Multisyringe Flow Injection Analysis of Tropomyosin Allergens in Shellfish Samples

This paper presents the development and the application of a multisyringe flow injection analysis system for the fluorimetric determination of the major heat-stable known allergen in shrimp, rPen a 1 (tropomyosin). This muscle protein, made up of 284 amino acids, is the main allergen in crustaceans and can be hydrolyzed by microwave in hydrochloric acid medium to produce glutamic acid, the major amino acid in the protein. Glutamic acid can then be quantified specifically by thermal conversion into pyroglutamic acid followed by chemical derivatization of the pyroglutamic acid formed by an analytical protocol based on an OPA-NAC reagent. Pyroglutamic acid can thus be quantified between 1 and 100 µM in less than 15 min with a detection limit of 1.3 µM. The method has been validated by measurements on real samples demonstrating that the response increases with the increase in the tropomyosin content or with the increase in the mass of the shrimp sample.

Protein-templated copper nanoclusters for fluorimetric determination of human serum albumin

Copper nanoclusters (CuNCs) are attractive for their unique optical properties, providing sensitive fluorescent detection of several kinds of targets even in complex matrices. Their ability in growing on suitable protein and nucleic acid templates make CuNCs efficient optical reporters to be exploited in bioanalysis. In this work, we report the specific and sensitive determination of human serum albumin (HSA) in human serum (HS) and urine via CuNCs fluorescence. HSA is the most abundant protein in plasma, and plays a key role in the early diagnosis of serious pathological conditions such as albuminuria and albuminemia. Recently, HSA has become clinically central also as a biomarker to assess severity, progression, and prognosis of various cancers. We report the controlled and reproducible growth of CuNCs directly on the target analyte, HSA, which results in a fine dose-dependent fluorescent emission at 405 nm. The protocol is optimized in water, and then applied to serum and urine specimens, without matrix pretreatment. The method linearly responds within the whole concentration of clinical interest, with a sensitivity of 1.8 ± 0.1 × 10−3 g L−1 and 0.62 ± 0.03 × 10−3 g L−1 in serum and urine, respectively, and excellent reproducibility (CVav% ca. 3% for both). The assay is designed to have a single protocol working for both matrices, with recovery of 95% (HS) and 96% (urine). The stability of the fluorescence after CuNCs formation was tested over 3 days, displaying good results (yet higher in urine than in serum). Graphical abstract