Rapid detection and identification of biological and chemical agents by immunoassay, gene probe assay and enzyme inhibition using a silicon-based biosensor

A rapid biosensor assay procedure that utilizes biotin streptavidin mediated filtration capture onto nitrocellulose membrane, in conjunction with a silicon-based light-addressable potentiometric sensor (LAPS) was developed for detection and identification of biological and chemical threat agents. Sandwich immunoassays, nucleic acid hybridization assays and enzyme inhibition assays are described. For immunoassays, the lower limits of detection (LOD) per 100-microl sample were approximately 5 pg/ml for protein (Staphylococcal enterotoxin B), 2 ng/ml for virus (Newcastle disease virus), and 20 ng/ml for vegetative bacteria (Brucella melitensis). In a dual gene probe assay format, the LOD was 0.30 fmol (1.8 x 10(8) copies per 60-microl) of single stranded target DNA. Enzyme inhibition assays on the LAPS using acetylcholinesterase were able to detect soman and sarin in aqueous samples at 2 and 8 pg (100 and 600 pM), respectively. The assays were easy to perform and required a total time equal to the reaction period plus about 15 min for filtering, washing and sensing. The assay format is suitable for detection of a wide range of infectious and toxic substances. New assays can be developed and optimized readily, often within 1 or 2 days.     

Array biosensor for detection of biohazards

A fluorescence-based biosensor has been developed for simultaneous analysis of multiple samples for multiple biohazardous agents. A patterned array of antibodies immobilized on the surface of a planar waveguide is used to capture antigen present in samples; bound analyte is then quantified by means of fluorescent tracer antibodies. Upon excitation of the fluorophore by a small diode laser, a CCD camera detects the pattern of fluorescent antibody:antigen complexes on the waveguide surface. Image analysis software correlates the position of fluorescent signals with the identity of the analyte. This array biosensor has been used to detect toxins, toxoids, and killed or non-pathogenic (vaccine) strains of pathogenic bacteria. Limits of detection in the mid-ng/ml range (toxins and toxoids) and in the 10(3)-10(6) cfu/ml range (bacterial analytes) were achieved with a facile 14-min off-line assay. In addition, a fluidics and imaging system has been developed which allows automated detection of staphylococcal enterotoxin B (SEB) in the low ng/ml range.     

Automated 10-channel capillary chip immunodetector for biological agents detection

The automated 10-channel capillary chip immunodetector (10K-IDWG) is a prototype, which has been developed for automatically operated biological agents (BA) point detection. The current technology uses a chemiluminescence capillary immunoassay (EIA) technique in combination with integrated microfluidics and allows the highly sensitive and rapid detection and preliminary identification of multiple BA in aqueous solutions in the laboratory. The chemiluminescence capillary EIA are performed within a disposable capillary chip containing 10 fused-silica capillaries arranged in parallel coated with selected capture antibodies. A multianode-photomultiplier array is used to detect chemiluminescence intensity in each capillary. Reservoirs for reagents and buffers and a waste disposal reservoir are integrated. This paper describes the technology of the 10K-IDWG and its evaluation with three different BA, the toxin staphylococcal enterotoxin B (SEB), the bacterial analyte Escherichia coli (E. coli) O157:H7 as a model for bacterial pathogens, and the bacteriophage M13 as a model for virus pathogens. The 10K-IDWG is able to detect the above mentioned three BA in an aqueous sample within 29 min (single analyte-detection and multiplexing). Limits of detection (LOD) are 0.1 ng/ml for SEB, 10(4)cfu/ml for E. coli O157:H7, and 5x10(5) pfu/ml for M13. Cross reactivities between the three assays were not observed.     

Development of surface plasmon resonance imaging for detection of Acidovorax avenae subsp. citrulli (Aac) using specific monoclonal antibody

An immunosensor based on surface plasmon resonance imaging (SPR imaging) using a specific monoclonal antibody 11E5 (MAb 11E5) was developed for the detection of the seed-borne bacterium Acidovorax avenae subsp. citrulli (Aac), which causes fruit blotch in watermelons and cantaloupes, and compared to the conventional ELISA technique. The 1:40 mixed self-assembled monolayer (mixed SAM) surface was used for the immobilized MAb 11E5 on sensor surface for the detection of Aac. Both whole cells and broken cells of Aac were tested by using direct and sandwich detection assay. The limit of detection (LOD) of Aac using the SPR imaging technique and a direct detection assay was 10(6)cfu/ml and a subsequent amplification of the SPR signal using a polyclonal antibody (PAb) lowered the LOD to 5×10(5) cfu/ml. The LOD for the ELISA technique was 5×10(4) cfu/ml for the detection of Aac, which was slightly better than that for the SPR technique. However, the sensor surface based on SPR imaging offered a major advantage in terms of surface regeneration, allowing at least five cycles with a shorter time assay, multi-channel analysis with an application on multiplex detection, and an ease of the surface usage for the detection of Aac in the naturally infected plant. The surface was tested against the naturally infected sample and showed good selectivity toward the Aac bacteria.     

Microbial fouling community analysis of the cooling water system of a nuclear test reactor with emphasis on sulphate reducing bacteria

Culture and molecular-based techniques were used to characterize bacterial diversity in the cooling water system of a fast breeder test reactor (FBTR). Techniques were selected for special emphasis on sulphate-reducing bacteria (SRB). Water samples from different locations of the FBTR cooling water system, in addition to biofilm scrapings from carbon steel coupons and a control SRB sample were characterized. Whole genome extraction of the water samples and SRB diversity by group specific primers were analysed using nested PCR and denaturing gradient gel electrophoresis (DGGE). The results of the bacterial assay in the cooling water showed that the total culturable bacteria (TCB) ranged from 10(3) to 10(5)?cfu?ml(-1); iron-reducing bacteria, 10(3) to 10(5)?cfu?ml(-1); iron oxidizing bacteria, 10(2) to 10(3)?cfu?ml(-1) and SRB, 2-29?cfu?ml(-1). However, the counts of the various bacterial types in the biofilm sample were 2-3 orders of magnitude higher. SRB diversity by the nested PCR-DGGE approach showed the presence of groups 1, 5 and 6 in the FBTR cooling water system; however, groups 2, 3 and 4 were not detected. The study demonstrated that the PCR protocol influenced the results of the diversity analysis. The paper further discusses the microbiota of the cooling water system and its relevance in biofouling.     

Antagonistic effect of enterocin CCM 4231 from Enterococcus faecium on "bryndza", a traditional Slovak dairy product from sheep milk

"Bryndza" is a traditional Slovak dairy product (type of soft cheese) made from sheep cheese which was ripened for 14 days. Because its manufacture, transporting and/or storing represent conditions which facilitate contamination, the effect of enterocin CCM4231 in "bryndza" was investigated with the aim to reduce the contaminant agents. "Bryndza" was divided into equal portions (50 g). The experimental sample (ES) as well as the control sample one (C1) were inoculated with Listeria innocua Li1 strain. The other control samples C2 and C3 were without Li1 strain. C3 control was selected as a reference control. ES and C2 portions were treated with purified enterocin CCM4231 in a concentration of 6400 AU/ml. Before the experimental inoculation, "bryndza" was checked for the presence of contaminant agents. The experiment lasted 1 week and the samples were stored in the refrigerator at 4 degrees C. Sampling was performed on day 1, on day 4 and on day 7. The control samples C2 and C3 were checked only on day 1 and then after 1 week. The following contaminant agents were detected in "bryndza" before its experimental inoculation with L. innocua Li1 strain: Escherichia coli in the amount 10(3) cfu/ml/g, Staphylococcus aureus (10(2) cfu/ml/g) and enterococci (10(4) cfu/ml/g). In the control sample C2, the number of E. coli was reduced to 10(2) cfu/ml/g. Enterococci and staphylococci were totally eliminated there. Concerning C3 control, natural decrease of bacteria was found and/or their unchanged counts. The value of pH (5) was stable during the whole experiment. In the experimental sample inoculated with Li1 strain, its counts were decreased immediately after enterocin CCM4231 addition approximately by one order of magnitude. This inhibitory effect was also detectable on day 4 by the difference of one order of magnitude between ES and C1. On day 7, 10(3) cfu/ml/g of Li1 strain were detected in both samples (ES, C1). The difference by one order of magnitude indicated, an inhibitory effect of enterocin CCM4231 in "bryndza". However, bacteriocin activity was not determined by laboratory analyses.     

Detection of low numbers of Salmonella in environmental water, sewage and food samples by a nested polymerase chain reaction assay

A polymerase chain reaction (PCR) assay with two nested pairs of primers selected from conserved sequences within a 2.3 kb randomly cloned DNA fragment from the Salmonella typhimurium chromosome was developed. The nested PCR assay correctly identified 128 of a total of 129 Salmonella strains belonging to subspecies I, II, IIIb and IV. One strain of Salm. arizona (ssp. IIIa) tested negative. No PCR products were obtained from any of the 31 non-Salmonella strains examined. The sensitivity of the assay was 2 cfu, as determined by analysis of proteinase K-treated boiled lysates of Salm. typhimurium. The performance of the assay was evaluated for environmental water, sewage and food samples spiked with Salm. typhimurium. Water and sewage samples were filtered and filters were enriched overnight in a non-selective medium. Prior to PCR, the broth cultures were subjected to a rapid and simple preparation procedure consisting of centrifugation, proteinase K treatment and boiling. This assay enabled detection of 10 cfu 100 ml(-1) water with background levels of up to 8700 heterotrophic organisms ml(-1) and 10000 cfu of coliform organisms 100 ml(-1) water. Spiked food samples were analysed with and without overnight enrichment in a non-selective medium using the same assay as above. Nested PCR performed on enriched broths enabled detection of <10 cfu g(-1) food. Variable results were obtained for food samples examined without prior enrichment and most results were negative. This rapid and simple assay provides a sensitive and specific means of screening drinking water or environmental water samples, as well as food samples, for the presence of Salmonella spp.     

Investigation of a simple amperometric electrode system to rapidly quantify and detect bacteria in foods

A two electrode system mounted as a single probe was developed to measure electrochemically the rate of reduction of a redox mediator (thionine) by bacteria. The system gave a rapid (2 min) bacterial-dependent current above 2.5 x 10(5) cfu/ml with pure cultures of bacteria, but when applied to the measurement of the bacterial contamination in samples of meat and milk it was unable to detect or quantify the contamination reliably. Incubation of samples for a few hours before examination enabled the system to detect bacteria in excess of 10(6) cfu/ml.     

Simultaneous assay of cocaine, heroin and metabolites in hair, plasma, saliva and urine by gas chromatography—mass spectrometry

As part of an ongoing research program on the development of drug detection methodology, we developed an assay for the simultaneous measurement of cocaine, heroin and metabolites in plasma, saliva, urine and hair by solid-phase extraction (SPE) and gas chromatography—mass spectrometry (GC—MS). The analytes that could be measured by this assay were the following: anhydroecgonine methyl ester; ecgonine methyl ester; ecgonine ethyl ester; cocaine; cocaethylene; benzoylecgonine; cocaethylene; norcocaethylene; benzoylnorecgonine; codeine; morphine; norcodeine; 6-acetylmorphine; normorphine; and heroin. Liquid specimens were diluted, filtered and then extracted by SPE. Additional handling steps were necessary for the analysis of hair samples. An initial wash procedure was utilized to remove surface contaminants. Washed hair samples were extracted with methanol overnight at 40°C. Both wash and extract fractions were collected, evaporated and purified by SPE. All extracts were evaporated, derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) and analyzed by GC—MS. The limit of detection (LOD) for cocaine, heroin and metabolites in biological specimens was approximately 1 ng/ml with the exception of norcodeine, normorphine and benzoylnorecgonine (LOD = 5 ng/ml). The LOD for cocaine, heroin and metabolites in hair was approximately 0.1 ng/mg of hair with the exception of norcodeine (LOD = 0.3 ng/mg) and normorphine and benzoylnorecgonine (LOD = 0.5 ng/mg). Coefficients of variation ranged from 3 to 26.5% in the hair assay. This assay has been successfully utilized in research on the disposition of cocaine, heroin and metabolites in hair, plasma, saliva and urine and in treatment studies.     

Investigation of a simple amperometric electrode system to rapidly quantify and detect bacteria in foods

A two electrode system mounted as a single probe was developed to measure electrochemically the rate of reduction of a redox mediator (thionine) by bacteria. The system gave a rapid (2 min) bacterial-dependent current above 2.5×10⁵ cfu/ml with pure cultures of bacteria, but when applied to the measurement of the bacterial contamination in samples of meat and milk it was unable to detect or quantify the contamination reliably. Incubation of samples for a few hours before examination enabled the system to detect bacteria in excess of 10⁶ cfu/ml.     

Antagonistic effect of enterocin CCM 4231 from Enterococcus faecium on “bryndza”, a traditional Slovak dairy product from sheep milk

“Bryndza” is a traditional Slovak dairy product (type of soft cheese) made from sheep cheese which was ripened for 14 days. Because its manufacture, transporting and/or storing represent conditions which facilitate contamination, the effect of enterocin CCM4231 in “bryndza” was investigated with the aim to reduce the contaminant agents. “Bryndza” was divided into equal portions (50 g). The experimental sample (ES) as well as the control sample one (C1) were inoculated with Listeria innocua Li1 strain. The other control samples C2 and C3 were without Li1 strain. C3 control was selected as a reference control. ES and C2 portions were treated with purified enterocin CCM4231 in a concentration of 6400 AU/ml. Before the experimental inoculation, “bryndza” was checked for the presence of contaminant agents. The experiment lasted 1 week and the samples were stored in the refrigerator at 4 °C. Sampling was performed on day 1, on day 4 and on day 7. The control samples C2 and C3 were checked only on day 1 and then after 1 week. The following contaminant agents were detected in “bryndza” before its experimental inoculation with L. innocua Li1 strain: Escherichia coli in the amount 10³ cfu/ml/g, Staphylococcus aureus (10² cfu/ml/g) and enterococci (10⁴ cfu/ml/g). In the control sample C2, the number of E. coli was reduced to 10² cfu/ml/g. Enterococci and staphylococci were totally eliminated there. Concerning C3 control, natural decrease of bacteria was found and/or their unchanged counts. The value of pH (5) was stable during the whole experiment. In the experimental sample inoculated with Li1 strain, its counts were decreased immediately after enterocin CCM4231 addition approximately by one order of magnitude. This inhibitory effect was also detectable on day 4 by the difference of one order of magnitude between ES and C1. On day 7, 10³ cfu/ml/g of Li1 strain were detected in both samples (ES, C1). The difference by one order of magnitude indicated, an inhibitory effect of enterocin CCM4231 in “bryndza”. However, bacteriocin activity was not determined by laboratory analyses.     

COMPARISON OF LUMINOL CHEMILUMINESCENCE WITH ATP BIOLUMINESCENCE FOR THE ESTIMATION OF TOTAL BACTERIAL LOAD IN PURE CULTURES

A rapid chemiluminescent assay of total bacterial load that is based on the oxidation of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) as catalyzed by bacterial iron protoporphyrins is described and compared to the ATP bioluminescent assay of microbial biomass. An assay format that elicits linear light output response to a range of analyte concentrations of model compounds such as hematin and various heme-containing enzymes within the dynamic range of a BioOrbit 1251 luminometer is presented. When the assay was applied to eight pure bacterial cultures, the sensitivity was typically in the range of 10⁴-10⁵ cfu/ml, and was comparable to that obtained by the ATP assay. Similar levels of sensitivity can be derived from estimates of average values of 2.8 × 10^-18 mole of heme/cfu and 1 × 10^-19 mole of ATP/cfu. The potential of the luminal assay as an alternative rapid test for the estimation of total bacterial count in food and environmental samples is discussed.     

A high-performance liquid chromatography-tandem mass spectrometry assay of the androgenic neurosteroid 3alpha-androstanediol (5alpha-androstane-3alpha,17beta-diol) in plasma

The testosterone metabolite 3alpha-androstanediol (5alpha-androstane-3alpha,17-diol) is a potential GABA(A) receptor-modulating neurosteroid with anticonvulsant properties and hence could act as a key neuromodulator in the central nervous system. However, there is no specific and sensitive assay for quantitative determination of the androgenic neurosteroid 3alpha-androstanediol in biological samples. We have established a liquid chromatography-tandem mass spectrometry (LC-MS-MS) assay to measure 3alpha-androstanediol in rat plasma. Standard 3alpha-androstanediol added to rat plasma has been successfully analysed with excellent linearity, specificity, sensitivity, and reproducibility. The sensitivity of the method was < 10 ng/ml with a detection limit of 2 ng/ml (6.8 nmol/l) and a linear range of 10-2000 ng/ml. The method was used for the analysis of testosterone-induced increase in plasma 3alpha-androstanediol levels in rats. Testosterone produced a dose-dependent elevation in plasma 3alpha-androstanediol, which was almost completely prevented by pretreatment with the 5alpha-reductase inhibitor finasteride, indicating that 3alpha-androstanediol is synthesized from testosterone via a 5alpha-reductase pathway. This LC-MS-MS method allows accurate, high-throughput analysis of 3alpha-androstanediol in small amounts (200 microl) of plasma and possibly other biological samples.     

Detection of ricin and other ribosome-inactivating proteins by an immuno-polymerase chain reaction assay

Ribosome-inactivating proteins (RIPs) are plant proteins with enzymatic activity, classified as type 1 (single chain) or type 2 (two chains). They are identified as rRNA N-glycosidases (EC 3.2.2.22) and cause an irreversible inhibition of protein synthesis. Among type 2 RIPs, there are potent toxins (ricin is the best known) that are considered as potential biological weapons. The development of a fast and sensitive method for the detection of biological agents is an important tool to prevent or deal with the consequences of intoxication. In this article, we describe a very sensitive immuno-polymerase chain reaction (IPCR) assay for the detection of RIPs-a type 1 RIP (dianthin) and a type 2 RIP (ricin)-that combines the specificity of immunological analysis with the exponential amplification of PCR. The limit of detection (LOD) of the technique was compared with the LODs of the conventional immunological methods enzyme-linked immunosorbent assay (ELISA) and fluorescent immunosorbent assay (FIA). The LOD of IPCR was more than 1 million times lower than that of ELISA, allowing the detection of 10 fg/ml of dianthin and ricin. The possibility to detect ricin in human serum was also investigated, and a similar sensitivity was observed (10 fg/ml). IPCR appears to be the most sensitive method for the detection of ricin and other RIPs.     

Development and performance of an enzyme-linked amperometric immunosensor for the detection of Staphylococcus aureus in foods

An amperometric enzyme-linked immunosensor was developed to detect and quantify levels of Staphylococcus aureus electrically in pure cultures and in foods. The assay was a modification of a 'sandwich' ELISA for the protein A of Staph. aureus, employing catalase-labelled anti-protein A antibody. On addition of hydrogen peroxide to the assay system the catalase released O2 which was monitored using an amperometric oxygen electrode. The rate of current increase was proportional to the antigen concentration (protein A or Staph. aureus). Protein A was detected reliably at 0.1 ng/ml representing a 20-fold increase in sensitivity over the conventional ELISA that used horseradish peroxidase. Pure cultures of Staph. aureus were detected at 10(-3)-10(-4) cfu/ml with the amperometric electrode (cf greater than 10(5)/ml for conventional ELISA). The same level of sensitivity was achieved for inoculated food samples. Low levels of contamination (1 cfu/g) of Staph. aureus were detected after incubation at 37 degrees C for 18 h, and the immunosensor could from the basis of a test for screening and identification of protein A-bearing Staph. aureus in 24 h, although natural variations in protein A content between different strains could make the system unreliable in accurate quantification of cell numbers.     

Automated immunoassay system for AFP-L3% using on-chip electrokinetic reaction and separation by affinity electrophoresis

Implementation of the on-chip immunoassay for α-fetoprotein (AFP)-L3% was achieved using a fully automated microfluidic instrument platform that will prepare the chip and run the assay with a total assay time of less than 10 min. Reagent/sample mixing, concentration, and reaction in microfluidic channels occur by the electrokinetic analyte transport assay (EATA) technique, enabling the integration of all assay steps on-chip. The determination of AFP-L3%, a biomarker for hepatocellular carcinoma, was achieved by the presence of Lens culinaris agglutinin in the separation channel, causing separation of the fucosylated isoform, AFP-L3, from the nonfucosylated AFP-L1 by lectin affinity electrophoresis. Laser-induced-fluorescence (LIF) detection was used to quantitate the labeled immunocomplexes. The limit of detection (LOD) was 0.1 ng/ml AFP, and assay precision of less than 2% coefficient of variation (CV) was obtained for quantitation from 24 to 922 ng/ml total AFP in spiked serum samples. Assay precision of less than 3% CV was obtained for AFP-L3% measurements from 8.5 to 81%. Furthermore, good correlation of test results for 68 patient serum samples with a commercially available reference method (LiBASys assay for AFP-L3%) was obtained, with r² = 0.981 and slope = 1.03.     

Multi-analyte interrogation using the fiber optic biosensor

The capabilities of the portable, automated fiber optic biosensor, RAPTOR, have recently been evaluated. Developed to perform rapid fluoroimmunoassays in the field, the RAPTOR was designed to test samples for up to four different target analytes simultaneously. Assay time could be varied from a 3-min rapid screen to a standard 10-min test. A trial of 203 blind samples tested for Staphylococcal enterotoxin B, ricin, Francisella tularensis, and Bacillus globigii has been conducted. Sensitivities obtained were 10, 50 ng/ml, 5 x 10(5), and 5 x 10(4) cfu/ml, respectively.     

Rapid high-throughput assay for the measurement of amino acids from microdialysates and brain tissue using monolithic C18-bonded reversed-phase columns

A rapid precolumn high-performance liquid chromatography method based on fluorescence detection has been developed for the measurement of multiple amino acids from both ex vivo and in vivo biological samples using monolithic C18 columns. A mixture of 18 primary amino acids were derivatised with napthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide. The resulting isoindole derivatives were resolved within 10 min using a linear binary gradient elution profile with Rs values in the range 1.2-9.0. The limit of detection (LOD) was found to be between 6.0 and 60 fmol for 5 microl injection with a signal to noise ratio of 3:1. The NDA derivatives were found to be stable for 9 h at 4 degrees C. This assay has been employed for the rapid analysis of amino acids from brain tissue and microdialysis samples. Examples of application of the method are given.     

Experimental enzyme-linked amperometric immunosensors for the detection of salmonellas in foods.

Two enzyme-linked amperometric immunosensors specific for salmonellas were developed as rapid methods for quantifying and detecting these organisms in pure cultures and foods. Both used alkaline phosphatase as the enzyme reporter molecule but one system used phenyl phosphate as the substrate followed by the electrochemical detection of phenol at a polarized platinum electrode. The other system incorporated an enzyme amplification step and relied on the electrochemical detection of a reduced mediator, ferrocyanide. Both assays were rapid (4 h) and specific and generated salmonella-dependent signals above 10(4) cfu/ml (phenyl phosphate system) or 10(5) cfu/ml (enzyme amplified system) in pure cultures and samples of several foods, although the results with beef samples showed considerable variation. Both systems were able to detect low (1-5 cfu/g or /ml) numbers of salmonellas in foods after non-selective (18 h) and selective (22 h) enrichment steps but four samples, out of 147, gave false positive results. False positive results were eliminated by reducing the enrichment steps to 6 h and 18 h respectively (90 samples).     

Experimental enzyme-linked amperometric immunosensors for the detection of salmonellas in foods

Two enzyme-linked amperometric immunosensors specific for salmonellas were developed as rapid methods for quantifying and detecting these organisms in pure cultures and foods. Both used alkaline phosphatase as the enzyme reporter molecule but one system used phenyl phosphate as the substrate followed by the electrochemical detection of phenol at a polarized platinum electrode. The other system incorporated an enzyme amplification step and relied on the electrochemical detection of a reduced mediator, ferrocyanide. Both assays were rapid (4 h) and specific and generated salmonella-dependent signals above 10⁴ cfu/ml (phenyl phosphate system) or 10⁵ cfu/ml (enzyme amplified system) in pure cultures and samples of several foods, although the results with beef samples showed considerable variation. Both systems were able to detect low (1–5 cfu/g or /ml) numbers of salmonellas in foods after non-selective (18 h) and selective (22 h) enrichment steps but four samples, out of 147, gave false positive results. False positive results were eliminated by reducing the enrichment steps to 6 h and 18 h respectively (90 samples).