The High Througth Put Screening Technology Based On Optical Self-encoding Microbeads

The discovery, screening and diagnostics of drug generally involve assay making on a great number of molecules. In conventional high throughput screening, each of the molecules is separated in the wells of a microtitre plate or separated by applying at a particular place in an array such as high-density DNA arrays or microchips at the price of expensive facilities and complicated practicing. In the second method, the reactions are carried out on individual microcarriers, for exemple, microbeads — each carrier having a particular molecule bound to its surface. The microbeads can be encoded for mixing the microbeads and subjecting them to assay simultaneously. When a microbead gives a positive reaction, reading the code can identify the substance on its surface. Obeviously this strategy is advantageously more flexibility because multiple discrete assays can be carried out simultaneously in mixing microvolume multi-samples. The present microbead encoding methods include chemical and physical encoding, but both of them are limited in application because of such problems as code stability, reaction compatibility and cost. The optical self-encoding microbeads are synthesized with styrene and its derivative co-monomers and they can be stably encoded by unique vibrational fingerprint of their spectra. Their chemical and physical properties are similar to the polystyrene resin. The reaction system, temperature, stirring speed, amounts of initiator and disperser are discussed in suspension polymerizing reaction of polystyrene. The latex polymerizing, disperse polymerizing and seeds polymerizing methods are compared with suspension polymerizing of polystyrene too. A novel dual initiators optimizing polymerizing way based on suspension and disperse polymerizing method is adopted. Twenty different self-encoding microbeads have been synthesized with styrene, and its six derivative co-monomers in combinatorial way. The identifiable and steady fingerprint of IR spectrum appears in 1400~1200cm-1 and 900~500cm-1. With GAUSS software in computer cluster server, the molecular vibration of styrene and its derivative co-monomers are simulated. The IR spectra of optical self-encoding microbeads are transformed to visible barcodes by identifying software. Based on optical self-encoding microbeads, two novel encoding combinatorial strategies named EPS(Encoding Position Scan) and PED(Position Encode Deconvolution) are put forward. The UV and fluorescence spectrophotometer analysis for measuring immunosorbent on polystyrene resins are compared. The influence of microbeads' size, coating temperature, content of IgG and pH value of coating buffer solution upon immunosorbent on polystyrene resins are discussed. Applying Ar+-laser fluorescence scan-imaging technology, immunofluorescence screening and fluorescence imaging on sub-millimeter class microbeads are explored successfully.The sensitivity of AP enzyme conjugated antibody in ELISA matrix tests on optical self-encoding microbeads proves 50 times higher than on 96-wells ELISA plate. The sensitivity of HRP enzyme conjugated antibody in ELISA matrix tests on optical self-encoding microbeads proves 62.5 times higher than on 96-wells ELISA plate.Twenty samples are coated on 20 different optical self-encoding microbeads respectively. All beads were mixed after coating and washing for multi-samples ELISA mix-screening. The sample that has positive reaction with assay is defined by identifying IR spectrum of the bead with the strongest signal.Twenty different optical self-encoding microbeads are coated with 20 different kinds of animal serum samples respectively. Three assays are added after mixing all beads for multi-assays & samples ELISA mix-screening. All beads are divided into three groups on average corresponding to three assays. The beads with strongest signal in each group are picked out for analyzing IR spectra. The samples that have positive reaction with different assays are selected and inspected in HPLC for quantitative analysis. The technology of multi-assays & sam...