BDT、BDTO分子结构修饰后高发光性能纳米材料的筛选及其在血清PCT检测中的应用

Screening of BDT and BDTO derivatives with high luminescence performance and the application in serum PCT detection

目的对苯并[1,2-b:4,5-b′]二噻吩(BDT)和苯并[1,2-b:4,5-b′]-二噻吩1,1,5,5-四氧化物(BDTO)的分子结构进行修饰后筛选高发光性能的纳米材料,并观察其在血清降钙素原(PCT)免疫荧光检测中的应用效果。方法对BDT和BDTO进行三苯胺(TPA)、3位连接咔唑(PcZ)、9位连接咔唑(CzP)及叔丁基(TCzP)分子结构修饰,根据其发光性能相关指标[最大吸收波长(λabs)、最大发射波长(λem)、Stokes位移及溶液态、聚集态的绝对量子产率]、在水中的粒径分布情况,筛选CzP-BDT作为进行后续实验的纳米材料。以DSPE-PEG2000-MAL为包覆基质,采用改进的共沉淀技术及酰胺缩合一步法对CzP-BDT纳米粒子进行包装及抗PCT检测抗体标记。取浓度分别为45、30、<0.05 ng/mL的血清PCT样品和生理盐水各100μL,分别采用荧光微球FM02的免疫荧光分析法(简称FM02法)及CzP-BDT的免疫荧光分析法(简称CzP-BDT法)对不同标本进行免疫荧光检测。结果在470 nm激发光波长下,FM02法检测的发射光谱为500~650 nm,在525 nm处测得最大荧光强度;其中检测45 ng/mL的血清PCT样品时在525 nm处荧光强度为415.5,检测30 ng/mL和<0.05 ng/mL的血清PCT样品以及生理盐水时在525 nm处荧光强度分别为197.5、197.5、146.8。在340 nm激发光波长下,CzP-BDT法检测的发射光谱为500~650 nm,在525 nm处测得最大荧光强度;其中检测45 ng/mL的血清PCT样品时在525 nm处荧光强度为1064.0,检测30 ng/mL和<0.05 ng/mL的血清PCT样品以及生理盐水时在525 nm处荧光强度分别为766.2、664.0、456.9。两种方法均能检出45 ng/mL的血清PCT样品,但对于30 ng/mL、<0.05 ng/mL的血清PCT样品,只有采用CzP-BDT纳米粒子进行免疫荧光检测能实现有效鉴别并与生理盐水进行区分。结论成功制备8种BDT、BDTO化合物,并从中筛选具有高发光性能的CzP-BDT纳米材料;其在低浓度血清PCT免疫荧光检测的灵敏度高于FM02荧光微球,有望成为血清PCT免疫荧光检测的新一代发光材料。

Objective To screen the derivatives of benzo[1,2-b:4,5-b′]dithio-phene(BDT)and benzo[1,2-b:4,5-b′]-dithiophene 1,1,5,5-tetraoxide(BDTO)with excellent luminescence properties through structure-activity relationship analysis and to observe the application effect in serum procalcitonin(PCT)immunofluorescence detection.Methods Based on a family of BDT and BDTO derivatives,according to the relevant indicators of their luminescence performance[λabs,λem,Stoke shift and absolute quantum yield]and particle size distribution,we screened CzP-BDT as the nanomaterial with high luminescence performance for subsequent experiments.Using DSPE-PEG2000-MAL as the coating matrix,the CzP-BDT nanoparticles were packaged and labeled with anti-PCT detection antibody by using an improved co-precipitation technology and one-step amide condensation method.We took PCT samples at the concentrations of 45,30,and<0.05 ng/mL and normal saline(each was 100μL),and used fluorescent microsphere FM02 immunofluorescence analysis method(FM02 method)and CzP-BDT method for immunofluorescence detection of different specimens.Results Under the excitation wavelength of 470 nm,the emission spectrum detected by the FM02 method was 500-650 nm,and the maximum fluorescence intensity was measured at 525 nm.The fluorescence intensity at 525 nm in the 45ng/mL serum PCT sample was 415.5,and the fluorescence intensity at 525nm in the 30ng/mL and<0.05 ng/mL PCT samples and physiological saline were 197.5,197.5,and 146.8 respectively.Under the excitation wavelength of 340 nm,the emission spectrum detected by the CzP-BDT method was 500-650 nm,and the maximum fluorescence intensity was measured at 525 nm.The fluorescence intensity at 525nm in the 45ng/mL serum PCT sample was 1064,and the fluorescence intensity at 525nm in the 30 ng/mL and<0.05 ng/mL PCT samples and physiological saline were 766.2,664,and 456.9 respectively.Both methods could detect PCT-positive serum at a concentration of 45 ng/mL,but for lower concentrations of 30 ng/mL and<0.05 ng/mL,only the method using CzP-BDT nanomaterials could achieve effective identification,which showed better sensitivity and lower detection limit as compared with that of FM02-based fluorescent beads.Conclusion We prepare 8 kinds of BDT and BDTO compounds and screen out the CzP-BDT nanomaterial with high luminescence performance,and its detection sensitivity is higher than that of FM02 fluorescent microspheres in the low-concentration serum PCT,which is expected to become a new generation of luminescent material for serum PCT immunofluorescence detection.