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.

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.

A Comparison of the Genotoxic Effects of Gold Nanoparticles Functionalized with Seven Different Ligands in Cultured Human Hepatocellular Carcinoma Cells.

Gold nanoparticles (GNPs) have shown great potential in diagnostic and therapeutic applications in diseases, such as cancer. Despite GNP versatility, there is conflicting data regarding the toxicity of their overall functionalization chemistry for improved biocompatibility. This study aimed to determine the possible genotoxic effects of functionalized GNPs in Human hepatocellular carcinoma (HepG2) cells. GNPs were synthesized and biofunctionalized with seven common molecules used for biomedical applications. These ligands were bovine serum albumin (BSA), poly(sodium 4-styrene sulfonate) (PSSNA), trisodium citrate (citrate), mercaptoundecanoic acid (MUA), glutathione (GSH), polyvinylpyrrolidone (PVP), and polyethylene glycol (PEG). Before in vitro genotoxicity assessment, inductively coupled plasma mass spectrometry was used to determine GNP cellular internalization quantitatively, followed by cell-based assays; WST-1 to find IC 30 and ApoPercentage for apoptotic induction time-points. The effect of the GNPs on cell growth in real-time was determined by using xCELLigence, followed by a comet assay for genotoxicity determination. The HepG2 cells experienced genotoxicity for all GNP ligands; however, they were able to initiate repair mechanisms and recover DNA damage, except for two functionalization chemistries.

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.

Facile immobilization of glucose oxidase onto gold nanostars with enhanced binding affinity and optimal function.

Gold nanoparticles provide a user-friendly and efficient surface for immobilization of enzymes and proteins. In this paper, we present a novel approach for enzyme bioconjugation to gold nanostars (AuNSs). AuNSs were modified with l-cysteine (Cys) and covalently bound to N-hydroxysulfosuccinimide (sulfo-NHS) activated intermediate glucose oxidase (GOx) to fabricate a stable and sensitive AuNSs-Cys-GOx bioconjugate complex. Such a strategy has the potential for increased attachment affinity without protein adsorption onto the AuNSs surface. Good dispersity in buffer suspension was observed, as well as stability in high ionic environments. Using the AuNSs-Cys-GOx bioconjugates showed greater sensitivity in the measuring of low concentrations of glucose based on plasmonic and colorimetric detection. Such a novel approach for enzyme immobilization can lead to AuNSs-Cys-GOx bioconjugate complexes that can be used as catalytic nanodevices in nanobiosensors based on oxidases in biomedical applications.

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.

Dynamic contrast enhanced CT aiding gross tumor volume delineation of liver tumors: an interobserver variability study.

PURPOSE: To evaluate the application of perfusion CT for gross tumor volume (GTV) delineation for radiotherapy of intrahepatic tumors. MATERIALS AND METHODS: 15 radiotherapy patients with confirmed liver tumors underwent contrast enhanced 4D-CT (Philips Brilliance Big-bore) as well as dynamic contrast enhanced (DCE) CT (GE 750HD). Perfusion maps were generated with CT perfusion v5 from GE. Five observers delineated GTVs of all intrahepatic foci on the 4D-CT, time-averaged DCE-CT and perfusion CT for every patient. STAPLE consensus contours were generated. Dice's coefficients were compared between GTVs generated by observers on each image set and the corresponding consensus GTVs. Comparisons were also performed with patients stratified by hepatocellular carcinoma (HCC) metastatic tumors, and by tumor volume. RESULTS: Overall, mean Dice's coefficients were 0.81±0.14, 0.84±0.10, and 0.81±0.14 for 4D-CT, DCECT and perfusion. DCE-CT performed significantly better than 4D-CT and perfusion (p=0.005 and p=0.01 respectively). For patients with HCC, DCE-CT reduced interobserver variability significantly compared to 4D-CT (Dice's coefficients 0.87 vs. 0.84, p<0.05). For patients with metastatic disease time-averaged DCE-CT images decreased variability compared to 4D-CT (Dice's coefficient 0.81 vs. 0.76, p<0.05), especially true for tumors<100cc. The smaller tumors results are important to be included here. CONCLUSIONS: DCE-CT imaging of liver perfusion reduced interobserver variability in GTV delineation for both HCC and metastatic liver tumors.