Development Of Dynamic Light Scattering Immunosensor Based On Boronic Acid Affinity Reaction For The Detection Of Procalcitonin And Lactoferrin

Procalcitonin（PCT）,a propeptide of serum calcitonin,has been considered an ideal biomarker for the diagnosis and management of bacterial sepsis.Accurate determination of procalcitonin（PCT）is highly crucial in bacterial infection diagnosis.Many biosensors previously developed suffer from large sample consumption or lengthy waiting time,which raise difficulties for more vulnerable patients,such as infants,old people,and other critically ill patients.As an important nutritional fortifier,Lf is one of the star nutrients in milk powder.Thus,the rapid and accurate quantitative detection of Lf content in milk powder is crucial to the evaluation of its quality.However,most reported immunoassay methods for Lf quantitation suffer from cumbersome sample pretreatment or lengthy testing time,and also do not meet the requirements for rapid Lf detection in milk powder.Dynamic light scattering（DLS）is a common optical technique for measuring particle size and size distribution.Due to its relatively limited binding affinity between antigen and antibody,the sensitivity of DLS immunosensor for protein detection usually falls in the ng/m L level,which is difficult to satisfy the requirement for the detection of trace targets.By contrast,boronate affinity materials exhibit higher binding affinity to cis-diol-containing compounds,such as glycoproteins,because boronic acid ligands can covalently bind to cis-diols to form a five-or six-membered cyclic ester.Both of the procalcitonin and lactoferrin are glycoprotein substances that structurally contain cis-diol.Herein,we present an innovative boronate affinity recognition（BAR）-enhanced dynamic light scattering（DLS）biosensor to achieve ultrasensitive PCT and Lf detection.In this biosensing system,monoclonal antibody-modified magnetic nanoparticles（MNP@mAb）are designed as probes to capture target analytes from samples.Polyvalent phenylboronic acid-labeled bovine serum albumin（BSA@PBA）is used as scaffold to aggregate MNP@mAb and target complex because of the specific interaction of cis-diol-containing target with boronic acid ligands on the surface of BSA@PBA.The BAR-enhanced DLS biosensor shows ultrahigh sensitivity for PCT and Lf determination because the BAR has high binding affinity to glycoprotein substances.Under the optimal detection conditions,the limit of detection（LOD）of proposed DLS sensor for PCT detection was 0.03 pg/m L.The dynamic linearity ranged from 0.035 pg/m L to 250 pg/m L.The regression equation was described asΔD_H=6.985ln(C_PCT)+175.24,with a correlation coefficient of 0.989.The average recoveries of intra-and inter-assays ranged from 96.08%to 117.63%with the variable coefficient varying from 2.93%to 14.96%.The LOD value of the developed DLS sensor for Lf detection was as low as 1.36 ng/m L.The dynamic linearity was in a range of 1 ng/m L to 10000 ng/m L.The regression equation was expressed as y=7.4199ln（x）+142.85（R²=0.96）.The average recoveries for intra-and inter-assays ranged from 90.13%to 108.64%,and the CV values ranged from5.03%to 14.83%.These results indicated the acceptable accuracy and precision of the BAR-DLS biosensor for PCT and Lf quantitative detection in real samples.Moreover,the DLS biosensor also showed the high specificity for PCT and Lf quantitative detection;and the total detection time of the proposed method is less than 15 min with small sample consumption（about 1μL）due to its high sensitivity,rapid magnetic separation and aggregation of MNP@mAb and target complex triggered by BSA@PBA.In addition,the test data obtained from proposed method for detecting PCT in real serum samples and Lf in real milk powder samples were compared with the data obtained from the commercial immunoassay kits,such as chemiluminescence immunoassay and enzyme-linked immunosorbent assay.The results showed that the data from the developed method are consistent with those from commercial immunoassay kits.Besides,the proposed method uses the phenylboric acid as an alternative of coupled antibody to form the sandwich immunoassay for glycoprotein determination.In a word,this work provides a simple and universal platform for rapid and ultrasensitive detection of trace amounts of glycoproteins or other cis-diol containing substances by DLS biosensors.