Label-Free Protein Glycosylation Analysis Using NanoMonitor-An Ultrasensitive Electrochemical Biosensor.

Glycans (oligosaccharide chains attached to glycoproteins) are a promising class of biomarkers, found in body fluids such as serum, saliva, urine, etc., that can be used for the diagnosis of disease conditions. Subtle changes in glycans resulting from altered glycosylation machinery have been reported during various diseases, including carcinogenesis. In this article, we detail protocols for the rapid, label-free analysis of glycans using a previously developed highly sensitive and selective electrochemical impedance spectroscopy-based biosensing diagnostic platform called "NanoMonitor." The glycosensor operation is based on the specific affinity capture of the target glycans on the sensor surface by glycan-binding proteins known as lectins. This glycan-lectin binding activity modulates the impedance of the electrical double layer at the buffer-electrode interface. Protocols for the preparation of glycoprotein samples and glycosylation analysis using NanoMonitor and lectin-based ELISA are described here. The data obtained using these protocols show that NanoMonitor is capable of distinguishing between glycoform variants of the glycoprotein fetuin and glycoproteins derived from cultured human pancreatic cancer cells with high sensitivity (orders of magnitude higher than lectin-based ELISA) and selectivity. The results obtained indicate that NanoMonitor protocols can be further developed to enable use of NanoMonitor as a handheld electronic biosensor device for routine multiplexed detection of glycan biomarkers from clinical samples. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparing the NanoMonitor surface for glycan biosensing Support Protocol: Synthesis of glycoform variants of fetuin Basic Protocol 2: Performing Electrochemical Impedance Spectroscopy (EIS) for analyzing glycoprotein structures.

Development and In Vitro Evaluation of Vitamin E-Enriched Nanoemulsion Vehicles Loaded with Genistein for Chemoprevention Against UVB-Induced Skin Damage.

There is a great need for effective protection against cutaneous pathologies arising from chronic exposure to harmful solar UVB radiations. A promising pharmaceutical strategy to improve the efficacy of chemotherapeutic/preventative natural compounds (e.g., soy isoflavone Genistein, Gen) is to enhance their dermal delivery using nanoemulsion (NE) formulations. This report investigates the development of nanoemulsified tocotrienol(T3)-rich fraction of red palm oil (Tocomin®), to yield an optimal NE delivery system for dermal photoprotection (z-average size <150 nm, ζ-potential ≈ -30 mV, polydispersity index < 0.25). Physicochemical characterization and photostability studies indicate NE formulations utilizing surfactant mixture (Smix) of Solutol® HS-15 (SHS15) blended with vitamin E TPGS (TPGS) as cosurfactant was significantly superior to formulations that utilized Lutrol® F68 (LF68) as the cosurfactant. A ratio of 60:40 of SHS15-TPGS-NE was further identified as lead Tocomin® NE topical platform using in vitro pharmaceutical skin reactivity studies that assess cutaneous irritancy and cytotoxicity. Prototype Tocomin® NE loaded with the antiphotocarcinogenic molecule Gen (Gen-Tocomin® NE) showed slow-release profile in both liquid and cream forms. Gen-Tocomin® NE also showed excellent biocompatibility, and provided substantial UVB protection to cultured subcutaneous L929 fibroblasts, indicating the great potential of our Tocomin® NE warranting further prototype development as topical pharmaceutical platform for skin photoprotection applications.

Single cells and intracellular processes studied by a plasmonic-based electrochemical impedance microscopy.

Electrochemical impedance spectroscopy is a crucial tool for the detection and study of various biological substances, from DNA and proteins to viruses and bacteria. It does not require any labelling species, and methods based on it have been developed to study cellular processes (such as cell spreading, adhesion, invasion, toxicology and mobility). However, data have so far lacked spatial information, which is essential for investigating heterogeneous processes and imaging high-throughput microarrays. Here, we report an electrochemical impedance microscope based on surface plasmon resonance that resolves local impedance with submicrometre spatial resolution. We have used an electrochemical impedance microscope to monitor the dynamics of cellular processes (apoptosis and electroporation of individual cells) with millisecond time resolution. The high spatial and temporal resolution makes it possible to study individual cells, but also resolve subcellular structures and processes without labels, and with excellent detection sensitivity (~2 pS). We also describe a model that simulates cellular and electrochemical impedance microscope images based on local dielectric constant and conductivity.

NanoMonitor: a miniature electronic biosensor for glycan biomarker detection.

AIM: The goal of our research is to develop an ultrasensitive diagnostic platform called 'NanoMonitor' to enable rapid label-free analysis of a highly promising class of biomarkers called glycans (oligosaccharide chains attached to proteins) with high sensitivity and selectivity. The glycosylation of fetuin - a serum protein - and extracts from a human pancreatic cancer line was analyzed to demonstrate the capabilities of the NanoMonitor. MATERIAL & METHODS: The NanoMonitor device consists of a silicon chip with an array of gold electrodes forming multiple sensor sites and works on the principle of electrochemical impedance spectroscopy. Each sensor site is overlaid with a nanoporous alumina membrane that forms a high density of nanowells on top of each electrode. Lectins (proteins that bind to and recognize specific glycan structures) are conjugated to the surface of the electrode. When specific glycans from a test sample bind to lectins at the base of each nanowell, a perturbation of electrical double-layer occurs, which results in a change in the impedance. Using the lectins Sambucs nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA), subtle variations to the glycan chains of fetuin were investigated. Protein extracts from BXPC-3, a cultured human pancreatic cancer cell line were also analyzed for binding to SNA and MAA lectins. The performance of the NanoMonitor was compared to a conventional laboratory technique: lectin-based enzyme linked immunosorbent assay (ELISA). RESULTS & DISCUSSION: The NanoMonitor was used to identify glycoform variants of fetuin and global differences in glycosylation of protein extracts from cultured human pancreatic cancerous versus normal cells. While results from NanoMonitor correlate very well with results from lectin-based ELISA, the NanoMonitor is rapid, completely label free, requires just 10 microl of sample, is approximately five orders of magnitude more sensitive and highly selective over a broad dynamic range of glycoprotein concentrations. CONCLUSION: Based on its performance metrics, the NanoMonitor has excellent potential for development as a point-of-care handheld electronic biosensor device for routine detection of glycan biomarkers from clinical samples.