APPLICATIONS OF TIME-RESOLVED FLUOROIMMUNOASSAY TO DETECT MAGNETIC BEAD CAPTURED ESCHERICHIA COLI O157:H7

A time-resolved fluorescence technique was developed to detect Escherichia coli O157:H7 in ground beef burger. After a 4.5 h enrichment period, streptavidin coated magnetic beads conjugated with biotin-labeled anti E. coli O157:H7 were used to capture the bacteria. The bacteria were, at the same time, also labeled by a nonfluorescent, europium (Eu)-tagged anti-E. coli O157:H7 antibody. The sandwiched bacterial complexes were then concentrated using a magnetic particle concentrator and washed to remove other solution components. Upon addition of an enhancement buffer, the Eu-labels were then released from the antibodies and chelated to nitrilo-triacetic acid (NTA) and trioctylphosphine oxide (TOPO) to form highly fluorescent Eu-(2-NTA)₃(TOPO)_2–3 micellar complexes. Delayed fluorescence associated with these complexes was measured and its intensity was used to estimate the original bacterial concentration spiked in hamburger. This approach was applied to detect E. coli O157:H7 spiked in hamburgers. The results indicated this method is able to detect 1 CFU/g of the bacteria after a brief enrichment for four and half hours at 37C. Specificity studies indicated that the approach exhibited no or limited cross reactivity to Salmonella typhimurium, E. coli K-12 or Shigella dysenteriae spiked in hamburgers. Thus, the developed approach may be used as a rapid screening procedure for E. coli O157 bacteria in foods.     

Liposome-based microcapillary immunosensor for detection of Escherichia coli O157:H7

Our group has previously reported a sandwich-based strip immunoassay for rapid detection of Escherichia coli O157:H7 [Anal. Chem. 75 (2003) 4330]. In the present study, a microcapillary flow injection liposome immunoanalysis (mFILIA) system was developed for the detection of heat-killed E. coli O157:H7. A fused-silica microcapillary with anti-E. coli O157:H7 antibodies chemically immobilized on the internal surface via protein A served as an immunoreactor/immunoseparator for the mFILIA system. Liposomes tagged with anti-E. coli O157:H7 and encapsulating a fluorescent dye were used as the detectable label. In the presence of E. coli O157:H7, sandwich complexes were formed between the immobilized antibodies in the column, the sample of E. coli O157:H7 and the antibody-tagged sulforhodamine-dye-loaded liposomes. Signals generated by lysing the bound liposomes with 30 mM n-octyl-beta-D-glucopyranoside were measured by a fluorometer. The detected signal was directly proportional to the amount of E. coli O157:H7 in the test sample. The mFILIA system successfully detected as low as 360 cells/mL (equivalent to 53 heat-killed bacteria in the 150 microL of the sample solution injected). MeOH (30%) was used for the regeneration of antibody binding sites in the capillary after each measurement, which allowed the immunoreactor/immunoseparator to be used for at least 50 repeated assays. The calibration curve for heat-killed E. coli O157:H7 has a working range of 6 x 10(3)-6 x 10(7)cells, and the total assay time was less than 45 min. A coefficient of variation for triplicate measurements was < or =8.9%, which indicates an acceptable level of reproducibility for this newly developed method.     

Real-time analysis of antibody binding interactions with immobilized E. coli O157:H7 cells using the BIAcore

The kinetic properties of antibody-antigen reaction and other interacting macromolecules can be analyzed in real-time using the surface plasmon resonance biosensor (BIAcore). The interactions of an antibody against Escherischia coli O157:H7 were studied using immobilized whole cells. The bacterial sensor surface was evaluated with anti-E. coli in a continuous flow system. Regeneration of the sensor surface with guanidine-HCl was more effective than with phosphoric acid and resulted in better binding reproducibility. The determined kinetic values, association and dissociation rate constants, can be used in the development of rapid immuno-techniques. This study also provides the basis to evaluate real-time interactions of macromolecules with immobilized cells.     

Solid-phase capture of proteins, spores, and bacteria

Current methods for the detection of pathogens in food and water samples generally require a preenrichment step that allows selective enrichment of the test organism. The objective of this research was to eliminate an enrichment step to allow detection of bacteria directly in food and water samples in 30 min. A high-flow-rate, fluidized bed to capture and concentrate large (bacteria and spores) and small (protein) molecules was developed. This format, ImmunoFlow, is volume independent and uses large beads (greater than 3 mm in diameter) when capturing bacteria to prevent sample clogging when testing food samples. Detection of bound targets was done using existing enzyme-linked immunosorbent assay (ELISA) protocols. Four antibodies (anti-Escherichia coli O157:H7, -Bacillus globigii, -bovine serum albumin [BSA], and -ovalbumin [OVA]) were covalently coupled to various glass and ceramic beads. Very small amounts of BSA (<1 ng) and OVA (0.2 to 4.0 microg) were detected. Various industrial and environmental samples were used to observe the effect of the sample composition on the capture of anti-B. globigii and anti-E. coli O157:H7 modified beads. The lower limit of detection for both E. coli O157:H7 and B. globigii was 1 spore/cell independent of the sample size. The activity of anti-B. globigii modified beads declined after 3 days. Anti-E. coli O157:H7 modified beads declined in their capture ability after 2 days in various storage buffers. Storage temperature (4 and 25 degrees C) did not influence the stability. The ImmunoFlow technology is capable of capturing bacteria and spores directly from samples, with subsequent detection in an ELISA format in 30 min.     

Detection of Escherichia coli 0157:H7 using a surface plasmon resonance biosensor

The BIAcore biosensor was used to detect binding of Escherichia coli O157:H7 with specific antibodies. Immobilized Protein A or Protein G captured antibodies which in turn bound to the bacteria. Alternatively, immobilized antibody captured the E. coli O157:H7 and the bacteria were further probed by a second antibody which enhanced the signal. The regenerated sensor surfaces were used for at least 50 separate analyses. The surface plasmon resonance biosensor has potential for use in rapid, real-time detection and identification of bacteria, and to study the interaction of organisms with different antisera or other molecular species. © Rapid Science Ltd. 1998     

Biotinylated steroid derivatives as ligands for biospecific interaction analysis with monoclonal antibodies using immunosensor devices

Systematic ligand-binding studies of the biospecific interaction between steroids and antisteroid antibodies can be performed in real time using biosensor techniques. In this study, quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) biosensor systems were applied. Different biotinylated testosterone (T) and 17beta-estradiol (E2) derivatives were preincubated with streptavidin and immobilized on the sensor surfaces. We obtained low matrix densities of antigen enabling the investigation of the binding kinetics and position specificities of various anti-E2 and anti-T monoclonal antibodies (mAbs) to these steroidal compounds. The highest immunoreactivity of anti-E2 and anti-T mAbs is not necessarily for the specific modified steroid that was used as a protein-coupled hapten for immunization. The kinetic data confirm that both 3- and 19-specific anti-T mAbs do not discriminate between the 3- and 19-biotinylated T derivatives, whereas the 7alpha-biotinylated T probe showed no affinity to these two anti-T mAbs. In the case of the 3-specific anti-E2 mAb, comparable interaction data were found for 3- and 6alpha-biotinylated E2 compounds. The 6-specific anti-E2 mAb showed comparable ligand binding, but a significant higher dissociation rate to the position-specific antigen. The QCM and SPR results correspond well to the data from cross-reactivity studies in solution as well as to enzyme immunoassay equilibrium measurements.     

A mixed self-assembled monolayer-based surface plasmon immunosensor for detection of E. coli O157:H7

The sensitivity and specificity of a polyethylene glycol terminated alkanethiol mixed self-assembled monolayers (SAM) on surface plasmon resonance (SPR) immunosensor to detect Escherichia coli O157:H7 is demonstrated. Purified monoclonal (Mabs) or polyclonal antibodies (PAbs) against E. coli O157:H7 were immobilized on an activated sensor chip and direct and sandwich assays were carried to detect E. coli O157:H7. Effect of Protein G based detection and effect of concentrations of primary and secondary antibodies in sandwich assay were investigated. The sensor surface was observed under an optical microscope at various stages of the detection process. The sensor could detect as low as 10(3)CFU/ml of E. coli O157:H7 in a sandwich assay, with high specificity against Salmonella Enteritidis. The detection limit using direct assay and Protein G were 10(6)CFU/ml and 10(4)CFU/ml, respectively. Results indicate that an alkanethiol SAM based SPR biosensor has the potential for rapid and specific detection of E. coli O157:H7, using a sandwich assay.     

A comparison of protocols for the optimisation of detection of bacteria using a surface acoustic wave (SAW) biosensor

A dual channel surface acoustic wave (SAW) device has been used as a biosensor to detect two different microorganisms, Legionella and Escherichia coli, simultaneously. A series of experiments was conducted to optimise the use of the SAW for bacterial detection using a novel protocol of coating bacteria on the sensor surface prior to addition of the antibody. Results were compared with an experiment in which a conventional protocol was utilised, where antibody was coated on the sensor surface prior to exposure to bacteria. The concentration of bacteria that attached to the surface of the SAW device was related to the antibody that specifically bound to it and therefore to frequency in a dose dependent fashion. Unlike conventional microbiological techniques quantitative results can be obtained for Legionella and E. coli down to 10(6) cells per ml within 3 h. In addition E. coli was detected down to 10(5) cells per ml in a modified protocol using sheep IgG as a blocking agent.     

A self-assembled monolayer-based piezoelectric immunosensor for rapid detection of Escherichia coli O157:H7

A piezoelectric immunosensor was developed for rapid detection of Escherichia coli O157:H7. It was based on the immobilization of affinity-purified antibodies onto a monolayer of 16-mercaptohexadecanoic acid (MHDA), a long-chain carboxylic acid-terminating alkanethiol, self-assembled on an AT-cut quartz crystal's Au electrode surface with N-hydroxysuccinimide (NHS) ester as a reactive intermediate. The binding of target bacteria onto the immobilized antibodies decreased the sensor's resonant frequency, and the frequency shift was correlated to the bacterial concentration. The stepwise assembly of the immunosensor was characterized by means of both quartz crystal microbalance (QCM) and cyclic voltammetry techniques. Three analytical procedures, namely immersion, dip-and-dry and flow-through methods, were investigated. The immunosensor could detect the target bacteria in a range of 10(3)-10(8)CFU/ml within 30-50 min, and the sensor-to-sensor reproducibility obtained at 10(3) and 10(5) colony-forming units (CFU)/ml was 18 and 11% R.S.D., respectively. The proposed sensor was comparable to Protein A-based piezoelectric immunosensor in terms of the amount of immobilized antibodies and detection sensitivity.     

Evaluation of different micro/nanobeads used as amplifiers in QCM immunosensor for more sensitive detection of E. coli O157:H7

Micro/nanobeads with different materials (magnetic, silica and polymer) and different sizes (diameters from 30nm to 970nm) were investigated for their use as amplifiers in a quartz crystal microbalance (QCM) immunosensor for more sensitive detection of Escherichia coli O157:H7. The micro/nanobeads were conjugated with anti-E. coli antibodies. E. coli O157:H7 cells were first captured by the first antibody immobilized on the electrode surface, and then micro/nanobeads labeled secondary antibodies attached to the cells, and finally the complexes of antibody-E. coli-antibody modified beads were formed. The results showed that antibody-labeled beads lead to signal amplification in both the change in frequency (ΔF) and the change in resistance (ΔR). Since the penetration depth of the oscillation-induced shear-waves for a ～8MHz crystal is limited to 200nm, the interpretation of how the signal is amplified by the adsorbed particles was represented in terms of the coupled-oscillator theory. The amplification is not sensed in terms of increase in mass on the sensor surface. Amplification is sensed as a change in bacterial resonance frequency when the spheres adsorb to the bacteria. The change in the values of ΔF caused by different micro/nanobeads (amplifiers) attaching on target bacterial cells is indicative of the ratio between the resonance frequency of the absorbed bacterial-particle complex (ω(s)), and the resonance frequency of the crystal (ω).     

Development of a screen-printed carbon electrochemical immunosensor for picomolar concentrations of estradiol in human serum extracts

Investigations into the development of a prototype electrochemical immunosensor for estradiol (E(2)) are described. After optimising reagent loadings in a 96-well enzyme-linked immunosorbent assay (ELISA), antibodies (rabbit anti-mouse IgG and monoclonal mouse anti-E(2)) were immobilised by passive adsorption onto the surface of screen-printed carbon electrodes (SPCEs). A competitive immunoassay was then performed using an alkaline-phosphatase (ALP)-labelled E(2) conjugate. Calibration plots for E(2) buffer standards, performed colorimetrically on the SPCEs using a para-nitrophenyl phosphate substrate solution, were in good agreement with ELISA calibration plots. Electrochemical measurements were then performed using differential pulse voltammetry (DPV) following the production of 1-naphthol from 1-naphthyl phosphate. The calibration plot of DPV peak current versus E(2) concentration showed a measurable range of 25-500 pg/ml with a detection limit of 50 pg/ml. A coefficient of variation of between 13.0 and 15.6% was obtained for repeat measurements. The immunosensor was applied to the determination of E(2) in spiked serum, following an extraction step with diethyl ether. A mean recovery for the method of 102.5% was obtained with a CV of 19.1%. The options available for further development of the sensor regarding precision, limit of detection and direct sample analysis are discussed.     

Interdigitated array microelectrode based impedance biosensor coupled with magnetic nanoparticle-antibody conjugates for detection of Escherichia coli O157:H7 in food samples

An impedance biosensor based on interdigitated array microelectrode (IDAM) coupled with magnetic nanoparticle-antibody conjugates (MNAC) was developed and evaluated for rapid and specific detection of E. coli O157:H7 in ground beef samples. MNAC were prepared by immobilizing biotin-labeled polyclonal goat anti-E. coli antibodies onto streptavidin-coated magnetic nanoparticles, which were used to separate and concentrate E. coli O157:H7 from ground beef samples. Magnitude of impedance and phase angle were measured in a frequency range of 10 Hz to 1 MHz in the presence of 0.1M mannitol solution. The lowest detection limits of this biosensor for detection of E. coli O157:H7 in pure culture and ground beef samples were 7.4 x 10(4) and 8.0 x 10(5)CFU ml(-1), respectively. The regression equation for the normalized impedance change (NIC) versus E. coli O157:H7 concentration (N) in ground beef samples was NIC=15.55 N-71.04 with R(2)=0.95. Sensitivity of the impedance biosensor was improved by 35% by concentrating bacterial cells attached to MNAC in the active layer of IDAM above the surface of electrodes with the help of a magnetic field. Based on equivalent circuit analysis, it was observed that bulk resistance and double layer capacitance were responsible for the impedance change caused by the presence of E. coli O157:H7 on the surface of IDAM. Surface immobilization techniques, redox probes, or sample incubation were not used in this impedance biosensor. The total detection time from sampling to measurement was 35 min.     

THE USE OF STREPTAVIDIN COATED MAGNETIC BEADS FOR DETECTING PATHOGENIC BACTERIA BY LIGHT ADDRESSABLE POTENTIOMETRIC SENSOR (LAPS)

A modified procedure for magnetic capture of antibody-conjugated bacteria for light addressable potentiometric sensor (LAPS) detection using the Threshold System was developed. Streptavidin coated magnetic beads, partially labeled with biotinylated anti Escherichia coli O157 antibodies, were used to capture Escherichia coli O157:H7. Captured bacteria were further labeled with fluorescein-conjugated anti -E. coli O157:H7 antibodies and urease-labeled. anti-fluorescein antibody. Magnetically concentrated bacteria-containing complexes were then immobilized through streptavidin-biotin interactions on 0.45 μ biotinylated nitro-cellulose membranes assembled as sample sticks for the Threshold instrument. The rate of pH change associated with the production of NH₃ by the urease in urea-containing solution was measured by a LAPS incorporated in the Threshold instrument. This approach allowed us to detect 10³ to 10⁴ CPU of cultured E. coli O157:H7 in PBS solutions. Furthermore, detectable LAPS signals of the sample sticks remained relatively constant for at least 24 h at 4C. The developed approach was applied to detect the E. coli in beef hamburger spiked with the bacteria. After a 5 to 6-h enrichment at 37C, as low as 1 CFU/g of E. coli O157:H7 in beef hamburger could be detected.     

Comparison of sensing strategies in SPR biosensor for rapid and sensitive enumeration of bacteria

Rapid and sensitive detections of microorganisms are very important for biodefence, food safety, medical diagnosis and pharmaceutics. The present study aims to find out the most proper bioactive surface preparation method to develop rapid, sensitive and selective bacteria biosensor, based on surface plasmon resonance (SPR) spectroscopy. Escherichia coli (E. coli) was used as a model bacterium and four sensing strategies in SPR were tested. Three of these strategies are antibody immobilization methods that are non-specific adsorption, specific adsorption via the avidin-biotin interaction, and immobilization of antibodies via self-assembled monolayer formation. The fourth strategy is a novel method for bacteria enumeration based on the combination of the SPR spectroscopy and immunomagnetic separation with using gold-coated magnetic nanoparticles. According to results, the most efficient SPR method is the one based on gold-coated magnetic nanoparticles. This method allows to specifically separate E. coli from the environment and to quantify rapidly without any labeling procedure. The developed method has a linear range between 30 and 3.0 × 10(4)cfu/ml, and a detection limit of 3 cfu/ml. The selectivity of the method was examined with Enterobacter aerogenes and Enterobacter dissolvens, which did not produce any significant response. The usefulness of the method to detect E. coli in real water samples was also investigated, and the results were compared with the results from plate-counting method. There was no significant difference between the methods (p>0.05).     

Production and specificity of monoclonal antibodies and polyclonal antibodies to Escherichia coli

Monoclonal antibodies were produced to whole cells of heat-treated Escherichia coli. Balb/c mice were immunized with a pool of five strains of heat-treated E. coli , and the resulting hybridomas were screened by indirect immunoassay. E. coli strains other than those used for immunization were used for screening to detect hybridomas producing antibody that reacted with a large number of E. coli strains. Of 864 hybridomas, 32 reacted strongly with either two or all three of the strains used for screening; 15 were successfully cloned. Antibody from hybridoma 6H2 reacted with 35 of 68 (51%) E. coli ; of 13 non- E. coli tested, only Enterobacter agglomerans was weakly positive. Hybridoma 9B12 antibody reacted with all six E. coli tested. Hybridoma 9B12, however, stopped producing antibody. Five hybridomas produced antibody which reacted with a majority of the bacteria tested whereas antibodies from two other hybridomas reacted with several E. coli and non- E. coli. Polyclonal antibodies produced to two strains of E. coli varied in the numbers of E. coli with which they reacted; both antisera cross-reacted with several non- E. coli.     

Expression of hepatitis C virus E2 ectodomain in E. coli and its application in the detection of anti-E2 antibodies in human sera

The second envelope glycoprotein (E2) of hepatitis C virus has been shown to bind human target cells and has become a major target for the development of anti-HCV vaccines. Anti-E2 antibodies have been suggested to be of clinical significance in diagnosis, treatment and prognosis of hepatitis C. However, large-scale expression and purification of E2 proteins in mammalian cells is difficult. As an alternative, E2 fragment (aa 385-730) with a four-amino-acid mutation (aa 568-571 PCNI to RVTS) was expressed as hexa-histidine-tagged full length protein [E2N730(m)] in E. coli and purified to over 85% purity. Purified E2N730(m) was specifically recognized by homologous hepatitis C patient serum in Western blot, suggesting that it displayed E2~specific antigenicity. Rabbit antiserum raised against E2N730(m) recognized E2 glyco-proteins expressed in mammalian cells in Western blot. Purified E2N730(m) was used to detect anti-E2 antibodies in human sera and showed better specificity and sensitivity than previousl     

QCM immunosensor detection of Escherichia coli O157:H7 based on beacon immunomagnetic nanoparticles and catalytic growth of colloidal gold

In this paper, we describe a novel method for detecting Escherichia coli (E. coli) O157:H7 by using a quartz crystal microbalance (QCM) immunosensor based on beacon immunomagnetic nanoparticles (BIMPs), streptavidin-gold, and growth solution. E. coli O157-BIMPs were magnetic nanoparticles loaded with polyclonal anti-E. coli O157:H7 antibody (target antibody, T-Ab) and biotin-IgG (beacon antibody, B-Ab) at an optimized ratio of 1:60 (T-Ab:B-Ab). E. coli O157:H7 was captured and separated by E. coli O157-BIMPs in a sample, and the streptavidin-gold was subsequently conjugated to E. coli O157-BIMPs by using a biotin-avidin system. Finally, the gold particles on E. coli O157-BIMPs were enlarged in growth solution, and the compounds containing E. coli O157:H7, E. coli O157-BIMPs, and enlarged gold particles were collected using a magnetic plate. The QCM immunosensor was fabricated with protein A from Staphylococcus aureus and monoclonal anti-E. coli O157:H7 antibody. The compounds decreased the immunosensor's resonant frequency. E. coli O157-BIMPs and enlarged gold particles were used as "mass enhancers" to amplify the frequency change. The frequency shift was correlated to the bacterial concentration. The detection limit was 23 CFU/ml in phosphate-buffered saline and 53 CFU/ml in milk. This method could successfully detect E. coli O157:H7 with high specificity and stability. The entire procedure for the detection of E. coli O157:H7 took only 4 h.     

Production and specificity of monoclonal antibodies and polyclonal antibodies to Escherichia coli

Monoclonal antibodies were produced to whole cells of heat-treated Escherichia coli. Balb/c mice were immunized with a pool of five strains of heat-treated E. coli, and the resulting hybridomas were screened by indirect immunoassay. E. coli strains other than those used for immunization were used for screening to detect hybridomas producing antibody that reacted with a large number of E. coli strains. Of 864 hybridomas, 32 reacted strongly with either two or all three of the strains used for screening; 15 were successfully cloned. Antibody from hybridoma 6H2 reacted with 35 of 68 (51%) E. coli; of 13 non-E. coli tested, only Enterobacter agglomerans was weakly positive. Hybridoma 9B12 antibody reacted with all six E. coli tested. Hybridoma 9B12, however, stopped producing antibody. Five hybridomas produced antibody which reacted with a majority of the bacteria tested whereas antibodies from two other hybridomas reacted with several E. coli and non-E. coli. Polyclonal antibodies produced to two strains of E. coli varied in the numbers of E. coli with which they reacted; both antisera cross-reacted with several non-E. coli.     

A portable surface plasmon resonance sensor system for real-time monitoring of small to large analytes

Many environmental applications exist for biosensors capable of providing real-time analyses. One pressing current need is monitoring for agents of chemical- and bio-terrorism. These applications require systems that can rapidly detect small organics including nerve agents, toxic proteins, viruses, spores and whole microbes. A second area of application is monitoring for environmental pollutants. Processing of grab samples through chemical laboratories requires significant time delays in the analyses, preventing the rapid mapping and cleanup of chemical spills. The current state of development of miniaturized, integrated surface plasmon resonance (SPR) sensor elements has allowed for the development of inexpensive, portable biosensor systems capable of the simultaneous analysis of multiple analytes. Most of the detection protocols make use of antibodies immobilized on the sensor surface. The Spreeta 2000 SPR biosensor elements manufactured by Texas Instruments provide three channels for each sensor element in the system. A temperature-controlled two-element system that monitors for six analytes is currently in use, and development of an eight element sensor system capable of monitoring up to 24 different analytes will be completed in the near future. Protein toxins can be directly detected and quantified in the low picomolar range. Elimination of false positives and increased sensitivity is provided by secondary antibodies with specificity for different target epitopes, and by sensor element redundancy. Inclusion of more than a single amplification step can push the sensitivity of toxic protein detection to femtomolar levels. The same types of direct detection and amplification protocols are used to monitor for viruses and whole bacteria or spores. Special protocols are required for the detection of small molecules. Either a competition type assay where the presence of analyte inhibits the binding of antibodies to surface-immobilized analyte, or a displacement assay, where antibodies bound to analyte on the sensor surface are displaced by free analyte, can be used. The small molecule detection assays vary in sensitivity from the low micromolar range to the high picomolar.

     

A portable surface plasmon resonance sensor system for real-time monitoring of small to large analytes

Many environmental applications exist for biosensors capable of providing real-time analyses. One pressing current need is monitoring for agents of chemical- and bio-terrorism. These applications require systems that can rapidly detect small organics including nerve agents, toxic proteins, viruses, spores and whole microbes. A second area of application is monitoring for environmental pollutants. Processing of grab samples through chemical laboratories requires significant time delays in the analyses, preventing the rapid mapping and cleanup of chemical spills. The current state of development of miniaturized, integrated surface plasmon resonance (SPR) sensor elements has allowed for the development of inexpensive, portable biosensor systems capable of the simultaneous analysis of multiple analytes. Most of the detection protocols make use of antibodies immobilized on the sensor surface. The Spreeta 2000 SPR biosensor elements manufactured by Texas Instruments provide three channels for each sensor element in the system. A temperature-controlled two-element system that monitors for six analytes is currently in use, and development of an eight element sensor system capable of monitoring up to 24 different analytes will be completed in the near future. Protein toxins can be directly detected and quantified in the low picomolar range. Elimination of false positives and increased sensitivity is provided by secondary antibodies with specificity for different target epitopes, and by sensor element redundancy. Inclusion of more than a single amplification step can push the sensitivity of toxic protein detection to femtomolar levels. The same types of direct detection and amplification protocols are used to monitor for viruses and whole bacteria or spores. Special protocols are required for the detection of small molecules. Either a competition type assay where the presence of analyte inhibits the binding of antibodies to surface-immobilized analyte, or a displacement assay, where antibodies bound to analyte on the sensor surface are displaced by free analyte, can be used. The small molecule detection assays vary in sensitivity from the low micromolar range to the high picomolar.