Antibody microarray detection of Escherichia coli O157:H7: Quantification, assay limitations, and capture efficiency

A sandwich fluorescent immunoassay in a microarray format was used to capture and detect E. coli O157:H7. Here, we explored quantitative aspects, limitations, and capture efficiency of the assay. When biotinylated capture antibodies were used, the signal generated was higher (over 5-fold higher with some cell concentrations) compared to biotinylated protein G-bound capture antibodies. By adjusting the concentration of reporter antibody, a linear fluorescent response was observed from approximately 3.0 x 10(6) to approximately 9.0 x 10(7) cells/mL, and this was in agreement with the number of captured bacteria as determined by fluorescence microscopy. Capture efficiency calculations revealed that, as the number of bacteria presented for capture decreased, capture efficiency increased to near 35%. Optimization experiments, with several combinations of capture and reporter antibodies, demonstrated that the amount of bacteria available for capture (10(6) versus 10(8) cells/mL) affected the optimal combination. The findings presented here indicate that antibody microarrays, when used in sandwich assay format, may be effectively used to capture and detect E. coli O157:H7.     

Procedures for preparing Escherichia coli O157:H7 immunoliposome and its application in liposome immunoassay

Although Escherichia coli serotype O157:H7 was identified as a human pathogen in the ninth decade of the twentieth century, it has become recognized as a major foodborne pathogen. In the United States, the severity of E. coli O157:H7 infection in the young and the elderly has had a tremendous impact on human health, the food industry, and federal regulations regarding food safety. In laboratory diagnosis, most microbiologic assays rely on a single phenotype to selectively isolate this pathogen. However, the process is labor- and time-consuming. It is important eventually to develop new assay procedures to detect them. Immunoliposomes, anti-E. coli O157:H7 antibody-tagged liposomes, encapsulating a visible dye, sulforhodamine B, were used in the present study for the development of a field-portable colorimetric immunoassay to detect E. coli O157:H7. The N-succinimidyl-S-acetylthioacetate derivative of the antibodies (anti-E. coli O157: H7) was first conjugated through the reactive N-(kappa-maleimidoundecanoyloxy) sulfosuccinimide ester derivative of dipalmitoylphosphatidylethanolamine and subsequently incorported into liposomes to form the immunoliposomes. A plastic-backed nitrocellulose strip with two immobilized zones is the basis for a sandwich assay to detect E. coli O157:H7. The first zone is the antigen capture zone (AC zone), which is used in a sandwich (noncompetitive) assay format; the other is the biotin capture zone (BC zone), which is used as a positive control for the strip. During the capillary migration of the wicking reagent containing 50 microL of immunoliposomes and 90 microL of the test sample, E. coli O157:H7 with surface-bound immunoliposomes is captured at the AC zone, while the unbound immunoliposomes migrate and bind to the antibiotin antibodies coated on BC zone. The color density of the AC zone were directly proportional to the amount of E. coli O157:H7 in the test sample. The detection limit of the current assay with heat-killed E. coli O157:H7 was approximately 2500 cells. The selectivity of the newly developed biosensor system was investigated, and pathogens, including Salmonella typhimurium and Listeria genus specific, were proven to have no interference with the detection of E. coli O157:H7.     

Magnetoelastic immunosensors: amplified mass immunosorbent assay for detection of Escherichia coli O157:H7

A mass-sensitive magnetoelastic immunosensor for detection of Escherichia coli O157:H7 is described, based on immobilization of affinity-purified antibodies attached to the surface of a micrometer-scale magnetoelastic cantilever. Alkaline phosphatase is used as a labeled enzyme to the anti-E. coli O157:H7 antibody, amplifying the mass change associated with the antibody-antigen binding reaction by biocatalytic precipitation of 5-bromo-4-chloro-3-indolyl phosphate in a pH 10.0 PBS solution. The detection limit of the biosensor is 10(2) E. coli O157:H7 cells/mL. A linear change in the resonance frequency of the biosensor was found to E. coli O157:H7 concentrations ranging from 10(2) to 10(6) cells/mL.     

Quantum dot biolabeling coupled with immunomagnetic separation for detection of Escherichia coli O157:H7

A sensitive, specific, and rapid method for the detection of E. coli O157:H7 was demonstrated using quantum dots (QDs) as a fluorescence marker coupled with immunomagnetic separation. Magnetic beads coated with anti-E. coli O157 antibodies were employed to selectively capture the target bacteria, and biotin-conjugated anti-E. coli antibodies were added to form sandwich immuno complexes. After magnetic separation, the immuno complexes were labeled with QDs via biotin-streptavidin conjugation. This was followed by a fluorescence measurement using a laptop-controlled portable device, which consisted of a blue LED and a CCD-array spectrometer. The peak intensity of the fluorescence emission was proportional to the initial cell concentration of E. coli O157:H7 in the range of 10(3)-10(7) CFU/mL with a detection limit at least 100 times lower than that of the FITC-based method. The total detection time was less than 2 h. Neither E. coli K12 nor Salmonella typhimurium interfered with the detection of E. coli O157:H7.     

Detection of pathogens using luminescent CdSe/ZnS dendron nanocrystals and a porous membrane immunofilter

A biosensor system for detection of pathogens was developed by using CdSe/ZnS core/shell dendron nanocrystals with high efficiency and stability as fluorescence labels and a flowing chamber with a microporous immunofilter. The antibody-immobilized immunofilter captured the targeted pathogens, Escherichia coli O157:H7 as an example for bacteria and hepatitis B being a model system for viruses. The CdSe/ZnS core/shell dendron nanocrystals were conjugated with the corresponding antibodies and then passed through the microporous membrane where they attached to the membrane-antigen-antibody. The efficient and stable photoluminescence (PL) of the CdSe/ZnS nanocrystals on the formed "sandwich" structure complexes (membrane-antigen-antibody conjugated with the nanocrystals) was used as the detection means. The effects of the pore size of the membranes, buffer pH, and assay time on the detection of E. coli O157:H7 were investigated and optimized. The detectable level of this new system was as low as 2.3 CFU/mL for E. coli O157:H7 and 5 ng/mL for hepatitis B surface Ag (HBsAg). The assay time was shortened to 30 min without any enrichment and incubation.     

An antibody-immobilized capillary column as a bioseparator/bioreactor for detection of Escherichia coil O157:H7 with absorbance measurement.

A capillary-column-based bioseparator/bioreactor was developed for detection of Escherichia coli O157:H7 by chemically immobilizing anti-E. coli O157:H7 antibodies onto the inner wall of the column, forming the "sandwich" immunocomplexes (immobilized antibody-E. coli O157: H7-enzyme-labeled antibody) after the sample and the enzyme-labeled antibody passed through the column and detecting the absorbance of the product in the bioreactor with an optical detector. The effects of the blocking agent, flow rate of samples and substrates, buffer, MgCl2, and pH on the detection of E. coli O157:H7 were investigated. The parameters, 2% BSA in 1.0 x 10-2 M, pH 7.4, PBS as the blocking agent, 0.5 mL/h as the sample flow rate, 1.0 x 10(-2) M MgCl2, and 2.0 x 10(-4) M p-nitrophenyl phosphate in 1.0 M, pH 9.0 Tris buffer as the substrate for the enzymatic reaction, and 1.0 mL/h as the substrate flow rate, were used in the bioseparator/bioreactor system for detection of E. coli O157:H7. The selectivity of the system was checked, and other pathogens, including Salmonella typhimurium, Campylobacterjejuni, and Listeria monocytogenes, had no interference with the detection of E. coli O157:H7. Its working range was from 5.0 x 10(2) to 5.0 x 10(6) cfu/mL, and the total assay time was < 1.5 h without any enrichment. The relative standard deviation was approximately 2.0-7.3%.     

Long-range surface plasmon-enhanced fluorescence spectroscopy biosensor for ultrasensitive detection of E. coli O157:H7

A new biosensor platform for the detection of bacterial pathogens based on long-range surface plasmon-enhanced fluorescence spectroscopy (LRSP-FS) is presented. The resonant excitation of LRSP modes provides an enhanced intensity of the electromagnetic field, which is directly translated to an increased strength of fluorescence signal measured upon the capture of target analyte at the sensor surface. LRSPs originate from a coupling of surface plasmons across a thin metallic film embedded in dielectrics with similar refractive indices. With respect to regular surface plasmon-enhanced fluorescence spectroscopy, the excitation of LRSPs offers the advantage of a larger enhancement of the evanescent field intensity and a micrometer probing depth that is comparable to the size of target bacterial pathogens. The potential of the developed sensor platform is demonstrated in an experiment in which the detection of E. coli O157:H7 was carried out using sandwich immunoassays. The limit of detection below 10 cfu mL(-1) and detection time of 40 min were achieved.     

Mid-IR biosensor: detection and fingerprinting of pathogens on gold island functionalized chalcogenide films

Antibody (human IgG, anti-E. coli O157:H7, and anti-Salmonella) complexes on the surface of IR-transparent Ge-containing chalcogenide glass films were formed via thiol chemistry on 20-nm-thick gold islands. As a first step, the protocol was validated by monitoring fluorescently tagged targets to validate binding. FT-IR spectroscopy confirmed that the coating of the films with 20-nm gold did not have a significant effect on the propagation and penetration of IR evanescent waves through the film. The films functionalized with anti-E. coli O157:H7 and anti-Salmonella antibodies were used to detect E. coli O157:H7 and S. enteriditis through label-free IR fingerprinting. Highly selective detection of bacterial targets was achieved at both the species (E. coli vs. S. enteriditis) and strain level (E. coli O157:H7 vs E. coli K12). A mid-infrared approach could thus be used as a biosensor as well as a molecular fingerprinting tool.     

Protocol development using an electronic nose for differentiating E. coli strains

The presence of Escherichia coli (E. coli) O157:H7 in the food industry continues to be a problem in America. The pathogenic bacteria, commonly associated with beef products, are the cause of numerous human health concerns. On the other hand, the nontoxic E. coli is present in the intestinal tracts of animals and humans and helps in digestion. Thus, the long-term research objective is to differentiate quickly the pathogen E. coli O157:H7 from the nontoxic strains of E. coli in the farm-to-fork industry chain before the infected food reaches the consumer. This paper describes a constructed gas-sensor electronic-nose system that detects the released gases (amines and alcohol compounds) occurring during the growth of the bacteria cultured in two types of media, namely nutrient broth and brain heart infusion (BHI) broth. The output is a gas signature for each bacterial sample. Eight different E. coli samples were used in testing; four O157:H7 and four non-O157:H7 isolates. Different concentrations of bacteria were used in the study, ranging from 10/sup 2/ to 10/sup 7/ colony-forming units per milliliter. The results of the research showed that E. coli O157:H7 had a distinctly different gas signature than that of the non-O157:H7 bacteria when grown in nutrient broth. Bacteria grown in BHI broth did not have differentiating results. The initiation of gas increase in the bacteria correlated directly with the initial cell concentration of the sample. The initial increase of gas released by the bacteria occurred during the exponential phase of bacterial growth. The electronic nose device was able to differentiate the gas signatures of non-O157:H7 E. coli and O157:H7 E. coli isolates grown in nutrient broth with excellent reproducibility. Potential applications include food and environmental safety from farm to fork.     

Surface stress‐induced membrane biosensor based on double‐layer stable gold nanostructures for E. coli detection

The surface stress‐based biosensor has been applied in fast and sensitive identification of Escherichia coli (E. coli)with significance for public health, food, and water safety. However, the stable sensitive element of flexible biosensor based on surface stress is still crucial and challengeable. Here, the authors reported surface stress‐induced biosensors based on double‐layer stable gold nanostructures (D‐AuNS‐SSMB) for E. coli O157:H7 detection. Bacterial detection demonstrates the high stability of the biosensor. The resistance change of biosensor is linear to the logarithmic value of the E. coli O157:H7 concentrations ranging from 10³  to 10⁷  CFU/mL with a limit of detection (LOD) of 43 CFU/mL. The captured signals of D‐AuNS‐SSMB comes from surface stress generated by antigen–antibody binding. In addition, the biosensor exhibits good stability, reproducibility and specificity in detection of E. coli O157:H7 as well. This study provides a new preparation method of stable sensitive element for the E. coli detection.Inspec keywords: microorganisms, biosensors, gold, membranes, stress measurement, nanostructured materials, nanosensorsOther keywords: double‐layer stable gold nanostructures, D‐AuNS‐SSMB, bacterial detection, surface stress‐induced membrane biosensor, escherichia coli detection, water safety, stability, E. coli O157:H7 concentration detection, antigen–antibody binding, Au     

Immunobiosensor chips for detection of Escherichia coil O157:H7 using electrochemical impedance spectroscopy

Impedance biosensor chips were developed for detection of Escherichia coli O157:H7 based on the surface immobilization of affinity-purified antibodies onto indium tin oxide (ITO) electrode chips. The immobilization of antibodies onto ITO chips was carried out using an epoxysilane monolayer to serve as a template for chemical anchoring of antibodies. The surface characteristics of chips before and after the binding reaction between the antibodies and antigens were characterized by atomic force microscopy (AFM). The patterns of the epoxysilanes monolayer, antibodies, and E. coli cells were clearly observed from the AFM images. Alkaline phosphatase as the labeled enzyme to anti-E. coli O157:H7 antibody was used to amplify the binding reaction of antibody-antigen on the chips. The biocatalyzed precipitation of 5-bromo-4-chloro-3-indolyl phosphate by alkaline phosphatase on the chips in pH 10 PBS buffer containing 0.1 M MgCl2 increased the electron-transfer resistance for a redox probe of Fe(CN)6(3-/4-) at the electrode-solution interface or the electrode resistance itself. Electrochemical impedance spectroscopy and cyclic voltammetric method were employed to follow the stepwise assembly of the systems and the electronic transduction for the detection of E. coli. The biosensor could detect the target bacteria with a detection limit of 6 x 10(3) cells/mL. A linear response in the electron-transfer resistance for the concentration of E. coli cells was found between 6 x 10(4) and 6 x 10(7) cells/mL.     

Improved sensitivity for the electrochemical biosensor with an adjunct probe

Despite their promising applications in the biomedical research, the development of electrochemical biosensors with improved sensitivity and low detection limit has remained a great challenge. Here, we demonstrate a new approach to improve the sensitivity of the electrochemical biosensor by simply introducing an adjunct probe into its construction. This signal-on biosensor consists of a thiol-functionalized capture probe attached on the gold electrode surface, an electrochemical sign (methyl blue, MB)-modified reporter probe which is complementary to the capture probe, and an adjunct probe attached nearby the capture probe. The adjunct probe functions as a fixer to immobilize the element of reporter probe which is displaced by the target DNA and protein, increasing the chance of the dissociative reporter probe to collide with the electrode surface and facilitating the electron transfer. The biosensor with an adjunct probe exhibits improved sensitivity and a large dynamic range for DNA and the thrombin assay and can even distinguish 1-base mismatched target DNA. Importantly, the use of this biosensor is not limited to such and is viable for sensitive detection of numerous biomolecules, including RNA, proteins, and small molecules such as cocaine.     

A universal nucleic acid sequence biosensor with nanomolar detection limits

A quantitative universal biosensor was developed on the basis of olignucleotide sandwich hybridization for the rapid (30 min total assay time) and highly sensitive (1 nM) detection of specific nucleic acid sequences. The biosensor consists of a universal membrane and a universal dye-entrapping liposomal nanovesicle. Two oligonucleotides, a reporter and a capture probe that can hybridize specifically with the target nucleic acid sequence, can be coupled to the universal biosensor components within a 10-min incubation period, thus converting it into a specific assay. The liposomal nanovesicles bear a generic oligonucleotide sequence on their outer surface. The reporter probes consist of two parts: the 3' end is complementary to the generic liposomal oligonucleotide, and the 5' end is complementary to the target sequence. Streptavidin is immobilized in the detection zone of the universal membranes. The capture probes are biotinylated at the 5' end and are complementary to another segment in the target sequence. Thus, by incubating the liposomal nanovesicles with the reporter probes, the target sequence, and the capture probes in a hybridization buffer for 20 min, a sandwich complex is formed. The mixture is applied to the membrane, migrates along the strip, and is captured in the detection zone via streptavidin-biotin binding. The biosensor assay was optimized with respect to hybridization conditions, concentrations of all components, and length of the generic probe. It was tested using synthetic DNA sequences and authentic RNA sequences isolated and amplified using nucleic acid sequence-based amplification (NASBA) from Escherichia coli, Bacillus anthracis, and Cryptosporidium parvum. Dose-response curves were carried out using a portable reflectometer for the instantaneous quantification of liposomal nanovesicles in the detection zone. Limits of detection of 1 fmol per assay (1 nM) and dynamic ranges between 1 fmol and at least 750 fmol (1-750 nM) were obtained. The universal biosensors were compared to specific RNA biosensors developed earlier and were found to match or exceed their performance characteristics. In addition, no changes to hybridization conditions were required when switching to the detection of a new target sequence or when using actual nucleic acid sequence-based amplified RNA sequences. Therefore, the universal biosensor described is an excellent tool for use in laboratories or at test sites for rapidly investigating and quantifying any nucleic acid sequence of interest.     

Flow cytometric analysis to detect pathogens in bacterial cell mixtures using semiconductor quantum dots

Compared to a common green organic dye, semiconductor quantum dots (QDs) composed of CdSe/ZnS core/shell bioconjugates display brighter fluorescence intensities, lower detection thresholds, and better accuracy in analyzing bacterial cell mixtures composed of pathogenic E. coli O157:H7 and harmless E. coli DH5alpha using flow cytometry. For the same given bacterial mixture, QDs display fluorescence intensity levels that are approximately 1 order of magnitude brighter compared to the analogous experiments that utilize the standard dye fluorescein isothiocyanate. Detection limits are lowest when QDs are used as the fluorophore label for the pathogenic E. coli O157:H7 serotype: limits of 1% O157:H7 in 99% DH5alpha result, corresponding to 106 cells/mL, which is comparable to other developing fluorescence-based techniques for pathogen detection. Finally, utilizing QDs to label E. coli O157:H7 in cell mixtures results in greater accuracy and more closely approaches the ideal fluorophore for pathogen detection using flow cytometry. With their broader absorption spectra and narrower emission spectra than organic dyes, QDs can make vast improvements in the field of flow cytometry, where single-source excitation and simultaneous detection of multicolor species without complicating experimental setups or data analysis is quite advantageous for analyzing heterogeneous cell mixtures, both for prokaryotic pathogen detection and for studies on eukaryotic cell characteristics.     

Engineering nanostructured porous SiO2 surfaces for bacteria detection via "direct cell capture"

An optical label-free biosensing platform for bacteria detection ( Escherichia coli K12 as a model system) based on nanostructured oxidized porous silicon (PSiO(2)) is introduced. The biosensor is designed to directly capture the target bacteria cells on its surface with no prior sample processing (such as cell lysis). The optical reflectivity spectrum of the PSiO(2) nanostructure displays Fabry-Pe?rot fringes characteristic of thin-film interference, enabling direct, real-time observation of bacteria attachment within minutes. The PSiO(2) optical nanostructure is synthesized and used as the optical transducer element. The porous surface is conjugated with specific monoclonal antibodies (immunoglobulin G's) to provide the active component of the biosensor. The immobilization of the antibodies onto the biosensor system is confirmed by attenuated total reflectance Fourier transform infrared spectroscopy, fluorescent labeling experiments, and refractive interferometric Fourier transform spectroscopy. We show that the immobilized antibodies maintain their immunoactivity and specificity when attached to the sensor surface. Exposure of these nanostructures to the target bacteria results in "direct cell capture" onto the biosensor surface. These specific binding events induce predictable changes in the thin-film optical interference spectrum of the biosensor. Our preliminary studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations. The current detection limit of E. coli K12 bacteria is 10(4) cells/mL within several minutes.     

Real-time potentiometric detection of bacteria in complex samples

Detecting and identifying pathogen bacteria is essential to ensure quality at all stages of the food chain and to diagnose and control microbial infections. Traditional detection methods, including those based on cell culturing, are tedious and time-consuming, and their further application in real samples generally implies more complex pretreatment steps. Even though state-of-the-art techniques for detecting microorganisms enable the quantification of very low concentrations of bacteria, to date it has been difficult to obtain successful results in real samples in a simple, reliable, and rapid manner. In this Article, we demonstrate that the label-free detection and identification of living bacteria in real samples can be carried out in a couple of minutes and in a direct, simple, and selective way at concentration levels as low as 6 colony forming units/mL (CFU) in complex matrices such as milk or 26 CFU/mL in apple juice where the pretreatment step of samples is extremely easy. We chose Escherichia coli ( E. coli ) CECT 675 cells as a model organism as a nonpathogenic surrogate for pathogenic E. coli O157:H7 to test the effectiveness of a potentiometric aptamer-based biosensor. This biosensor uses single-walled carbon nanotubes (SWCNT) as excellent ion-to-electron transducers and covalently immobilized aptamers as biorecognition elements. The selective aptamer-target interaction significantly changes the electrical potential, thus allowing for both interspecies and interstrain selectivity and enabling the direct detection of the target. This technique is therefore a powerful tool for the immediate identification and detection of microorganisms. We demonstrate the highly selective detection of living bacteria with an immediate linear response of up to 10(4) CFU/mL. The biosensor can be easily built and used, is regenerated without difficulty, and can be used at least five times with no loss in the minimum amount of detected bacteria.     

Design and fabrication of a microimpedance biosensor for bacterial detection

A biosensor for bacterial detection was developed based on microelectromechanical systems, heterobifunctional crosslinkers and immobilized antibodies. The sensor detected the change in impedance caused by the presence of bacteria immobilized on interdigitated gold electrodes and was fabricated from (100) silicon with a 2-/spl mu/m layer of thermal oxide as an insulating layer. The sensor active area is 9.6 mm/sup 2/ and consists of two interdigital gold electrode arrays measuring 0.8 /spl times/ 6 mm. Escherichia coli specific antibodies were immobilized to the oxide between the electrodes to create a biological sensing surface. The impedance across the interdigital electrodes was measured after immersing the biosensor in solution. Bacteria cells present in the sample solution attached to the antibodies and became tethered to the electrode array, thereby causing a change in measured impedance. The biosensor was able to discriminate between different cellular concentrations from 10/sup 5/ to 10/sup 7/ CFU/mL in pure culture. The sample testing process, including data acquisition, required 5 min. The design, fabrication, and testing of the biosensor is discussed along with the implications of these findings toward further biosensor development.     

Biosensor for dengue virus detection: sensitive, rapid, and serotype specific

A serotype-specific RNA biosensor was developed for the rapid detection of Dengue virus (serotypes 1-4) in blood samples. After RNA amplification, the biosensor allows the rapid detection of Dengue virus RNA in only 15 min. In addition, the biosensor is portable, inexpensive, and very easy to use, making it an ideal detection system for point-of-care and field applications. The biosensor is coupled to the isothermal nucleic acid sequence-based amplification (NASBA) technique with which small amounts of virus RNA are amplified using a simple water bath. During the NASBA reaction, a generic sequence is attached to all RNA molecules as described earlier (Wu, S. J.; Lee, E. M.; Putvatana, R.; Shurtliff, R. N.; Porter, K R.; Suharyono, W.; Watt, D. M.; King, C. C.; Murphy, G. S.; Hayes, C. G.; Romano, J. W. J. Clin. Microbiol. 2001, 39, 2794-2798.). It has been shown earlier that Dengue virus can be detected specifically using two DNA probes: a first probe hybridized with the attached generic sequence and, therefore, bound to every amplified RNA molecule; and a second probe either bound to all four Dengue virus serotypes or chosen to be specific for only one serotype. These probes were utilized in the biosensor described in this publication. For a generic Dengue virus biosensor, the second probe is complementary to a conserved region found in all Dengue serotypes. For identification of the individual Dengue virus serotypes, four serotype-specific probes were developed (Wu, S. J.; Lee, E. M.; Putvatana, R.; Shurtiff, R. N.; Porter, K. R.; Suharyono, W.; Watt, D. M.; King, C. C.; Murphy, G. S.; Hayes, C. G.; Romano, J. W. J. Clin. Microbiol. 2001, 39, 2794-2798.). The biosensor is a membrane-based DNA/RNA hybridization system using liposome amplification. The generic DNA probe (reporter probe) is coupled to the outside of dye-encapsulating liposomes. The conserved or Dengue serotype specific probes (capture probes) are immobilized on a polyethersulfone membrane strip. Liposomes are mixed with amplified target sequence and are then applied to the membrane. The mixture is allowed to migrate along the test strip, and the liposome-target sequence complexes are immobilized in the capture zone via hybridization of the capture probe with target sequence. The amount of liposomes present in the immobilized complex is directly proportional to the amount of target sequence present in the sample and can be quantified using a portable reflectometer. The different biosensor components have been optimized with respect to sensitivity and, foremost, specificity toward the different serotypes. An excellent correlation to a laboratory-based detection system was demonstrated. Finally, the assay was tested using a limited number of clinical human serum samples. Although Dengue serotypes 1, 2 and 4 were identified correctly, serotype 3 displayed low cross-reactivity with biosensors designed for detection of serotypes 1 and 4.     

Antimicrobial peptides for detection of bacteria in biosensor assays

Bacteria, plants, and higher and lower animals have evolved an innate immune system as a first line of defense against microbial invasion. Some of these organisms produce antimicrobial peptides (AMPs) as a part of this chemical immune system. AMPs exert their antimicrobial activity by binding to components of the microbe's surface and disrupting the membrane. The overall goal of this study was to apply the AMP magainin I as a recognition element for Escherichia coli O157:H7 and Salmonella typhimurium detection on an array-based biosensor. We immobilized magainin I on silanized glass slides using biotin-avidin chemistry, as well as through direct covalent attachment. Cy5-labeled, heat-killed cells were used to demonstrate that the immobilized magainin I can bind Salmonella with detection limits similar to analogous antibody-based assays. Detection limits for E. coli were higher than in analogous antibody-based assays, but it is expected that other AMPs may possess higher affinities for this target. The results showed that both specific and nonspecific binding strongly depend on the method used for peptide immobilization. Direct attachment of magainin to the substrate surface not only decreased nonspecific cell binding but also resulted in improved detection limits for both Salmonella and E. coli.     

Detection of single bacterial pathogens with semiconductor quantum dots

Semiconductor quantum dots (QDs) have been used in a simple fluorometric assay to detect single cells of the pathogenic Escherichia coli O157:H7 serotype. Composed of CdSe/ZnS core/shell QDs conjugated to streptavidin, this system exhibits 2 orders of magnitude more sensitivity than a similar assay using a common organic dye. Selectivity for this pathogenic bacterial strain over a common lab strain (E. coli DH5alpha), which is gained from the use of specific biotinylated antibodies, is also demonstrated for QD labeling. Under continuous excitation, these QDs retain high fluorescence intensities for hours, whereas a typical organic dye bleaches within seconds, allowing for more rapid and accurate identification of E. coli O157:H7 in single-cell fluorescence-based assays. This indirect QD labeling method, based on antibody-antigen and streptavidin-biotin interactions, is flexible enough to expand to other systems and has great potential for use in simultaneous multicolor detection schemes.