Gold nanoparticle formation as an indicator of enzymatic methods: colorimetric l-phenylalanine determination

Alba Martín-Barreiro,Susana De Marcos,Javier Galbán

doi:10.1007/s00216-022-03900-3

Alba Martín-Barreiro, Susana De Marcos + Show 1 more

Open Access

https://doi.org/10.1007/s00216-022-03900-3

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Abstract

An enzymatic-colorimetric method has been developed based on the reaction between l-phenylalanine (l-Phe) and the l-amino acid oxidase (LAAO) in the presence of Au(III), which has led to the formation of gold nanoparticles. The intensity of the localized surface plasmon resonance (LSPR) band of the generated nanoparticles (550 nm) can be related to the concentration of l-Phe in the sample. The mechanism of the LAAO-l-Phe enzyme reaction in the presence of Au(III) has been studied through the evaluation and optimization of experimental conditions. These studies have reinforced the hypothesis that the catalytic center of the enzyme helps the Au(III) reduction and, thanks to the protein, the Au0 form is stabilized as gold nanoparticles (AuNPs). In the calibration study, a sigmoidal relationship between the concentration of the substrate and the LSPR of the nanoparticles was observed. The linearization of the signal has allowed the determination of l-Phe in the range from 17 to 500 µM with an RSD% (150 μM) of 4.8% (n = 3). The method is free of other amino acid interference normally found in blood plasma. These highly competitive results open the possibility of further development of a rapid method for l-Phe determination based on colorimetry.Graphical abstract

Highlights

Colorimetric biosensors stand out for possessing several key factors such as simplicity and a rapid response through visual detection, without the need for instruments for such detection, and the possibility of quantification using simple equipment
Current strategies used for colorimetric determinations based on oxidase reactions need coupling with an enzyme HRP-based enzyme indicator reaction, which catalyzes the conversion of chromogenic substrates in colored products after the oxidation process [1, 2]
Quantitative determination of l-Phe in physiological fluids is crucial in the diagnosis and therapy of disorders of phenylalanine catabolism, such as phenylketonuria (PKU), a genetic disorder characterized by a deficiency in the liver of the hepatic enzyme phenylalanine hydroxylase that catalyzes the conversion of phenylalanine into tyrosine, leading to an excessive l-Phe accumulation in the serum (> 120 μM) [18]

Summary

Introduction

Colorimetric biosensors stand out for possessing several key factors such as simplicity and a rapid response through visual detection, without the need for instruments for such detection, and the possibility of quantification using simple equipment. Quantitative determination of l-Phe in physiological fluids is crucial in the diagnosis and therapy of disorders of phenylalanine catabolism, such as phenylketonuria (PKU), a genetic disorder characterized by a deficiency in the liver of the hepatic enzyme phenylalanine hydroxylase that catalyzes the conversion of phenylalanine into tyrosine, leading to an excessive l-Phe accumulation in the serum (> 120 μM) [18] These levels are most commonly monitored by chromatography methods (LOD 1 μM) [19] or bacteriological inhibition assays (BIA) such as the Guthrie methodology (LOD 120 μM) [20, 21]. A simple enzymatic-colorimetric method has been developed based on the reaction between l-Phe and the enzyme LAAO in the presence of Au(III), which leads to the formation of gold nanoparticles whose absorbance can be related to the concentration of l-Phe in the sample

Materials and methods

Results

Conclusions