Photobleaching: Improving the Sensitivity of Fluorescence‐Based Immunoassays by Photobleaching the Autofluorescence of Magnetic Beads (Small 3/2019)

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

doi:10.1002/smll.201970016

Cited by 4 publications

(3 citation statements)

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“…For example, the detection sensitivity of traditional immunoassay approaches is limited due to the low binding capacity. Magnetic beads have been widely used to increase the binding affinity. , 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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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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“…Magnetic beads have been widely used to increase the binding affinity. 46,47 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 (IMPACT) System for Rapid Viral DNA Sensing

Bao

et al. 2020

Preprint

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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, CRISPR Cas12a/crRNA complex is injected into the fully enclosed system. With the presence of double-stranded DNA target, the CRISPR enzyme is activated and non-specifically cleaves the ssDNA reporters initially immobilized on the micropillars. This collateral cleavage releases fluorescence dyes 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 traditional dye-quencher labeled probe thus eliminating the fluorescence background presented in the assay. Furthermore, our one-step detection protocol is performed 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 rapid, sensitive, and simple nucleic acid detection and is an ideal candidate for the next generation molecular diagnostic platform for point-of-care (POC) applications, responding to emerging and deadly pathogen outbreaks.

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“…Despite the high quantum yields provided by the QDs, fluorescence sensitivity remains an issue to be resolved. For low-concentrated analytes, the emission of the fluorescent signal is weak and interferes with the background noise [146]. As cited in Table 7, Roth et al proposed a solution to reduce the autofluorescence of the magnetic bead, limiting the analytical detection performance of the fluorescent immunoassays by overlapping the fluorescent dyes and reducing the autofluorescence to 1% of its initial value.…”

Section: Challenges and Solutions For Fluorescence Based Detectionmentioning

confidence: 99%

Immunoassays: Analytical and Clinical Performance, Challenges, and Perspectives of SERS Detection in Comparison with Fluorescent Spectroscopic Detection

Terzapulo,

Kassenova,

Bukasov

2024

IJMS

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Immunoassays (IAs) with fluorescence-based detection are already well-established commercialized biosensing methods, such as enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay (LFIA). Immunoassays with surface-enhanced Raman spectroscopy (SERS) detection have received significant attention from the research community for at least two decades, but so far they still lack a wide clinical commercial application. This review, unlike any other review that we have seen, performs a three-dimensional performance comparison of SERS IAs vs. fluorescence IAs. First, we compared the limit of detection (LOD) as a key performance parameter for 30 fluorescence and 30 SERS-based immunoassays reported in the literature. We also compared the clinical performances of a smaller number of available reports for SERS vs. fluorescence immunoassays (FIAs). We found that the median and geometric average LODs are about 1.5–2 orders of magnitude lower for SERS-based immunoassays in comparison to fluorescence-based immunoassays. For instance, the median LOD for SERS IA is 4.3 × 10−13 M, whereas for FIA, it is 1.5 × 10−11 M. However, there is no significant difference in average relative standard deviation (RSD)—both are about 5–6%. The analysis of sensitivity, selectivity, and accuracy reported for a limited number of the published clinical studies with SERS IA and FIA demonstrates an advantage of SERS IA over FIA, at least in terms of the median value for all three of those parameters. We discussed common and specific challenges to the performances of both SERS IA and FIA, while proposing some solutions to mitigate those challenges for both techniques. These challenges include non-specific protein binding, non-specific interactions in the immunoassays, sometimes insufficient reproducibility, relatively long assay times, photobleaching, etc. Overall, this review may be useful for a large number of researchers who would like to use immunoassays, but particularly for those who would like to make improvements and move forward in both SERS-based IAs and fluorescence-based IAs.

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Photobleaching: Improving the Sensitivity of Fluorescence‐Based Immunoassays by Photobleaching the Autofluorescence of Magnetic Beads (Small 3/2019)

Cited by 4 publications

References 0 publications

Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection System for Viral DNA Sensing

Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection System for Viral DNA Sensing

Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection (IMPACT) System for Rapid Viral DNA Sensing

Immunoassays: Analytical and Clinical Performance, Challenges, and Perspectives of SERS Detection in Comparison with Fluorescent Spectroscopic Detection

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