| 1. |
- Yakovleva, Maria, et al.
(author)
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A novel combined thermometric and amperometric biosensor for lactose determination based on immobilised cellobiose dehydrogenase.
- 2012
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In: Biosensors & Bioelectronics. - : Elsevier BV. - 1873-4235 .- 0956-5663. ; 31, s. 251-256
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Journal article (peer-reviewed)abstract
- A novel method for lactose determination in milk is proposed. It is based on oxidation of lactose by cellobiose dehydrogenase (CDH) from the basidiomycete Phanerochaete chrysosporium, immobilised in an enzyme reactor. The reactor was prepared by cross-linking CDH onto aminopropyl-silanised controlled pore glass (CPG) beads using glutaraldehyde. The combined biosensor worked in flow injection analysis (FIA) mode and was developed for simultaneous monitoring of the thermometric signal associated with the enzymatic oxidation of lactose using p-benzoquinone as electron acceptor and the electrochemically generated current associated with the oxidation of the hydroquinone formed. A highly reproducible linear response for lactose was obtained between 0.05mM and 30mM. For a set of more than 500 samples an R.S.D. of less than 10% was achieved. The assay time was ca. 2min per sample. The sensor was applied for the determination of lactose in dairy milk samples (milk with a fat content of 1.5% or 3% and also "lactose free" milk). No sample preparation except dilution with buffer was needed. The proposed method is rapid, suitable for repeated use and allows the possibility to compare results from two different detection methods, thus providing a built-in quality assurance. Some differences in the response observed between the methods indicate that the dual approach can be useful in mechanistic studies of redox enzymes. In addition, a dual system opens up interesting possibilities for studies of enzyme properties and mechanisms.
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| 2. |
- Larsson, Per Olof, et al.
(author)
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Seeing through cells : Rapid measurement of intracellular target proteins
- 2022
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In: Frontiers in Bioengineering and Biotechnology. - : Frontiers Media SA. - 2296-4185. ; 10
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Journal article (peer-reviewed)abstract
- We have studied a method for making microbial cells transparent by immersing them in a solution with a high refractive index (RI). When the RI of the solution was matching that of the cells, light scattering was greatly diminished (by a factor of up to about 100) and the cell suspension became transparent, facilitating the spectrophotometric determination of intracellular compounds such as hemoglobin. We investigated the properties of several compounds such as sucrose, glycerol, bovine serum albumin, FicollTM, and iodixanol (OptiprepTM), each with advantages and disadvantages. Particularly good overall properties were found for iodixanol at a concentration of around 36% (w/v) and bovine serum albumin at a concentration of about 30% (w/v). By using this RI-matching principle the production of intracellular compounds can easily be followed in near real-time during fermentation processes. For example, some conditions for producing plant hemoglobin in Escherichia coli were conveniently determined without the need of any cell disintegration or product purification.
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| 3. |
- Gustavsson, Per-Erik, et al.
(author)
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Direct measurements of convective fluid velocities in superporous agarose beads
- 1998
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In: Journal of Chromatography A. - 0021-9673. ; 795:2, s. 199-210
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Journal article (peer-reviewed)abstract
- Superporous agarose beads contain two sets of pores, diffusion pores and so-called superpores or flow pores, in which the chromatographic flow can transport substances to the interior of each individual bead [Gustavsson and Larsson, J. Chromatogr. A 734 (1996) 231]. The existence of pore flow may be proven indirectly by the chromatographic performance of beads but it has never been directly demonstrated in a chromatographic bed. In this report, pore flow was directly measured by following the movement of micro-particles (dyed yeast cells) in a packed bed. The passage of the micro-particles through the superpores and through the interstitial pores was followed by a microscope/video camera focused on beads which were situated four layers from the glass wall. The video recordings were subsequently used to determine the convective fluid velocities in both the superpores and the interstitial pores. Experiments were carried out with three different bead size ranges, all of which contained superporous beads having an average superpore diameter of 30 mu m. The superpore fluid velocity as % of interstitial fluid velocity was determined to be 2-5% for columns packed with 300-500-mu m beads (3% average value), 6-12% for columns packed with 180-300 mu m beads (7% average value) and 11-24% for columns packed with 106-180-mu m beads (17% average value). These data were compared to and found to agree with theoretically calculated values based on the Kozeny-Carman equation. In order to observe and accurately measure fluid velocities within a chromatographic bed, special techniques were adopted. Also, precautions were made to ensure that the experimental conditions used were representative of normal chromatography runs. (C) 1998 Elsevier Science B.V.
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| 4. |
- Gustavsson, Per-Erik, et al.
(author)
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Fast Chromatography of Proteins.
- 2003
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In: Isolation and Purification of proteins. (Biotechnology and Bioprocessing ; 27). - 0824707265 ; , s. 423-454
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Book chapter (other academic/artistic)abstract
- Abstract is not available
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| 5. |
- Gustavsson, Per-Erik, et al.
(author)
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Monolithic Polysaccharide Materials.
- 2003
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In: Monolithic Materials: Preparation, Properties and Applications (Journal of Chromatography Library ; 67). - 0444508791 ; 67
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Book chapter (other academic/artistic)abstract
- Abstract is not available
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| 6. |
- Gustavsson, Per-Erik, et al.
(author)
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Purification of plasmid DNA with a new type of anion-exchange beads having a non-charged surface
- 2004
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In: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1038:1-2, s. 131-140
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Journal article (peer-reviewed)abstract
- We have prepared a new type of anion exchanger, which effectively discriminates between RNA and plasmid DNA. The material is based on a Sephacryl S-500 HR matrix provided with quartenary amine anion-exchange groups. A distinguishing feature of the beads is that a thin (2-3 mum) outer layer of the beads lacks ion-exchange groups. In the synthesis of these beads the vinyl groups in the outer layer of vinylalkyl substituted Sephacryl S-500 HR beads are reacted with bromine. The resulting layer of bromoalkyl groups are hydrolysed, creating an inert outer layer of hydroxyalkyl groups. Finally, bromination and trimethylamine reactions of the inner vinyl groups provide the beads with a core of cationic groups. Large plasmid molecules will not bind to such beads since they are too large to enter the pores and therefore cannot come into contact with the charged matrix in the inner parts of the beads. RNA and protein molecules present in a cleared lysate, on the other hand, readily enter the pores and become adsorbed. A two-column strategy was developed for plasmid purification (recombinant pBluescript, 5.9 kilo base pairs, kbp). The first column was packed with the restricted access anion-exchanger beads (lid beads) and the second column with normal ion-exchange material (same ligand density as the lid beads). Diluted (3 x), cleared lysate was pumped through the tandem columns. The first column was subsequently disconnected from the system and the purified plasmid adsorbed on the second column was eluted in a concentrated form (6x) and with 89% recovery. The two-column procedure removed 99.5% of the RNA and 96% of the proteins. (C) 2004 Elsevier B.V. All rights reserved.
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| 7. |
- Gustavsson, Per-Erik, et al.
(author)
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Superporous agarose beads as a hydrophobic interaction chromatography support
- 1999
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In: Journal of Chromatography A. - 0021-9673. ; 830:2, s. 275-284
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Journal article (peer-reviewed)abstract
- Superporous agarose beads were used as a support for hydrophobic interaction chromatography. These beads have large connecting flow pores in addition to their normal diffusion pores. The flow pores, which are approximately one fifth of the overall diameter of the superporous agarose beads, were earlier shown to give the beads improved mass transfer properties relative to homogeneous agarose beads (Gustavsson and Larsson, J. Chromatogr. A, 734 (1996) 231-240). Superporous agarose beads and homogeneous agarose beads of the same particle size range (106-180 mu m) were derivatized with phenyl groups. The properties of the superporous beads were then compared with the homogeneous beads in the separation of a mixture of three model proteins (ribonuclease A, lysozyme and bovine serum albumin) at various superficial flow velocities from 30 to 600 cm/h. The superporous beads gave satisfactory separation at flow velocities five times higher than was possible for homogeneous beads. The performance of the two types of beads was also compared in the purification of lactate dehydrogenase from a beef heart extract at a superficial flow velocity of 150 cm/h. The superporous beads performed considerably better, leading to twice the purification factor and twice the concentration of the desired product. The results were interpreted using the theoretical treatment given by Carta and Rodrigues (Carta and Rodrigues, Chem. Eng. Sci., 48 (1993) 3927). (C) 1999 Elsevier Science B.V. All rights reserved.
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| 8. |
- Izumrudov, Vladimir, et al.
(author)
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Factors Controlling Phase Separation in Water-Salt Solutions of DNA and Polycations.
- 2003
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In: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 19:11, s. 4733-4739
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Journal article (peer-reviewed)abstract
- Factors affecting phase separation in water-salt solutions of polyelectrolyte complexes (PECs), formed by DNA and integral or pendant polycations with a quaternary amino group in every monomer unit, have been studied. When no salt was added, quantitative DNA precipitation occurred at a stoichiometric charge ratio, = [+]/[-] 1. In DNA mixtures with poly(N,N'-dimethyldiallylammonium chloride) (PDMDAAC, a pendant polycation), insoluble PECs formed in the range 0.7 < < 2. This suggests the formation of soluble, negatively charged PECs at 0 < < 0.7 and soluble, positively charged PECs at > 2. For different aliphatic ionene bromides (integral polycations), the range of corresponding to insoluble PECs was significantly broader, mainly due to the poor ability of the ionenes to form soluble, positively charged PECs. The range was also relatively broad for poly(N-ethyl-4-vinylpyridinium bromide) (a pendant polycation) and became broader with decreasing degree of polymerization of the polycation. The formation of insoluble PECs was favored by the addition of salt (NaCl), and the effect was more pronounced when decreasing the relative content of the solubilizing component, i.e., the nucleic acid at < 1 and the polycation at > 1. At moderate ionic strength, 0.12 M < [NaCl] < 0.6 M, quantitative precipitation of DNA was attained by addition of PDMAAC in the whole region studied: 1 < < 4.5. The data obtained strongly suggest that phase separation in solutions of DNA-containing PECs follows general rules revealed by studying PECs formed by flexible vinyl polyanions. However, the high rigidity of the DNA double helix appears to be responsible for the key feature revealed in the phase diagrams, i.e., significant broadening of the region for insoluble PECs at the expense of the region in which soluble DNA-containing PECs are formed. This feature may severely limit the application of DNA-containing PECs in medicine and biology but could be beneficial in the development of simple and effective procedures for DNA separation in biotechnology.
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| 9. |
- Khayyami, M, et al.
(author)
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Development of an amperometric biosensor based on acetylcholine esterase covalently bound to a new support material.
- 1998
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In: Talanta. - 1873-3573. ; 45:3, s. 557-563
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Journal article (peer-reviewed)abstract
- A new type of amperometric biosensor based on immobilised acetylcholine esterase was designed and constructed. The enzyme was immobilised on a flow-through working electrode, which was prepared from reticulated vitreous carbon (RVC) or from a composite material consisting of RVC and superporous agarose. The sensor was operated in FIA mode using acetylthiocholine as a substrate. The sensor responded to inhibitors such as paraoxon-10(-9) mol was detected by the sensor in a non-optimised configuration. The practical lifetime of the sensor was at least 1 month.
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| 10. |
- Khayyami, Masoud, et al.
(author)
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Flow-injection determination of trace hydrogen peroxide or glucose utilizing an amperometric biosensor based on glucose oxidase bound to a reticulated vitreous carbon electrode
- 1996
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In: Talanta. - : Elsevier BV. - 0039-9140. ; 43:6, s. 957-962
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Journal article (peer-reviewed)abstract
- An electron transfer mediator, 8-dimethylamino-2,3-benzophenoxazine (Meldola Blue), dissolved in the carrier solution in a flow-injection system, was found to reduce the oxidation potential for hydrogen peroxide from 600-1200 mV without mediator to - 100 mV vs. Ag/AgCl with the mediator present. The very low background current of reticulated vitreous carbon (RVC) at this potential makes it possible to detect very low levels of hydrogen peroxide or glucose. Glucose oxidase was covalently coupled with carbodiimide to RVC, and the RVC was formed into a column inserted in a flow-injection system. The calibration curve was linear from 30 nM to 10 μM glucose with 5 μM mediator. At higher mediator concentrations, the linear range was extended to 1000 μM, but with a much higher background current. The sample throughput was about 60 h-1. The current response decreased to 50% of the original response after 20 days. The coulometric yield was high because the sample was pumped through the pores of the RVC. It was 16% and 55% at a flow rate of 1 ml min -1 at mediator concentrations of 5 and 50 μM respectively.
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