Publications & Communications
Repair of oxidative damage of thymine by HeLa whole-cell extracts : simultaneous analysis using a micosupport and comparison with traditional PAGE analysis.
Guerniou V., Rapin D., Millau J.F., Bufflier E., Favier A., Cadet J., Sauvaigo S.
Biochimie, in press
Development of DNA and immunoenzymatic biochips for high-throughput or rapid testing-based analyses in diagnostic and agri-food applications.
Cleuziat Ph.
Nanointerface in Biosmart Tools, 17th Entretiens du Centre Jacques Cartier, Montreal, Canada, October 7-8, 2004
The use of biochips is progressively moving towards routine analysis application as technologies become mature and industrialization issues are being solved. Among many application fields so far potentially concerned by such tools, including diagnostic of infectious diseases, therapeutic monitoring, food safety and quality or environment control, product requirements can significantly differ. Therefore, complementary technologies or formats might be used depending on their selective and competitive advantages. In that perspective, we developed biochip supports and technologies that can address either high throughput-based applications using standard laboratory equipment, or rapid analysis of sample in an emergency or field testing situation. Both DNA and antibody biochip applications will be presented with OLISATM (OLIgo Sorbent Arrays) and ILISATM (Immuno Linked Sorbent Array) 96-well microtiterplate-based biochips, relying on densitometric reading with a high resolution imaging system. Fast and direct detection of label-free biomolecules with electronic biochips will also be presented, focusing on reading modes and properties of electroactive polymer layers.
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Laurent A., de Lamber B., Charreyre M.T., Mandrand B., Chaix C.
Tetrahedron Letters, 2004, Vol 45, 8883-8887
An oligonucleotide microarray for the monitoring of repair enzyme activity toward different DNA base damage
Sauvaigo S., Guerniou V., Rapin D., Gasparutto D., Caillat S.,Favier A.
Analytical Biochemistry, 2004, Vol 333(1), 182-192
Optimal experimental conditions for the measurement of repair activities of DNA-N-glycosylases contained in cell extracts on a biochip
Guerniou V., Rapin D., Millau J.F., Bufflier E., Favier A., Cadet J., Sauvaigo S.
Applied Nanoscience, 2004, Vol A (N°1), 39-46
Etude de la réparation enzymatique de lésion de l'ADN en utilisant des oligonucléotides fixés sur un support
Guerniou V.
Ph.D. thesis, Laboratoire de lésion des Acides Nucléiques (CEA Grenoble) - Université Joseph Fourier, Grenoble, 2003, 183p.
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Bufflier E.
AgroGene XIIth seminar: GMOs: after the moratorium-Safety,
acceptability, tracability and potential Paris, France, February 26-27, 2004
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Perrin A., Duracher D., Perret M., Cleuziat P., Mandrand B.
Reprinted from Analytical biochemistry, 2003, Vol 322/2, 148-155, with
permission from Elsevier SAS (ed).
Improved performance of a protein array using conjugated polymers as capture phase for HIV serodiagnosis
Perrin A., Duracher D., Allard L., Cleuziat Ph.,Theretz A., Mandrand B..
Polymer International, May 2004, vol. 53, no. 5, pp. 586-590(5).
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Lopez-Crapez E., Livache T., Caillat P., Zsoldos D.
Methods Mol Med. 2004, vol. 97, pp 337-354
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Perrin A., Duracher D., Cleuziat Ph., Mandrand B..
BioAnalytica. München, Germany, April 1 - 4, 2003.
A low complexity antibody microarray for the detection and quantification of human hormones associated to fertility is described. Four parameters (FSH, TSH, LH, HGC) covering a wide range of concentration in women blood were chosen. Antibodies were spotted in 96-well microtiter plates for a final complexity of 8+1 spots/well. After sample incubation, wells were exposed to alkaline phosphatase labelled antibodies. The addition of a precipitating substrate induced spots coloration those density was correlated to analyte concentration. Specificity was investigated by adding consecutively each purified diluted hormone. Sensitivities were studied with solutions of known protein concentration. Also, comparisons were made with these reached on VIDAS™ immunoassay automate for the same serum dilutions.Testing several tens of human blood samples allowed obtaining convincing correlations between ILISA™ and VIDAS™ for each parameter. The complete range of physiological variations was covered. ILISA™ dynamic range was also enhanced by exploiting enzymatic revelation kinetic and by taking into account spot diameter at the issue ofthe reaction. Reproducibility, function of spot density, was in all cases below 10%.
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Sauvaigo S., Guerniou V., Gasparutto D.,Millau J.F.
6th Winter Research Conference on Free Radicals in Biology: Oxydative Damage to DNA - Formation and Biological Features. Les Houches, France, March 22-28, 2003.
Repair of DNA damage involves a very complex network of interacting proteins. Glycosylases are in charge of eliminating double-helix non distorting DNA lesions. Determination of the substrate specificity for each known prokaryoticor eukaryotic enzyme as well as precise measurement of glycosylase activities within either purified preparations or crude cell extracts constitute the matter of more and more publications in the literature.Studies are usually performed with radio-labeled synthetic oligonucleotides containing targeted lesions. The digestion products are analyzed after electrophoresis on denaturing polyacrylamide gels.It seemed then interesting to set up a versatile and generic tool that could help to study the repair of different lesions at the same time, to get rid of the radio-labeled material, and sensitive enough to be used for clinical evaluation of the roleof DNA damage repair. We thus designed a low density microchip tool functionnalized with different lesions bearing oligonucleotides.We used commercial (OLIgoSorbent Array-Apibio™, Grenoble, France) microchips constituted of microwells functionnalized with different oligonucleotides to set up the appropriate hybrialization and enzymatic digestion conditions.Each well comprises 17 distinct spots including spotting and revelation controls. The lesion bearing oligonucleotides, that are also labeled by a 3’biotin, were indirectly hybridized. After incubation with the glycosylase containingmedium and washings, the modified oligonucleotides remaining on the support were revealed. The revelation was done by incubation with streptavidin-phycoerythrin and the different signals were integrated using an appropriate software (ApiAnalyser-Apibio™) after fluorescence microscope image analysis.
OLISA™ Microchips : versatile, flexible and high throughput DNA Chips for genotyping-based applications
Cleuziat Ph.
AgroGene's XIth Seminar: High Throughput Genotyping - Technology, Applications & Perspectives. Paris, France, February 27-28, 2003.
OLISA™ (OLIgo Sorbent Arrays) are microarrays of up to 17 probes, including internal controls, arrayed on the well bottom of a 96-well microtiter plate using a proprietary surface chemistry. Short oligonucleotide probes are designed and optimized to hybridize specifically to any single nucleotide polymorphism of an amplified DNA target. OLISA™ microarrays procedures rely on a standard and robust protocol based on a single temperature process and colorimetric detection methods. This system allows to accurately characterize genotypes, with high signal to noise ratios. This extremely flexible platform is suitable to custom development of applications, such as genetic disease monitoring, bacterial identification, Genetically-Modified Organisms screening. Together with its associated imaging system Apimager™, this versatile 96-well OLISA™ format is easy to use with standard lab equipment and can be automated to reach high throughput requirements. Arrays of arrays can be designed on demand using combinations of wells and/or 8-well strips to fit wide product development specifications. The same technological platform can also be applied to proteomics with antigen and antibodibody panels detection using adequate surface chemistry.
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Abaibou H., Baud M., Cuzin Y., Mandrand B., Cleuziat Ph.
Lab-on-a-Chip and Microarrays: Discovery to Development. Zurich, Switzerland, February 13-14, 2003.
A new versatile oligonucleotide microarray (OLISA™: OLIgo Sorbent Arrays) has been developed by Apibio to simultaneously monitor up to 1572 different genes. This technology uses the 96-well microtiter plate as a platform in which up to 17 different probes can be attached to the bottom of each well. Therefore, OLISA™ technology permits a high throughput multidetection assay. Using synthetic biotin-labeled targets (50-70 mers), OLISA™ was shown to detect a low number of targets (<107 copies per assay) with a dynamic range of 2.5 logs. Both low and high copy number messenger RNAs were detected from 0.2 µg of total RNA per array, as demonstrated on a yeast model for cadmium toxicity.
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Bufflier E., Nicod A., Marcotte S., Moret C., Cleuziat Ph.
Lab-on-a-Chip and Microarrays: Discovery to Development. Zurich, Switzerland, February 13-14, 2003.
OLISA™ (OLIgo Sorbent Arrays) are microarrays of up to 17 probes arrayed on the well bottom of a 96-well microtiter plate using a proprietary surface chemistry. It has been used to characterize two human SNPs. Short oligonucleotide probes were designed to hybridize specifically to each allele of amplified DNA targets, during a single temperature process. Detection was performed using a colorimetric method. This system allowed to accurately characterize genotypes.
Development of DNA Chips & Multi-detection Tools for Genotyping, Gene Expression Monitoring & Immunoassays
Cleuziat Ph., Plenary Session.
21st European Conference on Dermocosmetology. Lyon, France, January 16-17, 2003.
OLISA™ (OLIgo Sorbent Arrays) are microarrays of up to 17 probes, including internal controls, arrayed on the well bottom of a 96-well microtiter plate using a proprietary surface chemistry. OLISA™ has been used to characterize human SNPs. Short oligonucleotide probes were designed and optimized to hybridize specifically to each allele of an amplified DNA target. OLISA™ microarrays were used in a single temperature process and detection was performed using a colorimetric method. This system allowed to accurately characterize genotypes, with signal to noise level greater than 2. This extremely flexible platform is suitable to custom development of applications, from biological species identification to SNP characterization. This versatile 96-well OLISA™ format, easy to use with standard lab equipment, can be automated to reach high throughput requirements. Indeed, OLISA™ was used to simultaneously monitor the expression of gene panels. It was shown to detect a low number of targets (<107 copies per assay) with a dynamic range of 2.5 logs. Both low and high copy number messenger RNAs were detected from 0.2 µg of total RNA per array, as demonstrated on a yeast model for cadmium toxicity. This versatile technology can also be applied to immunoassay panel detection using adequate surface chemistry and identical system environment.
Microtechnologies for Bio-Pharmaceutical Industries
Cleuziat Ph.
19th Euro-Immunology Conference, CORATA / ACOMEN. Lyon, France, October 22 - 24, 2002.
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Cuzin M.
In Tranfus Clin Biol. Elsevier SAS (ed.), 2001; 8: 291 - 6.
DNA chips are miniaturized microsystems based on the ability of DNA to spontaneously find and bind its complementary sequence in a highly specific and reversible manner, known as hybridization. Labeled DNA molecules in a sample are analyzed by DNA probes tethered at distinct sites on a solid support. The composition of the DNA sample is then deduced by analyzing the signal generated by labels present at each probe site. Applications are widespread: fundamental research, cancer or microbiology diagnostics, genotyping, gene expression, pharmacogenomics, and environmental control. Medical application consists, for example, in the identification and detection of mutations in genes responsible for cancers, or DNA chip analysis of individual polymorphisms which may provide a guide towards the most efficient treatment. In the environmental and agro-industrial fields, DNA chips show great promise in rapidly testing microorganism content, contamination or pathogenicity. DNA chip dimensions offer hybridization sites in the 50-200 micron range, producing arrays ranging from 100 to 1,000,000 different probes per cm².
Apibio
15, rue des Martyrs - zone ASTEC
38054 Grenoble Cedex 9 - France
Phone: +33 (0) 4 38 78 62 33 - Fax: +33 (0) 4 38 78 53 74
www.apibio.com - E-mail: contact@apibio.com