| At present, cancer remains a major problem in the developed and most developing countries, and there are approximately 12 million people will be diagnosed with cancer and more 8 million were died every year. The focus of cancer therapy is the diagnosis, and the key question is detected and diagnosed at the early stage of cancer in good time. The early treatment is obviously superior than its advanced stage, and the costs are significantly lower too. People have been doing and exploring the tumor marker indicating the tumorgenesis so as to diagnose and provide effective treatment progressively in time. Tumor marker has been an effective basis for the diagnosis and effective treatment of cancer at its early stage in comparing with the changes of cytohistology at the early stage of tumor. And now, it has been recognized by most people that many tumor markers are one of the effective objective index in diagnosising at early stage of tumor.There are classified three kinds of tumor markers, the serum tumor marker, the cellular tumor markers, and the molecular and genetic tumor markers. There are many measurement methods to detect the tumor marker, including ELISA, RIA, FIA, RII, IHC, automatic immunoassay system and molecular biology techniques applied in the molecular biology research now. But all the methods sensitivity is limited, and it is difficult to achieve a certain degree to diagnose the cancer at its early stage effectively, or only detect at its advanced stage, or is short of the characteristics of high throughput and difficult to be applied on a large scale general census and screening because of its expensive costs.The bio-bar-code technology based on the single-strand oligonucleotide is researched applied very comprehensively at present. The bio-bar-code technology can improve the detection sensitivity greatly by its cascade amplification. The research on the the bio-bar-code technology combining with some other methods, such as the scanometric, silver stain and electrochemiluminescence, has made a great progress now, and the detection sensitivity is improved more than before methods mentioned above. The characteristics of genechip are of high throughput, and it can detect many target markers at the same time. It will be of very important significance to detect the concentration or content by the bio-bar-code technology with more high sensitivity combing with the high throughput of genechip.This research focuses on the detection the concentration of the protein antigen IgG applying with the bio-bar-code and genechip. It not only improves the detection sensitivity, but also be of the characteristics with more high throughput to detect many taget markers at one time, and this method improves the detection efficiency greatly. This research paves the way for the study and research on the detection of many protein target markers at the same time, and including the tumor marker.The major contents of this paper are shown as follows:1. The research and application of molecular beacon (MB) Molecular beacon is a special kind of bio-bar-code prober, and is thought to be a new type fluorescein-labeled bio-bar-code probes with the exquisite design. MB is of more strong ability to specifically identify the target sequence for its extraordinary hair-pin structure. And now it has became a powerful research tool in the molecular biology and biotechnology. We designed three different sequence MB for the experiment to detect the target antigen, and the detection limit is 6.67 fM. The research proved the method to be of the availability, sensitivity and speciality in measuring the target antigen.2. The research and application of the Quantum Dot (QD)Quantum Dot called semiconductor nanocrystal is a kind of stable, soluble nanocrystal made from the second to sixth group or third to fifth group element, and its diameter is between two to twenty nanometer. QD has been applied in many scopes comprehensively as the fluorescence probes now. There are many advantages of it comparing with the general fluorescence probes. We detected the target antigen applying with the bio-bar-code labeled QD, and the results are stable and reliable. But there are some questions on how to confirm the coupling quantities to be solved.3. The research and application of the oligonucleotide bio-bar-code assayDesigning three different sequence bio-bar-code, made of 60 bases, are prepared the genechip, hence there are three corresponding different sequence, consist of 40 bases, complementary to the sequence above. The monoclonal-antibody IgG was labeled on the amino-modified magnetic microparticles (MMPs) with C3 space arm, and the diameter of MMPs is two to three micrometer. And the single-strand DNA bio-bar-code was labeled the fluorescein Cy3 and the thiol-group on the terminal of its 5'and the 3'respectively. Then we modified gold nanoparticles (Au-NPs) with above dual-labeled bio-bar-codes and polyclonal-antibody IgG, Au-NPs diameter is about 13 nanometers. We detected the fluorescence intensity of the bio-bar-code to get the low limit of the target protein, and the minimum concentration is 133.4 fM. Ours research proved the availability, sensitivity and specificity of the bio-bar-code assay. It paved the way for the progressive research and application of the genechip combining with the bio-bar-code assay.4. The research and application of genechipWith the advent of post-genomic era, the developed high-throughput gene chip technology has the characteristics of analyzing and measuring many different target markers in the same sample at one time. We prepared genechip with the single-strand DNA bio-bar-code consisted of 60 bases designed by ours. The sensitivity of the method reached to 6.67 fM too, and the method is of more better specificity and high throughput. We applied with three different sequence bio-bar-code to detect the same target antigen and come to the consistent results, it proved the stability and reliability of the genechip technology.All in brief, the study proved the availability, stability, sensitivity and specificity of the microconcentration detection method of target protein antigen applying the bio-bar-code assay, quantum dot, molecular beacon and genechip technology with the biomaterials of MMPs and Au-NPs. It showed available and stable of applying the bio-bar-code assay with the genechip technology, the sensitivity could reach to fetomolar level, and about 6.67 fM. The method will be of full potential in the research and application in the future. And there are many questions to be recognized and solved in applying the MB and QD with the biomaterials mentioned above. The research have paved a well way for the next study and applying in screening and measuring the tumor markers in the clinic at future. |
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