New Methods For C-Reactive Protein Detection Based On Gold Nanoparticles

C-reactive protein(CRP)is a very important biomarker of infection and inflammation,its concentration will rapidly rises to more than 1000 times within 24-48 hours after infection.In addition,as an important indicators for cardiovascular disease risk assessment,there is an important correlation between CRP level and cardiovascular disease.Accurate CRP measurement plays an irreplaceable role in disease diagnosis and treatment intervention.Therefore,it is necessary to develop rapid,low-cost,high sensitive and selective methods for CRP detection.At present,there are many nano materials include gold nanoparticles,magnetic nanoparticles,quantum dots have been used for CRP detection in a variety of detection methods such as chemiluminescence detection,fluorescence detection,electrochemical detection and so on.Among them,gold nanoparticles are characterized by simple synthesis method,superior photoelectric performance,good biocompatibility and various modification methods,which can be used for the development of different detection methods.Based on the existing detection methods,this thesis further explored the application of gold nanoparticles in CRP detection,and successful y established two new methods for CRP detection by utilizing the optical properties and carrier functions of gold nanoparticles.The main contents are as follows:1.Taking aptamer of CRP as the recognition molecule,a rapid and simple colorimetric detection method was developed based on the interaction between the aptamer and Au NPs.The detection principle is as follows: free aptamers can form complexes with gold nanoparticles through non-specific adsorption,which plays a certain protective role to the gold nanoparticles.After adding CRP into the system,due to the stronger affinity between the aptamer and CRP,some aptamers adsorb on the surface of gold nanoparticles will dissociate and bind to CRP,and the protective effect to gold nanoparticles is weakened,and the dissociation amount is positively correlated with CRP content.After adding a certain concentration of salt solution,the change of the protective effect of the aptamer to gold nanoparticles can be reflected in the change of the uv-visible absorption spectra of the solution.Then CRP can be quantified after the determination by the microplate reader.Under optimal conditions,the linear range of CRP detection was 0.1-1.0 ?g /m L with a detection limit of 0.06 ?g/m L.This method first applied ss DNA adsorption characteristics of gold nanoparticles to the detection of CRP,and successful y established a modify-free,low-cost and rapid CRP detection method,which has potential application value in the rapid diagnosis of inflammation.2.Using CRP monoclonal antibody as the recognition molecule and gold nanoparticles as the carrier,a double signal amplified CRP detection method was developed.CRP was detected by co-loading detection antibody CRPAb2 and signal molecule HRP on the surface of gold nanoparticles.Compared with the use of the enzyme-labled antibody HRP-CRPAb2,this co-loading method avoided direct labeling of antibody and improved the relative ratio between HRP and CRPAb2.Thus the same amount of CRP molecules to be tested could correspond to more HRP molecules which lead to the detection signal was improved obviously.Through this way,the detection sensitivity can be improved without the need to replace the antibody with stronger specificity.Under the optimal detection conditions,the linear range of CRP detection by this method is 0.625-40 ng/m L with a detection limit of0.45 ng/m L.Through the introduction of interfering protein,it is proved that this detection method has a high specific recognition ability for CRP.The precision of the assay has been confirmed for low coefficient of variation(CV),satisfying less than 10%(intra-assay and inter-assay),and the accuracy of assay meets the requirements with the mean recovery of the control was 100.94%.These results indicated that a high sensitivity and selectivity CRP detection method was successfully established.Here in this work,we designed two CRP detection methods,the colorimetric assay can achieve rapid detection of CRP with simple operation which is suitable for the on-site rapid detection of CRP.However,as the experiment process did not involve detection signal amplification steps,the concentration range of CRP detection is relatively high.In order to realize more sensitive detection of CRP,we developed another detection method of double signal amplification by utilizing the catalytic function of HRP and the load of it on gold nanoparticles,which significantly reduced the detection limit and could be used to detect CRP under the condition of higher detection sensitivity requirements.