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Microfluidic bases sample preparation for blood stream infections
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. (Nanobiotechnology)
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Microfluidics promises to re-shape the current health-care system by transferring diagnostic tools from central laboratories to close vicinity of the patient (point-of-care). One of the most important operational steps in any diagnostic platform is sample preparation, which is the main subject in this thesis. The goal of sample preparation is to isolate targets of interest from their surroundings. The work in this thesis is based on three ways to isolate bacteria:  immune-based isolation, selective cell lysis, size-based separation.

The first sample-preparation approach uses antibodies against lipopolysaccharides (LPS), which are surface molecules found on all gram-negative bacteria. There are two characteristics that make this surface molecule interesting. First, it is highly abundant: one bacterium has approximately a million LPS molecules on its cell-wall. Second, the molecule has a conserved region within all gram-negative bacteria, so using one affinity molecule to isolate disease-causing gram-negative bacteria is an attractive option, particularly from the point of view of sample preparation. The main challenge, however, is antigen accessibility. To address this, we have developed a treatment protocol that improves the capturing efficiency.

The strategy behind selective cell lysis takes advantage of the differences between the blood-cell membrane and the bacterial cell-wall. These fundamental differences make it possible to lyse (destroy) blood-cells selectively while keeping the target of interest, here the bacteria, intact and, what is more important alive. Viability plays an important role in determining antibiotic susceptibility.

Difference in size is another well-used characteristic for sample- separation. Inertial microfluidics can focus size-dependent particle at high flow-rates. Thus, particles of 10 µm diameter were positioned in precise streamlines within a curved channel.  The focused particles can then be collected at defined outlets.  This approach was then used to isolate white blood cells, which account for approximately 1% of the whole blood.  In such a device particles of 2µm diameter (size of bacteria) would not be focused and thereby present at every outlet. To separate bacteria from blood elasto-inertial microfluidics was used. Here, e blood components are diverted to center of the channels while smaller bacteria remain in the side streams and can subsequently be separated.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. , 95 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:19
Keyword [en]
sample-preparation, microfluidics, sepsis, size-based separation, selective cell-lysis, immune-based isolation
National Category
Other Biological Topics
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-157688ISBN: 978-91-7595-385-4 (print)OAI: oai:DiVA.org:kth-157688DiVA: diva2:771186
Public defence
2014-12-19, Air & Fire, Scilifelab, Tomtebodavägen 23A,, Solna, 13:00
Opponent
Supervisors
Funder
EU, FP7, Seventh Framework Programme, 223932
Note

QC 20141212

Available from: 2014-12-12 Created: 2014-12-12 Last updated: 2015-10-20Bibliographically approved
List of papers
1. Epitope unmasking for improved immuno-magnetic isolation of Gram-negative bacteria
Open this publication in new window or tab >>Epitope unmasking for improved immuno-magnetic isolation of Gram-negative bacteria
(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:kth:diva-157709 (URN)
Note

QS 2014

Available from: 2014-12-12 Created: 2014-12-12 Last updated: 2014-12-12Bibliographically approved
2. Microfluidic-based isolation of bacteria from whole blood for sepsis diagnostics
Open this publication in new window or tab >>Microfluidic-based isolation of bacteria from whole blood for sepsis diagnostics
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2015 (English)In: Biotechnology letters, ISSN 0141-5492, E-ISSN 1573-6776, Vol. 37, no 4, 825-830 p.Article in journal (Refereed) Published
Abstract [en]

Blood-stream infections (BSI) remain a major health challenge, with an increasing incidence worldwide and a high mortality rate. Early treatment with appropriate antibiotics can reduce BSI-related morbidity and mortality, but success requires rapid identification of the infecting organisms. The rapid, culture-independent diagnosis of BSI could be significantly facilitated by straightforward isolation of highly purified bacteria from whole blood. We present a microfluidic-based, sample-preparation system that rapidly and selectively lyses all blood cells while it extracts intact bacteria for downstream analysis. Whole blood is exposed to a mild detergent, which lyses most blood cells, and then to osmotic shock using deionized water, which eliminates the remaining white blood cells. The recovered bacteria are 100 % viable, which opens up possibilities for performing drug susceptibility tests and for nucleic-acid-based molecular identification.

Keyword
Bacteria; Cells; Cytology; Deionized water; Microfluidics, Bacteria isolation; Blood streams; Cell lysis; Drug susceptibility; Isolation of bacteria; Microfluidic-based; Molecular identification; Rapid identification, Blood
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-157710 (URN)10.1007/s10529-014-1734-8 (DOI)000351535300010 ()2-s2.0-84925535660 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20141212

Available from: 2014-12-12 Created: 2014-12-12 Last updated: 2017-12-05Bibliographically approved
3. Dean flow-coupled inertial focusing in curved channels
Open this publication in new window or tab >>Dean flow-coupled inertial focusing in curved channels
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2014 (English)In: Biomicrofluidics, ISSN 1932-1058, E-ISSN 1932-1058, Vol. 8, no 3, 034117- p.Article in journal (Refereed) Published
Abstract [en]

Passive particle focusing based on inertial microfluidics was recently introduced as a high-throughput alternative to active focusing methods that require an external force field to manipulate particles. In inertial microfluidics, dominant inertial forces cause particles to move across streamlines and occupy equilibrium positions along the faces of walls in flows through straight micro channels. In this study, we systematically analyzed the addition of secondary Dean forces by introducing curvature and show how randomly distributed particles entering a simple u-shaped curved channel are focused to a fixed lateral position exiting the curvature. We found the lateral particle focusing position to be fixed and largely independent of radius of curvature and whether particles entering the curvature are pre-focused (at equilibrium) or randomly distributed. Unlike focusing in straight channels, where focusing typically is limited to channel cross-sections in the range of particle size to create single focusing point, we report here particle focusing in a large cross-section area (channel aspect ratio 1: 10). Furthermore, we describe a simple u-shaped curved channel, with single inlet and four outlets, for filtration applications. We demonstrate continuous focusing and filtration of 10 mu m particles (with > 90% filtration efficiency) from a suspension mixture at throughputs several orders of magnitude higher than flow through straight channels (volume flow rate of 4.25ml/min). Finally, as an example of high throughput cell processing application, white blood cells were continuously processed with a filtration efficiency of 78% with maintained high viability. We expect the study will aid in the fundamental understanding of flow through curved channels and open the door for the development of a whole set of bio-analytical applications.

Keyword
Continuous Particle Separation, Microfluidic Device, Poiseuille Flow, Tumor-Cells, Microchannels, Filtration, Filter
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-149227 (URN)10.1063/1.4884306 (DOI)000339004500017 ()
Funder
EU, FP7, Seventh Framework ProgrammeSwedish Research CouncilScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20140819

Available from: 2014-08-19 Created: 2014-08-18 Last updated: 2017-12-05Bibliographically approved
4. Elasto-Inertial microfluidics for bacteria separation from whole blood for sepsis diagnostics
Open this publication in new window or tab >>Elasto-Inertial microfluidics for bacteria separation from whole blood for sepsis diagnostics
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(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:kth:diva-157715 (URN)
Note

QS 2014

Available from: 2014-12-12 Created: 2014-12-12 Last updated: 2014-12-12Bibliographically approved
5. Recombinant Shigella flexneri apyrase enzyme for bioluminescence based diagnostic applications
Open this publication in new window or tab >>Recombinant Shigella flexneri apyrase enzyme for bioluminescence based diagnostic applications
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(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:kth:diva-157716 (URN)
Note

QS 2014

Available from: 2014-12-12 Created: 2014-12-12 Last updated: 2014-12-12Bibliographically approved

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