Improving the sensitivity of fluorescence-based immunoassays by photobleaching the autofluorescence of magnetic beads

Roth, Shira; Hadass, Orr; Cohen, Meir; Verbarg, Jasenka; Wilsey, Jennifer; Danielli, Amos

doi:10.1117/12.2507597

Cited by 4 publications

(4 citation statements)

References 22 publications

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“…However, the fluorescence intensity from the Qdot TM 585 channel (yellow bar) was minimal in comparison with the intensity from the Qdot TM 525 channel (blue bar) such that it would not influence the overall protein concentration determination. In the GFAP detection band (right pair of bars in Figure 3 B), the signal from the Qdot TM 525 channel was due to strong autofluorescence of the ~2.8 µm assay beads at 525 nm rather than contamination by unbound quantum dots from the IL-6 detection band [ 35 ]. It is physically impossible for ~4.5 µm diameter assay beads to become trapped in the GFAP detection band due to the channel height gradient.…”

Section: Resultsmentioning

confidence: 99%

A Variable Height Microfluidic Device for Multiplexed Immunoassay Analysis of Traumatic Brain Injury Biomarkers

2021

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Traumatic brain injury (TBI) is a leading cause of global morbidity and mortality, partially due to the lack of sensitive diagnostic methods and efficacious therapies. Panels of protein biomarkers have been proposed as a way of diagnosing and monitoring TBI. To measure multiple TBI biomarkers simultaneously, we present a variable height microfluidic device consisting of a single channel that varies in height between the inlet and outlet and can passively multiplex bead-based immunoassays by trapping assay beads at the point where their diameter matches the channel height. We developed bead-based quantum dot-linked immunosorbent assays (QLISAs) for interleukin-6 (IL-6), glial fibrillary acidic protein (GFAP), and interleukin-8 (IL-8) using DynabeadsTM M-450, M-270, and MyOneTM, respectively. The IL-6 and GFAP QLISAs were successfully multiplexed using a variable height channel that ranged in height from ~7.6 µm at the inlet to ~2.1 µm at the outlet. The IL-6, GFAP, and IL-8 QLISAs were also multiplexed using a channel that ranged in height from ~6.3 µm at the inlet to ~0.9 µm at the outlet. Our system can keep pace with TBI biomarker discovery and validation, as additional protein biomarkers can be multiplexed simply by adding in antibody-conjugated beads of different diameters.

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Section: Resultsmentioning

confidence: 99%

A Variable Height Microfluidic Device for Multiplexed Immunoassay Analysis of Traumatic Brain Injury Biomarkers

2021

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“…Magnetic beads have been widely used to increase the binding affinity. 56,57 However, it is difficult to incorporate beads into the microfluidic channel as they easily settle down and get stuck in the channel. In vivo detection is also challenging as the beads emit strong auto-fluorescence.…”

Section: ■ Discussionmentioning

confidence: 99%

Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection System for Viral DNA Sensing

Hass

Bao

et al. 2020

ACS Omega

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A fully Integrated Micropillar Polydimethylsiloxane Accurate CRISPR deTection (IMPACT) system is developed for viral DNA detection. This powerful system is patterned with high-aspect-ratio micropillars to enhance reporter probe binding. After surface modification and probe immobilization, the CRISPR-Cas12a/crRNA complex is injected into the fully enclosed microchannel. With the presence of a double-stranded DNA target, the CRISPR enzyme is activated and denatures the single-stranded DNA reporters from the micropillars. This collateral cleavage releases fluorescence reporters into the assay, and the intensity is linearly proportional to the target DNA concentration ranging from 0.1 to 10 nM. Importantly, this system does not rely on the traditional dye-quencher-labeled probe, thus reducing the fluorescence background presented in the assay. Furthermore, our one-step detection protocol is performed on-chip at isothermal conditions (37 °C) without using complicated and time-consuming off-chip probe hybridization and denaturation. This miniaturized and fully packed IMPACT chip demonstrates sensitive and accurate DNA detection within 120 min and paves ways to the next-generation point-of-care diagnostics, responding to emerging and deadly pathogen outbreaks.

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“…Upon completion of the final amplification step, the reaction products (20 μL) were transferred to a 96-well plate pre-loaded with ∼25,000 streptavidin-coupled magnetic beads\well in 80 μL of x1 PBS buffer (Biological Industries, Beit HaEmek, Israel) with 0.05% (v/v) of Tween 20 (P9416, Sigma-Aldrich, MO, USA). Prior to their use in the assay, the magnetic beads were photobleached for 18 hours 25 . The total volume of 100 μL in each well was mixed by pipetting, and the plate was incubated under constant shaking (RH-24 3D Gyratory Rocker, MIULAB, Hangzhou, China) for 3 minutes at room temperature.…”

Section: Methodsmentioning

confidence: 99%

A Magnetic Modulation Biosensing-Based Molecular Assay for Rapid and Highly Sensitive Clinical Diagnosis of Coronavirus Disease 2019 (COVID-19)

Margulis

Erster

Roth

et al. 2021

The Journal of Molecular Diagnostics

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Rapid and sensitive detection of human pathogens, such as the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is an urgent and challenging task for clinical laboratories. Currently, the gold standard test for SARS-CoV-2-specific ribonucleic acid (RNA) is based on reverse transcription quantitative polymerase chain reaction (RT-qPCR), which relies on target amplification by Taq polymerase and uses a fluorescent resonance energy transfer (FRET)-based hydrolysis probe. Although this method is accurate and specific, it is also time consuming. To rapidly detect the presence of the viral RNA in clinical samples, we describe a new molecular assay that combines a highly sensitive magnetic modulation biosensing (MMB) system, rapid thermal cycling, and a modified double-quenched hydrolysis probe. Using in vitro transcribed SARS-CoV-2 RNA targets spiked in PCR-grade water, we found that the calculated limit of detection of the MMB-based molecular assay was 1.6 copies per reaction. Testing 309 RNA extracts from 170 confirmed RT-qPCR SARS-CoV-2-negative individuals (30 of which are positive to other respiratory viruses) and 139 RT-qPCR SARS-CoV-2-positive patients ( ) resulted in 97.8% sensitivity, 100% specificity, and 0% cross-reactivity. The total turnaround time of the MMB-based assay is 30 minutes, which is 3–4 times faster than a standard RT-qPCR. By adjusting the primers and the probe set, the platform can be easily adapted to detect most of the pathogens that are currently being diagnosed by RT-qPCR.

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Improving the sensitivity of fluorescence-based immunoassays by photobleaching the autofluorescence of magnetic beads

Cited by 4 publications

References 22 publications

A Variable Height Microfluidic Device for Multiplexed Immunoassay Analysis of Traumatic Brain Injury Biomarkers

A Variable Height Microfluidic Device for Multiplexed Immunoassay Analysis of Traumatic Brain Injury Biomarkers

Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection System for Viral DNA Sensing

A Magnetic Modulation Biosensing-Based Molecular Assay for Rapid and Highly Sensitive Clinical Diagnosis of Coronavirus Disease 2019 (COVID-19)

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