The Novel Immobilization Methods Of Indicators For Fluorescence Sensor And Probe Fabrication

There has been considerable interest in studying fluorescence sensors and probes, because they can be applied to a wide range of analytical tasks, such as clinical diagnosis, bioassay, environmental monitoring, etc. In the design and fabrication of fluorescence sensors or probes, immobilizing indicators is a crucial step. The immobilization methods play an important role in the property of the obtained sensors or probes. Aiming at the problems existed in present fluorescence sensors or probes such as dye leaching, photobleaching, this research developed several new strategies of immobilizing indicators in order to improve the ability to resist photobleaching, prevent dye leakage and shorten response time. The obtained sensors or probes were used to determine some medicines and dangerous substrands. The detail contents are as follows:1. A novel optical sensor was prepared by UV photopolymerization. A terminal double bond was attached to 3-amino-9-ethylcarbazole (AEC) via methacryloyl chloride. The resultant compound, 3-(N-methacryloyl) amino-9-ethylcarbazole (MAEC) was copolymerized with 2-hydroxypropyl methacrylate on surface-modified quartz glass plates by UV irradiation. The MAEC-immobilized sensor was obtained and used to determine picric acid based on fluorescence quenching. It shows a linear response toward picric acid in the range of 9.33×10-8 - 9.33×10-5 mol l-1 with high stability. Moreover, the leaching of dye from the optical sensor was hindered due to covalent immobilization. The response time of the sensor is about 3 min. The recovery of picric acid is 96.3% - 104.5%.2. Ag nanoparticles were used as bridges and carriers to covalently immobilize indicators. 3-Amino-9-ethylcarbazole (AEC) was anchored on the outmost surface of quartz glass slides as following procedures: cleaning of the glass slides, silanization in order to bring mercapto group, attachment of Ag nanoparticles, self-assembly of HS-(CH2)16-COOH, activation of the carboxyl group by using EDC and NHS, and covalent binding of 3-amino-9-ethylcarbazole (AEC). As a result, the indicator dye was covalently immobilized on the outermost layer of the optical sensor, which significantly shortens response and recovery time, enhances lifetime and effectively prevents leakage of the dye. In addition, the proposed optical sensor, which was used to rutin assay, showed long-term stability, high selectivity, sufficient reproducibility and reversibility. The linear range and detection limit are 2.0×10–6 - 1.5×10–4 mol l-1 and 8.0×10-7 mol l-1, respectively. In the present work, we have only explored the covalent immobilization of AEC; however, this approach should be readily adaptable to other indicators with primary amino groups.3. A novel technique of immobilization of indicators is investigated. Au nanoparticles are used as bridges and carriers for anchoring indicators. AEC was attached to the outmost surface of the glass slide via silanization, glutaric dialdehyde cross-linking procedure, action with chitosan, attachment of Au particles, self-assembly of HS-(CH2)11-OH, and coupling of AEC using cyanuric chloride. Thus, an AEC fluorescence sensor was obtained and employed to determine 2-nitrophenol. This sensor shows very rapid response, high selectivity, good reproducibility, reversibility and no dye leaching. The linear response to picric acid covers the range of 9.55×10-7 - 1.91×10-4 mol l-1 with a detection limit of 7.8×10-7 mol l-1. All of its merits make it adequate for practical application. This methodology can be readily adaptable to other indicators with primary amino groups.4. The polymer particles were prepared by copolymerization of phthalic aldehyde, allyl mercaptan and ethylene glycol dimethacrylate (EDMA) under heating. The fluorescence intensity of the polymer particles is very weak. A compound, which has isoindole unit in its molecule with strong fluorescence intensity, will form when the polymer particles are reacted with a compound with primary-amine group. This property can be used to assay primary amines. Aminoacetic acid was determined as an example. Experiments indicate that the fluorescence intensity will reach a stable value when the polymer particles (1.5 mg ml-1) are reacted with aminoacetic acid for 3 h in the mixed medium of ethanol and pH 8.0 phosphate buffer (1:4, V/V). The linear response range to dopamime was from 6.0×10-6 mol l-1 to 1.0×10-4 mol l-1 with a detection limit of 5.0×10-6 mol l-1. The probe had high selectivity to dopamime and could resist to the interference from ascorbic acid. This point might be a salient advantage of this method compared to electrochemical methods.