Investigation On New Enzyme-Linked Chromogenic Systems And Their Application In High-performance Colorimetric Immunoassay

Enzyme-linked immunosorbent assay?ELISA?has emerged as the most popular analytical technique with the detection of a color change since its development in 1971.It has been attracting more and more attentions in a variety of fields due to its naked eye visual inspection and less dependence on sophisticated instrument as compared with the assays based on fluorescence,chemiluminescence,surface-enhanced Raman spectroscopy.Many chromogenic substrates such as 3,3?,5,5?-tetramethylbenzidine?TMB?,o-phenylene diamine?OPD?,2,2?-azino-bis?3-ethylbenzothiazoline-6-sulphonic acid??ABTS?and so on have been used in ELISA.Although intense color and reasonable sensing performance are achieved in traditional ELSA,these substrates are expensive or highly toxic,and their products are not stable enough under the operation conditions.Therefore,it is still desirable for a new color develop system to overcome the above-mentioned limitations of the conventional substrates.As a matter of the fact,many efforts have been devoted to overcoming the shortcomings of traditional ELISA.Recently,a variety of plasmonic nanomaterials such as gold nanoparticles/nanorods and silver nanoplates are employed as color developing reagents for colorimetric immunoassays.However,these nanomaterials-based color development is not reliable enough because their stability in solution is apt to be affected by many parameters including electrolyte concentration,pH and interfering molecules in samples.Therefore,it is desired to develop simple,fast and stable chromogenic substrates for ELISA.The work intends to investigate new chromogenic systems,which are further employed to develop high-performance colorimetric immunoassay platform for fundamental and practical applications.The experimental works and results are as follows:1.An ultrasensitive colorimetric immunoassay based on glucose oxidase?GOx?catalyzed cascade formation of blue-black iodine-starch complexAs we all known,H₂O₂ is one of the end product of GOx catalyzed glucose oxidation?2?7)?80?+₂?^?3?27)?89?4)(8(6?84?(9+₂₂).H2O2 can oxidize I^-to iodine?I2?and then I^3-,which result in intense blue-black color development due to the formation of I₂-starch complex.Based on this principle,GOx can be used as an enzyme conjugation to design a colorimetric ELISA with KI-starch as a chromogenic substrate.We further combined the GOx-catalyzed KI-starch color development system with sandwich immunoassay to accomplish an ultrasensitive colorimetric detection of PSA.In this design,the more PSA is in the sample,the more GOx-conjugated avidin can be captured on the bottom of 96-well polystyrene plates,resulting in more H₂O₂ and subsequently more I₂-starch complex.Thus,the color intensity is highly correlated with the PSA concentration.The developed blue-black color could be easily distinguished from the colorlessness obtained at lower analyte concentrations with the naked eye.The lowest PSA concentration where the blue-black color can be clearly discriminated by the naked eye is 1 pg mL^-1.A broad linear dynamic range?R²=0.9945?is achieved in the range of 0 pg mL^-11 to 1?g mL^-1.An LOD is determined to be 0.46 pg mL^-1,which is much lower than the PSA cut-off value 4 ng mL^-1 in blood.Therefore,this work provides an attractive alternative for ultrasensitive biomarker detection in early cancer diagnosis,in particularly in those rural areas with constrained health care facilities.2.Metalionchelation-basedcolorgenerationforalkaline phosphatase?ALP?-linked high-performance visual immunoassaysIn the work,we have developed a novel colorimetric immunoassay strategy based on metal ion chelation-induced color generation and alkaline phosphatase?ALP?-catalyzed signal amplification.The intense purple-colored Cu?I?-bicinchoninic acid?BCA?complex is utilized as a promising chromogenic reporter for the visual ALP-linked immunoassay.In the presence of target,ALP is introduced to catalyze the cascade conversion of L-ascorbic acid 2-phosphate?AAO?to L-ascorbic acid?AA?,which results in Cu²⁺reduction to Cu⁺and subsequently in situ formation of purple-colored Cu?I?-BCA complex.The resulted complex is water-soluble and exhibits a strong absorbance at 562 nm due to the ligand-to-metal charge-transfer?LMCT?.The absorbance value is in turn proportional to the level of target analyte.Based on this concept,an ALP-linked colorimetric immunoassay is established with Cu?II?-BCA mixture as a color developing reagent for rabbit IgG detection,achieving a linear range from 0.1 ng mL^-11 to 25 ng mL^-1 and a LOD of 0.05 ng mL^-1.Its potentiality for practical application is also investigated by detection of a cancer biomarker-prostate specific antigen?PSA?in spiked human serum.A linear range from 0.5 ng mL^-1 to 25 ng mL^-1and a LOD of 0.38 ng mL^-1 are achieved,which is much lower than the cut-off value of PSA in human blood.The proposed method holds many advantages including low cost,good color stability,reliability and excellent compatibility with the existing ELISA platform,providing a promising colorimetric immunoassay platform for bio-chemical analysis in a variety of fields.In summary,we have developed two high-performance colorimetric immunoassay platforms.Very low LODs and wide dynamic ranges have been achieved by using PSA and IgG as the model target proteins.It is well compatible with the conventional ELISA platforms and all required reagents are commercially available in the market,the color is very intense and can be easily discriminated by a naked eye even at a very low level of targets.A quantitative detection can be accomplished with a very common UV-Vis spectrophotometer.Thus,the methods provide a new and attractive colorimetric immunoassay platform for fundamental and practical applications in a variety of fields.