Why does signal-to-noise ratio matter in toehold switch validation?

A high on/off signal ratio indicates specific target engagement with minimal background, which is critical for distinguishing true positives in complex samples. This metric was used to identify sensor 27B as the optimal candidate due to its superior specificity over other switches. Reliable signal separation supports confident biomarker detection in patient-derived materials.

How does NASBA amplification improve detection limits in this workflow?

NASBA pre-amplification increases the concentration of target RNA, enabling detection of as few as 124 molecules per microliter when coupled with the toehold switch sensor. This amplification step was essential for achieving clinically relevant sensitivity in patient sample testing. It bridges the gap between low-abundance biomarkers and the sensor’s dynamic range.

What role does the PLUM portable plate reader play in assay execution?

The PLUM reader measures absorbance at 570 nm over time to capture the colorimetric response of the paper-based reactions, enabling real-time monitoring of switch activation. It was used to track reaction kinetics and determine positivity based on exceeding a predefined threshold. This portable readout supports decentralized testing without requiring laboratory infrastructure.

Why is reproducibility emphasized before advancing to patient trials?

Reproducibility across biological triplicates ensures that sensor performance is not due to stochastic variation or reagent inconsistency, which is essential for generating trustworthy diagnostic data. The protocol stresses optimizing NASBA primer sensitivity and switch performance prior to patient sample testing. Consistent results build confidence in the assay’s reliability for clinical decision support.

What specific output determines a positive sample in the toehold switch assay?

A sample is considered positive if its colorimetric signal exceeds the threshold established from negative controls, as measured by absorbance at 570 nm over time. This threshold-based call was validated against RT-qPCR, where positivity was defined as a cycle threshold ≤ 38. The method provides a quantitative, instrument-readable output suitable for automated interpretation.

Исследование от проектирования до внедрения для разработки и проверки пациентом бумажной диагностики toehold Switch

3.3K просмотров

Процитировано: 7

10:42 мин

17 июня 2022 г.

10.3791/63223-v

17 июня 2022 г.

3.3K просмотров

Katariina Jaenes^*¹ , Severino Jefferson Ribeiro da Silva^*¹^,² , Justin R. J. Vigar^*¹ , Kaiyue Wu³^,⁴ , Masoud Norouzi¹ , Pouriya Bayat¹ , Margot Karlikow¹ , Seray Cicek¹ , Yuxiu Guo¹ , Alexander A. Green³^,⁴ , Lindomar Pena² , Keith Pardee¹^,⁵

¹Leslie Dan Faculty of Pharmacy, University of Toronto, ²Laboratory of Virology and Experimental Therapy (LAVITE), Department of Virology, Aggeu Magalhães Institute (IAM), Oswaldo Cruz Foundation (Fiocruz), ³Department of Biomedical Engineering, Boston University, ⁴Molecular Biology, Cell Biology & Biochemistry Program, Graduate School of Arts and Sciences, Boston University, ⁵Department of Mechanical and Industrial Engineering, University of Toronto

^* These authors contributed equally

Доступ к децентрализованной, недорогой и высокопроизводительной диагностике, которая может быть развернута в сообществе для децентрализованного тестирования, имеет решающее значение для борьбы с глобальными кризисами в области здравоохранения. В этой рукописи описывается, как построить бумажную диагностику для последовательностей вирусных РНК, которые могут быть обнаружены с помощью портативного оптического считывателя.