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Microtiter Plates (microtiter + plate)
Selected AbstractsRapid Diversity-Oriented Synthesis in Microtiter Plates for In Situ Screening of HIV Protease InhibitorsCHEMBIOCHEM, Issue 11 2003Ashraf Brik Dr. Click and go: By using click chemistry based on a new triazole forming reaction condition (see scheme), over 100 triazole compounds generated in microtiter plates from a core structure were screened for HIV protease inhibition in situ without product isolation. Potent inhibitors, active at nanomolar concentrations, against the wild type and drug resistant mutants were identified. [source] A new multiplex assay allowing simultaneous detection of the inhibition of cell proliferation and induction of cell deathJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2005Józefa W, sierska-G Abstract The efficacy of distinct anti-cancer drugs used in the chemotherapy of human malignancies varies between tumor tissues and depends largely on the ability of the therapeutic agents to simultaneously inhibit cell proliferation and to eliminate malignant cells by apoptosis. Especially, detection of early apoptotic changes seems to be important because early stages of apoptosis differ from those of necrosis. Therefore, the development of a novel test allowing fast and concomitant screening of the anti-proliferative and pro-apoptotic action of a number of anti-cancer drugs is of great interest. For this purpose, we choose as an experimental model a well characterized anti-proliferative and pro-apoptotic effect of cisplatin (CP) on human cervical carcinoma HeLaS3 cells. As previously reported, exposure of HeLaS3 to CP resulted in a concomitant inhibition of cell proliferation and induction of apoptosis in a dose- and time-dependent manner. In the present study we performed two independent approaches. In the first approach, we examined the cell proliferation and activity of caspases-3/7 in two separate microtiter plates using the CellTiter-GloÔ Luminescent Cell Viability Assay and the Caspase-GloÔ 3/7 Assay, respectively. In the second approach, we determined the same parameters sequentially in one microtiter plate by a mutiplexing assay using CellTiter-BlueÔ Cell Viability Assay and Caspase-GloÔ 3/7 Assay. The both approaches gave very similar results indicating that this new multiplexing assay offers an important advantage for simultaneous detection of cell number and activation of caspases-3/7. The new multiplexing assay offers a range of benefits over standard assays. © 2005 Wiley-Liss, Inc. [source] ANTIMICROBIAL ACTIVITY OF MELANOIDINSJOURNAL OF FOOD QUALITY, Issue 2 2007JOSÉ A. RUFIÁN-HENARES ABSTRACT Melanoidins are high molecular weight compounds formed during the final stage of the Maillard reaction. Melanoidins have been studied in recent years because of their nutritional, biological and health implications, apart from their role on the stability during processing and shelf life of foods. A fast and robust microtiter plate-based assay for a quantitative screening of the antimicrobial activity of melanoidins was applied. Oxytetracyclin was used as reference for assessing the antimicrobial activity of different melanoidins isolated from model systems. The minimum inhibitory concentration was calculated, and activity was related to the antimicrobial activity of an oxytetracyclin solution (100 µg/L). Glucose,lysine melanoidin exerted the highest antimicrobial activity, being at a concentration of 1 mg/mL, equivalent to an oxytetracyclin solution of 170 µg/L. [source] Immunoliposomes Sandwich Fluorometric Assay (ILSF) for Detection of Escherichia coli O157:H7JOURNAL OF FOOD SCIENCE, Issue 6 2004Sungsu Park ABSTRACT: We report the development of automated flourometric immunoassay for the detection of Escherichia coli O157:H7, using antibody-directed liposomes (immunoliposomes) encapsulating fluorophore as an analytical reagent. Thiolated antibodies (anti- E. coli O157:H7) were coupled to malemide-tagged liposomes encapsulating dye. To automate the assay, a fluorescence plate reader was included in the assay system to detect fluorophore released from lysed liposomes in a microplate. The detection limit of the current assay with pure cultures of the serotype was about 104 colony-forming units (CFU)/mL. The assay can detect E. coli O157 in ground beef samples inoculated with as few as 0.8 CFU/mL after a 12-h enrichment. These results demonstrate the feasibility of using fluorophore-encapsulated immunoliposomes in a microtiter plate for the rapid and automated detection of molecules with multivalent antigenic sites. [source] Evaluation of drug precipitation of solubility-enhancing liquid formulations using milligram quantities of a new molecular entity (NME)JOURNAL OF PHARMACEUTICAL SCIENCES, Issue 11 2007Wei-Guo Dai Abstract A precipitation screening method using a 96-well microtiter plate was developed to evaluate in vitro drug precipitation kinetics of liquid formulations for poorly water-soluble compounds, using milligram quantities of compounds and milliliter volumes of biorelevant media. By using this method we identified three formulations showing distinct in vitro precipitation kinetics (fast, slow, and no precipitation) for a model new molecular entity (JNJ-25894934). The in vitro precipitation profiles in simulated intestinal fluid (SIF), fasted state simulated intestinal fluid (FaSSIF), and fed state simulated intestinal fluid (FeSSIF) were compared with those measured by a USP dissolution method, and with in vivo absorption at the fasted and fed states in canine pharmacokinetic (PK) studies. The precipitation kinetics of all three formulations in the initial hours measured by the screening method correlated to those determined by the USP method (R2,=,0.96). The PK results showed that the fast-precipitation formulation had the lowest bioavailability. However, a similar bioavailability was observed for the slow- and no-precipitation formulations. The oral bioavailability of JNJ-25894934 at the fed state was also significantly higher than that at the fasted state for all three formulations (p,<,0.05). In addition, the in vitro precipitation profiles in FeSSIF correlated better with in vivo absorption than those in SIF and FaSSIF. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96: 2957,2969, 2007 [source] Ink-Jet Printing of Luminescent Ruthenium- and Iridium-Containing Polymers for Applications in Light-Emitting DevicesMACROMOLECULAR RAPID COMMUNICATIONS, Issue 4 2005Emine Tekin Abstract Summary: Defined films of luminescent ruthenium(II) polypyridyl-poly(methyl methacrylate) (PMMA) and iridium(III) polypyridyl-polystyrene (PS) copolymers could be prepared by ink-jet printing. The copolymers were deposited on photoresist-patterned glass substrates. Films as thin as 120 nm could be printed with a roughness of 1 to 2%. In addition, the film thickness could be varied in a controlled way through the number of droplets deposited per unit area. The topography of the ink-jet printed films was analyzed utilizing an optical profilometer. The absorbance and emission spectra were measured using fast parallel UV-vis and fluorescence plate reader. Photo of the solutions of luminescent ruthenium (left) and iridium (right) containing polymers in a glass microtiter plate (top). The subsequently prepared films using ink-jet dispensing techniques are shown below. [source] Aerobic batch cultivation in micro bioreactor with integrated electrochemical sensor arrayBIOTECHNOLOGY PROGRESS, Issue 1 2010Michiel van Leeuwen Abstract Aerobic batch cultivations of Candida utilis were carried out in two micro bioreactors with a working volume of 100 ,L operated in parallel. The dimensions of the micro bioreactors were similar as the wells in a 96-well microtiter plate, to preserve compatibility with the current high-throughput cultivation systems. Each micro bioreactor was equipped with an electrochemical sensor array for the online measurement of temperature, pH, dissolved oxygen, and viable biomass concentration. Furthermore, the CO2 production rate was obtained from the online measurement of cumulative CO2 production during the cultivation. The online data obtained by the sensor array and the CO2 production measurements appeared to be very reproducible for all batch cultivations performed and were highly comparable to measurement results obtained during a similar aerobic batch cultivation carried out in a conventional 4L bench-scale bioreactor. Although the sensor chip certainly needs further improvement on some points, this work clearly shows the applicability of electrochemical sensor arrays for the monitoring of parallel micro-scale fermentations, e.g. using the 96-well microtiterplate format. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Development of a system for the on-line measurement of carbon dioxide production in microbioreactors: Application to aerobic batch cultivations of Candida utilisBIOTECHNOLOGY PROGRESS, Issue 3 2009Michiel van Leeuwen Abstract We developed and applied a conductometric method for the quantitative online measurement of the carbon dioxide (CO2) production during batch cultivations of Candida utilis on a 100-,L scale. The applied method for the CO2 measurement consisted of absorption of the produced CO2 from the exhaust gas of the microbioreactor in an alkali solution, of which the conductivity was measured on-line. The measured conductivity change of the alkali solution showed a linear relation with the total amount of CO2 absorbed. After calibration of the CO2 measurement system, it was connected to a well of a 96-well microtiter plate. The mixing in the well was achieved by a magnetic stirrer. Using online measurement of the CO2 production during the cultivation, we show reproducible exponential batch growth of C. utilis on a 100-,L scale. The CO2 production measurements obtained from the microcultivation were compared with the CO2 production measurement in a 4-L bioreactor equipped with a conventional off-gas analyzer. The measurements showed that on-line measurement of the CO2 production rate in microbioreactors can provide essential data for quantitative physiological studies and provide better understanding of microscale cultivations. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Research Letter: New Potent Indole Derivatives as Hyaluronidase InhibitorsCHEMICAL BIOLOGY & DRUG DESIGN, Issue 6 2007Süreyya Ölgen Because of the physiologic importance of hyaluronidases, the identification of potent and selective inhibitors of hyaluronidases has become increasingly important. A variety of assay methods have been used for such a purpose, i.e. classical turbidimetric, viscometric and colorimetric. In this study, a modified enzymatic assay has been used to obtain a microtiter plate-based sensitive activity screening. All inhibitors were tested in a stains-all assay at pH 7 and in a Morgan-Elson assay at pH 3.5. Among the tested compounds, 1, 2, 3, 6, 7, 8, 16, 17 and 18 showed good inhibition of more than 50%, so the IC50 values of these derivatives were determined in the range of 25,41 ,m. The IC50 value of the most active hyaluronidase inhibitor Vcpal (6-palmitoyl- l -ascorbic acid) was measured as 8.36 ,m. All inhibitors including Vcpal showed twofold less activity at pH 3.5 in a Morgan-Elson assay. Examination of substituent effects on the activity showed that para -positions of benzamide needs to be chlorinated or fluorinated to obtain good inhibitory effect. It was found that the introduction of a p -fluoro benzyl ring in the indole nitrogen has a positive effect for the inhibitory effects of both indole-2- and 3-carboxamide derivatives. [source] An epidemiologic analysis of staphylococcus aureus-associated keratitis in BostonACTA OPHTHALMOLOGICA, Issue 2009I BEHLAU Purpose S. aureus is a normal commensal of the human skin and nasopharynx. It is therefore of interest to determine whether S. aureus keratitis is caused by a subset of these organisms. In this study, the phenotypic and genotypic characteristics of S.aureus keratitis isolates were analyzed. Methods All S. aureus clinical isolates were prospectively collected over a 24 month period at the MEEI (2006-2008). The diagnosis of clinical keratitis and associated risk factors was by medical record review. Keratitis-associated S. aureus strains were assessed for: 1) antibiotic susceptibility, 2) biofilm robustness by gentian violet staining using an in vitro microtiter plate assay, and 3) genetic lineage by multi-locus sequence typing (MLST). Results 26 cases of keratitis were identified from the 600 S. aureus clinical isolates. Risk factors associated with S.aureus keratitis included trauma, prior surgery, soft contact lens wear, and the presence of a foreign body. Ocular surface disease does not appear to be an independent risk factor. All 26 isolates were tetracycline- and trimethoprim-sulfamethoxazole- sensitive. All the MRSA strains were found to be ciprofloxacin-resistant (10/26). Nearly one-half of all the S.aureus keratitis-associated isolates were caused by a single clone, ST5. Both methicillin sensitive and resistant S. aureus strains were represented within ST5. Conclusion These results suggest that there may be specific S.aureus lineages which possess phenotypic and genotypic characteristics that enable S. aureus to more effectively cause sight-threatening keratitis. Future work will examine their virulence traits and a comparison to commensal S.aureus strains. [source] Enhancement of antibiotic and secondary metabolite detection from filamentous fungi by growth on nutritional arraysJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2008G.F. Bills Abstract Aims:, We asked to what extent does the application of the OSMAC (one strain, many compounds) approach lead to enhanced detection of antibiotics and secondary metabolites in fungi? Protocols for bacterial microfermentations were adapted to grow fungi in nutritional arrays. Methods and Results:, Protocols for microfermentations of non-sporulating fungi were validated using known antifungal-producing fungi. Detection of antifungal activity was often medium dependent. The effects of medium arrays and numbers of strains on detection of antifungal signals were modelled by interpolation of rarefaction curves derived from matrices of positive and negative extracts. Increasing the number of fermentation media for any given strain increased the probability of detection of growth inhibition of Candida albicans. Increasing biodiversity increased detection of antifungal phenotypes, however, nutritional arrays could partly compensate for lost antibiotic phenotypes when biodiversity was limiting. Conclusions:, Growth and extraction in microtiter plates can enable a discovery strategy emphasizing low-cost medium arrays that can better exploit the metabolic potential of strains. Significance and Impact of the Study:, Increasing fermentation parameters raise the probability of detecting bioactive metabolites from strains. The protocols can be used to pre-select strains and their growth conditions for scale up that will most likely yield antibiotics and secondary metabolites. [source] Chlorhexidine release and antibacterial properties of chlorhexidine-incorporated polymethyl methacrylate-based resin cementJOURNAL OF BIOMEDICAL MATERIALS RESEARCH, Issue 1 2010N. Hiraishi Abstract This study evaluated chlorhexidine release from experimental, chlorhexidine-incorporated polymethyl methacrylate (PMMA)-based resin cements prepared from Super-Bond C&B (Sun Medical) and examined the antimicrobial activity against Streptococcus mutans and Enterococcus faecalis. Chlorhexidine diacetate was added into PMMA polymer to obtain chlorhexidine concentration of 0.0, 1.0, 2.0, 3.0, and 4.0 wt %. Chlorhexidine-incorporated, cured resin disks were immersed in distilled water at 37°C for 5 weeks, and the chlorhexidine release was analyzed by high-performance liquid chromatography. The antibacterial effect of freshly mixed resin cements was examined using the agar diffusion test. For the direct contact test, the wells (n = 6) of microtiter plates were coated with cements. The coated wells were aged up to 3 weeks prior to the placement of bacterial suspensions directly on cured cements. The 3.0 and 4.0% chlorhexidine-incorporated cement exhibited chlorhexidine release for 5 weeks; however, more than 98% of chlorhexidine was retained in resin matrix. No release was detected from the 1.0 and 2.0% incorporated cement at 1 week and 2 weeks, respectively. The agar diffusion test failed to detect antibacterial effects against Enterococcus faecalis, whereas the direct contact test revealed the antibacterial effect of 3.0 and 4.0% incorporated cements against each microbe for 2 weeks. The 3.0 and 4.0% chlorhexidine-incorporated resin cement possessed prolonged chlorhexidine release and antibacterial properties for 2 weeks. © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2010 [source] A new multiplex assay allowing simultaneous detection of the inhibition of cell proliferation and induction of cell deathJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2005Józefa W, sierska-G Abstract The efficacy of distinct anti-cancer drugs used in the chemotherapy of human malignancies varies between tumor tissues and depends largely on the ability of the therapeutic agents to simultaneously inhibit cell proliferation and to eliminate malignant cells by apoptosis. Especially, detection of early apoptotic changes seems to be important because early stages of apoptosis differ from those of necrosis. Therefore, the development of a novel test allowing fast and concomitant screening of the anti-proliferative and pro-apoptotic action of a number of anti-cancer drugs is of great interest. For this purpose, we choose as an experimental model a well characterized anti-proliferative and pro-apoptotic effect of cisplatin (CP) on human cervical carcinoma HeLaS3 cells. As previously reported, exposure of HeLaS3 to CP resulted in a concomitant inhibition of cell proliferation and induction of apoptosis in a dose- and time-dependent manner. In the present study we performed two independent approaches. In the first approach, we examined the cell proliferation and activity of caspases-3/7 in two separate microtiter plates using the CellTiter-GloÔ Luminescent Cell Viability Assay and the Caspase-GloÔ 3/7 Assay, respectively. In the second approach, we determined the same parameters sequentially in one microtiter plate by a mutiplexing assay using CellTiter-BlueÔ Cell Viability Assay and Caspase-GloÔ 3/7 Assay. The both approaches gave very similar results indicating that this new multiplexing assay offers an important advantage for simultaneous detection of cell number and activation of caspases-3/7. The new multiplexing assay offers a range of benefits over standard assays. © 2005 Wiley-Liss, Inc. [source] Bioprocess scale-up: quest for the parameters to be used as criterion to move from microreactors to lab-scaleJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 9 2010Marco P. C. Marques Abstract Advances in high-throughput process development and optimization involve the rational use of miniaturized stirred bioreactors, instrumented shaken flasks and microtiter plates. As expected, each one provides different levels of control and monitoring, requiring a compromise between data quantity and quality. Despite recent advances, traditional shaken flasks with nominal volumes below 250 mL and microtiter plates are still widely used to assemble wide arrays of biotransformation/bioconversion data, because of their simplicity and low cost. These tools are key assets for faster process development and optimization, provided data are representative. Nonetheless, the design, development and implementation of bioprocesses can present variations depending on intrinsic characteristics of the overall process. For each particular process, an adequate and comprehensive approach has to be established, which includes pinpointing key issues required to ensure proper scale-up. Recently, focus has been given to engineering characterization of systems in terms of mass transfer and hydrodynamics (through gaining insight into parameters such as kLa and P/V at shaken and microreactor scale), due to the widespread use of small-scale reactors in the early developmental stages of bioconversion/biotransfomation processes. Within this review, engineering parameters used as criteria for scaling-up fermentation/bioconversion processes are discussed. Particular focus is on the feasibility of the application of such parameters to small-scale devices and concomitant use for scale-up. Illustrative case studies are presented. Copyright © 2010 Society of Chemical Industry [source] Competitive ELISA studies of neural thread protein in urine in Alzheimer's diseaseJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 1 2007Susanna Levy Abstract A specific and reliable competitive affinity assay kit has been developed to quantitatively measure neural thread protein (NTP) in first morning urine samples. This assay, called the urine neural thread protein test (UNTP), is a competitive enzyme-linked immunosorbent assay (ELISA) format affinity assay using 32-well microtiter plates. The assay detects UNTP in the 10,60,µg/mL range (an improvement over earlier assays of 103 × ), is linear and more reproducible (average coefficient of variation [CV] 6.2% in precision studies). The utility of the assay has been demonstrated in urine samples from patients with Alzheimer's disease (AD) and controls (sensitivity of 90% and specificity of 91%). Test,retest assays of subjects with AD and controls were comparatively stable at intervals of 2 days to 4.5 years, which suggests that positive (elevated) or negative (normal) NTP levels do not fluctuate significantly over time with respect to the cutoff. J. Clin. Lab. Anal. 21:24,33, 2007. © 2007 Wiley-Liss, Inc. [source] Development of a Monoclonal Antibody for Grouper (Epinephelus marginatus) and Wreck Fish (Polyprion americanus) Authentication Using an Indirect ELISAJOURNAL OF FOOD SCIENCE, Issue 6 2003L. Asensio ABSTRACT: A monoclonal antibody generated against soluble muscle proteins from grouper (Epinephelus marginatus) has been used in 2 indirect ELISA formats (microtiter plates and immunostick tubes) for the rapid authentication of grouper and wreck fish (Polyprion americanus). This monoclonal antibody (1A4-MAb) was tested against native and heat-treated (cooked and sterilized) soluble muscle protein extracts from several commonly marketed fish species and only reacted with grouper and wreck fish samples. [source] An Electrochemical Robotic System for the Optimization of Amperometric Glucose Biosensors Based on a Library of Cathodic Electrodeposition PaintsMACROMOLECULAR RAPID COMMUNICATIONS, Issue 1 2004Sabine Reiter Abstract Summary: A library of 148 cathodic electrodeposition paints was synthesized and the properties of related amperometric glucose biosensors were evaluated. For this a novel automatic electrochemical robotic system was designed. The automatic biosensor fabrication and characterization sequence involves the electrochemically induced precipitation of the cathodic paint on the electrode surface in the presence of glucose oxidase, the conditioning of the obtained polymer layer, the recording of a glucose calibration graph and the quantitative dissolution of the polymer film and cleaning of the electrode surface. Schematic representation of the developed electrochemical robotic system for electrochemical screening in microtiter plates. [source] Synthesis and Quality Control of Thiol Tagged Compound Libraries for Chemical MicroarraysMOLECULAR INFORMATICS, Issue 11 2006Sabine Maier Abstract A method for the synthesis and quality control of compound collections containing reactive thiol functions was developed. Such libraries form the basis for the construction of chemical microarrays to be used in fragment-based screening. Amino-modified polymer membranes fixed into the wells of microtiter plates were used as the solid phase for the nanomole-scale synthesis of a thiol-tagged small molecule library using a spatial one-compound/one-well strategy. A thiolselective Liquid Chromatography-Mass Spectroscopy (LC-MS) protocol of each compound before attachment to the microarray surface was established, allowing an exact determination of compound purity and concentration. The established synthesis and quality control method is an important prerequisite for an accurate read-out of the array compound,target interaction data, and simplifies the usage of small molecule microarrays for low affinity screening. [source] Quantification of biofilm in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci,APMIS, Issue 8 2007SRDJAN STEPANOVI The details of all steps involved in the quantification of biofilm formation in microtiter plates are described. The presented protocol incorporates information on assessment of biofilm production by staphylococci, gained both by direct experience as well as by analysis of methods for assaying biofilm production. The obtained results should simplify quantification of biofilm formation in microtiter plates, and make it more reliable and comparable among different laboratories. [source] Microfluidic biolector,microfluidic bioprocess control in microtiter platesBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2010Matthias Funke Abstract In industrial-scale biotechnological processes, the active control of the pH-value combined with the controlled feeding of substrate solutions (fed-batch) is the standard strategy to cultivate both prokaryotic and eukaryotic cells. On the contrary, for small-scale cultivations, much simpler batch experiments with no process control are performed. This lack of process control often hinders researchers to scale-up and scale-down fermentation experiments, because the microbial metabolism and thereby the growth and production kinetics drastically changes depending on the cultivation strategy applied. While small-scale batches are typically performed highly parallel and in high throughput, large-scale cultivations demand sophisticated equipment for process control which is in most cases costly and difficult to handle. Currently, there is no technical system on the market that realizes simple process control in high throughput. The novel concept of a microfermentation system described in this work combines a fiber-optic online-monitoring device for microtiter plates (MTPs),the BioLector technology,together with microfluidic control of cultivation processes in volumes below 1,mL. In the microfluidic chip, a micropump is integrated to realize distinct substrate flow rates during fed-batch cultivation in microscale. Hence, a cultivation system with several distinct advantages could be established: (1) high information output on a microscale; (2) many experiments can be performed in parallel and be automated using MTPs; (3) this system is user-friendly and can easily be transferred to a disposable single-use system. This article elucidates this new concept and illustrates applications in fermentations of Escherichia coli under pH-controlled and fed-batch conditions in shaken MTPs. Biotechnol. Bioeng. 2010;107: 497,505. © 2010 Wiley Periodicals, Inc. [source] Scaling-up of complex whole-cell bioconversions in conventional and non-conventional mediaBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2010Marco P.C. Marques Abstract The use of whole cells is becoming a more common approach in pharmaceutical and agrochemical industries in order to obtain pure compounds with fewer production steps, higher yields, and cleaner processes, as compared to those achieved with traditional strategies. Whole cells are often used as enzymes pools, in particular when multi-step reactions and/or co-factor regeneration are envisaged. Nonetheless, published information on the scale-up of such systems both in aqueous and in two-phase aqueous,organic systems is relatively scarce. The present work aims to evaluate suitable scale-up criteria in conventional and non-conventional medium for a whole-cell bioconversion that uses resting cells of Mycobacterium sp. NRRL B-3805 to cleave the side chain of ,-sitosterol, a poorly water-soluble substrate. The experiments were performed in 24-well microtiter plates and in 250,mL shaken flasks as orbital stirred systems, and in 300,mL stirred tanks as mechanically stirred systems. Results show that productivity yields were similar in all scales tested, when maintaining oxygen mass transfer coefficients constant in aqueous systems, or when maintaining constant volumetric power consumption in aqueous,organic two-phase systems. Biotechnol. Bioeng. 2010;106: 619,626. © 2010 Wiley Periodicals, Inc. [source] "Enzyme Test Bench," a high-throughput enzyme characterization technique including the long-term stabilityBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2009Kirill Rachinskiy Abstract A new high throughput technique for enzyme characterization with specific attention to the long term stability, called "Enzyme Test Bench," is presented. The concept of the Enzyme Test Bench consists of short term enzyme tests in 96-well microtiter plates under partly extreme conditions to predict the enzyme long term stability under moderate conditions. The technique is based on the mathematical modeling of temperature dependent enzyme activation and deactivation. Adapting the temperature profiles in sequential experiments by optimal non-linear experimental design, the long term deactivation effects can be purposefully accelerated and detected within hours. During the experiment the enzyme activity is measured online to estimate the model parameters from the obtained data. Thus, the enzyme activity and long term stability can be calculated as a function of temperature. The engineered instrumentation provides for simultaneous automated assaying by fluorescent measurements, mixing and homogenous temperature control in the range of 10,85,±,0.5°C. A universal fluorescent assay for online acquisition of ester hydrolysis reactions by pH-shift is developed and established. The developed instrumentation and assay are applied to characterize two esterases. The results of the characterization, carried out in microtiter plates applying short term experiments of hours, are in good agreement with the results of long term experiments at different temperatures in 1 L stirred tank reactors of a week. Thus, the new technique allows for both: the enzyme screening with regard to the long term stability and the choice of the optimal process temperature regarding such process parameters as turn over number, space time yield or optimal process duration. The comparison of the temperature dependent behavior of both characterized enzymes clearly demonstrates that the frequently applied estimation of long term stability at moderate temperatures by simple activity measurements after exposing the enzymes to elevated temperatures may lead to suboptimal enzyme selection. Thus, temperature dependent enzyme characterization is essential in primary screening to predict its long term behavior. Biotechnol. Bioeng. 2009;103: 305,322. © 2008 Wiley Periodicals, Inc. [source] Biotec Visions July 2009BIOTECHNOLOGY JOURNAL, Issue 7 2009Article first published online: 17 JUL 200 News: Mutagenic biodiesel blends , Technicolor cancer imaging , Anticancer nanoparticle , Increased oxygen transfer in baffled microtiter plates , Transgenic barley growing on acid soil , Brain music , Laser light-induced brain waves , First genome sequence of ruminant species Special issues: Cytometry of microbes , Food-borne Mycotoxins Book highlights: Biotech funding trends , Biotechnology in Flavor Production Opinion: Another biofuel blunder? Tips and tricks: Good to know: Gel Electrophoresis Test your knowledge. Do you recognize this? Most read Writing tips Briefs: A hypothetical new model of LDL , Gaden Award , Patenting hES cells in Europe [source] High-throughput system for determining dissolution kinetics of inclusion bodiesBIOTECHNOLOGY JOURNAL, Issue 5 2009Astrid Dürauer Dr. Abstract Efficient solubilization is a crucial step during inclusion body processing and dissolving conditions were usually empirically established. Here we describe a new methodology for rapid screening of solubilization conditions and evaluation of dissolution kinetics in microtiter plates. Increase of protein in solution over time was directly related to decrease of turbidity measured by absorbance at 600 nm. Dissolution kinetics of inclusion bodies were described by a first-order reaction kinetics, which was used for drug dissolution modeling. Reaction constants were in the range of 0.01,0.03 s,1 for buffer conditions providing sufficient solubilization power. This method is not limited to the screening of optimal buffer conditions for solubilization and can be applied for studying other parameters involved in the solubility of IBs, such as pI of the protein, influence of fermentation conditions, influence of initial protein concentration, and more. [source] Rapid Diversity-Oriented Synthesis in Microtiter Plates for In Situ Screening of HIV Protease InhibitorsCHEMBIOCHEM, Issue 11 2003Ashraf Brik Dr. Click and go: By using click chemistry based on a new triazole forming reaction condition (see scheme), over 100 triazole compounds generated in microtiter plates from a core structure were screened for HIV protease inhibition in situ without product isolation. Potent inhibitors, active at nanomolar concentrations, against the wild type and drug resistant mutants were identified. [source] | |||||||||||||||||||