an-electroporation-cytometry-protocol-for-live-cell-fluorescence

An Electroporation Cytometry Protocol for Live-Cell, Fluorescence Microscopy Using U2 OS Cell Culture

Pulsed electric fields (PEFs) have a wide range of applications in medical research and clinical applications. A key area of research focuses on ...

Toronto Metropolitan University

Science and Technology (iBEST), a partnership between St. Michael's Hospital, a site of Unity Health Toronto, and Toronto Metropolitan University

Keenan Research Centre for Biomedical Science at St. Michael's Hospital

Neutralizing antibodies (nAbs) produced from infection or vaccination play an important role in acquired immunity. Determining virus-specific nAb titers is a useful tool for measuring aquired immunity in an individual. The standard methods to do so rely on titrating serum samples against live virus and monitoring viral infection in cultured cells which requires high biosafety level containment. The surrogate virus neutralization test (sVNT) reduces the biohazards and it is suitable for designing rapid test device in a lateral flow assay (LFA) format. Here, we introduce the fabrication and development of a unique paper-based LFA device for determining the level of SARS-CoV-2 nAb in a sample with a semiquantitative direct colorimetric readout. A LFA-based gradient assay design was used to facilitate the sVNT, where the spike glycoprotein receptor binding domain (RBD) and angiotensin-converting enzyme 2 (ACE2) stand in as proxies for viruses and cells, respectively. The gradient assay employed multiple test dots of ACE2 spotted in increasing concentration along the sample flow path and gold nanoparticle-conjugated RBD for readout. In this way, the number of developed spots is inversely proportional to the concentration of nAbs present in the sample. The assay was tested with both standard solutions of nAb as well as human serum samples. We have demonstrated that the device can effectively provide semiquantitative test results of nAbs by direct instrument-free colorimetric detection.

Semiquantitative Paper-Based Microfluidic Surrogate Virus Neutralization Test for SARS-CoV-2 Neutralizing Antibodies.

Electric fields are used in biology to address a broad range of questions and through a variety of techniques, including electroporation, gene electrotransfer (GET), electrostimulation (ES), and electrochemotherapy. Each of these modalities requires specific conditions and has drastically different target outcomes on the cell. ES has demonstrated that non-pore forming electric fields alter cell cycle progression. However, pore forming electric fields such as with GET have not been as widely explored despite major clinical advancements. Additionally, the real-time visual analysis of electrical field effects on mammalian cell culture is currently lacking among most commercial systems. To facilitate investigations into these research areas, an electroporation cytometry system was developed including a custom chamber compatible with live cell imaging and exponential decay pulse generator for live cell analysis. The functionality of the system was demonstrated using a recombinant cell line using U-2 OS cells and FUCCI(CA)5 cell cycle reporter. The exposure of the cells to a 180 V pulse in both unsynchronized and synchronized populations revealed an effect on the cell cycle.

An electroporation cytometry system for long-term, live cell cycle analysis.

Microfluidic technologies based on droplets provide discrete volumes within which chemical and/or biological processes can take place. Two major platforms in this space include droplet microfluidics (emulsions within channels) and digital microfluidics (discrete droplet manipulation by electric fields). The integration of electrochemical sensing with both microfluidic platforms offers advantages in miniaturization and portability, as sensors can be integrated directly within the microfluidic devices and instrumentation is relatively compact.

Article	Year
Semiquantitative Paper-Based Microfluidic Surrogate Virus Neutralization Test for SARS-CoV-2 Neutralizing Antibodies. Analytical chemistry\| PubMed ID: 38980117	2024
An electroporation cytometry system for long-term, live cell cycle analysis. Biomicrofluidics\| PubMed ID: 39219593	2024
A review of electrochemical sensing in droplet systems: Droplet and digital microfluidics. Analytica chimica acta\| PubMed ID: 40024652	2025

Darius G. Rackus

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