Enhancing Target Detection: A Fluorescence-Based Streptavidin-Bead Displacement Assay

Sireethorn Tungsirisurp; Nunzianda Frascione

doi:10.3390/bios14100509

Enhancing Target Detection: A Fluorescence-Based Streptavidin-Bead Displacement Assay

Sireethorn Tungsirisurp, Nunzianda Frascione^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

2 Citations (Scopus)

Abstract

Fluorescence-based aptasensors have been regarded as innovative analytical tools for the detection and quantification of analytes in many fields, including medicine and therapeutics. Using DNA aptamers as the biosensor recognition component, conventional molecular beacon aptasensor designs utilise target-induced structural switches of the DNA aptamers to generate a measurable fluorescent signal. However, not all DNA aptamers undergo sufficient target-specific conformational changes for significant fluorescence measurements. Here, the use of complementary ‘antisense’ strands is proposed to enable fluorescence measurement through strand displacement upon target binding. Using a published target-specific DNA aptamer against the receptor binding domain of SARS-CoV-2, we designed a streptavidin-aptamer bead complex as a fluorescence displacement assay for target detection. The developed assay demonstrates a linear range from 50 to 800 nanomolar (nM) with a limit of detection calculated at 67.5 nM and a limit of quantification calculated at 204.5 nM. This provides a ‘fit-for-purpose’ model assay for the detection and quantification of any target of interest by adapting and functionalising a suitable target-specific DNA aptamer and its complementary antisense strand.

Original language	English
Article number	509
Journal	Biosensors
Volume	14
Issue number	10
DOIs	https://doi.org/10.3390/bios14100509
Publication status	Published - Oct 2024

Keywords

antisense strands
biosensor
DNA aptamers
fluorescence-based

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.3390/bios14100509

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title = "Enhancing Target Detection: A Fluorescence-Based Streptavidin-Bead Displacement Assay",

abstract = "Fluorescence-based aptasensors have been regarded as innovative analytical tools for the detection and quantification of analytes in many fields, including medicine and therapeutics. Using DNA aptamers as the biosensor recognition component, conventional molecular beacon aptasensor designs utilise target-induced structural switches of the DNA aptamers to generate a measurable fluorescent signal. However, not all DNA aptamers undergo sufficient target-specific conformational changes for significant fluorescence measurements. Here, the use of complementary {\textquoteleft}antisense{\textquoteright} strands is proposed to enable fluorescence measurement through strand displacement upon target binding. Using a published target-specific DNA aptamer against the receptor binding domain of SARS-CoV-2, we designed a streptavidin-aptamer bead complex as a fluorescence displacement assay for target detection. The developed assay demonstrates a linear range from 50 to 800 nanomolar (nM) with a limit of detection calculated at 67.5 nM and a limit of quantification calculated at 204.5 nM. This provides a {\textquoteleft}fit-for-purpose{\textquoteright} model assay for the detection and quantification of any target of interest by adapting and functionalising a suitable target-specific DNA aptamer and its complementary antisense strand.",

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author = "Sireethorn Tungsirisurp and Nunzianda Frascione",

note = "Publisher Copyright: {\textcopyright} 2024 by the authors.",

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PY - 2024/10

Y1 - 2024/10

N2 - Fluorescence-based aptasensors have been regarded as innovative analytical tools for the detection and quantification of analytes in many fields, including medicine and therapeutics. Using DNA aptamers as the biosensor recognition component, conventional molecular beacon aptasensor designs utilise target-induced structural switches of the DNA aptamers to generate a measurable fluorescent signal. However, not all DNA aptamers undergo sufficient target-specific conformational changes for significant fluorescence measurements. Here, the use of complementary ‘antisense’ strands is proposed to enable fluorescence measurement through strand displacement upon target binding. Using a published target-specific DNA aptamer against the receptor binding domain of SARS-CoV-2, we designed a streptavidin-aptamer bead complex as a fluorescence displacement assay for target detection. The developed assay demonstrates a linear range from 50 to 800 nanomolar (nM) with a limit of detection calculated at 67.5 nM and a limit of quantification calculated at 204.5 nM. This provides a ‘fit-for-purpose’ model assay for the detection and quantification of any target of interest by adapting and functionalising a suitable target-specific DNA aptamer and its complementary antisense strand.

AB - Fluorescence-based aptasensors have been regarded as innovative analytical tools for the detection and quantification of analytes in many fields, including medicine and therapeutics. Using DNA aptamers as the biosensor recognition component, conventional molecular beacon aptasensor designs utilise target-induced structural switches of the DNA aptamers to generate a measurable fluorescent signal. However, not all DNA aptamers undergo sufficient target-specific conformational changes for significant fluorescence measurements. Here, the use of complementary ‘antisense’ strands is proposed to enable fluorescence measurement through strand displacement upon target binding. Using a published target-specific DNA aptamer against the receptor binding domain of SARS-CoV-2, we designed a streptavidin-aptamer bead complex as a fluorescence displacement assay for target detection. The developed assay demonstrates a linear range from 50 to 800 nanomolar (nM) with a limit of detection calculated at 67.5 nM and a limit of quantification calculated at 204.5 nM. This provides a ‘fit-for-purpose’ model assay for the detection and quantification of any target of interest by adapting and functionalising a suitable target-specific DNA aptamer and its complementary antisense strand.

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KW - DNA aptamers

KW - fluorescence-based

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Enhancing Target Detection: A Fluorescence-Based Streptavidin-Bead Displacement Assay

Abstract

Keywords

UN SDGs

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