Detection Of CD10and Lysozyme Biosensor Based On Signal Amplification Technique

Quick and easy detection of low concentration of tumor antigens has very important significance. Now there are many electrochemicaj detection methods being used for the analysis of antigen detection, however, the morphology and the kinetic parameters on the binding of antigen and antibody are restricted by these methods. Thus, in order to study more about the tumor antigen, we have done mainly two parts of work about the CD10antigen in this work. First, we studied the CD10Ramos antigen on tumor cells by using scanning electrochemical microscopy (SECM) Second, we used gold nanoparticles and magnetic bead as carrier signal amplification to study the CDIO antigen by quartz crystal microbalance (QCM). Besides,This paper also constructed a high sensitive biosensor using DNA rolling circle amplification method and realized the detection of lysozyme by using QCM. Introduce as follows now:I. A novel topography and image observing strategy of the recognition and combination of CDIO antigen and CDIO antibody (anti-CD10) by applying scanning electrochemical microscopy (SECM) was proposed. Based on a sandwich strategy, the sensor was fabricated by assembling anti-CD10on the gold electrode surface with the help of the protein thiol group firstly, then followed by the immobilization of CD10 antigen and anti-CD10/HRP modified gold nanoparticals (AuNPs) step by step. When it was immersed in the working solution (1.0mM Fe2+/Fe3+), a current signal would be detected under the potential of0.5V and then the topography of the specific image of the CD10antigen and anti-CD10was formed automatically. The AuNPs was modified on the sensor for its favorable biocompatibility. And horseradish peroxidase (HRP), a kind of enzyme, because of its diminutive size and remarkable stability, was used for catalyzing the oxidation of Fe2+to Fe3+by H2O2. Under the optimized conditions, the non linked analyte regions and the linked analyte regions of the modified gold electrode showed statistically distinguishable current expression levels. The linear range of the standard curve of antigen CD10was1×10-11～6.0×10-11M. The equation is Y=a+bX=0.0313+5.134X (X concentration of the CD10antigen10-10M, Y is the current growth range,△Icurrent), R=0.9998. The limited detection of antigen CD10is4.38×10-12M. It provided a visible method for observing the special nonrecogn’ition of antigen and antibody using CD10as the model.2. Based on the preliminary experiment with antigen CD10and gold nanoparticles, we proposed a new method of using QCM to detect the antigen. In this method, we compared the the differences between using gold nanoparticles and magnetic beads as carrier.We modified the DNA and antigen CD10on the gold nanoparticles, by DNA hybridization, the hairpin DNA continually turned on and connected to the gold nanoparticles, so the signal increased in the reaction. The experimental results showed that, under the optimal reaction conditions, when the other conditions were the same, the frequency change which used gold nanoparticles to connect to the DNA was higher, suggesting this method have the higher sensitivity to detect antigen if the quantity of the antigen connected to the chip was the same.3. On the basis of the above work, we explored a new method for detection of lysozyme. Compared with the previous two experiments, the focus of the experiment was no longer the detection of antigens, but the DNA rolling circle amplification and detection of lysozyme signal by QCM. The principle was as follows:after the capture DNA was modified on the chip, we dropped the ap tamer DNA chain to make a part of hybrid complementary, then dropped lysozyme in the solution chain, thus the capture DNA and aptamer DNA were partial dissociated. At the same time, we joined the ring-like initiated DNA. Under the action of the polymerase, with the capture DNA as the template, the continuous rolling circle replicated. When the reaction to a certain degree, we putted the chip into QCM, and then the streptavidin modified DNA was added. Because the modified DNA has long complementary strand, therefore, it would continue to bind to the chip, further improved the detection signal. When the adsorption layer on a chip increased, changing frequency of the detected signal value would change accordingly, therefore, we could realize the detection of lysozyme.