| Nanocarrier systems for drug delivery combined with therapeutic and diagnostic capabilities,present an exciting new chance to deliver drugs to specific cells and identify disease sites with the rapid development and application of nanomedicine.Silica nanoparticles as ideal drug carriers based on its chemical stability,non-toxic,good biocompatibility,extraordinary drug loading capacity and high encapsulation efficiency and a series of excellent material properties are expected to be used in clinical applications.Although most nanocarriers are passive,active targeting patterns based on molecular recognition are rapidly advancing using antibodies or ligands,providing a highly effective route for targeted delivery of anticancer drugs.Hence,we synthesized a novel targeted nano-drug carriers based on silica nanoparticles as carriers and folic acid as ligands—Folate-functionalized silica nanoparticles（SiO2-FO）.In order to further promote the clinical application of SiO2-FO targeted nano-drug carriers,a full assessment should be evaluated with administration to address the clinical dosages and frequency of drugs for achieving the best therapeutic effect for humans.Therefore,the development of sensitive,rapid and cost-effective methods for SiO2-FO detection is the focus of this paper.In this paper,the synthesized SiO2-FO as hapten was firstly coupled to the carrier protein to become immunogen.Then the immunogen was injected into rabbits to produce highly specific antibodies.The antigen and antibody were as core reagents,three kinds of highly sensitive and selective immunoassays were established for trace analysis of SiO2-FO.The main contents of this paper are as follows:1.Preparation of SiO2-FO antigen and anti-SiO2-FO polyclonal antibodiesAccording to the sol-gel method,SiO2-FO was successfully synthesized as the hapten.The immunogen（SiO2-FO-BSA）and coating antigen（SiO2-FO-OVA）were obtained by respectively coupling the carrier proteins（BSA and OVA）through glutaraldehyde cross-linking method.The obtained SiO2-FO-BSA was then applied to immunize New Zealand white rabbits.Finally,the anti-SiO2-FO polyclonal antibodies were obtained when the titers reached 1:128,000.2.Indirect competitive enzyme-linked immunosorbent assay by antigen coated for quantitative detection of SiO2-FOIn this experiment,an indirect competitive enzyme-linked immunoassay for SiO2-FO analysis was developed based on the high specific reaction of the antigen-antibody and the amplified signal of the HRP-labeled goat anti-rabbit IgG,and the experiment conditions were optimized.The standard curve was established under the optimum conditions,the linear equation was A/Amax×100%=73–9.53logC(A and Amaxax is the absorbance value in the presence of standards and absence of standards,respectively;C is the concentration of SiO2-FO)with a correlation coefficient R2 is 0.998.The linear range was 0.18104ng/mL,the limit of detection(LOD,IC10)was 16pg/m L and the detection sensitivity(IC50)was258.8ng/m L.The recoveries of the samples were 97.3%111%and the cross-reactivities of two analogs were less than 0.01%.The results showed that the method is highly selective and accurate,which is suitable for the quantitative detection of SiO2-FO.3.FITC labeled fluoroimmunoassay for quantitative detection of SiO2-FOIn this experiment,fluorescein isothiocyanate（FITC）was chosen as fluorescent probe to couple with anti-SiO2-FO antibody.Based on the specificity of immunoassays and the sensitivity of fluorescence,a direct competitive fluoroimmunoassay using FITC as label was developed for the quantitative detection of SiO2-FO,and the experiment conditions were optimized.The standard curve was established under the optimum conditions,the linear equation was（F–F0）/(Fmax–F0)×100%=68–10.61logC(F and Fmax is the fluorescence intensity in the presence of standards and absence of standards,respectively;F0 is the fluorescence background value;C is the concentration of SiO2-FO)with a correlation coefficient R2 is 0.9991.The linear range was 0.074104ng/m L,the limit of detection(LOD,IC10)was 8.4pg/mL and the detection sensitivity(IC50)was50.12ng/m L.The recoveries of the samples were 93.3%108.8%and the cross-reactivities of two analogs were less than 0.01%.Compared with enzyme-linked immunoassay,this method has the advantages of simple,rapid and higher sensitivity,which provides another effective method for the quantitative detection of SiO2-FO.4.BODIPY-embedded polymer nanoprobe labeled direct competitive fluoroimmunoassay for SiO2-FO detectionIn this study,A novel boron dipyrromethene（BODIPY）-embedded polymer fluorescent nanoprobe（BPFNP）with strong luminescence,excellent photostability and easy modification was firstly fabricated via precipitation polymerization method by using acrylamide（AM）as monomer,ethylene glycol dimethacrylate（EGDMA）as cross-linker,boron dipyrromethene（BODIPY）as fluorophores and azobisisobutyronitrile（AIBN）as initiator.Then,a highly sensitive immunological quantitative detection system for SiO2-FO was developed based on the stability of fluorescence and the amplified signal of BPFNP,and the experiment conditions were optimized.The standard curve was established under the optimum conditions,the linear equation was（F–F0）/(Fmax–F0)×100%=58–12.63logC(F and Fmax is the fluorescence intensity in the presence of standards and absence of standards,respectively;F0 is the fluorescence background value;C is the concentration of SiO2-FO)with a correlation coefficient R2 is 0.997.The linear range was 0.018102ng/mL,the limit of detection(LOD,IC10)was 2.9pg/m L and the detection sensitivity(IC50)was 4.3ng/m L.The recoveries of the samples were 91%104%and the cross-reactivities of two analogs were less than 0.01%.Compared with the ic-ELISA and the FITC-labeled FIA,the sensitivity of the assays was improved by 60times and 12 times,respectively.The results showed that this method has higher sensitivity and lower detection limit,which provides a sensitive and stable detection technique for trace analysis of SiO2-FO. |
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