US-12618835-B2 - Multiplex microelectrode array for detection of proteases as biomarkers

US12618835B2US 12618835 B2US12618835 B2US 12618835B2US-12618835-B2

Abstract

An electrochemical method for measuring the activity of biomarkers using microelectrode arrays functionalized with peptide consensus sequences and redox reporter moieties. Contact of the arrays with a biological sample containing one or more target biomarkers results in cleavage of the peptides and changes the electric current across the array in a quantifiable manner indicating not just the presence of the target biomarker in the sample, but its activity.

Inventors

Jun Li
Duy H. Hua
Morgan James Anderson
Jessica Erin Koehne
Meyya Meyyappan

Assignees

KANSAS STATE UNIVERSITY RESEARCH FOUNDATION
UNITED STATES OF AMERICA AS REPRESENTED BY THE ADMINISTRATOR OF NASA

Dates

Publication Date: 20260505
Application Date: 20220426

Claims (13)

1 . A microelectrode array for simultaneous detection of two or more target protease biomarkers, said array comprising: a plurality of individually addressable microelectrodes spaced apart from one another and separated by an insulating layer, each microelectrode comprising an electrically conductive surface consisting of gold, an alkanethiol monolayer on said gold surface having a plurality of peptides extending therefrom, said alkanethiol monolayer comprising thiol moieties adsorbed to said gold surface and a plurality of terminal carboxylic acid groups and hydroxyl groups distal from the gold surface in a ratio, each peptide comprising a proximal end having an amino group covalently attached to a respective carboxylic acid group in said alkanethiol monolayer and a distal end that is spaced apart from said surface, said distal end of each peptide comprising a redox moiety attached thereto, each of said peptides comprising a consensus sequence for a target protease biomarker, wherein the ratio of carboxylic acid to hydroxyl groups in the alkanethiol monolayer yields a spacing of 2.46 nm to 7.9 nm between each peptide; wherein each of said microelectrodes comprising a plurality of said peptides extending therefrom; wherein at least one microelectrode comprises a plurality of peptides comprising first consensus sequences for detection of a first target protease biomarker and wherein at least a second microelectrode comprises a plurality of peptides comprising second consensus sequences different from said first consensus sequences for detection of a second target protease biomarker different from said first target protease biomarker; said array being configured to simultaneously detect the activity of two or more target protease biomarkers present within a biological sample through cleavage of a respective consensus sequence by a target protease biomarker, if present, which releases said redox moiety effecting a detectable decrease in alternating current (AC) peak current across said array over time, as measured by continuously repeated AC voltammetry, due to release of said redox moiety in proximity to said electrode surface.
2 . The microelectrode array of claim 1 , wherein each microelectrode is spaced A apart by 500 μm to 2 mm.
3 . The microelectrode array of claim 1 , wherein each of said microelectrodes is configured to detect a different target protease biomarker.
4 . The microelectrode array of claim 1 , wherein said redox moiety is selected from the group consisting of ferrocenes, methylene blues, viologens, anthraquinones, ethidium bromide, daunomycin, ruthenium, bis-pyridine, tris-pyridine, bis-imidizole, and analogs thereof.
5 . The microelectrode array of claim 1 , each of said peptides comprising a consensus sequence for a target protease biomarker selected from the group consisting of: Target Protease SEQ ID NO: Cathepsin B residues 2-5 of 1 Cathepsin B 4 Cathepsin B 1 Cathepsin B 6 Cathepsin B 7 Cathepsin B 8 Cathepsin B 9 ADAM-10 10 ADAM-10 residues 2-5 of SEQ 11 ADAM-10 residues 2-7 of SEQ 11 ADAM-10 residues 2-8 of SEQ 11 ADAM-10 11 ADAM-10 11 ADAM-17 11 Cathepsin B 2 Cathepsin B 3 ADAM-17 residues 3-8 of SEQ 11 Cathepsin D 12 ADAM-17 13 ADAM-17 14 Cathepsin D 15 Cathepsin D 16 MMP-9 17 MMP-9 18 MMP-9 19 MMP-9 20 MMP-9 21
6 . An electronic chip comprising a microelectrode array according to claim 1 , further comprising contact pads, each contact pad being connected to a respective microelectrode via a respective conductive lead configured for continuous ACV interrogation and detection of AC electrical current and decrease of AC peak current over time upon contact of a microelectrode with a target protease biomarker.
7 . A system for simultaneous electrochemical detection of two or more target protease biomarkers, said system comprising an electronic chip according to claim 6 , wherein said chip is positioned within an electrochemical cell, said electrochemical cell being electrically connected via a breakout box to a potentiostat, said system further comprising a microfluidic channel in fluid communication with a sample inlet and sample outlet and configured to direct a biological sample into contact with said microelectrode array, said system further comprising one counter electrode and one reference electrode, wherein said counter electrode and said reference electrode are each printed on said chip or positioned or deposited on a coverslip or cover aligned over said microelectrode array and in contact with the microfluidic channel and biological sample flowing therethrough.
8 . A method of simultaneously detecting two or more protease biomarkers within a biological sample comprising contacting a microelectrode array according to claim 1 with a biological sample containing or suspected of containing two or more protease biomarkers and detecting a decrease in the AC peak electric current across said array over time, as measured by continuously repeated AC voltammetry.
9 . The method of claim 8 , wherein cleavage of a consensus sequence by a target protease biomarker results in a detectable exponential decrease in the peak current as measured by AC voltammetry.
10 . The method of claim 9 , wherein an inverse of the exponential decrease time constant is correlated with activity of the target protease biomarker in the biological sample.
11 . The method of claim 8 , wherein the biological sample is selected from the group consisting of blood, serum, urine, saliva, sweat, exhaled breath condensate, cell lysate, tissue lysate, and tissue biopsies.
12 . The method of claim 8 , wherein aliquots of a collected biological sample can be directly contacted with said array without dilution.
13 . The method of claim 8 , further comprising diluting said biological sample in a modified buffer before contacting with said array, wherein said modified buffer has a pH ranging from 7 to 7.5, wherein said modified buffer is a phosphate-based buffer that is essentially free of chloride salts.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims the priority benefit of U.S. Provisional Patent Application Ser. No. 63/179,848, filed Apr. 26, 2021, entitled MULTIPLEX MICROELECTRODE ARRAY FOR DETECTION OF PROTEASES AS BIOMARKERS, incorporated by reference in its entirety herein. FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under R01 CA217657 awarded by the National Cancer Institute of the National Institutes of Health. The government has certain rights in the invention. SEQUENCE LISTING This application contains a sequence listing in computer readable format (CRF), submitted via EFS-Web as an ASCII text file entitled “SequenceListing,” created on Apr. 26, 2022, as 4,126 bytes, to serve as both the paper copy and CRF in compliance with 37 C.F.R. 1.821. The content of the ASCII text file is hereby incorporated by reference herein. BACKGROUND Field Electronic chips, micro- and nanoelectrode arrays, and assays for simultaneous, multiplex profiling of the protease biomarkers, which can be used for advanced medical diagnosis, treatment monitoring and protease inhibitor screening. Description of Related Art Proteases play important roles as protein-degrading enzymes in many metabolic processes, including immune response, wound healing, food digestion, cell cycle, and protein recycling. Proteases hydrolyze proteins based on recognition of specific peptide sequences. Two viral cysteine proteases, the main protease (Mpro, also called 3C like protease, 3CLpro) and the papain like protease (PLpro) were also found to play critical mediating role in viral production and transcription in the coronavirus SARS-COV-2 which caused the COVID-19 global pandemic. Proteases also act as key signaling molecules in progression of many diseases such as cancer, neurodegenerative diseases, cardiovascular diseases, diabetes, and other inflammatory diseases. For example, aberrant overexpression of proteases has been reported in breast cancer, colorectal cancer, gastric cancer, and prostate cancer. It is well-known that proteases play diverse roles in tumor growth, invasion, and metastasis. Several matrix metalloproteinases (MMPs), such as MMP-2 and MMP-9, can affect signaling pathways and growth factors to enhance tumor growth. Numerous proteases, including cathepsins, kallikreins, and other serine proteases, facilitate the spread of cancer to distant organs by degrading the extracellular matrix. For example, proteases can degrade E-cadherin, a tumor suppressor and an essential protein in the formation of adherent junctions to bind cells with each other. Small molecule drugs targeting protease inhibition have attracted great attentions for cancer treatment. However, it remains a great challenge to understand the complex protease signaling and develop specific inhibitors. First, there are about 600 known proteases in humans, and they interact with each other in a complex network. Second, individual proteases have very limited indicative value because they only represent one aspect of carcinogens. Third, the protease levels in humans are extremely low. Hence, there is a strong demand for developing highly sensitive and highly specific bioanalytical techniques that can detect a group of protease biomarkers in parallel, rather than a single one each time. Particularly, the activity of proteases in extracellular space is significantly reduced and subject to rapid inactivation. Developing new techniques that can directly detect extracellular protease activity is critical for disease diagnostics. Cathepsin B is a member of the cysteine cathepsin family consisting of 11 lysosomal hydrolases. It is linked to general protein degradation in lysosomes. Initially, cathepsin B is synthesized on the rough endoplasmic reticulum (RER) as a proenzyme consisting of 339 amino acids with a signal peptide of 17 amino acids. After post-translational modification, proenzyme cathepsin B undergoes autocatalytic activation (normally in mild acidic conditions) and converts into mature cathepsin B by proteolytic cleavage and dissociation of the blocking peptide. Increased cathepsin B levels have been observed in many types of cancer such as prostate cancer, melanomas cancer and breast cancer. Its activity is essential for tumor migration, invasion, and metastases. However, the link between the cathepsin B concentration and activity is not clear. Developing a rapid, sensitive and specific method for detecting cathepsin B activity is critical for cancer diagnosis and therapeutic efficacy assessment. Currently, protease detection can be classified into two broad categories, i.e., affinity-based and activity-based techniques. The affinity-based technique detects the protease concentration by capturing proteases using the specific probe-target affinity such as enzyme-linked immunosorbent assay (ELISA) and aptamer sensors. Although ELISA has very high selectivity and sensitivity, it is time-consuming,