scholarly journals Quantitative E. coli Enzyme Detection in Reporter Hydrogel-Coated Paper Using a Smartphone Camera

Biosensors ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 25
Author(s):  
Kawaljit Kaur ◽  
Winny Chelangat ◽  
Sergey I. Druzhinin ◽  
Nancy Wangechi Karuri ◽  
Mareike Müller ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Equilibrium Constants ◽  
Enzyme Concentration ◽  
Blue Color ◽  
Coated Paper ◽  
E Coli ◽  
Substrate Conversion ◽  
Enzyme Detection

There is a growing demand for rapid and sensitive detection approaches for pathogenic bacteria that can be applied by non-specialists in non-laboratory field settings. Here, the detection of the typical E. coli enzyme β-glucuronidase using a chitosan-based sensing hydrogel-coated paper sensor and the detailed analysis of the reaction kinetics, as detected by a smartphone camera, is reported. The chromogenic reporter unit affords an intense blue color in a two-step reaction, which was analyzed using a modified Michaelis–Menten approach. This generalizable approach can be used to determine the limit of detection and comprises an invaluable tool to characterize the performance of lab-in-a-phone type approaches. For the particular system analyzed, the ratio of reaction rate and equilibrium constants of the enzyme–substrate complex are 0.3 and 0.9 pM−1h−1 for β-glucuronidase in phosphate buffered saline and lysogeny broth, respectively. The minimal degree of substrate conversion for detection of the indigo pigment formed during the reaction is 0.15, while the minimal time required for detection in this particular system is ~2 h at an enzyme concentration of 100 nM. Therefore, this approach is applicable for quantitative lab-in-a-phone based point of care detection systems that are based on enzymatic substrate conversion via bacterial enzymes.

scholarly journals A newly developed polydopamine-coated paper-based microchip for rapid and highly selectivity detection of foodborne pathogens

2021 ◽  
Author(s):  
Jinfeng Liu ◽  
Mimi Zhang ◽  
Zhiqiang Shen ◽  
Yongxin Liu ◽  
Yang Song ◽  
...  
Keyword(s):  
Foodborne Pathogens ◽  
Pathogen Detection ◽  
Lower Cost ◽  
Limit Of Detection ◽  
Fluorescence Labeling ◽  
Economic Losses ◽  
Great Promise ◽  
Salmonella Spp ◽  
Coated Paper ◽  
E Coli

Abstract Background In 2020, Covid-19 pneumonia has had a great impact on human health in although the countries around the world, it brings serious threaten to people’s lives and resulted in serious economic losses. At the same time, a lot news about the detection of Covid-19 in food emerges endlessly, a rapid and high selectivity detection method or technology is in urgent need for its ability to help relevant departments effectively control the epidemic situation and ensuring people’s lives and property safety. In recent years, loop-mediated isothermal amplification (LAMP) has been certified as a quick and highly selective technique to detect foodborne microorganisms. Results In this paper, a newly developed microchip with polydopamine-coated paper based on LAMP was fabricated. This microchip consists of nine chambers for sampling and reactions, the targeted nucleic acid of foodborne pathogens was labeled by calcein fluorescence rather than SYBR. The microchip is advantageous of lower cost of materials and simple pretreated methods, and is easy to operate without the need for complex controlled fluid flow. The LAMP procedure and fluorescence detection of pathogens can be carried on the chip without opening the lid, preventing aerosol contamination and reducing the probability of false positives. In experiments, the LAMP reaction conditions including the optimal reaction temperature and reaction time are thoroughly discussed and have been executed for various foodborne bacteria samples, including Escherichia coli O157:H7 (E. coli O157:H7), Salmonella spp., Staphylococcus aureus (S. aureus), and Vibrio parahaemolyticus (V. parahaemolyticus). Testing of E. coli O157:H7 proved to be highly selective and sensitive (as low as 0.0134 ng µL− 1). Additionally, experimental test of real milk sample was figured, the complete detection duration time was within 68 min, the limit of detection(LOD) for Salmonella spp. was determined to be lower than 12 CFU mL− 1. Conclusion In summary, a newly developed LAMP microchip with polydopamine-coated and calcein fluorescence labeling paper-based provides a lower cost, easy to use, highly selective, and multiplexable pathogen detection capability with great promise as a rapid, highly efficient, and economical solution for future foodborne pathogen testing.

scholarly journals Detection of Listeria innocua by Acoustic Aptasensor

Proceedings ◽  
2020 ◽  
Vol 60 (1) ◽  
pp. 18
Author(s):  
Veronika Oravczová ◽  
Marek Tatarko ◽  
Judit Süle ◽  
Milan Hun ◽  
Zoltán Kerényi ◽  
...  
Keyword(s):  
Foodborne Pathogens ◽  
Pathogenic Bacteria ◽  
Limit Of Detection ◽  
Low Cost ◽  
Positive Sample ◽  
Listeria Innocua ◽  
Serological Tests ◽  
E Coli ◽  
Develop Technology ◽  
Quartz Crystal Microbalances

Early detection of foodborne pathogens is significant for ensuring food safety. Nowadays, the detection of pathogens found in food can take up to 72 h and it might take a week to confirm a positive sample. While standardized methods give test results in a shorter period, the reoccurring costs for each measurement are high. Therefore, it is necessary to develop technology that will be low-cost, fast, simple and accurate enough. Biosensors in combination with nucleic acid aptamers offer such possibilities. This work is focused on the development and testing of a biosensor based on DNA aptamers for detection of pathogenic bacteria Listeria innocua using the method of multi-harmonic quartz crystal microbalances (QCM). The aptasensor was prepared on the surface of a piezo crystal, whose frequency was affected by deposited mass. An aptamer specific to the genus Listeria spp. was used for the detection of this pathogen, which includes 16 subspecies, out of which 3 are excluded as their antigen structure differs from other species (L. murrayi, L. grayi, L. ivanovii). We found that addition of the pathogens at the surface of QCM transducer modified by aptamers resulted in the decrease of the resonant frequency in concentration depending manner. We also confirmed the specificity of the aptamer used for Listeria innocua, as neglected response of the sensor took place for E. coli for which Listeria spp. has some partial antigens identical and thus can cause cross-reactions in serological tests. The developed aptasensor showed promising sensitivity and specificity for real-time detection of Listeria innocua, with a detection time of 30 min. The achieved limit of detection was approximately 1.6 × 103 CFU/mL.

scholarly journals A Fast, Reliable, and Sensitive Method for Detection and Quantification ofListeria monocytogenesandEscherichia coliO157:H7 in Ready-to-Eat Fresh-Cut Products by MPN-qPCR

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Pasquale Russo ◽  
Giuseppe Botticella ◽  
Vittorio Capozzi ◽  
Salvatore Massa ◽  
Giuseppe Spano ◽  
...  
Keyword(s):  
Shelf Life ◽  
Pathogenic Bacteria ◽  
Limit Of Detection ◽  
Raw Materials ◽  
Minimally Processed ◽  
E Coli ◽  
Biochemical Assays ◽  
Fresh Cut ◽  
Minimally Processed Vegetables ◽  
Detection And Quantification

In the present work we developed a MPN quantitative real-time PCR (MPN-qPCR) method for a fast and reliable detection and quantification ofListeria monocytogenesandEscherichia coliO157:H7 in minimally processed vegetables. In order to validate the proposed technique, the results were compared with conventional MPN followed by phenotypic and biochemical assays methods. WhenL. monocytogenesandE. coliO157:H7 were artificially inoculated in fresh-cut vegetables, a concentration as low as 1 CFU g−1could be detected in 48 hours for both pathogens. qPCR alone allowed a limit of detection of 101 CFU g−1after 2 hours of enrichment forL. monocytogenesandE. coliO157:H7. Since minimally processed ready-to-eat vegetables are characterized by very short shelf life, our method can potentially address the consistent reduction of time for microbial analysis, allowing a better management of quality control. Moreover, the occurrences of both pathogenic bacteria in mixed salad samples and fresh-cut melons were monitored in two production plants from the receipt of the raw materials to the early stages of shelf life. No sample was found to be contaminated byL. monocytogenes. One sample of raw mixed salad was found positive to an H7 enterohemorrhagic serotype.

scholarly journals Sustainable, Alginate-Based Sensor for Detection of Escherichia coli in Human Breast Milk

Sensors ◽  
2020 ◽  
Vol 20 (4) ◽  
pp. 1145
Author(s):  
Nicholas Kikuchi ◽  
Margaret May ◽  
Matthew Zweber ◽  
Jerard Madamba ◽  
Craig Stephens ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Breast Milk ◽  
Pathogen Detection ◽  
Protein Extraction ◽  
Limit Of Detection ◽  
Pcr Amplification ◽  
Human Breast Milk ◽  
Blue Color ◽  
E Coli ◽  
The Cost

There are no existing affordable diagnostics for sensitive, rapid, and on-site detection of pathogens in milk. To this end, an on-site colorimetric-based sustainable assay has been developed and optimized using an L16 (54) Taguchi design to obtain results in hours without PCR amplification. To determine the level of Escherichia coli (E. coli) contamination, after induction with 150 µL of breast milk, the B-Per bacterial protein extraction kit was added to a solution containing an alginate-based microcapsule assay. Within this 3 mm spherical novel sensor design, X-Gal (5-Bromo-4-Chloro-3-Indolyl β-d-Galactopyranoside) was entrapped at a concentration of 2 mg/mL. The outward diffusing X-Gal was cleaved by β-galactosidase from E. coli and dimerized in the solution to yield a blue color after incubation at 40 °C. Color intensity was correlated with the level of E. coli contamination using a categorical scale. After an 8 h incubation period, a continuous imaging scale based on intensity normalization was used to determine a binary lower limit of detection (LOD), which corresponded to 102 colony forming unit per mL (CFU/mL) and above. The cost of the overall assay was estimated to be $0.81 per sample, well under the $3 benchmark for state-of-the-art immune-based test kits for pathogen detection in biofluids. Considering the reported binary LOD cutoff of 102 CFU/mL and above, this proposed hydrogel-based assay is suited to meet global requirements for screening breast milk or milk for pathogenic organisms of 104 CFU/mL, with a percentage of false positives to be determined in future efforts.

Bacterial detection and differentiation via direct volatile organic compound sensing with surface enhanced Raman spectroscopy

2017 ◽  
Author(s):  
Caitlin S. DeJong ◽  
David I. Wang ◽  
Aleksandr Polyakov ◽  
Anita Rogacs ◽  
Steven J. Simske ◽  
...  
Keyword(s):  
Raman Spectroscopy ◽  
Bacterial Infections ◽  
Direct Detection ◽  
Point Of Care ◽  
Surface Enhanced Raman Spectroscopy ◽  
E Coli ◽  
Recent Emergence ◽  
Surface Enhanced ◽  
Volatile Organic ◽  
Surface Enhanced Raman

Through the direct detection of bacterial volatile organic compounds (VOCs), via surface enhanced Raman spectroscopy (SERS), we report here a reconfigurable assay for the identification and monitoring of bacteria. We demonstrate differentiation between highly clinically relevant organisms: <i>Escherichia coli</i>, <i>Enterobacter cloacae</i>, and <i>Serratia marcescens</i>. This is the first differentiation of bacteria via SERS of bacterial VOC signatures. The assay also detected as few as 10 CFU/ml of <i>E. coli</i> in under 12 hrs, and detected <i>E. coli</i> from whole human blood and human urine in 16 hrs at clinically relevant concentrations of 10<sup>3</sup> CFU/ml and 10<sup>4</sup> CFU/ml, respectively. In addition, the recent emergence of portable Raman spectrometers uniquely allows SERS to bring VOC detection to point-of-care settings for diagnosing bacterial infections.

Ferromagnetic Resonance Biosensor for Homogeneous and Volumetric Detection of DNA

2017 ◽  
Author(s):  
Bo Tian ◽  
Peter Svedlindh ◽  
Mattias Strömberg ◽  
Erik Wetterskog
Keyword(s):  
Magnetic Nanoparticles ◽  
Ferromagnetic Resonance ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Rolling Circle Amplification ◽  
Resonance Field ◽  
Isothermal Amplification ◽  
Detection Sensitivity ◽  
Serum Samples ◽  
Rolling Circle

In this work, we demonstrate for the first time, a ferromagnetic resonance (FMR) based homogeneous and volumetric biosensor for magnetic label detection. Two different isothermal amplification methods, <i>i.e.</i>, rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) are adopted and combined with a standard electron paramagnetic resonance (EPR) spectrometer for FMR biosensing. For RCA-based FMR biosensor, binding of RCA products of a synthetic Vibrio cholerae target DNA sequence gives rise to the formation of aggregates of magnetic nanoparticles. Immobilization of nanoparticles within the aggregates leads to a decrease of the net anisotropy of the system and a concomitant increase of the resonance field. A limit of detection of 1 pM is obtained with an average coefficient of variation of 0.16%, which is superior to the performance of other reported RCA-based magnetic biosensors. For LAMP-based sensing, a synthetic Zika virus target oligonucleotide is amplified and detected in 20% serum samples. Immobilization of magnetic nanoparticles is induced by their co-precipitation with Mg<sub>2</sub>P<sub>2</sub>O<sub>7</sub> (a by-product of LAMP) and provides a detection sensitivity of 100 aM. The fast measurement, high sensitivity and miniaturization potential of the proposed FMR biosensing technology makes it a promising candidate for designing future point-of-care devices.<br>

A Point of Care Lateral Flow Assay for Rapid and Colorimetric Detection of Interleukin 6 and Perspectives in Bedside Diagnostics

2020 ◽  
Author(s):  
Carla Eiras
Keyword(s):  
Interleukin 6 ◽  
Limit Of Detection ◽  
Inflammatory Diseases ◽  
Point Of Care ◽  
Colorimetric Detection ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Aggregation Assay ◽  
Before And After ◽  
Bedside Diagnosis

Interleukin-6 (IL-6) is a multifunctional cytokine and high bloodstream levels of which have been associated with severe inflammatory diseases, such as dengue fever, sepsis, various cancers, and visceral leishmaniasis (VL). Rapid tests for the quantification of IL-6 would be of great assistance for the bedside diagnosis and treatment of diseases such as VL. We have developed a lateral flow assay (LFA) for rapid and colorimetric IL-6 detection, consisting of anti-IL-6 antibodies conjugated to gold nanoparticles (AuNPs). The optimal concentration of anti-IL-6 used in the conjugate was determined to be 800.0 μg/mL, based on an aggregation assay using LFA. A linear relationship between IL-6 standard concentration and color intensity was observed after 20 min, with a linear range between 1.25 ng/mL and 9,000 ng/mL. The limit of detection for this method was estimated a t0.38 ng/mL. The concentration of IL-6 in five patients with severe VL was measured using LFA, and the results were consistent with those obtained using the cytometric bead array (CBA) method. A thorough analysis of the LFA membranes’ surface morphology, before and after sample contact, was performed using atomic force microscopy (AFM).The prototype described here is still being tested and improved, but this LFA will undoubtedly be of great help in the clinical quantification of IL-6.

A Point of Care Lateral Flow Assay for Rapid and Colorimetric Detection of Interleukin 6 and Perspectives in Bedside Diagnostics

2020 ◽  
Author(s):  
Carla Eiras
Keyword(s):  
Interleukin 6 ◽  
Limit Of Detection ◽  
Inflammatory Diseases ◽  
Point Of Care ◽  
Colorimetric Detection ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Aggregation Assay ◽  
Before And After ◽  
Bedside Diagnosis

Interleukin-6 (IL-6) is a multifunctional cytokine and high bloodstream levels of which have been associated with severe inflammatory diseases, such as dengue fever, sepsis, various cancers, and visceral leishmaniasis (VL). Rapid tests for the quantification of IL-6 would be of great assistance for the bedside diagnosis and treatment of diseases such as VL. We have developed a lateral flow assay (LFA) for rapid and colorimetric IL-6 detection, consisting of anti-IL-6 antibodies conjugated to gold nanoparticles (AuNPs). The optimal concentration of anti-IL-6 used in the conjugate was determined to be 800.0 μg/mL, based on an aggregation assay using LFA. A linear relationship between IL-6 standard concentration and color intensity was observed after 20 min, with a linear range between 1.25 ng/mL and 9,000 ng/mL. The limit of detection for this method was estimated at a t0.38 ng/mL. The concentration of IL-6 in five patients with severe VL was measured using LFA, and the results were consistent with those obtained using the cytometric bead array (CBA) method. A thorough analysis of the LFA membranes’ surface morphology, before and after sample contact, was performed using atomic force microscopy (AFM). The prototype described here is still being tested and improved, but this LFA will undoubtedly be of great help in the clinical quantification of IL-6.

Probiotics Slow the Growth of Pathogenic Bacteria

2019 ◽  
Vol 14 (1) ◽  
pp. 28-31 ◽  
Author(s):  
Rowles H. L.
Keyword(s):  
Growth Rate ◽  
Bacterial Infections ◽  
Pathogenic Bacteria ◽  
Diarrheal Disease ◽  
Growth Curves ◽  
Mortality Rates ◽  
Controlled Delivery ◽  
E Coli ◽  
Multiple Strains ◽  
Commercial Probiotics

Probiotics are live microorganisms, which when ingested in sufficient amounts, confer health benefits to the host by improving the gut microflora balance. The purpose of this research was to determine whether commercial probiotic products containing multitude of commensal bacteria would reduce the growth rate of pathogenic bacteria, specifically Escherichia coli and Salmonella typhimurium. Growth curves were established, and the growth rates were compared for samples of E. coli, S. typhimurium, Nature’s Bounty Controlled Delivery probiotic, Sundown Naturals Probiotic Balance probiotic, and cocultures of the pathogenic bacteria mixed with the probiotics. The findings of this research were that the commercial probiotics significantly reduced the growth rate of E. coli and S. typhimurium when combined in cocultures. Probiotics containing multiple strains may be taken prophylactically to reduce the risk of bacterial infections caused by E. coli and S. typhimurium. Probiotics could be used to reduce the high global morbidity and mortality rates of diarrheal disease.

Bioenhancing Effect of Cow Urine Distillate and Pepper Extract on Antibacterial Activity of Azadirachta Indica Leaves

1970 ◽  
Vol 9 (2) ◽  
pp. 3212-3214
Author(s):  
Pramod Dhakal ◽  
Ankit a Achary ◽  
Vedamurthy Joshi
Keyword(s):  
Antibacterial Activity ◽  
Azadirachta Indica ◽  
Pathogenic Bacteria ◽  
Ethanol Extract ◽  
Watery Diarrhea ◽  
Diffusion Method ◽  
E Coli ◽  
Drug Molecules ◽  
Cow Urine

Bioenhancers are drug facilitator which do not show the typical drug activity but in combination to enhance the activity of other molecule in several way including increase the bioavailability of drug across the membrane, potentiating the drug molecules by conformational interaction, acting as receptor for drug molecules and making target cell more receptive to drugs and promote and increase the bioactivity or bioavailability or the uptake of drugs in combination therapy. The objective of the present study was to evaluate the antibacterial and activity of combination in Azadirachta indica extract with cow urine distillate and pepper extract against common pathogenic bacteria, a causative agent of watery diarrhea. It has been found that Indian indigenous cow urine and its distillate also possess bioenhancing ability. Bioenhancing role of cow urine distillate (CUD) and pepper extract was investigated on antibacterial activity of ethanol extract of Azadirachta indica. Antibacterial activity of ethanol extract neem alone and in combination with CUD and pepper extract were determined the ATCC strains against Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and E-coli by cup plate diffusion method. Ethanol extract of neem has showed more effect on P. aeruginosa, E-coli than S. aureus and K. pneumonia with combination of CUD and pepper extract. CUD and pepper did not show any inhibition of test bacteria in low concentration. The antibacterial effect of combination of extract and CUD was higher than the inhibition caused by extract alone and is suggestive of the bioenhancing role of cow urine distillate and pepper. Moreover, inhibition of test bacteria was observed with less concentration of extract on combining with CUD

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