Outer‐Membrane Protease (OmpT) Based <i>E. coli</i> Sensing with Anionic Polythiophene and Unlabeled Peptide Substrate

Sinsinbar, Gaurav; Gudlur, Sushanth; Wood, Sarah; Ammanath, Gopal; Yildiz, Hakan U.; Palaniappan, Alagappan; Mrksich, Milan; Liedberg, Bo

doi:10.1002/ange.202008444

Cited by 3 publications

(12 citation statements)

References 39 publications

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“…Sinsinbar et al recently reported an optical assay for the detection of bacterial pathogens based on the activity of the bacterial membrane proteases and interaction of PTs with protease substrate. 131 They used an optimized peptide substrate for an outer membrane protease, OmpT, present on E. coli, and polythiophene acetic acid (PTAA) as the reporter PT. 131 The protease substrate, when interacting with PTAA, results in a blue shift in the absorbance spectrum and an increase in fluorescence (Figure 24).…”

Section: Analytesmentioning

confidence: 99%

“…131 They used an optimized peptide substrate for an outer membrane protease, OmpT, present on E. coli, and polythiophene acetic acid (PTAA) as the reporter PT. 131 The protease substrate, when interacting with PTAA, results in a blue shift in the absorbance spectrum and an increase in fluorescence (Figure 24). However, in the presence of E. coli, the peptide substrate was cleaved by OmpT, and the cleaved peptide fragments were not capable of bringing about any change in the absorbance and fluorescence spectra.…”

Section: Analytesmentioning

confidence: 99%

Section: Applications Of Pt Reportersmentioning

confidence: 99%

“…They used an optimized peptide substrate for an outer membrane protease, OmpT, present on E. coli , and polythiophene acetic acid (PTAA) as the reporter PT . The protease substrate, when interacting with PTAA, results in a blue shift in the absorbance spectrum and an increase in fluorescence (Figure ).…”

Section: Applications Of Pt Reportersmentioning

confidence: 99%

Section: Applications Of Pt Reportersmentioning

confidence: 99%

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A Perspective on Polythiophenes as Conformation Dependent Optical Reporters for Label-Free Bioanalytics

et al. 2022

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Poly(3-alkylthiophene) (PT)-based conjugated polyelectrolytes (CPEs) constitute an important class of responsive polymers with excellent optical properties. The electrostatic interactions between PTs and target analytes trigger complexation and concomitant conformational changes of the PT backbones that produce distinct optical responses. These conformation-induced optical responses of the PTs enable them to be utilized as reporters for detection of various analytes by employing simple UV−vis spectrophotometry or the naked eye. Numerous PTs with unique pendant groups have been synthesized to tailor their interactions with analytes such as nucleotides, ions, surfactants, proteins, and bacterial and viral pathogens. In this perspective, we discuss PTtarget analyte complexation for bioanalytical applications and highlight recent advancements in point-of-care and field deployable assays. Subsequently, we highlight a few areas of critical importance for future applications of PTs as reporters, including (i) design and synthesis of specific PTs to advance the understanding of the mechanisms of interaction with target analytes, (ii) using arrays of PTs and linear discriminant analysis for selective and specific detection of target analytes, (iii) translation of conventional homogeneous solution-based assays into heterogeneous membrane-based assay formats, and finally (iv) the potential of using PT as an alternative to conjugated polymer nanoparticles and dots in bioimaging.

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Section: Analytesmentioning

confidence: 99%

Section: Analytesmentioning

confidence: 99%

Section: Applications Of Pt Reportersmentioning

confidence: 99%

Section: Applications Of Pt Reportersmentioning

confidence: 99%

Section: Applications Of Pt Reportersmentioning

confidence: 99%

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A Perspective on Polythiophenes as Conformation Dependent Optical Reporters for Label-Free Bioanalytics

et al. 2022

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Smartphone and handheld fluorometer enable rapid point of care testing of Escherichia coli in urinary tract infections via specific proteolytic cleavage and cascade amplifications

Lin,

Wu,

Wang

et al. 2024

Sensors and Actuators B: Chemical

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Rapid Detection of Escherichia coli: Optimized Peptide-Polythiophene Interactions Help Reduce Assay Time and Improve Naked-Eye Detection

Sinsinbar

Gudlur

Liedberg

2022

ACS Appl. Mater. Interfaces

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Recent improvements in methods for rapid detection of microbial contamination in food and water samples have aided in the development of on-site and point-of-care testing. Early detection, made possible via on-site testing, can help limit the spread of food and waterborne illnesses. Recently, we reported a novel fluorescence-based Omptin-Polythiophene assay (the assay) to detect Escherichia coli in contaminated water samples. The assay targets OmpT�an E. coli outer membrane protease�and exploits the protease's ability to cleave at dibasic sites within a peptide. By combining a peptide substrate optimized for OmpT with a conjugated polythiophene reporter molecule whose optical properties vary upon interaction with the intact or cleaved peptide, we demonstrated the detection of 1−10 CFU/mL and 10 5 CFU/mL E. coli in 5.5 and 1 h, respectively. In comparison, most microbial detection methods that rely on culturing and plating techniques take anywhere between 8 and 24 h to report their results. Herein we report significant improvements in the assay which include reducing the assay time from an already short 1 h to a mere 10 min for detecting E. coli in highly contaminated samples and augmenting the assay with colorimetric sensing capability for naked eye discernment under normal visible light or under UV-A light. These improvements were made possible by characterizing the optical changes resulting from the interaction of the peptide with five carboxylate-functionalized polythiophene variants carrying different 3-side chain carboxylic acids and by identifying preferential peptide substrates via the screening of ten peptide sequence variants for OmpT activity.

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Outer‐Membrane Protease (OmpT) Based E. coli Sensing with Anionic Polythiophene and Unlabeled Peptide Substrate

Cited by 3 publications

References 39 publications

A Perspective on Polythiophenes as Conformation Dependent Optical Reporters for Label-Free Bioanalytics

A Perspective on Polythiophenes as Conformation Dependent Optical Reporters for Label-Free Bioanalytics

Smartphone and handheld fluorometer enable rapid point of care testing of Escherichia coli in urinary tract infections via specific proteolytic cleavage and cascade amplifications

Rapid Detection of Escherichia coli: Optimized Peptide-Polythiophene Interactions Help Reduce Assay Time and Improve Naked-Eye Detection

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