Study And Application Of Four Immunochromatographic Assays Based On Novel Signal Output

Immunochromatographic assay(ICA)is a rapid immunoassay method combining the development of immunoassay,chromatography,and nanomaterials technology.Compared with the conventional instrument method,this method has many advantages,such as rapidity,simplicity,no expensive instrument,on-site detection and so on.The signal output of conventional ICA is absorption spectrum type signal output-colloidal gold,resulting in the sensitivity of this method is often low and qualitative.Improving the sensitivity of rapid detection method is a growing practical requirement.At present,there are three modes to improve the sensitivity of ICA:high affinity antigen and antibody,signal amplification,and novel signal output.Among them,the highly sensitive ICA technology based on the novel signal output can fundamentally solve the problem of low sensitivity caused by the poor conventional signal output,which is the research hotspot in the field of ICA.Based on the new signal output,this study has constructed four new ICAs,and applies them to the actual detection to evaluate their performance.In the first chapter of this study,the development and the prospect of ICA were reviewed.And the detection of the targets(Escherichia coli O157:H7(E.coli O157:H7),Salmonella Choleraesuis(S.Choleraesuis),human chorionic gonadotrophin(HCG),and β2-adrenergic agonist)was summarized.In the second chapter,an ICA based on gold-silver double test lines(T lines)was constructed to detect E.coli O157:H7 and S.Choleraesuis.In this study,the red colloidal gold of the conventional immunochromatography and a novel color signal output-blue triangle silver were used as the signal output.By spraying two T lines on the test strip,the purpose of simultaneous detection of E.coli O157:H7 and S.Choleraesuis was achieved.The limits of detection(LOD)of the double T lines ICA simultaneously detecting E.coli O157:H7 and S.Choleraesuis were 2.15×104 and 1.18×105 CFU/mL,respectively.Because conventional chromogenic lables were used,the sensitivity is not improved.Meanwhile,make full use of the color difference between the two lables,the "interference" phenomenon between T lines in the double T lines ICA for the simultaneous detection of pathogenic bacteria was verified and explained.In the third chapter,a novel chrysanthemum-like Au@polydopamine(AuNC@PDA)prepared via one-pot method was compared with the conventional flower-like Au@polydopamine(AuNF@PDA)prepared via three-step method.AuNC@PDA had more branch structures with more uniform petaloid shape and narrower particle size distribution compared with AuNF@PDA.In solution,the absorbance of visible light(400-800 nm)of AuNC@PDA was 3 folds higher than that of AuNF@PDA;On the nitrocellulose(NC)membrane,the grayscale of AuNC@PDA was approximately 2.5 times higher than that of AuNF@PDA.These results indicated that AuNC@PDA had superior morphological and optical performance.The novel AuNC@PDA was utilized as a lable compared with conventional AuNF@PDA in ICA.The LOD of ICA based on AuNC@PDA and AuNF@PDA were 1.59 and 5.13 mIU/mL,respectively,when HCG was detected as the target.The ICA based on AuNC@PDA had threefold improvement in sensitivity compared with that based on AuNF@PDA.In the fourth chapter,based on the property of colloidal gold can quench fluorescent microsphere,a novel fluorescence quenching ICA had been constructed to detect β2-adrenergic agonist,upgrading the conventional ICA to fluorescent ICA.In this study,fluorescent microsphere was sprayed and fixed on the T line and C line(control line)of the test strip,and then colloidal gold was used as its quenching agent.When β2-adrenergic agonist present in the sample,colloidal gold labeled antibody could not combine with the complete antigen coated on T line,and the colloidal gold could not quench the fluorescent microsphere on T line,resulting in a strong fluorescence signal of T line.On the contrary,when β2-adrenergic agonist is absent in the sample,colloidal gold could quench the fluorescent microsphere on T line,which could quench the fluorescence of the T line,resulting in a weak fluorescence signal of T line.In other words,fluorescence quenching ICA transformed the traditional "turn off" mode of small molecule competition inhibition detection into a more favorable "turn on" mode,which improved the detection sensitivity.When clorprenaline,a β2-adrenergic agonist,was detected as the target,the LOD of this method is 0.12 ng/mL.In the fifth chapter,the conventional fluorescent microsphere was upgraded to new aggregation induced emission(AIE)fluorescent microsphere based on the AIE materials,and the AIE-ICA was constructed for the first time.The microencapsulation method was used to entrap a large number of AIE dyes into the microspheres,ensuring uniform and surface modification of the spheres.Then it was applied to the detection of E.coli O157:H7 by ICA platform.Generally,the fluorescence emission performance of microspheres is in direct proportion to the amount of fluorescent dye embedded in the microspheres.While a large number of fluorescent dyes embedded in a single microsphere is unfavorable to the conventional fluorescent microsphere,because the traditional fluorescent dyes have aggregation-caused quenching(ACQ),resulting in a dilemma between the performance of conventional fluorescent microspheres and the amount of embedded dye.However,for AIE microspheres,the fluorescence performance is only proportional to the amount of dye embedded in the microspheres,which makes the fluorescence performance of AIE fluorescent microspheres theoretically much better than that of conventional fluorescent microspheres.The maximum fluorescence intensity of AIE fluorescent microspheres prepared in this study was 9 times of that of conventional fluorescent microspheres,and the quantum yield of AIE fluorescent microspheres was 4.5 times of that of conventional fluorescent microspheres.When E.coli O157:H7 was detected as the target,the LOD of AIE-ICA was 3.98×103 CFU/mL,which was 11 times and 7 times lower than the two commercial fluorescent microspheres(Ocean microspheres and Merck microspheres),respectively.In the sixth chapter,the work of this paper was summarized.The lable and sensitivity were regarded as context cues,carding the logical relationship of work ideas.And the prospect of improving the sensitivity of ICA in the future was anticipated.