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- Lilliehorn, Tobias, et al.
(författare)
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Bioassays on ultrasonically trapped microbead clusters in microfluidic systems
- 2004
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Ingår i: Micro Total Analysis Systems 2004. - 0854048960 ; 2, s. 327-329
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Konferensbidrag (refereegranskat)abstract
- The handling of biochemically functionalised beads or particles is becoming increasingly important in µTAS. Bead-based analysis of e.g. proteins can be made sensitive due to the large active surface area and flexible by chemical design of the bead surface. We have developed a microfluidic device utilising an array of integrated and individually controlled ultrasonic microtransducers for particle trapping [1]. Particles inserted in the device are subjected to acoustic radiation forces [2] confining them at localised trapping sites. We would now, for the first time at an international conference, like to present a technique for performing bioassays on such ultrasonically trapped beads in microfluidic systems. The microfluidic device is shown in Fig. 1, where the piezoceramic ultrasonic transducers can be seen in the channel crossings in the insert. The device is designed as an acoustic resonator, to obtain localised standing acoustic waves at each transducer with essentially one pressure node in the middle of the 72 µm deep channel when operated near 10 MHz. This configuration is chosen to keep trapped particles away from the interior surfaces of the device, thus enabling fast switching of beads with a minimum in carry-over between assays. The fluidic chip, shown in Fig. 2, is designed to allow injection of microbeads, washing fluid and sample to the three trapping sites. It has been shown that the microbead clusters, as shown in Fig. 3, can be trapped at considerably high perfusion rates, up to 10 µl/min, Fig 4. As a model bioassay, 6.7 µm biotin-covered beads (PC-B-6.0, Gerlinde Kisker, Germany) were injected and transported to one tapping site using washing fluid (water). Activating the transducer trapped the beads. A solution of FITC-tagged avidin was perfused over the bead bed at 3 µl/min, using the corresponding orthogonal sample channel. After 100 s the sample flow was turned off and the bead trap was washed by perfusing water at 3 µl/min. The fluorescence response from the trapped bead clusters was monitored during the assay, and the result is shown in Fig. 5. After excess avidin was washed from the bead trap, a measured step response . indicated that avidin had bound to the beads. Finally the possibility of moving trapped microbeads between the individually controlled trapping sites in the device is shown in Fig. 6, where the transducers are activated sequentially while keeping the bead carrying washing fluid at 3 µl/min during the experiment. Work in the near future will be focused on optimising the device with respect to the bioassay performance, and in a longer perspective on expanding the concept to two dimensions to enable a new dynamic mode of generating bioanalytical arrays.
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- Melke, Jonas, 1971, et al.
(författare)
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A polymorphism in the serotonin receptor 3A (HTR3A) gene and its association with harm avoidance in women.
- 2003
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Ingår i: Archives of general psychiatry. - : American Medical Association (AMA). - 0003-990X. ; 60:10, s. 1017-23
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The brain neurotransmitter serotonin is known to affect various aspects of human behavior, including personality traits. Serotonin receptor type 3 is a ligand-gated channel encoded by 2 different subunit genes, HTR3A and HTR3B. A polymorphism (C178T) in the 5' region of the HTR3A gene has recently been identified and suggested to be of functional importance. OBJECTIVE: To elucidate the possible association between the C178T polymorphism in the HTR3A gene and personality traits in women. DESIGN: Two independent samples of 35- to 45-year-old Swedish women were recruited using the population register. Sample 1 (n = 195) was assessed via the Karolinska Scales of Personality and the Temperament and Character Inventory; sample 2 (n = 175) was assessed using the latter only. Both samples were genotyped with respect to the C178T polymorphism in the HTR3A gene. The A1596G polymorphism in the same gene was also investigated. RESULTS: A significant association between C178T genotype and the Temperament and Character Inventory factor harm avoidance was observed in sample 1 (corrected for multiple comparisons P =.04); this finding was subsequently replicated in sample 2 (P =.004) (pooled populations: P<.001). In the pooled sample, all harm avoidance subscales were found to be significantly associated with the C178T polymorphism: anticipatory worry (P =.001), fear of uncertainty (P<.001), shyness (P<.001), and fatigability and asthenia (P =.008). In addition, a significant association was found in sample 1 between the C178T polymorphism and the Karolinska Scales of Personality nonconformity factor (corrected P =.002), including the subscales of social desirability (P<.001), indirect aggression (P =.002), verbal aggression (P =.05), and irritability (P<.001). Participants homozygous for the less common T allele (<4%) differed from the remaining women by displaying lower ratings on harm avoidance and nonconformity. CONCLUSION: The C178T polymorphism in the HTR3A gene may affect the personality trait of harm avoidance in women.
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- Nilsson, Mikael, 1970-
(författare)
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Protein–DNA Recognition : In Vitro Evolution and Characterization of DNA-Binding Proteins
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- DNA-recognizing proteins are involved in a multitude of important life-processes. Therefore, it is of great interest to understand the underlying mechanisms that set the rules for sequence specific protein–DNA interactions. Previous attempts aiming to resolve these interactions have been focused on naturally occurring systems. Due to the complexity of such systems, conclusions about structure–function relationship in protein–DNA interactions have been moderate. To expand the knowledge of protein–DNA recognition, we have utilized in vitro evolution techniques. A phage display system was modified to express the DNA-binding, helix-turn-helix protein Cro from bacteriophage λ. A single-chain variant of Cro (scCro) was mutated in the amino acid residues important for sequence-specific DNA-binding. Three different phage-libraries were constructed. Affinity selection towards a synthetic ORas12 DNA-ligand generated a consensus motif. Two clones containing the motif exhibited high specificity for ORas12 as compared to control ligands. The third library selection, based on the discovered motif, generated new protein variants with increased affinity for ORas-ligands. Competition experiments showed that Arg was important for high affinity, but the affinity was reduced in presence of Asp or Glu. By measuring KD values of similar variant proteins, it was possible to correlate DNA-binding properties to the protein structure.mRNA display of scCro was also conducted. The system retained the wild-type DNA-binding properties and allowed for functional selection of the mRNA–scCro fusion. Selected species was eluted and the gene encoding the scCro was recovered by PCR. The two in vitro selection methods described in this thesis can be used to increase the knowledge of the structure–function relationship regarding protein–DNA recognition. Furthermore, we have also shown that new helix-turn-helix proteins exhibiting novel DNA-binding specificity can be constructed by phage display. The ability to construct proteins with altered DNA-specificity has various important applications in molecular biology and in gene therapy.
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- Nilsson, Mikael T.I., et al.
(författare)
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Functional expression and affinity selection of single-chain Cro by phage display: isolation of novel DNA-binding proteins
- 2000
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Ingår i: Protein Engineering. - 0269-2139 .- 1460-213X. ; 13:7, s. 519-526
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Tidskriftsartikel (refereegranskat)abstract
- A robust selection system affording phage display of the DNA-binding helix–turn–helix protein Cro is presented. The aim of the work was to construct an experimental system allowing for the construction and isolation of Cro-derived protein with new DNA-binding properties. A derivative of the phage Cro repressor, scCro8, in which the protein subunits had been covalently connected via a peptide linker was expressed in fusion with the gene 3 protein of Escherichia coli filamentous phage. The phage-displayed single-chain Cro was shown to retain the DNA binding properties of its wild-type Cro counterpart regarding DNA sequence specificity and binding affinity. A kinetic analysis revealed the rate constant of dissociation of the single-chain Cro-phage/DNA complex to be indistinguishable from that of the free single-chain Cro. Affinity selection using a biotinylated DNA with a target consensus operator sequence allowed for a 3000-fold enrichment of phages displaying single-chain Cro over control phages. The selection was based on entrapment of phage/DNA complexes formed in solution on streptavidin-coated paramagnetic beads. The expression system was subsequently used to isolate variant scCro8 proteins, mutated in their DNA-binding residues, that specifically recognized new, unnatural target DNA ligands.
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