Postgraduate students
(positions funded by the Ministry of Education and Culture and by the Academy of Finland)
2010-2013
FM Huovinen Tuomas University of Turku, Biotechnology
FM Lehmusvuori Ari Abacus Diagnostica Oy
FM Mäntymaa Anne Tampere University of Technology, Automation Science and Engineering
FM Nummela Marika VTT, Medical Biotechnology
FM Räsänen Markus University of Turku, Materials Chemistry and Chemical Analysis
FM Savukoski Tanja University of Turku, Biotechnology
FM Ylihärsilä Minna University of Turku, Virology
2012-2015
DI Arola Henri VTT, Molecular diagnostics & immunotechnology
FM Arppe Riikka University of Turku, Biotechnology
FM Blom, Sami Institute for Molecular Medicine Finland (FIMM)
FM Sjöblom Liisa Institute of Biomedical Technology (IBT)
Abstracts
Anne Mäntymaa
Microfluidics in dry-reagent based immunoassays
Supervisor: Prof. Pasi Kallio
Background of the research: General trends of in vitro diagnostics include high sensitivity and precision [1], a decrease in sample and reagent volumes, and increasing functionality by integrating the protocol steps onto a single microfluidic cartridge. Typically the goal is to shorten the analysis times and decrease the involvement of the user, which could lead to reduced total costs. [2] Polystyrene nanoparticles coated with antibodies, and incorporated with europium labels are known to enhance the immunoassay performance [3, 4]. A sandwich immunoassay with integrated dry-reagent storage in the cartridge enables a convenient ready-to-use setting, long-term storage, and lower consumption of reagents.
Objectives of the research: The objective of the research is to study and optimize microfluidics in dry-reagent based sandwich immunoassays. The goal is to study and optimize the solubility of dried reagents, to study and reduce the nonspecific binding of nanoparticle labels, and to study and optimize the immunoreactions in a microfluidic cartridge.
Nonspecific binding of nanoparticles in the detection area results in an unwanted background signal. The first goal is to reduce the background signal, which will be studied from different approaches, such as computer assisted dispensing of dry reagents, the shape and geometries of the reaction chamber, effective washing steps, different materials, and surface treatments.
Experimental: Fluorescence detection of europium(III) –chelate dyed nanoparticles are widely used in various experiments of this study.
References:
[1] Warsinke A. Point-of-care testing of proteins. Anal Bioanal Chem 2009; 393, 1393-1405.
[2] Ducrée J & Zengerle R. 2004. FlowMap – Microfluidics Roadmap for the Life Sciences. Books on Demand GmbHm Norderstedt, Germany. www.microfluidic s-roadmap.com. 2004.
[3] Härmä H, Soukka T & Lövgren T. Europium nanoparticles and time-resolved fluorescence for ultrasensitive detection of prostate-specific antigen. Clin. Chem. 2001; 47, 561-568.
[4] Kokko L, Sandberg K, Lövgren T & Soukka T. Europium(III) chelate-dyed nanoparticles as donors in a homogeneous proximity-based immunoassay for estradiol. Anal. Chim. Acta 2004; 503, 155-162.
Ari Lehmusvuori
Rapid closed-tube PCR assay for Chlamydia trachomatis utilizing novel lanthanide chelate complementation based reporter technology
Ari Lehmusvuori, Ulla Karhunen, Urpo Lamminmäki & Tero Soukka
The Graduate School of Advanced Diagnostic Technologies and Applications (DIA-NET)
Department of Biotechnology, University of Turku, Finland
Chlamydia trachomatis infection is the most common bacterial sexually transmitted disease and a major public health problem worldwide. Untreated C. trachomatis infection can lead to serious complications such as infertility and pelvic inflammatory disease. Asymptomatic nature of the disease and the lack of rapid, sensitive, easy-to-use and inexpensive diagnostics complicates the therapy and the prevention of C. trachomatis transmission especially in the countries with limited resources and infrastructure. Currently C. trachomatis diagnosis is time consuming (sample to results several days) and is performed in central laboratories requiring sophisticated laboratory instruments. The aim of the study is to develop a rapid homogenous C. trachomatis PCR assay utilizing novel supersensitive lanthanide chelate complementation based reporter technology where the results are available in 30 minutes from the sampling.
Marika Nummela
Array-based Serodiagnostics and HLA-genotyping in Autoimmune Diseases and Allergy
Marika Nummela, M.Sc., Research Scientist
Supervisor: Petri Saviranta, Ph.D., Senior Research Scientist
VTT Technical Research Centre of Finland, Medical Biotechnology
Background: The study and diagnosis of complex diseases on large clinical sample panels requires rapid, simple and high throughput assays. To meet these requirements VTT has developed a novel microarray platform, the Arrays-in-Wells (AIW), which can be used for both genetic and protein-based multiplex profiling studies. In this project AIW will be applied for the study and diagnosis of autoimmune diseases and allergy.
Association of HLA genotypes and autoantibody profiles with autoimmune diseases: Autoimmune diseases, where the immune system attacks the body’s own tissues, posses a predisposing genetic background which lies mainly in the polymorphism of the HLA (human leukocyte antigen) locus. AIW platform provides a robust way to combine the serodiagnostics with genetic screening for large-scale clinical association studies of autoimmune diseases. An oligonucleotide array will be developed to screen for the HLA risk alleles and allele combinations. Serum autoantibody levels, on the other hand, will be profiled using an array containing a panel of autoantigens. Together with clinical collaborators large patient sample panels are available from studies on known autoimmune diseases (e.g. type 1 diabetes) and diseases with suspected autoimmunity involvement (e.g. schizophrenia).
Serum IgE/IgG profiling to predict/monitor the efficacy of hypoallergenic therapy: Identification and production of recombinant allergenic proteins has provided means to develop specific and sensitive IgE tests for the diagnosis of allergies. AIW platform possess great potential in IgE testing as a panel of allergens can be assayed in just a single reaction well. An array containing different types and forms of allergens (e.g. food allergens and birch pollen) will be developed to profile the sera of allergic persons for the presence of specific IgE and IgG antibodies. The goal is to identify the exact allergens against which the persons have IgE antibodies, and to see whether various modified formulations of the allergens (i.e. hypoallergens) will have lost the IgE reactivity. This would enable robust and sensitive allergy testing and also help in the development and monitoring of the hypoallergen therapy, which is ongoing at VTT.
Figure 1. Arrays-in-Wells (AIW) platform. A microarray is printed in the wells of 96-well microtiter plate. Depending on assay concept the array can contain either oligonucleotides, antibodies, or antigens (proteins, peptides, haptens).
Markus Räsänen
Diagnostiikkaan soveltuvien lantanidikelaattien luminesenssitutkimus
Markus Räsänen, Keijo Haapakka, Harri Takalo* ja Jouko Kankare
Turun Yliopisto, Kemian laitos, Materiaalikemian ja kemiallisen analyysin laboratorio, 20014 Turun yliopisto,
* Turun yliopisto, Biokemian ja elintarvikekemian laitos/bioteknologia, 20014 Turun yliopisto
Lantanidien luminesenssiominaisuudet (suuri Stokesin siirtymä, kapeat emissiovyöt ja pitkä virittyneen tilan elinikä) mahdollistavat aikaerotteisen fluorometrian ja täten siihen perustuvat erittäin herkät diagnostiset määritykset. Lantanideilla on heikko molaarinen absorptiviteetti, mutta se voidaan korvata käyttämällä viritykseen lantanidi-ionien kanssa kompleksoituvia orgaanisia ligandeja. Ligandien tehtävänä on energian keräämisen ja siirron lisäksi toimia kelaatin kiinnittimenä analyyttiin.
Näiden diagnostiikassa leimayhdisteenä käytettävien lantanidikelaattien luminesenssiominaisuuksiin vaikuttavat ainakin energiansiirron mekanismi, suhteelliset energiatasot ja sekä ulkoiset että sisäiset sammutusprosessit. Jo pienet muutokset ligandien rakenteessa vaikuttavat näihin tekijöihin ja näin ollen kelaatin luminesenssiominaisuuksiin. Sen vuoksi ideaalisen leimana käytettävän lantanidikelaatin valmistaminen ei ole helppoa, ja yhä parempien leimamolekyylien valmistamiseksi on syvällisesti tutkittava niitä tekijöitä, jotka vaikuttavat luminesenssiominaisuuksiin vaikuttaviin tekijöihin.
Tässä työssä tutkitaan niitä tekijöitä, jotka vaikuttavat lantanidikelaattien luminesenssiin ja stabiilisuuteen. Työssä selvitetään esimerkiksi kelaatin kromoforin rakenteen, lukumäärän ja etäisyyden vaikutusta kelaatin luminesenssiominaisuuksiin sekä kelatoivan rakenneosan vaikutusta kelaatin luminesenssiominaisuuksiin ja stabiilisuuteen. Työssä tutkitaan europium- ja terbiumionien lisäksi samarium- ja dysprosiumkomplekseja sen selvittämiseksi, mitkä ovat keskeisimmät tekijät korkean Sm- ja Dy-luminesenssin aikaansaamiseksi.
Minna Ylihärsilä
Array-in-well platforms for detection and serotyping human respiratory track infection causing viruses by utilizing upconverting phosphor label technology
M.Sc. Minna Ylihärsilä
Supervisors professor Tero Soukka and docent Matti Waris
The aim is to study a robust array-in-well test platform based on oligonucleotide and protein arrays combining advantages of simple instrumentation and upconverting phosphor reporter technology. Upconverting phosphors are lanthanide-doped crystals that have a unique feature of being capable to convert infrared to visible light. No autofluorescence is produced from the sample under infrared excitation enabling a highly sensitive assay. Microarrays will permit multiplexed, rapid and accurate detection of viruses and their serotypes causing respiratory track infections. In this study the high-quality spotting method developed by VTT for oligonucleotide and protein arrays-in-well is employed. The study provides a verification of the advantages and potential of the upconverting phosphor-based reporter technology in multianalyte assays and anti-Stokes photoluminescence detection with a new anti-Stokes photoluminescence imager.
Tanja Savukoski
Autoantibodies to cardiac troponin - a new potential cardiac disease risk marker
Supervisors: M.Sc. Noora Ristiniemi and Ph.D. Saara Wittfooth, Department of Biochemistry and Food Chemistry / Biotechnology, University of Turku
Cardiac diseases are one of the major human health problems causing disability and death worldwide. Screening and diagnosing the cardiac diseases can be challenging and it depends on the availability of specific, accurate and effective disease or risk markers. While increasing evidence indicates that organ-specific autoimmunity may play a pivotal role in the etiology of cardiac diseases, autoantibodies to cardiac troponins (cTn) have been found in a high proportion of individuals with or without cardiac diseases. Even though the functional role of these autoantibodies is not yet clear, it is suspected that these autoantibodies are connected with clinical conditions by either playing an active role in the disease process or reflecting an inflammatory response to myocyte necrosis. In both cases, they could be used in diagnosing the clinical condition.
The aim of this thesis work is to study the molecular diversity of circulating cTn-specific autoantibodies and to design new assay concepts for autoantibody detection that would combine the correct specificity and high sensitivity while being suitable for implementation into simple and robust routine use. The applicability of these assays will be evaluated with clinical materials and the frequency of cTn autoantibodies will be studied in healthy controls and different disease conditions. The role and mechanism of autoantibodies in the pathogenesis of chronic idiopathic dilated cardiomyopathy following acute myocarditis will also be examined with a murine model of chronic heart failure.
Tuomas Huovinen
Tools for diagnostics from recombinant antibody libraries
Principal supervisor: FT Urpo Lamminmäki
Phage displayed antibody libraries have become an inviting alternative to the traditional way of generating antibodies by animal immunization and hybridoma technology. The main research interests are to develop mutagenesis methods to create functional diversity on antibody genes, display the antibodies on phage in an efficient and unbiased way and select antibodies against targets for which antibody generation by traditional methods is difficult and time-consuming. For the last task, a focused antibody library for small molecule recognition is constructed and studied with various antigens.
Riikka Arppe
Single-crystal study of upconverting nanoparticles and their applications
M.Sc. Riikka Arppe
Principal supervisor: Professor Tero Soukka, Department of Biochemistry and Food Chemistry / Biotechnology, University of Turku
Upconversion of low-energy light to visible wavelengths is a new advanced detection technology capable of sensitive bioassays without background interference from autofluorescence. Recently the high specific activity of these upconverting particles has been demonstrated on numeral different bioanalytical assays. However, these assays were performed with particular labels of submicrometer size. Reducing the particle size to just tens of nanometers can be foreseen to improve their performance and applicability further. The aim of this research is to study single upconverting nanoparticles and their elemental composition to learn more about the capabilities of this label technology. Also the surface chemistry is going to be improved for strong as possible luminescence without quenching effects of the surroundings. Advanced separation techniques for purification of water-soluble nanoparticles are going to be investigated. And finally the performance of the nanosized upconverting phosphor particles is going to be demonstrated in homogeneous resonance energy transfer assays which measure either the intensity of acceptor emission or the decreasing lifetime of the nanoparticle donors. When completed, this research sets the grounds for inexpensive and highly sensitive future diagnostic assays.