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ENZYME ELECTRODES FOR IN VIVO APPLICATION; KINETIC PROPERTIES; STERILIZATION, AND GEOMETRY
No full textUsing glucose oxidase/hydrogen peroxide electrodes, covered with
Cuprophane and sealed with cellulose acetate or polyurethane, there
are three major difficulties in practical application of subcutaneous
implantable glucose electrodes. These are the lack of knowledge
concerning kinetic properties between different sensor preparations
and their influence on the response characteristics in vivo, the
suitable sterilization procedure, and the sensor geometry. Response
times of the sensors in vitro were between 1 and 5 min (testgeie 3 of
according to nonlinear regression analysis, NLRA), in dependence on
qualities and thickness of the covering layer. The time constants T
resulting from in vivo measurings subjected to NLRA at increases and
decreases of glucose were 28+8 and 15+2 min, in blood, 26.5+5 and 18+2
min in plasma, and 53+10 and 2644 min in subcutaneous tissue.
As a practicable methode to sterilize enzyme sensors, gamma irradation
in the presence of hydrogen peroxide has been used at doses of 0.6 kGy
+ 0.1 % H202 for safe killing Pseudomonas aeruginosa. Increasing H202-
concentration (at about 1 %), however, reduces the sensitivity by
influencing the enzyme activity.
Overcoming problems at sensors implantation site caused by the size of
the sensor (d = 2 mm, 1 = 20 mm) most probably, a miniaturized dualelectrode
was constructed, where the working electrode (Pt-anode,
d < 0.5 mm) has been prepared as an enzyme electrode, only. This
electrode arrangement has shown excellent electrochemical
characteristics as well as in the pOz - and in the H202-polarogran
CONTINUOUS FLOW IMMUNOASSAY: USE OF A NOVEL TRIFUNCTIONAL CARRIER MOLECULE FOR THE SYNTHESIS OF FLUOROPHORE-LABELED ANTIGENS
No full textWe developed a fluorescent immunosensor operating in continuous flow and capable of
detecting low molecular weight antigens. The approach differs from previously described
continuous flow assays by not requiring incubation steps or the introduction of reagents
following the loading of the sample into the system. Detection of the antigen is rapid, occurring
within three minutes in the system described. The assay is based on the binding of labeled
antigen to an immobilized antibody, with subsequent displacement of the labeled antigen when
antigen is present in the buffer flow. In order to increase the sensitivity of the assay, we
developed a novel trifunctional carrier molecule for the fluorescent labeling of the antigen. The
backbone of the carrier consists of the 21 amino acid residues of the insulin A-chain, which
provides a single site (terminal amino group) for covalent coupling of the antigen, three carboxyl
groups for the attachment of fluorophores, and four sulfhydryl groups for derivatization with
hydrophilic residues to compensate for the hydrophobic effect of the fluorophores. In this study,
the model antigen 2,4-dinitrophenol (DNP) was coupled to the terminal amino group, the
sulfhydryl groups were oxidized to S-sulfonates, and the carboxyl groups were derivatized with
fluorescein using carbohydrazide as spacer. The properties of the DNP-insulin A-chainfluorescein
conjugate (DNP-Ins-Fl) were compared to those of a DNP derivative labeled with a
single fluorescein residue via a small lysine spacer (DNP-Lys-Fl). At equimolar concentrations
the DNP-Ins-Fl generated a 2.6-fold higher fluorescent signal than the DNP-Lys-Fl, and
exhibited a three-fold lower nonspecific adsorption to immobilized nonimmune IgG. Due to
these properties of DNP-Ins-Fl,as little as 50 pmol of DNP-lysine could be detected in the
fluorescent continuous flow immunoassay
Title, Preface, Contents, List of authors
No full textThe term bioinformatics has two quite distinct meanings. It may describe information handling in
living organisms, and it is widely used for the application of computer science to biological
problems. It is this second area which is covered in this book. Theseriesof articles presented
here represents a selection of the papers given at an invigorating conference on
Bioinformatics/Computer Application in the Biosciences, held in October 1995 in Braunschweig
at the German National Laboratory for Biotechnology.
The development and use of computer applications in the biological sciences, thoughinitiated
rather late compared to the situation in physics and chemistry, has reached a high standard
nowadays and has becomean indispensable part of any research in this area. A strong impetus
has come from modern gene sequencing projects and also from the rapid developmentin the field
of structural biochemistry,i.e. the determination of protein and DNA/RNA3D-structures as well
as rational protein engineering and design.
This is reflected in the subjects coveredin the articles in this book. They describe the present
state in this field, in particular the following facts become obvious:
- The use and developmentof biological data bases has becomean essential foundation for
research in protein science and molecular biology.
- Whereas the coding regions of DNA have been the main target of research in the past,
nowadays the non-coding regions and RNAare receiving closer attention.
- The sequence comparison and correctalignment of protein sequencesis a prerequisite for
any protein engineering. Although routinely used in almost all biochemistry laboratories,
alignment of sequences with low homology still requiresfurther intensive research so that
significantly better results can be producedthan those currently available.
- The description and simulation of the interactions between different biological molecules
will be one of the fascinating areas of future research.
- In addition to understanding the biological processes on a molecular level, we have to
simulate the metabolism in the living cell in order to achieve real metabolic design for the
optimal biotechnological production of compounds.
Whereasthe first development of these methods stems from the sixties and seventies,it is only
recently that biologists, chemists and computer scientists have channelled their expertize into
large scale collaborative projects aimed at the advancementin this exciting area. Government
programs started, for example in Germany and the UK, have provided extra moneyfor joint
projects involving computerscientists and biologists. Together with the rapid progress in modern
biology and biotechnology, we can expect to see wide-ranging new developments in
bioinformatics in the years to come
An Algorithm for the Protein Docking Problem
No full textWe have implemented a parallel distributed geometric docking algorithm that
uses a new measureforthe size of the contact area of two molecules. The measure
is a potential function that counts the “van der Waals contacts” between the
atoms of the two molecules ( the algorithm does not compute the Lennard-Jones
potential). An integer constant c, is added to the potential for each pair of atoms
whose distance is in a certain interval. For each pair whose distance is smaller
than the lower boundofthe interval an integer constant c, is subtracted from the
potential (cg < c,). The number of allowed overlapping atom pairs is handled
by a third parameter N. Conformations where more than N atom pairs overlap
are ignored. In our “real world” experiments we have used a small parameter
N that allows small local penetration. The algorithm almost always found good
(rms) approximations of the real conformations that were among the best five
geometric dockings. In 42 of 52 test examples the best conformation with respect
to the potential function was an approximation of the real conformation. The
running time of our sequential algorithm is in the order of the running time of
the algorithm of Norel et al. [NLW+]. Theparallel version of the algorithm has a
reasonable speedup and modest communication requirements
Force Field Minimization: Domain Decomposition, Positive Definite Functions, and Wavelets
No full textIn force field calculations the 3D-structure of macromolecules is computed by minimization
of the total internal energy. The large numberof degrees of freedom causes numerical
problems in the optimization procedure evenfor relatively small molecules. The number
of free variables is reduced by a domain decomposition method assembling certain groups
of atoms into configurational structures with considerably less degrees of freedom. To
reduce the amount of computations necessary for a prescribed accuracy, approximations
to the energy function with respect to these variables are constructed using methods from
the theory of splines, wavelets, and positive definite functions
Marktdaten zur Biotechnologie - Produkte und Reaktoren
No full textAufgrund der hohen Wachstumsraten, die vielfach fiir den Biotechnologiemarkt
prognostiziert werden, und gefördert durch das Biotechnologie-Programm
der Bundesregierung, in dem die Entwicklung der Bioverfahrenstechnik
einen hohen Stellenwert einnimmt, kann man heute beobachten, daß zahlreiche
Anlagen- und Apparatebaufirmen erwägen, sich auf dem Gebiet der Biotechnologie
zu betätigen und zu investieren, um so langfristig an diesem Markt
teilzunehmen. Auch der Verband Deutscher Maschinen- und Anlagenbau eV
(VDMA) ist sich der wachsenden Bedeutung der Biotechnologie bewußt und
hat 1986 und 1987 Workshops zu diesem Problemkreis organisiert.
Allerdings differieren die Erwartungen und Prognosen über den zukünftigen
Biotechnologiemarkt außerordentlich stark. Selbst die Erfassung des derzeitigen
Marktvolumens ist schwierig. Die Diskrepanzen ergeben sich teilweise
durch unklare Definitionen darüber, welche Produkte der Biotechnologie
zugeordnet werden. Bei der Abschätzung des Potentials der neuen
Biotechnologieprodukte ist besonders deren Markteintritt unsicher. Viele
Prognosen sind spekulativ und nicht nachvollziehbar. "Bioprognostik"
stellt zur Zeit einen eigenen Markt mit nicht unbeträchtlichem Marktvolumen
dar. Nach einem Bericht von N. Rau in BTF-Biotech-Forum 3 (1986) 121 sind
über 300 Marktstudien, -analysen und -forschungsberichte über den Bereich
der Biotechnologie erhältlich, deren Preis sich aufsummiert auf etwa 2 Mio
DM beläuft. Für einzelne Studien sind bis zu 35.000 US $ zu zahlen, Seitenpreise
bis zu 100 DM sind keine Seltenheit. Die Vielzahl der Prognosen
allein schon verdeutlicht die Verwirrung.
Mit der Herausgabe dieses Hefts bemüht sich die GBF, die Diskussion zu
versachlichen, indem in Teil I - selbstverständlich ohne Anspruch auf
Vollständigkeit - Daten aus Marktstudien und Analysen zusammengestellt
wurden, auf deren Basis einige, wie wir meinen, konservative Schlußfolgerungen
über den erwarteten Bioboom gezogen werden können.
Im Teil II der vorliegenden Studie wird nur der Bioreaktormarkt betrachtet,
da viele Anlagen- und Apparatebauer, besonders die, die für die Pharma-,
Lebens- und Genußmittelindustrie tätig sind, hier einen günstigen Einstieg
in einen lukrativen Markt vermuten
Algal fluorescence bioassay - with correlation of spontanions and delayed fluorescence
No full textA new algal fluorence bioassay is sugguested. The toxicity will be
derived from the quotient of spontaneous and delayed fluorescence. The
inverse behaviour of spontaneous and delayed fluorescence gives a
square effect of toxicity and the time derivativ of the toxicity can be
classified in dangerous processes
Biosensors for non-medical applications (fermentation control, food and environmentalanalysis)
No full textIn the GBF - the national centre for biotechnological research in W. Germany - a task force
for biosensor development was set up in mid-1987 (1). In the two years which have elapsed
since then, various prototypes of biosensors for non-medical applications have been developed
based mainly on the concepts of flow injection analysis (FIA), fibre optics and amperometric
devices (2). In addition, investigations in protein engineering and the screening of enzymes and
antibodies pertinent to biosensor application havebeeninitiated (2)
BIOSENSORS ON-LINE IN HPLC
No full textAnalysis in clinical chemistry, food chemistry or biochemistry are
often concerned with the difficulty to determine special substances
with high sensitivity (e.g. in the pmole-range). Often the commonly
known methods for detection are not sensitive enough or complicated
sample preparations are necessary. To solve such problems in analysis
we have developed on-line-Biosensors for HPLC.
The Biosensor in this system is defined by two parts:
immobilized enzymes creating H202 and a Pt-working electrode in an
electrochemical detector.
We present here two examples of the method. Sensitive and rapid
determination of acetylcholine and choline and of hypoxanthine and
xanthine. By combining the high resolving power of HPLC with the
specificity of immobilized enzymes and the high sensitivity of
electrochemical detection, HPLC/Biosensor-technology is a simple and
economic method for many problens in analysis and will have widespread
further applications
FLOW INJECTION ZYMOGRAPHY FOR THE ON-LINE DETECTION OF ENZYMEACTIVITY
No full textnew method - Flow Injection Zymography - has been developed for the detection
of enzymeactivity during purification procedures usingflow injection analysis (FIA). Four oxidases
and a lipase were used as model enzymes. The method involves frequent sampling ofeluents via
a flow injection system and enables swift localization of the active fractions. Furthermore, the
dilution technique of zone sampling enables estimation of the purification effect