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Genotyping of single nucleotide polymorphisms by melting curve analysis using thin film semi-transparent heaters integrated in a lab-on-foil system
KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
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2013 (English)In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 13, no 11, 2075-2082 p.Article in journal (Refereed) Published
Abstract [en]

The recent technological advances in micro/nanotechnology present new opportunities to combine microfluidics with microarray technology for the development of small, sensitive, single-use, point-of-care molecular diagnostic devices. As such, the integration of microarray and plastic microfluidic systems is an attractive low-cost alternative to glass based microarray systems. This paper presents the integration of a DNA microarray and an all-polymer microfluidic foil system with integrated thin film heaters, which demonstrate DNA analysis based on melting curve analysis (MCA). A novel micro-heater concept using semi-transparent copper heaters manufactured by roll-to-roll and lift-off on polyethylene naphthalate (PEN) foil has been developed. Using a mesh structure, heater surfaces have been realized in only one single metallization step, providing more efficient and homogenous heating characteristics than conventional meander heaters. A robust DNA microarray spotting protocol was adapted on Parylene C coated heater-foils, using co-polymer poly(DMA-NAS-MAPS) to enable covalent immobilization of DNA. The heaters were integrated in a microfluidic channel using lamination foils and MCA of the spotted DNA duplexes showed single based discrimination of mismatched over matched target DNA-probes. Finally, as a proof of principle, we perform MCA on PCR products to detect the Leu7Pro polymorphism of the neutropeptide Y related to increased risk of Type II diabetes, BMI and depression.

Place, publisher, year, edition, pages
2013. Vol. 13, no 11, 2075-2082 p.
Keyword [en]
article, body mass, depression, devices, DNA determination, DNA microarray, film, foil, heating, melting curve analysis, non insulin dependent diabetes mellitus, nucleic acid analysis, polymerase chain reaction, priority journal, single nucleotide polymorphism, thin film heater
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-123643DOI: 10.1039/c3lc50171jISI: 000318514400008Scopus ID: 2-s2.0-84877682690OAI: oai:DiVA.org:kth-123643DiVA: diva2:628568
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20130614

Available from: 2013-06-14 Created: 2013-06-13 Last updated: 2017-04-27Bibliographically approved
In thesis
1. 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

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