| The core of proteomics includes large-scale discovery and high-throughput validation. Thelarge-scale discovery technology based on mass spectrometry has been rapidly developedrecently, but the accurate validation of target proteins remains a challenge. The existingvalidation methods are mainly the multiple reaction monitoring （MRM） mass spectrometrymethod and the immune detection method. MRM method has a low sensitivity, though it has agood capability to verify the target proteins in large-scale. So the preferred verificationtechnology is mainly the immune detection method as the sensitivity of immunoassay method isrelatively high. However, because this method relies on the expensive antibodies and itssensitivity is not enough to detect low abundance proteins, novel immunoassay methods areurgently needed.In this thesis, the new western blotting method and enzyme linked immunosorbent assay（ELISA） methods were developed by introducing gold nanoparticles and graphene oxide,respectively. Both types of nano materials have large surface area and can be used to amplifysignals by appropriate chemical modifications. The introduction of two primary antibodies andhigh proportion of HRP to second antibody reduce the cost greatly.The new immune detection methods include the dual signal amplification western blottingmethod and the triple signal amplification ELISA method based on graphene oxide and goldnanoparticles.In the study of new western blotting method, the gold nanoparticles and graphene oxide wereintroduced simultaneously. First, the proportion of primary antibody to antigen was increased bythe combination of gold nanoparticles modified with the primary antibody. Then, the proportionof second antibody to primary antibody was increased by the combination of graphene oxidemodified with the second antibody.The signal is amplified and the limit of detection was greatly lowered two times. And in theprocess of gold nanoparticles modified with the primary antibody, two kinds of primaryantibodies （the specific primary antibody and the assistant primary antibody） are introduced. Thecost is reduced and the signal is amplified at the same time because the specific primary antibodyhas a higher price compared to the assistant primary antibody.There are several advantages of the new western blotting method: amplifying the signal,reducing the cost, expanding the applications and enhancing detection ability. The results using β-actin protein in HeLa cell lysate as a model show that this new western blotting method has alimit of detection of2ng mL-1 with a good reproducibility, while in the traditional westernblotting method, the limit of detection is64ng mL-1.In the new ELISA method, Gold nanoparticles, graphene oxide and gold nanoparticles areintroduced sequentially. First, the gold nanoparticles are modified with two primary antibodies ina certain proportion to achieve first signal amplification and cost reduction. Then, the secondantibody and HRP in a certain proportion （the proportion of HRP to second antibody is100:1）are attached to gold nanoparticles to achieve a second signal amplification and cost reduction.Finally, graphene oxide is combined with the intermediate in the second step to achieve a thirdsignal amplification by increasing the proportion of second antibody to primary antibody.The new ELISA method is named as3G-ELISA （for the introduction of gold nanoparticles,graphene oxide and gold nanoparticles sequentially） method which is able to amplify signalsthree times and reduce cost twice compared to traditional ELISA method. The results usingHSP70protein as a model show that3G-ELISA method can detect HSP70antigen in serum witha limit of detection of64times lower than that of the traditional ELISA method, and the cost isnear one tenth of that of the traditional ELISA method. In addition, this method has a goodstability, and can be used to verify diagnostic markers, drug targets and low abundance proteins,and applied to clinical tests. |
PDF Full Text Request