Quantitative Galactose Colorimetric Competitive Assay Based on Galactose Dehydrogenase and Plasmonic Gold Nanostars.

We describe a competitive colorimetric assay that enables rapid and sensitive detection of galactose and reduced nicotinamide adenine dinucleotide (NADH) via colorimetric readouts and demonstrate its usefulness for monitoring NAD+-driven enzymatic reactions. We present a sensitive plasmonic sensing approach for assessing galactose concentration and the presence of NADH using galactose dehydrogenase-immobilized gold nanostars (AuNS-PVP-GalDH). The AuNS-PVP-GalDH assay remains turquoise blue in the absence of galactose and NADH; however, as galactose and NADH concentrations grow, the reaction well color changes to a characteristic red color in the presence of an alkaline environment and a metal ion catalyst (detection solution). As a result, when galactose is sensed in the presence of H2O2, the colored response of the AuNS-PVP-GalDH assay transforms from turquoise blue to light pink, and then to wine red in a concentration-dependent manner discernible to the human eye. This competitive AuNS-PVP-GalDH assay could be a viable analytical tool for rapid and convenient galactose quantification in resource-limited areas.

Feasibility of NAD(P)/NAD(P)H as redox agents in enzymatic plasmonic gold nanostar assays for galactose quantification.

Plasmonic colorimetric sensors have emerged as powerful analytical tools in biochemistry due to their localized surface plasmon resonance extinction in the visible range. Here, we describe the feasibility of NAD(P)/NAD(P)H as redox agents in enzymatic plasmonic gold nanostar (AuNS) assays for galactose quantification using three model enzymes, GalDH, AR and GalOx, immobilized separately on polyvinylpyrrolidone-capped AuNS scaffolds. These highly specific, sensitive and selective bioassays induce the transformation of AuNS into quasi-spherical nanoparticles during the biorecognition of galactose in water and synthetic blood matrices. As a result, using our inexpensive and simple AuNS plasmon bioassays, the presence of galactose may be detected spectrophotometrically and by the naked eye.

Miniaturization and Automation Protocol of a Urinary Organic Acid Liquid-Liquid Extraction Method on GC-MS.

The aim of this study was to improve the extraction method for urinary organic acids by miniaturizing and automating the process. Currently, manual extraction methods are commonly used, which can be time-consuming and lead to variations in test results. To address these issues, we reassessed and miniaturized the in-house extraction method, reducing the number of steps and the sample-to-solvent volumes required. The evaluated miniaturized method was translated into an automated extraction procedure on a MicroLab (ML) Star (Hamilton Technologies) liquid handler. This was then validated using samples obtained from the ERNDIM External Quality Assurance program. The organic acid extraction method was successfully miniaturized and automated using the Autosampler robot. The linear range for most of the thirteen standard analytes fell between 0 to 300 mg/L in spiked synthetic urine, with low (50 mg/L), medium (100 mg/L), and high (500 mg/L) levels. The correlation coefficient (r) for most analytes was >0.99, indicating a strong relationship between the measured values. Furthermore, the automated extraction method demonstrated acceptable precision, as most organic acids had coefficients of variation (CVs) below 20%. In conclusion, the automated extraction method provided comparable or even superior results compared to the current in-house method. It has the potential to reduce solvent volumes used during extraction, increase sample throughput, and minimize variability and random errors in routine diagnostic settings.

The Effect of Capping Agents on Gold Nanostar Stability, Functionalization, and Colorimetric Biosensing Capability.

Capping agents (organic ligands, polymers, and surfactants) are pivotal for stabilizing nanoparticles; however, they may influence the surface chemistry, as well as the physico-chemical and biological characteristics, of gold nanostar (AuNS)-based biosensors. In this study, we proved that various capping agents affected capped and bioconjugated AuNS stability, functionality, biocatalysis, and colorimetric readouts. Capped and bioconjugated AuNSs were applied as localized surface plasmon resonance (LSPR)-based H2O2 sensors using glucose oxidase (GOx) as a model enzyme. Furthermore, our analyses revealed that the choice of capping agent influenced the properties of the AuNSs, their stability, and their downstream applications. Our analyses provide new insights into factors governing the choice of capping agents for gold nanostars and their influences on downstream applications with conjugated enzymes in confined environments.

Serum Metabolomic and Lipoprotein Profiling of Pancreatic Ductal Adenocarcinoma Patients of African Ancestry.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with a characteristic dysregulated metabolism. Abnormal clinicopathological features linked to defective metabolic and inflammatory response pathways can induce PDAC development and progression. In this study, we investigated the metabolites and lipoproteins profiles of PDAC patients of African ancestry. Nuclear Magnetic Resonance (NMR) spectroscopy was conducted on serum obtained from consenting individuals (34 PDAC, 6 Chronic Pancreatitis, and 6 healthy participants). Seventy-five signals were quantified from each NMR spectrum. The Liposcale test was used for lipoprotein characterization. Spearman's correlation and Kapan Meier tests were conducted for correlation and survival analyses, respectively. In our patient cohort, the results demonstrated that levels of metabolites involved in the glycolytic pathway increased with the tumour stage. Raised ethanol and 3-hydroxybutyrate were independently correlated with a shorter patient survival time, irrespective of tumour stage. Furthermore, increased levels of bilirubin resulted in an abnormal lipoprotein profile in PDAC patients. Additionally, we observed that the levels of a panel of metabolites (such as glucose and lactate) and lipoproteins correlated with those of inflammatory markers. Taken together, the metabolic phenotype can help distinguish PDAC severity and be used to predict patient survival and inform treatment intervention.

Gold Nanostar Colorimetric Detection of Fructosyl Valine as a Potential Future Point of Care Biosensor Candidate for Glycated Haemoglobin Detection.

Diabetes Mellitus is a growing global concern. The current methods used to detect glycated haemoglobin are precise, however, utilise expensive equipment, reagents and consumables. These are luxuries which rural communities cannot access. The nanotechnology methods which have been developed for glycated haemoglobin detection are predominantly electrochemically based, have complicated lengthy fabrication processes and utilise toxic chemicals. Here a fructosyl amino acid oxidase gold nanostar biosensor has been developed as a potential future point of care biosensor candidate for glycated haemoglobin detection. The workup done on this biosensor showed that it was able to give a spectrophotometric readout and colorimetric result with naked eye detection in blank serum spiked with fructosyl valine.

Modified HEPES one-pot synthetic strategy for gold nanostars.

Gold nanostars are being used more regularly in the biosensing field. Despite their useful attributes, there is still a need to optimize aspects of the synthesis and stability. The seedless, synthetic method comprising 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) is a facile, rapid method; however, it produces heteromorphic nanostars. The modification of a HEPES method resulted in a silver-assisted, seedless gold nanostar synthesis method. The nanostars resulting from this method were monodispersed, multi-branched and approximately 37 ± 2 nm in diameter. It proved to be a repeatable method that produced homogeneous and robust nanostars. Once functionalized with polyvinylpyrrolidone 10 000, the new nanostars were observed to be stable in various environments such as salt, ionic strength and cell culture medium. In conclusion, the addition of the silver nitrate improved the morphology of the reported HEPES nanostars for the purpose of nanobiosensor development.

Plasmonic Detection of Glucose in Serum Based on Biocatalytic Shape-Altering of Gold Nanostars.

Nanoparticles have been used as signal transducers for optical readouts in biosensors. Optical approaches are cost-effective with easy readout formats for clinical diagnosis. We present a glucose biosensor based on the biocatalytic shape-altering of gold nanostars via silver deposition. Improved sensitivity was observed due to the nanostars clustering after being functionalised with glucose oxidase (GOx). The biosensor quantified glucose in the serum samples with a 1:1000 dilution factor, and colorimetrically distinguished between the concentrations. The assay demonstrated good specificity and sensitivity. The fabricated glucose biosensor is a rapid kinetic assay using a basic entry level laboratory spectrophotometric microplate reader. Such a biosensor could be very useful in resource-constrained regions without state-of-the-art laboratory equipment. Furthermore, naked eye detection of glucose makes this a suitable biosensor for technology transfer to other point-of-care devices.

Seedless gold nanostars with seed-like advantages for biosensing applications.

Gold nanostars (AuNSs) are seen as promising building blocks for biosensors with potential for easy readouts based on naked-eye and ultraviolet-visible spectroscopy detection. We present a seedless synthesis strategy for AuNSs that has the advantages of the seeded methods. The method used ascorbic acid as a reducing agent and silver nitrate as an anisotropic growth control assisting agent. AuNSs with multiple branches and a diameter of 59 nm were produced. They showed good stability when capped with PVP and modified with an enzyme in relatively strong ionic conditions. We investigated their application in plasmonic sensing by modifying them with glucose oxidase and detection of glucose. The AuNSs were found to be a good scaffold for the enzyme, proved to be stable and sensitive as transducers. Thus, the AuNSs showed good promise for further applications in plasmonic biosensing for in vivo biomedical diagnosis.