scholarly journals A Low-Cost, 3D-Printed Biosensor for Rapid Detection of Escherichia coli

2022 ◽  
Author(s):  
Samir Malhotra ◽  
Dang Song Pham ◽  
Michael P.H. Lau ◽  
Anh H. Nguyen ◽  
Hung Cao
Keyword(s):  
Escherichia Coli ◽  
Rapid Detection ◽  
Pathogen Detection ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Bacterial Pathogens ◽  
Cross Reactivity ◽  
Potential Candidate ◽  
Limit Of Quantification ◽  
3D Printed

Detection of bacterial pathogens is significant in the fields of food safety, medicine, public health, etc. If bacterial pathogens are not treated promptly, antimicrobial resistance is possible and can lead to morbidity and mortality. Current bacterial detection methodologies rely on laboratory-based techniques that pose limitations such as long turnaround detection times, expensive costs, in-adequate accuracy, and required trained specialists. Here, we describe a cost-effective and port-able 3D-printed electrochemical biosensor that facilitates rapid detection of certain Escherichia coli (E. coli) strains (DH5α, BL21, TOP10, and JM109) within 15 minutes using 500 μL of sample and costs $2.50 per test. The sensor displayed an excellent limit of detection (LOD) of 53 CFU, limit of quantification (LOQ) of 270 CFU, and showed cross-reactivity with strains BL21 and JM109 due to shared epitopes. This advantageous diagnostic device is a potential candidate for high-frequency testing at the point of care as well as applicable to various fields where pathogen detection is of interest.

scholarly journals SaQuant: a real-time PCR assay for quantitative assessment of Staphylococcus aureus

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Colin Wood ◽  
Jason Sahl ◽  
Sara Maltinsky ◽  
Briana Coyne ◽  
Benjamin Russakoff ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Sensitivity And Specificity ◽  
Pathogen Detection ◽  
Limit Of Detection ◽  
Future Research ◽  
Limit Of Quantification ◽  
Pathogen Prevalence ◽  
Single Well ◽  
Effective Public Health ◽  
Detection And Quantification

Abstract Background Molecular assays are important tools for pathogen detection but need to be periodically re-evaluated with the discovery of additional genetic diversity that may cause assays to exclude target taxa or include non-target taxa. A single well-developed assay can find broad application across research, clinical, and industrial settings. Pathogen prevalence within a population is estimated using such assays and accurate results are critical for formulating effective public health policies and guiding future research. A variety of assays for the detection of Staphylococcus aureus are currently available. The utility of commercial assays for research is limited, given proprietary signatures and lack of transparent validation. Results In silico testing of existing peer-reviewed assays show that most suffer from a lack of sensitivity and specificity. We found no assays that were specifically designed and validated for quantitative use. Here we present a qPCR assay, SaQuant, for the detection and quantification of S. aureus as might be collected on sampling swabs. Sensitivity and specificity of the assay was 95.6 and 99.9 %, respectively, with a limit of detection of between 3 and 5 genome equivalents and a limit of quantification of 8.27 genome equivalents. The presence of DNA from non-target species likely to be found in a swab sample, did not impact qualitative or quantitative abilities of the assay. Conclusions This assay has the potential to serve as a valuable tool for the accurate detection and quantification of S. aureus collected from human body sites in order to better understand the dynamics of prevalence and transmission in community settings.

scholarly journals A Smartphone Camera Colorimetric Assay of Acetylcholinesterase and Butyrylcholinesterase Activity

Sensors ◽  
2021 ◽  
Vol 21 (5) ◽  
pp. 1796
Author(s):  
Miroslav Pohanka ◽  
Jitka Zakova
Keyword(s):  
Limit Of Detection ◽  
Colorimetric Assay ◽  
Point Of Care ◽  
Specific Treatment ◽  
Point Of Care Testing ◽  
Analytical Instruments ◽  
Colorimetric Test ◽  
3D Printed ◽  
Butyrylcholinesterase Activity ◽  
Additional Education

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) can serve as biochemical markers of various pathologies like liver disfunction and poisonings by nerve agents. Ellman’s assay is the standard spectrophotometric method to measure cholinesterase activity in clinical laboratories. The authors present a new colorimetric test to assess AChE and BChE activity in biological samples using chromogenic reagents, treated 3D-printed measuring pads and a smartphone camera as a signal detector. Multiwell pads treated with reagent substrates 2,6-dichlorophenolindophenyl acetate, indoxylacetate, ethoxyresorufin and methoxyresorufin were prepared and tested for AChE and BChE. In the experiments, 3D-printed pads containing indoxylacetate as a chromogenic substrate were optimal for analytical purposes. The best results were achieved using the red (R) channel, where the limit of detection was 4.05 µkat/mL for BChE and 4.38 µkat/mL for AChE using a 40 µL sample and a 60 min assay. The major advantage of this method is its overall simplicity, as samples are applied directly without any specific treatment or added reagents. The assay was also validated to the standard Ellman’s assay using human plasma samples. In conclusion, this smartphone camera-based colorimetric assay appears to have practical applicability and to be a suitable method for point-of-care testing because it does not require specific manipulation, additional education of staff or use of sophisticated analytical instruments.

scholarly journals Label-Free Electrochemical Biosensor Based on Au@MoS₂–PANI for Escherichia coli Detection

Chemosensors ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 49
Author(s):  
Pushap Raj ◽  
Man Hwan Oh ◽  
Kyudong Han ◽  
Tae Yoon Lee
Keyword(s):  
Escherichia Coli ◽  
Bacterial Infections ◽  
Limit Of Detection ◽  
Bacterial Pathogens ◽  
Cost Effective ◽  
Covalent Immobilization ◽  
Label Free ◽  
Self Assembled Monolayer ◽  
Detection Range ◽  
Linear Detection

Bacterial infections have become a significant challenge in terms of public health, the food industry, and the environment. Therefore, it is necessary to address these challenges by developing a rapid, cost-effective, and easy-to-use biosensor for early diagnosis of bacterial pathogens. Herein, we developed a simple, label-free, and highly sensitive immunosensor based on electrochemical detection using the Au@MoS₂–PANI nanocomposite. The conductivity of the glassy carbon electrode is greatly enhanced using the Au@MoS₂–PANI nanocomposite and a self-assembled monolayer of mercaptopropionic acid on the gold nanoparticle surface was employed for the covalent immobilization of antibodies to minimize the nonspecific adsorption of bacterial pathogens on the electrode surface. The biosensor established a high selectivity and sensitivity with a low limit of detection of 10 CFU/mL, and detected Escherichia coli within 30 min. Moreover, the developed biosensor demonstrated a good linear detection range, practical utility in urine samples, and electrode regenerative studies.

scholarly journals Rapid immunoassays for detection of anabolic nortestosterone in dietary supplements

2013 ◽  
Vol 31 (No. 5) ◽  
pp. 514-519 ◽  
Author(s):  
B. Holubová ◽  
S. Göselová ◽  
L. Ševčíková ◽  
M. Vlach ◽  
M. Blažková ◽  
...  
Keyword(s):  
Dietary Supplements ◽  
Rapid Detection ◽  
Visual Detection ◽  
Limit Of Detection ◽  
Polyclonal Antibodies ◽  
Cross Reactivity ◽  
Rapid Analysis ◽  
Experimental Conditions ◽  
Immunochromatographic Strip ◽  
Strip Test

An enzyme immunoassay (ELISA) and an immunochromatographic strip were designed for a rapid detection of nortestosterone in dietary supplements. Two polyclonal antibodies and two types of nortestosterone-protein coating conjugates were tested to develop the most appropriate method. Under optimal experimental conditions, the most sensitive ELISA achieved the IC<sub>50 </sub>and the limit of detection values of 6.41 and 0.09 ng/ml, respectively. The assay specificity was tested measuring cross-reactivity of several steroids. The interference with the assay was negligible (&lt; 0.1%), except for cross-reactivity with another frequently abused steroid testosterone (23%). The optimised gold particle-based immunochromatographic strip provided in semi-quantitative test a visual detection limit of 1&nbsp;ng/ml. None of these methods showed the interference using a filtrate of the suspension of non-contaminated sample. After the validation for particular matrices, the ELISA and the strip test could be useful tools for a rapid analysis of nortestosterone in crude extracts of dietary supplements.

scholarly journals A Droplet Digital PCR Assay to Detect SARS-CoV-2 RNA

2020 ◽  
Author(s):  
Martin J. Romeo ◽  
Christian P. DiPaola ◽  
Cassidy Mentus ◽  
Cynthia D. Timmers
Keyword(s):  
Limit Of Detection ◽  
Digital Pcr ◽  
The United States ◽  
Cross Reactivity ◽  
Droplet Digital Pcr ◽  
Molecular Testing ◽  
Guidance Document ◽  
Respiratory Pathogens ◽  
Limit Of Quantification ◽  
United States Food

AbstractWe describe a quantitative droplet digital PCR (ddPCR) assay for detection of SARS-CoV-2 viral ribonucleic acid (RNA) in total RNA extracted from human sputum. This method was validated using the guidance of the United States Food and Drug Administration’s Accelerated Emergency Use Authorization (EUA) Template for SARS-CoV-2 that Causes Coronavirus Disease (COVID-19) Molecular Testing of Respiratory Speciment in CLIA Certified High-Complexity Laboratories. Though our laboratory is not CLIA certified, this method met all criteria specified by the guidance document with a Limit of Detection (LOD) of 0.25 copies/μL in the final ddPCR (at least 19/20 replicates reactive), which we consider to be a Lower Limit of Quantification (LLOQ); inclusivity of all known annotated SARS-CoV-2 genomes; no cross-reactivity with other respiratory pathogens; and reactivity of all contrived positives at or above the LOD.

scholarly journals 2019 Novel Coronavirus Disease (COVID-19): Paving the Road for Rapid Detection and Point-of-Care Diagnostics

Micromachines ◽  
2020 ◽  
Vol 11 (3) ◽  
pp. 306 ◽  
Author(s):  
Trieu Nguyen ◽  
Dang Duong Bang ◽  
Anders Wolff
Keyword(s):  
Rapid Detection ◽  
Point Of Care ◽  
Potential Candidate ◽  
The Road ◽  
Point Of Care Diagnostics ◽  
Novel Coronavirus

We believe a point-of-care (PoC) device for the rapid detection of the 2019 novel Coronavirus (SARS-CoV-2) is crucial and urgently needed. With this perspective, we give suggestions regarding a potential candidate for the rapid detection of the coronavirus disease 2019 (COVID-19), as well as factors for the preparedness and response to the outbreak of the COVID-19.

scholarly journals The Effect of Synthesis Procedure on Hydrogen Peroxidase-Like Catalytic Activity of Iron Oxide Magnetic Particles

2020 ◽  
Vol 10 (19) ◽  
pp. 6756
Author(s):  
Atripan Mukherjee ◽  
Amir M. Ashrafi ◽  
Pavel Svec ◽  
Lukáš Richtera ◽  
Jan Přibyl ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Rapid Detection ◽  
Magnetic Particles ◽  
Limit Of Detection ◽  
Cathodic Potential ◽  
Limit Of Quantification ◽  
Figures Of Merit ◽  
Satisfactory Analysis ◽  
Synthesis Procedure

A comparative study was carried out using magnetic nanoparticles (MNPs) for the fabrication of non-enzymatic sensors for the continuous and rapid detection and monitoring of H2O2. Various MNPs, differing in terms of their synthesis procedure and modification, were synthesized and characterized by different techniques. The electrochemical catalytic activity of the synthesized MNPs toward the reduction in H2O2 was investigated by cyclic voltammetry. The naked MNPs showed the highest catalytic activity among all the synthesized MNPs. The biosensor based on the naked MNPs was then applied in the determination of H2O2 using chronoamperometry. The parameters such as the applied cathodic potential and the amount of MNPs on the developed biosensor were optimized. Moreover, the analytical figures of merit, including reproducibility (RSD = 6.14%), sensitivity (m = 0.0676 µA µM−1), limit of detection (LOD) = 27.02 µmol L−1, and limit of quantification (LOQ) = 89.26 µmol L−1 of the developed biosensor indicate satisfactory analysis. Finally, MNPs were successfully utilized for the determination of H2O2 in milk.

scholarly journals Detection of ESKAPE Bacterial Pathogens at the Point of Care Using Isothermal DNA-Based Assays in a Portable Degas-Actuated Microfluidic Diagnostic Assay Platform

2016 ◽  
Vol 83 (4) ◽  
Author(s):  
Lars D. Renner ◽  
Jindong Zan ◽  
Linda I. Hu ◽  
Manuel Martinez ◽  
Pedro J. Resto ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Low Cost ◽  
Bacterial Pathogens ◽  
Diagnostic System ◽  
Molecular Diagnostic ◽  
Technology Gap ◽  
Content Type ◽  
Antimicrobial Drug Resistance ◽  
Industrial Processing

ABSTRACT An estimated 1.5 billion microbial infections occur globally each year and result in ∼4.6 million deaths. A technology gap associated with commercially available diagnostic tests in remote and underdeveloped regions prevents timely pathogen identification for effective antibiotic chemotherapies for infected patients. The result is a trial-and-error approach that is limited in effectiveness, increases risk for patients while contributing to antimicrobial drug resistance, and reduces the lifetime of antibiotics. This paper addresses this important diagnostic technology gap by describing a low-cost, portable, rapid, and easy-to-use microfluidic cartridge-based system for detecting the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) bacterial pathogens that are most commonly associated with antibiotic resistance. The point-of-care molecular diagnostic system consists of a vacuum-degassed microfluidic cartridge preloaded with lyophilized recombinase polymerase amplification (RPA) assays and a small portable battery-powered electronic incubator/reader. The isothermal RPA assays detect the targeted ESKAPE pathogens with high sensitivity (e.g., a limit of detection of ∼10 nucleic acid molecules) that is comparable to that of current PCR-based assays, and they offer advantages in power consumption, engineering, and robustness, which are three critical elements required for the point-of-care setting. IMPORTANCE This paper describes a portable system for rapidly identifying bacteria in resource-limited environments; we highlight the capabilities of the technology by detecting different pathogens within the ESKAPE collection, which cause nosocomial infections. The system is designed around isothermal DNA-based assays housed within an autonomous plastic cartridge that are designed with the end user in mind, who may have limited technological training. Displaying excellent sensitivity and specificity, the assay systems that we demonstrate may enable future diagnoses of bacterial infection to guide the development of effective chemotherapies and may have a role in areas beyond health where rapid detection is valuable, including in industrial processing and manufacturing, food security, agriculture, and water quality testing.

scholarly journals Multiplex Detection of Three Select Agents Directly from Blood by Use of the GeneXpert System

2019 ◽  
Vol 57 (5) ◽  
Author(s):  
Padmapriya P. Banada ◽  
Srinidhi Deshpande ◽  
Sukalyani Banik ◽  
Darshini Shah ◽  
Ranie Koshy ◽  
...  
Keyword(s):  
Rapid Detection ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Severe Disease ◽  
High Sensitivity ◽  
Negative Control ◽  
Blood Samples ◽  
Content Type ◽  
Clinical Patient

ABSTRACT Francisella tularensis, Bacillus anthracis, and Yersinia pestis are tier 1 select agents with the potential to rapidly cause severe disease. Rapid detection of these bacteria from patient samples at the point of care could contribute to improved clinical outcomes in the event of a bioterrorism attack. A multiplex nested PCR assay for detection of F. tularensis, B. anthracis, and Y. pestis directly from patient blood samples was developed using the GeneXpert system. The multiplex GeneXpert cartridge-based assay includes all necessary sample processing and amplification reagents. Blood samples spiked with different numbers of CFU were used to measure the analytical limit of detection (LOD) and dynamic range. Sensitivity was determined by testing spiked blood samples and negative-control blood in a blind manner. Specificity was determined by testing against nontarget pathogens and blood samples from clinical patients. The assay LOD was 8.5 CFU/ml for F. tularensis, 10 CFU/ml for B. anthracis, and 4.5 CFU/ml for Y. pestis. The sensitivity was 100% at the LOD for all three select agent bacteria in spiked patient blood samples. The assay specificity was 100% when it was tested against both nontarget pathogens and clinical patient blood samples. The total assay time was approximately 100 min. This automated assay, which is suitable for use at the point of care, identifies three select agents directly in blood without the need for enrichment with a high sensitivity within 100 min. This assay may enable rapid detection and treatment of patients infected with the target organisms in the event of a bioterrorism attack.

scholarly journals Multiple Bacteria Identification in the Point-of-Care: an Old Method Serving a New Approach

Sensors ◽  
2020 ◽  
Vol 20 (12) ◽  
pp. 3351
Author(s):  
Sara Viveiros ◽  
Mónica Rodrigues ◽  
Débora Albuquerque ◽  
Sofia A. M. Martins ◽  
Susana Cardoso ◽  
...  
Keyword(s):  
Bacterial Infections ◽  
Limit Of Detection ◽  
Detection System ◽  
Point Of Care ◽  
Bovine Mastitis ◽  
Cross Reactivity ◽  
Superparamagnetic Nanoparticles ◽  
Nucleic Acid Amplification ◽  
Streptococcus Uberis ◽  
Detection Assay

The accurate diagnosis of bacterial infections is of critical importance for effective treatment decisions. Due to the multietiologic nature of most infectious diseases, multiplex assays are essential for diagnostics. However, multiplexability in nucleic acid amplification-based methods commonly resorts to multiple primers and/or multiple reaction chambers, which increases analysis cost and complexity. Herein, a polymerase chain reaction (PCR) offer method based on a universal pair of primers and an array of specific oligonucleotide probes was developed through the analysis of the bacterial 16S ribosomal RNA gene. The detection system consisted of DNA hybridization over an array of magnetoresistive sensors in a microfabricated biochip coupled to an electronic reader. Immobilized probes interrogated single-stranded biotinylated amplicons and were obtained using asymmetric PCR. Moreover, they were magnetically labelled with streptavidin-coated superparamagnetic nanoparticles. The benchmarking of the system was demonstrated to detect five major bovine mastitis-causing pathogens: Escherichia coli, Klebsiella sp., Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae. All selected probes proved to specifically detect their respective amplicon without significant cross reactivity. A calibration curve was performed for S. agalactiae, which demonstrates demonstrating a limit of detection below 30 fg/µL. Thus, a sensitive and specific multiplex detection assay was established, demonstrating its potential as a bioanalytical device for point-of-care applications.

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