constructed for verifying the results from the solid phase PCR microarray. Finally, the solid phase PCR microarray for DMD and oligonucleotide microarray were used to test the product of whole genome amplification from a DMD fetus with deletion of exon 13 to test the clinical practicability of the constructed SP-PCR microarray.Results:Sample preparation: By continuous concentration gradient experiments, the optimal concentration of red cell agglutinative agent was determined to be 2 %, whose advantage was to avoid loss of nucleated red blood cells at most while agglutinating the mature red blood cells. During the step of removing white blood cells by magnetic sorting, CD50 antibody could remove white blood cells with the same efficiency as commonly used CD45 antibody, meanwhile decreasing the loss of NRBC by 10~15%. Mimic maternal bloodexperiments confirmed that the newly established concentration method of fetal cells had a enrichment efficiency of 65% or above while that of the commonly used method was less than 30%. The background nucleated cells were both 105""6 in the two methods. Experiments on real maternal blood samples showed that extra harvest of NRBCs 93.5 nucleated red blood cells per sample could be obtained with the new method as compared to the commonly used method.Biochemical reaction: The internal surface of PCR microarray chamber composed of glass, plastic coverslip, Gene Frame double-sided gum frame did not inhibit biochemical reactions. After the reaction conditions of in the constructed microarray PCR reaction chamber DOP-PCR were optimized, we found that the best amplification efficiency was obtained when adding 2.5U AmpliTaq Gold enzyme and 0.1 %BSA into the reaction system. This system enabled DOP-PCR microarray to perform single cell amplification, whose product length was about 200-2000bp and amplification efficiency was 104. Amplification of multiple genes could be further conducted using the product as the templates.Outcome detection: Compared PLL slides with silylated slides, PLL microarray was adopted as the base film for its better thermostability to the fixed probe. The best ultravoilet cross-linking conditions, truss arm lengths and spotting concentrations of the probe on PLL base film were 180mJ, 25-T and 20umol/L of respectively. The solid phase PCR microarray for detecting deletion of 9 exons in DMD was constructed and was further verified to diagnose a DMD fetus with a deletion of exon 13. The results were coincided with that detected by oligonucleotide microarray and clinical diagnosis.Conclusions: (1) A method of enrichment of fetal nucleated red blood cells from maternal blood has been established. This method has the advantages of avoiding the centrifugation step used in conventional methods, removing white blood cells with CD50 antibody, and filtrating mature red blood cells using the miniature silicon membrane filtration microarray, which is superior to the conventional methods in enrichment efficiency and speed and is easier to achieve miniaturization and automation. (2) Whole genome amplification of single fetal cell can be conducted in the constructed non-microprocessed PCR reaction chamber of microarray, whose amplification efficiency is as high as 104. The amplification product can be used for subsequent genetic analysis. Meanwhile, this easily made chamber is also easy to be integrated with thermo-controllingsystem into the PCR microarray. (3) The solid phase diagnostic PCR microarray for deletion form of DMD constructed according to the optimized experimental conditions combines genes amplification with outcome detection, which can increase the detection sensitivity of microarray and establish a technical platform for the solid phase diagnostic PCR microarray. 4. The optimized sample preparation methods, biochemical reaction conditions and disease diagnosis techniques provide a basis for establishing miniaturized microarray laboratory for prenatal diagnosis.