An electrochemical immunosensor based on enzyme-encapsulated liposomes and biocatalytic metal deposition

A novel electrochemical immunosensor based on double signal amplification of enzyme-encapsulated liposomes and biocatalytic metal deposition was developed for the detection of human prostate specific antigen (PSA). Alkaline phosphatase (ALP)-encapsulated and detection antibody-functionalized liposomes were first prepared and used as the detection reagent. In the sandwich immunoassay, the model analyte PSA was first captured by anti-PSA capture antibody immobilized on the electrode and then sandwiched with the functionalized liposomes. The bound liposomes were then lysed with surfactant to release the encapsulated ALP, which served as secondary signal amplification means. ALP on the electrode surface initiated the hydrolysis of ascorbic acid 2-phosphate (AA-p) to produce ascorbic acid. The latter, in turn, reduced silver ions on the electrode surface, leading to deposition of the metal silver on the electrode surface. Linear sweep voltammetry (LSV) was chosen to detect the amount of the deposited silver. The results showed that the anodic stripping peak current was linearly dependent on the PSA concentration in the range of 0.01-100 ng mL⁻¹, and a detection limit as low as 0.007 ng mL⁻¹ can be obtained. Since the cut-off value of human PSA is 4 ng mL⁻¹, the proposed electrochemical immunosensor would be expected to gain widespread applications for the detection of PSA in clinical diagnosis.     

A fast and sensitive enzyme immunoassay for brain natriuretic peptide based on micro-magnetic probes strategy

In this paper, a simple, rapid and low-cost method for the high-sensitivity detection of brain natriuretic peptide (BNP) was developed, which adopted three amplification steps: (a) biotin-streptavidin amplification; (b) micro-magnetic probe amplification; (c) HRP (horseradish peroxidase) signal amplification. In the present strategy, the streptavidin-coated micro-magnetic particles (MMPs) were first conjugated with biotin-labeled capture antibody via the biotin-streptavidin interaction, which formed bio-functional micro-magnetic probes. Then, the analyte (antigen) is sandwiched by HRP-labeled antibody and capture antibody bound to MMPs. Finally, the HRP at the surface of sandwich structures catalytically oxidized the substrate and generated optical signals that reflected the amount of the target BNP. The influence of some important parameters such as the size of magnetic particles, the working concentration of HRP-labeled BNP antibody, the stability of magnetic probes, and the assay medium of serum BNP, etc. on the detection ability of present method was investigated in details. It is found that the detection limit of the proposed method could reach 10 pg/mL for BNP, which is much lower than that of sandwich-type ELISA. Furthermore, this detection time for the proposed method just takes about 30 min (two reaction steps and one wash step), which is faster than that of conventional sandwich-type ELISA (taking about 4 h, three reaction steps and three wash steps). Inspired by these advantages, it is expected that this method can probably be applicable to the detection of other hormones and tumor markers that are present in only low concentrations within the human body.     

Dopamine-loaded Liposomes-amplified Electrochemical Immunoassay Based on MXene (Ti3C2)−AuNPs

A simple and novel electrochemical immunoassay based on MXene (Ti₃C₂)−Au nanoparticles (AuNPs) was designed for sensitive screening of a disease-related biomarker, prostate-specific antigen (PSA), by using dopamine-loaded liposomes (DLL) for signal amplification. The system involves two parts, namely, sandwich-type immunoreaction to capture DLL and electrochemical measurement of dopamine. The target PSA can cause a specific antigen-antibody reaction and DLL are enriched in the enzyme-labeled pores. After Triton X-100 is injected into the detection cell, the carried DLL was quickly cracked to release dopamine wrapped in the cavity. A nanocomposite consisting of MXene (Ti₃C₂) support to immobilize Au nanoparticles (Ti₃C₂−Au) was utilized to modify a glassy carbon electrode, which gives a strongly enhanced differential pulse voltammetric (DPV) signals for dopamine. In this case, the change of DPV signal depends on the amount of dopamine released by liposomes, which is further positively correlated with the concentration of the analyte PSA. Combining the of MXene (Ti₃C₂)−AuNPs nanomaterials (large specific surface area, excellent electrical conductivity, and good electrocatalytic properties) with the liposome signal amplification strategy, the electrochemical immunoassay exhibited excellent performance toward PSA determination with a broad linear range of 1 pg/mL to 50 ng/mL and limit of detection down to 0.31 pg/mL (S/N=3) under the optimized testing conditions. High specificity for PSA over other disease-related biomarkers and acceptable nanocomposite/electrode stability were acquired. The excellent analytical performance shows that the current strategy provides an effective detection platform for clinical sample analysis.     

The study of a chemiluminescence immunoassay using the peroxyoxalate chemiluminescent reaction and its application

The paper presented a novel chemiluminescence (CL) immunoassay method, which combines the advantages of traditional enzyme-linked immunosorbent assays (ELISA) and bis (2,4,6-trichlorophenyl) oxalate (TCPO)-hydrogen peroxide CL detection system. A fluorescent product 2,3-diaminophenazine (DAPN) was produced by reaction between o-phenylenediamine (OPDA, 1,2-diaminobenzene) and H₂O₂ catalyzed by horseradish peroxidase (HRP). DAPN was excited by the reactive intermediate of TCPO-H₂O₂ chemiluminescent reaction, and led to CL. The dependence of the CL intensity on the concentrations of antigen was studied. As analytical application, the proposed method was used for determination of recombinant human interleukin 6 (rHu IL-6) and β-human chorionic gonadotropin (β-HCG). Under the selected experimental conditions, a linear relationship was obtained between the CL intensity and the concentration of rHu IL-6 in the range of 4.0-625.0 pg/ml, and β-HCG in the range of 12.5-400.0 mIU/ml. The detection limits were 0.5 pg/ml for rHu IL-6 and 3 mIU/ml for β-HCG with relative standard deviation of 2.3 for 78.0 pg/ml rHu IL-6, and 3.9 for 50.0 mIU/ml β-HCG. This method has been applied to the determination of rHu IL-6 in human serum and β-HCG in urine with satisfactory results.     

Construction of supported lipid membrane modified piezoelectric biosensor for sensitive assay of cholera toxin based on surface-agglutination of ganglioside-bearing liposomes

A novel piezoelelctric biosensor has been developed for cholera toxin (CT) detection based on the analyte-mediated surface-agglutination of ganglioside (GM1)-functionalized liposomes. To achieve a CT-specific agglutination at the surface, the gold electrode is modified by a GM1-functionalized supported lipid membrane via spontaneous spread of the liposomes on a self-assembled monolayer of a long-chain alkanethiol. In the presence of CT, the GM1-incorporated liposomes in assay medium will rapidly specifically agglutinate at the electrode surface through the binding of CT to GM1 on the electrode surface and the liposome interface. This results in an enormous mass loading on the piezoelelctric crystal as well as a significant increase of density and viscosity at the interface, thereby generating a decrease in frequency of the piezoelelctric crystal. The combination of mass loading with interfacial change in the surface-agglutination reaction allows the developed piezoelelctric biosensor to show substantial signal amplification in response to the analyte CT. The detection limit can be achieved as low as 25 ng mL⁻¹ CT. This is the first demonstration on CT detection based on specific surface-agglutination of GM1-modified liposomes. The supported lipid layer based sensing interface can be prepared readily and renewably, making the developed technique especially useful for simple, reusable and sensitive determination of proteins.     

Determination of peroxidase encapsulated in liposomes using homogentisic acid y-lactone chemiluminescence.

Homogentisic acid gamma-lactone (HAL) chemiluminescence (CL) was applied to the determination of horseradish peroxidase (HRP) encapsulated in liposomes. HRP was detected after the lysis of HRP-trapped liposomes with Triton X-100. CL response rate, detection limit and linear range of calibration curve for HRP in HAL CL were compared with those in piodophenol (p-IP)-enhanced luminol CL. Maximal light emission in HAL CL appeared more rapidly compared to that in p-IP enhanced luminol CL, thus resulting in remarkable reduction of CL measurement time. The detection limit for HRP in HAL CL was the same as that in p-IP-enhanced luminol CL. The linear range of calibration curve for HRP in HAL CL was improved by a factor of 50 compared with that in p-IP-enhanced luminol CL. From these results, it was found that HAL CL were superior to p-IP-enhanced luminol CL for the determination of HRP encapsulated in liposomes.     

Liposome-based flow injection enzyme immunoassay for theophylline

Preliminary results are presented on the development of a sensitive, quantitative immunoassay based on a regenerable, flow injection analysis system incorporating a double-amplification approach. The double amplification is achieved by means of liposome-encapsulated peroxidase enzyme molecules which are released subsequent to a competitive immunological reaction with analyte molecules for immobilized antibodies. The released peroxidase enzymatically cleaves, from an organofluorine substrate, fluoride ions which are then potentiometrically measured. The entire process is carried out in a flow injection analysis system. The competition between the analyte molecules (theophylline) and theophylline-derivatized liposomes for immobilized antibody sites in flow-through immunoreactor column results in unbound liposomes being carried downstream where they are ruptured in the presence of hydrogen peroxide andp-fluorophenol. The peroxidase molecules released react enzymatically to produce fluoride ions which are measured with an ion-selective electrode. The immunoreactor column is then regenerated with a chaotropic agent and the next sample or calibration solution is injected. By means of column regeneration and calibration, accurate quantitation can be achieved; a feature missing from conventional batch-type immunoassays. By means of this liposome/enzyme double-amplification approach, theophylline was determined over a range of concentrations from 0.2 to 4000 ng/ml. The detection limit of 200 pg/ml corresponds to about 100 femtomole of theophylline measured in the 100 l sample injected.Dedicated to Professor W. Simon on the occasion of his 60th birthday     

Aptamer sandwich assays: human α-thrombin detection using liposome enhancement

Fluorescent dye-encapsulating liposomes tagged with aptamers were developed and used as reporting signals in an aptamer-based sandwich assay. α-Thrombin was utilized as a prototypical analyte as two well-studied aptamers binding distinct epitopes are available to form a sandwich complex. Cholesteryl–TEG-modified aptamers were embedded into the liposomal lipid bilayer while the interior cavity of the liposomes encapsulated fluorescent sulforhodamine B dye. Such liposomes successfully formed a sandwich complex with α-thrombin and a microtiter plate immobilized aptamer, proving that aptamers retain their ability to fold when anchored to the liposome surface. Parameters studied included liposomal aptamer coverage, sandwich aptamer orientation, aptamer label orientation, aptamer spacer length and type, incubation buffer, and aptamer concentration. The optimized conditions found here in the fluorescence assay led to a limit of detection of 64 pM or 2.35 ng/mL, corresponding to 6.4 fmol or 235 pg, respectively, in a 100 μL volume. This is an order of magnitude lower than previous sandwich aptamer assays using the same sequences with lowest reported limits of detection of 0.45 nM. In addition, the assay was applied successfully to the detection of α-thrombin in human plasma. The success of this method in a standard microtiter plate format and the relatively facile functionalization of liposomes with aptamers suggest that this approach provides a versatile option for routine analytical applications.     

Determination of umbilical cord and maternal plasma concentrations of fentanyl by using novel spectrophotometric and chemiluminescence enzyme immunoassays

Two novel enzyme immunoassays of fentanyl have been developed using Horseradish Peroxidase (HRP) as an enzyme, 3,3′,5,5′tetramethylbenzidine (TMB) and luminol as its colorimetric and its chemiluminescence substrate, respectively. A fentanyl polyclonal antibody was used as a capture antibody for fentanyl and fentanyl-bovine serum albumin (BSA) conjugate. The latter was first biotinylated and then bound by streptavidin labeled with HRP, resulting in the development of two novel competitive immunoassays. The detection limits were 0.045 and 0.0048 ng ml⁻¹ for spectrophotometric and chemiluminescence detection, respectively, and were much lower than existing HRP-fentanyl based kits. Intra- and inter-assay CVs were 2.6-4.5 and 5.4-11.2%, respectively, at concentrations of 0.050-5.000 ng ml⁻¹ for the colorimetric assay, whilst for the chemiluminescent assay intra- and inter-assay CVs were 3.7-6.2 and 6.2-12.3%, respectively, for the linear range of the assay at concentrations of 0.008-0.800 ng ml⁻¹. The methods in this study were developed in order to measure maternal and neonatal fentanyl plasma samples during cesarean section, after the application of a novel subarachnoid analgesia technique. The 28 maternal and neonatal plasma samples were measured by both assays, providing data for subarachnoid administration of fentanyl that had never been presented before.     

Microfluidic bead-based multienzyme-nanoparticle amplification for detection of circulating tumor cells in the blood using quantum dots labels

This study reports the development of a microfluidic bead-based nucleic acid sensor for sensitive detection of circulating tumor cells in blood samples using multienzyme-nanoparticle amplification and quantum dot labels. In this method, the microbeads functionalized with the capture probes and modified electron rich proteins were arrayed within a microfluidic channel as sensing elements, and the gold nanoparticles (AuNPs) functionalized with the horseradish peroxidases (HRP) and DNA probes were used as labels. Hence, two signal amplification approaches are integrated for enhancing the detection sensitivity of circulating tumor cells. First, the large surface area of Au nanoparticle carrier allows several binding events of HRP on each nanosphere. Second, enhanced mass transport capability inherent from microfluidics leads to higher capture efficiency of targets because continuous flow within micro-channel delivers fresh analyte solution to the reaction site which maintains a high concentration gradient differential to enhance mass transport. Based on the dual signal amplification strategy, the developed microfluidic bead-based nucleic acid sensor could discriminate as low as 5 fM (signal-to-noise (S/N) 3) of synthesized carcinoembryonic antigen (CEA) gene fragments and showed a 1000-fold increase in detection limit compared to the off-chip test. In addition, using spiked colorectal cancer cell lines (HT29) in the blood as a model system, the detection limit of this chip-based approach was found to be as low as 1 HT29 in 1 mL blood sample. This microfluidic bead-based nucleic acid sensor is a promising platform for disease-related nucleic acid molecules at the lowest level at their earliest incidence.     

Magnetic beads-based immunoassay as a sensitive alternative for atrazine analysis

A new immunoassay strategy for sensitive atrazine determination based on magnetic beads is reported. The immuno-method is a competitive solid-phase immunoassay where the anti-atrazine antibody is immobilized on the magnetic beads surface and fixed at the reaction cell bottom using a simple magnet, which generates a magnetic field. Analyte and HRP (horseradish peroxidase) tracer compete for active sites of antibody. After the immunointeractions antibody-analyte and antibody-tracer, atrazine quantification from the sample is performed by injection of the chemiluminescence substrate (luminol, hydrogen peroxide and p-iodophenol). Different antibodies (polyIgG anti-atrazine Ab I and affinity purified polyIgG anti-atrazine AbI) were tested in this configuration. Also, optimum concentration of antibody-covered magnetic beads was set up (8 mg/l Ab II). Finally, the performance of magnetic beads-based immunoassay for atrazine determination was evaluated demonstrating that the magnetic beads-based immunoassay is one of the most sensitive method for atrazine determination (LoD = 3 pg/l, IC₅₀ = 37 pg/l, DR = 10-1000 pg/l).     

偶合反应化学发光酶免疫分析研究: I. HRP及其标记物的化学发 光测定

本文将辣根过氧化物酶(HRP)催化过氧化氢氧化KI生成I~2的反应与Luminol-I~2的化学发光反应相偶合,提出了测定HRP及其抗原、抗体标记物的高灵敏度的化学发光法。检测下限为7pg,线性范围为10-6000pg。通常使用的HRP直接催化Luminol-H~2O~2化学发光法灵敏度高10-100倍;克服了固相吸附法直接测定HRP标记物的缺陷,提高了测定的选择性。文中还对该偶合反应的机理及其动力学特性进行了研究。     

A repeatable assembling and disassembling electrochemical aptamer cytosensor for ultrasensitive and highly selective detection of human liver cancer cells

In this work, a repeatable assembling and disassembling electrochemical aptamer cytosensor was proposed for the sensitive detection of human liver hepatocellular carcinoma cells (HepG2) based on a dual recognition and signal amplification strategy. A high-affinity thiolated TLS11a aptamer, covalently attached to a gold electrode through Au–thiol interactions, was adopted to recognize and capture the target HepG2 cells. Meanwhile, the G-quadruplex/hemin/aptamer and horseradish peroxidase (HRP) modified gold nanoparticles (G-quadruplex/hemin/aptamer–AuNPs–HRP) nanoprobe was designed. It could be used for electrochemical cytosensing with specific recognition and enzymatic signal amplification of HRP and G-quadruplex/hemin HRP-mimicking DNAzyme. With the nanoprobes as recognizing probes, the HepG2 cancer cells were captured to fabricate an aptamer-cell-nanoprobes sandwich-like superstructure on a gold electrode surface. The proposed electrochemical cytosensor delivered a wide detection range from 1 × 10² to 1 × 10⁷ cells mL⁻¹ and high sensitivity with a low detection limit of 30 cells mL⁻¹. Furthermore, after the electrochemical detection, the activation potential of −0.9 to −1.7 V was performed to break Au–thiol bond and regenerate a bare gold electrode surface, while maintaining the good characteristic of being used repeatedly. The changes of gold electrode behavior after assembling and desorption processes were investigated by electrochemical impedance spectroscopy and cyclic voltammetry techniques. These results indicate that the cytosensor has great potential in disease diagnostic of cancers and opens new insight into the reusable gold electrode with repeatable assembling and disassembling in the electrochemical sensing.     

Simultaneous electrochemical detection of multiple tumor markers based on dual catalysis amplification of multi-functionalized onion-like mesoporous graphene sheets

In this work, a sandwich-type electrochemical immunosensor for simultaneous sensitive detection of prostate specific antigen (PSA) and free prostate specific antigen (fPSA) is fabricated. Gold nanoparticles (AuNPs) modified Prussian blue and nickel hexacyanoferrates nanoparticles were firstly prepared, respectively, and then decorated onion-like mesoporous graphene sheets (denoted as Au@PBNPs/O-GS and Au@NiNPs/O-GS) as distinguishable signal tags to label different detection antibodies. Subsequently, streptavidin and biotinylated alkaline phosphatase (bio-AP) were employed to block the possible remaining active sites. With the employment of the as prepared nanohybrids, the dual catalysis amplification can be achieved by catalysis of the ascorbic acid 2-phosphate to in situ produce AA in the presence of bio-AP, and then AA was further catalyzed by Au@PBNPs/O-GS and Au@NiNPs/O-GS nanohybrids, respectively, to obtain the higher signal responses. The experiment results show that the linear range of the proposed immunosensor for simultaneous determination of fPSA is from 0.02 to 10 ng mL⁻¹ with a detection limit of 6.7 pg mL⁻¹ and PSA is from 0.01 to 50 ng mL⁻¹ with a detection limit of 3.4 pg mL⁻¹ (S/N = 3). Importantly, the proposed method offers promise for rapid, simple and cost-effective analysis of biological samples.     

Determination of clenbuterol by capillary electrophoresis immunoassay with chemiluminescence detection

A competitive immunoassay for clenbuterol (CLB) based on capillary electrophoresis with chemiluminescence (CL) detection was established. The method was based on the competitive reaction of horseradish peroxidase (HRP)-labeled CLB (CLB-HRP) and free CLB with anti-CLB antiserum. The factors affecting the electrophoresis and CL detection were systematically investigated with HRP as a model sample. Under the optimal conditions, the tracer CLB-HRP and the immunoassay complex were separated, and the linear range and the detection limit (S/N = 3) for CLB were 5.0-40 nmol l⁻¹ and 1.2 nmol l⁻¹, respectively. The proposed method has been applied satisfactorily in the analysis of urine sample.     

Direct determination of horseradish peroxidase encapsulated in liposomes by using luminol chemiluminescence.

Horseradish peroxidase (HRP) encapsulated in liposomes was directly detected by using luminol chemiluminescence (CL) with H2O2 without lysis of liposomes. At a low concentration of H2O2, the initial rate of HRP-catalyzed luminol CL in liposomes was slower than that of HRP-catalyzed luminol CL in a lipid-free bulk solution. The decrease in the initial rate of the CL reaction in liposomes was due to the membrane permeation of luminol and H2O2. At a high concentration of H2O2, the initial rate of the CL reaction in liposomes was the same as that in a lipid-free bulk solution. The CL measurement conditions in both a lipid-free bulk solution and in liposomes were optimized in the concentrations of luminol and H2O2 by measuring the CL response curves, in which only one peak appeared and the CL intensity was maximal. The CL intensity observed in HRP-catalyzed luminol CL in liposomes was a factor of seven greater than that observed in a lipid-free bulk solution. The CL intensity was dependent on the amount of HRP-encapsulated liposomes used. The detection limit in the direct detection of HRP encapsulated in liposomes was sensitive by a factor of 3 compared with that in HRP-catalyzed luminol CL in a lipid-free bulk solution.     

Development of an automatic multi-channel ink-jet ejection chemiluminescence system and its application to the determination of horseradish peroxidase

In this work, an automatic multi-channel ink-jet for chemiluminescence (CL) analysis was developed. The four-channel ink-jet device was controlled by a home-made circuit. Differing from the classic flow injection CL, the whole procedure for CL analysis was automatically completed on a hydrophobic glass side. CL reaction of luminal and hydrogen peroxide for the determination of horseradish peroxidase (HRP) was selected as an application to automatic CL analysis platform. All solutions delivered by different channels were precisely ejected to the same position of the glass slide for the CL analysis. The consumption of reaction solution was reduced to nanoliter level. The whole CL analysis could be completed in less than 4 min, which was benefited from the prompt solution mixing in small size of droplet. The CL intensity increased linearly with HRP concentration in the range from 0.01 to 0.5 μg mL⁻¹. The limit of detection (LOD) (S/N = 3) was 0.005 μg mL⁻¹. Finally, the automatic CL system could also be used for the detection of HRP in HRP–protein conjugates, which showed its practical application in immunoassay.     

Trace determination of airborne polyfluorinated iodine alkanes using multisorbent thermal desorption/gas chromatography/high resolution mass spectrometry

A novel gas chromatography/high resolution mass spectrometry method coupled with multisorbent thermal desorption cartridges has been developed for the determination of volatile neutral polyfluorinated iodine alkanes (PFIs) in airborne samples. It allows, for the first time, simultaneous analysis of four mono-iodized perfluorinated alkanes, three diiodized perfluorinated alkanes and four mono-iodized polyfluorinated telomers in ambient air samples. 3.75 L air sample was passed through a sorbent tube packed with 150 mg of Tenax TA and 200 mg of Carbograph 1TD for analyte adsorption. Important factors during the analysis procedures, such as safe sampling volume, air sampling rate, analyte desorption and transfer strategies, were optimized and good thermal desorption efficiencies were obtained. The method detection limit (MDL) concentration ranged from 0.04 pg/L for 1H,1H,2H,2H-perfluorododecyl iodide to 1.2 pg/L for perfluorohexyl iodide, and instrument response of a seven-point calibration was linear in the range of 10–1000 pg. Travel spike recoveries ranged from 83% to 116%. Small variabilities of less than 36% were obtained near the MDLs and the differences between triplicates were even smaller (2.1–7.3%) at 200 pg spiked level. The method was successfully applied to analyze ambient air samples collected near a point source, and five PFIs were identified (10.8–85.0 pg/L), with none of the analytes detectable at the background site.     

Application of ZnO quantum dots dotted carbon nanotube for sensitive electrochemiluminescence immunoassay based on simply electrochemical reduced Pt/Au alloy and a disposable device

We report on a disposable microdevice suitable for sandwich-type electrochemiluminescence (ECL) detection of prostate specific antigen (PSA). The method is making use of ZnO quantum dots dotted carbon nanotube (ZnO@CNT) and simply electrochemical reduced Pt/Au alloy. The latter was selected as immunosensing probe to modify screen-printed carbon electrode, due to its excellent electrical property. For further ultrasensitive, low-potential and stable ECL detection, ZnO@CNT composite was first synthesized using a facile solvothermal method, and employed as signal amplification label. In this work, two working electrodes in one device were used for one determination to obtain more exact results based on screen-print technique. Taking advantage of dual-amplification effects of the Pt/Au and ZnO@CNT, this immunosensor could detect the PSA quantitatively, in the range of 0.001–500 ng mL⁻¹, with a low detection limit of 0.61 pg mL⁻¹. The resulting versatile immunosensor possesses high sensitivity, satisfactory reproducibility and regeneration. This simple and specific strategy has vast potential to be used in other biological assays.     

Chemiluminescence multichannel immunosensor for biodetection

An improved portable detector for biological compounds, the chemiluminescence multichannel immunosensor (CL-MADAG), has been developed and characterised. The device is based on a capillary ELISA technique in combination with a miniaturised fluidics system and uses chemiluminescence as the detection principle. The fluidics system construction allows three chemiluminescence immunoassays to be performed simultaneously within three fused silica capillaries (FSC). The CL-MADAG was characterised in a series of experiments with staphylococcal enterotoxin B (SEB) as a model toxin, the bacterial phage virus M13 as a virus simulant, and a pathogenic strain of Escherichia coli as simulant for bacteria. It was shown that the CL-MADAG can assay liquid samples for these substances within 24 min. The detection limits were 5 ng/ml for SEB, 10⁵ cfu/ml for E. coli O157:H7 and 10⁷ pfu/ml for M13.