Petr Skládal

Micromechanical cantilever-based biosensors

Abstract The merging of silicon microfabrication techniques with surface functionalization biochemistry offers new exciting opportunities in developing microscopic biomedical analysis devices with unique characteristics. Micro-mechanical transducers for chemical and biosensing applications represent one possibility. Microcantilevers can transduce a chemical signal into a mechanical motion with high sensitivity. In this review we summarize how cantilever-based sensors can be operated, and their working principle is presented in few selected biosensing experiments which have been performed recently in our groups in the study of biotin–streptavidin and antigen–antibody interactions, and specific surface charge development of organic molecules. We also discuss the advantages of this novel technique as well as its potentials.

Nanoparticle-Based Immunochemical Biosensors and Assays: Recent Advances and Challenges

We review the progress achieved during the recent five years in immunochemical biosensors (immunosensors) combined with nanoparticles for enhanced sensitivity. The initial part introduces antibodies as classic recognition elements. The optical sensing part describes fluorescent, luminescent, and surface plasmon resonance systems. Amperometry, voltammetry, and impedance spectroscopy represent electrochemical transducer methods; electrochemiluminescence with photoelectric conversion constitutes a widely utilized combined method. The transducing options function together with suitable nanoparticles: metallic and metal oxides, including magnetic ones, carbon-based nanotubes, graphene variants, luminescent carbon dots, nanocrystals as quantum dots, and photon up-converting particles. These sources merged together provide extreme variability of existing nanoimmunosensing options. Finally, applications in clinical analysis (markers, tumor cells, and pharmaceuticals) and in the detection of pathogenic microorganisms, toxic agents, and pesticides in the environmental field and food products are summarized.

Sensitive detection of pesticides using amperometric sensors based on cobalt phthalocyanine-modified composite electrodes and immobilized cholinesterases

The determination of organophosphate and carbamate pesticides was carried out using cobalt phthalocyanine-modified carbon epoxy composite electrodes coupled with acetylcholinesterase or butyrylcholinesterase. Covalent immobilization of enzymes on Immobilon membranes or nylon nets was examined; the highest sensitivity to inhibitors was found for the nylon net containing low enzyme loading and this was subsequently used for the construction of an amperometric biosensor for pesticides. Analyses were done using acetyl- or butyrylthiocholine as substrates; thiocholine produced by hydrolysis in the enzyme membrane was electrochemically oxidized at +300 mV (vs. Ag/AgCl reference). The decrease of substrate steady-state current caused by the addition of pesticide was used for evaluation. With this approach, 1.5 and 8.4 micrograms l-1 of paraoxon and heptenophos, respectively, can be detected in less than 3 min. These detection limits are similar as those obtained when analyses were performed using free cholinesterase and 10 min incubation with inhibitor.

Detection of organophosphate and carbamate pesticides using disposable biosensors based on chemically modified electrodes and immobilized cholinesterase

An amperometric biosensor for the detection of organophosphate and carbamate pesticides was constructed as a disposable strip containing a cobalt phthalocyanine-modified carbon composite electrode and a cross-linked cholinesterase layer. With butyrylthiocholine as substrate, enzymatically produced thiocholine was oxidized at +250 mV. The steady-state current, Iss, was used as a measure of the enzyme activity. In the presence of pesticides, an irreversible inhibition of cholinesterase occurred, resulting in a decrease in the rate of current change, dI/dt. The ratio (dI/dt)/Iss was used for evaluations. The influence of the cholinesterase loading and the cholinesterase to glutaraldehyde ratio on the biosensors response was studied and the measuring conditions (pH, temperature, substrate concentration) were optimized. Detection limits of 0.30, 1.2 and 11 nmol l−1 for paraoxon, dichlorvos and carbaryl, respectively, were achieved. The time of inhibition varied from a few seconds (high pesticide concentrations) to 6 min required for reliable measurements at levels close to the detection limit. When the analysis was performed by 10 min preincubation of the biosensor or free cholinesterase with sample, paraoxon concentrations above 3.0 nmol l−1 could be detected.

Piezoelectric quartz crystal sensors applied for bioanalytical assays and characterization of affinity interactions

This review presents piezoelectric quartz crystals as transducers suitable for development of different types of bioanalytical assays. The components of measuring systems for piezosensors are described together with providers of commercial equipment. The piezoelectric biosensors are summarized for determination of viruses, bacterial and other cells, proteins, nucleic acids and small molecules as drugs, hormones and pesticides. In addition to mass changes, some agglutination assays employing viscosity effects are addressed. Finally, the direct label-free and real-time monitoring of affinity interactions using piezosensors is presented. The theoretical background for determination of appropriate kinetic rate and equilibrium constants is shown and the approach is demonstrated on the interaction of antibody with the corresponding antigen (protein secalin). Several examples of affinity studies are provided, including interactions of proteins (antibody and antigens, receptors and ligands), nucleic acids (hybridization, intercalation of metal complexes), lipids and saccharide-based layers.

A disposable amperometric immunosensor for 2,4-dichlorophenoxyacetic acid

Abstract A disposable immunochemical biosensor for the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was developed using a screen-printed electrode system as an amperometric transducer and monoclonal antibodies against 2,4-D as a biospecific part. At first, the electrochemical system for determination of peroxidase activity was studied. For measurements, the peroxidase substrates were hydrogen peroxide together with either hydroquinone, 1,2-phenylenediamine or pyrocatechol as electron donors, which were amperometrically detected using platinum, gold and graphite composite layers as working electrode materials. The optimal concentrations of reaction components were found, the highest signals being achieved for hydroquinone with gold or graphite electrodes. The immunochemical system makes use of the competitive assay principle with 2,4-D covalently immobilized either directly on the surface of the gold electrode or on a separate nylon membrane. The peroxidase-antibody conjugate was synthesized and used as a tracing reagent. By using 2,4-D immobilized through its carboxylic group directly to the silanized surface of the gold working electrode, a detection limit close to 0.1 μg/1 of free 2,4-D was achieved. One analysis was completed in 1 h (30 min for the immunochemical reaction, washing, 25 min incubation with substrate, measurement).

Determination of organophosphate and carbamate pesticides using a cobalt phthalocyanine-modified carbon paste electrode and a cholinesterase enzyme membrane

Abstract An amperometric biosensor for the detection and determination of organophosphate and carbamate pesticides is based on a cobalt phthalocyanine-modified carbon paste electrode covered with a cholinesterase enzyme membrane. The electrode poised at +400 mV (vs. Ag/AgCl/3 M KCl) oxidizes thiocholine formed by butyrylthiocholine hydrolysis in the glutaraldehyde-cross-linked cholinesterase layer. The activity of cholinesterase is non-competitively inhibited in the presence of pesticides. A linear relationship exists between the relative decrease in steady-state current after the addition of a sample and the inhibitor concentration. This method of evaluation allows the repetitive use of an enzyme membrane; twenty analyses can be carried out with a single cholinesterase membrane. The sensitivity varies from 0.181 (Hostaquick) to 0.00136 s −1 g −1 1(Seedox); detection limits are 0.3 and 80 mg 1 −1 , respectively, for these two pesticides. The determination of eleven pesticides is demonstrated.

Construction and characterization of the direct piezoelectric immunosensor for atrazine operating in solution

The direct immunosensor for determination of the herbicide atrazine was studied. The gold electrodes of the piezoelectric quartz crystal were silanized and activated using glutaraldehyde. The bioaffinity ligand atrazine was linked through albumin as a spacer molecule. The modified piezoelectric crystal was placed in a flow cell and all measurements were performed directly in flowing solution. The interaction of the anti atrazine monoclonal antibody (MAb, clone D6F3) with the immobilized atrazine was characterized using both crude ascitic fluid and Protein A-purified MAb preparates. The association and dissociation rate constants were determined, ka = 1.21 x 10(5) M-1S-1 and kd = 4.0 x 10(-4)S-1. The competitive determination of free atrazine was studied using different dilutions (100x, 250x and 1000x) of the ascitic fluid containing MAb. MAb was preincubated with atrazine (concentrations 0-1 microgram/l) for 15 min and the mixture was then introduced to the flow cell. As a signal, either the rate of frequency decrease or the relative change of frequency after the fixed binding period (10 min) was evaluated. As expected, the higher dilutions of MAb provided improved sensitivity for the analyte. For the 1000x diluted ascitic fluid, 0.1 and 1 microgram/l atrazine caused 5 and 30% decreases of the relative binding of MAb, respectively. Repeated use of the crystals was achieved using a 5 min flow of 100 mM NaOH for regeneration. The results obtained seem to be promising for determination of atrazine in drinking water using direct piezoelectric immunosensors.

Improved direct piezoelectric biosensors operating in liquid solution for the competitive label-free immunoassay of 2,4-dichlorophenoxyacetic acid

Abstract The development of a label-free direct piezoelectric immunosensor is reported. The piezoelectric crystals were modified with 2,4-dichlorophenoxyacetic acid (2,4-D) using coupling procedures based on self-assembled monolayers of suitable thiocompounds (aminothiophenol, cysteamine, cystine and dithiobis(succinimidyl)propionate) on the surface of gold electrodes of the crystals. The piezoelectric biosensor has been placed in a flow-through cell and the affinity binding of two monoclonal antibodies against 2,4-D on the modified piezoelectric crystals was studied in real time without any additional labels. For the interactions, the kinetic equilibrium association constants Ka were 12.6 and 25.0 μM−1 for the antibodies E2/G2 and E4/C2, respectively. The system was used for competitive determination of free 2,4-D in water. For the antibodies E2/G2 and E4/C2 (both in concentration of 15 μg/ml), the limits of detection (10% decrease of relative binding of the antibody) for free 2,4-D were 0.27 and 0.24 μg/l, respectively. The total time for one measurement was below 25 min (10 min preincubation of antibody with sample, 10 min binding reaction, 5 min regeneration).

A multichannel immunochemical sensor for determination of 2,4-dichlorophenoxyacetic acid

Abstract A disposable multichannel immunochemical sensor based on the electrochemical transducer is reported. The basic sensor consists of the array of 8 working electrodes (gold) and one common reference electrode (silver) placed at a ceramic substrate. On top, a piece of plastic plate with drilled holes is sealed to obtain 8 microwells with electrodes at their bottoms. An electronic computer-controlled switch is constructed which enables to use the multichannel sensor together with a usual amperometric measurement system. To prepare an immunochemical biosensor, circular pieces of nitrocellulose membrane with adsorbed antibodies against the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) are inserted into the microwells. For the competitive immunochemical assay of 2,4-D, the 2,4-D molecule conjugated to horse radish peroxidase is used as a tracer. The amount of bound tracer is determined amperometrically using hydrogen peroxide and hydroquinone as substrates for peroxidase. The detection limit for free 2,4-D in water is 0.1 μg 1 , the total time of analysis being less than 1 h. The multichannel sensor is potentially suitable for analysis of several samples and standards using one biorecognition system, moreover, different biospecific layers may be used to determine several different analytes in the same sample using only one multichannel sensor.