Allosteric split trehalase biosensor
AssigneeCreative Protein Solutions Inc.
InventorJeroen De Buck
Life of this patent
Estimating the term…
Abstract
The present disclosure relates to a method referred to herein as the “split trehalase assay biosensor” (also referred to herein as “STIGA”) is based on the use of engineered E. coli trehalase to detect analytes such as antibodies in a sample. The trehalase is engineered in a way such that the enzyme is split into two inactive fragments (N-terminal fragment H and C-terminal fragment A) with antigens fused to both fragments. When bivalent antibodies react specifically with the fused antigens, two inactive trehalase fragments are brought in close proximity to restore the activity of trehalase. The restored trehalase will hydrolyze trehalose into two glucose molecules which can be measured using existing glucose detection methods such as glucometer, Benedict's reagent, or ACCU-CHEK AVIVA® glucose test strips.
Claims8
A composition for use to detect an analyte in a biological sample, said composition comprising:
- a first component consisting of a TreAN fragment of a trehalase enzyme split into two fragments, said TreAN fragment consisting of an amino acid sequence set forth in anyone of SEQ ID Nos:
- 20 and 25, said amino acid sequence excluding the HIS-tag, said TreAN fragment fused to a first complexing domain wherein the first complexing domain is a first conserved stable protein sequence selected for fusion with said TreAN fragment;
- and
- a second component consisting of a TreAC fragment of the split trehalase enzyme, said TreAC fragment consisting of an amino acid sequence set forth in anyone of SEQ ID NOs. 22 and 23, said amino acid sequence excluding the HIS-tag, said TreAC fragment fused to a second complexing domain wherein the second complexing domain is a second conserved stable protein sequence selected for fusion with said TreAC;
- wherein the first component and second component are mixed with the biological sample and a trehalose substrate;
- whereby a presence of the analyte in the biological sample will fuse the first component and the second component to thereby provide a trehalase-catalyzed generation of glucose moieties from the trehalose substrate, said generated glucose moieties detectable and/or measurable by a glucose-detecting technology selected from an electrochemical assay, a colorimetric assay, a fluorometric assay, and a luminescent assay;
- and
- whereby a lack of presence of the analyte in the biological sample will not generate said glucose moieties.
A biosensor for detecting an analyte in a biological sample, the biosensor comprising:
- a test strip or a test chip communicable with a test strip reader;
- a first component engaged with a surface of the test strip or the test chip, wherein the first component consists of a TreAN fragment of a trehalase enzyme, said TreAN fragment consisting of an amino acid sequence set forth in anyone of SEQ ID NOs. 20 and 25, said amino acid sequence excluding the HIS-tag, said TreAN fragment fused to a first complexing domain wherein the first complexing domain is a first conserved stable protein sequence selected for fusion with said TreAN fragment;
- and
- a second component engaged with the surface of the test strip or the test chip, wherein the second component consists of a TreAC fragment of the trehalase enzyme, said TreAC fragment consisting of an amino acid sequence set forth in anyone of SEQ ID Nos:
- 22 and 23, said amino acid sequence excluding the HIS-tag, said TreAC fragment fused to a second complexing domain
- wherein the second complexing domain is a second conserved stable protein sequence selected for fusion with said TreAC fragment;
- wherein the test strip or the test chip is contactable with the biological sample and with a trehalose substrate whereby a presence of the analyte in the biological sample will complex the first component and the second component to thereby produce a trehalase-catalyzed generation of glucose moieties from the trehalose substrate, said generated glucose moieties detectable and/or measurable by a glucose-detecting technology selected from one of an electrochemical assay, a colorimetricassay, a fluorometricassay, and a luminescent assay;
- and whereby a lack of presence of the analyte in the biological sample will not produce said glucose moieties.
Dependencies are inferred from the claim text.