Resonant nano-cluster devices.

The resonance-enhanced absorption (REA) by metal clusters on a surface is an effective technique on which to base bio-optical devices. A four-layer device consisting of a metal mirror, a polymer or glass-type distance layer, a biomolecule interaction layer and a sub-monolayer of biorecognitively bound metal nano-clusters is reported. Experiments indicate a strong influence of the resonator homogeneity on the absorption maximum. Layer stability plays an important role in the overall performance of the device. Techniques and optimised lab protocols to set up biochips that use the REA process in the detection are presented. The sensors show one to three narrow reflection minima in the visible and or infra-red (IR) part of the spectrum and therefore they do not suffer from the spectral limitations associated with spherical gold colloids. Metal clusters (synthesised by thermal step reduction) as well as metal- dielectric shell clusters (synthesised by various shell deposition processes) are used to precisely shift the readout of the device to any frequency in the visible and near IR range. Disposable single-step protein chips, DNA assays as well as complex biochip arrays are established that use various DNARNA, antigen-antibody and protein-protein interaction systems.

Design and peptide-based validation of phage display antibodies for proteomic biochips.

To validate potential application of phage display-antibody arrays for high-throughput screening on a novel proteomics biochip, we examined the epitopes versus the full protein of glucose-6-phosphate-dehydrogenase (G6PD) from yeast. In a predictive approach, we used the Hopp-Woods method and compared the results with antibodies directed against the entire enzyme. In total, 16 peptides of a length of 11 amino acids each fulfilling the desired criteria were identified and synthesized. Subsequently, antibodies against G6PD were raised using a phage display library. Selective interaction of the antibodies with certain peptides facilitated the identification of epitopes predicted by the hydropathic profile. The setup was adapted to a novel biochip system based on surface-enhanced absorption for direct CCD-camera based screening.

Slide-format proteomic biochips based on surface-enhanced nanocluster-resonance.

The most fundamental properties of metal nanoclusters, namely the high local-field enhancement and nanoscale resonance behavior of the cluster electron plasma when exited by electromagnetic radiation, have been used to set up a variety of sensors transducing biorecognitive interactions into optical signals. This paper focuses on applications of resonant-cluster technology, which enabled us to monitor biorecognitive binding of a variety of proteins on a chip, thus constructing high-throughput interaction-screening devices. Decisive for this type of sensor is the nanometric distance from the local field surrounding a cluster to other parts of the sensor interacting with this field. In particular, the cluster-mirror or cluster-fluorophore distance gives rise to a variety of enhancement phenomena. Depending on the desired application this "resonance"- distance is approximately 5-400 nm. All types of sensor can be set up on photolithographically constructed microchips, but microscopic glass slides can also be employed; this also enables the use of standard devices for dotting and read out. Using slide based chips a standard format of 3,200 microdots (125 microm in diameter) was the basis of either microassays applying direct optical transduction via surfaceenhanced absorption or striking for more sensitivity via surface-enhanced fluorescence.

Capillary electrophoresis with on-chip four-electrode capacitively coupled conductivity detection for application in bioanalysis.

Microchip capillary electrophoresis (CE) with integrated four-electrode capacitively coupled conductivity detection is presented. Conductivity detection is a universal detection technique that is relatively independent on the detection pathlength and, especially important for chip-based analysis, is compatible with miniaturization and on-chip integration. The glass microchip structure consists of a 6 cm etched channel (20 microm x 70 microm cross section) with silicon nitride covered walls. In the channel, a 30 nm thick silicon carbide layer covers the electrodes to enable capacitive coupling with the liquid inside the channel as well as to prevent interference of the applied separation field. The detector response was found to be linear over the concentration range from 20 microM up to 2 mM. Detection limits were at the low microM level. Separation of two short peptides with a pI of respectively 5.38 and 4.87 at the 1 mM level demonstrates the applicability for biochemical analysis. At a relatively low separation field strength (50 V/cm) plate numbers in the order of 3500 were achieved. Results obtained with the microdevice compared well with those obtained in a bench scale CE instrument using UV detection under similar conditions.

Miniaturized electrospraying as a technique for the production of microarrays of reproducible micrometer-sized protein spots.

Electrospraying in a stable cone-jet mode at <400 microm above a substrate is shown to be a powerful technique to produce arrays of identical micrometer-sized spots consisting of biologically active substances. Aqueous solutions with a surface tension of 0.04 N m(-1) and conductivities ranging from 0.04 to 2.2 S m(-1) were sprayed at ultralow flow rates ranging from 100 to 300 pL s(-1). The charged jet that emanates from the cone tip breaks up into a spray of charged droplets that are deposited in the form of a uniform spot of 130-350 microm in diameter by spraying during 0.5-3 s at 220-400 microm above a substrate, respectively. After a spot was deposited, spraying was stopped instantaneously by increasing the distance between the capillary tip and the substrate by an additional 100 microm using a computer-controlled x-y-z table. This was immediately followed by a rapid shift of the substrate 400 microm sideways and 100 microm upward, thus causing spraying to resume instantaneously because of the increased electric field strength, which resulted in the deposition of the next spot. It is shown here that spraying of lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G6P-DH), and pyruvate kinase (PK) on a liquid layer resulted in the complete preservation of their activities despite the high solution conductivity of 3.3 S m(-1) and high currents ranging from 300 to 500 nA. LDH and PK activities were fully preserved after spraying onto dry aluminum by adding 0.05 M buffer and 0.5 and 1 wt % of trehalose, respectively, to the spray solutions. Electrospraying allows for accurate dispensing of liquid volumes as small as 50 pL. Enzymatic activities of LDH and PK are fully preserved after spraying.

High-throughput assays on the chip based on metal nano-cluster resonance transducers.

High throughput transducers using metal cluster resonance technology are based on surface-enhancement of metal cluster light absorption. These devices can be used for detection of biorecognitive binding, as well as structural changes of nucleic acids, proteins or any other polymer. The optical property for the analytical application of metal cluster films is the so-called anomalous absorption. An absorbing film of clusters positioned 10--400 nm to a mirror surface reacts in a similar way to a reflection filter. At a certain distance of the absorbing layer to the mirror the reflected electromagnetic field has the same phase at the position of the absorbing cluster as the incident fields. This feedback mechanism strongly enhances the effective cluster absorption coefficient. The system is characterised by a narrow reflection minimum whose spectral position shifts sensitively with the interlayer thickness, because a given cluster-mirror distance and wavelength defines the optimum phase. Based on this principle a set of novel tools including biochips and micro arrays is presented, which enabled us to transduce binding, as well as changes of protein-, DNA- and polymer-conformation, quantitatively into an optical signal which can be observed directly as a colour change of a sensor-chip surface.

Nanofilms and nanoclusters: energy sources driving fluorophores of biochip bound labels.

Nanoclusters and nanofilms have the potential to amplify fluorescence and thus to enhance the signal of labeled biomolecules on biochip surfaces. Fluorescent molecules are bound at a certain distance to a resonant layer of a metal or a semiconductor or both, resulting in enhanced absorption and emission of the fluorophore within the electromagnetic near-field. This property makes the system highly useful for interaction studies, including those of DNA and proteins. Due to the amount of data, derived from various sequencing projects and from Proteomic interaction studies within the next years, microarrays (or biochips) will represent a central technology in every lab facilitating high-throughput screening and being easily interfaced with computer databases. However, most chips suffer from the disadvantage of insufficient signal-to-noise (background) ratio and are thus limited to molecules of medium-to-high abundance. Novel approaches are needed for identification of, e.g., low copy RNAs or regulatory proteins. Here we present a study, using novel surface enhanced chips in the standard glass-slide-formats. Applying surface-enhanced fluorescence (SEF), the chips turned out to be useful for interaction studies, such as DNA hybridization, thereby strongly enhancing the on-chip-signals. Compared to standard glass-slide-DNA chips, both the fluorescent signals as well as signal-to-noise ratio were considerably higher.

Enzyme-based flow injection analysis system for glutamine and glutamate in mammalian cell culture media.

We present the setup of a flow injection analysis system designed for on-line monitoring of glutamate and glutamine. These amino acids represent a major energy source in mammalian cell culture. A cycling assay consisting of glutamate dehydrogenase and aspartate aminotransferase produces NADH proportional to the glutamate concentration in the sample. NADH is then measured spectrophotometrically. Glutamine is determined by conversion to glutamate which is fed into the cycling assay. The conversion of glutamine to glutamate is catalyzed by asparaginase. Asparaginase was used in place of glutaminase due to its relatively high reactivity with glutamine and a pH optimum similar to that of glutamate dehydrogenase. The enzymes were immobilized covalently to activated controlled pore glass beads and integrated into the flow injection analysis system. The application of the immobilized enzymes and the technical setup are presented in this paper.

Immobilization of active facilitated glucose transporters (GLUT-1) in supported biological membranes.

Membrane fragments or membrane proteins within a lipid mixture were immobilized over metal electrodes. This procedure has been developed to study biological membranes without interference from cell machinery. To obtain a smooth hydrophilic biomembrane support and a mode of binding of the membrane, either a crosslinked gel or an aromatic polyamine-polymer doped with avidin was deposited at the metal electrode by electropolymerization. This layer (less than 10 nm thick) also served as a submembrane compartment. The facilitated glucose transporter (GLUT-1) purified from human erythrocytes was integrated into a lipid membrane containing artificial biotinylated lipids and reacted with the activated surface of the glucose sensitive electrode. It was demonstrated that the lipid layer was attached to the polymer-containing avidin and could only be removed by detergent extraction. The presence of an active membrane transporter was demonstrated by electrochemical detection of glucose in the submembrane compartment, and by inhibition of glucose transport with the specific inhibitor Cytochalasin-B.

Novel method for coupling of poly(ethyleneglycol) to carboxylic acid moieties of proteins.

Lack of toxicity, excellent solubility and superb biocompatibility make polyethylene glycol (PEG) one of the most popular modifiers of biologicals. The most common method for attachment of PEG is based on modification of amino groups of proteins with methoxy- or succinimide-derivatives of PEG. In the case of proteins with amino groups important for biological activity, this modification can lead to inactivation of proteins. A new strategy for covalent attachment of PEG to carboxylic groups of proteins using O,O-bis-(2-aminopropyl)polyethylene glycol and carbodiimide/N-hydroxysuccinimide-mediated reaction was developed. The reaction is carried out under mild aqueous conditions. The attached PEG serves as a hydrophilic spacer for further bioconjungation with biomolecules and haptens. Lipase from Candida rugosa was used as a model protein. Characteristic data of the modified protein such as activity, isoelectric points and stability were compared with that of the unmodified protein.

Overexpression and purification of a recombinant chimeric HIV type 2/HIV type 1 envelope peptide and application in an accelerated immunobased HIV type 1/2 antibody detection system (AIBS): a new rapid serological screening assay.

A chimeric HIV-2/HIV-1 envelope sequence containing an immunodominant region of HIV-2 gp36 and the corresponding region of HIV-1 gp41 was constructed and overexpressed in Escherichia coli. The recombinant product (rp21/18) was purified and applied in a novel antibody-screening assay. Characteristics in the design of this new principle are as follows: (1) the overall assay time is about 30 min; (2) the assay procedure includes three manipulation steps; and (3) the test shows a reliable performance with respect to sensitivity and specificity. The diluted serum sample and the protein G-horseradish peroxidase conjugate are added simultaneously into a coated (hybrid antigen HIV-1/2) and blocked microtiter plate well. The in-batch incubation of serum sample with protein G-horseradish peroxidase saves two manipulation steps that are normally necessary in the five-step procedure of a classical ELISA. AIBS was evaluated with commercially available seroconversion panels and with random negative serum samples from a blood bank. Seroconversion results demonstrated that AIBS has equivalent sensitivity to ELISAs and the third generation assays. The specificity was determined on a total blood donor population of 5012 (Red Cross Vienna, Austria). The repeat reactive rates for donor population were 0.02%. AIBS represents a general immunometric system (not only HIV antibodies). The entire assay procedure of AIBS evaluated for HIV-1/2 screening, including result reporting, can be performed automatically by several commercially available systems. Depending on these systems AIBS is potentially useful in laboratories or blood banks that have both high- and low-volume testing.

Photostructurized electrochemical biosensors for bioreactor control and measurement in body fluids.

This article details a new type of biosensor for the simultaneous analysis of glucose, glutamate and glutamine in complex biological fluids like fermentation broths and blood. Simultaneous analysis was made possible by the application of different enzyme layers onto different electrodes of one photostructurized sensor. Photostructuring was done by means of a new developed photopolymer. Preparation of the photopolymer and the enzyme layers as well as the characterization of the sensors thus constructed with respect to linearity, response time and sensitivity are described.

Oligonucleotide labelled lipase as a new sensitive hybridization probe and its use in bio-assays and biosensors.

Radiolabelled polynucleotide probes have been employed extensively for the detection of complementary nucleic acids by specific hybridization. Within the last few years, various methods have been developed using enzyme-labelled probes to avoid unstable and hazardous isotopes. These assays, based on photometry, fluorescence, and chemiluminescence, have helped to overcome the use of radioactive probes. To increase the performance of a non-radioactive DNA detection system, the labelling enzyme should remain stable under hybridization conditions which allow the formation of a 15-25 bp long DNA-DNA duplex (Tm = 50-70 degrees C). Therefore, the use of unstable phosphatase and peroxidase conjugates must be avoided due to the composition of the hybridization mixture and the high temperature. By screening various hydrolytic enzymes to fit the special demands, fungal lipases turned out to be the most practical. They offer high sensitivity due to an extremely high turnover number, stability at room temperature for several years, thermostability under working conditions and an easy design of various chromogenic, fluorescent and electrochemical active substrates. Several types of silanized, oxidized and unmodified metal sensors and also standard microtitre plates modified with amino groups were used for the immobilization of oligonucleotides. A sandwich hybridization using the lipase-labelled oligonucleotide probe and a terminal immobilized capture DNA on a microtitre plate or sensor surface combined with a rapid hybridization in solution simplifies and improves the performance of the DNA detection system.

Construction and use of two alpha-human atrial natriuretic peptide-fragment affinity chromatography columns in the isolation of C- and N-terminal epitope-specific antibodies for use in a prototype alpha-hANP biosensor.

Two alpha-human atrial natriuretic peptide (alpha-hANP) based affinity chromatography columns were produced by covalently immobilizing the C- and N-terminal epitopes of alpha-hANP. The stationary phase was made from a controlled-pore-glass bead solid support, which was silanized and treated with sulphosuccinimidyl 4-(maleimidomethyl)cyclohexyl carboxylate before the individual fragments were immobilized by substitution at their thiol groups. These columns were used to isolate alpha-hANP-specific antibodies from a goat anti-alpha-hANP serum, which were then further sorted according to their epitope specifity. These C- and N-terminal epitope-specific antibodies were in turn used as components in the construction of an alpha-hANP biosensor based on an enzyme-linked immunosorbent assay (ELISA) sandwich principle. Initial in vitro testing of the sensor using a physiological alpha-hANP solution showed a reproducible response to the peptide. There is to date no other equally fast, sensitive and precise method available to detect this peptide. This alpha-hANP sensor may prove to be an invaluable aid in human medicine as a monitor of patient status during transplant surgery, for example, an area inaccessible to radioimmunoassay and normal ELISA techniques.