Comparison of static and microfluidic protease assays using modified bioluminescence resonance energy transfer chemistry.

<h4>Background</h4>Fluorescence and bioluminescence resonance energy transfer (F/BRET) are two forms of Förster resonance energy transfer, which can be used for optical transduction of biosensors. BRET has several advantages over fluorescence-based technologies because it does not requir...

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Autores principales:	Nan Wu, Helen Dacres, Alisha Anderson, Stephen C Trowell, Yonggang Zhu
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/14a10281f72d4204bebcaec911d9215b
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spelling	oai:doaj.org-article:14a10281f72d4204bebcaec911d9215b2021-11-18T08:32:29ZComparison of static and microfluidic protease assays using modified bioluminescence resonance energy transfer chemistry.1932-620310.1371/journal.pone.0088399https://doaj.org/article/14a10281f72d4204bebcaec911d9215b2014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24551097/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>Fluorescence and bioluminescence resonance energy transfer (F/BRET) are two forms of Förster resonance energy transfer, which can be used for optical transduction of biosensors. BRET has several advantages over fluorescence-based technologies because it does not require an external light source. There would be benefits in combining BRET transduction with microfluidics but the low luminance of BRET has made this challenging until now.<h4>Methodology</h4>We used a thrombin bioprobe based on a form of BRET (BRET(H)), which uses the BRET(1) substrate, native coelenterazine, with the typical BRET(2) donor and acceptor proteins linked by a thrombin target peptide. The microfluidic assay was carried out in a Y-shaped microfluidic network. The dependence of the BRET(H) ratio on the measurement location, flow rate and bioprobe concentration was quantified. Results were compared with the same bioprobe in a static microwell plate assay.<h4>Principal findings</h4>The BRET(H) thrombin bioprobe has a lower limit of detection (LOD) than previously reported for the equivalent BRET(1)-based version but it is substantially brighter than the BRET(2) version. The normalised BRET(H) ratio of the bioprobe changed 32% following complete cleavage by thrombin and 31% in the microfluidic format. The LOD for thrombin in the microfluidic format was 27 pM, compared with an LOD of 310 pM, using the same bioprobe in a static microwell assay, and two orders of magnitude lower than reported for other microfluidic chip-based protease assays.<h4>Conclusions</h4>These data demonstrate that BRET based microfluidic assays are feasible and that BRET(H) provides a useful test bed for optimising BRET-based microfluidics. This approach may be convenient for a wide range of applications requiring sensitive detection and/or quantification of chemical or biological analytes.Nan WuHelen DacresAlisha AndersonStephen C TrowellYonggang ZhuPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 2, p e88399 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Nan Wu Helen Dacres Alisha Anderson Stephen C Trowell Yonggang Zhu Comparison of static and microfluidic protease assays using modified bioluminescence resonance energy transfer chemistry.
description	<h4>Background</h4>Fluorescence and bioluminescence resonance energy transfer (F/BRET) are two forms of Förster resonance energy transfer, which can be used for optical transduction of biosensors. BRET has several advantages over fluorescence-based technologies because it does not require an external light source. There would be benefits in combining BRET transduction with microfluidics but the low luminance of BRET has made this challenging until now.<h4>Methodology</h4>We used a thrombin bioprobe based on a form of BRET (BRET(H)), which uses the BRET(1) substrate, native coelenterazine, with the typical BRET(2) donor and acceptor proteins linked by a thrombin target peptide. The microfluidic assay was carried out in a Y-shaped microfluidic network. The dependence of the BRET(H) ratio on the measurement location, flow rate and bioprobe concentration was quantified. Results were compared with the same bioprobe in a static microwell plate assay.<h4>Principal findings</h4>The BRET(H) thrombin bioprobe has a lower limit of detection (LOD) than previously reported for the equivalent BRET(1)-based version but it is substantially brighter than the BRET(2) version. The normalised BRET(H) ratio of the bioprobe changed 32% following complete cleavage by thrombin and 31% in the microfluidic format. The LOD for thrombin in the microfluidic format was 27 pM, compared with an LOD of 310 pM, using the same bioprobe in a static microwell assay, and two orders of magnitude lower than reported for other microfluidic chip-based protease assays.<h4>Conclusions</h4>These data demonstrate that BRET based microfluidic assays are feasible and that BRET(H) provides a useful test bed for optimising BRET-based microfluidics. This approach may be convenient for a wide range of applications requiring sensitive detection and/or quantification of chemical or biological analytes.
format	article
author	Nan Wu Helen Dacres Alisha Anderson Stephen C Trowell Yonggang Zhu
author_facet	Nan Wu Helen Dacres Alisha Anderson Stephen C Trowell Yonggang Zhu
author_sort	Nan Wu
title	Comparison of static and microfluidic protease assays using modified bioluminescence resonance energy transfer chemistry.
title_short	Comparison of static and microfluidic protease assays using modified bioluminescence resonance energy transfer chemistry.
title_full	Comparison of static and microfluidic protease assays using modified bioluminescence resonance energy transfer chemistry.
title_fullStr	Comparison of static and microfluidic protease assays using modified bioluminescence resonance energy transfer chemistry.
title_full_unstemmed	Comparison of static and microfluidic protease assays using modified bioluminescence resonance energy transfer chemistry.
title_sort	comparison of static and microfluidic protease assays using modified bioluminescence resonance energy transfer chemistry.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/14a10281f72d4204bebcaec911d9215b
work_keys_str_mv	AT nanwu comparisonofstaticandmicrofluidicproteaseassaysusingmodifiedbioluminescenceresonanceenergytransferchemistry AT helendacres comparisonofstaticandmicrofluidicproteaseassaysusingmodifiedbioluminescenceresonanceenergytransferchemistry AT alishaanderson comparisonofstaticandmicrofluidicproteaseassaysusingmodifiedbioluminescenceresonanceenergytransferchemistry AT stephenctrowell comparisonofstaticandmicrofluidicproteaseassaysusingmodifiedbioluminescenceresonanceenergytransferchemistry AT yonggangzhu comparisonofstaticandmicrofluidicproteaseassaysusingmodifiedbioluminescenceresonanceenergytransferchemistry
_version_	1718421667391209472

Comparison of static and microfluidic protease assays using modified bioluminescence resonance energy transfer chemistry.

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