Development of fluorescent sensors-based methodologies for the application of food contamination and diagnosis

This thesis presents the development of a selective and sensitive fluorescent method for formalin in food and non-transferrin-bound iron determining in human plasma. The first approach, nitrogen-doped carbon dots (N-CDs) combined with the silver mirror reaction, served as the foundation for the development of a new fluorometric assay for the sensitive and selective measurement of formalin (FA). Formalin has been found to be used illegally to extend the shelf life and preservation of food. Consequently, the presence of formalin in food poses health risks to humans. The use of formalin as a food additive is prohibited by the Ministry of Public Health of Thailand. The first approach started with the synthesis of N-CDs using ethylene glycol and ammonia as carbon and nitrogen precursors via the hydrothermal method. For the detection mechanism, the strong fluorescence intensity of N-CDs was quenched after incorporating the silver ion (Ag+) and Tollens' reagent, which [Ag(NH3)2]+ complex on the surface of N-CDs was formed. Then, in the presence of FA, the reduction reaction of Ag+ formed metallic Agº and was released from the N-CDs surface, resulting in N-CDs' fluorescence being switched back on. The fluorescence intensity of N-CDs increased linearly with FA concentrations from 5 to 100 mg L-1, with a detection limit of 1.5 mg L-1. This method offers rapid, simple, sensitive, and selective detection of FA in food samples, consistent with the standard acetylacetone method. In addition, this proposed method is an up-and-coming technique for detecting FA in food samples. The approach extended to developing multispectral portable fluorometer-like fluorescence instruments for FA detection based on silver mirror reaction. The portable multispectral fluorometer, with twin excitation components and a detection unit capable of sensing six wavelengths, performs comparably to a traditional fluorometer for FA detection using N-CDs as a fluorescence probe. The advantages of the developed system include its portability, multispectral operation, and its affordability (that it is inexpensive). Subsequently, the development of the sample holder of a portable fluorometer for the detection chamber in the future analytical applications.
In the second study, free iron in human serum or non-transferrin-bound iron (NTBI), can generate free radicals and lead to oxidative damage to cells. Additionally, it is highly hazardous to a variety of tissues and a crucial indicator for thalassemia and Alzheimer's patients' iron-loading status. NTBI levels in healthy persons are often less than 1 µM; sophisticated technology and a multi-step sample preparation process are typically required for modern NTBI measurement. To overcome this limitation, we used tapioca starch to trap our BODIPY-PH onto the microcentrifuge tube lid as a fluorescent probe. As the concentration of NTBI grew (turn-on), so did the intensity of BODIPY-PH's fluorescence. Rapid analysis (about 5 minutes) with tiny sample quantities (10 µL sample in a total volume of 600 µL) is made possible by the created portable reaction chamber. Under optimum conditions, using the portable fluorescence device and fluorescence spectrometer, we could attain remarkable detection (LOD) limits of 0.003 and 0.0015 IM, respectively, under ideal circumstances.
The developed sensors show high selectivity for Fe3+ over other biomolecules and metal ions (Fe2+, C13+, Cu2+, and glucose). In serum samples from thalassemia patients, sensor performance matched the labeled value from standard methods. This fluorescent sensor offers excellent selectivity, sensitivity, and a short 5-minute incubation time, making it ideal for NTBI determination. The method is user-friendly, requiring minimal chemicals and low-cost equipment. This thesis also covers the development of fluorescence-based techniques for detecting formalin in food and free iron, as well as portable detection devices that are convenient, sensitive, and specific. We hope these techniques can be adapted for detecting a wide range of substances.