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Microfluidic blood sample preparation for rapid sepsis diagnostics
KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.ORCID iD: 0000-0001-8531-5607
2012 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Sepsis, commonly referred to as blood poisoning, is a serious medical condition characterized by a whole-body inflammatory state caused by microbial infection. Rapid treatment is crucial, however, traditional culture-based diagnostics usually takes 2-5 days.  The overall aim of the thesis is to develop microfluidic based sample preparation strategies, capable of isolating bacteria from whole blood for rapid sepsis diagnostics. 

Although emerging technologies, such as microfluidics and “lab-on-a-chip” (LOC) devices have the potential to spur the development of protocols and affordable instruments, most often sample preparation is performed manually with procedures that involve handling steps prone to introducing artifacts, require skilled technicians and well-equipped, expensive laboratories.  Here, we propose the development of methods for fast and efficient sample preparation that can isolate bacteria from whole blood by using microfluidic techniques with potential to be incorporated in LOC systems.

We have developed two means for high throughput bacteria isolation: size based sorting and selective lysis of blood cells. To process the large blood samples needed in sepsis diagnostics, we introduce novel manufacturing techniques that enable scalable parallelization for increased throughput in miniaturized devices.

The novel manufacturing technique uses a flexible transfer carrier sheet, water-dissolvable release material, poly(vinyl alcohol), and a controlled polymerization inhibitor to enable highly complex polydimethylsiloxane (PDMS) structures containing thin membranes and 3D fluidic networks.

The size based sorting utilizes inertial microfluidics, a novel particles focusing method that operates at extremely high flow rates. Inertial focusing in flow through a single inlet and two outlet, scalable parallel channel devices, was demonstrated with filtration efficiency of >95% and a flowrate of 3.2 mL/min.

Finally, we have developed a novel microfluidic based sample preparation strategy to continuously isolate bacteria from whole blood for downstream analysis. The method takes advantage of the fact that bacteria cells have a rigid cell wall protecting the cell, while blood cells are much more susceptible to chemical lysis. Whole blood is continuously mixed with saponin for primary lysis, followed by osmotic shock in water. We obtained complete lysis of all blood cells, while more than 80% of the bacteria were readily recovered for downstream processing.

Altogether, we have provided new bacteria isolation methods, and improved the manufacturing techniques and microfluidic components that, combined offer the potential for affordable and effective sample preparation for subsequent pathogen identification, all in an automated LOC format.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2012. , ix, 40 p.
Series
Trita-FYS, ISSN 0280-316X ; 2012:39
Keyword [en]
3D fluidic networks, bacteria isolation, inertial microfluidics, lab-on-chip, microfluidics, particle filtration, PDMS membrane, selective cell lysis. sepsis
National Category
Engineering and Technology
Identifiers
URN: urn:nbn:se:kth:diva-96313ISBN: 978-91-7501-428-9 (print)OAI: oai:DiVA.org:kth-96313DiVA: diva2:530388
Presentation
2012-06-15, FD5 (The Svedberg Hall), Albanova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 14:00 (English)
Opponent
Supervisors
Funder
EU, FP7, Seventh Framework Programme
Note
QC 20120611Available from: 2012-06-11 Created: 2012-06-01 Last updated: 2012-06-11Bibliographically approved
List of papers
1. Fabrication and transfer of fragile 3D PDMS microstructures
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2012 (English)In: Journal of Micromechanics and Microengineering, ISSN 0960-1317, E-ISSN 1361-6439, Vol. 22, no 8, 1-9 p.Article in journal (Refereed) Published
Abstract [en]

We present a method for PDMS microfabrication of fragile membranes and 3D fluidic networks, using a surface modified water-dissolvable release material, poly(vinyl alcohol), as a tool for handling, transfer and release of fragile polymer microstructures. The method is well suited for the fabrication of complex multilayer microfluidic devices, here shown for a PDMS device with a thin gas permeable membrane and closely spaced holes for vertical interlayer connections fabricated in a single layer. To the authors knowledge, this constitutes the most advanced PDMS fabrication method for the combination of thin, fragile structures and 3D fluidics networks, and hence a considerable step in the direction of making PDMS fabrication of complex microfluidic devices a routine endeavour.

Keyword
Microfluidic Devices, Layer
National Category
Electrical Engineering, Electronic Engineering, Information Engineering
Identifiers
urn:nbn:se:kth:diva-95357 (URN)10.1088/0960-1317/22/8/085009 (DOI)000306649000009 ()2-s2.0-84864447006 (Scopus ID)
Note

QC 20150624

Available from: 2012-05-23 Created: 2012-05-23 Last updated: 2017-12-07Bibliographically approved
2. Inertial Particle Focusing In Parallel Microfluidic Channels For High-Throughput Filtration
Open this publication in new window or tab >>Inertial Particle Focusing In Parallel Microfluidic Channels For High-Throughput Filtration
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2011 (English)In: 16th International  Solid-State Sensors, Actuators and Microsystems Conference (TRANSDUCERS), 2011, IEEE conference proceedings, 2011, 1777-1780 p.Conference paper, Published paper (Refereed)
Abstract [en]

In this study, we introduce inertial microfluidics in straight, parallel channels for high-throughput particle filtration. We show that particles flowing through low aspect ratio rectangular microchannels can be focused into four particle streams, distributed at the centers of each wall face, or into two particle streams, at the centers of the longest channel walls, depending on the particles' size. For high-throughput filtration, we fabricated scalable, single inlet and two outlet, parallel channel microdevices, using a high-density 3D microfluidic PDMS channel manufacturing technology, in a design that allows for easy integration with other downstream on-chip functions we recently described. We demonstrate filtration of 24 μm particles from a suspension mixture in a microdevice with four parallel channels. The filtration efficiency at a non-optimized flow rate of 0.8 ml/min was 82%.

Place, publisher, year, edition, pages
IEEE conference proceedings, 2011
Keyword
Filtration, Focusing, Force, Manufacturing, Microchannel, Microfluidics, Three dimensional displays
National Category
Engineering and Technology
Identifiers
urn:nbn:se:kth:diva-48371 (URN)10.1109/TRANSDUCERS.2011.5969458 (DOI)2-s2.0-80052116772 (Scopus ID)978-1-4577-0157-3 (ISBN)
Conference
16th International Solid-State Sensors, Actuators and Microsystems Conference (TRANSDUCERS), 2011
Note

QC 20111118

Available from: 2011-11-18 Created: 2011-11-17 Last updated: 2014-06-03Bibliographically approved
3. Inertial microfluidics in parallel channels for high-throughput applications
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2012 (English)In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 12, no 22, 4644-4650 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 this study, we introduce inertial microfluidics in flows through straight, multiple parallel channels. The scalable, single inlet and two outlet, parallel channel system is enabled by a novel, high-density 3D PDMS microchannel manufacturing technology, mediated via a targeted inhibition of PDMS polymerization. Using single channels, we first demonstrate how randomly distributed particles can be focused into the centre position of the channel in flows through low aspect ratio channels and can be effectively fractionated. As a proof of principle, continuous focusing and filtration of 10 μm particles from a suspension mixture using 4- and 16-parallel-channel devices with a single inlet and two outlets are demonstrated. A filtration efficiency of 95-97% was achieved at throughputs several orders of magnitude higher than previously shown for flows through straight channels. The scalable and low-footprint focusing device requiring neither external force fields nor mechanical parts to operate is readily applicable for high-throughput focusing and filtration applications as a stand-alone device or integrated with lab-on-a-chip systems.

Place, publisher, year, edition, pages
Royal Society of Chemistry, 2012
Keyword
article, filtration, force, fractionation, inertial microfluidics, lab on a chip, microfluidics, priority journal
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-95364 (URN)10.1039/c2lc40241f (DOI)000310865200008 ()2-s2.0-84867518462 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceEU, European Research Council
Note

QC 20121115. Updated from accepted to published.

Available from: 2012-05-23 Created: 2012-05-23 Last updated: 2017-12-07Bibliographically approved
4. Microfluidic selective cell lysis for bacteria isolation from whole blood
Open this publication in new window or tab >>Microfluidic selective cell lysis for bacteria isolation from whole blood
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(English)Manuscript (preprint) (Other academic)
National Category
Engineering and Technology
Identifiers
urn:nbn:se:kth:diva-96608 (URN)
Note

QS 2012

Available from: 2012-06-07 Created: 2012-06-07 Last updated: 2014-12-12Bibliographically approved

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