Label-free Detection Of Bacteria Research Articles

Optical Methods for Label-Free Detection of Bacteria

Pathogenic bacteria are the leading causes of food-borne and water-borne infections, and one of the most serious public threats. Traditional bacterial detection techniques, including plate culture, polymerase chain reaction, and enzyme-linked immunosorbent assay are time-consuming, while hindering precise therapy initiation. Thus, rapid detection of bacteria is of vital clinical importance in reducing the misuse of antibiotics. Among the most recently developed methods, the label-free optical approach is one of the most promising methods that is able to address this challenge due to its rapidity, simplicity, and relatively low-cost. This paper reviews optical methods such as surface-enhanced Raman scattering spectroscopy, surface plasmon resonance, and dark-field microscopic imaging techniques for the rapid detection of pathogenic bacteria in a label-free manner. The advantages and disadvantages of these label-free technologies for bacterial detection are summarized in order to promote their application for rapid bacterial detection in source-limited environments and for drug resistance assessments.

Fabrication of ZnO Nanoparticle-Based FET Device for Label-Free Bacteria Detection.

This paper reports fabrication and characterization of ZnO nanoparticle based Field Effect Transistor (FET) device and its application for simple, rapid and label-free bacteria detection. [Formula: see text] FET devices were fabricated by standard UV lithography technique on Si wafers. The fabricated devices consisted of ZnO nanoparticles as channel material. Characterization of such devices resulted in threshold voltage of -2 V and trans-conductance of [Formula: see text]. The interaction between ZnO nanoparticles and bacteria sample has been exploited in this study to utilize ZnO nanoparticle based FET device to successfully differentiate between gram positive and gram-negative bacteria. Gram negative bacteria sample resulted in higher output characteristics compared to that obtained with gram positive bacteria sample. This study reports sensitivity and Limit of Detection (LOD) of 9.48 nA/CFU/mL and 776 CFU/mL respectively for gram negative bacteria and 6.96 nA/CFU/mL and 665 CFU/mL for gram positive bacteria respectively.

Planar single and dual-resonant microwave biosensors for label-free bacteria detection

Label-free microwave resonator-type biosensors covered with polyclonal anti-Escherichia coli antibodies are presented. The sensors` construction provides high sensitivity on the bacteria detection, whereas the utilization of specific antibodies ensures selective bacteria binding. Two types of biosensors are studied to explore the benefits of microwave sensing: the first one, in which increased sensitivity on the bacteria detection is obtained due to the differential excitation of the resonating conductors, and the second one, in which a multi-resonant character increases the probability for the bacteria detection. The exemplary sensors were designed to operate in the frequency range of 4.4 – 4.8 GHz or 15 – 25 GHz and physically realized on a silicon wafer using standard microelectronic processor using the United Monolithic Semiconductor (UMS) PH25 process on a 100 µm thick GaAs substrate, respectively. The sensors were experimentally validated by measurements of various concentrations of Escherichia coli. Both types of sensors were compared to show their advantages and disadvantages in terms of future use for biomedical applications. Both biosensors were shown to enable bacteria detection at concentrations reaching as low as 103 CFU/ml. Interestingly, a monotonic response for bacteria concentration was observed for a multi-resonant sensor, which can be utilized for estimation of bacteria content using the calibration curve.

Sensitive and Label-free Detection of Bacteria in Osteomyelitis through Exo III-Assisted Cascade Signal Amplification.

Rapid and sensitive pathogenic bacterial identification and isolation from complicated clinical specimens are of great importance for the early diagnosis and prevention of osteomyelitis. Herein, we proposed a novel methicillin-resistant Staphylococcus aureus (MRSA) detection strategy through two specially designed streptavidin magnetic bead-based probes, including a capture probe and a report probe. In detail, the capture probe takes the responsibility to specially bind with the surface protein of MRSA and leads to the liberation of the promoter which could subsequently initiate report probe-based signal amplification. Afterward, the hybridization of the promoter probe with the report probe could then transform the protruding 3′ terminus of template DNA in the report probe into a blunt end. With the assistance of Exo III, the template could be digested to liberate the promoter to form a recycle and to liberate the biprobe to induce the following rolling circle amplification (RCA)-based signal amplification. Through the integration of the Exo III-assisted recycle and RCA-based signal amplification, the proposed method exhibited a favorable detection performance.

Optical fiber aptasensor for label-free bacteria detection in small volumes

Highly sensitive devices for fast bacteria detection are sought to be developed with the task of quantifying the worldwide problem of pathogenic bacteria and thus helping to take control over spreading bacterial infections. This work concerns a sensing solution based on microcavity in-line Mach-Zehnder interferometer (μIMZI) induced in an optical fiber. Such a device exhibits ultrahigh sensitivity to refractive index changes in the microcavity. We show that for well-adjusted chemical surface functionalization combined with highly specific low-molecular-weight bioreceptor the μIMZI can also be used for efficient label-free bacteria detection. The detection capability has been shown for peptide aptamers and pathogenic E. coli O157:H7 bacteria used as a bioreceptor and a target, respectively. We have found that the sensor is capable of detecting bacteria concentrations as low as 10 colony-forming units per mL in a sample which volume may be as small as hundreds of picoliters. The selectivity of the synthesized peptides is additionally confirmed by Enzyme-Linked Immunosorbent Assay (ELISA). Based on the presented performance, the μIMZI sensor is one of the most sensitive and lowest-analyte-volume label-free biosensors among fiber optical platforms to date.

Dispersible Conjugated Polymer Nanoparticles as Biointerface Materials for Label-Free Bacteria Detection

Fast and efficient identification of bacterial pathogens in water and biological fluids is an important issue in medical, food safety, and public health concerns that requires low-cost and efficient sensing strategies. Impedimetric sensors are promising tools for monitoring bacteria detection because of their reliability and ease-of-use. We herein report a study on new biointerface-based amphiphilic poly(3-hexylthiophene)-b-poly(3-triethylene-glycol-thiophene), P3HT-b-P3TEGT, for label-free impedimetric detection of Escherichia coli (E. coli). This biointerface is fabricated by the self-assembly of P3HT-b-P3TEGT into core-shell nanoparticles, which was further decorated with mannose, leading to an easy-to-use solution-processable nanoparticle material for biosensing. The hydrophilic block P3TEGT promotes antifouling and prevents nonspecific interactions, while improving the ionic and electronic transport properties, thus enhancing the electrochemical-sensing capability in aqueous solution. Self-assembly and micelle formation of P3HT-b-P3TEGT were analyzed by 2D-NMR, Fourier transform infrared, dynamic light scattering, contact angle, and microscopy characterizations. Detection of E. coli was characterized and evaluated using electrochemical impedance spectroscopy and optical and scanning electron microscopy techniques. The sensing layer based on the mannose-functionalized P3HT-b-P3TEGT nanoparticles demonstrates targeting ability toward E. coli pili protein with a detection range from 103 to 107 cfu/mL, and its selectivity was studied with Gram(+) bacteria. Application to real samples was performed by detection of bacteria in tap and the Nile water. The approach developed here shows that water/alcohol-processable-functionalized conjugated polymer nanoparticles are suitable for use as electrode materials, which have potential application in fabrication of a low-cost, label-free impedimetric biosensor for the detection of bacteria in water.

A Nanostructured Gold/Graphene Microfluidic Device for Direct and Plasmonic-Assisted Impedimetric Detection of Bacteria.

Hierarchical 3D gold nano-/microislands (NMIs) are favorably structured for direct and probe-free capture of bacteria in optical and electrochemical sensors. Moreover, their unique plasmonic properties make them a suitable candidate for plasmonic-assisted electrochemical sensors, yet the charge transfer needs to be improved. In the present study, we propose a novel plasmonic-assisted electrochemical impedimetric detection platform based on hybrid structures of 3D gold NMIs and graphene (Gr) nanosheets for probe-free capture and label-free detection of bacteria. The inclusion of Gr nanosheets significantly improves the charge transfer, addressing the central issue of using 3D gold NMIs. Notably, the 3D gold NMIs/Gr detection platform successfully distinguishes between various types of bacteria including Escherichia coli (E. coli) K12, Pseudomonas putida (P. putida), and Staphylococcus epidermidis (S. epidermidis) when electrochemical impedance spectroscopy is applied under visible light. We show that distinguishable and label-free impedimetric detection is due to dissimilar electron charge transfer caused by various sizes, morphologies, and compositions of the cells. In addition, the finite-difference time-domain (FDTD) simulation of the electric field indicates the intensity of charge distribution at the edge of the NMI structures. Furthermore, the wettability studies demonstrated that contact angle is a characteristic feature of each type of captured bacteria on the 3D gold NMIs, which strongly depends on the shape, morphology, and size of the cells. Ultimately, exposing the platform to various dilutions of the three bacteria strains revealed the ability to detect dilutions as low as ∼20 CFU/mL in a wide linear range of detection of 2 × 101-105, 2 × 101-104, and 1 × 102-1 × 105 CFU/mL for E. coli, P. putida, and S. epidermidis, respectively. The proposed hybrid structure of 3D gold NMIs and Gr, combined by novel plasmonic and conventional impedance spectroscopy techniques, opens interesting avenues in ultrasensitive label-free detection of bacteria with low cost and high stability.

Bioinspired Carbohydrate-Decorated Long-Period Fiber Grating for Label-Free Bacteria Detection

Pathogenic bacteria cause thousands of human infections every year. Their effective detection can prevent diseases, decrease the cost of patient hospitalizations, and thus enhance the standard of people’s life. Here, we present a bioinspired sensor for bacteria detection in water, which is based on long-period fiber grating (LPFG) and $\alpha $ -mannoside receptor. Embedding of p-aminophenyl- $\alpha $ -D-mannopyranoside, sensitive to E. coli strain, into the biosensor, allows mimicking natural bacteria adhesion to the host cell. The receptor has been covalently immobilized by a thiourea bond to the LPFG platform to ensure stable and reproducible work. The lowest detectable concentration of bacteria was equal to 103 colony forming unit ml−1.

Label-Free Detection of Bacteria in Fruit Juice by Nano-Flow Cytometry.

Rapid quantification of microbial contamination in fruit juice is highly desired for food safety control. Yet, the complex sample matrix and the diversity of bacterial contaminants present a great challenge. Employing a laboratory-built nano-flow cytometer (nFCM), here we report the development of a label-free approach for the detection of bacteria population in fruit juice. The weak autofluorescence of bacterial cells was used as a hallmark for the identification of bacteria. The sample pretreatment protocol was optimized to reduce fluorescence background, lyse residual plant cells and debris, and attain a good recovery of bacteria from juice samples. It was demonstrated that the nFCM was able to enumerate individual bacteria of very weak autofluorescence, and a clear differentiation from residual juice particulates was achieved. For bacteria spiked in the orange juice, the recovery rate was around 95% and a linear correlation between nFCM analysis and plate counting was acquired in the range of 3 × 104 to 3 × 108 cfu/mL. The assay, including sample pretreatment and instrument analysis, can be accomplished within 1 h, which is far more efficient than plate counting. Using a 40 mL sample volume, the detection limit in apple juice was ∼102 cells/mL. The as-developed method was successfully applied to bacterial measurement of freshly made orange juice and apple juice purchased from grocery stores. We believe it could also have potential practical application in microbial control analysis of other juices and water.

Electrochemical sensing of bacteria via secreted redox active compounds using conducting polymers

Bacterial infections and antibiotic resistance represent major global threats to public health. Current diagnostics use culture based assays that are reliable but slow, hence appealing for new rapid methods. Here we describe redox sensing as a novel concept for rapid, label-free detection of bacteria. We utilize a two-electrode poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS) based sensor for detection of bacterially secreted redox-active compounds. Using purified redox-active compounds, we show the ability of the sensor to detect and quantify compounds in micromolar concentrations within minutes. When applied for detection and quantification of Salmonella, we show that secreted, low molecular weight redox compounds cause reduction of the PEDOT:PSS electrode. A potential role of redox sensing in infection diagnostics was demonstrated as uropathogenic strains of E. coli, Staphylococcus, Enterococcus, Pseudomonas, Proteus, and Klebsiella spp., major causes of complicated urinary tract infections, were successfully detected in complex media or processed urine. Since numerous bacterial species are capable of extracellular electron transfer, redox sensing may find use as a generic method for bacterial detection with applications in research laboatories, the clinic and industry alike.

Rapid and label-free bacteria detection using a hybrid tri-layer dielectric integrated n-type organic field effect transistor

An ultra-low voltage operated n-type OFET device that could detect and distinguish live bacteria with an LOD of 103 cfu mL−1 is developed.

Escherichia coli Fiber Sensors Using Concentrated Dielectrophoretic Force with Optical Defocusing Method.

A sensitive tapered optical fiber tip combined with dielectrophoretic (DEP) trapping was used for rapid and label-free detection of bacteria in water. The angular spectrum of the optical field at the fiber tip was changed with the surrounding refractive index (RI). By measuring far-field intensity change at the defocus plane, the intensity sensitivity was up to 95 200%/RIU (RI unit), and the detection limit was 5.2 × 10-6 RIU at 0.5% intensity stability. By applying an AC voltage to a Ti/Al coated fiber tip and an indium-tin-oxide glass, the DEP force effectively trapped the Escherichia coli ( E. coli) near the fiber tip. Those bacteria can be directly measured from optical intensity change due to the increase of surrounding RI. By immobilizing the antibody on the Ti/Al fiber tip, the tests for specific K12 bacteria and nonspecific BL21 bacteria verified the specificity. The antibody-immobilized Ti/Al coated fiber tip with DEP trapping can detect bacteria at a concentration about 100 CFU/mL.

Octupolar Metastructures for a Highly Sensitive, Rapid, and Reproducible Phage-Based Detection of Bacterial Pathogens by Surface-Enhanced Raman Scattering.

The development of fast and ultrasensitive methods to detect bacterial pathogens at low concentrations is of high relevance for human and animal health care and diagnostics. In this context, surface-enhanced Raman scattering (SERS) offers the promise of a simplified, rapid, and high-sensitive detection of biomolecular interactions with several advantages over previous assay methodologies. In this work, we have conceived reproducible SERS nanosensors based on tailored multilayer octupolar nanostructures which can combine high enhancement factor and remarkable molecular selectivity. We show that coating novel multilayer octupolar metastructures with proper self-assembled monolayer (SAM) and immobilized phages can provide label-free analysis of pathogenic bacteria via SERS leading to a giant increase in SERS enhancement. The strong relative intensity changes of about 2100% at the maximum scattered SERS wavelength, induced by the Brucella bacterium captured, demonstrate the performance advantages of the bacteriophage sensing scheme. We performed measurements at the single-cell level thus allowing fast identification in less than an hour without any demanding sample preparation process. Our results based on designing well-controlled octupolar coupling platforms open up new opportunities toward the use of bacteriophages as recognition elements for the creation of SERS-based multifunctional biochips for rapid culture and label-free detection of bacteria.

Label-free bimodal waveguide immunosensor for rapid diagnosis of bacterial infections in cirrhotic patients

Spontaneous bacterial peritonitis is an acute bacterial infection of ascitic fluid; it has a high incidence in cirrhotic patients and it is associated with high mortality. In such a situation, early diagnosis and treatment is crucial for the survival of the patient. However, bacterial analysis in ascitic fluid is currently based on culture methods, which are time-consuming and laborious. We report here the application of a photonic interferometer biosensor based on a bimodal waveguide (BiMW) for the rapid and label-free detection of bacteria directly in ascitic fluid. The device consists of a straight waveguide in which two modes of the same polarization interfere while interacting with the external medium through their evanescent fields. A bimolecular event occurring on the sensor area of the device (e.g. capturing bacteria) will differently affect each light mode, inducing a variation in the phase of the light exiting at the output of the waveguide. In this work, we demonstrate the quantitative detection of Bacillus cereus in buffer medium and Escherichia coli in undiluted ascitic fluid from cirrhotic patients. In the case of Bacillus cereus detection, the device was able to specifically detect bacteria at relevant concentrations in 12.5min and in the case of Escherichia coli detection, the analysis time was 25min. Extrapolation of the data demonstrated that the detection limits of the biosensor could reach few bacteria per milliliter. Based on the results obtained, we consider that the BiMW biosensor is positioned as a promising new clinical tool for user-friendly, cost-effective and real-time microbiological analysis.

Rapid and label-free detection and assessment of bacteria by terahertz time-domain spectroscopy.

Here we demonstrated the potential and applicability of terahertz (THz) spectroscopy to detect four commonly found bacteria in the infectious diseases. Besides the different spectral characteristics between bacterial species, THz absorption differences for living bacteria, dead bacteria and bacterial powder of the same species were also investigated. Our results revealed that small differences in water contents between bacterial cells account for distinct discrepancies of the absorption coefficients, which can be used for bacterial species identification. Furthermore, living and dead bacteria showed different absorption coefficients as a result of their different hydration levels, suggesting that THz spectroscopy can be used to rapidly assess the living state of bacteria under test. Our results clearly demonstrated the ability of THz spectroscopy for time-saving and label-free detection of bacteria with minimal sample preparation, potentially to be utilized for point-of-care tests in the near future. Schematic representation of bacterial detection by THz spectroscopy. Different bacteria have distinctive absorption coefficients as a result of their different water contents.

Integrated optical biosensor for rapid detection of bacteria

AbstractIn medical diagnostics, rapid detection of pathogenic bacteria from body fluids is one of the basic issues. Most state-of-the-art methods require optical labeling, increasing the complexity, duration and cost of the analysis. Therefore, there is a strong need for developing selective sensory devices based on label-free techniques, in order to increase the speed, and reduce the cost of detection. In a recent paper, we have shown that an integrated optical Mach-Zehnder interferometer, a highly sensitive all-optical device made of a cheap photopolymer, can be used as a powerful lab-on-a-chip tool for specific, labelfree detection of proteins. By proper modifications of this technique, our interferometric biosensor was combined with a microfluidic system allowing the rapid and specific detection of bacteria from solutions, having the surface of the sensor functionalized by bacterium-specific antibodies. The experiments proved that the biosensor was able to detect Escherichia coli bacteria at concentrations of 106 cfu/ml within a few minutes, that makes our device an appropriate tool for fast, label-free detection of bacteria from body fluids such as urine or sputum. On the other hand, possible applications of the device may not be restricted to medical microbiology, since bacterial identification is an important task in microbial forensics, criminal investigations, bio-terrorism threats and in environmental studies, as well.

Microorganisms recognition and quantification by lectin adsorptive affinity impedance

Lectin-based screen-printed gold electrodes are reported for the impedimetric label-free detection of bacteria. The selective interaction of lectins with carbohydrate components from microorganisms surface was used as the recognition principle for their detection and identification. Electrochemical impedance spectroscopy (EIS) was employed for the direct label-free transduction of the bacteria–lectin binding. Biotinylated Concanavalin A (Con A) and Escherichia coli were used for the evaluation of the lectin–bacteria complex formation. This complex was formed in solution, and then adsorbed onto the gold SPE surface. No bacteria immobilization was observed on the sensor prepared in the absence of ConA, demonstrating the absence of non-specific bacteria adsorption onto the gold SPE. On the contrary, the changes in electron transfer resistance allowed monitoring of E. coli–biotinylated Con A complex formation without any amplification step. Experimental variables such as the biotinylated-Con A concentration and the bacteria–lectin incubation time were optimized. The electron transfer resistance varied linearly with the logarithmic value of E. coli concentration over four orders of magnitude, 5.0 × 10 3 and 5.0 × 10 7 cfu mL −1. The selectivity of the approach was evaluated by checking the impedimetric responses of gold SPE modified with the complexes formed between nine lectins and three different bacteria ( E. coli, Staphylococcus aureus and Mycobacterium phlei). Different response profiles were found when the different lectins were used as recognition elements. principal component analysis (PCA) allowed classification and distinction among bacteria. Finally, electrochemical monitoring of β-galactosidase activity for the surface attached bacteria was demonstrated to be useful to distinguish between E. coli and S. aureus, which exhibit a similar affinity towards biotinylated-Con A.

Label-Free Detection of Bacteria by Electrochemical Impedance Spectroscopy: Comparison to Surface Plasmon Resonance

The low but known risk of bacterial contamination has emerged as the greatest residual threat of transfusion-transmitted diseases. Label-free detection of a bacterial model, Escherichia coli, is performed using nonfaradic electrochemical impedance spectroscopy (EIS). Biotinylated polyclonal anti-E. coli is linked to a mixed self-assembled monolayer (SAM) on a gold electrode through a strong biotin-neutravidin interaction. The binding of one antibody molecule for 3.6 neutravidin molecules is determined using the surface plasmon resonance (SPR). The detection limit of E. coli found by SPR is 10(7) cfu/mL. After modeling the impedance Nyquist plot of E. coli/anti-E. coli/mixed SAM/gold electrode for increasing concentrations of E. coli (whole bacteria or lysed bacteria), the main parameter that is modified is the polarization resistance RP. A sigmoid variation of RP is observed when the log concentration of bacteria (whole or lysed) increases. A concentration of 10 cfu/mL whole bacteria is detected by EIS measurements while 103 cfu/mL is detected for lysed E. coli.