Detection Of Whole Cells Research Articles

Allosteric probe-initiated double rolling circle amplification in tandem for sensitive whole-cell detection of E. coli O157:H7

The development of facile, reliable nucleic acid amplification assays for pathogenic bacteria is paramount but still poses a challenge. Here we present a molecular amplification system that integrates the whole-cell recognition of allosteric probes with cascaded rolling circle amplification. Allosteric probe-initiated double rolling circle amplification (AID-RCA) assay involves a three-step process that utilizes allosteric probes, dumbbell templates, and circular templates to recognize, amplify, and detect target bacteria, E. coli O157:H7. The allosteric probes are designed to recognize E. coli O157:H7 without bacterial isolation and nucleic acid extraction. Subsequently, the two-stage in-tandem amplification, that is, dumbbell template-based exponential-rolling circle amplification (DT-RCA) and circular template-based linear-rolling circle amplification (CT-RCA), is initiated and yields powerful fluorescence signals using molecular beacon as a signal probe. The symmetric structure of the dumbbell template endows the AID-RCA assay with good specificity. Cascaded signal amplification boosts its sensitivity. The AID-RCA assay demonstrated high sensitivity in the quantitative detection of E. coli O157:H7(limit of detection 1.6 CFU·mL−1). Compared to real-time PCR, the AID-RCA system exhibited higher sensitivity for low concentrations of E. coli O157:H7. Furthermore, the AID-RCA system can selectively differentiate E. coli O157:H7 from other pathogenic bacteria in complex samples such as drinking water and milk. In a blind validation study using milk samples, the AID-RCA system effectively distinguished between contaminated and pasteurized milk samples. Our method is expected to hold great potential for developing precise, reliable, and efficient analytical platforms to combat pathogenic bacterial pollution and safeguard human health.

A novel method for the whole-cell detection of environmental microorganisms using hemin and tyramide signal amplification (Hemin-TSA) with a desired fluorescent dye

A method called hemin-tyramide signal amplification (Hemin-TSA) was developed for visualization of environmental microorganisms using hemin and tyramide signal amplification. In Hemin-TSA, hemin, which has peroxidase activity, is bound to microbial cells, and a desired fluorescent dye is deposited on the microbial cells by a hemin-catalyzed TSA reaction. The protocol was initially optimized in terms of hemin concentration, hemin binding time and repeated reaction times of TSA. Hemin-TSA showed a comparative or improved signal-to-noise ratio compared to DAPI staining. The shapes of fluorescent signals obtained from microbial cells were almost morphologically identical to those observed in phase contrast microscopy. Hemin-TSA staining provided more accurate cell counts than DAPI staining, especially for actively growing cells, for which two or three spotty DAPI signals were obtained from a single cell. In addition, microbial cells that were not detected by DAPI staining were detected by Hemin-TSA with fluorescein, which enabled us to avoid high non-specific fluorescence under UV excitation. The method developed in this study allows us to visually detect microbial cells in various environments with the characteristics of better cell morphological identification, improved enumeration accuracy and selectivity of fluorescent dyes.

Harnessing split fluorescent proteins in modular protein logic for advanced whole-cell detection

Whole-cell biosensors have demonstrated promising capabilities in detecting target molecules. However, their limited selectivity and precision can be attributed to the broad substrate tolerance of natural proteins. In this study, we aim to enhance the performance of whole-cell biosensors by incorporating of logic AND gates. Specifically, we utilize the HrpR/S system, a widely employed hetero-regulation module from Pseudomonas syringae in synthetic biology, to construct an orthogonal AND gate in Escherichia coli. To accomplish this, we compare the HrpR/S system with self-associating split fluorescent proteins using the Spy Tag/Spy Catcher system. Our objective is to selectively activate a reporter gene in the presence of both IPTG and Hg(II) ions. Through systematic genetic engineering and evaluation of various biological parts under diverse working conditions, our research demonstrates the utility of self-associating split fluorescent proteins in developing high-performance whole-cell biosensors. This approach offers advantages such as engineering simplicity, reduced basal activity, and improved selectivity. Furthermore, the comparison with the HrpR/S system serves as a valuable control model, providing insights into the relative advantages and limitations of each approach. These findings present a systematic and adaptable strategy to overcome the substrate tolerance challenge faced by whole-cell biosensors.

Carbon dots and graphene oxide based FRET immunosensor for sensitive detection of Helicobacter pylori

We report a novel Fluorescence Resonance Energy Transfer (FRET) immunosensor for sensitive detection of whole cell H. pylori, a causative organism for gastric carcinoma. Highly fluorescent and well-dispersed functionalized carbon dots (FCDs) were synthesized and chemically conjugated with anti-H. pylori antibody to fabricate a fluorescent probe (FCDs-Ab). The fluorescence of FCDs-Ab gets quenched upon interaction with graphene oxide (GO), whereas in presence of H. pylori, the interaction between the FCD-Ab and GO gets interrupted and gradual restoration of fluorescence was observed due to the specific affinity and binding of Ab towards H. pylori. The immunosensor was characterized at each step of fabrication. The assay exhibits a linear detection range of 5-107 cells mL−1 with limit of detection (LOD) of 10 cells mL−1 with high specificity and selectivity to target pathogen. The analytical performance of the developed immunosensor was evaluated with different spiked food samples and the substantial recovery validates its potential for risk assessment in food testing, thus ensuring general safety of public health.

Analytical methods for the characterization and diagnosis of infection with Pseudomonas aeruginosa: A critical review

The recent increase in outbreaks of pathogenic bacteria and antimicrobial resistance represent major public health problems. Being the leading cause of death in humans, the bacterial infections need to be accurately and quickly diagnosed. Hence, the development of fast, cost-effective, sensitive and specific strategies for the detection of the targeted bacterium is of utmost importance.This review presents a systematic, critical evaluation of the recent analytical methods for the characterization and diagnosis of infections caused by Pseudomonas aeruginosa. The clinical manifestations, incidence and treatment of the P. aeruginosa infection and the associated quorum sensing, biofilm formation and virulence factors are also discussed. An overview of a variety of analytical methods for the detection of P. aeruginosa is provided, including whole-cell detection (microbiological methods, biosensors), antigens, DNA, and relevant markers (quorum sensing molecules, virulence factors) detection. The latest trends in analytical methods, especially sensors, are the orientation towards portability and on-site detection. The efforts made so far to achieve these goals in the detection of P. aeruginosa and its markers are also presented and discussed in this review. The strengths and weaknesses of the current detection methods are evaluated, while exploring potential routes for further development.

Electrochemical-based ‘‘antibiotsensor’’ for the whole-cell detection of the vancomycin-susceptible bacteria

In this study, we aim to develop an antibiotic-based biosensor platform ‘Antibiotsensor’ for the specific detection of gram-positive bacteria using vancomycin modified Screen Printed Gold Electrodes (SPGEs). Through this pathway, vancomycin molecules were first functionalized with thiol groups and characterized with quadrupole time of flight (q-TOF) mass spectroscopy analysis. Immobilization of thiolated vancomycin molecules (HS-Van) onto SPGEs was carried out based on self-assembled monolayer (SAM) phenomenon. Electrochemical impedance spectroscopy (EIS) was employed to test the detection and showed a considerable change in impedance value upon the binding of HS-Van molecules onto the electrode surface. Atomic Force Microscopy analysis indicated that SPGE was successfully modified upon the treatment with HS-Van molecules based on the shift in surface roughness from 173 ± 2 nm to 301 ± 3 nm. Fourier Transform Infrared Spectroscopy (FTIR) spectroscopy proved the EIS and AFM results as well by showing characteristic peaks of immobilized HS-Van molecule. As a proof-of-concept, EIS-based susceptibility testing was performed using Escherichia coli, Staphylococcus aureus and Mycobacterium smegmatis bacteria to prove the specificity of obtained SPGE-Van. EIS data showed that the charge transfer resistance (Rct) values changed from 1.08, 1.18 to 26.5, respectively, indicating that vancomycin susceptible S. aureus was successfully attached onto SPGE-Van surface strongly, while vancomycin resistance E. coli and M. smegmatis did not show any significant attachment properties. In addition, different concentration (108-10 CFU/mL) of S. aureus was performed to investigate sensitivity of proposed sensor platform. Limit of detection and limit of quantitation was calculated as 101.58 and 104.81 CFU/mL, respectively. Scanning electron microscopy (SEM) analysis also confirmed that only S. aureus bacteria was attached to the surface in a dense monolayer distribution. We believe that the proposed approach is selective and sensitive towards the whole-cell detection of vancomycin-susceptible bacteria and can be modified for different purposes in the future.

Microfluidic integration for electrochemical biosensor applications

Electrochemical biosensors are particularly suitable for miniaturization and integration in microfluidic devices. Applications include the detection of whole cells, cell components, proteins, and small molecules to address tasks in the fields of diagnostics and food and environmental control. Microfluidic setups range from simple channels for sample transport to channels with integrated sensing electrodes to highly sophisticated platforms with additional elements for sample preparation. The design of the microfluidics depends on both the type of detection and on the application and sample material. This review summarizes recent work on electrochemical biosensors with integrated microfluidics with the focus on developments for real sample applications, particularly those including measurements with real sample media.

Highly sensitive and label-free digital detection of whole cell E. coli with Interferometric Reflectance Imaging

Bacterial infectious diseases are a major threat to human health. Timely and sensitive pathogenic bacteria detection is crucial in bacterial contaminations identification and preventing the spread of infectious diseases. Due to limitations of conventional bacteria detection techniques there have been concerted research efforts towards developing new biosensors. Biosensors offering label-free, whole bacteria detection are highly desirable over those relying on label-based or pathogenic molecular components detection. The major advantage is eliminating the additional time and cost required for labeling or extracting the desired bacterial components. Here, we demonstrate rapid, sensitive and label-free Escherichia coli (E. coli) detection utilizing interferometric reflectance imaging enhancement allowing visualizing individual pathogens captured on the surface. Enabled by our ability to count individual bacteria on a large sensor surface, we demonstrate an extrapolated limit of detection of 2.2 CFU/ml from experimental data in buffer solution with no sample preparation. To the best of our knowledge, this level of sensitivity for whole E. coli detection is unprecedented in label-free biosensing. The specificity of our biosensor is validated by comparing the response to target bacteria E. coli and non-target bacteria S. aureus, K. pneumonia and P. aeruginosa. The biosensor’s performance in tap water proves that its detection capability is unaffected by the sample complexity. Furthermore, our sensor platform provides high optical magnification imaging and thus validation of recorded detection events as the target bacteria based on morphological characterization. Therefore, our sensitive and label-free detection method offers new perspectives for direct bacterial detection in real matrices and clinical samples.

An Integrated Whole-Cell Detection Platform for Heavy Metal Ions

Heavy metal ions, such as Pb2+ and Cd2+, are main sources of water quality contaminants. Whole-cell detection has recently been actively researched to use genetically modified bacteria to sense the presence of heavy metal ions in water or soil. Compared to common cell-free methods, such as immunosensor and electrochemical sensor, whole-cell sensors need simple sample preparation and can continuously sense metal contaminants in the environment with the cell culture. Nonetheless, whole-cell sensors so far have demonstrated sensitivity too low for water quality monitoring. In this work, we developed an integrated platform based on whole-cell detection. Our genetically-modified E. coli bacteria has a good specificity on lead and cadmium. The platform integrates the light source, cultured cells and the detector for the first time. The integrated platform shows good sensitivity. Limits of detection for Pb2+ and Cd2+ are 518 ppb and 44.8 ppb respectively.

A Single-Layer PDMS Chamber for On-Chip Bacteria Culture.

On-chip cell culture devices have been actively developed for both mammalian cells and bacteria. Most designs are based on PDMS multi-layer microfluidic valves, which require complicated fabrication and operation. In this work, single-layer PDMS microfluidic valves are introduced in the design of an on-chip culture chamber for E. coli bacteria. To enable the constant flow of culturing medium, we have developed a (semi-)always-closed single-layer microfluidic valve. As a result, the growth chamber can culture bacteria over long duration. The device is applied for the whole-cell detection of heavy metal ions with genetically modified E. coli. The platform is tested with culturing period of 3 h. It is found to achieve a limit-of-detection (LoD) of 44.8 ppb for Cadmium ions.

Whole-Cell Detection of C-P Bonds in Bacteria.

Naturally produced molecules possessing a C-P bond, such as phosphonates and phosphinates, remain vastly underexplored. Although success stories like fosfomycin have reinvigorated small molecule phosphonate discovery efforts, bioinformatic analyses predict an enormous unexplored biological reservoir of C-P bond-containing molecules, including those attached to complex macromolecules. However, high polarity, a lack of chromophores, and complex macromolecular association impede phosphonate discovery and characterization. Here we detect widespread transcriptional activation of phosphonate biosynthetic machinery across diverse bacterial phyla and describe the use of solid-state nuclear magnetic resonance to detect C-P bonds in whole cells of representative Gram-negative and Gram-positive bacterial species. These results suggest that phosphonate tailoring is more prevalent than previously recognized and set the stage for elucidating the fascinating chemistry and biology of these modifications.

Application of Long-Range Surface Plasmon Resonance for ABO Blood Typing.

In this study, we demonstrate a long-range surface plasmon resonance (LR-SPR) biosensor for the detection of whole cell by captured antigens A and B on the surface of red blood cells (RBCs) as a model. The LR-SPR sensor chip consists of high-refractive index glass, a Cytop film layer, and a thin gold (Au) film, which makes the evanescent field intensity and the penetration depth longer than conventional SPR. Therefore, the LR-SPR biosensor has improved capability for detecting large analytes, such as RBCs. The antibodies specific to blood group A and group B (Anti-A and Anti-B) are covalently immobilized on a grafting self-assembled monolayer (SAM)/Au surface on the biosensor. For blood typing, RBC samples can be detected by the LR-SPR biosensor through a change in the refractive index. We determined that the results of blood typing using the LR-SPR biosensor are consistent with the results obtained from the agglutination test. We obtained the lowest detection limits of 1.58 × 105 cells/ml for RBC-A and 3.83 × 105 cells/ml for RBC-B, indicating that the LR-SPR chip has a higher sensitivity than conventional SPR biosensors (3.3 × 108 cells/ml). The surface of the biosensor can be efficiently regenerated using 20 mM NaOH. In summary, as the LR-SPR technique is sensitive and has a simple experimental setup, it can easily be applied for ABO blood group typing.

Whole-cell detection of live lactobacillus acidophilus on aptamer-decorated porous silicon biosensors.

This work describes the design of optical aptamer-based porous silicon (PSi) biosensors for the direct capture of Lactobacillus acidophilus. Aptamers are oligonucleotides (single-stranded DNA or RNA) that can bind their targets with high affinity and specificity, making them excellent recognition elements for biosensing applications. Herein, aptamer Hemag1P, which specifically targets the important probiotic L. acidophilus, was utilized for direct bacteria capture onto oxidized PSi Fabry-Pérot thin films. Monitoring changes in the reflectivity spectrum (using reflective interferometric Fourier transform spectroscopy) allows for bacteria detection in a label-free, simple and rapid manner. The performance of the biosensor was optimized by tuning the PSi nanostructure, its optical properties, as well as the immobilization density of the aptamer. We demonstrate the high selectivity and specificity of this simple "direct-capture" biosensing scheme and show its ability to distinguish between live and dead bacteria. The resulting biosensor presents a robust and rapid method for the specific detection of live L. acidophilus at concentrations relevant for probiotic products and as low as 10(6) cells per mL. Rapid monitoring of probiotic bacteria is crucial for quality, purity and safety control as the use of probiotics in functional foods and pharmaceuticals is becoming increasingly popular.

Detection of whole cells using reflectometric interference spectroscopy

The detection of clinically relevant bacteria as whole cells is subject of intense research. Besides established methods like ELISAs, even optical biosensors as surface plasmon resonance spectroscopy (SPR) have been proven to be applicable for such purposes. However, detection limit seems to be only at 105 cells mL−1, which is not sufficient for many applications. A possible reason for this limitation is that particles like bacteria could only immerse to a very restricted degree into the evanescent field. As an alternative to SPR, reflectometric interference spectroscopy (RIfS) can be used for whole cell detection. It is expected that this method can overcome the aforementioned limitation because the space for detection is only, theoretically, limited to the dimensions of the flow cell. Experiments were conducted using Legionella pneumophila as model organism. In this work, preliminary results are presented demonstrating that detection of whole cells using a RIfS‐device combined with a flow‐through system is generally possible. Legionella cells were either captured on the sensor surface by hydrophobic interaction or alternatively by specific antibodies. However, the results indicate that the detection limit is in the same range as observed for SPR.

Simple Whole-Cell Biodetection and Bioremediation of Heavy Metals Based on an Engineered Lead-Specific Operon

A lead-specific binding protein, PbrR, and promoter pbr from the lead resistance operon, pbr, of Cupriavidus metallidurans CH34 was incorporated into E. coli in conjunction with an engineered downstream RFP (red fluorescence protein), which allowed for highly sensitive and selective whole-cell detection of lead ions. The subsequent display of PbrR on the E. coli cell surface permitted selective adsorption of lead ions from solution containing various heavy metal ions. The surface-engineered E. coli bacteria effectively protected Arabidopsis thaliana seed germination from the toxicity of lead ions at high concentrations. Engineering the E. coli bacteria harboring these lead-specific elements from the pbr operon may potentially be a valuable general strategy for biodetection and bioremediation of toxic heavy metal ions in the environment.

Alternating Current Electrohydrodynamics Induced Nanoshearing and Fluid Micromixing for Specific Capture of Cancer Cells

We report a new tuneable alternating current (ac) electrohydrodynamics (ac-EHD) force referred to as “nanoshearing” which involves fluid flow generated within a few nanometers of an electrode surface. This force can be externally tuned via manipulating the applied ac-EHD field strength. The ability to manipulate ac-EHD induced forces and concomitant fluid micromixing can enhance fluid transport within the capture domain of the channel (e.g., transport of analytes and hence increase target–sensor interactions). This also provides a new capability to preferentially select strongly bound analytes over nonspecifically bound cells and molecules. To demonstrate the utility and versatility of nanoshearing phenomenon to specifically capture cancer cells, we present proof-of-concept data in lysed blood using two microfluidic devices containing a long array of asymmetric planar electrode pairs. Under the optimal experimental conditions, we achieved high capture efficiency (e.g., approximately 90%; %RSD=2, n=3) with a 10-fold reduction in nonspecific adsorption of non-target cells for the detection of whole cells expressing Human Epidermal Growth Factor Receptor 2 (HER2). We believe that our ac-EHD devices and the use of tuneable nanoshearing phenomenon may find relevance in a wide variety of biological and medical applications.

Approaches to the detection of whole cells using reflectometric interference spectroscopy

AbstractMethods for rapid and reliable detection of clinically relevant bacteria are of highest interest. For such purposes, many analytical methods, e.g. ELISA, have been developed. Also optical biosensors like reflectometric interference spectroscopy (RIfS) are suitable analytical platforms. Here, results are described to capture and detect Legionella pneumophila serogroup 1 on differently functionalized surfaces of RIfS glass chips. As a major problem, availability of suitable antibodies was found. Immobilization of capturing antibodies on the chip surface followed by trapping of L. pneumophila gave only insufficient results. For this approach, the chip surface was partially blocked with bovine serum albumin (BSA) in different ratios between BSA and the Legionella specific antibody in order to obtain a site‐directed immobilization. Further on, antibodies were immobilized by hydrophobic interaction followed by BSA treatment and exposition to L. pneumophila. However, also this approach did not allow sufficient capturing of bacteria cells. It can be assumed that binding forces between the antibody and bacteria are too low. Alternatively, L. pneumophila bacteria were directed immobilized on a hydrophobic chip surface. A sufficient sensor signal for quantification was obtained in a range between 8.25 × 106 and 8.25 × 108 bacteria cells/mL. This approach might open up the possibility for safe identification of L. pneumophila after subsequent addition of specific antibodies.

Engineering a gold-specific regulon for cell-based visual detection and recovery of gold

A gold-specific sensory protein GolS from Samonella gol regulon was incorporated into E. coli, which in conjunction with an engineered downstream red fluorescence protein allowed the highly sensitive and selective whole-cell detection of gold(III) ions by naked eyes. The putative gold-chaperone, GolB, in the gol regulon was next verified to be specific to gold(I) ions over other metal ions including copper(I). The subsequent display of GolB on E. coli cell surface permitted selective enrichment of gold ions from media containing various thiophilic metal ions. The cell surface-enriched gold(I) was further shown to be easily recovered and the gold-deprived bacteria were capable for re-usage. E. coli bacteria harboring these gold-specific elements from the gol regulon could be a valuable tool for visual detection and facile recycling of gold ions from environmental resources.

The trace analysis of microorganisms in real samples by combination of a filtration microcartridge and capillary isoelectric focusing

Trace analysis of microorganisms in real biological samples needs very sensitive methods for their detection. Most procedures for detecting and quantifying pathogens require a sample preparation step including concentrating microorganisms from large sample volumes with high and reproducible efficiency. Electromigration techniques have great potential to include the preconcentration, separation, and detection of whole cells and therefore they can rapidly indicate the presence of pathogens. The preconcentration and separation of microorganisms from real suspensions utilising a combination of filtration and capillary isoelectric focusing was developed and the possibility for its application to real samples was verified. For our experiments, spores of Monilinia species and of Penicillium expansum were selected as model bioparticles, as they cause major losses in agrosystems. The isoelectric points of the spores of M. laxa, M. fructigena, M. fruticola, and P. expansum were determined and the method was verified using real samples taken directly from infected apples. The coupling of a filtration cartridge with a separation capillary can improve the detection limit of isoelectric focusing with UV detection by at least 4 orders of magnitude. Spores of M. fructigena and of M. laxa in numbers of hundreds of particles per milliliter were detected on a visually noninfected apple surface which was cross-contaminated during handling and storage. The efficiency of preconcentration and a preliminary identification was verified by the phenotyping technique after cultivation of the spores sampled from the apple surface.

Nanomaterial Enabled Biosensors for Pathogen Monitoring - A Review

One promising, but currently underexplored, area for the future of drinking water pathogen monitoring stems from the development of nanomaterial-enabled detection strategies. The nanoscience literature contains numerous reports of nanoenabled biosensors; however, to date only a small percentage have focused on the detection of whole cells, in general, and waterborne pathogens, in particular. There are significant opportunities for the use of nanoenabled biosensors for environmental monitoring, and this review is intended to both illustrate the state of this field and to spur additional research in this area.