On-chip automation of cell-free protein synthesis: new opportunities due to a novel reaction mode.

Many pharmaceuticals are proteins or their development is based on proteins. Cell-free protein synthesis (CFPS) is an innovative alternative to conventional cell based systems which enables the production of proteins with complex and even new characteristics. However, the short lifetime, low protein production and expensive reagent costs are still limitations of CFPS. Novel automated microfluidic systems might allow continuous, controllable and resource conserving CFPS. The presented microfluidic TRITT platform (TRITT for Transcription - RNA Immobilization & Transfer - Translation) addresses the individual biochemical requirements of the transcription and the translation step of CFPS in separate compartments, and combines the reaction steps by quasi-continuous transfer of RNA templates to enable automated CFPS. In detail, specific RNA templates with 5' and 3' hairpin structures for stabilization against nucleases were immobilized during in vitro transcription by newly designed and optimized hybridization oligonucleotides coupled to magnetizable particles. Transcription compatibility and reusability for immobilization of these functionalized particles was successfully proven. mRNA transfer was realized on-chip by magnetic actuated particle transfer, RNA elution and fluid flow to the in vitro translation compartment. The applicability of the microfluidic TRITT platform for the production of the cytotoxic protein Pierisin with simultaneous incorporation of a non-canonical amino acid for fluorescence labeling was demonstrated. The new reaction mode (TRITT mode) is a modified linked mode that fulfills the precondition for an automated modular reactor system. By continual transfer of new mRNA, the novel procedure overcomes problems caused by nuclease digestion and hydrolysis of mRNA during TL in standard CFPS reactions.

Enhancement of immunoassay sensitivity by molecular modification of competitors.

The potential of weak competitors to enhance the sensitivity of competitive immunoassays is described. Several triazine derivatives have been analyzed for their use as competitors. Their binding properties were determined using an enzyme-linked immunosorbent assay (ELISA) with a monoclonal antibody specific to triazines. Selected derivatives were immobilized onto the surface of a fibre optic sensor and atrazine was determined in a competitive manner using a fluorescein-labelled antibody. Using the weak binding competitor 11-(4-ethylamino-6-methylthio-s-triazine-2-yl)undecanoic acid (TE11S) the detection limit for atrazine could be lowered 100-fold in comparison to 2-aminohexylamino-4-ethylamino-6-isopropylamino-s-triazine (AHA), the previously used competitor.

Changing functionality of surfaces by directed self-assembly using oligonucleotides--the Oligo-Tag.

A method is presented to modify surfaces for biotechnological applications. Oligonucleotides have been coupled covalently to a pre-activated surface. Complementary oligonucleotides hybridize to the surface, which are conjugated with functionalities. The oligonucleotides serve as "Oligo-Tags" for these functionalities that now are linked specifically and reversibly. The approach might be used to change DNA-arrays into arrays of arbitrary ligands. We demonstrate the method with an optical wave guide grating coupler as a sensing surface using two different haptens as examples for a variety of functionalities. The haptens were 2,4-dichlorophenoxyacetic acid and atrazin and are recognized by specific antibodies. The surface created was completely regenerable by alkaline washing or temperature increase without any loss of binding capacity. Specificity was demonstrated by competitive binding of antibody in presence and absence of analyte; unspecific binding has not been observed.

Nucleic acid based sensors.

Nucleic acids may be analyte or molecular recognition elements in biosensors. Both aspects merge in the genosensor approach, where detection of special sequences is facilitated by hybridization of a target nucleic acid to a complementary immobilized template. All three roles of nucleic acids in biosensors are discussed and the state of sensor development reviewed. With the invention of evolutionary synthesis strategies applied to nucleic acids new types of biomolecular receptors are accessible. The impact of aptamers and ribozymes on biosensor development is discussed.

Label-free observation of DNA-hybridisation and endonuclease activity on a wave guide surface using a grating coupler.

Hybridisation of nucleic acid oligomers to an immobilised target has been observed in real time using evanescent field technology. A biotinylated 24-mer with random sequence including the EcoRI recognition site was immobilised via streptavidin onto a grating coupler wave guide surface. Hybridisation of 22-mer, 15-mer and 8-mer was observed. Activity of restriction endonuclease EcoRI was visualised by measurement of the loss of bound DNA after incubation.

Denaturation behaviour of DNA-protein-complexes detected in situ in metaphase chromosomes in suspension by Hoechst 33258 fluorescence.

The denaturation behaviour of DNA-protein complexes in metaphase chromosomes in suspension was analysed in situ by Hoechst 33258 fluorescence. The results indicate that due to the stability of the dye molecule and the product of the molecular extinction coefficient and the quantum yield at different temperatures, Hoechst 33258 is a suitable probe for the detection of double-stranded DNA. Thus, it is possible to monitor the concentration of double-stranded DNA in a suspension by measuring the total fluorescence intensity. The fluorescence denaturation profiles of DNA (calf thymus) were found to be comparable to absorption measurements. The decrease in fluorescence of metaphase chromosomes in suspension with increasing temperature may therefore be used to detect conformational changes of DNA in situ.

High sensitive competitive immunodetection of 2,4-dichlorophenoxyacetic acid using enzymatic amplification with electrochemical detection.

The amplification cycle consisting of NADH independent oligosaccharide dehydrogenase (ODH) and laccase has been recently reported to be highly sensitive to several catecholamines and p-aminophenol. A competitive immunoassay for 2,4-dichlorophenoxyacetic acid has been developed by combining this amplification cycle with beta-galactosidase as enzyme label resulting in p-aminophenol as product. The combination of enzymatic amplification cycles with a competitive immunoassay yields a highly sensitive measurement of 2,4-dichlorophenoxyacetic acid. Using a monoclonal antibody the linear range of the assay was between 0.02 and 100 ng/l and the c(50) was found at 0.2 ng/l; the detection limit was at 5 pg/l (25 fmol/l) corresponding to 5 amol.

Functional evaluation of candidate ice structuring proteins using cell-free expression systems.

Ice structuring proteins (ISPs) protect organisms from damage or death by freezing. They depress the non-equilibrium freezing point of water and prevent recrystallization, probably by binding to the surface of ice crystals. Many ISPs have been described and it is likely that many more exist in nature that have not yet been identified. ISPs come in many forms and thus cannot be reliably identified by their structure or consensus ice-binding motifs. Recombinant protein expression is the gold standard for proving the activity of a candidate ISP. Among existing expression systems, cell-free protein expression is the simplest and gives the fastest access to the protein of interest, but selection of the appropriate cell-free expression system is crucial for functionality. Here we describe cell-free expression methods for three ISPs that differ widely in structure and glycosylation status from three organisms: a fish (Macrozoarces americanus), an insect (Dendroides canadensis) and an alga (Chlamydomonas sp. CCMP681). We use both prokaryotic and eukaryotic expression systems for the production of ISPs. An ice recrystallization inhibition assay is used to test functionality. The techniques described here should improve the success of cell-free expression of ISPs in future applications.

A novel handheld fluorescent microarray reader for point-of-care diagnostic.

A novel handheld optical sensor for quantification of fluorescent microarrays, the so-called portMD-113 has been developed. On the surface of a planar waveguide, the spots of different fluorescently labeled biological complexes are excited by the evanescent field of the guided light. The emitted fluorescence signals of the spots are independently and simultaneously detected applying our system, which consists of a pinehole array, a microlens array, an interference filter and a detector array. As it is demonstrated in comparative measurements, the detection limit of this sensor is close to that of commercial top microarray readers, e.g. of modern laser scanners, while it has remarkable and important advantages over them. Namely, the device comprises only a few low-cost, lightweight and small components without applying any moving or energy-intensive elements, which results in turn in a commercially competitive, handheld and compact design and in the possibility to be supplied simply by a battery or a personal computer. These advantageous properties open prospects e.g. for point-of-care medical checks, as well.

Dielectrophoretic manipulation of DNA.

The characterisation and spatial manipulation of cells by AC electrokinetic methods such as dielectrophoresis and electrorotation is well established. However, applications to submicroscopical objects like viruses and molecules have been rare. Only recently has the number of such studies risen more quickly due to the availability of suitable electrodes and a growing need for single molecule techniques. Of special interest is the spatial control of single DNA molecules for genetic investigations as well as for the building of well defined structures with nanometre resolution. Here a review is given of dielectrophoretic studies dealing with single and double stranded DNA emphasising single molecule aspects.

Feature-size limitations of microarray technology--a critical review.

The appeal of microarray technology is the possibility of large-scale parallel determination of a variety of variables simultaneously. Hence, microarray technologies attract the interest of both the scientific and business worlds alike. High-throughput screening has been the major focus of the utilization of microarray technologies in recent years, and has provided the strong driving force for developments in this field. DNA chip and biochip technologies have been developed as a consequence of worldwide activity in genome research. This review focuses on microarray-based analysis and emphasizes some of its principal constraints, especially detection limits.

Real-time observation of affinity reactions using grating couplers: determination of the detection limit and calculation of kinetic rate constants.

The use of integrated optical grating couplers for the analysis of bioaffinity reactions in order to calculate kinetic rate constants was investigated. The specificity of the sensor surface was determined by adsorptive or covalent attachment of the specific ligands. As an evanescent field sensor, the specific interaction of the corresponding ligand could be observed in real time and without labels. The detection limit in terms of the molecular weight of the analyte was studied by the specific binding of biotinylated proteins of different molecular weights to avidin-loaded sensors. It was shown that grating coupler sensors allowed detection of compounds of at least 2000 daltons using high-affinity receptors, while the direct sensing of low molecular analytes, such as biotin, could not be significantly achieved. Association rate constants were calculated for the interaction of the different biotinylated proteins to avidin-covered sensors from single binding curves. Due to the strong binding between avidin and biotin, the dissociation of the formed complex could not be observed. Kinetic rate constants and equilibrium constants were determined by studying the interaction of human immunoglobulin with the immobilized receptor, protein G. For the four human immunoglobulin subclasses a high affinity to protein G was determined with affinity constants ranging from 3.3 to 8.4 x 10(8) M-1.

Improved preparative separation of human B and T lymphocytes and determination of relative mobilities of B and T lymphoblast cell lines by free flow electrophoresis.

The improved quantitative separation of human B and T lymphocytes by free flow electrophoresis is presented. Buffer systems with different compositions are tested analytically and partly preparatively for their effect on the separation profile. The best result is achieved with a modified phosphate-buffered saline and allows the enrichment of unstimulated, functionally intact B and T lymphocytes as well as monocytes in three distinct fractions. Furthermore, the electrophoretic mobilities of six established acute lymphoblastic leukemia cell lines are determined with regard to the mobilities of the healthy cells.

On-line monitoring of monoclonal antibodies in animal cell culture using a grating coupler.

A grating coupler was used for the on-line determination of monoclonal antibodies produced in perfused animal cell bioreactor. The device was connected with the culture vessel via a flow-injection analysis (FIA) system, which was controlled automatically. Specific antimouse lgG antibodies were immobilized on the surface of the sensor-chip. After injection of the sample, the binding of mouse lgG was observed in real time. The regeneration of the binding sites of the immobilized antibodies using an acidic solution allowed the on-line detection of produced monoclonal antibodies in the range of 10 to 150 microg/mL. In contrast to other techniques coupled to bioprocesses, the developed method represents a regenerable direct immunosensor. Results were compared with standard ELISA techniques (off-line) and a competitive immunochemical assay using the grating coupler (off-line).

Zeptomole-detecting biosensor for alkaline phosphatase in an electrochemical immunoassay for 2,4-dichlorophenoxyacetic acid.

A bienzyme substrate-recycling biosensor in a flow injection analysis system is described for the sensitive measurement of alkaline phosphatase (ALP) and applied to the fast readout of a competitive immunoassay for the widely used pesticide 2,4-dichlorophenoxyacetic acid (2,4-D). The phenol-indicating biosensor consists of a Clark-type electrode covered by a membrane with coentrapped tyrosinase and quinoprotein glucose dehydrogenase. ALP dephosphorylates phenyl phosphate to phenol (K(m) = 36 microM) outside the flow system. Phenol is oxidized in the sensor membrane by the oxygen-consuming tyrosinase via catechol to o-quinone. The quinone is reconverted to catechol by glucose dehydrogenase. This substrate cycling results in a 350-fold amplified sensor response to phenol. The oxygen consumption of the enzyme couple in the presence of phenol is monitored as a decrease in current. A total of 3.2 fM ALP (320 zmol/ 100 microL) has been detected after a 57.5 min incubation with phenyl phosphate. All involved reagents are stable over the time of measurement. The sensor does not produce any measurable blank signals. The immunoassay detects 0.1 microgram/L 2,4-D, the maximum concentration for pesticides allowed in drinking water by European Community regulations. The applicability of this biosensor for fast immunoassay readout is demonstrated by a 2 min incubation. By comparison, a standard photometric method (p-nitrophenyl phosphate) requires overnight incubation.

Determination of the electrophoretic mobility of chromosomes by free flow electrophoresis. I. Morphology and stability.

Isolated metaphase chromosomes of several fibroblastoid cell lines (Chinese hamster, Chinese hamster x human hybrid) were subjected to free flow electrophoresis (FFE) to study their electrophoretic mobility (EM). The morphology and stability of the chromosomes were unaffected by FFE as examined by cytogenetic methods and flow cytometry. The chromosomes of the complement all showed similar EM under most of the conditions applied. At neutral pH the EM of the chromosomes had the same sign as free DNA and about 2/3 of its magnitude. The variation of EM with buffer parameters such as ionic strength, valence of counterions, buffer capacity and dielectric constant of the solvent were investigated. Thermal denaturation increased the EM of the chromosomes by 20%. Partial denaturation might offer a possibility to separate or enrich large amounts of chromosomes by FFE.