| Glycoprotein is a class of important biomolecules,which play the important roles in many physiological and pathological processes.The aberrant expression of glycoprotein in vivo is closely associated with various diseases,and is widely considered as one of the biomarkers for clinical diagnosis.Thus,it is of great significance to realize the point of care test(POCT)of glycoprotein for early diagnosis of disease and improve the survival rate of patients.At present,enzyme-linked immunosorbent assay(ELISA)is the most popular method for detecting glycoprotein markers in clinical diagnosis due to its unique advantages such as high throughput and excellent robustness.Moreover,conventional sandwich ELISA uses enzyme-antibody conjugate for catalyzing the chromogenic substrate to produce the color product as the output signal,whose sensitivity is generally within ng/m L toμg/m L.In addition,the long detection time of the conventional ELISA is difficult to meet the POCT requirement.Moreover,the sandwich ELISA needs a pair of matched antibodies,which is costly and difficult to preparation.The study proposed a sandwich immunoassay based on magnetic beads labeled the single antibody combined with boronate-affinity reaction as the recognition components,and a novel method was developed for the glycoprotein detection with high sensitivity and specificity.The immuno-magnetic nanobeads(MNB@m Ab)functionalized with monoclonal antibodies against human chorionic gonadotropin(HCG-m Ab)were synthesized,which can separate and concentrate HCG(glycoprotein)from complex samples.QB@4-APBA formed by modifing4-aminophenylboronic acid(4-APBA)on quantum beads(QBs)was used as the detection probe to bind to the cis-diol on the glycoprotein.Under the optimal conditions,the limit of detection(LOD)of HCG by this method was 0.19 m IU/m L,while that of traditional ELISA was 7.6 m IU/m L;the dynamic linear range for quantitative HCG was from 0.24 to 62.5 m IU/m L,which can be expressed as the following equation of y=21.858ln(x)+35.832(R~2=0.9747).The total detection time for HCG in serum by this method was 40 min,which was about half of that by traditional ELISA.The specificity experiment results showed that the proposed method has no obvious cross-reaction with other common biomarkers,saccharides and their derivatives.The results of inter-and intra-assays showed that the average recovery for determing HCG spiked serum samples by this method were from 94.77%to 114.9%,and the variation coefficients were from 0.41%to 9.19%,indicating an accepted precision and accuracy.The detection results by this method were further compared with those by the chemiluminescence immunoassay kit(CLIA),and the results showed that the results from two methods had good consistency.Additionally,a phenylboronic acid-induced nanoaggregate based dynamic light scattering sensor(PINA-DLS sensor)was further established for the ultra-sensitive detection of glycoprotein in serum samples.The immuno-magnetic nanoparticle(MNP@m Ab)functionalized with monoclonal antibodies against Alpha fetoprotein(AFP-m Ab)were synthesized.Firstly,the MNP@m Ab can separate and concentrate AFP from complex samples to form MNP@m Ab-AFP,and then,the phenylboronic acid crosslinker(PAC)of 8 arm PBA was used to bind glycoprotein.The PAC was prepared by coupling the phenylboronic acid onto the 8arm PEG polymers.which can induce the aggregation of MNP@m Ab-AFP via the cross-linking reaction between the MNP@m Ab-AFP and PAC molecular.The hydrodynamic diameter(D_H)before and after the reaction was recorded by a particle size analyzer,and then the quantitative detection of glycoprotein was conducted according to the D_H changes of positive and negative samples.Under the optimum conditions,the LOD of PINA-DLS sensor for the detection of AFP was 0.33 pg/m L and the quantitative linear range was from 0.48to 125 pg/m L.The average recoveries of the inter-and intra-assays were from 87.86%to 119.48%and the coefficient of variation was from 1.91%to 13.56%.Moreover,this method was further extended for the detection of other glycoprotein(NT-pro BNP and HCG).The results showed that the LOD for NT-pro BNP by the PINA-DLS sensor was 14 fg/m L and the quantitative linear range was from 0.0156 to 8 pg/m L.The LOD for HCG detection was 0.17 m IU/m L and the linear range of quantification was from 0.24 to 50 m IU/m L.Specificity experiment results showed that the above method was no significant cross-reaction with other common biomarkers,saccharides and their derivatives.Furthermore,the detection results by the PINA-DLS sensor were compared with those of the commercial kits,and the results showed that the two methods had good consistency.In addition,the overall detection process of the PINA-DLS sensor only needs 15 min.Since the proposed method had good detection sensitivity,specificity,and short detection time,it shows a broad application prospect for the POC detection of trace concentration of glycoprotein in real serum,and even whole blood samples. |
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