HIV And HCV Detection In Blood By Microfluidics Technology Based On Nano Material

Background:On November21,2011, the2011world AIDS day report,which published by the United Nations AIDS program, showed that in2010, the global number of live HIV infection is34million, with2.7million new cases of HIV infection. Blood Epidemic investigations revealed that50%of HIV infections are co-infected with HCV. Blood transfusion is one of the main ways that HIV, HCV spread all over the world. The latest statistics from The world health organization (WHO) shows that globally nearly ten thousand people a year are infected with HIV/AIDS, hepatitis virus, syphilis for accepting unhealthy blood or blood products; Every year,5%～10%of the new HIV carriers are infected after blood transfusion or blood products acception. Blood safety problems are listed by the WHO as one of seven key works of the world health. After long-term unremitting efforts, the detection method of pathogens in blood was continuously improved and studied. The accuracy and sensitivity of these detection methods are greatly improved. The detection and diagnosis of HIV and HCV has made a great progress. But so far, no economic HIV and HCV detection technology has been developed that is suitable for wide application in developing and undeveloped countries. The development of a new kind of high throughput HIV and HCV testing technology is of very importance to prevent the spread of the two diseases.Objectives:In this study, we use nanometer material named polydimethylsiloxane (PDMS) to make microfluidic chips. By the use of the special physical characteristics of the fluid in micro channels on the microfluidic chips and the ability on macromolecule adsorption of the PDMS material surface, we perform the reaction of antigens and antibodies on the tiny cross regional of two micro channels on different microfluidic chips. The HIV and HCV antibodies in the plasma can be detected and evaluated qualitatively and half quantitatively. And assess the effects of microfluidic chip system on detecting HIV and HCV antibodies in human plasma samples. Explore the advantages and the best conditions of this new test system. By testing a lot of plasma samples, we verify the accuracy and stability of this new method. And we comparing the results with that of WB experiments.Methods:According to the size and shape designed by AutoCAD, we print the photoresist on the silicon plates by using Soft Lithography technology to make a mould for microfluidic chips. Pour the mixture of PDMS and curing agent liquid into the mould and heat at60℃for2hours to make a PDMS microfluidic chip. Using the same material to make a base chip by curing the mixture on a flat plate and make sure the substrate are formed uniformly thickness. Assemble the chip with channels and the substrate. Import the variety antigens of HIV and HCV into the micro channel and coat them onto the base flat plate. After that, import the plasma samples and the controls into channels vertical to the antigen stripes by another chip. Target antibodies being tested in the samples will be captured by the antigen stripes on the substrate. Fluorescein Conjugated monoclonal secondery antibodies are dropwise added to the cross area of reaction and specifically target to the antibodies being tested. After.incubation and wash, catch the result by useing a fluorescence microscope. The fluorescence intensity of the reaction region are related positively to the target antibodies in the sample. Analyse the result by Image Pro Plus6.0Image processing software to count fluorescence intensity of all the reaction points and compare with the controls (strong positive, weakly positive and negative) so as to process the qualitative and quantitative detection.Results:Analysis sensitivity experiments are carried out by using HIV and HCV recombinant antigens and monoclonal antibodies. When detecting the antibodies by the microfluidic chips system, the fluorescence intensity of the reaction point is positively related to the monoclonal antibody concentration. The dose response curves are set up and the Correlation coefficient is up to0.89. The lower limit of detecting antibodies is up to10ng/mL under the condition of without seal up after the antigen coating procedure, the testing result of29both HIV and HCV infected plasma sample are coincide with that of nucleic acid test; The conformance of108HIV positive samples and12plasma samples from normal people detecting result was100%; When detecting HCV antibodies within160samples, the conformance rate is94.5%, and there are false negatives and false positives. Ten copies of HIV positive, including strong positive, weak positive and negative samples were detected by the new method and simultaneously detected by WB. Tthe accordance rate is up to80%Conclusions:The microfluidic chips detection system based on rianotechnology is feasible when detecting HIV and HCV antibodies in human plasma sample. High degree of accuracy are showed in this experiment. There are many advantages when test by Microfluidic chip system. Such as, high throughput and time saving when used as blood screening tools, low cost and portable when used as tools to make a definite diagnosis, etc. This new detecting system has broad prospect in application of blood screening and clinical diagnosis. Especially in undeveloped and developing countries, where there are high popularity of HIV and HCV, the microfluidic chip detection system has a huge advantage.