| Brain damage including cerebrovascular disease and traumatic brain injury is causing terrible suffering to millions of people every year and the cases of severe brain injury exhibit poor prognosis and high death rates.Conventional methods in diagnosis of brain injury,such as Computed Tomography(CT)and Nuclear Magnetic Resonance(MRI)mainly based on imaging analysis are not applicable in basic medical institution due to the low efficiency and high-tech device dependence,especially for those patients with acute brain damage.Early and rapid diagnosis played a critical role in brain damage screening,timely treatment and prognosis.S100B protein(S100B)and neuron-specific enolase(NSE)have been reported as valuable biomarkers in clinical detection of brain damage.Thus,it is of a desperate need to develop a rapid detection method of S100B and NSE that with high sensitivity and accurate quantification.Lateral flow immunoassays(LFIA)have been widely used in the field of biological detection because of simplicity,rapidity and low cost.However,traditional LFIA using tracer like colloidal gold can only provide qualitative("yes/no" signal)or semi-quantitative information,meanwhile,the performance such as accuracy and sensitivity do not meet current requirement for clinical detection anymore.In this study,Fe3O4-Au composite nanoparticles(core/shell)(GoldMag)was modified with copolymers poly(4-styrenesulfonic acide-co-maleic acid)(PSS-MA),further conjugated with specific antibody.By using these antibody-nanoparticle composite,a rapid and sensitive LFIA system based on magnetic quantification was established for S100B and NSE detection.And the viability of this detection system in brain injury scanning was also evaluated through clinical trials.This study is mainly as follows:1.Surface-Modification of GoldMag.PSS-MA was firstly used in GoldMag nanoparticles modification.PSS-MA-GoldMag have superparamagnetic and good magnetic field response.And there are excellent dispersibility and stability of PSS-MA-GoldMag in buffer solution from pH 6.0 to pH 9.0.2.Establishment of LFIA system for quantitative analysis of S100B.The condition of antibody conjugation and LFIA establishment were optimized,followed by evaluation of the detection system performance.This LFIA system had detection range of S100B from 0.1 ng/mL to 3 ng/mL with correlation coefficient of 0.97,with significant correlation between MAR and concentrations of S100B.The value of the detection limit for S100B was 0.065 ng/mL.The recoveries were between 99.6 and 103.7%.The coefficient of variation of intra-batch precision and inter-batch precision of the detection system were less than 15%.The results of the interference experiment showed that there is no interference from serum with the S100B test strips.3.Establishment of LFIA strip test system for NSE determination.Under the optimal condition of antibody-nanoparticles conjugation and LFIA system,the detection rangeof NSE was from 5 ng/mL to 160 ng/mL with correlation coefficient of 0.97 and detection limit of 3.59 ng/mL.The recoveries were between 95.6 and 99.7%.The coefficient of variation of intra-batch precision and inter-batch precision of the detection system were less than 15%.The results of the interference experiment showed that there is no interference from serum with the NSE test strips.4.Application of the LFIA in clinical trial,that based on PSS-MA-GoldMag for S100B and NSE magnetic quantitative detection,we provide a quantitative The comparison of the S100B and NSE magnetic quantitative detection system with Electrochemiluminescence immunoassay(ECLI)was carried out by measuring the concentration of S100B and NSE in 216 clinical samples,respectively.The results show shown good conformance between two different methods with correlation coefficient of 0.9550 and 0.9534 respectively.The results indicate that this LFIA system is comparable with electrochemical luminescence detection system in terms of sensitivity and accuracy.Through statistical analysis of clinical samples,combined with the clinical manifestations of patients,we scored NIHSS for neurological deficit.The result shows a significant correlation between S100B and NSE(p<0.05).The level of S100B and NSE in serum were not related to age and sex.S100B and NSE levels were significantly correlated with NIHSS(p<0.05,respectively),indicating that the level of S100B and NSE in serum were related to the development of brain damage which can be used as biomarker in the diagnosis of brain damage.The ROC curve reveal that S100B and NSE are valuable outcome predictor for patients with brain damage with cutoff value of 0.140 ng/mL and 9.33 ng/mL for S100B and NSE respectively.Application of magnetic nano-materials as a tracer in development of full quantitative chromatography detection system is a difficult problem that scientists are looking forward to breakthrough for these years.The results show that PSS-MA-GoldMag is more suitable for coupling antibody of S100B and NSE comparied with other polymer used in our team previously.The magnetic quantitative lateral flow immunoassay system based on PSS-MA-GoldMag for S100B and NSE detection possess many advantages such as simple,rapid,sensitive and portable which can provide the final results in 30 minutes.This LFIA system could be served as a very promising point of care testing in early diagnosis of brain damage. |
PDF Full Text Request