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Biosensors which make use of the high specificity of enzymes, antibodies, and nucleic acids have been described for detection of numerous metabolites, hormones, and nucleic acid sequences. In addition to biological components nowadays biomimetic recognition molecules are also used. Especially antibodies, aptamers, and molecular imprints are promising biomimetics. They could broaden the range of detectable analytes and could increase the functional stability of the sensor. In this publication we describe the generation of biomimetic antibodies and biomimetic molecular imprints for binding creatinine and for hydrolyzing phenylcarbamates to be used in electrochemical sensors.
Electrochemical immunoassays
(2000)
Biosensors for food analysis
(1997)
Point-of-care testing (POCT) is a fast developing area in clinical diagnostics that is considered to be one of the main driving forces for the future in vitro diagnostic market. POCT means decentralized testing at the site of patient care. The most important POCT devices are handheld blood glucose sensors. In some of these sensors, after the application of less than 1 A mu l whole blood, the results are displayed in less than 10 s. For protein determination, the most commonly used devices are based on lateral flow technology. Although these devices are convenient to use, the results are often only qualitative or semiquantitative. The review will illuminate some of the current methods employed in POCT for proteins and will discuss the outlook for techniques (e.g., electrochemical immunosensors) that could have a great impact on future POCT of proteins.
Biosensoren
(1998)
Characterization of a monoclonal antibody and its Fab fragment against diphenylurea hapten with BIA
(1998)
This paper describes the principle of a homogeneous indirect fluorescence quenching immunoassay that uses monoclonal antibodies. It is a carrier-free assay system that is performed completely in solution. The assay system was established for the determination of a low molecular weight substance (hapten), the herbicide diuron, used as a model analyte. A fluorescein-monuron conjugate together with a fluorescence-quenching monoclonal anti-fluorescein antibody and an anti-analyte antibody (here an anti-diuron/monuron monoclonal antibody) were used as central components of the assay. The fluorescein-monuron conjugate can be bound either by the anti-fluorescein monoclonal antibody or by the anti-diuron/ monuron monoclonal antibody. Due to steric hindrance, binding of both antibodies to the conjugate was not possible at the same time. By selecting the antibody concentrations appropriately, a dynamic equilibrium can be established that permits the preferential binding of the anti-diuron/monuron antibody to the conjugate, which allows the fluorescein in the conjugate to fluoresce. This equilibrium can be easily altered by adding free analyte (diuron), which competes with the conjugate to bind to the anti-diuron/monuron antibody. A reduction of anti-diuron/monuron antibody binding to the conjugate results in an increase in the binding of the anti-fluorescein antibody, which leads to a decrease in the fluorescence of the conjugate. The fluorescence is therefore a direct indicator of the state of equilibrium of the system and thus also the presence of free unconjugated analyte. The determination of an analyte based on this test principle does not require any washing steps. After the test components are mixed, the dynamic equilibrium is rapidly reached and the results can be obtained in less than 5 min by measuring the fluorescence of the fluorescein. We used this test principle for the determination of diuron, which was demonstrated for concentrations of approximately 5 nM.
Biosensorik / Bioanalytik
(2004)
Detection of subicomolar concentrations of human matrix metalloproteinase-2 by an optical biosensor
(2004)
We describe in this paper the development of a one-step sandwich assay for the highly sensitive and fast detection of human matrix metalloproteinase (MMP)-2 (EC 3.4.24.24), using surface plasmon resonance (SPR). For the assay, two ligands were selected: monoclonal anti-MMP-2 antibody Ab-2 and the tissue inhibitor of metalloproteinases (TIMP)-2. They were chosen on the basis of (1) their affinities to MMP-2, (2) the efficiency of immobilization to the sensor chip, (3) the efficiency of adsorption to colloidal gold, and (4) the stability of these protein-coated gold particles. The assay included mixing of MMP-2 with antibody Ab-2 adsorbed to colloidal gold with a diameter of about 20 rim and injection into the flowcell of the SPR instrument containing immobilized TIMP-2. By using colloidal gold particles an amplification factor of 114 and a detection limit of 0.5 pM for MMP-2 were obtained. The precision of the assay was high even at low analyte concentrations, the standard deviation being 8.3% for five determinations of 1 pM MMP- 2. No significant binding was observed with the structurally related MMP-9. The assay is far more sensitive and faster than commonly used methods for MMP-2 detection. As TIMP-bound MMP-2 is not detected by this method, the assay can be applied for measuring free MMP-2, reflecting the imbalance of free and inhibitor-bound enzyme in various pathological situations. (C) 2004 Elsevier Inc. All rights reserved
Electrochemical bioassay utilizing encapsulated electrochemical active microcrystal biolabels
(2005)
A new approach to perform electrochemical immunoassay based on the utilization of encapsulated microcrystal was developed. The microcrystal labels create a "supernova effect" upon exposure to a desired releasing agent. The microcrystal cores dissolve, and large amounts of signal-generating molecules diffuse across the capsule wall into the outer environment. Layer-by-Layer (LbL) technology was employed for the encapsulation of electrochemical signal- generating microcrystals (ferrocene microcrystals). The encapsulated microcrystals were conjugated with antibody molecules through the adsorption process. The biofunctionalized microcrystals were utilized as a probe for immunoassays. The microcrystal-based label system provided a high-signal molecule to antibody (SIP) ratio of 10(4)-10(5). Microcrystal biolabels with different antibody surface coverage (1.60-5.05 mg m(-2)) were subjected to a solid-phase immunoassay for the detection of mouse immunoglobulin G (M-IgG) molecules. The microcrystal-based immunoassay for the detection of M-IgG performed with microcrystals having antibody surface coverage of 5.05 mg m(-2) showed a sensitivity of 3.93 nA g(- 1) L-1 with a detection limit of 2.82 g L-1
Leukotriene B4 as an inflammatory mediator is an important biomarker for different respiratory diseases like asthma, chronic obstructive pulmonary disease or cystic lung fibrosis. Therefore the detection of LTB4 is helpful in the diagnosis of these pulmonary diseases. However, until now its determination in exhaled breath condensates suffers from problems of accuracy. Reasons for that could be improper sample collection and preparation methods of condensates and the lack of consistently assay specificity and reproducibility of the used immunoassay detection system. In this study we describe the development and the characterization of a specific monoclonal antibody (S27BC6) against LTB4, its use as molecular recognition element for the development of an enzyme-linked immunoassay to detect LTB4 and discuss possible future diagnostic applications.
Affinity interaction betwen phenylboronic acid-carrying self-assembled monolayers and FAD or HRP
(2005)
A method is provided for the recognition of glycated molecules based on their binding affinities to boronate- carrying monolayers. The affinity interaction of flavin adenine dinucleotide (FAD) and horseradish peroxidase (HRP) with phenylboronic acid monolayers on gold was investigated by using voltammetric and microgravimetric methods. Conjugates of 3-aminopherrylboronic acid and 3,3'-dithiodipropionic acid di(N-hydroxysuccinimide ester) or 11-mercaptoundecanoic acid were prepared and self-assembled on gold surfaces to generate monolayers. FAD is bound to this modified sur-face and recognized by a pair of redox peaks with a formal potential of -0.433 V in a 0.1 m phosphate buffer solution, pH 6.5. Upon addition of a sugar to the buffer, the bound FAD could be replaced, indicating that the binding is reversible. Voltammetric, mass measurements, and photometric activity assays show that the HRP can also be bound to the interface. This binding is reversible, and HRP can be replaced by sorbitol or removed in acidic solution. The effects of pH, incubation time, and concentration of H2O2 were studied by comparing the catalytic reduction of H2O2 in the presence of the electron-donor thionine. The catalytic current of the HRP-loaded electrode was proportional to HRP concentrations in the incubation solution in the range between 5 mu g mL(-1) and 0.4 mg mL(-1) with a linear slope of 3.34 mu A mL mg(-1) and a correlation coefficient of 0.9945
In this work we describe a new preparation method for an esterolytic imprinted polymer with catalytic sites on the surface. A template was prepared by immobilizing a transition state analogue (phosphoramidic acid derivative) of an esterolytic reaction within porous silica particles. Polymerization within the pores was carried out using 4- vinylimidazole as a functional monomer and divinylbenzene as a cross-linker. The polymer was released by dissolution of the silica support with hydrofluoric acid and catalytic properties were studied by incubation with three different 4- nitrophenylesters and spectrophotometric determination of the released 4-nitrophenol. For 4-nitrophenyl acetate an activity of 211 nmol min(-1) mg(-1) and a K-m value of 2.2 mmol L-1 was obtained
Binding or catalysis? Both can be distinguished with a molecularly imprinted polymer (MIP) by the different patterns of heat generation. The catalytically active sites, like in the corresponding enzyme, generate a steady-state temperature increase. Thus, enzyme-like catalysis and antibody-analogue binding are analyzed simultaneously in a bifunctional MIP for the first time (see scheme).
A novel electrochemical immunoassay based on the multiple affinity labeling of the indicator antibody with an electro-active tag is presented. The concept is illustrated for the determination of the glycated hemoglobin HbA1c in hemoglobin samples. Hemoglobin is adsorbed to the surfactant-modified surface of a piezoelectric quartz crystal. Whereas the quartz crystal nanobalance is used to validate the total Hb binding, the HbA1c on the sensor surface is recognized by an antibody and quantified electrochemically after the sugar moieties of the antibody have been labeled in-situ with ferroceneboronic acid. The sensitivity of this sensor is about threefold higher than the sensitivity of a hemoglobin sensor, where the ferroceneboronic acid is bound directly to HbA1c.
In this work we present a CMOS high frequency direct immunosensor operating at 6 GHz (C-band) for label free determination of creatinine. The sensor is fabricated in standard 0.13 mu m SiGe:C BiCMOS process. The report also demonstrates the ability to immobilize creatinine molecules on a Si3N4 passivation layer of the standard BiCMOS/CMOS process, therefore, evading any further need of cumbersome post processing of the fabricated sensor chip. The sensor is based on capacitive detection of the amount of non-creatinine bound antibodies binding to an immobilized creatinine layer on the passivated sensor. The chip bound antibody amount in turn corresponds indirectly to the creatinine concentration used in the incubation phase. The determination of creatinine in the concentration range of 0.88-880 mu M is successfully demonstrated in this work. A sensitivity of 35 MHz/10 fold increase in creatinine concentration (during incubation) at the centre frequency of 6 GHz is gained by the immunosensor. The results are compared with a standard optical measurement technique and the dynamic range and sensitivity is of the order of the established optical indication technique. The C-band immunosensor chip comprising an area of 0.3 mm(2) reduces the sensing area considerably, therefore, requiring a sample volume as low as 2 mu l. The small analyte sample volume and label free approach also reduce the experimental costs in addition to the low fabrication costs offered by the batch fabrication technique of CMOS/BiCMOS process.
This work describes a method for surface regeneration of microfluidic microarray printheads through plasma techniques. Modification procedures were chosen in a way to obtain high reproducibility with a minimum of time consumption. The idea behind this is a complete regeneration of a microarray printhead before or after usage to achieve best printing results over a typical print job. A sequence of low-pressure oxygen-plasma and plasma polymerization with hexamethyldisiloxane (HMDSO) was used to regenerate printheads. Proof of the concept is given through quality control performed with a spotter implemented CCD camera, contact angle measurements and a typical hybridization experiment. Stable printing results were obtained over 3000 activations showing that the presented method is suitable for treatment of microarray printheads.