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Abstract Nucleic acid amplification testing has great potential for point-of-need diagnostic testing with high detection sensitivity and specificity. Current sample preparation is limited by a tedious workflow requiring multiple steps, reagents and instrumentation, hampering nucleic acid testing at point of need. In this study, we present the use of mixed cellulose ester (MCE) paper for DNA binding by ionic interaction under molecular crowding conditions and fluid transport by wicking. The poly(ethylene) glycol-based (PEG) reagent simultaneously provides the high pH for alkaline lysis and crowding effects for ionic binding of the DNA under high salt conditions. In this study, we introduce Paper-based Abridged Solid-Phase Extraction with Alkaline Poly(ethylene) Glycol Lysis (PASAP). The anionic mixed cellulose ester (MCE) paper is used as solid phase and allows for fluid transport by wicking, eliminating the need for pipetting skills and the use of a magnet to retain beads. Following the release of DNA from the cells due to the lytic activity of the PASAP solution, the DNA binds to the anionic surface of the MCE paper, concentrating at the bottom while the sample matrix is transported towards the top by wicking. The paper was washed by dipping it in 40% isopropanol for 10 s. After air-drying for 30 s, the bottom section of the paper (3 mm × 4 mm) was snapped off using the cap of a PCR tube and immersed in the colourimetric loop-mediated isothermal amplification (cLAMP) solution for direct amplification and colourimetric detection. The total sample processing was completed in 15 min and ready for amplification. cLAMP enabled the detection of 102 CFU/mL of Escherichia coli (E. coli) from culture media and the detection of E. coli in milk

Nucleic acid amplification testing has great potential for point-of-need diagnostic testing with high detection sensitivity and specificity. Current sample preparation is limited by a tedious workflow requiring multiple steps, reagents and instrumentation, hampering nucleic acid testing at point of need. In this study, we present the use of mixed cellulose ester (MCE) paper for DNA binding by ionic interaction under molecular crowding conditions and fluid transport by wicking. The poly(ethylene) glycol-based (PEG) reagent simultaneously provides the high pH for alkaline lysis and crowding effects for ionic binding of the DNA under high salt conditions. In this study, we introduce Paper-based Abridged Solid-Phase Extraction with Alkaline Poly(ethylene) Glycol Lysis (PASAP). The anionic mixed cellulose ester (MCE) paper is used as solid phase and allows for fluid transport by wicking, eliminating the need for pipetting skills and the use of a magnet to retain beads. Following the release of DNA from the cells due to the lytic activity of the PASAP solution, the DNA binds to the anionic surface of the MCE paper, concentrating at the bottom while the sample matrix is transported towards the top by wicking. The paper was washed by dipping it in 40% isopropanol for 10 s. After air-drying for 30 s, the bottom section of the paper (3 mm × 4 mm) was snapped off using the cap of a PCR tube and immersed in the colourimetric loop-mediated isothermal amplification (cLAMP) solution for direct amplification and colourimetric detection. The total sample processing was completed in 15 min and ready for amplification. cLAMP enabled the detection of 102 CFU/mL of Escherichia coli (E. coli) from culture media and the detection of E. coli in milk < 103 CFU/mL (10 CFU) after incubation at 68 °C for 60 min, demonstrating applicability of the method to complex biological samples. [ABSTRACT FROM AUTHOR]

A dual-signal readout has been designed detecting platform based on a cascade reaction for Escherichia coli (E. coli) detection by using colorimetric approach and a handheld pH meter. The immunoreaction was conducted using polydopamine@copper ferrite-Ag nanoparticles (PDA@CuFe2O4-Ag NP) and a glucose oxidase (GOD)–conjugated graphene oxide-gold nanosheet composite (GOD-GO/Au NS) to synthesize a sandwich complex mode between targets. Together with the formation of immune complexes, the GOD-GO/Au NS can catalyze glucose to produce gluconic acid and hydrogen peroxide (H2O2). The gluconic acid produced altered the pH of the detection solution. Since the PDA@CuFe2O4-Ag NP have good peroxidase-like activity, they can catalyze the oxidation of TMB to the blue product oxTMB once H2O2 is produced in the reaction system, and the absorbance change of oxTMB at 652 nm can be recorded using ultraviolet–visible (UV–Vis) spectroscopy. Interestingly, the PDA@CuFe2O4-Ag NP composites can consume the generated H2O2, and can create a reaction cycle that promotes glucose oxidation. Under optimal conditions, the proposed dual-channel signal platform is proportional to the logarithm of the E. coli concentration within a range of 102–107 cfu mL−1. Additionally, the devised approach was successfully used to detect E. coli at the required levels in real samples. This dual-mode detection method notably enhances the accuracy and diversity of detection, and curbs the false negative and positive rates. [ABSTRACT FROM AUTHOR]

As one class of molecular imprinted polymers (MIPs), surface imprinted polymer (SIP)-based biosensors show great potential in direct whole-bacteria detection. Micro-contact imprinting, that involves stamping the template bacteria immobilized on a substrate into a pre-polymerized polymer matrix, is the most straightforward and prominent method to obtain SIP-based biosensors. However, the major drawbacks of the method arise from the requirement for fresh template bacteria and often non-reproducible bacteria distribution on the stamp substrate. Herein, we developed a positive master stamp containing photolithographic mimics of the template bacteria (E. coli) enabling reproducible fabrication of biomimetic SIP-based biosensors without the need for the "real" bacteria cells. By using atomic force and scanning electron microscopy imaging techniques, respectively, the E. coli -capturing ability of the SIP samples was tested, and compared with non-imprinted polymer (NIP)-based samples and control SIP samples, in which the cavity geometry does not match with E. coli cells. It was revealed that the presence of the biomimetic E. coli imprints with a specifically designed geometry increases the sensor E. coli -capturing ability by an "imprinting factor" of about 3. These findings show the importance of geometry-guided physical recognition in bacterial detection using SIP-based biosensors. In addition, this imprinting strategy was employed to interdigitated electrodes and QCM (quartz crystal microbalance) chips. E. coli detection performance of the sensors was demonstrated with electrochemical impedance spectroscopy (EIS) and QCM measurements with dissipation monitoring technique (QCM-D). [Display omitted] • Biomimetic surface imprinted polymer-based biosensor for E. coli detection • Development of a positive master stamp as photolithographic mimics of E. coli • Biomimetic E. coli imprints increase E. coli -capturing ability of the sensor • Showing the importance of geometry-dependent physical recognition in SIP-based sensors • E. coli detection performance of the sensors was demonstrated with EIS and QCM-D [ABSTRACT FROM AUTHOR]

Conventional detection methods require the isolation and enrichment of bacteria, followed by molecular, biochemical, or culture-based analysis. To address some of the limitations of conventional methods, this study develops a machine learning (ML) approach to analyze the excitation-emission matrix (EEM) fluorescence data generated based on bacteriophage T7 and E. coli interactions for in-situ detection of live bacteria in the presence of fresh produce homogenate. We trained classification models using various ML algorithms based on the 3-D EEM data generated with bacteria and their interactions with a T7 phage. These ML algorithms, including linear Support Vector Classifier and Random Forest, demonstrate high accuracy (>0.85) for detecting E. coli at 10 2 CFU/ml concentration within 6 hours. Additionally, these ML models can differentiate among different E. coli concentration levels. For example, the Gaussian Process model achieved an accuracy of 92% in detecting different concentration levels of live E. coli. Application of these ML methods to detect E. coli in spinach homogenate yielded an accuracy of 89% using the linear-SVC model. Furthermore, feature selection techniques were employed to reduce the dimensionality of the data, revealing that only six features were necessary for achieving classification accuracy (>0.85) of spinach homogenate samples containing 10 2 CFU/ml of E. coli. These findings highlight the potential of this novel bacterial detection methodologies, offering rapid, specific, and efficient solutions for applications in food safety and environmental monitoring., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Nitin Nitin reports financial support was provided by USDA-National Institute of Food and Agriculture. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.]., (Copyright © 2024. Published by Elsevier Inc.)

A PCR-free, optics-free device is used for the detection of Escherichia coli (E. coli) 16S rRNA at 10 fM, which corresponds to ~100-1000 colony forming units/mL (CFU/mL) depending on cellular rRNA levels. The development of a rapid, sensitive, and cost-effective nucleic acid detection platform is sought for the detection of pathogenic microbes in food, water and body fluids. Since 16S rRNA sequences are species specific and are present at high copy number in viable cells, these nucleic acids offer an attractive target for microbial pathogen detection schemes. Here, target 16S rRNA of E. coli at 10 fM concentration was detected against a total RNA background using a conceptually simple approach based on electromechanical signal transduction, whereby a step change reduction in ionic current through a pore indicates blockage by an electrophoretically mobilized bead-peptide nucleic acid probe conjugate hybridized to target nucleic acid. We investigated the concentration detection limit for bacterial species-specific 16S rRNA at 1 pM to 1 fM and found a limit of detection of 10 fM for our device, which is consistent with our previous finding with single-stranded DNA of similar length. In addition, no false positive responses were obtained with control RNA and no false negatives with target 16S rRNA present down to the limit of detection (LOD) of 10 fM. Thus, this detection scheme shows promise for integration into portable, low-cost systems for rapid detection of pathogenic microbes in food, water and body fluids.

A nanophotonic structure with electric control based photocatalytic nanocomposite is proposed to realize label-free optical detection of foodborne pathogens. The photocatalytic response of Cu-ZnO/TiO2 (CZT) nanocomposite, in terms of the photocurrent, is utilized to detect E. coli in the surrounding media of Luria Bertani broth (LB) solution. An engineered nanophotonic structure based on an optical slot waveguide is presented to provide a two-fold benefit - enhanced light-matter interaction and flexibility to accommodate bio-sample. CZT nanocomposite is synthesised and prepared by a cost-effective sol-gel process, acting as an active layer in the structure, is responsible for the generation of photo-excited carriers which provide photocurrent on the application of a voltage. The fabricated structure with a synthesised nanocomposite slot waveguide exhibits a measured photocurrent of 22 μA with LB media, decreasing to a value of 13 μA in the presence of E. coli. The fabricated biosensor is capable of detecting E. coli bacteria concentrations of 5000 CFU/ml. Altogether a change in photocurrent of 9 μA is obtained with E. coli on applying a low voltage of 3.5 volts. The combination of photocatalytic nanocomposite with engineered optical waveguide carries great potential for applications in biochemical detection and in realizing other integrated photonic devices with slotted nanocomposites. [ABSTRACT FROM AUTHOR]

The high sensitivity of chalcogenide surface plasmon resonance (SPR) structures with chalcogenide films is fructified to elaborate the salinity sensor for the seawater. A four layers configuration was taken into consideration, comprising a BK7 prism, a gold thin film, a chalcogenide As2S3 film and the ambient medium, which is the ß-Galactosidase solution. The transfer matrix formalism was used in our study to determine the reflectances characterizing the plasmonic structure. The two polarization modes (TE and TM) were taken into consideration, in order to determine the best configuration that allows an optimal sensitivity. The structure used as optical sensor has a high sensitivity. A 0.05 % change in concentration causes an 18-arc seconds change in plasmonic resonance incident angle and it can be resolved. [ABSTRACT FROM AUTHOR]

In this study a sensitive electrochemical sensor for the detection of E.coli has been developed using silver nanoparticle (Ag) embedded poly (9, 9-dioctylfluorene-ran-phenylene) (CFP) nanocomposite as a conductive platform and DNA hybridization technique. The new polymer was synthesized from 9, 9-dioctylfluorene and 1, 3-dichlorobenzene and biphenyl through Friedel Crafts alkylation reaction and the synthesized polymer as well as the Ag nanoparticles loaded composite were characterized using Fourier-transform infrared spectroscopy (FTIR), Nuclear Magnetic Resonance (NMR), X-ray powder diffraction (XRD), Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) analysis. For accurate and rapid label-free electrochemical detection of pathogenic bacteria such as E.coliwas studied by spin coating the nanocomposite suspension into indium tin oxide electrode (ITO) followed by the immobilization of aminoterminated oligonucleotide (pDNA), as probe. The resultant pDNA/Ag-CFP/ITO biosensor was then used to detect ssDNA, cleaved from genomic DNA of E.coli, using differential pulse voltammetry (DPV) technique. Under optimal experimental conditions, the biosensor could detect ssDNA in a wide linear range from 1 × 10-15 M to 1 × 10-22 M with a lowest detection limit of 1 × 10-22 M. [ABSTRACT FROM AUTHOR]

We develop a multivalent aptamer meshed open pore membrane with a signal amplification method as a simple and robust platform for sensitive, robust, and high-flux detection of E. coli O157:H7 whole cells in food samples. Nylon open pore membranes are surface modified by multi-handled poly(amidoamine) dendrimers as templates that are subsequently functionalized with multiple branches of capturing RCA (cRCA). The resulting cRCA networks are composed of tandem repeating aptamers that can specifically recognize and capture E. coli O157:H7 cells. The captured cells are then conjugated with signaling RCA (sRCA) to amplify the fluorescent detection signal. Our results demonstrate that the cRCA-based aptamer meshwork significantly enhances the surface-capturing performance of E. coli O157:H7 cells while achieving high-fluxes. Moreover, the membrane surface modification using dendrimers renders the resulting membrane nonfouling thus significantly reducing the background noises by approximately 96 times while the sRCA signal intensification enhances the fluorescent detection signals by approximately 27 times. We show that the membranes exhibit excellent performances in detecting E.coli O157:H7 in milk and orange juice samples with limit of detection of 10 cells per 250 mL with excellent specificity, offering a robust and practical approach for rapid and sensitive detection of foodborne bacteria. • Membrane allows high flux detection of bacteria in complex food matrices. • Dendrimer surface modification decreases detection background noises. • Dendrimer multi-handle templates allow formation of aptamer network on the membrane. • cRCA products mesh the membrane open pores to enhance target capturing. • sRCA signal intensification amplifies detection signals for sensitive detection. [ABSTRACT FROM AUTHOR]

A novel label-free electrochemical DNA biosensor was constructed for the determination of Escherichia coli bacteria in environmental water samples. The aminated DNA probe was immobilized onto hollow silica microspheres (HSMs) functionalized with 3-aminopropyltriethoxysilane and deposited onto a screen-printed electrode (SPE) carbon paste with supported gold nanoparticles (AuNPs). The biosensor was optimized for higher specificity and sensitivity. The label-free E. coli DNA biosensor exhibited a dynamic linear response range of 1 × 10−10 µM to 1 × 10−5 µM (R2 = 0.982), with a limit of detection at 1.95 × 10−15 µM, without a redox mediator. The sensitivity of the developed DNA biosensor was comparable to the non-complementary and single-base mismatched DNA. The DNA biosensor demonstrated a stable response up to 21 days of storage at 4 ℃ and pH 7. The DNA biosensor response was regenerable over three successive regeneration and rehybridization cycles.

In designing a bacteria biosensor, various issues must be addressed: the specificity of bacteria recognition, the immobilization of biomolecules that act as the bacteria receptor, and the selectivity of sensor surface. The aim of this paper was to examine how the biofunctionalized surface of Ti, Au, and Ru metals reacts in contact with strains of Escherichia coli (E. coli). The focus on metal surfaces results from their future use as electrodes in high frequency biosensors, e.g., resonant circuits or transmission-line sections. First, the surfaces of different metals were chemically functionalized with 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde or with 3-glycidylooxypropyltrimethoxysilane (GPTMS) followed by N-(5-amino-1-carboxypentyl) iminodiacetic acid (AB-NTA) and NiCl2. Secondly, the lipopolysaccharide binding protein (LBP), polyclonal anti-Escherichia coli antibody and bacteriophage protein gp37 were tested as bacteria receptors. The selectivity and specificity have been confirmed by the Enzyme-Linked Immunosorbent Assay (ELISA) and visualized by scanning electron microscopy at low landing energies. We noticed that LBP, polyclonal antibody, and gp37 were successfully immobilized on all studied metals and recognized the E. coli bacteria selectively. However, for the antibody, the highest reactivity was observed when Ti surface was modified, whereas the bacteria binding was comparable between LBP and gp37 on the functionalized Ru surfaces, independent from modification. Thus, all surfaces were biocompatible within the scope of biosensor functionality, with titanium functionalization showing the best performance. [ABSTRACT FROM AUTHOR]

Herein, we introduced a molecular imprinting based capacitive biosensor for real-time and highly sensitive bacteriophage detection. The sensing mechanism was based on the binding of target phage into the specific cavities on the electrode surface which resulted in a measurable change in the total capacitance of the system. Phage detection was investigated in the concentration range of 1.0 × 10 1 –1.0 × 10 5 plaque forming units (pfu)/mL and the limit of detection (LOD) was measured as 10 pfu/mL which shows the high sensitivity of the system compared to results reported for previous studies. The system also allowed the detection of phages in river water samples which is very important for the usability of the system as in-field analysis for different applications e.g. investigating the contamination of drinking water via wastewater or reservoir water in the future. Recently, due to their high specificity towards their host bacteria, being cost-effective and also stable in harsh environments, bacteriophages have been used as biorecognition elements in many studies. Due to this reason, the applicability of the phage imprinted biosensor was also investigated for host bacteria detection. E. coli detection has been performed in the concentration range of 1.0 × 10 2 –1.0 × 10 7 colony forming units (cfu)/mL with a LOD value of 100 cfu/mL. This system offers direct, real-time, very sensitive and rapid detection of bacteriophage and its host bacteria. [ABSTRACT FROM AUTHOR]

The supply of safe potable water, free from pathogens and chemicals, requires routine analyses and the application of several diagnostic techniques. Apart from being expensive, many of the detection methods require trained personnel and are often time-consuming. With drastic climate changes, severe droughts, increases in population and pollution of natural water systems, the need to develop ultrasensitive, low-cost and hand-held, point-of-use detection kits to monitor water quality is critical. Although Escherichia coli is still considered the best indicator of water quality, cell numbers may be below detection limits, or the cells may be non-culturable and thus only detected by DNA amplification. A number of different biosensors have been developed to detect viable, dead or non-culturable microbial cells and chemicals in water. This review discusses the differences in these biosensors and evaluates the application of microfluidics in the design of ultra-sensitive nano-biosensors. [ABSTRACT FROM AUTHOR]

In this paper, we report a single step solid phase synthesis strategy for colistin modified fluorescent carbon dots (CDs@colistin) for the selective detection of gram-negative bacteria like Escherichia coli . Colistin is a well known cyclic polypeptide antibiotic drug but here we have used as a binding ligand for E. coli. Here also, the synthesised CDs@colistin are used for selective Fe 3+ metal ion detection. The detection limits are 460 cfu/mL for E. coli and 56 nM for Fe 3+ with a linear range of 3.81 × 10 2 –2.44 × 10 4 cfu/mL and 0–48 μM, respectively. The synthesised CDs@colistin are well dispersed in water with an uniform particle size ranging from 2 nm to 5 nm executing high quantum yield of 7.56%. The synthesis process of CDs@colistin is simple, cheap, and less time consuming. Simultaneously, we have applied CDs@colistin as a novel probe for E. coli detection in three different real samples like human urine, apple juice, and tap water to evaluate the practical applicability of the synthesised colistin conjugated carbon dots. [ABSTRACT FROM AUTHOR]

Nano-biosensors are excellent monitoring tools for rapid, specific, sensitive, inexpensive, in-field, on-line, and/or real-time detection of pathogens in foods, soil, air, and water samples. A variety of nano-materials (metallic, polymeric, and/or carbon-based) were employed to enhance the efficacy, efficiency, and sensitivity of these nano-biosensors, including graphene-based materials, especially graphene oxide (GO)-based materials. GO bears many oxygen-bearing groups, enabling ligand conjugation at the high density critical for sensitive detection. We have fabricated GO-modified nano-porous polycarbonate track-etched (PCTE) membranes that were conjugated to an Escherichia coli -specific antibody (Ab) and used to detect E. coli . The random distribution of nanopores on the PCTE membrane surface and the bright coating of the GO onto the membrane were confirmed by scanning electron microscope. Anti- E. coli β-gal Abs were conjugated to the GO surface via 1-ethyl-3,3-dimethylaminopropyl carbodiimide hydrochloride– N -hydroxysuccinimide chemistry; antibody coating was confirmed by the presence of a characteristic IR peak near 1600 cm − 1 . A non-corresponding Ab (anti- Pseudomonas ) was used as a negative control under identical conditions. When E. coli interacted anti- E.coli β-gal with Ab-coated GO-nano-biosensor units, we observed a clear shift in the IR peak from 3373.14 to 3315 cm − 1 ; in contrast, we did not observe any shift in IR peaks when the GO unit was coated with the non-corresponding Ab (anti- Pseudomonas) . Therefore, the detection of E. coli using the described GO-nano-sensor unit is highly specific, is highly selective and can be applied for real-time monitoring of E. coli with a detection limit between 100 μg/mL and 10 μg/mL, similar to existing detection systems. [ABSTRACT FROM AUTHOR]

In designing a bacteria biosensor, various issues must be addressed: the specificity of bacteria recognition, the immobilization of biomolecules that act as the bacteria receptor, and the selectivity of sensor surface. The aim of this paper was to examine how the biofunctionalized surface of Ti, Au, and Ru metals reacts in contact with strains of Escherichia coli (E. coli). The focus on metal surfaces results from their future use as electrodes in high frequency biosensors, e.g., resonant circuits or transmission-line sections. First, the surfaces of different metals were chemically functionalized with 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde or with 3-glycidylooxypropyltrimethoxysilane (GPTMS) followed by N-(5-amino-1-carboxypentyl) iminodiacetic acid (AB-NTA) and NiCl2. Secondly, the lipopolysaccharide binding protein (LBP), polyclonal anti-Escherichia coli antibody and bacteriophage protein gp37 were tested as bacteria receptors. The selectivity and specificity have been confirmed by the Enzyme-Linked Immunosorbent Assay (ELISA) and visualized by scanning electron microscopy at low landing energies. We noticed that LBP, polyclonal antibody, and gp37 were successfully immobilized on all studied metals and recognized the E. coli bacteria selectively. However, for the antibody, the highest reactivity was observed when Ti surface was modified, whereas the bacteria binding was comparable between LBP and gp37 on the functionalized Ru surfaces, independent from modification. Thus, all surfaces were biocompatible within the scope of biosensor functionality, with titanium functionalization showing the best performance.

• A novel method for label-free detection of Escherichia coli introduced relying on microwave resonator array biosensor for improved sensitivity and probability of bacteria detection. • Low-Temperature Co-Fired Ceramics technology is employed for fabrication due to its quasi-3D structure capability and ease of biofunctionalization with polyclonal antibodies for increased sensing robustness and selective response. • A demonstrator developed exhibiting five distinct resonances in a frequency range of 4 – 6 GHz operating in differential transmission mode. • Experiment carried out for of various concentrations of Escherichia coli and a non-specific high concentration of Lactobacillus rhamnosus LOCK 0919 proved the low detection threshold and selective response capability. A novel method for label-free detection of Escherichia coli relying on microwave sensing is proposed. A transmission-type differential resonator array biosensor covered with polyclonal anti- Escherichia coli antibodies is introduced providing a robust response with high sensitivity for detecting bacteria presence due to resonant operation combined. The high selectivity is given by the presence of specific bacteria-binding antibodies. Low-Temperature Co-Fired Ceramics technology is proposed to be used for implementation to take advantage of its quasi-three-dimensional fabrication capability. The proposed sensing structure operates in a frequency range of 4 – 6 GHz with five distinct resonances increasing the probability of bacteria detection presence. The developed sensor was experimentally validated by measurements of various concentrations of Escherichia coli along with measurement of a non-specific high concentration of Lactobacillus rhamnosus LOCK 0919. The obtained results prove the performance and suitability of the proposed biosensor for biomedical applications. [ABSTRACT FROM AUTHOR]

As a first stage toward the development of an impedimetric biosensor to quantify viable bacterial cells, this work defines the relationships between microbial growth and impedance changes associated with acidification of the culture medium. Escherichia coli bacterium was used as a model of a common pathogenic bacterium found in a foodstuff. Impedance was measured at a fix frequency of 10 kHz using interdigitated microelectrodes. Impedance changes were found to depend on the number of microorganisms in a sample. However, as the kinetics greatly depends on the bacterial concentration, it was not possible to obtain a single calibration curve in a wide concentration range at a fixed incubation time. To resolve this, a novel calibration method was proposed by measuring the sensor response at 270 and 390 min of incubation and taking a mean value. Obtained calibration plots were used to determine E. coli in a spiked milk samples. Performed experiments demonstrated the capacity of the method to detect E.coli concentrations in a range between 10 2 and 10 6 cfu mL −1 in milk in only 6 hours with 5–12% error margin. [ABSTRACT FROM AUTHOR]

It is well known that the metabolic activity of some microorganisms results in changes of pH of the culture medium, a phenomenon that can be used for detection and quantification of bacteria. However, conventional glass electrodes that are commonly used for pH measurements are bulky, fragile and expensive, which hinders their application in miniaturized systems and encouraged to the search for alternatives. In this work, two types of metal oxide pH sensors have been tested to detect the metabolic activity of the bacterium Escherichia coli ( E. coli ). These pH sensors were produced on silicon chips with platinum metal contacts, onto which thin layers of IrO x or Ta 2 O 5 were incorporated by two different methods (electrodeposition and e-beam sputtering, respectively). In order to facilitate measurement in small sample volumes, an Ag/AgCl pseudo-reference was also screen-printed in the chip and was assayed in parallel to an external Ag/AgCl reference electrode. As it is shown, the developed sensors generated results indistinguishable from those provided by a conventional glass pH-electrode but could be operated in significantly smaller sample volumes. After optimization of the detection conditions, the metal oxide sensors are successfully applied for detection of increasing concentrations of viable E. coli, with detection of less than 10 3 cfu mL −1 in undiluted culture medium in just 5 h. [ABSTRACT FROM AUTHOR]

In this work, a simple, label-free, low cost electrochemical biosensor for highly sensitive and selective detection of Escherichia coli has been developed on the basis of rolling circle amplification (RCA) coupled peroxidase-mimicking DNAzyme amplification. A aptamer-primer probe (APP) containing anti- E. coli aptamer and a primer sequence complementary to a circular probe, which includes two G-quadruplex units, is used for recognizing target and triggering RCA-based polymerase elongation. Due to RCA coupled DNAzyme amplification strategy, the presence of target E. coli leads to the formation of numerous G-quadruplex oligomers on electrode, which folds into G-quadruplex/hemin complexs with the help of K + and hemin, thus generating extremely strong catalytic activity toward H 2 O 2 and giving a remarkably strong electrochemical response. As far as we know, this work is the first time that RCA coupled peroxidase-mimicking DNAzyme amplification technique have been integrated into electrochemical assay for detecting pathogenic bacteria. Under optimal conditions, the proposed biosensor exhibits ultrahigh sensitivity toward E. coli with detection limits of 8 cfu mL −1 and a detection range of 5 orders of magnitude. Besides, our biosensor also shows high selectivity toward target E. coli and has the advantages in its rapidness, low cost, simplified operations without the need of electrochemical labeling steps and additional labile reagents. Hence, the RCA coupled peroxidase-mimicking DNAzyme amplification-based electrochemical method might create a useful and practical platform for detecting E. coli and related food safety analysis and clinical diagnosis. [ABSTRACT FROM AUTHOR]

Graphene is an allotrope of carbon with two‐dimensional (2D) monolayer honeycombs. A larger detection area and higher sensitivity can be provided by graphene‐based nanosenor because of its 2D structure. In addition, owing to its special characteristics, including electrical, optical and physical properties, graphene is known as a more suitable candidate compared to other materials used in the sensor application. A novel model employing a field‐effect transistor structure using graphene is proposed and the current–voltage (I –V) characteristics of graphene are employed to model the sensing mechanism. This biosensor can detect Escherichia coli (E. coli) bacteria, providing high levels of sensitivity. It is observed that the graphene device experiences a drastic increase in conductance when exposed to E. coli bacteria at 0–105 cfu/ml concentration. The simple, fast response and high sensitivity of this nanoelectronic biosensor make it a suitable device in screening and functional studies of antibacterial drugs and an ideal high‐throughput platform which can detect any pathogenic bacteria. Artificial neural network and support vector regression algorithms have also been used to provide other models for the I –V characteristic. A satisfactory agreement has been presented by comparison between the proposed models with the experimental data. [ABSTRACT FROM AUTHOR]

A PCR-free, optics-free device is used for the detection of Escherichia coli (E. coli) 16S rRNA at 10 fM, which corresponds to ~100–1000 colony forming units/mL (CFU/mL) depending on cellular rRNA levels. The development of a rapid, sensitive, and cost-effective nucleic acid detection platform is sought for the detection of pathogenic microbes in food, water and body fluids. Since 16S rRNA sequences are species specific and are present at high copy number in viable cells, these nucleic acids offer an attractive target for microbial pathogen detection schemes. Here, target 16S rRNA of E. coli at 10 fM concentration was detected against a total RNA background using a conceptually simple approach based on electromechanical signal transduction, whereby a step change reduction in ionic current through a pore indicates blockage by an electrophoretically mobilized bead-peptide nucleic acid probe conjugate hybridized to target nucleic acid. We investigated the concentration detection limit for bacterial species-specific 16S rRNA at 1 pM to 1 fM and found a limit of detection of 10 fM for our device, which is consistent with our previous finding with single-stranded DNA of similar length. In addition, no false positive responses were obtained with control RNA and no false negatives with target 16S rRNA present down to the limit of detection (LOD) of 10 fM. Thus, this detection scheme shows promise for integration into portable, low-cost systems for rapid detection of pathogenic microbes in food, water and body fluids.

We report on a new fluorimetric assay for β-galactosidase (β-gal) and faecal coliform bacteria that utilizes a long-wavelength dye, chlorophenol red-β- d-galactopyranoside (CPRG), that has been widely used for colorimetric assays. The novel feature of this new assay is the unexpected development of a large fluorescence response from liberated chorophenol red (CPR) upon complexation with poly- l-arginine (pR) in solution. The binding of CPR to pR occurs through the sulphonate group of CPR, causing formation of a charge-transfer complex and up to a 70-fold increase in emission intensity. A major advantage of the assay is the ability to utilize excitation and emission wavelengths in the red end of the spectrum, which avoids common interferences obtained when using UV-absorbing dyes such as 4-methylumbelliferyl-β- d-galactopyranoside. We provide data on the utility of CPRG as a fluorimetric reporter for both β-gal and Escherichia coli ATCC 25922 and demonstrate optimized reaction conditions for rapid and sensitive detection of E. coli at a level of 1 colony-forming unit (cfu)/10 mL after 12 h of culture followed by a 1-h assay, which is below the regulatory limit for testing of recreational water. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Magneto-resistive biosensors have been found to be useful because of their high sensitivity, low cost, small size, and direct electrical output. They use super-paramagnetic beads to label a biological target and detect it via sensing the stray field. In this paper, we report a new setup for magnetic biosensors, replacing the conventional “sandwich” concept with an electromagnetic trap. We demonstrate the capability of the biosensor in the detection of E. coli. The trap is formed by a current-carrying microwire that attracts the magnetic beads into a sensing space on top of a tunnel magneto-resistive sensor. The sensor signal depends on the number of beads in the sensing space, which depends on the size of the beads. This enables the detection of biological targets, because such targets increase the volume of the beads. Experiments were carried out with a 6µm wide microwire, which attracted the magnetic beads from a distance of 60μm, when a current of 30mA was applied. A sensing space of 30µm in length and 6µm in width was defined by the magnetic sensor. The results showed that individual E. coli bacterium inside the sensing space could be detected using super-paramagnetic beads that are 2.8µm in diameter. The electromagnetic trap setup greatly simplifies the device and reduces the detection process to two steps: (i) mixing the bacteria with magnetic beads and (ii) applying the sample solution to the sensor for measurement, which can be accomplished within about 30min with a sample volume in the µl range. This setup also ensures that the biosensor can be cleaned easily and re-used immediately. The presented setup is readily integrated on chips via standard microfabrication techniques. [ABSTRACT FROM AUTHOR]

ABSTRACT Mannose-rhodamine (Rh) conjugate (80% yield) was synthesized in a one-pot reaction and immobilized onto magnetic polymeric nanoparticles (MPNP; 43% magnetic content) of poly(styrene/divinyl benzene/acrylic acid). The resulting nanoparticles contained MPNP as a substrate, mannose as an E. coli receptor and Rh as a fluorescent signaling unit. TEM imaging clearly demonstrated that multiple mannose-Rh MPNPs could be captured by E. coli strain ORN178. The fluorescent signal from captured nanoparticles was emitted at 580 nm. These results indicate that mannose-Rh MPNP offers a simple and rapid strategy for bacterial detection. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2014, 131, 40012. [ABSTRACT FROM AUTHOR]

Abstract: Intensely fluorescent, colistin-functionalised CdSe/ZnS QDs (Colis–QDs) nanoparticles, are synthesized and used as sensitive probes for the detection of Escherichia coli, a Gram-negative bacteria. Colistin molecules are attached to the terminal carboxyl of the mercaptoacetic acid-capped QDs in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as amide bond promoters. The TEM analysis of bacteria treated with Colis–QDs conjugates showed the accumulation of Colis–QDs in the cell wall of E. coli. Under the recommended working conditions, the method provides a detection limit as few as 28 E. coli cells per mL, which is competitive which more elaborate detection systems. The simplicity of the method together with short analysis time (<15min, without including preparation and photoactivation of the Colis–QDs conjugate) make the proposed approach useful as quick bacteria screening system. [Copyright &y& Elsevier]

Recently, the β-galactosidase assay has become a key component in the development of assays and biosensors for the detection of enterobacteria and E. coli in water quality monitoring. The assay has often performed below its maximum potential, mainly due to a poor choice of conditions. In this study we establish a set of optimal conditions and provide a rough estimate of how departure from optimal values reduces the output of the assay potentially decreasing its sensitivity. We have established that maximum response for detecting low cell concentrations requires an induction of the samples using IPTG at a concentration of 0.2 mM during 180 min. Permeabilization of the samples is mandatory as lack of it results in an almost 60% reduction in assay output. The choice of enzyme substrate is critical as different substrates yield products with different extinction coefficients or fluorescence yields. The concentration of substrate used must be high enough (around 3 to 4 times K m) to ensure that the activity measured is not substrate limited. Finally, as the color/fluorescence of the reaction products is highly dependent on pH, care must be taken to ensure that pH at the time of reading is high enough to provide maximum signal. [Display omitted] • Optimization the of β-galactosidase for bacterial detection and biosensor development. • Lack of permeabilization reduces 60% the sensitivity of the assay. • The concentration of galactoside substrates should be 3–4 times the value of their Km. • High concentrations of IPTG inhibit the β-galactosidase assay. [ABSTRACT FROM AUTHOR]

This paper reports an experimental study of E. coli sensing based on surface-skimming-bulk-wave delay line device fabricated on ST-cut quartz. The sensor configuration consisted of an 87.7 MHz oscillator with the delay line as control element in its feedback loop. The antigen–antibody immunochemical reaction was employed on the acoustic wave device surface to generate mass loading and frequency shift. The sensor output was monitored by an RF spectrum analyzer. Highly specific sensing is demonstrated for E. coli cells up to 0.4 cells/μl in water. The methodology offers possibility for developing highly sensitive and fast E. coli sensors for testing of water potability. [Copyright &y& Elsevier]

A simple method of detecting Escherichia coli on retail beef was developed. Diluent used in spongesampling of meat was added to a tube containing double-strength lauryl sulfate tryptone broth + 5-bromo-4-chloro-3-indolyl β-D-glucuronide (X-GIcA) and a Durham tube. Tubes with blue color and gas after 24 h incubation at 35 °C were presumptive-positive. Chi-squared analysis showed a significant (P < 0.05) relationship between the method and the Petrifilm™ E. coil/Coliform Count Plate method for E. coli in analysis of 66 retail beef samples and the 2 methods had a similar proportion of confirmed isolates (61 and 63%). The method developed is qualitatively comparable to the PetrifllmTM method and provides small-scale beef processors a simple way to monitor sanitary conditions in fabrication and grinding operations. [ABSTRACT FROM AUTHOR]

Inhibitors are an important means of reducing the growth of foodborne microorganisms. However, excessively using inhibitors will cause great harm to human beings and organisms. Therefore, quantitatively evaluating the effect of low-dose inhibitors on foodborne bacteria growth is very important. Electrochemical impedance spectroscopy (EIS) is a commonly used biosensor technique for detecting foodborne bacteria. In this paper, we show a machine learning-based EIS biosensor method that can be used to detect the effect of a low-dose inhibitor (e.g., hydrogen peroxide) on Escherichia coli (E. coli). After obtaining the minimum inhibitory concentration (MIC), the inhibitor concentration and below was applied to E. coli solution. EIS data were obtained by binding the target bacteria to the electrode surface through antibodies, and then, the impedance parameters were fitted by the Randles model. XGBoost and a support vector regression (SVR) machine learning models were compared to establish a quantitative relationship between multiple impedance parameters and the bacterial concentration under the effect of inhibitors. The results showed that XGBoost improved the quantitative analysis accuracy toward inhibitors (0.175–0.375 μL/mL), based on the bacteria growth in the solution. After different low concentrations of the inhibitor were added into a standard bacterial solution for 1 h, 2 h and 3 h, the maximum prediction error of the inhibitor concentration was 4.95%, 1.03% and 0.46%, respectively. The prediction error decreased as the incubation time of the E. coli culture was extended. These results pave the way for the automation of an accurate EIS biosensor for analyzing foodborne microorganisms under various low doses of inhibitors or drugs [ABSTRACT FROM AUTHOR]

By combining a functionalized microwire sensor with a dielectrophoresis (DEP) technique, a rapid and accurate detection method for bacterial pathogens was developed and demonstrated. The gold-tungsten microsensor (sensing diameter, 25 μm) was functionalized with specific Escherichia coli (E. coli) antibodies against surface antigens to capture E. coli bacterial cells from phosphate buffer saline (PBS) and ground beef. The DEP technique consisted of the application of a pin-type microwire and large base electrode under an alternating current (AC) electric field of 10 Vpp at a frequency of 3 MHz. Additionally, bovine serum albumin (BSA) was utilized as a blocking agent for the attachment of nonspecific proteins to the microwire surface to prevent false positive signals due to nonspecific binding. The resistance and fluorescent intensity (FI) demonstrated a linear relationship with the concentration of E. coli cells ranging from 103 to 107 CFU/mL. These results showed that DEP can be used as a reliable alternative for the detection of E. coli without a blocking effect minimizing the significant difference between PBS and ground beef. • A functionalized microwire biosensor with DEP was developed. • The biosensor demonstrated the accurate detection of E. coli in protein-based food. • The resistance values indicated a linear correlation with increasing E. coli concentration. • A detection limit of 103-107 CFU/mL with a detection time of 25 min was feasible. [ABSTRACT FROM AUTHOR]

A novel label-free electrochemical DNA biosensor was constructed for the determination of Escherichia coli bacteria in environmental water samples. The aminated DNA probe was immobilized onto hollow silica microspheres (HSMs) functionalized with 3-aminopropyltriethoxysilane and deposited onto a screen-printed electrode (SPE) carbon paste with supported gold nanoparticles (AuNPs). The biosensor was optimized for higher specificity and sensitivity. The label-free E. coli DNA biosensor exhibited a dynamic linear response range of 1 × 10−10 µM to 1 × 10−5 µM (R2 = 0.982), with a limit of detection at 1.95 × 10−15 µM, without a redox mediator. The sensitivity of the developed DNA biosensor was comparable to the non-complementary and single-base mismatched DNA. The DNA biosensor demonstrated a stable response up to 21 days of storage at 4 ℃ and pH 7. The DNA biosensor response was regenerable over three successive regeneration and rehybridization cycles. [ABSTRACT FROM AUTHOR]

In spite of the clinical need, there is a major gap in rapid diagnostics for identification and quantitation of E. coli and other pathogens, also regarded as the biggest bottleneck in the fight against the spread of antimicrobial resistant bacterial strains. This study reports for the first time an optical, smartphone-based microfluidic fluorescence sandwich immunoassay capable of quantifying E. coli in buffer and synthetic urine in less than 25 min without sample preparation nor concentration. A limit of detection (LoD) up to 240 CFU/mL, comensurate with cut-off for UTIs (103–105 CFUs/mL) was achieved. Replicas of full response curves performed with 100–107 CFUs/mL of E. coli K12 in synthetic urine yielded recovery values in the range 80–120%, assay reproducibility below 30% and precision below 20%, therefore similar to high-performance automated immunoassays. The unrivalled LoD was mainly linked to the 'open fluidics' nature of the 10-bore microfluidic strips used that enabled passing a large volume of sample through the microcapillaries coated with capture antibody. The new smartphone based test has the potential of being as a rapid, point-of-care test for rule-in of E. coli infections that are responsible for around 80% of UTIs, helping to stop the over-prescription of antibiotics and the monitoring of patients with other symptomatic communicable diseases caused by E. coli at global scale. • Smartphone quantitation of E. coli in synthetic urine using FEP microfluidic strips. • Fluorescence, ELISA detection in less than 25 min without sample preparation. • A limit of detection, LoD of 240 CFU/mL, compatible with cut-off for UTIs. • Recovery, reproducibility and precision comparable to high-performance immunoassays. [ABSTRACT FROM AUTHOR]

A portable kinetics fluorometer is developed to detect viable cells which may be contaminating various samples. The portable device acts as a single-excitation, single-emission photometer that continuously measures fluorescence intensity of an indicator dye and plots it. The slope of the plot depends on the number of colony forming units per milliliter. The device uses resazurin as the indicator dye. Viable cells reduce resazurin to resorufin, which is more fluorescent. Photodiode is used to detect fluorescence change. The photodiode generated current proportional to the intensity of the light that reached it, and an op-amp is used in a transimpedance differential configuration to ensure amplification of the photodiode's signal. A microfluidic chip is designed specifically for the device. It acts as a fully enclosed cuvette, which enhances the resazurin reduction rate. In tests, the E. coli-containing media are injected into the microfluidic chip and the device is able to detect the presence of E. coli in LB media based on the fluorescence change that occurred in the indicator dye. The device provides fast, accurate, and inexpensive means to optical detection of the presence of viable cells and could be used in the field in place of more complex methods, i.e., loop-meditated isothermal amplification of DNA (LAMP) to detect bacteria in pharmaceutical samples (Jimenez et al., J Microbiol Methods 41(3):259-265, 2000) or measuring the intrinsic fluorescence of the bacterial or yeast chromophores (Estes et al., Biosens Bioelectron 18(5):511-519, 2003).