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A vertical flow paper-microarray assay with isothermal DNA amplification for detection of Neisseria meningitidis
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-1510-5927
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0003-0344-8049
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2018 (English)In: Talanta: The International Journal of Pure and Applied Analytical Chemistry, ISSN 0039-9140, E-ISSN 1873-3573, Vol. 183, p. 192-200Article in journal (Refereed) Published
Abstract [en]

Paper-based biosensors offer a promising technology to be used at the point of care, enabled by good performance, convenience and low-cost. In this article, we describe a colorimetric vertical-flow DNA microarray (DNA-VFM) that takes advantage of the screening capability of DNA microarrays in a paper format together with isothermal amplification by means of Recombinase Polymerase Amplification (RPA). Different assay parameters such as hybridization buffer, flow rate, printing buffer and capture probe concentration were optimized. A limit of detection (LOD) of 4.4 nM was achieved as determined by tabletop scanning. The DNA-VFM was applied as a proof of concept for detection of Neisseria meningitidis, a primary cause of bacterial meningitis. The LOD was determined to be between 38 and 2.1Å~106 copies/VFM assay, depending on the choice of DNA capture probes. The presented approach provides multiplex capabilities of DNA microarrays in a paper-based format for future point-of-care applications.

Place, publisher, year, edition, pages
Elsevier, 2018. Vol. 183, p. 192-200
Keyword [en]
Microarray, Paper-based biosensor, Point-of-care, Gold nanoparticles, Neisseria meningitidis
National Category
Diagnostic Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-227158DOI: 10.1016/j.talanta.2018.02.070Scopus ID: 2-s2.0-85042419551OAI: oai:DiVA.org:kth-227158DiVA, id: diva2:1203233
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20180503

Available from: 2018-05-02 Created: 2018-05-02 Last updated: 2018-05-03Bibliographically approved
In thesis
1. Development of array systems for molecular diagnostic assays
Open this publication in new window or tab >>Development of array systems for molecular diagnostic assays
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

For molecular diagnostics, assays detecting biomarkers can be used to provide information to medical questions. Array formats such as microarrays and suspension bead arrays allow for multiplex assays, analyzing hundreds or thousands of analytes simultaneously in one single sample. There is a growing demand of multiplex assays, using panels of biomarkers, for the use in Point-of-Care (POC) diagnostic tests. These diagnostic tests require little or no equipment and relies on easy read-out systems. Lateral flow assays (LFAs) are well-known, paper-based POC assays, with advantageous features, which include performing the assay by capillary forces, stable reagents storage and naked-eye read-out. Vertical Flow Microarrays (VFMs) have previously been presented as an alternative to LFAs, circumventing downstream effects by vertically applying sample and reagents. In Paper I and II of this thesis, VFM arrays have been applied in the development of assays for molecular diagnosis, while Paper III explores Suspension Bead Array (SBA) format for biomarker discovery.

 

In Paper I, we have developed a DNA-VFM assay towards POC testing of Neisseria meningitidis, one of the major meningitis causing bacteria. Here, the target gene of N. meningitidis was amplified using Recombinase Polymerase Amplification (RPA). The amplified DNA was digested into ssDNA and hybridized to multiple VFM probes, for multiplex detection of different segments of the target gene. Optimization of the assay resulted in a Limit Of Detection (LOD) of 4.4 nM for amplification of synthetic DNA.

 

In Paper II, a VFM for reverse phase detection of IgE was developed. The assay was optimized using IgE spiked into IgE-negative serum, resulting in a LOD of 1.9 μg/mL. Optimized conditions were then used to screen a cohort of serum samples, including patients with rare primary immunodeficiency Hyper IgE syndrome and healthy controls. A comparative, traditional reverse phase assay was also performed on the same serum samples, showing comparable results to the reverse phase VFM.

 

In Paper III, a pediatric cohort from Plasmodium falciparum endemic Rwanda was analyzed using SBAs for proteins involved during various stages of malaria infection. Large, significant differences between cases and controls were found for 22 of the analyzed proteins. The majority of the candidate proteins presented validated previous work, nevertheless, several proteins were identified with no previously known link to malaria pathogenesis. Proteins discriminating between mild and severe malaria infection were also identified, showing minor separation between the two sample groups.

 

In Paper IV, a focus group discussion of using POC tests with Ugandan health care personnel was conducted. Health care personnel at different levels were interviewed about their perception of using POC tests in the health care system, including strengths and weaknesses. This study was designed to bridge some of the knowledge gap between the POC test developers and end-users.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2018. p. 71
Series
TRITA-CBH-FOU ; 2018:5
National Category
Medical Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-227159 (URN)978-91-7729-717-8 (ISBN)
Public defence
2018-05-25, Gardaulan, Folkhälsomyndigheten, Nobels väg 18, Solna, 10:00 (English)
Opponent
Funder
EU, European Research Council, 615458
Note

QC 20180503

Available from: 2018-05-03 Created: 2018-05-03 Last updated: 2018-05-03Bibliographically approved

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