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Microfluidic-based isolation of bacteria from whole blood for sepsis diagnostics
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0001-8531-5607
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0001-5199-0663
Show others and affiliations
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.

Place, publisher, year, edition, pages
2015. Vol. 37, no 4, 825-830 p.
Keyword [en]
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: urn:nbn:se:kth:diva-157710DOI: 10.1007/s10529-014-1734-8ISI: 000351535300010Scopus ID: 2-s2.0-84925535660OAI: oai:DiVA.org:kth-157710DiVA: diva2:771227
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
In thesis
1. Microfluidic bases sample preparation for blood stream infections
Open this publication in new window or tab >>Microfluidic bases sample preparation for blood stream infections
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
sample-preparation, microfluidics, sepsis, size-based separation, selective cell-lysis, immune-based isolation
National Category
Other Biological Topics
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-157688 (URN)978-91-7595-385-4 (ISBN)
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
2. From Lab to Chip – and back: Polymer microfluidic systems for sample handling in point-of-care diagnostics
Open this publication in new window or tab >>From Lab to Chip – and back: Polymer microfluidic systems for sample handling in point-of-care diagnostics
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis contributes to the development of Lab-on-a-Chip systems that enables reliable, rapid medical diagnostics at the point-of-care. These contributions are focused on microfluidic Lab-on-a-Chip systems for sepsis diagnosis, autonomous sample-to-answer tests, and dried blood spot sampling.

Sepsis is a serious condition with high mortality and high costs for society and healthcare. To facilitate rapid and effective antibiotic treatment, improved sepsis diagnostics is needed. Diagnosis of sepsis requires the processing of relatively large blood volumes, creating a need for novel and effective techniques for the handling of large volume flows and pressures on chip. Components, materials, and manufacturing methods for pneumatically driven Lab-on-a-Chip systems have therefore been developed in this thesis. Microvalves, an essential component in many Lab-on-a-Chip systems have been the focus on several of the advances: a novel elastomeric material (Rubbery Off-Stoichiometric-Thiol-Ene-Epoxy) with low gas and liquid permeability; the first leak-tight vertical membrane microvalves, allowing large channel cross-sections for high volumetric flow throughput; and novel PDMS manufacturing methods enabling their realization. Additionally, two of the new components developed in this thesis focus on separation of bacteria from blood cells based on differences in particle size, and cell wall composition: inertial microfluidic removal of large particles in multiple parallel microchannels with low aspect ratio; and selective lysis of blood cells while keeping bacteria intact. How these components, materials and methods could be used together to achieve faster sepsis diagnostics is also discussed.

Lab-on-a-Chip tests can not only be used for sepsis, but have implications in many point-of-care tests. Disposable and completely autonomous sampleto- answer tests, like pregnancy tests, are capillary driven. Applying such tests in more demanding applications has traditionally been limited by poor material properties of the paper-based products used. A new porous material, called “Synthetic Microfluidic Paper”, has been developed in this thesis. The Synthetic Microfluidic Paper features well-defined geometries consisting of slanted interlocked micropillars. The material is transparent, has a large surface area, large porous fraction, and results in low variability in capillary flowrates. The fact that Synthetic Microfluidic Paper can be produced with multiple pore sizes in the same sheet enables novel concepts for self-aligned spotting of liquids and well-controlled positioning of functional microbeads.

Diagnostic testing can also be achieved by collecting the sample at the point-of-care while performing the analysis elsewhere. Easy collection of finger-prick blood in paper can be performed by a method called dried blood spots. This thesis investigates how the process of drying affects the homogeneity of dried blood spots, which can explain part of the variability that has been measured in the subsequent analysis. To reduce this variability, a microfluidic sampling chip has been developed in this thesis. The chip, which is capillary driven, autonomously collects a specific volume of plasma from a drop of blood, and dry-stores it in paper. After sampling, the chip can be mailed back to a central lab for analysis.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2016. xiii, 75 p.
Series
TRITA-EE, ISSN 1653-5146 ; 2016:002
National Category
Other Electrical Engineering, Electronic Engineering, Information Engineering
Research subject
Electrical Engineering
Identifiers
urn:nbn:se:kth:diva-180740 (URN)978-91-7595-844-6 (ISBN)
Public defence
2016-02-05, F3, Lindstedtsvägen 26, KTH, Stockholm, 09:00 (English)
Opponent
Supervisors
Note

QC 20160122

Available from: 2016-01-22 Created: 2016-01-22 Last updated: 2016-01-22Bibliographically approved

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