Biology subjects

Kotnala, A.

Publications and source records attributed to Kotnala, A..

4 recordsLinked to original sources

bioRxiv2026

Isolation and Purification-Free Digital Single-Small Extracellular Vesicle Biosensing with Scalable Plasmonic Arrays

Recent advances in plasmonic biosensing and imaging have enabled label-free analysis of single biological nanoparticles. We previously developed PlAsmonic NanOapeRture lAbel-free iMAging (PANORAMA) for isolation and purification-free, digital counting and precise localization of small extracellular vesicles (sEVs), with complementary fluorescence interrogation of surface and intravesicular biomarkers for quantitative molecular profiling. The fact that no isolation and purification or isolation is needed represents a crucial advantage because various specificity, efficiency, and time-consumption issues hinder quantitatively reproducible extraction of sEVs from biological fluids. PANORAMA achieves ultrahigh refractive-index sensitivity through arrayed gold nanodisks on invisible substrates (AGNIS) fabricated by nanosphere lithography (NSL). However, despite its simplicity and low cost, NSL is frequently constrained by poor large-area uniformity, which hinders scalable fabrication. Here, we introduce nanosphere settling lithography (NSSL) as an alternative to the gold-standard Langmuir-Blodgett trough (LBT) process, enabling highly uniform, large-area monolayers with reduced process stringency. AGNIS fabricated via NSSL exhibits high refractive-index sensitivity with low spatial variability across 60 mm x 24 mm substrates, sufficient for 60-well in standard 384-well plate format. The platform demonstrates exquisite sensitivity through PANORAMA digital counting and sizing of 25, 50, and 100 nm polystyrene beads, as well as single-vesicle characterization of sEVs derived from H460 lung cancer cells. For the first time, combined PANORAMA and fluorescence imaging enables quantitative analysis of microRNA-21 (miR-21) expression in sEVs to identify "cancer-suspicious" sub-population from liver cancer patient plasma in an unbiased fashion allowing both highly sensitive detection of individual sEVs and simultaneous molecular profiling. Collectively, NSSL enables uniform, high-performance plasmonic biosensing over large areas, providing a scalable and economical pathway for high-throughput, digital single-sEV analysis and translational liquid biopsy applications.

bioengineering↗

bioRxiv2025

Microfluidic Nano-Plasmonic Imaging Platform for Purification- and Label-Free Single Small Extracellular Vesicle Counting

Tumor-derived circulating small extracellular vesicles (sEVs) is a promising non-invasive biomarker for disease diagnosis. However, their quantitative detection remains challenging due to their small size and the complexity of blood plasma. Typically, sample preparation like purification is required. This study presents a purification-free approach using a microfluidic chip integrated with PANORAMA (Plasmonic nano-aperture label-free imaging) for label-free single sEV counting in plasma. CD63, CD9, and CD81 antibodies, specific biomarkers for most sEVs, are functionalized on AGNIS (arrayed gold nanodisks on invisible substrate) for selective capture. The automated microfluidic platform minimizes manual errors and allows precise programming of flow rates, directions, and media for optimization. Only 20 {micro}L of plasma is required, and the analysis is completed within 60 minutes. This platform shows great potential as a sensitive and efficient tool for detecting circulating sEVs without purification or labeling.

bioengineering↗

bioRxiv2021

Tissue fixation effects on human retinal lipid analysis by MALDI imaging and LC-MS/MS technologies

Imaging mass spectrometry (IMS) allows the location and abundance of lipids to be mapped across tissue sections of human retina. For reproducible and accurate information, sample preparation methods need to be optimized. Paraformaldehyde fixation of a delicate multilayer structure like human retina facilitates the preservation of tissue morphology by forming methylene bridge cross-links between formaldehyde and amine/ thiols in biomolecules; however, retina sections analyzed by IMS are typically fresh-frozen. To determine if clinically significant inferences could be reliably based on fixed tissue, we evaluated the effect of fixation on analyte detection, spatial localization, and introduction of artefactual signals. Hence, we assessed the molecular identity of lipids generated by matrix-assisted laser desorption ionization (MALDI-IMS) and liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) for fixed and fresh-frozen retina tissues in positive and negative ion modes. Based on MALDI-IMS analysis, more lipid signals were observed in fixed compared to fresh-frozen retina. More potassium adducts were observed in fresh-frozen tissues than fixed as the fixation process caused displacement of potassium adducts to protonated and sodiated species in ion positive ion mode. LC-MS/MS analysis revealed an overall decrease in lipid signals due to fixation that reduced glycerophospholipids and glycerolipids and conserved most sphingolipids and cholesteryl esters. The high quality and reproducible information from untargeted lipidomics analysis of fixed retina informs on all major lipid classes, similar to fresh-frozen retina, and serves as a steppingstone towards understanding of lipid alterations in retinal diseases.

biochemistry↗

bioRxiv2020

Lipid Landscape of the Human Retina and Supporting Tissues Revealed by High Resolution Imaging Mass Spectrometry

The human retina evolved to facilitate complex visual tasks. It supports vision at light levels ranging from starlight to sunlight, and its supporting tissues and vasculature regulate plasma-delivered lipophilic essentials for vision, including retinoids (vitamin A derivatives). The human retina is of particular interest because of its unique anatomic specializations for high-acuity and color vision that are also vulnerable to prevalent blinding diseases. The retinas exquisite cellular architecture is composed of numerous cell types that are aligned horizontally, giving rise to structurally distinct cell, synaptic, and vascular layers that are visible in histology and in diagnostic clinical imaging. Suitable for retinal investigations, MALDI imaging mass spectrometry (IMS) technologies are now capable of providing images at low micrometer spatial resolution with high levels of chemical specificity. In this study, a multimodal imaging approach combined with a recently developed method of high accuracy multi-image registration was used to define the localization of lipids in human retina tissue at laminar, cellular, and sub-cellular levels. Data acquired by IMS combined with autofluorescence and bright-field microscopy of human retina sections in macular and peripheral regions indicate differences in distributions and abundances of lipid species across and within single cell types. Of note is localization of signals within specific layers of macula, localization within different compartments of photoreceptors and RPE, complementarity of signals between macular retina and non-macular RPE, and evidence that lipids differing by a single double bond can have markedly different distributions.

biochemistry↗