Biology subjects

Rallapalli, Y.

Publications and source records attributed to Rallapalli, Y..

3 recordsLinked to original sources

bioRxiv2026

Inexpensive High-Throughput Multiplexed Cytokine Detection for Tuberculosis Diagnostics Using Amplified Enzymatic Metallization

Lack of accurate yet inexpensive diagnostics remains a critical bottleneck in the control and elimination of tuberculosis (TB). Interferon-gamma release assay (IGRA), which involve measurement of the release of the cytokine, interferon-gamma (IFN-{gamma}), in blood samples stimulated with Mycobacterium tuberculosis antigens, is used to detect latent TB infection (LTBI). Use of IGRA in resource-poor settings, in which TB is endemic, is however hindered by the need for specialized equipment for sensitive detection of low amounts of cytokine released. Additionally, recent research has shown the advantage of multiplexed detection of other non-IFN-{gamma} cytokines in improving diagnostic accuracy. However, this requires even more expensive instrumentation and there are no inexpensive or point-of-care compatible multiplexed cytokine detection tools available yet. Here we develop and demonstrate a low-cost, high-throughput, multiplexed cytokine detection platform based on amplified enzymatic silver metallization on a plastic substrate. The assay is performed in microwells formed on a commonly available plastic petri dish and the dry readout of the deposited silver is obtained using a cellphone camera, thus significantly reducing overall cost and complexity of multiplexed cytokine detection. We demonstrate the ability to measure clinically relevant sub-picomolar levels of multiple cytokines, including IFN-{gamma}, interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-) from a small volume (<5{micro}L) of the same blood sample used in an IGRA. Furthermore, we demonstrate the use of this assay to distinguish IGRA+ and IGRA-participant samples from a TB endemic setting.

bioengineering↗

bioRxiv2025

Microparticle-Enabled Single Cell Multiparameter Electronic Immunophenotyping for Selective Electroporation

Electroporation (EP) is one of the leading non-viral intracellular delivery methods used in various applications across research and cell therapy development and manufacturing. Currently widely used bulk EP methods, while they offer scalability, cost efficiency and simplicity, cannot be used for targeted or selective delivery to a defined subset of a input cell population. Here, we present a Microparticle-Enabled Selectively Permeabilizing Impedance Cytometer (ME-SPICy), a microfluidic single-cell EP platform that enables targeted EP of selected cell subpopulations based on their surface markers. Antibody conjugated microparticles (MPs) are used to label selected cell subpopulations within a larger heterogenous sample. Using multifrequency impedance detection, ME-SPICy discriminates, in real-time, non-labeled and labeled cells within the mixed sample as they flow through a 3D printed biconical micro-aperture. This allows for the system to analyze if a cell is a target cell and selectively apply a low voltage (<16 V) for targeted single-cell EP. Simulations and experimental validation demonstrate that MP binding substantially alters cell impedance and phase signature, enabling accurate label-based discrimination. We demonstrated selective EP first using Jurkat cells by targeting either the labeled or non-labeled populations. Then we demonstrated targeted delivery to primary human lymphocytes within peripheral blood mononuclear cells. ME-SPICy achieved high precision, with 98% purity and >5 fold enrichment of lymphocytes in the electroporated cell population. This approach expands the capabilities of EP, offering a promising solution to decrease manufacturing complexity in both research and clinical cell engineering workflows

bioengineering↗

bioRxiv2025

Label-Free Targeted High Efficiency Electroporation with Single-Cell Feedback Control Using Focused Microscale Electric Fields

Efficient, safe, and cell-selective intracellular delivery remains a bottleneck for scalable and cost-effective manufacturing of cell therapies. Here, we introduce Selective Permeabilization using Impedance Cytometry (SPICy) that couples multifrequency single-cell impedance cytometry with real-time, feedback-controlled, low-voltage single-cell electroporation. Electric field focusing in a 3-D printed biconical micro-aperture confines both sensing and electroporation to a microscale zone, enabling continuous-flow operation and the use of low voltages (<15 V) for electroporation. Impedance spectra are captured for each single cell and machine-learning based analysis enabled accurately distinguishing cells in a label-free manner. Selectively triggered low-voltage electroporation achieved simultaneous high delivery efficiency (>80 %) and high (>90 %) cell viability. Delivery of a range of different cargo sizes (4-500 kDa), GFP mRNA expression, CRISPR-Cas9 based knock-out and delivery to a variety of different cell lines, primary human T cells and peripheral blood mononuclear cells (PBMCs) was also demonstrated. Using heterogenous or mixed samples, selective delivery to both cell lines, and primary immune cell subpopulations, from PBMCs, was demonstrated. SPICy thus provides a label-free, continuous flow, targeted non-viral platform for precision cell engineering.

bioengineering↗