| 1. |
- Evander, Mikael, et al.
(författare)
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Study of ATP-release from acoustically levitated eryhrocytes
- 2007
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Ingår i: The proceedings of micro total analysis systems 2007. - 9780979806407 ; 2, s. 1372-1374
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Konferensbidrag (refereegranskat)abstract
- Erythrocytes (red blood cells) are known to produce large amounts of Adenosine Triphosphate (ATP). It has recently become clear that the ATP-release is part of a mechanism controlling the dilation of the body’s blood vessels. The study of the erythrocyte’s behaviour is complicated by the fact that they respond easily to any physical contact. In this paper we propose a new method for studying the dynamics of the ATP-release by combining non-contact acoustic trapping in a microfluidic chip with a chemiluminiscent assay. Sensitivity levels down to 10 pM were achievable and the ATP-release from a cluster of levitated live erythrocytes was recorded.
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| 2. |
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| 3. |
- Hernández-Neuta, Iván, et al.
(författare)
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Microfluidic magnetic fluidized bed for DNA analysis in continuous flow mode
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Magnetic solid phase substrates for biomolecule manipulation have become a valuable tool for simplification and automation of molecular biology protocols. However, the handling of magnetic particles inside microfluidic chips for miniaturized assays is often challenging due to inefficient mixing, aggregation, and the advanced instrumentation required for effective actuation. Here, we describe the use of a microfluidic magnetic fluidized bed approach that enables dynamic, highly efficient and simplified magnetic bead actuation for DNA processing in a continuous flow platform with minimal technical requirements. We evaluate the performance of this approach by testing the efficiency of individual steps of a DNA assay based on padlock probes and rolling circle amplification (RCA). This assay comprises common nucleic acid analysis principles, such as hybridization, ligation, amplification and restriction digestion. We obtained efficiencies of up to 90% for these reactions and high throughput capabilities, with flow rates up to 5 L/min without compromising performance. The obtained efficiency values using the fluidized bed were superior to a commercially available solution for microfluidic manipulation of magnetic beads. Moreover, to demonstrate the potential of this approach for integration into micro-total analysis systems, we optimized the production of a low-cost polymer based micro arrayand tested its analytical performance for integrated single-molecule digital read-out. Finally, we provide the proof-of-concept for a single-chamber microfluidic chip that combines the fluidized bed with the polymer microarray for a highly simplified and integrated magnetic bead-based DNA analyzer, with potential applications in diagnostic systems.
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| 4. |
- Hernández-Neuta, Iván, et al.
(författare)
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Microfluidic magnetic fluidized bed for DNA analysis in continuous flow mode
- 2018
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Ingår i: Biosensors & bioelectronics. - : Elsevier BV. - 0956-5663 .- 1873-4235. ; 102, s. 531-539
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Tidskriftsartikel (refereegranskat)abstract
- Magnetic solid phase substrates for biomolecule manipulation have become a valuable tool for simplification and automation of molecular biology protocols. However, the handling of magnetic particles inside microfluidic chips for miniaturized assays is often challenging due to inefficient mixing, aggregation, and the advanced instrumentation required for effective actuation. Here, we describe the use of a microfluidic magnetic fluidized bed approach that enables dynamic, highly efficient and simplified magnetic bead actuation for DNA analysis in a continuous flow platform with minimal technical requirements. We evaluate the performance of this approach by testing the efficiency of individual steps of a DNA assay based on padlock probes and rolling circle amplification. This assay comprises common nucleic acid analysis principles, such as hybridization, ligation, amplification and restriction digestion. We obtained efficiencies of up to 90% for these reactions with high throughput processing up to 120 mu L of DNA dilution at flow rates ranging from 1 to 5 mu L/min without compromising performance. The fluidized bed was 20-50% more efficient than a commercially available solution for microfluidic manipulation of magnetic beads. Moreover, to demonstrate the potential of this approach for integration into micro-total analysis systems, we optimized the production of a low-cost polymer based microarray and tested its analytical performance for integrated single-molecule digital read-out. Finally, we provide the proof-of-concept for a single-chamber microfluidic chip that combines the fluidized bed with the polymer microarray for a highly simplified and integrated magnetic bead-based DNA analyzer, with potential applications in diagnostics.
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| 5. |
- Kalm, Frida
(författare)
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Studies of cell-on-chip technology and basophil regulation for improved allergy diagnostics
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Allergic diseases affect approximately 30% of adults and has an impact on both the individual’s quality of life as well as an economic impact on society. Two effector cells involved in allergic disease are mast cells and basophils, where basophils are more readily available in blood and therefore of great interest when studying allergy. Basophils can be recruited into the tissue during inflammation originating from for example allergic reactions or parasite infections. Allergy diagnostics starts with evaluation of the patient’s medical history followed by in vivo and/or in vitro testing. All diagnostic tests have different advantages and disadvantages are chosen depending on the patient and the circumstances. In vivo tests include the gold standard of allergy diagnostics, which is the challenge tests, but also the commonly used skin prick test (SPT). Allergy diagnostics can also be done in vitro using allergen-specific IgE antibody assays and the basophil activation test (BAT). BAT is useful to study the cellular response to an allergen but is only available at university hospitals and therefore often require long transportations of blood samples. A research field that is growing fast is microfluidics which can miniaturize and improve existing methods and diagnostic tests. The aims of this thesis were to improve the existing BAT using microfluidic techniques to enable fast and cheap point-ofcare (POC) diagnostics as well as to further study basophil adhesion and activation for a better understanding of basophil behavior and regulation both in vivo and in vitro in a microfluidic chip. In paper I, we developed a novel microfluidic immunoaffinity-based basophil activation test (miBAT) assay to investigate whether it was possible to capture and activate basophils from whole blood in a microfluidic chip. The yield of captured basophils from whole blood was 64% at a capture flow rate of 3 µl/min. The captured basophils were activated using an anti-FceRI antibody and the basophil identification marker CD203c and the activation marker CD63 were detected using fluorescence microscopy. This was done using blood from both healthy donors and allergic patients and showed comparable results between BAT and miBAT. In paper II, we further investigated whether it was possible to detect a dose-dependent allergen activation for basophils captured in a microfluidic chip. We detected a significant difference in CD63-expression between the negative control and allergen-activated basophils from allergic patients but no difference between the negative control and the non-relevant allergen (an allergen to which the patient had no IgE antibodies). The healthy donors showed no significant difference in activation between the negative control and the allergens. The miBAT results were comparable to BAT. In paper III, we studied basophil adhesion and activation to better understand both basophil function as well as the effect that basophil capture and stimulation in a microfluidic chip has on the cell. The basophil capture in a microfluidic chip could potentially mimic basophil adhesion to the endothelium and was therefore of interest due to the elevated background activation seen in unstimulated basophils captured in a microfluidic chip, reported in paper I and II. Basophils did not upregulate CD63 after passage through a microfluidic chip, but there was a slight but significant activation after crosslinking of CD203c, which is the surface marker used for basophil-specific capture in miBAT, giving one potential factor for the background activation. IgE-dependent (anti-IgE) basophil degranulation after crosslinking of adhesion molecules, to mimic adhesion before transmigration into tissue, showed a significant decrease in CD63-expression compared to anti-IgE activation, which indicate a regulatory function. Cytokine stimulation followed by IgE-independent (fMLP) basophil degranulation on the other hand showed a significantly increased CD63-expression compared to non-primed fMLP activation. In conclusion, we have developed a novel microfluidic-based technique (miBAT) able to detect basophil activation (CD63-expression) using allergens in allergic patients. miBAT was also able to discriminate between relevant and non-relevant allergen activation as well as between allergic patients and healthy controls. miBAT has the potential to be used at POC for allergy diagnostics. We have also shown that crosslinking of CD203c is a potential contributor to the basophil activation seen in the negative controls in miBAT but also that IgE-dependent activation is downregulated from crosslinking of some adhesion molecules, which is of interest both in the microfluidic chip but also in vivo to better understand basophil functions
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| 6. |
- Lenshof, Andreas, et al.
(författare)
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Improved ultrasonic micro array separation using far field ultrasonic excitation
- 2007
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Ingår i: [Host publication title missing]. - 9780979806407 ; 2, s. 1417-1419
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Konferensbidrag (refereegranskat)abstract
- This paper reports improved separation efficiency of an ultrasonic standing wave separator microchannel array by means of ultrasonic far field excitation. By putting an aluminium spacer between the transducer and the channel array, the acoustic power transmission region is moved into the ultrasonic far field decreasing the local intensity variations which is induced in close vicinity to the transducer. This generates a more stable acoustic separation, which enables better separation performance and higher separation efficiency.
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| 7. |
- Matthiesen, Isabelle
(författare)
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Recreating the microenvironment of the neurovascular unit
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The neurovascular unit (NVU) comprises the blood-brain-barrier (BBB) and its surrounding astrocytes, pericytes and neurons that are embedded in the extracellular matrix (ECM). As the main function of the BBB is to protect the brain from inlet of pathogens and toxins, the specialized endothelial cells that keep the barrier tight will also hinder the passage of pharmaceuticals. Understanding the detailed microenvironment and cellular interactions involved in the development of the neurovascular unit is, therefore, an important step towards designing CNS-targeting pharmaceuticals that can pass into the brain. At the same time, the initial steps of pharmaceutical development often involve the use of animal based in vitro models with poor human translation; thus, there is a great need for novel methods to better mimic the complexity of the human NVU. Apart from conventional cell culture models, the use of micro-engineered devices, microphysiological systems (MPS), have gained popularity. The use of MPS allows for fabrication of tissue-like structures using stem cells and provide more in vivo-like parameters in terms of physical cues and dynamic flow. Various materials have been explored for chip fabrication, and biological and synthetic ECM-mimicking hydrogels have been developed for cell encapsulation. Unfortunately, models developed to date often lack either: i) relevant and reproducible cell sources, ii) materials that allow for easy chip fabrication where sensors can be integrated to understand metabolic effects and barrier integrity, or iii) animal-free defined ECM-mimicking scaffolds that support the culture of sensitive cells. This thesis presents an isogenic model of the BBB using iPSC-derived endothelial cells and astrocytes cultured in a MPS made from the non-absorbing polymer OSTE+ that allows for easy fabrication and integration of interdigitated gold electrodes for continuous barrier integrity monitoring. The model presents barrier-protective effects of the BBB-penetrating drug NACA. To better understand the metabolic attributes of astrocytes, a flow-cell sensor is evaluated for the measurement of glucose and lactate turnover during a ketogenic diet. The results imply that such a sensor is valuable for the measurement of metabolic changes and can, in the future, be integrated into MPSs.Furthermore, a model of early neuronal development is realized by using defined copper-free click chemistry to conjugate laminin to a hyaluronic-based hydrogel system for the differentiation of neuroepithelial stem cells. The use of the hydrogel is validated for bioprinting, and the first-ever printed neuroepithelial stem cells are presented. In another study astrocyte 3D culture and bioprinting is evaluated in peptide conjugated hyaluronic-based hydrogels. Unique attachment and spreading of human fetal astrocytes is observed while the common glioblastoma U87 cells display a rounded up morphology. The results of the hydrogel study imply that the defined chemistry of the hydrogel is suitable for both neuroepithelial stem cells, U87 and fetal primary astrocytes, and can in the future be integrated into MPS to circumvent the use of animal derived matrices. In summary, these results provide solutions to some of the problems to date and lay the ground work for the continuation of the development of human-relevant MPS of the NVU.
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| 8. |
- Ressine, Anton, et al.
(författare)
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Ultrahydrophobic properties of porous silicon for surface based bioanalysis
- 2007
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Ingår i: Micro Total Analysis Systems 2007, Proceedings of µTAS 2007 Conference. - 9780979806407 ; 2, s. 1046-1047
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Konferensbidrag (refereegranskat)abstract
- Previously we reported the extreme water repellency of randomly organized macro-/nanoporous silicon surfaces [1]. The texturized silicon with hierarchical porous structure in micrometer and nanometer scale was shown to exhibit the superhydrophobic behaviour. Contact angles as high as 176º can be easily achieved with further fluoroalkylsilane modification of the porous surfaces. Porous silicon technology is compatible with the silicon patterning techniques allowing easy integration of the ultrahydrophobic properties into microfluidic and lab-on-a-chip devices for a new generation of surface based bioanalysis. In this paper we report the fabrication of ultrahydrophobic porous silicon channels and ultrahydrophobic nanovial arrays. Also the MALDI-TOF MS (matrix assisted laser desorption/ionization time-of-flight mass-spectrometry) target anchor chip composed of the hydrophilic anchor points for sample deposition surrounded by the ultrahydrophobic porous area is reported. Such anchor chip allowed the deposition of large sample volumes providing the strategy to improve MALDI-TOF MS sensitivity.
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| 9. |
- Salieb-Beugelaar, Georgette, et al.
(författare)
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DNA movement in sub-20 nm nanoslits
- 2007
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Ingår i: Proceedings of the 11th International Conference on Miniaturized Systems for Chemistry and Life Sciences, uTAS 2007. - 9780979806407 ; , s. 1201-1203
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Konferensbidrag (refereegranskat)abstract
- The movement of XbaI digested O-DNA in 20 nanometer and O-DNA in 12 nanometer high slits was investigated. We found that DNA moved intermittently and following preferential pathways, indicating an important influence of surface roughness. From these intermittent movements two different mobilities were calculated, the total averaged mobilities and averaged mobilities between the intermittent sticking events. The friction coefficient per unit length was calculated from the latter mobilities. A three order of magnitude increase was found for the 12 nm slits compared to the theoretical value. The mobility furthermore differs less than one order of magnitude between 20 nm and 12 nm slits, and the influence of varying the ionic strength of the buffer was not significant. This work is the first time DNA movement in such shallow constrictions is investigated.
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