Nanoparticle-based lipase biosensor utilizing a custom-synthesized peptidyl-ester substrate
Inventors
MEDINTZ, Igor • Breger, Joyce A. • Susumu, Kimihiro • Diaz, Sebastian • Brask, Jesper
Assignees
Publication Number
US-11718868-B2
Publication Date
2023-08-08
Expiration Date
2041-01-29
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Abstract
Lipase activity can be detected with a biosensor that includes a quantum dot adhered to a construct having a lipase-cleavable ester to attach a fluorophore acceptor configured as a Förster resonance energy transfer (FRET) acceptor to the QD when the construct is bound thereto. Cleavage of the ester by a lipase results in a measurable reduction in FRET. In further embodiments, the cleavable ester can be used to detect esterase activity, or the ester could be replaced with a glycosidic linkage to detect glycoside activity.
Core Innovation
The invention is a nanoparticle-based lipase biosensor that detects lipase activity using a construct adhered to a quantum dot (QD). The construct comprises a peptide with a nanoparticle association domain, a cleavable ester positioned between the peptide and a fluorophore acceptor configured as a Förster resonance energy transfer (FRET) acceptor to the QD. Cleavage of the ester bond by lipase results in dissociation of the fluorophore acceptor from the QD, thereby altering the FRET signal, which is measurable and correlates with lipase activity.
The problem addressed by the invention is the need for specific and sensitive assays to monitor and quantitate lipase activity. Lipases are enzymes with important biological and commercial roles, including in food digestion, virulence of pathogens, industrial food preparation, biofuels preparation, and detergent additives. Existing methods lack the specificity and sensitivity necessary for precise lipase activity measurement.
The biosensor uses photoluminescent semiconductor quantum dots to which a custom-synthesized peptidyl-ester substrate is ratiometrically self-assembled via a terminal hexahistidine motif for metal coordination with the Zn-rich QD surface. This assembly allows a high rate of FRET between the QD and the substrate's dye acceptor. In the presence of lipase, hydrolysis of the ester releases the dye acceptor, altering FRET activity in a concentration-dependent manner. The assay can determine enzyme characteristics using integrated Michaelis-Menten kinetics. Alternative embodiments include replacing the cleavable ester with a glycosidic linkage to detect glycosidase activity.
Claims Coverage
The patent describes two independent claims covering a biosensor apparatus and a method for measuring esterase activity, each with inventive features related to the biosensor construction and use.
Biosensor construct with peptide-linked cleavable ester and FRET acceptor
A biosensor comprising a quantum dot adhered to at least one construct that includes a peptide with the amino acid sequence of SEQ ID NO:1, a cleavable ester positioned between the peptide and a fluorophore acceptor configured as a Förster resonance energy transfer (FRET) acceptor to the QD. Cleavage of the ester results in dissociation of the fluorophore acceptor from the QD and a change in FRET activity. The peptide includes a nanoparticle association domain effective for binding to the QD.
Method of measuring esterase activity using the biosensor
A method involving contacting a sample with the biosensor as described, measuring changes in FRET activity of the biosensor following contact, and correlating the change to esterase activity. The peptide has the amino acid sequence of SEQ ID NO:1 and includes a nanoparticle association domain for QD binding. The ester cleavage causes dissociation of the fluorophore acceptor and alters FRET.
The claims cover a lipase biosensor constructed with a peptide-linked cleavable ester and fluorophore acceptor adhered to a quantum dot, and a method for measuring esterase activity by detecting changes in FRET following ester cleavage. The features emphasize the specific peptide sequence and nanoparticle association domain facilitating biosensor assembly and activity detection.
Stated Advantages
Luminescent quantum dots and nanoparticles can be functionalized with various surface ligands to chemically influence the nanoparticle's environment.
Lipase peptidyl substrates are modularly synthesized to include a polyhistidine sequence for metal affinity coordination, spacers, charged sequences, and dye acceptors, allowing flexible peptide reconfiguration for different biosensing formats.
Binding substrates to quantum dots through a simple hexahistidine tag enables efficient assembly.
Other bioconjugation chemistries such as thiol attachment or biotin-avidin interactions can be used, and substrates can be chemically modified during synthesis for different designs.
Displaying multiple substrate copies on the nanoparticle surface in a dense localized format enhances sensitivity over single-substrate probe systems.
Increased local density of substrates enables greater relative enzymatic activity detection at lower enzyme concentrations.
Documented Applications
Monitoring and quantitation of lipase activity in biological and commercial contexts.
Detection of esterase activity using cleavable esters in the biosensor construct.
Potential detection of glycosidase activity by replacing the cleavable ester with a glycosidic linkage.
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