Nanostructured biosensing platforms for the detection of food- and water-borne pathogenic Escherichia coli.

Pathogenic bacterial infection is one of the principal causes affecting human health and ecosystems. The accurate identification of bacteria in food and water samples is of significant interests to maintain safety and health for humans. Culture-based tests are practically tedious and may produce false-positive results, while viable but non-culturable microorganisms (NCMs) cannot be retrieved. Thus, it requires fast, reliable, and low-cost detection strategies for on-field analysis and point-of-care (POC) monitoring. The standard detection methods such as nucleic acid analysis (RT-PCR) and enzyme-linked immunosorbent assays (ELISA) are still challenging in POC practice due to their time-consuming (several hours to days) and expensive laboratory operations. The optical (surface plasmon resonance (SPR), fluorescence, and surface-enhanced Raman scattering (SERS)) and electrochemical-based detection of microbes (early stage of infective diseases) have been considered as alternative routes in the emerging world of nanostructured biosensing since they can attain a faster and concurrent screening of several pathogens in real samples. Moreover, optical and electrochemical detection strategies are opening a new route for the ability of detecting pathogens through the integration of cellphones, which is well fitted for POC analysis. This review article covers the current state of sensitive mechanistic approaches for the screening and detection of Escherichia coli O157:H7 (E. coli) pathogens in food and water samples, which can be potentially applied in clinical and environmental monitoring.

Cryo-EM structure of Shiga toxin 2 in complex with the native ribosomal P-stalk reveals residues involved in the binding interaction.

Shiga toxin 2a (Stx2a) is the virulence factor of enterohemorrhagic Escherichia coli. The catalytic A1 subunit of Stx2a (Stx2A1) interacts with the ribosomal P-stalk for loading onto the ribosome and depurination of the sarcin-ricin loop, which halts protein synthesis. Because of the intrinsic flexibility of the P-stalk, a structure of the Stx2a-P-stalk complex is currently unknown. We demonstrated that the native P-stalk pentamer binds to Stx2a with nanomolar affinity, and we employed cryo-EM to determine a structure of the 72 kDa Stx2a complexed with the P-stalk. The structure identifies Stx2A1 residues involved in binding and reveals that Stx2a is anchored to the P-stalk via only the last six amino acids from the C-terminal domain of a single P-protein. For the first time, the cryo-EM structure shows the loop connecting Stx2A1 and Stx2A2, which is critical for activation of the toxin. Our principal component analysis of the cryo-EM data reveals the intrinsic dynamics of the Stx2a-P-stalk interaction, including conformational changes in the P-stalk binding site occurring upon complex formation. Our computational analysis unveils the propensity for structural rearrangements within the C-terminal domain, with its C-terminal six amino acids transitioning from a random coil to an α-helix upon binding to Stx2a. In conclusion, our cryo-EM structure sheds new light into the dynamics of the Stx2a-P-stalk interaction and indicates that the binding interface between Stx2a and the P-stalk is the potential target for drug discovery.

New PEPTIR-2.0 Peptide Designed for Use as Recognition Element in Electrochemical Biosensors with Improved Specificity towards E. coli O157:H7.

The detection of pathogens through alternative methodologies based on electrochemical biosensors is being studied. These devices exhibit remarkable properties, such as simplicity, specificity, and high sensitivity in monitoring pathogens. However, it is necessary to continue conducting studies that adequately improve these characteristics, especially the recognition molecule. This work aims to design and evaluate a new peptide, named PEPTIR-2.0, as a recognition molecule in electrochemical biosensors to detect E. coli O157:H7 in water. PEPTIR-2.0 was obtained from modifications of the PEPTIR-1.0 peptide sequence, which was previously reported and exhibited excellent properties for detecting and quantifying this pathogenic microorganism. PEPTIR-1.0 is a peptide analogous to the TIR (Translocated Intimin Receptor) protein capable of interacting with the Intimin outer membrane. The basis of this study was to obtain, by using bioinformatics tools, a molecule analogous to PEPTIR-1.0 that maintains its three-dimensional structure but increases the hydrophobic interactions between it and Intimin, since these intermolecular forces are the predominant ones. The designed PEPTIR-2.0 peptide was immobilized on screen-printed electrodes modified with gold nanoparticles. The detection capacity of E. coli O157:H7 in water was evaluated using electrochemical impedance spectroscopy in the presence of other microorganisms, such as P. aeruginosa, S. aureus, and non-pathogenic E. coli. The results showed that PEPTIR-2.0 confers remarkable specificity to the biosensor towards detecting E. coli, even higher than PEPTIR-1.0.

Electrochemical genosensor based on gold nanostars for the detection of Escherichia coli O157:H7 DNA.

Escherichia coli O157:H7 (E. coli O157:H7) is an enterohemorrhagic E. coli (EHEC), which has been issued as a major threat to public health worldwide due to fatal contamination of water and food. Thus, its rapid and accurate detection has tremendous importance in environmental monitoring and human health. In this regard, we report a simple and sensitive electrochemical DNA biosensor by targeting Z3276 as a genetic marker in river water. The surface of the designed gold electrode was functionalized with gold nanostars and an aminated specific sensing probe of E. coli O157:H7 to fabricate the genosensor. Cyclic voltammetry (CV) and square wave voltammetry (SWV) techniques were applied for electrochemical characterization and detection. The synthesized gold nanostars were characterized using different characterization techniques. The fabricated DNA-based sensor exhibited a high selective ability for one, two, and three-base mismatched sequences. Regeneration, stability, selectivity, and kinetics of the bioassay were investigated. Under optimal conditions, the fabricated genosensor exhibited a linear response range of 10-5 to 10-17 µM in the standard sample and 7.3 to 1 × 10-17 µM in water samples with a low limit of quantification of 0.01 zM in water samples. The detection strategy based on silver plated gold nanostars and DNA hybridization improved the sensitivity and specificity of the assay for E. coli O157:H7 detection in real water samples without filtration. The detection assay has the advantages of high selectivity, sensitivity, low amounts of reagents, short analysis time, commercialization, and potential application for the determination of other pathogenic bacteria.

A Bioinspired Peptide in TIR Protein as Recognition Molecule on Electrochemical Biosensors for the Detection of E. coli O157:H7 in an Aqueous Matrix.

Currently, the detection of pathogens such as Escherichia coli through instrumental alternatives with fast response and excellent sensitivity and selectivity are being studied. Biosensors are systems consisting of nanomaterials and biomolecules that exhibit remarkable properties such as simplicity, portable, affordable, user­friendly, and deliverable to end­users. For this, in this work we report for the first time, to our knowledge, the bioinformatic design of a new peptide based on TIR protein, a receptor of Intimin membrane protein which is characteristic of E. coli. This peptide (named PEPTIR­1.0) was used as recognition element in a biosensor based on AuNPs­modified screen­printed electrodes for the detection of E. coli. The morphological and electrochemical characteristics of the biosensor obtained were studied. Results show that the biosensor can detect the bacteria with limits of detection and quantification of 2 and 6 CFU/mL, respectively. Moreover, the selectivity of the system is statistically significant towards the detection of the pathogen in the presence of other microorganisms such as P. aeruginosa and S. aureus. This makes this new PEPTIR­1.0 based biosensor can be used in the rapid, sensitive, and selective detection of E. coli in aqueous matrices.

Using a safe and effective fixative to improve the immunofluorescence staining of bacteria.

The emerging and development of green chemistry has once again drawn the researchers' attention to eliminating the use and generation of hazardous materials. Here we report the use of a safe and effective fixative, chlorine dioxide (ClO2), instead of traditional hazardous fixatives for the cross-linking of cellular proteins to improve immunofluorescence staining of bacteria. The concentration of ClO2needed for 100% fixation is 50µg ml-1, which is much lower than that of traditional fixatives (1000-10000µg ml-1). The ClO2mediated cross-linking can preserve the integrity of bacterial cells and prevent cell loss through lysis. Meanwhile, lysozyme can permeabilize the bacterial cells, allowing the labelled antibodies to diffuse to their intracellular target molecules. By usingE. coliO157:H7/RP4 as a gram-negative bacteria model, immunofluorescence staining assays for both intracellular protein and surface polysaccharide were carried out to investigate the effect of ClO2fixation on the staining. The results demonstrated that ClO2fixation could prevent the target antigens from cracking off the bacteria without damage on the interaction between the antibodies and antigens (either for polysaccharide or protein). As a safe and effective fixative, ClO2has potential practical applications in immunofluorescence staining and fluorescencein situhybridization for single bacteria/cell analysis.

MMM: Integrative ensemble modeling and ensemble analysis.

Proteins and their complexes can be heterogeneously disordered. In ensemble modeling of such systems with restraints from several experimental techniques the following problems arise: (a) integration of diverse restraints obtained on different samples under different conditions; (b) estimation of a realistic ensemble width; (c) sufficient sampling of conformational space; (d) representation of the ensemble by an interpretable number of conformers; (e) recognition of weak order with site resolution. Here, I introduce several tools that address these problems, focusing on utilization of distance distribution information for estimating ensemble width. The RigiFlex approach integrates such information with high-resolution structures of ordered domains and small-angle scattering data. The EnsembleFit module provides moderately sized ensembles by fitting conformer populations and discarding conformers with low population. EnsembleFit balances the loss in fit quality upon combining restraint subsets from different techniques. Pair correlation analysis for residues and local compaction analysis help in feature detection. The RigiFlex pipeline is tested on data simulated from the structure 70 kDa protein-RNA complex RsmE/RsmZ. It recovers this structure with ensemble width and difference from ground truth both being on the order of 4.2 Å. EnsembleFit reduces the ensemble of the proliferating-cell-nuclear-antigen-associated factor p15PAF from 4,939 to 75 conformers while maintaining good fit quality of restraints. Local compaction analysis for the PaaA2 antitoxin from E. coli O157 revealed correlations between compactness and enhanced residual dipolar couplings in the original NMR restraint set.

Induction of Escherichia coli O157:H7 into a viable but non-culturable state by high temperature and its resuscitation.

Escherichia coli O157:H7, a causative agent of haemolytic uremic syndrome, can enter into a viable but non-culturable (VBNC) state in response to harsh stress. Bacteria in this state can retain membrane integrity, metabolic activity and virulence expression, which may present health risks. However, virulence expression and resuscitation ability of the VBNC state are not well understood. Here, we induced E. coli O157:H7 into a VBNC state by high temperature, which is commonly used to prevent the proliferation of pathogens in process of soil solarization, composting and anaerobic digestion of organic wastes. The virulence genes were highly expressed in the VBNC state and resuscitated daughter cells. The resuscitation of VBNC cells occurred after the removal of heat stress in Luria-Bertani medium. In addition, E. coli O157: H7 cells can leave the VBNC state and resuscitate with the clearance of protein aggregates. Notably, with the accumulation of protein aggregation and increased levels of reactive oxygen species, cells lost their ability to resuscitate. The results of this study not only can facilitate a better understanding of the health risks associated with the VBNC state but also have the potential to provide a theoretical basis for thermal disinfection processing.

Simultaneous detection of three zoonotic pathogens based on phage display peptide and multicolor quantum dots.

With the rapid development of human's exploitation of nature and animal husbandry, zoonoses have become a major public health problem worldwide. It is necessary to establish a rapid, specific and sensitive detection method to screen several zoonotic pathogenic bacteria simultaneously. In this study, phage display technology was used to screen specific peptide of three common zoonotic pathogens, E. coli O157:H7, L. monocytogenes and B. melitensis 16 M. Then, three peptide were obtained, named E2, L4 and B4, which can identify the three pathogens respectively. Three peptide modified with biotin were synthesized and were coupled to streptavidin magnetic beads (MBs) to form peptide-MBs, which enriched the above three pathogens from the samples. Three quantum dot (QD) probes were constructed by coupling three polyclonal antibodies to different fluorescent QD surfaces (QD540, QD580 and QD630). The simultaneous detection method based on peptide-MBs and QDs multicolor fluorescent labeling was established and could detect E. coli O157:H7, L. monocytogenes and B. melitensis 16 M simultaneously. The detection method took about 100 min with the detection limits of 103, 102 and 102 CFU/mL, respectively. The detection method could be also well utilized in real samples.

Electrochemical aptasensor using boron-carbon nanorods decorated by nickel nanoparticles for detection of E. coli O157:H7.

The development of a label-free impedimetric aptasensor is reported for rapid and sensitive detection of Escherichia coli O157:H7 employing boron-carbon nanorods decorated by nickel nanoparticles (BC-Ni) nanostructured platform. These highly electroactive BC-Ni nanorods were synthesized to increase the sensitivity of the sensor surface and subsequently functionalized with a specific anti-E. coli O157:H7 aptamer (Kd = 69 nM) as bio-recognition moiety. This fully characterized high-affinity DNA aptamer against the bacteria was selected using a facile microtiter plate-based cell-SELEX methodology. The fabricated electrochemical aptasensor is demonstrated to detect E. coli O157:H7 selectively with a detection limit of 10 cfu and a dynamic detection range of 100 to 105 cfu in water, juice, and fecal samples. Graphical abstract.

An electrochemical biosensor based on methylene blue-loaded nanocomposites as signal-amplifying tags to detect pathogenic bacteria.

A sandwich-type electrochemical biosensor was successfully constructed for the sensitive detection of pathogenic bacteria. In this biosensor platform, methylene blue (MB) organic-inorganic nanocomposites (MB@MI) were synthesized from magainin I (MI, antimicrobial peptide specific to Escherichia coli O157:H7), Cu3(PO4)2 and MB via a one-pot method, and were explored as a novel electrochemical signal label of biosensors generating amplified electrochemical signals by differential pulse voltammetry (DPV). E. coli O157:H7 specifically sandwich bound to the aptamers on the electrode surface and MB@MI nanocomposites, and the changes in the current signal generated on the electrode surface were used for the quantitative determination of E. coli O157:H7. Under optimum conditions, the proposed biosensor showed excellent performance with a wide linear range of 102-107 CFU mL-1 and a low detection limit of 32 CFU mL-1, featuring favorable selectivity, repeatability and stability. According to the experiments conducted on real samples, the proposed approach is capable of detecting pathogenic bacteria in clinical diagnostics.

Recent Progress on the Electrochemical Biosensing of Escherichia coli O157:H7: Material and Methods Overview.

Escherichia coli O157:H7 (E. coli O157:H7) is a pathogenic strain of Escherichia coli which has issued as a public health threat because of fatal contamination of food and water. Therefore, accurate detection of pathogenic E. coli is important in environmental and food quality monitoring. In spite of their advantages and high acceptance, culture-based methods, enzyme-linked immunosorbent assays (ELISAs), polymerase chain reaction (PCR), flow cytometry, ATP bioluminescence, and solid-phase cytometry have various drawbacks, including being time-consuming, requiring trained technicians and/or specific equipment, and producing biological waste. Therefore, there is necessity for affordable, rapid, and simple approaches. Electrochemical biosensors have shown great promise for rapid food- and water-borne pathogen detection. Over the last decade, various attempts have been made to develop techniques for the rapid quantification of E. coli O157:H7. This review covers the importance of E. coli O157:H7 and recent progress (from 2015 to 2020) in the development of the sensitivity and selectivity of electrochemical sensors developed for E. coli O157:H7 using different nanomaterials, labels, and electrochemical transducers.

Using gadolinium ions as affinity probes to selectively enrich and magnetically isolate bacteria from complex samples.

Magnetic trapping has been employed in the development of analytical methods owing to its ease and simplicity in handling samples. Nevertheless, the generation of functional probes is usually time consuming. A new and simple affinity method that uses gadolinium ion (Gd3+), a magnetic ion, as affinity probe for magnetic tapping of pathogenic bacteria was demonstrated in the present study. Escherichia coli O157:H7, Staphylococcus aureus, and Acinetobacter baumannii were selected as model bacteria. The model bacteria were magnetically isolated after incubation in Tris buffer (pH 8) containing Gd3+ (0.1 M) under microwave heating (power: 180 W, 90 s × 3). The resultant Gd3+-bacterium conjugates possessed sufficient magnetism, resulting in magnetic aggregations by an external magnet (∼4,000 Gauss). For ease of magnetic isolation, the sample containing Gd3+-bacterium complexes was stirred by a small magnet. After 1 h, the magnet attached with precipitates, i.e., Gd3+-bacterium conjugates, was readily removed using a pair of tweezers. The bacteria in the resultant conjugates were characterized by matrix-assisted laser desorption/ionization mass spectrometry. The limits of detection of the current approach toward E. coli O157:H7, S. aureus, and A. baumannii in complex samples were ∼104-105 cells mL-1.

Gold Nanobones Enhanced Ultrasensitive Surface-Enhanced Raman Scattering Aptasensor for Detecting Escherichia coli O157:H7.

Sensitive, robust, and highly specific detection of Escherichia coli O157:H7, one of the most hazardous foodborne pathogens and the cause of numerous diseases, is needed to ensure public health. Herein, a one-pot step method is reported for the preparation of multifunctional gold nanobones (NBs) (GNRApt-1+RhB) from gold nanorods (GNRs) comediated by an aptamer (Apt-1) and the signal molecule rhodamine B (RhB) for surface-enhanced Raman scattering detection of E. coli O157:H7. The characterized result showed that Apt-1 and RhB were embedded in the gold NBs, and then, this combination exhibited good recognition, excellent stability, and significant Raman signal intensity enhancement. The Raman enhancement derived from a strong electromagnetic field distribution with the locations at the apex of both ends of the GNRApt-1+RhB and the signal stability was because of the firm embedment of Apt-1 (poly A20 + E. coli O157:H7 aptamers) and RhB on the surface of the GNRApt-1+RhB. Optimization experiments established that surface-enhanced Raman-scattered RhB absorption at 1350 cm-1 had a strong linear relationship (y = 180.30x - 61.49; R2 = 0.9982) with E. coli O157:H7 concentration over the range of 10-10,000 cfu/mL with a limit of detection of 3 cfu/mL. This novel aptasensor sensitively detects E. coli O157:H7 and has great promise for food pathogenic bacteria detection.

A capacitive DNA sensor for sensitive detection of Escherichia coli O157:H7 in potable water based on the z3276 genetic marker: fabrication and analytical performance.

We report a label-free biosensor for the detection of Escherichia coli O157:H7 ATCC 43895 in potable water using a newly designed DNA sensing probe targeting the z3276 genetic marker. The surface of indium tin oxide (ITO) was functionalized with the novel sensing probe by covalent coupling using APTES as a crosslinker to fabricate the DNA sensor (dubbed ZEC [z[combining low line]3276 gene of E[combining low line]. c[combining low line]oli O157:H7 ATCC 43895]). The electrochemical characterization of the fabricated ZEC sensor was performed using cyclic voltammetry and electrochemical impedance spectroscopy. Atomic force microscopy and scanning electron microscopy revealed significant changes in the surface topographies of the fabricated ZEC sensor chip. Equivalent circuit analyses suggested the capacitive nature of the ZEC sensor chip, which demonstrated a declining trend of the capacitance value from 1.568 µF (Bare ITO) to 1.221 µF (after DNA hybridization). Non-faradaic sensing measurements revealed systematically declining capacitance values upon DNA hybridization, with a 10 min response time at 10 Hz frequency and 10 mV applied potential. The ZEC sensor chip exhibited linearity in the range of 0.5 to 25 pg per 10 mL for E. coli O157:H7, with ubiquitous cross-validation of each DNA concentration using quantitative PCR prior to the analyses of real water samples. The limit of detection (LOD) at 95% confidence estimated by logistic regression was 0.1 pg DNA per 10 mL of E. coli O157:H7 (equivalent to 13.67 CFU per 10 mL) with a p-value of 0.0237. Consequently, the obtained results demonstrate the possible application of the developed ZEC sensor chip for E. coli O157:H7 detection in real water samples.

Physiological and proteomic response of Escherichia coli O157:H7 to a bioprotective lactic acid bacterium in a meat environment.

The enterohemorrhagic Escherichia (E.) coli (EHEC) is a pathogen of great concern for public health and the meat industry all over the world. The high economic losses in meat industry and the high costs of the illness highlight the necessity of additional efforts to control this pathogen. Previous studies have demonstrated the inhibitory activity of Enterococcus mundtii CRL35 towards EHEC, showing a specific proteomic response during the co-culture. In the present work, additional studies of the EHEC-Ent. mundtii interaction were carried out: i) differential protein expression of E. coli O157:H7 NCTC12900 growing in co-culture with Ent. mundtii in a meat environment, ii) the reciprocal influence between these two microorganisms in the adhesion to extracellular matrix (ECM) proteins and iii) the possible induction of the phage W933, coding for Shiga toxin (Stx1), by Ent. mundtii CRL35. Proteomic analysis showed a significant repression of a number of E. coli NCTC12900 proteins in co-culture respect to its single culture, these mostly related to the metabolism and transport of amino acids and nucleotides. On the other hand, statistically significant overexpression of EHEC proteins involved in stress, energy production, amino acid metabolism and transcription was observed at 30 h respect to 6 h when EHEC grew in co-culture. Data are available via ProteomeXchange with identifier PXD014588. Besides, EHEC showed a decreased adhesion capacity to ECM proteins in the presence of the bioprotective strain. Finally, Ent. mundtii CRL35 did not induce the lytic cycle of W933 bacteriophage, thus indicating its potential safe use for eliminating this pathogen. Overall, this study expands the knowledge of EHEC- Ent. mundtii CRL35 interaction in a meat environment, which will certainly contribute to find out effective biological strategies to eliminate this pathogen.

Aptamer-based SERS biosensor for whole cell analytical detection of E. coli O157:H7.

Infectious outbreaks caused by foodborne pathogens such as E. coli O157:H7 are still imposing a heavy burden for global food safety, causing acute illnesses and significant industrial impact worldwide. Despite the growth of biosensors as a research field, continuous innovation on detection strategies, novel materials and enhanced limits of detection, most of the platforms developed at the laboratory scale never will get to meet the market. The use of aptamers as capture biomolecules has been proposed as a promising alternative to overcome the harsh environmental conditions of industrial manufacturing processes, and to enhance the performance under real, complex, conditions. In this work, we present the feasibility of using aptameric DNA sequences, covalently conjugated to 4-aminothiophenol-gold nanoparticle complexes for the sensitive and highly specific detection of E. coli O157:H7 via surface enhanced Raman spectroscopy (SERS) analysis. Low concentrations of E. coli O157:H7 were detected and quantified within 20 min in both pure culture (∼101 CFU mL-1) and ground beef samples (∼102 CFU mL-1). The SERS intensity response showed a strong negative linear correlation (r2 = 0.995) with increasing concentrations of E. coli O157:H7 (ranging from 102 to 106 CFU mL-1). High specificity was achieved at genus (L. monocytogenes, S. aureus S. typhimurium) species (E. coli B1201) and serotype (E. coli O55:H7) level, demonstrating with 95% of confidence that the interferent microorganisms tested generated a Raman signal response not significantly different from the background (p = 0.786). This work evaluates the incorporation of aptameric DNA sequences as bio capture molecules exclusively. The successful performance presented using non-modified citrate reduced GNPs, is promising for potential low-cost, high-throughput applications. The findings might be applied simultaneously to the detection of a wide variety of foodborne pathogens in a multiplexed fashion employing unique Raman probes and strain-specific aptamer sequences.

Evaluation of structural changes and intracellular substance leakage of Escherichia coli O157:H7 induced by ohmic heating.

AIMS: To investigate the effects of ohmic heating (OH) and water bath heating (WB) on the membrane permeability, membrane structure, intracellular organization and leakage of intracellular substances of Escherichia coli O157:H7 at the same inactivation level and at a heating temperature of 72°C. METHODS AND RESULTS: Flow cytometry analysis indicated that membrane permeability of E. coli O157:H7 by OH was comparable to WB at 72°C. Scanning electron microscopy analysis showed that the OH-treated E. coli O157:H7 had greater morphological changes than those of WB-treated ones both at the same inactivation level and the same heating temperature. Transmission electron microscopy analysis showed that both OH and WB caused severe damage on the intracellular organization of E. coli O157:H7 at 72°C. Moreover, OH-treated E. coli O157:H7 had more leakage of intracellular substances than those treated with WB due to the electroporation caused by OH. CONCLUSION: OH presents considerable potential in inactivation of E. coli O157:H7, especially OH at 10 V cm-1 with a much shorter heating time. SIGNIFICANCE AND IMPACT OF THE STUDY: The nonthermal effect of OH had a greater effect on the cell membrane of E. coli O157:H7, resulting in more pores and more leakage of intracellular substances out of the E. coli O157:H7 cells. These results could promote the application of OH in food processing.

Nucleic acid lateral flow assays using a conjugate of a DNA binding protein and carbon nanoparticles.

Nucleic acid lateral flow assays (NALFA) are often performed with gold nanoparticles. These are typically associated with ligand-labeled PCR amplicons via affinity interactions of adsorbed/conjugated proteins. Otherwise, they are conjugated to specific ssDNA sequences that hybridize to the target sequence. To avoid the need to generate ssDNA and to reduce the costs associated with primer labeling and antibody use, NALFA assays were developed that allow the direct detection of PCR amplicons using conjugates of a DNA binding protein with carbon nanoparticles (CNPs). The target gene encoding 16S ribosomal RNA of Escherichia coli was amplified by PCR using a single fluorophore-labeled forward primer and a reverse primer extended with the binding sequence of the bacteriophage lambda Cro repressor protein. Three different detection approaches were evaluated: (a) scCro/CNPs conjugate (black color), (b) HRP-scCro enzyme conjugate (red color), and (c) HRP-scCro/CNPs conjugate for dual color development. The limits of detection were between 6.9 and 10.4 ng of PCR product for all three approaches. These correspond to 3.0 to 4.5 × 103 CFU·mL-1. The single-step scCro/CNP approach proved to be the fastest one to perform and gave no false-positive signals. It also showed a broad dynamic range even though the signal intensities were lower compared to the enzyme-amplified tests. However, the latter ones produced some background signal. In our perception, the application of scCro in lateral flow assays to bind dsDNA appears to be an excellent alternative to the use of small tags that have to be chemically linked to synthetic primers. Finally, the approach is generic because any primer sequence can be extended with the specific scCro binding sequence. Graphical abstract Schematic presentation of the lateral flow-based fluorometric detection of DNA amplicons using conjugates of scCro DNA binding protein with (A) carbon nanoparticles, (B) HRP and (C) HRP and carbon nanoparticles.

Disposable syringe-based visual immunotest for pathogenic bacteria based on the catalase mimicking activity of platinum nanoparticle-concanavalin A hybrid nanoflowers.

Disposable syringes were used in a novel point-of-care visual test for detecting pathogenic bacteria (Escherichia coli O157:H7 and Salmonella typhimurium). Hybrid nanoflowers composed of platinum nanoparticles and concanavalin A (Pt-nanoflowers) were prepared through a one-pot reaction and were found to be viable catalase mimics. They catalyze the decomposition of hydrogen peroxide (H2O2) to generate O2. When used as labels in immunoassays, they integrate both the functions of biological recognition and signal amplification. The disposable syringe pressure readout was combined with Pt-nanoflower signal conversion and successfully applied to a visual bacteria detection scheme. Both Escherichia coli O157:H7 and Salmonella typhimurium can be quantified with detection limits of as low as 15 and 7 CFU·mL-1, respectively. Graphical abstract One-pot synthetic platinum nanoparticle (PtNP)-concanavalin A hybrid nanoflowers (Pt-nanoflowers), have been used as ideal signal labels for immunoassays and integrating both essential functions of biological recognition and signal amplification. Disposable syringes were used as a readout to detect pathogenic bacteria.