Study On Signal Amplification Strategy For The Construction Of Electrochemical And Colorimetric Immunoassay

With the rapid progress of technology,there are many emerging immunological technique to develop.The determination of tumor marker plays an important role in clinical diagnoses,cure evaluation,and prognosis for the patients with certain tumor associated diseases.Similarly,the detection of biotoxin also is important and imperative due to the present serious environmental problems.The signal amplified strategy plays a key role in improving the sensitivity of the electrochemical and colorimetric immunoassay.In this thesis,a series of electrochemical immunoassays and colorimetric immunoassays were developed based on signal amplification technologies including enzyme catalyzed signal amplification,nano signal amplification and so on,which could improve the sensitivity and decrease the detection limit of target of the immunoassay.Hence,numerous of biomarkers containing alpha fetoprotein,carcinoembryonic antigen,biotoxin were effectively detected by those electrochemical and colorimetric immunoassays.Furthermore,we investigated the practical application of these methods,which open a new situation for the development of electrochemical and colorimetric immunoassay.There are six chapters in this thesis,and main contents are listed as follows:In the first chapter,a general introduction of tumor marker and biotoxin was made.Then,the detection principle,categories and application of the electrochemical and colorimetric immunoassay were described in detail,particularly,the several signal amplification strategies.Meanwhile,the research purpose and signification of this thesis were summarized.In the second chapter,a new electrochemical immunoassay based on nanogold/poly(o-phenylenediamine)/MnO2 hybrid nanostructures(GNPM)coupled with anti-CEA as nanotags and peroxidase mimics was designed with nano signal amplification.GNPM was not only used as carrier for assembling of nanogold and protein,but also used as peroxidase mimic for amplifying signal with the aid of hydrogen peroxide(H2O2).The assay was carried out by using differential pulse voltammetry(DPV)measurement method.The electrochemical immunoassay exhibited a wide linear range of 0.01?80 ng mL-1 with a low detection limit of 6.0 pg mL-1 carcinoembryonic(CEA).In the third chapter,a novel colorimetric immunoassay based on redox cycling signal amplification was proposed.Using sandwich-type mode,the producing signal of ferrocene labelled on detection antibody was greatly amplified with the aid of squaric acid and tris(2-carboxyethyl)phosphine(TCEP)redox cycling.Under optimal conditions,the absorbance increased with the increment of target alpha-fetoprotein(AFP,as a model)concentration in the sample.The dynamic concentration range spanned from 0.001?200 ng mL-1 with a detection limit of 0.6 pg mL-1.In the fourth chapter,a simple and feasible colorimetric immunoassay accompanying magnetic bead-based enzyme-chromogenic substrate system for sensitive determination of prostate-specific antigen(PSA,used as a model)was reported.Using a competitive-type immunoassay format,the carried glucose oxidase(GOx)-catalyzed oxidization of glucose leads to the formation of gluconic acid and H2O2.The latter can catalytically oxidize iron(?)to iron(?),which can rapidly(<1 min)coordinate with the SQA.Formation of the iron-squarate complex causes the color of the solution to change from bluish purple to bluish red accompanying the increasing absorbance with the increment of iron(?)concentration.On the basis of the SQA-iron(?)system,the absorbance intensity shows to be dependent on the concentration of target PSA.A linear dependence between the absorbance and target PSA concentration is obtained under optimal conditions in the range from 1.0 pg mL-1 to 30 ng mL-1 with a detection limit(LOD)of 0.5 pg mL-1 estimated at the 3Sbiank level.In the fifth chapter,a new colorimetric immunosensing platform accompanying enzyme cascade amplification strategy was fabricated for quantitative screening of small-molecular mycotoxins(aflatoxin B1,AFB1 used in this case)coupling with enzyme-controlled dissolution of MnO2 nanoflakes.The visual colored assay was executed by high-efficient MnO2-3,3',5,5'-tetramethylbenzidine(TMB)system(blue).In the presence of ascorbic acid,MnO2 nanoflakes were dissolved into Mn2+ ions,thus resulting in a perceptible color change from blue to colorless.The reaction could be weakened through ascorbate oxidase to catalyze ascorbic acid(AA)into dehydroascorbic acid,which indirectly depended on the concentration of ascorbate oxidase.Under optimal conditions,the absorbance decreased with increasing target AFB1 within the dynamic range of 0.05?150 ng mL-1 with a detection limit of 6.5 pg mL-1 at the 3Sblank level.In the sixth chapter,on the basis of the above experiments,a template-free just-in-time generation of copper hexacyanoferrate(?)strategy as oxidase mimic-based on colorimetric platform was designed with nano signal amplification and enzyme cascade signal amplification.The generated nanomaterial of the reaction between potassium hexacyanoferrate(?)and Cu2u can possess enzyme activity,catalyzing chromogenic agent for the development of coloration.The strategy,for the first time serve as oxidase mimic,for the first time is used in colorimetric filed for highly sensitive bioanalysis.In the presence of target aflatoxin B1(AFB1 as a model analyte),the capture AFB1-bovine serum albumin(AFB,-BSA)bound on the magnetic bead(MB)will compete with target for the detection antibodies,which are conjugated on AuNPs with alkaline phosphatase(ALP).The carried ALP on AuNPs initially catalyzes substrate(i.e.L-Ascorbic acid 2-phosphate,AAP)to AA,which could reduce K3[Fe(CN)6]to K4[Fe(CN)6],leading to decreasing of generated copper hexacyanoferrate(?).Then the solution can display a color change from blue to colorless.Under optimal conditions,the absorbance increased with increasing targetAFB1 within the dynamic range of 0.01?100 ng mL-1 with a detection limit of 5 pg mL-1 at the 3Sblank level.