Biology subjects

Miklos, A. E.

Publications and source records attributed to Miklos, A. E..

3 recordsLinked to original sources

bioRxiv2025

A Rapid and Modular Nanobody Assay for Plug-and-Play Antigen Detection

Rapid and portable antigen detection is essential for managing infectious diseases and responding to toxic exposures, yet current methods face significant limitations. Highly sensitive platforms like the Enzyme-Linked Immunosorbent Assay (ELISA) are time- and cost-prohibitive for point-of-need detection, while portable options like lateral flow assays (LFAs) require systemic overhauls for new targets. Furthermore, the complex infrastructure, high production costs, and extended timelines for assay development constrain manufacturing of traditional diagnostic platforms in low-resource settings. To address these challenges, we describe the Rapid and Modular Nanobody Assay (RAMONA) as a versatile antigen detection platform that leverages nanobody-coiled coil fusion proteins for modular integration with downstream readout methods. RAMONA merges the portability of LFAs with the benefits of nanobodies, such as their smaller size, improved solubility, and compatibility with cell-free protein synthesis systems, enabling on-demand biomanufacturing and rapid adaptation for diverse targets. We demonstrate assay generalizability through the detection of three distinct protein targets, robustness across various temperatures and incubation periods, and compatibility with saliva samples and cell-free synthesis. Detection occurs in under 30 minutes, with results strongly and positively correlating to ELISA data while requiring minimal resources. Moreover, RAMONA supports multiplexed detection of three antigens simultaneously using orthogonal capture probes. By overcoming several limitations of traditional immunoassays, RAMONA represents a significant advancement in rapid, adaptable, and field-deployable antigen detection technologies.

bioengineering↗

bioRxiv2024

Semi-automated Production of Cell-free Biosensors

Cell-free synthetic biology biosensors have potential as effective in vitro diagnostic technologies for the detection of chemical compounds such as toxins and human health biomarkers. They have several advantages over conventional laboratory-based diagnostic approaches, including being able to be assembled, freeze-dried, distributed, and then used at the point-of-need. This makes them an attractive platform for cheap and rapid chemical detection across the globe. Though promising, a major challenge is scaling up biosensor manufacturing to meet the needs of their multiple uses. Currently, cell-free biosensor assembly during lab-scale development is mostly performed manually by the operator, leading to quality control and performance variability issues. Here we explore the use of liquid handling robotics to manufacture cell-free biosensor reactions. We compare both manual and semi-automated reaction assembly approaches using the Opentrons OT-2 liquid handling platform on two different cell-free gene expression assay systems that constitutively produce colorimetric (LacZ) or fluorescent (GFP) signals. We test the designed protocol by constructing an entire 384-well plate of fluoride sensing cell-free biosensors and demonstrate that they perform closely to expected detection outcomes.

synthetic biology↗

bioRxiv2024

High-Throughput Optimization of Paper-Based Cell-Free Biosensors

Cell-free expression systems maintain core cellular processes without intact cells and offer attractive properties as point-of-need biosensors. The ability to lyophilize, store, and use on-demand make these sensors usable in the field, and the lack of membranes means that there are no analyte transport issues and that new sensors can be deployed by simply adding a different DNA molecule. The lack of membranes also means that sensor designs and reaction optimizations can be screened in high throughput. While shelf stability has been demonstrated in specific cases using additives, these approaches are not universal to the myriad cell-free expression methods and formats. Here, we present new high-throughput screening methods to optimize cell-free expression formulations when embedded into paper for use as sensors. Our method leverages acoustic liquid handling to dispense reactions onto 384-well paper ticket formats and machine vision to quantify reaction performance from a colorimetric reporter enzyme. The throughput enabled shifts the bottleneck from experimental execution to selecting the experiments to execute; we therefore implement design-of-experiments to optimize the information gained from each design-build-test-learn cycle. We used these approaches to first optimize the performance of a low-cost cell-free expression formulation that was initially non-functional when embedded in paper, then further optimize it for tolerance to exposure to heat. With only 2 rounds of experimentation lasting 4 days total for each goal, the result are an energy mixture with 8% of the materials cost of a commonly used version and a formulation of excipients that maintain 60% of activity after 6 hours of storage at 50 {degrees}C and. Finally, we showcase the use of the cost-optimized formulation in a 3D-printed paperfluidic device where it outperforms the standard formulation at much lower cost.

synthetic biology↗