Membrane protease-based methods for detection of bacteria
Inventors
Liedberg, Bo • ALAGAPPAN, Palaniappan • Gudlur, Sushanth • SINSINBAR, Gaurav • AMMANATH, Gopal • Mrksich, Milan • WOOD, Sarah Elizabeth
Assignees
Nanyang Technological University • Northwestern University
Publication Number
US-12359240-B2
Publication Date
2025-07-15
Expiration Date
2040-04-27
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Abstract
The invention provides a method for detecting bacteria. The method utilises a peptide that forms a complex with a conjugated reporter polymer and is susceptible to cleavage by one or more proteases on the surface of a bacteria. Presence or absence of the bacteria can be determined by assessing the optical absorption and/or colour and/or photoluminescence (e.g. fluorescence) of the conjugated reporter polymer, which may undergo a conformational change after binding with the to the cleaved peptide substrate. Specifically, the peptide substrate may comprise a cleavage site for digestion by the protease, and the protease may be an omptin protease. The conjugated reporter polymer may be selected from a polythiophene, a poly(1,4-phenylene vinylene) (PPV), a poly(1,4-phenylene) (PPP), a polyfluorenes (PFO), a nitrogen-containing polymer such as polyquinoline, poly(2,5-pyridinevinylene) (PPyV), 1,3,4-oxadiazole, and poly(9-vinylcarbazole) (PVK), and a polypyrrole. The method may be used to detect contamination in food or water, or as a clinical and/or diagnostic test.
Core Innovation
The invention provides a method for detecting bacteria expressing at least one outer membrane protease by using a peptide substrate and a conjugated reporter polymer. The peptide substrate is susceptible to cleavage by one or more proteases on the surface of the bacteria, causing a change in the optical absorption and/or colour and/or photoluminescence of the conjugated reporter polymer due to changes in its conformation after interacting with intact peptide substrate versus cleaved peptide fragments.
The problem being solved is the need for a rapid, sensitive, and easy to use bacterial detection method that does not require sophisticated equipment or highly trained personnel. Existing gold standard tests, such as biochemical assays and 16S rRNA sequencing, require lengthy preprocessing and culturing steps taking from 12 hours to 7 days, which is not suitable for home testing or food industry needs. Rapid nucleic acid and immunoassays reduce the assay time to about 24 hours but are laboratory-based and require specialized instruments. Existing polymer-based biosensors lack specificity and have higher detection limits.
Claims Coverage
The patent contains 13 claims comprising multiple inventive features related to a bacterial detection method, peptide substrates, conjugated reporter polymers, and associated processes.
Method for detecting bacteria expressing outer membrane protease
A method comprising contacting a test sample with a peptide substrate containing two adjacent basic amino acid residues cleavable by bacteria-expressed outer membrane protease, simultaneously or sequentially contacting with a conjugated reporter polymer wherein intact peptide substrate forms a complex and changes polymer conformation, while cleaved fragments do not, followed by comparing optical absorption and/or colour and/or photoluminescence with a control.
Sequential contacting steps for detection method
A method comprising contacting the test sample first with the peptide substrate for a predetermined period, then contacting with conjugated reporter polymer under conditions where intact peptide induces polymer conformation change, followed by comparing optical/fluorescence characteristics with a control.
Targeted bacteria types
The bacteria detected are pathogenic and/or gram-negative, specifically selected from Escherichia coli, Salmonella enterica, Yersinia pestis, and Shigella flexneri.
Selection of conjugated reporter polymer
The polymer is selected from polythiophene, poly(1,4-phenylene vinylene), poly(1,4-phenylene), polyfluorenes, nitrogen-containing polymers (like polyquinoline, poly(2,5-pyridinevinylene), 1,3,4-oxadiazole, poly(9-vinylcarbazole)) and polypyrrole; or more specifically a conjugated polythiophene such as polythiophene acetic acid (PTAA), PT6, PT4, or their combinations.
Use of omptin protease as target outer membrane protease
The outer membrane protease targeted is an omptin protease, including OmpT, OmpP, PgtE, PgtE2, Pla, PlaA SopA series, IscP, Q8ZGQ6, Staphylococcal peptidase I, and Protease 7; with OmpT and Escherichia coli as a preferred example.
Peptide substrate characteristics
The peptide substrate contains two adjacent basic amino acid residues independently selected from Lysine, Arginine, or Histidine and comprises cleavage sites from a defined group of sequences including FRRV, FRRY, FRRA, YRRA, and ARRA.
Peptide substrate sequence similarity and structure
The substrate has at least 40% sequence similarity with peptides represented by SEQ ID NOs 6 to 54 or functional fragments thereof, and can be a peptide comprising amino acid sequences from SEQ ID NOs 1-17 or 49-54, or represented by formula X1-A-X2 with X1 and X2 being 0-10 amino acid residues.
Combined preferred components for detection
The method preferably uses bacteria selected from Escherichia coli, Salmonella enterica, Yersinia pestis, and Shigella flexneri; conjugated reporter polymer being PTAA, PT6, PT4 or combinations; and a peptide substrate with at least 40% sequence similarity to specific peptides including SEQ ID NOs 6-17 and 49-54.
Optional pre-processing steps and sample sources
The method can involve a pre-culturing and/or pre-concentration step before applying the peptide and conjugated polymer, with pre-culturing for not more than 12 hours; samples are derived from water, food, clinical or biological fluids; and the method is applicable for detecting contamination in water/food or for clinical/diagnostic testing.
Predetermined incubation time
The contacting with conjugated reporter polymer is conducted for a predetermined period ranging from 1 minute to 2 hours.
The claims cover a bacterial detection method utilizing a peptide substrate cleavable by outer membrane protease and a conjugated reporter polymer whose conformation changes upon interaction with intact peptide to produce an optical signal, targeting specific bacteria with defined peptides and polymers, optionally including sample pre-processing and applicable to various sample types.
Stated Advantages
Significant reduction in assay time allowing detection within minutes to hours instead of days.
High sensitivity capable of detecting contamination as low as 1 CFU/mL.
High specificity customizable to detect specific bacteria or strains by varying peptide substrates.
Room temperature storage and stability due to use of synthetic peptides and polythiophenes instead of antibodies.
Ease of use enabling performance by untrained users without sophisticated equipment.
Documented Applications
Detection of bacterial contamination in food and water sources.
Clinical and diagnostic tests including detection of urinary tract infections by Escherichia coli in urine samples.
Home-use kits for monitoring drinking water quality and pool water contamination.
Testing and monitoring contamination on fresh leafy vegetables.
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