| Background and objectivesPHC (Primary Hepatocellular Carcinoma) is one of the common malignant tumors occurring in hepatocytes and intrahepatic cholangiocytes, whose mortality rate ranks second only to gastric cancer and esophagus cancer, and its disease incidence shows ascendant tendency. In our country, about 110,000 people die from liver cancer each year, which accounts for 45% of the liver cancer deaths around the world. At the present time, surgical therapy is still the best treatment for PHC, but it also brings the patients great suffering and heavy financial burden. To reduce the disease incidence and mortality rate of PHC and lessen patients'physical and psychological burden of surgery, it is urgently necessary to institute an effective cancer screening program. The most practical and simplest way of tumor screening is to detect the tumor markers.Tumor marker is one substance produced by cancer cells, which usually exists in the tumor cells or the body fluid of hosts in the form of antigen, enzyme, hormone or metabolin, and we can identify and diagnose tumor according to the biochemical and immune characters of them. Ideal tumor marker should be highly specific and suitable for detecting. The serological techniques can easily diagnose cancer. In terms of hepatoma, alpha fetoprotein (AFP), with the highest specificity, is the main index to diagnose liver cancer and is widely applied to cancer screening, cancer diagnosing, evaluating therapeutic effect and predicting recurrence. In general, the content of AFP in human blood serum is very low, it will increase the risk of having liver cancer if the content is greater than 20ng/ml. So, it is very important to evaluate the trace amount of AFP.For the same reason, we took the early HIV screening into consideration. Acquired immune deficiency syndrome (AIDS) is a kind of severe infectious disease caused by infection of human immune deficiency virus (HIV). In China, there are 370,000 patients and infestors in total by the end of October 2010, of which 130,000 are infected and 68,000 have died according to the Ministry of Health's statistics. The severe epidemic situation of some topical regions and some certain groups and the trend of the disease from high-risk groups to the general population put the prevention and cure in a grim and complex situation. There is no effective vaccine or medicine to prevent and cure the HIV yet because of its high mutability, so preventing infections has become the major method to control its spread. And the laboratory diagnosis of HIV infection is the predominant method for HIV prevention and control, so it is a must to set up a sensitive and practical program for detecting, diagnosing and blood screening.HIV can be categorized as HIV-1 and HIV-2 according to the different reaction of blood serum. What's more, HIV-1 exists widely all over the world and is the major cause of the wide-spreading of HIV. HIV, which is spherical and a cubically symmetrical icosahedron, has three kinds of structures like gag, pol and env which separately synthesize these three kinds of structural proteins:core protein, multimerin and envelope protein. The variability of HIV is very strong, and the extent of heteromorphosis of different genes is different. The env has the highest heteromorphosis rate, while gag and pol are relatively conservative. The p24 covers on the surface of the viral nucleic acid, which forms the outer covering of the virus nucleocapsid, then gene gag codes precursor protein, gene pol codes protease. So, the amino acid sequence of p24 is highly conservative, which makes it reveals nice regularity in the HIV detection. Antigen p24 occurs in the early stage of HIV infection which is earlier than the occurrence of antibody, which results from that the viruses copy quickly after being infected, and they combine with the highly contagious viruses. Early detection of p24 is of great significance in early HIV detection, blood screening, and diagnosis of HIV infection of new infants, treatment effect and process. But after an acute infection period of HIV, the specific antibody occurs in the body, and the p24 antigen and antibody compose immune complex which makes the concentration of free antigen lower, then common methods can't be used to detect the p24 antigen. So we need develop new methods with high sensitivity to detect directly the p24 antigen at low antigen concentration.As a conclusion of the work mentioned above, to evaluate and detect the AFP and HIV-p24 antigen is of great significance, it is also a hot topic of clinical examinations. In recent years, immunoassay has attracted the interests of many researchers because of its high sensitivity.Immunoassay is a major method to fulfill the rapid diagnostic of tumors and infectious diseases. It can easily recognize the patients' condition through obtaining the content and sorts of the diagnostic protein (DP, antigen/antibody) of certain disease and making analysis. In recent years, immune analysis method has got considerable development, a lot of immune analysis methods have been developed, such as agglutination reaction, Fluorescence immune assays (FLA), radio immune assays technique (RIA), enzyme-linked immune sorbent assay (ELISA, western blot (WB), chemiluminescence immune assay, electrochemiluminescence immune assay, immunochip, immunohistochemistry and so on. These methods are widely used in clinical diagnosis and greatly promote the update, automation, intelligentization and networking of clinical immune projects. But there are still quite a few deficiencies. For example, RIA is always accompanied with more or less radioactive pollutions; WB needs ionophortic separation, which is time-consuming and complex; immunohistochemistry needs microscope to observe and its preparation is complex; chemiluminescence and electrochemiluminescence needs expensive instruments; the other methods all have low sensitivity and specificity. Basic medical institutions especially the remote areas are limited by varieties of conditions. The common methods can not meet the needs of quick screening for the patients due to their low sensitivity, bad specificity and complex process. For fearing of being pried about privacy and discriminated, HIV high-risk groups are not willing to go to hospital or the centers for disease control. In conclusion, the now available methods can not satisfy the needs of quick screening of liver cancer and HIV. So it is urgent to develop a new technique and associated instruments which are highly sensitive, specific, cheap, quick, convenient and portable. It will be of great significance in preventing and controlling the spread of HIV.In 1990, Herry put forward the conception of immunosensor. It makes use of the high affinity and molecular recognition ability of antigen and antibody and combines traditional immune test technique with biosensor technique, which makes it be quick, highly sensitive and specific, convenient and cheap. It has been widely applied to food clinical diagnosis and food security, environmental monitoring and many other fields. This thesis aims to prepare a new self-assembled amperometric immunosensor which is sensitive to AFP, then, prepare a new HIV-p24 amperometric immunosensor through plating gold on the surface of the carbon electrode and the sandwich method. Methods1. Research on the new enzymefree amperometric immunosensor decorated with carbon nanotubesFirst, coat a layer of carboxylic carbon nanotubes (CNTs) on the surface of the glassy carbon electrode (GCE); then decorate the thionine with self-assembled monolayer to strengthen the detecting signals by making use of the electrostatic reaction between the DNA molecule with negative charge and the thionine with positive charge; and then use the amino of thionine to fix the nano-Au so as to fix the antigen; finally, use the BSA (Bovine Serum Albumin) to seal up the points which are not linked. Incubate the prepared electrode and the AFP standard, and then quantify the AFP through detecting the sensor's current.2. Research on the new sandwich HIV-p24 amperometric immunosensor decorated with glassy carbon electrode plated with goldPlace the polished electrode into the 1% chloroaurate solution, and exert constant potential to it for 30s by chronoamperometric method at -0.2V. Then dip the electrode into the anti-p24 Ab1, and incubate them at 4℃for one night. After that, wash it and eliminate the unspecific antibody, use the BSA to seal up the rest points which are not linked. Then the p-24 sensor is prepared. Incubate the sensor and p24 antigen which formates immune complex, wash it, and then incubate the sensor and the HRP-Ab2 marked with peroxidase, and formates sandwicn immune complex on the surface of the electrode. Take the hydroquinone as the signal molecule, and use HRP (horse radish peroxidase) to catalyze the hydroquinone and peroxide. The changing values of the current before and after the reaction is proportional to the content of enzyme on the surface of the electrode, while the changing value is connected with the amount of antigen, so we quantify the p24 according to the changing of the current.Results1. We successfully construct a new enzymefree amperometric immunosensor decorated with carbon nanotubes, which decorates the thionine by layer through making use of the electrostatic reaction between the DNA molecule with negative charge and the thionine with positive charge. When the thionine is decorated with five layers, the signal reaches the highest. At pH7.0, after being incubated for 30min, the sensor has nice detecting linear between 0.5ng to 25ng.2. We successfully construct a new sandwich HIV-p24 amperometric immunosensor decorated with glassy carbon electrode plated with gold. After plating gold on the surface of the electrode, the electrical conductivity and reversibility of electrode improve greatly, and nano-Au can nicely fix numbers of antibody so as to keep its bioactivity. The sensor reacts successively with antigen and Ab2, sandwich immune complex forms on the surface of the electrode. The reaction catalyzed by HRP between peroxide and hydroquinone shows that the oxydic current decreases and the reduced current increases. This sensor has nice detecting linear between 0.01ng/ml to 100ng/ml.ConclusionsThis research prepared the highly sensitive amperometric immunosensor used for detecting AFP and p24 in two different electrode-decorated methods. This sensor is expected to provide a new method to detect liver cancer and HIV. These two methods show:1) the DNA molecule can decorate the thionine multilayerly, and thus improving the signal intensity of the sensor.2) Plating gold on the surface of the electrode can form a layer of nano-Au on te glassy carbon electrode, which can conveniently fix the antibody. These two methods use the same principle that we can construct different amperometric immunosensors which detect differnent proteins by changing the decorating antibodies. It can provide reference for the other cancer markers and infectious diseases. |
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