| 1. |
- Gabig-Ciminska, Magdalena, et al.
(författare)
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Electric chips for rapid detection and quantification of nucleic acids
- 2004
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Ingår i: Biosensors & bioelectronics. - : Elsevier BV. - 0956-5663 .- 1873-4235. ; 19:6, s. 537-546
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Tidskriftsartikel (refereegranskat)abstract
- A silicon chip-based electric detector coupled to bead-based sandwich hybridization (BBSH) is presented as an approach to perform rapid analysis of specific nucleic acids. A microfluidic platform incorporating paramagnetic beads with immobilized capture probes is used for the biorecognition steps. The protocol involves simultaneous sandwich hybridization of a single-stranded nucleic acid target with the capture probe on the beads and with a detection probe in the reaction solution, followed by enzyme labeling of the detection probe, enzymatic reaction, and finally, potentiometric measurement of the enzyme product at the chip surface. Anti-DIG-alkaline phosphatase conjugate was used for the enzyme labeling of the DIG-labeled detection probe. p-Aminophenol phosphate (pAPP) was used as a substrate. The enzyme reaction product, p-aminophenol (pAP), is oxidized at the anode of the chip to quinoneimine that is reduced back to pAP at the cathode. The cycling oxidation and reduction of these compounds result in a current producing a characteristic signal that can be related to the concentration of the analyte. The performance of the different steps in the assay was characterized using in vitro synthesized RNA oligonucleotides and then the instrument was used for analysis of 16S rRNA in Escherichia coli extract. The assay time depends on the sensitivity required. Artificial RNA target and 16S rRNA, in amounts ranging from 10(11) to 10(10) molecules, were assayed within 25 min and 4 h, respectively.
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| 2. |
- Johansson, Louise, et al.
(författare)
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Shikimic acid production by a modified strain of E-coli (W3110.shik1) under phosphate-limited and carbon-limited conditions
- 2005
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 92:5, s. 541-552
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Tidskriftsartikel (refereegranskat)abstract
- Shikimic acid is one of several industrially interesting chiral starting materials formed in the aromatic amino acid pathway of plants and microorganisms. In this study, the physiology of a shikimic acid producing strain of Escherichia coli (derived from W31 10) deleted in aroL (shikimic acid kinase II gene), was compared to that of a corresponding control strain W31 10) under carbonand phosphate-limited conditions. For the shikimic acid producing strain (referred to as W31 10.shik1), phosphate limitation resulted in a higher yield of shikimic acid (0.059 +/- 0.012 vs. 0.024 +/- 0.005 c-mol/c-mol) and a lower yield of by-products from the shikimate pathway, when compared to carbon-limited condition. The yield of the by-product 3-clehydroshikimic acid (DHS) decreased from 0.076 +/- 0.028 to 0.022 +/- 0.001 c-mol/c-mol. Several other by-products were only detected under carbon-limited conditions. The latter group included 3-dehydroquinic acid (0.021 +/- 0.021 c-mol/c-mol), quinic acid (0.012 +/- 0.005 c-mol/c-mol), and gallic acid (0.002 +/- 0.001 c-mol/c-mol). For both strains, more acetate was produced under phosphate than the carbon-limited case. Considerable cell lysis was found for both strains but was higher for W3110.shikl ' and increased for both strains under phosphate limitation. The advantages of the latter condition in terms of an increased shikimic acid yield was thus counteracted by an increased cell lysis, which may make downstream processing more difficult. (c) 2005 Wiley Periodicals, Inc.
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