Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Genotyping by dynamic heating of monolayered beads on a microheated surface
KTH, Superseded Departments, Signals, Sensors and Systems.
KTH, Superseded Departments, Signals, Sensors and Systems.ORCID iD: 0000-0002-9327-2544
KTH, Superseded Departments, Signals, Sensors and Systems.
KTH, Superseded Departments, Signals, Sensors and Systems.
Show others and affiliations
2004 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 25, no 21-22, 3712-3719 p.Article in journal (Refereed) Published
Abstract [en]

 A miniaturized bead-based dynamic allele-specific hybridization (DASH) approach for sing le-nucleotide polymorphism analysis is presented. Chips with integrated heater and temperature sensors for open-surface DNA analysis were microfabricated. Microcontact printing using a poly(dimethylsiloxane) (PDMS) stamp was employed to create monolayers of immobilized beads on the surface of the chip. This chip allows fast, well-controllable temperature ramping. The temperature distribution was homogeneous over the entire heater area. All three possible variants of an SNP site of a synthesized oligonucleotide were accurately scored using the bead-based DASH approach. Our assay has a nonoptimized temperature ramping rate of 4degreesC-6degreesC/min compared to earlier reported values of 2degreesC-3degreesC/min, thereby reducing the total analysis time by a factor of 2. Reliable DASH measurement data from areas as small as 12 x 13 mum was achieved. Our bead-based DASH approach has enabled a dramatic volume reduction and is a step towards developing a cost-effective high-throughput DASH method on arrays of single beads.

Place, publisher, year, edition, pages
2004. Vol. 25, no 21-22, 3712-3719 p.
Keyword [en]
beads, dynamic allele-specific hybridization, microcontact printing, microheater, miniaturization, single-nucleotide polymorphism
National Category
Medical Genetics
Identifiers
URN: urn:nbn:se:kth:diva-5011DOI: 10.1002/elps.200406065ISI: 000225679000021Scopus ID: 2-s2.0-10944219819OAI: oai:DiVA.org:kth-5011DiVA: diva2:7465
Note

QC 20100916 20110915

Available from: 2005-04-01 Created: 2005-04-01 Last updated: 2017-04-26Bibliographically approved
In thesis
1. Microfluidic bead-based methods for DNA analysis
Open this publication in new window or tab >>Microfluidic bead-based methods for DNA analysis
2005 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

With the completion of the human genome sequencing project, attention is currently shifting toward understanding how genetic variation, such as single nucleotide polymorphism (SNP), leads to disease. To identify, understand, and control biological mechanisms of living organisms, the enormous amounts of accumulated sequence information must be coupled to faster, cheaper, and more powerful technologies for DNA, RNA, and protein analysis. One approach is the miniaturization of analytical methods through the application of microfluidics, which involves the manipulation of fluids in micrometer-sized channels. Advances in microfluidic chip technology are expected to play a major role in the development of cost-effective and rapid DNA analysis methods.

This thesis presents microfluidic approaches for different DNA genotyping assays. The overall goal is to combine the potential of the microfluidic lab-on-a-chip concept with biochemistry to develop and improve current methods for SNP genotyping. Three genotyping assays using miniaturized microfluidic approaches are addressed.

The first two assays are based on primer extension by DNA polymerase. A microfluidic device consisting of a flow-through filter chamber for handling beads with nanoliter liquid volumes was used in these studies. The first assay involved an allelespecific extension strategy. The microfluidic approach took advantage of the different reaction kinetics of matched and mismatched configurations at the 3’-ends of a primer/template complex. The second assay consisted of adapting pyrosequencing technology, a bioluminometric DNA sequencing assay based on sequencing-bysynthesis, to a microfluidic flow-through platform. Base-by-base sequencing was performed in a microfluidic device to obtain accurate SNP scoring data on nanoliter volumes. This thesis also presents the applications of monolayer of beads immobilized by microcontact printing for chip-based DNA analysis. Single-base incorporation could be detected with pyrosequencing chemistry on these monolayers.

The third assay developed is based on a hybridization technology termed Dynamic Allele-Specific Hybridization (DASH). In this approach, monolayered beads containing DNA duplexes were randomly immobilized on the surface of a microheater chip. DNA melting-curve analysis was performed by dynamically heating the chip while

simultaneously monitoring the DNA denaturation profile to determine the genotype. Multiplexing based on single-bead analysis was achieved at heating rates more than 20 times faster than conventional DASH provides.

Place, publisher, year, edition, pages
Stockholm: KTH, 2005. x, 52 p.
Series
Trita-ILA, ISSN 0281-2878 ; 0502
Keyword
Genetics, single nucleotide polymorphism, DNA analysis, SNP, microfluidics, pyrosequencing, beads, lab on a chip, hybridization, DASH, microsystem, micro totat analysis system, allele-specific extension, DASH, microcontact printing, Genetik
National Category
Medical Genetics
Identifiers
urn:nbn:se:kth:diva-155 (URN)91-7283-992-9 (ISBN)
Public defence
2005-04-08, Q2, Osquldas v 10, KTH, 13:00
Opponent
Note
QC 20101008Available from: 2005-04-01 Created: 2005-04-01 Last updated: 2010-10-08Bibliographically approved
2. Assembly of microsystems for optical and fluidic applications
Open this publication in new window or tab >>Assembly of microsystems for optical and fluidic applications
2005 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

This thesis addresses assembly issues encountered in optical and fluidic microsystem applications.

In optics, the first subject concerns the active alignment of components in optical fibersystems. A solution for reducing the cost of optical component assembly while retaining submicron accuracy is to integrate the alignment mechanism onto the optical substrate. A polymer V-shaped actuator is presented that can carry the weight of the large components - on a micromechanical scale - and that can generate movement with six degrees of freedom.

The second subject in optics is the CMOS-compatible fabrication of monocrystalline silicon micromirror arrays that are intended to serve as CMOS-controlled high-quality spatial light modulators in maskless microlithography systems. A wafer-level assembly method is presented that is based on adhesive wafer bonding whereby a monocrystalline layer is transferred onto a substrate wafer in a CMOS-compatible process without needing bond alignment.

In fluidics, a hybrid assembly method is introduced that combines two separately micromachined structures to create hotwire anemometers that protrude from a surface with minimum interference with the air flow. The assembled sensor enables one to make accurate time-resolved measurements of the wall shear stress, a quantity that has previously been hard to measure with high time resolution. Also in the field of hotwire anemometers, a method using a hotwire anemometer array is presented for measuring the mass flow, temperature and composition of a gas in a duct.

In biochemistry, a bio-analysis chip is presented. Single nucleotide polymorphism scoring is performed using dynamic allele-specific hybridization (DASH). Using monolayers of beads, multiplexing based on single-bead analysis is achieved at heating rates more than 20 times faster than conventional DASH provides.

Space and material e±ciency in packaging are the focus of the other two projects in fluidics. The first introduces an assembly based on layering conductive adhesives for the fabrication of miniature polymer electrolyte membrane fuel cells. The fuel cells made with this low-cost approach perform among the best of their type to date. The second project concerns a new cross-flow microvalve concept. Intended as a step towards the mass production of large-flow I/P converters, the silicon footprint area is minimized by an out-of-plane moving gate and in-plane, half-open pneumatic channels.

Place, publisher, year, edition, pages
Stockholm: KTH, 2005. xiv, 70 p.
Series
Trita-ILA, ISSN 0281-2878 ; 0501
Keyword
Applied mechanics, microsystem technology, micromachining, assembly, active alignment, BCB, Teknisk mekanik
National Category
Mechanical Engineering
Identifiers
urn:nbn:se:kth:diva-120 (URN)91-7283-958-9 (ISBN)
Public defence
2005-02-11, Kollegiesalen, Valhallavägen 79, Stockholm, 14:00
Opponent
Supervisors
Note
QC 20101019Available from: 2005-02-09 Created: 2005-02-09 Last updated: 2010-10-19Bibliographically approved
3. Foil-based Lab-on-Chip technologies for advanced Point-of-Care molecular diagnostics
Open this publication in new window or tab >>Foil-based Lab-on-Chip technologies for advanced Point-of-Care molecular diagnostics
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Infectious diseases pose a serious threat to global health. Molecular diagnostics provide solutions for effective control and prevention of infections, however suffers from expensive laboratory equipment, and infrastructure to be fully implemented at point of care (POC), especially at low-resource settings. Lab-on-a-chip that aims to integrate complex biochemical analyses into automated systems is promising for POC analysis. A major challenge is the integration of a complete molecular diagnostic assay, generally translating into complex microfluidics, with the requirement of low fabrication cost. This thesis explores the use of flexible electronics, plastic foils and roll-to-roll manufacturing to enable low-cost microfluidic systems, for molecular diagnostic assays especially targeted towards infectious diseases. Many biochemical assays rely on heat; hence a first aspect in this thesis is the integration of a microheater into microfluidics. In a first project a system for SNP-genotyping is presented using solid phase melting curve analysis to discriminate mutations at a single base resolution. Starting with a glass based concept (paper I) which is further developed to a foil based system (paper II), detection of the polymorphism in the neuropeptide Y associated with increased risk of type II diabetes is demonstrated as a proof of principle. Further development and optimization of the microheater concept has enabled roll-to-roll manufacturing compatibility and multiplexing of targets (paper III). A bacterial sub-typing and multiresistance detection in clinical Staphylococcus Aureus samples is demonstrated for applications in infectious diseases diagnostics. Finally, the microheater concept is further developed to enable μPCR (paper IV). Detection of genomic HIV-1 is demonstrated and a portable detection setup based on an LED light source and low cost CMOS camera for detection was developed. A second aspect of this thesis is integration of light sources and optical detection (paper V-VI). A multilayer system integrating an electroluminescent light source, reactive sensor dyes and organic semiconductor transistor for detection is demonstrated. The system could be used for amine detection in gases (paper V). System was made further roll-to-roll compatible. The system uses an external LED light source and a photodetector processed in only one screen printing- and one dispensing step (paper VI). As a proof of principle, absorbance based DNA hybridization was detected. Collectively, roll-to-roll manufacturing compatible “lab on foil” systems have the potential to improve our ability to diagnose at POC especially at resource-limited settings.

Place, publisher, year, edition, pages
KTH: KTH Royal Institute of Technology, 2017. 83 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2017:12
National Category
Medical Engineering Medical Laboratory and Measurements Technologies
Research subject
Biotechnology; Medical Technology; Materials Science and Engineering
Identifiers
urn:nbn:se:kth:diva-205933 (URN)978-91-7729-372-9 (ISBN)
Public defence
2017-05-19, Air and Fire, Scilife lab, Tomtebodav.23A, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

QC 20170426

Available from: 2017-04-26 Created: 2017-04-25 Last updated: 2017-04-28Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full textScopus

Authority records BETA

Haasl, SjoerdStemme, Göran

Search in DiVA

By author/editor
Russom, AmanHaasl, SjoerdOhlander, AnnaAndersson, HeleneStemme, Göran
By organisation
Signals, Sensors and Systems
In the same journal
Electrophoresis
Medical Genetics

Search outside of DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 156 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf