Development And Application Of Novel Chemiluminescent Immunosensors

Immunosensors are a kind of biosensors which are constructed based on the recognition machanism between antibodies and antigens.They show great application prospects in the fields of clinical diagnosis,food safety and environmental monitoring.As a combination of traditional immunoassay and biosensing technology,immunosensors possess high specificity as well as simple analytical process,which have attracted intense research interest.At present,the research focus and hotspots of immunosensors are mainly aimed at improving detection sensitivity,expanding detection range,and implementing point-of-care testing(POCT).In recent years,immunosensors based on silica capillary and optical fiber have developed rapidly.As solid-phase carriers for biorecognition molecules,silica capillary and optical fiber have unique advantages: 1)they are mainly composed of silica,which can be easily modified with antibodies or antigens via mature silicon chemistry methods;2)they have excellent light guiding properties,making them ideal materials for construction of optical immunosensors,such as chemiluminescent immunosensor;3)the consumption of reagents and samples for immunoassays will be greatly reduced,which is especially important for the analysis of trace-amount biological samples.However,due to the inherent one-dimensional structure of silica capillary and optical fiber,the immobilization of biorecognition molecules is greatly limited,which further affects the analysis performance of immunosensors,resulting in low sensitivity and insufficient linear range.In our study,we focus on the construction and application of novel chemiluminescence capillary or optical fiber immunosensors,and propose several effective strategies to improve their analytical performance,and carry out integrated research on the instrumental platform.The details are listed as follows:(1)By combining signal amplification system with capillary based immunoassay,a chemiluminescent capillary immunosensor with ultra-high sensitivity was constructed.In this study,by using the silica capillary as the solid-phase carrier for biorecognition molecules,and the SA-B-HRP nanocomplex formed via self-assembly of streptavidin(SA)and biotinylated horseradish peroxidase(B-HRP)for chemiluminescent signal amplification,we achieved the quantitative detection of procalcitonin(PCT)in human serum.Besides high specificity,the constructed capillary immunosensor has advantages such as minimal sample consumption and ultra-high sensitivity.Compared with the conventional capillary immunosensor,the sensitivity is improved by more than two orders of magnitude.In addition,the results of PCT determination in human serum samples using the proposed method are well consistent with the clinical method,showing high accuracy.Therefore,the proposed signal amplified chemiluminescent capillary immunosensor has potential application in ultra-sensitive determination of PCT and other low abundance biomarkers.(2)A chemiluminescent optical fiber immunosensor(OFIS)with tunable detection range was constructed,which achieved sensitive and fast detection of three veterinary drug residues of chloramphenicol,sulfadiazine and neomycin in milk samples.In this study,optical fiber serves as both solid-phase carrier for biorecognition molecules and signal transduction element,which greatly reducing the complexity of the instrument.More importantly,the synergy between modulation of the length of the optical fiber sensing region and the number of fibers allows performing multiplexed immunoassays in an easily controllable manner over a tunable detection range from pg/mL to ?g/mL analyte concentrations.In addition,the combination of SA-B-HRP nanocomplex and OFIS has further improved the detection sensitivity.The constructed chemiluminescent OFIS with tunable detection range adopts an integrated design,which is suitable for field operation and can be used for multiple quantitative detection of both low-abundance and high-abundance analytes.(3)A portable pencil-like immunosensor(PPS)analytical platform was constructed for POCT of inflammatory biomarkers.The integrated PPS platform consists of four parts: a unique pencil-like optical-fiber-based sensor,a reagent strip consisting of a series of pencil-cap-like wells,a battery-powered photon counting detector and a data processing system.The pencillike optical-fiber-based sensor can realize the controllable extanding and retracting of the fiber probe via rotation;by immersing the fiber probe into the wells on the reagent strip sequentially,all reactions involved in the immunoassay can be completed;the compact and battery-powered photon counting detector can be used for chemiluminescent signal detection in the field;a touch screen computer was used for data processing.The whole PPS platform is 32 cm ~* 23 cm ~* 11 cm in size and only 3 kg in weight.In addition,the fiber probe in the PPS platform can be used up to 10 times sequentially,which reduces the experimental cost and avoids frequent replacement of probes.The PPS platform was portable,universal and operable with minimal technical skills,which meets the requirements of POCT and has broad application prospects in early diagnosis and screening of diseases,as well as monitoring clinical treatment effects.(4)An optical fiber immunosensor with all all-directional chemiluminescent collection was developed for ultra-sensitive detection of trace biomarkers.In this study,an all-optical chemiluminescent collection vial(CC vial)was constructed using a concave mirror and a coaxial tubular mirror as the bottom and the wall,respectively,which significantly increases the chemiluminescent collection efficiency,thus improves the sensitivity of optical fiber immunosensor.The detection limit of the proposed method is about two orders of magnitude lower than that of a conventional chemiluminescent optical fiber immunosensor.In addition,the proposed strategy to improve the sensitivity does not rely on complicated instruments or additional chemical reagents.It is economical and practical,and provides a powerful tool for ultra-sensitive detection of trace biomarkers.