Abstract
Precise quantitative and highly sensitive detection of small molecules (haptens) is highly demanded in medicine, food quality control, in vitro diagnostics, criminalistics, environmental monitoring, etc. In the present work, the magnetic method of particle quantification and the optical methods of spectral correlation and spectral phase interferometry complement each other for optimization of a quantitative assay for measuring concentrations of small molecules. The assay employs magnetic nanoparticles as labels in rapid immunochromatographic format. The approach was demonstrated with fluorescein as a model molecule. The interferometric label-free biosensors were employed for selection of optimal reagents that produced high specificity and sensitivity. The method of magnetic particle quantification counted the magnetic labels over the entire volume of the immunochromatographic membrane to provide their distribution along the test strip. Such distribution was used for optimization of such parameters as concentrations of the used reagents and of antibody immobilized on the labels, amount of the labels and conjugates of haptens with protein carriers to realize the advanced quantitative immunochromatographic assay.
Highlights
Small molecules, i.e., toxins, hormones, drugs, antibiotics and vitamins exhibit biological activity even in extremely low concentrations
The single-channel spectral-phase interferometry (SPI) and 3-channel spectral-correlation interferometry (SCI) biosensors were used for real-time monitoring of immunochemical reactions of the reagents, as well as to control reproducibility of Magnetic nanoparticles (MP) functionalization, of accessibility of antigen determinants in synthesized conjugates, etc
The carboxylated glass cover slip was activated with ethylcarbodiimide hydrochloride (EDC) solution, and fluorescein isothiocyanate (FITC)/bovine serum albumin (BSA) was covalently immobilized to it
Summary
Small molecules (haptens), i.e., toxins, hormones, drugs, antibiotics and vitamins exhibit biological activity even in extremely low concentrations. Development of methods for ultrasensitive quantitative detection of the small molecules is among the high-priority research tasks in many fields of science and industry [1, 2]. Liquid chromatography and mass-spectrometry traditionally used for detection of small molecules [3, 4] offer very high sensitivity and specificity. Long sample preparation, expensive equipment and reagents significantly limit their use
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