Novel Signal Transduction Systems For Improving The Detection Sensitivity Of Immunoassay

Immunoassays have attracted much attention in the field of analytical testing owing to their unique advantages.Immunochromatography assay?ICA?and enzyme-linked immunosorbent assay?ELISA?are the two most commonly used and most mature screening platforms,which have been widely applied in clinical diagnosis,environmental monitoring,as well as food safety detection due to their rapidity,simplicity,convenience,strong specificity,high throughput and low cost.These two methods have become the major directions of industrialization and commercialization in the field of rapid detection.However,the biggest challenge of these two methods is their low detection sensitivities,making them impossible to enable accurate qualitative and quantitative analysis in detecting trace amounts of target analytes.As a result,the two methods cannot meet the requirements of certain practical applications.Therefore,to design the strategies for improving the sensitivities of these two methods are urgently required.Previous studies have shown that optimizing the sensitivity of detection signal is one of the effective ways to improve the detection sensitivity of analytical methods.This study intends to demonstrate the feasibility in improving the detection sensitivity of these two conventional immunoassays by optimizing signal generation mechanisms.In conventional ICA,gold nanoparticles?AuNPs?have been used as the most classical nano-label materials because of their advantages such as simple synthesis,low cost,and being easily recognized by naked eye.However,the biggest drawback is the weak color intensity of commonly used AuNPs with size of 30-40 nm,which results in relatively low detection sensitivity of AuNP-based ICA.To overcome this issue,Ru?phen?₃²⁺-doped silica nanoparticles?Ru@SiO₂?with ultrastrong luminescence were first prepared and used as novel fluorescent detection probes to fabricate ultrasensitive ICA for detecting enrofloxacin?ENR?residues in chicken tissues.Results indicated that the resulting Ru@SiO₂ exhibited a 23,000-fold improvement in luminescent intensity compared with free Ru?phen?₃²⁺.Through the conjugation of anti-ENR monoclonal antibodies,the as-prepared conjugates were applied as fluorescent probes to develop fluorescence ICA for screening detection of OTA in chicken tissues.Under the optimal condition,the linear detection range of this method was 0.025 ng/mL-3.500 ng/mL with a detection limit?LOD?of 0.02ng/mL in chicken extract,which corresponded to 0.12?g/kg in chicken meat.Additionally,the average recoveries of ENR-spiked chicken tissues ranged from78.54%to 118.63%,with coefficient variations of less than 13%.Moreover,a good linear correlation was achieved between our proposed fluorescence ICA and a commercial ENR ELISA kit in detecting ENR-spiked chicken samples.Secondly,a supermolecular self-assembly mediated cycle signal amplification system was introduced into conventional AuNP-based ICA to develop an ultrasensitive detection method for measuring carcinoembryonic antigen?CEA?in serum.Amantadine?ADA?labeled bovine serum albumen?BSA?was used to block the surface of anti-CEA detected mAb-AuNP conjugates,which were then applied to fabricate the sandwich ICA for detecting CEA;and then,the?-cyclodextrin coated AuNPs??-CD@AuNPs?were introduced to achieve the first signal amplification on the test line;later,tetrakis?4-carboxyphenyl?porphyrin?TCPP?and?-CD@AuNPs were introduced to achieve the secondary signal amplification;finally,TCPP and?-CD@AuNPs were repeatedly added to achieve cyclic signal amplification.Through the combination of TCPP-mediated supramolecular self-assembly to induce layer-by-layer AuNP accumulation on the test line,the optical densities on the T and C lines were significantly amplified,thus largely increasing the detection sensitivity of traditional AuNP-based ICA.Under the optimal conditions,results showed that the LOD decreased with increasing the numbers of cycle amplification,in which the LOD with naked eyes declined from 5 ng/mL to 0.1 fg/mL,whereas the LOD with strip reader also decreased from 0.5 ng/mL to 0.1 fg/mL.These results indicated that the detection sensitivity exhibited 6-7 orders of magnitude improvement than traditional AuNP-based ICA without any amplification.Besides,this method shows a wide detection range of 1×10^-7ng/mL-50 ng/mL.Compared with the clinically used Abcam CEA chemiluminescence kit,results indicated that the two methods have no significant difference in detecting different concentrations of CEA-spiked serums with a good linear correlation.Moreover,no significant cross-reaction was observed with other common disease protein biomarkers in serum.Conventional ELISA methods usually employ horseradish peroxidase?HRP?or alkaline phosphatase?ALP?to catalyze the chromogenic substrates for the generation of detection signal.However,the relatively weak signal intensity and single tonality result in poor detection sensitivity and are not suitable for naked-eye detection.To address these problems,AuNPs with strong light scattering ability were first synthesized and applied as detection probes to fabricate ultrasensitive dynamic light scattering?DLS?-based homogenous immunoassay for determining Listeria monocytogenes?LM?in lettuces coupled with large-volume immunomagnetic separation?IMS?.Under the optimal conditions,the LODs in PBS solution and LM-spiked lettuces were 3.5×10¹ CFU/m L and 2.2×10¹ CFU/g,respectively.The linear detection range was 3.5×10¹ CFU/g-3.5×10⁴ CFU/g with the average recoveries of 74.4%–123.4%.In addition,there were no significant cross-reactions with other common food-borne pathogens.Secondly,hydrogen peroxide?H₂O₂?-sensitive mercaptopicuric acid modified CdTe quantum dots?QDs?were prepared and used as immunoassay signal output to build a novel direct competitive fluorescence ELISA?FLISA?for determining ochratoxin A?OTA?in various foods.Results revealed that the synthesized MPA-modified CdTe QDs were sensitive to H₂O₂ concentrations with the minimum quenching concentration of 0.136?M.Through the combination of catalase?CAT?-mediated H₂O₂ decomposition,the H₂O₂-sensitive CdTe QDs were used as the detection signal output for developing direct competition FLISA method.Under the optimal conditions,the half inhibitory concentration(IC₅₀)and LOD of this method reached up to 0.53 pg/mL and 0.05pg/mL,which were about 283-and 300-fold than conventional ELISA,respectively.The linear detection range of this method was 0.05 pg/m L-10 pg/m L with the average recoveries of 87.7%-116.75%.In addition,a negligible cross-reaction was observed with other common mycotoxins.Moreover,there was an excellent linear correlation between our proposed FLISA and a commercial OTA ELISA kit in detecting OTA-spiked food samples.Finally,SiO₂ carrying poly?acrylic acid?brushes?Si O₂@PAA?were synthesized by reversible addition-fragmentation chain transfer polymerization?RAFT?method and employed as carriers to conjugate CAT and OTA haptens for preparing SiO₂@PAA@CAT@OTA as competitive antigen to construct an ultrasensitive direct competitive plasmonic ELISA?pELISA?for measuring OTA in foods and serum.Results showed that the resultant SiO₂@PAA exhibited an extremely high CAT loading level of 440.5±8.4?g per mg of SiO₂@PAA?corresponding to 1900 CAT coupling per SiO₂@PAA?.By optimizing the coupling ratio of OTA hapten on SiO₂@PAA@CAT surface,the nano-antigen of SiO₂@PAA@CAT@OTA_1:1:1 was obtained and exhibited small equilibrium dissociation constant of 3.4×10^-55 M to anti-OTA mAbs.Subsequently,a novel direct competitive pELISA for ultrasensitive detection of OTA was constructed using the composite nano-antigen of SiO₂@PAA@CAT@OTA_1:1:1 as a competitive antigen.Under the optimum conditions,the LOD for naked-eye detection was 10^-1818 g/m L,whereas the LOD for microplate reader was as low as 5×10^-2020 g/mL,which were about 7-8 orders of magnitudes than conventional ELISA.The linear detection range was from 10^-2020 g/m L to 10^-1818 g/mL.In addition,the average recoveries in three different OTA-spiked food samples?wheat,corn,rice?and serum samples ranged from 80.9%to 124.0%with a negligible cross-reaction with other common mycotoxins.Moreover,an excellent linear correlation was acquired between our proposed pELISA and a commercial OTA ELISA kit in detecting OTA-spiked real samples.