Abstract
A reagent-free colorimetric method for galactose quantification using a composite of cerium oxide nanoparticles (nanoceria) and galactose oxidase (Gal Ox) entrapped in an agarose gel was developed. In the presence of galactose, the Gal Ox entrapped within the agarose gel catalyzed the oxidation of galactose to generate H2O2, which induced a color change from white to intense yellow. This reaction occurred without any chromogenic substrate. This color transition is presumed to be due to the H2O2-mediated alteration of the oxidation state of cerium ions present on the surface of the nanoceria. The intensity of color change was quantified by acquiring an image with a conventional smartphone, converting the image to cyan-magenta-yellow-black (CMYK) mode, and subsequently analyzing the image using the ImageJ software. Using this strategy, galactose concentration was specifically determined with excellent sensitivity of as low as 0.05 mM. The analytical utility of the assay was successfully verified by correctly determining diverse levels of galactose in human serum, which is enough to diagnose galactosemia, a genetic disorder characterized by the malfunctioning of enzymes responsible for galactose metabolism. The assay employing a hydrogel composite with entrapped nanoceria and Gal Ox, is a simple, cost-effective, and rapid colorimetric assay for galactose quantification, without using any chromogenic reagent. This cost-effective method has great potential for the diagnosis of galactosemia and is highly promising in comparison to the laborious instrumentation-based methods currently in use.
Highlights
Galactose, one of the breakdown products of lactose in milk, is an essential nutrient in the human body
A composite system for galactose quantification, containing nanoceria and galactose oxidase (Gal Ox) simultaneously entrapped within an agarose gel matrix, was established (Figure 1)
In the presence of galactose, Gal Ox in the composite catalyzed the oxidation of galactose to produce H2O2, which subsequently interacted with the nanoceria to induce the oxidation of surface-exposed Ce3+ to Ce4+ species, and produced peroxide complexes at the nanoceria surface [18,19]
Summary
One of the breakdown products of lactose (a disaccharide composed of one glucose and one galactose molecule) in milk, is an essential nutrient in the human body. Galactose levels are usually determined using diverse analytical instruments, such as high-performance liquid chromatography (HPLC), gas chromatography/mass spectrometry (GC/MS), and tandem mass spectrometry (MS/MS) [9,10,11] Assays employing these techniques enable sensitive and accurate analyses for galactose, but are expensive and often complicated to operate due to many pre-/post-treatment steps; they are not suitable for rapid screening of newborns. In the presence of galactose, Gal Ox generates H2O2, which subsequently activates peroxidase-mimicking magnetic nanoparticles and converts a colorimetric substrate of 2,2 -azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS) into a green product These enzymatic methods are quite sensitive and quantitative, but multiple components, including the colorimetric substrate and complicated procedures, still limit their widespread use. Essential analytical features of the system, such as selectivity, sensitivity, and detection precision, can be assessed using images acquired with a smart cellular phone, which is quite suitable for instrumentation-free, point-of-care testing (POCT) environments
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