| Dynamic light scattering technology is a widely used method to characterize the particle size and distribution of particles（such as proteins,nanoparticles,etc.）.Because of its high sensitivity,simple operation,no washing,and easy data processing,it has attracted more and more researchers’attention in the field of analysis and detection.DLS immunoassay combines DLS technology and immunoassay technology.It has the advantages of high sensitivity,good specificity,and rapid response.It has been widely used in clinical diagnosis,food safety testing and environmental monitoring,etc.The existing DLS immunoassay mainly depends on the manipulation of the dispersion and aggregation of nanoparticles by targets to induce the change of average hydrodynamic diameter（HD）that is readily measured by DLS analyzer,thus achieving the quantitative relationship between particle size and the concentration of the target and the sensitive detection of the target.It has been reported that DLS immunoassay has higher detection sensitivity than traditional ELISA.However,the low immunoreaction efficiency and aggregation reaction efficiency of DLS probes in the existing DLS sensing system result in the limited sensitivity,which is difficult to meet the needs of high sensitivity target detection.Therefore,how to effectively amplify the aggregation reaction efficiency and immunoreaction efficiency of DLS probes in the immunorecognition process is the key to improve the detection sensitivity of DLS immunoassay technology.This study intends to systematically study new ideas and methods to improve the detection sensitivity of DLS immunoassays from the perspective of improving the immunoreaction efficiency and aggregation reaction efficiency of DLS probe.Development of strategies to amplify the nanoparticle aggregation for improving DLS signal transduction contributes to highly sensitive detection.Due to the specific interaction of boronic acid ligands with cis-diols,boronate affinity materials have attracted increasing attention in many important fields,such as disease diagnosis,cell targeting,and bacterial identification and killing.Different from traditional antigen-antibody reactions,boric acid affinity reactions can achieve polyvalent binding between boric acid ligands and cis-diol-containing molecules.Therefore,boric acid affinity materials can efficiently amplify the cross-linking of nanoparticles through specific recognition of cis-diol substances to achieve the sensitive DLS signal transduction.Glycoproteins are a class of cis-diol-containing biomolecules with important physiological functions,which are involved in various physiological and pathological processes and play an important role in the early diagnosis of diseases.As a glycoprotein in serum,amino-terminal pro-brain natriuretic peptide（NT-pro BNP）has been regarded as a clinically recognized biomarker for early diagnosis of heart failure.In this study,we successfully developed an ultrasensitive DLS immunosensor for the NT-pro BNP by combining boric acid affinity amplified nanoparticle cross-linking aggregation with DLS immunoassay technology.The detection principle is to use monoclonal antibody-functionalized magnetic nanoparticles（MNP@m Ab）to separate and enrich the target NT-pro BNP from serum,followed by the addition of phenylboronic acid cross-linking agent to induce the aggregation of MNP probes through boronic acid affinity reaction,thus achieving the concentration-dependent average hydrodynamic diameter change for the quantitative detection of targets.By virtues of the multivalent and fast boronate affinity recognition between glycoprotein NT-pro BNP and Si O2@PBA,the developed DLS immunosensor showed the advantages of ultrahigh sensitivity（7.4 fg/m L）,rapid response time（<20 min）,and small sample consumption（1μL）.Besides,the selectivity,accuracy,precision,reproducibility,and practicability of this immunosensor were well demonstrated by an assay of NT-pro BNP in human serum.The above results show that DLS immunosensor based on boronic acid affinity amplification has a good application prospect in the rapid and highly sensitive detection of glycoproteins in complex samples such as serum.Antigen-antibody reaction has the advantages of good specificity,but traditional antigen-antibody recognition mainly depends on the monovalent or bivalent binding of target antigen and antibody,which has the defects of low immunoreaction efficiency.The multivalent recognition strategy of antibodies can overcome the shortcomings of traditional antibody recognition and make the target recognition more sensitive and efficient.In addition,multivalent antibodies can simultaneously provide multiple antigen-binding sites,so they can act as cross-linking agents to induce aggregation of target antigens,thus triggering the nanoprobe aggregation.Nanobody multimerization is currently one of the main strategies to achieve multivalent recognition of antibodies.Compared with traditional monoclonal antibodies,multimeric nanobodies have higher affinity and stability,which helps to improve the sensitivity and reproducibility of detection methods.C4b binding protein（C4bp）is a common soluble glycoprotein with a multimerization domain.Taking advantage of the multimerization characteristics of C4bp,here we constructed a heptamer（A1-C4bpα）with seven antigen-binding sites that specifically recognize alpha-fetoprotein（AFP）.On this basis,the multivalent recognition strategy assisted by multimerization nanobody was further introduced into the DLS immunoassay sensing system,and the DLS immunosensor was successfully established with heptamer as biometric element and cross-linking agent,and antibody functionalized MNPs as labeled probe,which was used for rapid and sensitive detection of AFP in serum.The multivalent recognition between the nanobody heptamer and the target antigen and the magnetic enrichment of the analyte in complex samples make this method have the advantages of high sensitivity,strong specificity,and simple operation.Under the optimal conditions,our proposed DLS immunosensor provides excellent linear detection of AFP in the range of 0.06-512 ng/m L with a detection limit of 15 pg/m L.In addition,this method was used to detect AFP-spiked serum samples,and the results showed that the spiked recovery was 80.80%-107.08%,and the intra-assay and inter-assay coefficients of variation were 3.23%-15.77%,indicating that the method has good precision and accuracy.Moreover,there is no cross-reaction with other interfering serum proteins,indicating that the method has good specificity.The above results indicate that this method is suitable for the accurate quantitative analysis of AFP in actual serum.This method is of great significance for the early diagnosis,treatment and prognosis evaluation of primary liver cancer. |
