Detection Of Single Bacteria Research Articles

Label-free detection and identification of single bacteria via terahertz near-field imaging.

In recent years, terahertz (THz) imaging has attracted much attention because of its ability to obtain physical and chemical information in a label-free, noninvasive and nonionizing manner. However, the low spatial resolution of traditional THz imaging systems and the weak dielectric response of biological samples hinder the application of this technology in the biomedical field. In this paper, we report a new THz near-field imaging method for a single bacteria, through the coupling effect of nanoscale radius of probe and platinum gold substrate, which greatly enhances THz near-field signal of biological samples. A THz super-resolution image of bacteria has been successfully obtained by strictly controlling the relevant test parameters such as tip parameters and driving amplitude. By analyzing and processing the THz spectral image, the morphology and inner structure of bacteria have been observed. The method has been used to detect and identify Escherichia coli represented by Gram-negative bacteria and Staphylococcus aureus represented by Gram-positive bacteria. This application provides a new label-free, noninvasive and nonionizing testing protocol for the detection of single bacteria.

Single bacteria detection by droplet DNAzyme-coupled rolling circle amplification.

We described a new system termed droplet DNAzyme-coupled rolling circle amplification (dDRCA) that can selectively detect bacteria from clinical urine samples with single-cell sensitivity within 1.5 h compared with the several hours needed for traditionally used culture-based methods.

Trypsin-Amplified Aerolysin Nanopore Amplified Sandwich Assay for Attomolar Nucleic Acid and Single Bacteria Detection.

Nanopore technology is promising for the next-generation of nucleic acid-based diagnosis. However, sequence reservation could still be hardly achieved in low-concentration. Herein, we propose a trypsin-activated catalysis reaction for amplified detection, which substantially improves the sensitivity of nanopore technique. The proposed trypsin-amplified nanopore amplified sandwich assay (tNASA) could contribute to a sensitivity approximately 100 000 times higher based on nucleic acid probe design. Remarkably, tNASA is capable of attomolar nucleic acid and single cell detection by using a miniaturized current amplifier without alignment algorithm. Also it allows 10 pathogenic species in serum to be accurately and robustly profiled, thus be utilized for the diagnosis of infectious diseases. tNASA may evolve the construction of nanopore techniques for nucleic acid detection and would facilitate its translation for pocket diagnosis and precision medicine.

Simultaneous Sensing of Seven Pathogenic Bacteria by Guanidine-Functionalized Upconversion Fluorescent Nanoparticles.

The method capable of simultaneously detecting multiple target bacterial pathogens is necessary and of great interest. In this research, we demonstrated our initial effort to simultaneously detect seven common foodborne bacteria by developing a straightforward upconversion fluorescence sensing approach. The fluorescent nanosensor was constructed from a designed guanidine-functionalized upconversion fluorescent nanoparticles (UCNPs@GDN), tannic acid, and hydrogen peroxide (HP) and could quantify pathogenic bacteria in a nonspecific manner because the luminescence of the upconversion fluorescent nanoparticle was effectively strengthened in the presence of bacteria. When the developed nanosensor was applied to quantify multiple bacteria including Escherichia coli, Salmonella, Cronobacter sakazakii, Shigella flexneri, Vibrio parahaemolyticus, Staphylococcus aureus, and Listeria monocytogenes, a linear range of 103 to 108 cfu mL–1 and a detection limit of 1.30 × 102 cfu mL–1 have been obtained for the seven model mixture bacteria. In addition, the similar linear range and detection limit were also obtained for the detection of single bacteria. The present approach also exhibited acceptable recovery values ranging from 70.0 to 118.2% for bacteria in real samples (water, milk, and beef). All these results suggested that the guanidine-functionalized upconversion fluorescent nanosensor could be considered as a promising candidate for the rapid detection and surveillance of microbial pollutants in food and water.

Gain-Assisted Transition Metal Ternary Nitrides (Ti1−xZrxN) Core–Shell Based Sensing of Waterborne Bacteria in Drinking Water

We propose gain-assisted Ti1 − xZrxN-based multilayered core–shell nanoparticles (MCSNPs) as refractive index (RI) sensor for real-time and label-free detection of waterborne bacteria. RI sensor designs optimized for diseases caused by pathogen Escherichia coli (E. coli), Serratia marcescens (S. marcescens), and Mierococcus lysodeikticus (M. lysodeikticus) are presented. Mie theory–based analysis shows that the precise incorporation of optical gain can compensate plasmonic losses at resonant wavelength and enhance sensor’s figure of merit (FOM) for bacteria detection up to ~ 106–107. The use of Ti1 − xZrxN ternary alloy as plasmonic material is advantageous as (I) Ti1 − xZrxN offers an alternative to the conventional plasmonic materials (e.g., Au, Ag, Cu, etc.); (II) specific Ti/Zr fractions in ternary Ti1 − xZrxN reduces the critical gain requirement compared to binary TiN; and (III) spectrum can be fine-tuned by controlling Ti/Zr fraction. The present work can led to the development of ultrasensitive plasmonic sensors capable of single bacteria detection as well as level of concentration of bacteria in water.

Asymmetric Membrane for Digital Detection of Single Bacteria in Milliliters of Complex Water Samples.

In this work, we introduce an asymmetric membrane as a simple and robust nanofluidic platform for digital detection of single pathogenic bacteria directly in 10 mL of unprocessed environmental water samples. The asymmetric membrane, consisting of uniform micropores on one side and a high density of vertically aligned nanochannels on the other side, was prepared within 1 min by a facile method. The single membrane covers all the processing steps from sample concentration, purification, and partition to final digital loop-mediated isothermal amplification (LAMP). By simple filtration, bacteria were enriched and partitioned inside the micropores, while inhibitors typically found in the environmental samples (i.e., proteins, heavy metals, and organics) were washed away through the nanochannels. Meanwhile, large particles, indigenous plankton, and positively charged pollutants in the samples were excluded by using a sacrificial membrane stacked on top. After initial filtration, modified LAMP reagents, including NaF and lysozyme, were loaded onto the membrane. Each pore in the asymmetric membrane functioned as an individual nanoreactor for selective, rapid, and efficient isothermal amplification of single bacteria, generating a bright fluorescence for direct counting. Even though high levels of inhibitors were present, absolute quantification of Escherichia coli and Salmonella directly in an unprocessed environmental sample (seawater and pond water) was achieved within 1 h, with sensitivity down to single cell and a dynamic range of 0.3–10000 cells/mL. The simple and low-cost analysis platform described herein has an enormous potential for the detection of pathogens, exosomes, stem cells, and viruses as well as single-cell heterogeneity analysis in environmental, food, and clinical research.

Simple and Precise Counting of Viable Bacteria by Resazurin-Amplified Picoarray Detection.

Simple, fast, and precise counting of viable bacteria is fundamental to a variety of microbiological applications such as food quality monitoring and clinical diagnosis. To this end, agar plating, microscopy, and emerging microfluidic devices for single bacteria detection have provided useful means for counting viable bacteria, but they also have their limitations ranging from complexity, time, and inaccuracy. We present herein our new method RAPiD (Resazurin-Amplified Picoarray Detection) for addressing this important problem. In RAPiD, we employ vacuum-assisted sample loading and oil-driven sample digitization to stochastically confine single bacteria in Picoarray, a microfluidic device with picoliter-sized isolation chambers (picochambers), in <30 s with only a few minutes of hands-on time. We add AlamarBlue, a resazurin-based fluorescent dye for bacterial growth, in our assay to accelerate the detection of "microcolonies" proliferated from single bacteria within picochambers. Detecting fluorescence in picochambers as an amplified surrogate for bacterial cells allows us to count hundreds of microcolonies with a single image taken via wide-field fluorescence microscopy. We have also expanded our method to practically test multiple titrations from a single bacterial sample in parallel. Using this expanded "multi-RAPiD" strategy, we can quantify viable cells in E. coli and S. aureus samples with precision in ∼3 h, illustrating RAPiD as a promising new method for counting viable bacteria for microbiological applications.

Blocking electrochemical collisions of single E. coli and B. subtilis bacteria at ultramicroelectrodes elucidated using simultaneous fluorescence microscopy

The adsorption events and movements of individual E. coli and B. subtilis bacteria were detected in real time using ultramicroelectrodes(UMEs). Electrochemical detection of single bacteria was enabled by their inhibition of diffusive flux of a redox species when near the UME. With the UME biased for steady state oxidation of the redox species, each bacteria–electrode collision event at the UME resulted in a transient in the oxidation current. The two bacteria species show differences in their collision transient shapes, with E. coli showing “step” shaped transients and B. subtilis showing predominantly “blip” (spike) shaped transients. Simultaneous fluorescence microscopy of the UME surface during bacteria detection experiments allowed each current transient in the electrochemical data to be correlated with a specific collision event of fluorescently labeled bacteria. It was established that step-shaped transients observed for E. coli were due to adsorption of bacteria to the UME surface, while blip-shaped transients observed for B. subtilis were due to short-lived bounce events of the bacteria. The frequency of recorded transients varied linearly over bacteria concentrations of 1–10 fM for E. coli and 15–60 fM for B. subtilis, establishing the blocking collision method as a route for in situ determinations of bacteria concentration. This work demonstrates for the first time differences in blocking collision transient shapes between two bacterial analytes, and suggests applications of the method for selective electrochemical bacteria sensing.

Capacitive detection of single bacterium from drinking water with a detailed investigation of electrical flow cytometry

Pathogenic contamination of drinking water is critical in regard to human health. In this study, we investigated the electrical detection of single bacterium from drinking water. A microfluidics chip consisting of polydimethylsiloxane (PDMS) microchannel and gold microelectrodes was fabricated with conventional microfabrication techniques. Electrical characterizations were done with an LCR meter and the measurements were in good agreement with simulation results. The impact of channel and electrode dimensions was studied for the different type and size of particles, using both experimental and simulation techniques. In addition, the effect of excitation signal frequency and solution conductivity was analyzed employing both simulation and experimental methods. Finally, capacitive detection of a single Escherichia coli (E. coli) from drinking water was successfully carried out under optimum parameters and design geometries.

Culture-Free Detection of Crop Pathogens at the Single-Cell Level by Micro-Raman Spectroscopy.

The rapid and sensitive identification of invasive plant pathogens has important applications in biotechnology, plant quarantine, and food security. Current methods are far too time‐consuming and need a pre‐enrichment period ranging from hours to days. Here, a micro‐Raman spectroscopy‐based bioassay for culture‐free pathogen quarantine inspection at the single cell level within 40 min is presented. The application of this approach can readily and specifically detect plant pathogens Burkholderia gladioli pv. alliicola and Erwinia chrysanthemi that are closely related pathogenically. Furthermore, the single‐bacterium detection was able to discriminate them from a reference Raman spectral library including multiple quarantine‐relevant pathogens with broad host ranges and an array of pathogenic variants. To show the usefulness of this assay, Burkholderia gladioli pv. alliicola and Erwinia chrysanthemi are detected at single‐bacterium level in plant tissue lesions without pre‐enrichment. The results are confirmed by the plate‐counting method and a genetic molecular approach, which display comparable recognition ratios to the Raman spectroscopy‐based bioassay. The results represent a critical step toward the use of micro‐Raman spectroscopy in rapid and culture‐free discrimination of quarantine relevant plant pathogens.

Highly Sensitive Bacteriophage-Based Detection of Brucella abortus in Mixed Culture and Spiked Blood.

For decades, bacteriophages (phages) have been used for Brucella species identification in the diagnosis and epidemiology of brucellosis. Traditional Brucella phage typing is a multi-day procedure including the isolation of a pure culture, a step that can take up to three weeks. In this study, we focused on the use of brucellaphages for sensitive detection of the pathogen in clinical and other complex samples, and developed an indirect method of Brucella detection using real-time quantitative PCR monitoring of brucellaphage DNA amplification via replication on live Brucella cells. This assay allowed the detection of single bacteria (down to 1 colony-forming unit per milliliter) within 72 h without DNA extraction and purification steps. The technique was equally efficient with Brucella abortus pure culture and with mixed cultures of B. abortus and α-proteobacterial near neighbors that can be misidentified as Brucella spp., Ochrobactrum anthropi and Afipia felis. The addition of a simple short sample preparation step enabled the indirect phage-based detection of B. abortus in spiked blood, with the same high sensitivity. This indirect phage-based detection assay enables the rapid and sensitive detection of live B. abortus in mixed cultures and in blood samples, and can potentially be applied for detection in other clinical samples and other complex sample types.

Rapid electrochemical detection of single influenza viruses tagged with silver nanoparticles.

Using a state of the art nano-electrochemical technique, we show that a single virus 'tagged' with silver nanoparticles can be rapidly detected in real time at the single virus level. A solution containing a low concentration of influenza virus is exposed to silver nanoparticles which are adsorbed onto the virus surface, as revealed by UV-Vis spectroscopy and transmission electron microscopy. With sufficient potential applied to a carbon electrode introduced into the solution, current spikes are observed which correspond to the oxidation of the nanoparticles decorating the virus. The frequency of the current spikes and their magnitude are linearly proportional to the virus concentration and to the surface coverage of the nanoparticles, respectively. Differences observed from single bacterium detection are discussed and a comparison with existing detection methods is made, with emphasis on the favourability of the proposed technique towards the realization of point of care test devices.

Detection of single bacteria – causative agents of meningitis using raman microscopy

Early diagnostics of meningitis is a very topical problem as it is a fulminant disease with a high level of mortality. The progress of this disease is, as a rule, accompanied by the appearance of bacteria in the cerebrospinal fluid (CSF) composition. The examination of the CSF is well known to be the only reliable approach to the identification of meningitis. However, the traditional biochemical analyses are time consuming and not always reliable, simple, and inexpensive, whereas the optical methods are poorly developed. This work is devoted to the study of Raman spectra of several bacterial cultures which are mainly present during meningitis. Raman microscopy is a prompt and noninvasive technique capable of providing reliable information about molecular-level alterations of biological objects at their minimal quantity and size. It was shown that there are characteristic lines in Raman spectra which can be the reliable markers for determination of bacterial form of meningitis at a level of a single bacterium.

Rapid detection of single bacteria in unprocessed blood using Integrated Comprehensive Droplet Digital Detection.

Blood stream infection or sepsis is a major health problem worldwide, with extremely high mortality, which is partly due to the inability to rapidly detect and identify bacteria in the early stages of infection. Here we present a new technology termed ‘Integrated Comprehensive Droplet Digital Detection’ (IC 3D) that can selectively detect bacteria directly from milliliters of diluted blood at single-cell sensitivity in a one-step, culture- and amplification-free process within 1.5–4 h. The IC 3D integrates real-time, DNAzyme-based sensors, droplet microencapsulation and a high-throughput 3D particle counter system. Using Escherichia coli as a target, we demonstrate that the IC 3D can provide absolute quantification of both stock and clinical isolates of E. coli in spiked blood within a broad range of extremely low concentration from 1 to 10,000 bacteria per ml with exceptional robustness and limit of detection in the single digit regime.

Resonance-enhanced microfluidic impedance cytometer for detection of single bacteria.

This paper reports on a novel impedance-based cytometer, which can detect and characterize sub-micrometer particles and cells passing through a microfluidic channel. The cytometer incorporates a resonator, which is constructed by means of a discrete inductor in series with the measurement electrodes in the microfluidic channel. The use of a resonator increases the sensitivity of the system in comparison to state-of-the-art devices. We demonstrate the functionality and sensitivity of the cytometer by discriminating E. coli and B. subtilis from beads of similar sizes by means of the resonance-enhanced phase shift of the current through the microfluidic channel. The phase shift can be correlated to size and dielectric properties of the measured objects.

Real-time Measurement of Single Bacterium's Refractive Index Using Optofluidic Immersion Refractometry

This paper presents a biophysical method to characterize single bacterium in water by using an on-chip optofluidic immersion refractometer. Water safety is a major factor in the well-being of people, but the presence of bacteria such as Escherichia coli (E. coli), bacillus subtilis, Shigella flexneri and vibrio cholera in drinking water can lead to infectious diseases such as typhoid fever. Hence, it is crucial to detect and identify bacteria to prevent bacterial outbreaks. In this paper, an optofluidic immersion refractometer is developed to measure three biophysical parameters, i.e. size, shape and refractive index. The refractive index of a single bacterium is measured in high sensitivity of 0.005 RIU. This system is an innovative method to allow on-site real-time detection of single bacterium in water. It significantly reduces the amount of detection time and do not require trained personnel or additional chemical and biological reagents.

Bacteria detection utilizing electrical conductivity

Real-time and specific detection of single bacterium remains a fundamental challenge and draws very much attention. Using test patterns composed of interdigitated Au-electrode arrays modified with antibody, the specific and quantitative detection of the electrical conductivity of a single Escherichia coli ( E. coli, JM109) has been carried out in this work. The key is to ensure low background current of the antibody-modified test patterns before bacteria detection (<0.7 pA in this case) and minimize the residual moisture or hydration after E. coli immobilization, such as via the use of 1-min bake at 50 °C prior to electrical measurement. This method holds great potential for future application in the real-time, specific, and quantitative bacterium detection down to a single bacterium cell.

A compact CMOS biochip immunosensor towards the detection of a single bacteria

Recent use of biological warfare (BW) agents has led to a growing interest in the rapid and sensitive detection of pathogens. Therefore, the development of field-usable detection devices for sensitive and selective detection of BW agents is an important issue. In this work, we report a portable biochip system based on complementary metal oxide semiconductor (CMOS) technology that has great potential as a device for single-bacteria detection. The possibility of single-bacteria detection is reported using an immunoassay coupled to laser-induced fluorescence (LIF) detection. Bacillus globigii spores, which are a surrogate species for B. anthracis spores, were used as the test sample. Enzymatic amplification following immunocomplex formation allowed remarkably sensitive detection of B. globigii spores, and could preclude a complicated optical and instrumental system usually required for high-sensitive detection. Atomic force microscopy (AFM) was employed to investigate whether B. globigii spores detected in the portable biochip system exist in single-cell or multicellular form. It was found that B. globigii spores mostly exist in multicellular form with a small minority of single-cell form. The results showed that the portable biochip system has great potential as a device for single-particle or possibly even single-organism detection.

Bionanotechnology Based on Silica Nanoparticles

AbstractFor Abstract see ChemInform Abstract in Full Text.

Bionanotechnology based on silica nanoparticles.

We have developed uniform core/shell nanoparticles, consisting of a silica layer coating and pigments or magnetite core, using a water-in-oil microemulsion method. The nanoparticles are highly luminescent and photostable with the size ranging from 5 nm to 400 nm. Bioconjugation of these silica nanoparticles adds unique biofunctions with various molecules such as enzymes, antibodies, and DNA molecules. Significant advantages have been shown in using bioconjugated nanoparticles for biosensing and bioimaging, such as cell staining, DNA detection and separation, rapid single bacterium detection, and biotechnological application in DNA protection.