MOF Mimic Enzyme-Based Label-Free Chemiluminescence Imaging Multiplexed Immunoassay

Chemiluminescence(CL)multiplexed immunoassay effectively obtain multiple analytes content based on the quantitative relationship between the light signal generated by the CL system and the concentration of multiple antigens or antibodies in the immune system.It has the characteristics of fast analysis speed and high detection throughput.However,the current CL multiplexed immunoassay generally uses labeling method,which selects natural enzymes to pre-label antibodies or antigens,and labeling steps are complicated.It is inevitable that the activity of antibodies or antigen molecules is easily reduced or even inactivated.Moreover,the stability of natural enzymes is poor,which greatly affects the detection results.Therefore,it is urgent to find artificial mimic enzymes that have strong stability and low cost,and can be applied to CL multiplexed immunoassay.Among many mimic enzymes that have been developed,MOF(Metal Organic Framework)materials with endogenous peroxidase activity have the advantages of simple synthesis,large specific surface area and good stability.MOF mimic enzymes have been widely used in the field of colorimetric sensors.At present,the application of MOF mimic enzyme in the field of CL imaging immunoassay has not been reported.In this thesis,three kinds of octahedral monometallic Cu-MOF,regular tetrahedral bimetallic CoCu-MOF,and spherical trimetallic CoCuFe-MOF were synthesized.Three MOF materials all have good peroxidase activity and were used as a mimetic enzyme to construct multiple label-free CL imaging immunosensors.The label-free multiple CL immunosensors show low cost,low sample consumption,high sensitivity,fast and accurate detection,and can realize the simultaneous detection of multiple disease markers.The detialed research work is as follows:(1)Through simple magnetic stirring at room temperature,a single metal Cu-MOF material with high mimic enzyme-like activity was successfully synthesized.It can be seen that the material has a regular octahedral structure with a size of about 500-600 nm through scanning electron microscope(SEM)and transmission electron microscope(TEM).In addition,UV characterization and colorimetric experiment have confirmed that the Cu-MOF material has good peroxidase activity.Cu-MOF material was dispersed into good biocompatibility of chitosan,and then droped into the array micropores of the epoxy silanized glass chip.Next,streptavidin solution was droped on the surface of the Cu-MOF-chitosan array film.Through the specific recognition of avidin to biotin,a variety of biotinylated antibodies were effectively immobilized on array chip.After blocking non-specific sites of array chip with bovine serum albumin,a multiplexed label-free CL imaging array immunosensor was successfully prepared.When the inbubation of corresponding antigen with array immunosensor,the formed immunocomplex will inhibit the contact between the CL substrate and the Cu-MOF mimic enzyme,thus resulting in the decreaese of CL imaging signals.The concentration of the antigen was different,and the value of the CL imaging signal was also different.Through collecting and analyzing the CL imaging signals value of each microwell in the array chip with CCD detector and the supporting software,the quantitative detection of multi-componen lung cancer markers(CEA,CA 125)can be realized.It has been proved that the CEA and CA 125 immunosensors show good linearity in the range of 0.010-15 ng/mL and 0.010-40 U/mL,and the detection limits of CEA and CA 125 were 1.0 pg/mL and 1.0×10-3 U/mL at the signal-to-noise ratio of 3.At the same time,the immunosensor array also exhibits good stability,specificity and acceptable reproducibility.And the results for detection of patient serum show the reliability in practical applications.The research provides a promising platform for the early diagnosis of lung cancer.(2)The bimetallic organic framework material(CoCu-MOF)was successfully synthesized by a one-pot synthesis method.It is observed that CoCu-MOF has a regular tetrahedral structure with uniform size of about 550 nm through TEM and SEM characterization.UV characterization and colorimetric experiment confirm that CoCu-MOF material has peroxidase activity.A CoCu-MOF-based label-free arary CL immunosensor was proposed for simultaneously detecting Corona Virus Disease 2019(COVID-19)antibody markers(IgG,IgM).First,the CoCu-MOF material was moidifed in the micropores of the array sensor by aid of good biocompatibility of chitosan.Next,streptavidin and biotinylated antibodies(IgG,IgM)were dropped sequentially to CoCu-MOF modified micropores of the array sensor.After online incubating corresponding antigens,the calibration curves of the two antigens were finally obtained through the CCD detector and supporting analysis software.The linear ranges of two markers are 0.0010 ng/mL-10 μg/mL(IgG)and 0.0010 ng/mL-15 μg/mL(IgM),and the detection limits for IgG and IgM are as low as 0.31 pg/mL and 0.36 pg/mL,respectively.The above results prove that the label-free COVID-19 immunosensor array based on CoCu-MOF has a wide linear range and high sensitivity.In addition,the sensor can still maintain high stability after being stored in PBS solution for six weeks.The specificity studies and spike recovery experiments were carried out on different interfering substances and actual serums of patients,which confirmed that the label-free immunosensor has good selectivity and practical ability for detectoion of actual samples,and shows a wide prospect in related fields.(3)Using the synthesis of bimetallic CoCu-MOF as a template,a trimetallic organic framework material(CoCuFe-MOF)was prepared by doping another metal(Fe).The material has a spherical structure with a size of about 500 nm.Utilizing the excellent peroxidase activity and long-term catalytic activity of CoCuFe-MOF material,a label-free arary CL immunosensor was proposed for simultaneously detecting COVID-19 antibody markers(IgG,IgM).Firstly,the material was dropped into the epoxy silanized microporous array to form a mimic enzyme sensor array.Then,a variety of biotinylated antibodies were successfully immobilized on mimic enzyme sensor array by streptavidin-biotin interaction.After blocking the unreacted active site of sensor array with bovine serum albumin,the label-free COVID-19 arary immunosensor was obtained.The formed immunocomplex will hinder the catalysis reaction of the CL substrate by CoCuFe-MOF minic enzyme,which in turn leads to a decrease in the value of CL imaging signal.The CCD detector was used to collect the CL imaging signals of each reaction microwell,and the data was analyzed through the supporting software to realize the high-sensitivity,high-throughput and rapid detection of the COVID-19 antibody markers(IgG,IgM).The label free CL immunosensor array exhibits a good linear relationship in the range of 0.0010 ng/mL-80μg/mL(IgG)and 0.00100 ng/mL-100 μg/mL(IgM),and the detection limits can reach 0.17 pg/mL and 0.21 pg/mL for IgG and IgM,respectively.After storing the immunosensor for six weeks,its signal value only decreased by 2.4%and 5.7%,which confirmed good storage stability.The proposedlabel freee COVID-19 CL immunosensor has the advantages of wide linear range,high sensitivity,good specificity and fast detection speed,and can be successfully applied to the detection of IgG and IgM in human serum samples.