A Regenerative Immunoaffinity Layer Based on the Outer Membrane of Z-Domains Autodisplaying E. coli for Immunoassays and Immunosensors

Through orientation control of antibodies, Z-domains autodisplaying Escherichia coli outer cell membrane (OM) may be utilized to improve the sensitivity and limit of detection (LOD) of immunoassays and immunosensors. A regenerative immunoaffinity layer based on Z-domains autodisplaying E. coli OM was developed for the surface plasmon resonance (SPR) biosensor. Regeneration conditions for the Z-domains autodisplaying E. coli OM-based immunoassays and immunosensors were optimized by varying pH and detergent concentration. An E. coli cell-based HRP immunoassay was tested and validated in three sequential regenerative immunoassays under optimal conditions. The OM of Z-domains autodisplaying E. coli was isolated and coated on the two-dimensional substrate (microplate). The OM-based HRP immunoassay was tested and validated in four regenerative immunoassays. This regenerative OM layer was applied to the SPR biosensor. Z-domains autodisplaying OM layered onto the gold surface of SPR biosensors was developed, and the OM-based regenerative immunoaffinity layer with orientation control was tested using CRP analyte. The SPR biosensor regenerative immunoaffinity layer demonstrated that CRP biosensing was repeated for five regeneration cycles with less than 2% signal difference. Therefore, the newly developed regenerative immunoaffinity layer with antibody orientation control may improve biosensing sensitivity and reduce the cost of medical diagnosis.

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Covalently Immobilized Regenerable Immunoaffinity Layer with Orientation-Controlled Antibodies Based on Z-Domain Autodisplay

International Journal of Molecular Sciences ◽

10.3390/ijms23010459 ◽

2021 ◽

Vol 23 (1) ◽

pp. 459

Author(s):

Jong-Min Park ◽

Mi Yeon Kim ◽

Joachim Jose ◽

Min Park

Keyword(s):

Antibody Binding ◽

Chemical Fixation ◽

C Reactive Protein ◽

Poly Ethylene Glycol ◽

E Coli ◽

Reactive Protein ◽

Spr Biosensor ◽

Antibody Orientation ◽

Z Domain ◽

Poly Ethylene

A regenerable immunoaffinity layer comprising covalently immobilized orientation-controlled antibodies was developed for use in a surface plasmon resonance (SPR) biosensor. For antibody orientation control, antibody-binding Z-domain-autodisplaying Escherichia coli (E. coli) cells and their outer membrane (OM) were utilized, and a disuccinimidyl crosslinker was employed for covalent antibody binding. To fabricate the regenerable immunoaffinity layer, capture antibodies were bound to autodisplayed Z-domains, and then treated with the crosslinker for chemical fixation to the Z-domains. Various crosslinkers, namely disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS) and poly (ethylene glycol)-ylated bis (sulfosuccinimidyl)suberate (BS(PEG)5), were evaluated, and DSS at a concentration of 500 μM was confirmed to be optimal. The E. coli-cell-based regenerable HRP immunoassay was evaluated employing three sequential HRP treatment and regeneration steps. Then, the Oms of E. coli cells were isolated and layered on a microplate and regenerable OM-based HRP immunoassaying was evaluated. Five HRP immunoassays with four regeneration steps were found to be feasible. This regenerable, covalently immobilized, orientation-controlled OM-based immunoaffinity layer was applied to an SPR biosensor, which was capable of quantifying C-reactive protein (CRP). Five regeneration cycles were repeated using the demonstrated immunoaffinity layer with a signal difference of <10%.

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

Sensors ◽

10.3390/s20041145 ◽

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.

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Confirming putative variants at ≤ 5% allele frequency using allele enrichment and Sanger sequencing

Scientific Reports ◽

10.1038/s41598-021-91142-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yan Helen Yan ◽

Sherry X. Chen ◽

Lauren Y. Cheng ◽

Alyssa Y. Rodriguez ◽

Rui Tang ◽

...

Keyword(s):

Sanger Sequencing ◽

Limit Of Detection ◽

High Sensitivity ◽

Sequencing Errors ◽

Whole Exome ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Fresh Frozen ◽

The Cost ◽

Allelic Fraction

AbstractWhole exome sequencing (WES) is used to identify mutations in a patient’s tumor DNA that are predictive of tumor behavior, including the likelihood of response or resistance to cancer therapy. WES has a mutation limit of detection (LoD) at variant allele frequencies (VAF) of 5%. Putative mutations called at ≤ 5% VAF are frequently due to sequencing errors, therefore reporting these subclonal mutations incurs risk of significant false positives. Here we performed ~ 1000 × WES on fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue biopsy samples from a non-small cell lung cancer patient, and identified 226 putative mutations at between 0.5 and 5% VAF. Each variant was then tested using NuProbe NGSure, to confirm the original WES calls. NGSure utilizes Blocker Displacement Amplification to first enrich the allelic fraction of the mutation and then uses Sanger sequencing to determine mutation identity. Results showed that 52% of the 226 (117) putative variants were disconfirmed, among which 2% (5) putative variants were found to be misidentified in WES. In the 66 cancer-related variants, the disconfirmed rate was 82% (54/66). This data demonstrates Blocker Displacement Amplification allelic enrichment coupled with Sanger sequencing can be used to confirm putative mutations ≤ 5% VAF. By implementing this method, next-generation sequencing can reliably report low-level variants at a high sensitivity, without the cost of high sequencing depth.

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Changes in Aerobic Plate and Escherichia coli–Coliform Counts and in Populations of Inoculated Foodborne Pathogens on Inshell Walnuts during Storage

Journal of Food Protection ◽

10.4315/0362-028x.jfp-15-553 ◽

2016 ◽

Vol 79 (7) ◽

pp. 1143-1153 ◽

Cited By ~ 8

Author(s):

JOHN C. FRELKA ◽

GORDON R. DAVIDSON ◽

LINDA J. HARRIS

Keyword(s):

Escherichia Coli ◽

Listeria Monocytogenes ◽

Relative Humidity ◽

Low Temperature ◽

Foodborne Pathogens ◽

Limit Of Detection ◽

Sampling Time ◽

E Coli ◽

Plate Counts ◽

Forced Air

ABSTRACT After harvest, inshell walnuts are dried using low-temperature forced air and are then stored in bins or silos for up to 1 year. To better understand the survival of bacteria on inshell walnuts, aerobic plate counts (APCs) and Escherichia coli–coliform counts (ECCs) were evaluated during commercial storage (10 to 12°C and 63 to 65% relative humidity) over 9 months. APCs decreased by 1.4 to 2.0 log CFU per nut during the first 5 months of storage, and ECCs decreased by 1.3 to 2.2 log CFU per nut in the first month of storage. Through the remaining 4 to 8 months of storage, APCs and ECCs remained unchanged (P > 0.05) or decreased by <0.15 log CFU per nut per month. Similar trends were observed on kernels extracted from the inshell walnuts. APCs and ECCs were consistently and often significantly higher on kernels extracted from visibly broken inshell walnuts than on kernels extracted from visibly intact inshell walnuts. Parameters measured in this study were used to determine the survival of five-strain cocktails of E. coli O157:H7, Listeria monocytogenes, and Salmonella inoculated onto freshly hulled inshell walnuts (~8 log CFU/g) after simulated commercial drying (10 to 12 h; 40°C) and simulated commercial storage (12 months at 10°C and 65% relative humidity). Populations declined by 2.86, 5.01, and 4.40 log CFU per nut for E. coli O157:H7, L. monocytogenes, and Salmonella, respectively, after drying and during the first 8 days of storage. Salmonella populations changed at a rate of −0.33 log CFU per nut per month between days 8 and 360, to final levels of 2.83 ± 0.79 log CFU per nut. E. coli and L. monocytogenes populations changed by −0.17 log CFU per nut per month and −0.26 log CFU per nut per month between days 8 and 360, respectively. For some samples, E. coli or L. monocytogenes populations were below the limit of detection by plating (0.60 log CFU per nut) by day 183 or 148, respectively; at least one of the six samples was positive at each subsequent sampling time by either plating or by enrichment.

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Preliminary Estimates of Costs of Foodborne Disease in the United States

Journal of Food Protection ◽

10.4315/0362-028x-52.8.595 ◽

1989 ◽

Vol 52 (8) ◽

pp. 595-601 ◽

Cited By ~ 139

Author(s):

EWEN C. D. TODD

Keyword(s):

United States ◽

Food Industry ◽

The United States ◽

Control Measures ◽

Effective Control ◽

Foodborne Diseases ◽

Hemorrhagic Colitis ◽

E Coli ◽

The Cost ◽

Total Impact

Although the full economic impact of foodborne diseases has yet to be measured, preliminary studies show that the cost of illness, death, and business lost is high indeed. This impact is probably greatest in developing countries, but few facts are known. For the United States, preliminary estimates are 12.6 million cases costing $8.4 billion. These may seem excessive but other authors have postulated even higher case and dollar figures. Microbiological diseases (bacterial and viral) represent 84% of the United States' costs, with salmonellosis and staphylococcal intoxication being the most economically important diseases (annually $4.0 billion and $1.5 billion, respectively). Other costly types of illnesses are toxoplasmosis ($445 million), listeriosis ($313 million), campylobacteriosis ($156 million), trichinosis ($144 million), Clostridium perfringens enteritis ($123 million), and E. coli infections including hemorrhagic colitis ($223 million). Botulism has a high cost per case ($322,200), but its total impact is only $87 million because relatively few cases occur (270). This is because the food industry has been able to introduce effective control measures. Salmonellosis, however, is much more widespread (2.9 million cases) and affects all sectors of the food industry.

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Development of an Immunoassay for Detection of Staphylococcal Enterotoxin-Like J, A Non-Characterized Toxin

Toxins ◽

10.3390/toxins10110458 ◽

2018 ◽

Vol 10 (11) ◽

pp. 458 ◽

Cited By ~ 1

Author(s):

Hisaya Ono ◽

Nobuaki Hachiya ◽

Yasunori Suzuki ◽

Ikunori Naito ◽

Shouhei Hirose ◽

...

Keyword(s):

Limit Of Detection ◽

Expression System ◽

Sandwich Elisa ◽

Food Poisoning ◽

High Sensitivity ◽

Enzyme Linked Immunosorbent Assay ◽

E Coli ◽

Detection Systems ◽

C Terminus ◽

Culture Supernatants

Staphylococcal enterotoxins (SEs) are the cause of staphylococcal food poisoning (SFP) outbreaks. Recently, many new types of SEs and SE-like toxins have been reported, but it has not been proved whether these new toxins cause food poisoning. To develop an immunoassay for detection of SE-like J (SElJ), a non-characterized toxin in SFP, a mutant SElJ with C-terminus deletion (SElJ∆C) was expressed and purified in an E. coli expression system. Anti-SElJ antibody was produced in rabbits immunized with the SElJ∆C. Western blotting and sandwich enzyme-linked immunosorbent assay (ELISA) detection systems were established and showed that the antibody specifically recognizes SElJ without cross reaction to other SEs tested. The limit of detection for the sandwich ELISA was 0.078 ng/mL, showing high sensitivity. SElJ production in S. aureus was detected by using the sandwich ELISA and showed that selj-horboring isolates produced a large amount of SElJ in the culture supernatants, especially in that of the strain isolated from a food poisoning outbreak in Japan. These results demonstrate that the immunoassay for detection of SElJ is specific and sensitive and is useful for determining the native SElJ production in S. aureus isolated from food poisoning cases.

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Alleviation of C⋅C Mismatches in DNA by the Escherichia coli Fpg Protein

Frontiers in Microbiology ◽

10.3389/fmicb.2021.608839 ◽

2021 ◽

Vol 12 ◽

Author(s):

Almaz Nigatu Tesfahun ◽

Marina Alexeeva ◽

Miglė Tomkuvienė ◽

Aysha Arshad ◽

Prashanna Guragain ◽

...

Keyword(s):

Escherichia Coli ◽

Excision Repair ◽

Dna Lesions ◽

Base Pairs ◽

E Coli ◽

Thymine Glycol ◽

Base Excision ◽

The Cost ◽

Primary Substrate

DNA polymerase III mis-insertion may, where not corrected by its 3′→ 5′ exonuclease or the mismatch repair (MMR) function, result in all possible non-cognate base pairs in DNA generating base substitutions. The most thermodynamically unstable base pair, the cytosine (C)⋅C mismatch, destabilizes adjacent base pairs, is resistant to correction by MMR in Escherichia coli, and its repair mechanism remains elusive. We present here in vitro evidence that C⋅C mismatch can be processed by base excision repair initiated by the E. coli formamidopyrimidine-DNA glycosylase (Fpg) protein. The kcat for C⋅C is, however, 2.5 to 10 times lower than for its primary substrate 8-oxoguanine (oxo8G)⋅C, but approaches those for 5,6-dihydrothymine (dHT)⋅C and thymine glycol (Tg)⋅C. The KM values are all in the same range, which indicates efficient recognition of C⋅C mismatches in DNA. Fpg activity was also exhibited for the thymine (T)⋅T mismatch and for N4- and/or 5-methylated C opposite C or T, Fpg activity being enabled on a broad spectrum of DNA lesions and mismatches by the flexibility of the active site loop. We hypothesize that Fpg plays a role in resolving C⋅C in particular, but also other pyrimidine⋅pyrimidine mismatches, which increases survival at the cost of some mutagenesis.

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Conjugation of Carboxylated Graphene Quantum Dots with Cecropin P1 for Bacterial Biosensing Applications

10.26434/chemrxiv.12116175.v2 ◽

2020 ◽

Author(s):

Jonathan Bruce ◽

Jude Clapper

Keyword(s):

Quantum Dots ◽

Structural Modification ◽

Light Emission ◽

Limit Of Detection ◽

Graphene Quantum Dots ◽

Strong Light ◽

E Coli ◽

Cecropin P1 ◽

Compact Field ◽

Test Kit

Quantum dots have proven to be strong candidates for biosensing applications in recent years, due to their strong light emission properties and their ability to be modified with a variety of functional groups for the detection of different analytes. Here, we investigate the use of conjugated carboxylated graphene quantum dots (CGQDs) for the detection of E. coli, using a biosensing procedure that focuses on measuring changes in fluorescence quenching. We have also further developed this biosensing assay into a compact, field-deployable test kit focused on rapidly measuring changes in absorbance to determine bacterial concentration. Our CGQDs were conjugated with cecropin P1, a naturally-produced antibacterial peptide that facilitates the attachment of CGQDs to E. coli cells. We also confirm the structural modification of these conjugated CGQDs in addition to analyzing their optical characteristics. Our findings have the potential to be used in situations where rapid, reliable detection of bacteria in liquids, such as drinking water, is required, especially given our biosensor’s relatively low observed limit of detection (LOD).

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Programmable receptors enable bacterial biosensors to detect pathological biomarkers in clinical samples

Nature Communications ◽

10.1038/s41467-021-25538-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Hung-Ju Chang ◽

Ana Zúñiga ◽

Ismael Conejero ◽

Peter L. Voyvodic ◽

Jerome Gracy ◽

...

Keyword(s):

High Performance ◽

Limit Of Detection ◽

Clinical Samples ◽

Monitoring And Control ◽

Modular Assembly ◽

E Coli ◽

Naked Eye ◽

Synthetic Receptor ◽

And Control ◽

Do So

AbstractBacterial biosensors, or bactosensors, are promising agents for medical and environmental diagnostics. However, the lack of scalable frameworks to systematically program ligand detection limits their applications. Here we show how novel, clinically relevant sensing modalities can be introduced into bactosensors in a modular fashion. To do so, we have leveraged a synthetic receptor platform, termed EMeRALD (Engineered Modularized Receptors Activated via Ligand-induced Dimerization) which supports the modular assembly of sensing modules onto a high-performance, generic signaling scaffold controlling gene expression in E. coli. We apply EMeRALD to detect bile salts, a biomarker of liver dysfunction, by repurposing sensing modules from enteropathogenic Vibrio species. We improve the sensitivity and lower the limit-of-detection of the sensing module by directed evolution. We then engineer a colorimetric bactosensor detecting pathological bile salt levels in serum from patients having undergone liver transplant, providing an output detectable by the naked-eye. The EMeRALD technology enables functional exploration of natural sensing modules and rapid engineering of synthetic receptors for diagnostics, environmental monitoring, and control of therapeutic microbes.

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Mechanistic Understanding of the Interactions of Cationic Conjugated Oligo- and Polyelectrolytes with Wild-type and Ampicillin-resistant Escherichia coli

Scientific Reports ◽

10.1038/s41598-019-56946-2 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Ehsan Zamani ◽

Shyambo Chatterjee ◽

Taity Changa ◽

Cheryl Immethun ◽

Anandakumar Sarella ◽

...

Keyword(s):

Escherichia Coli ◽

Surface Charge ◽

Cell Envelope ◽

Drug Binding ◽

Outer Cell ◽

Binding Modes ◽

Wild Type ◽

Bacterial Surface ◽

E Coli ◽

Future Design

AbstractAn in-depth understanding of cell-drug binding modes and action mechanisms can potentially guide the future design of novel drugs and antimicrobial materials and help to combat antibiotic resistance. Light-harvesting π-conjugated molecules have been demonstrated for their antimicrobial effects, but their impact on bacterial outer cell envelope needs to be studied in detail. Here, we synthesized poly(phenylene) based model cationic conjugated oligo- (2QA-CCOE, 4QA-CCOE) and polyelectrolytes (CCPE), and systematically explored their interactions with the outer cell membrane of wild-type and ampicillin (amp)-resistant Gram-negative bacteria, Escherichia coli (E. coli). Incubation of the E. coli cells in CCOE/CCPE solution inhibited the subsequent bacterial growth in LB media. About 99% growth inhibition was achieved if amp-resistant E. coli was treated for ~3–5 min, 1 h and 6 h with 100 μM of CCPE, 4QA-CCOE, and 2QA-CCOE solutions, respectively. Interestingly, these CCPE and CCOEs inhibited the growth of both wild-type and amp-resistant E. coli to a similar extent. A large surface charge reversal of bacteria upon treatment with CCPE suggested the formation of a coating of CCPE on the outer surface of bacteria; while a low reversal of bacterial surface charge suggested intercalation of CCOEs within the lipid bilayer of bacteria.

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