!!

Advances in DNA Detection on

Paper Chips

Yajing Song KTH Royal Institute of Technology

School of Biotechnology

Stockholm 2013 !!!!!!!

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Yajing Song, 2013: Advances in DNA Detection on Paper Chips Division of Gene Technology, School of Biotechnology, Royal Institute of Technology (KTH), Stockholm, Sweden. ISBN 978-91-7501-922-2

Abstract

DNA detection has an increasing importance in our everyday lives, with applications ranging from microbial diagnostics to forensic analysis. Currently, as the associated costs decrease, DNA diagnostic techniques are routinely used not only in research laboratories, but also in clinical and forensic practice. The present thesis aims to unravel the potential of cellulose filter paper to be a viable candidate for DNA array support. There are two papers in this study. In Paper I, we studied the method of functionalizing the surface of filter paper and the possibility to detect DNA on acitve paper using fluorescence. In Paper II, we investigated visualization and throughput of DNA detection with magnetic beads on active filter papers, an assay which requires no instrumentation (scanner). The findings in Paper I show that XG-NH2 and PDITC can functionalize the cellulose filter paper and that the activated filter papers can covalently bind oligonucleotides modified with amino groups to detect DNA. The detection limit of the assay is approximately 0.2 pmol. In Paper II, visualization of DNA detection on active paper is achieved without instrumentation, based on the natural color of magnetic beads. Furthermore, successful multiplex detection supports the potential to increase the throughput of DNA detection on active papers. In summary, these studies show that active cellulose filter paper is a good DNA array support candidate as it provides a user-friendly and cost-efficient DNA detection assay. The methods described in Paper I and II are possible sources of development to a point-of-care device for on-site analysis of DNA contents in a sample. Keywords: DNA detection, active filter papers, visualization, throughput, fluorescence, superparamagnetic beads © Yajing Song, 2013

Svensk sammanfattning

Detektion av DNA har fått en allt ökad betydelse i våra liv, inom allt från diagnostik av bakterier till kriminalanalys. I takt med att kostnaderna för detta har sjunkit, används nu diagnostiska DNA-tekniker rutinmässigt inte bara i forskningslaboratorier, utan också i klinisk och kriminalteknisk verksamhet. Föreliggande avhandling syftar till att tydliggöra huruvida cellulosafilterpapper har potential att vara en kandidat för DNA-matrissupport. Denna studie omfattar två artiklar. I artikel I (Paper I) studerade vi en metod för att funktionalisera ytan på filterpapper samt möjligheten att på aktiverat papper detektera DNA medelst fluorescens. I artikel II (Paper II) undersökte vi både kapaciteten för och möjligheterna till visualisering av DNA-detektion medelst magnetiska kulor på aktiverat filterpapper, vilket är en metod som inte kräver ett instrument (skanner) för detektionen. Resultaten i artikel I visar att XG-NH2 och PDITC kan funktionalisera cellulosafilterpapper och att de aktiverade filterpapprena kan bilda kovalenta bindningar med aminogruppsmodifierade oligonukleotider för DNA-detektion. Detektionsgränsen för metoden är cirka 0,2 pmol. I artikel II påvisas att visualisering av DNA-detektion kan erhållas på aktivt papper utan instrument, genom att använda sig av den naturliga färgen hos magnetkulorna. Därutöver påvisas framgångsrik detektion av s.k. multiplexade prover, vilket ytterligare styrker metodens potential att öka kapaciteten av DNA-detektion på aktivt papper. Sammantaget visar dessa studier att aktivt cellulosafilterpapper är en duglig kandidat för DNA-matrissupport, då det möjliggör en användarvänlig och kostnadseffektiv metod för DNA-detektion. Metoderna som beskrivs i artiklarna I och II är möjliga utgångspunkter för utveckling av ett patient-nära instrument för direkt analys av DNA-innehåll i ett prov. ! !

© Yajing Song, 2013

Contents List of Publications Abbreviations 1 Introduction 2 DNA Science 2.1 DNA History 2.2 DNA Structure 2.2.1 Primary Structure of DNA 2.2.2 Secondary Structure of DNA 2.2.2.1 A-, B-, Z-DNA 2.2.2.2 Bent DNA 2.2.2.3 Cruciform DNA 2.2.2.4 Triplex and Quadruplex Forms of DNA 2.2.3 Stability of Double-helical DNA 2.2.4 Separation of Double-helical DNA 2.2.4.1 Denaturation 2.2.5 Buffer 2.3 DNA Function 2.3.1 DNA Replication 2.3.2 Transcription 2.3.3 Translation 2.4 DNA Detection 2.4.1 Background 2.4.2 DNA Cloning and Library 2.4.3 Polymerase Chain Reaction (PCR) 2.4.3.1 Basic Principles of PCR 2.4.3.2 PCR Variants 2.4.3.2.1 Hot-start PCR 2.4.3.2.2 Nested PCR 2.4.3.3 Quantitative Real-time PCR 2.4.4 Isothermal Amplification 2.4.5 DNA Microarray 2.4.5.1 Background 2.4.5.2 Principle 2.4.5.3 Classification Based on Dimensions of Support Surfaces 2.4.5.3.1 Two Dimensional Surfaces 2.4.5.3.1.1 Glass Surfaces 2.4.5.3.1.1.1 Development 2.4.5.3.1.1.2 Support Surface Treatment for Glass Slides 2.4.5.3.1.1.3 5’ Amino Modifiers 2.4.5.3.1.1.4 Attachment of 5’ Amino-modified Oligonucleotides 2.4.5.3.1.1.5 Fabrication: Spotted or in Situ Synthesized Microarray 2.4.5.3.1.2 Silicon Chips

2.4.5.3.1.3 Paper Chips 2.4.5.3.1.3.1 Paper Test Strips 2.4.5.3.1.3.2 Bioactive Filter Paper Detection 2.4.5.3.2 Three Dimensional Surfaces 2.4.5.3.2.1 Microbeads (Illumina): Silica Beads 2.4.5.3.2.2 Microbeads (Lumimex): Polystyrene Beads 2.4.5.4 Microfluidic DNA Microarray 3 Specific Aims of the Studies 3.1 Paper I 3.2 Paper II 4 Results and Discussion 4.1 Paper I 4.1.1 Activation of Cellulose Filter Papers 4.1.2 DNA Detection on Functionalized Papers 4.1.2.1 Synthetic Oligonucleotides 4.1.2.2 Human and Canine Samples 4.2 Paper II 4.2.1 DNA Visualization 4.2.2 Inspiration from Porous Structure of Cellulose Paper Chips 4.2.3 Multiplex Detection 4.2.4 Parameter Optimization 4.2.5 Future Development 5 Conclusions References

List of Publications I Araújo, A.A. *, Song Y. *, Lundeberg, J., Ståhl P.L., Brumer H, 3rd. (2012) Ac-tivated paper surfaces for the rapid hybridization of DNA through capillary transport. Anal Chem. 84(7): 3311-3317. (* Araújo, A.A., Song Y. contributed ea-qually to this work.) II Song, Y., Gyarmati, P., Araújo, A.A., Lundeberg, J., Brumer H, 3rd., Ståhl P.L. (2013) Visual Detection of DNA on Paper Chips. Submitted . Paper I is reprinted with permission of Analytical Chemistry. Copyright 2013 American Chemical Society.

Abbreviations A adenine APTES aminopropyltriethoxysilane C cytosine cDNA complementary DNA DNA deoxyribonucleic acid dsDNA double-stranded DNA E. coli Escherichia coli FP filter paper FDA food and drug administration G guanine HDA helicase-dependent amplification HGP the Human Genome Project mRNA messenger RNA -OH hydroxyl group PDITC phenylenediisothiocyanate PE phycoerythrin PCR polymerase chain reaction RCA rolling cycle amplification RNA ribonucleic acid rRNA ribosomal RNA SSB Single-stranded DNA binding protien ssDNA single-stranded DNA T thymine tRNA transfer RNA XG xyloglucan

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1 Introduction What is life (Schrödinger 1944, Ernberg 2010)? Where are we coming from and where are we going to go? The curiosity and exploration of life has promoted discovery of DNA and development of DNA technologies. From the end of 19th century, DNA was found in white blood cells (Dahm, 2005; Dahm & Miescher, 2008); to the mid of 20th century, it was realized that DNA molecules possess a unique duplex structure and carry genetic information (Hershey & Chase, 1952; Watson & Crick, 1953); and to the 21st century, the first human genome draft was announced and more different species genome drafts were accomplished. At present, DNA science is facing a good opportunity to make great progress due to improvements and developments on related fields such as chemistry, biotechnology, physics, etc. Microarray technology has turned to be a unique and important tool to analyze biomolecules because of its high-throughput capacity. Thousands to millions of features can be analysed simultaneously on a microarray. This property meets a variety of omics needs e.g., proteomics, genomics. In pursuit of more potentials and possibilities, there is a great interest in improving and further developing microarray systems. The objective of the microarray technology is to enable scientists to study biological information and to understand and serve life better. Researchers can design and fabricate arrays they need or this technology can be developed to a commercial product to serve researchers and clinicians. How to discover a new DNA microarray method that is user friendly, cost-effective, and timesaving, is the goal of this thesis.

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2 DNA Science 2.1 DNA History What is life? In the long run to pursue the answers, DNA was discovered from the nuclei of white blood cells by Friedrich Miescher, a German docotor in 1869, which was termed 'nuclein' (Dahm, 2005; Dahm & Miescher, 2008). In 1952, DNA was proved to be the molecule of heredity by the Hershey-Chase Blender experiment. This study included two parts and was done side-by-side by Alfred Hershey and his assistant Martha Chase. In this experiment, the protein capsule and the DNA core of a phage were labeled with radioactive sulfur (35S) and phosphorus (32P), respectively. After the phage infected the bacteria and the bacterial cell culture, centrifugation separated the phage which was left in the supernatant from the infected bacterial cell fraction. 35S was detected mainly in the suspernatant, while 32P from phage was found predominantly in the infected bacterial cells, from which the new generation of phage generated. It evidenced that the DNA, but not protein was the molecule carrying hereditary information (Hershey & Chase, 1952). What is the structure of this molecule of heredity? This chemical puzzle, accumulating for more than 80 years, finally was solved by James Watson and Francis Crick in 1953 (Watson & Crick, 1953), who announced the "double helix" structure of DNA (Figure 1). It is the most essential discovery in biology in the 20th century. The physical basis of DNA was revealed via X-ray crystallography. Maurice Wilkins and Rosalind Franklin obtained X-ray diffraction photographs of DNA in 1951, which strongly suggested the helical nature of DNA structure and promoted Watson and Crick, published their Nature paper about the first duplex DNA model (alfa helix). In 1990, the Human Genome Project (HGP) was initiated with the goal to understand human genome and the genetic background of human diseases. Further objective was to serve human health and human evolution by providing the human genome publicly available for researchers. In 2001, the first draft of DNA codes of the human genome was published, representing a start of the DNA era, and with the announcement of the complete genome in April 2003, HGP finished. However, the issue of the accurate number of genes in human genome is still going on (IHGSC, 2004).

Figure 1 DNA double helix structure. Two strands are antiparallel and complementary to each other.

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2.2 DNA Structure 2.2.1 Primary Structure of DNA By 1900s, the basic chemistry of DNA polymer had been worked out. The basic building block of DNA is the nucleotide and is composed of three distinct subunits: 2'- deoxyribose, a nitrogenous base and an acidic phosphate group. 2'- deoxyribose is a pentose with a hydrogen group on the 2'-carbon of ribose. 1' carbon of sugar is the position for a base to attach to. There are four nitrogenous bases, two single-ring pyrimidines (cytosine, C and thymine, T) and two double-ring purines (Guanine, G and Adenine, A). Following the base-pairing, DNA double helical structure forms with two base pairing combination via hydrogen bond inside of DNA: C base-paired with G by three hydrogen bonds and T base-pired with A by two hydrogen bonds. Thus, in each sample of double helical DNA, the amount of G equals the amount of C; the amount of A eaquals the amount of T and the ratio of purines and pyrimidines is 1:1 that is the famous "Chargaff’s rules" (Chargaff et al. 1952; Elson & Chargaff 1952). DNA helix is formed with deoxyribose-phosphate backbone by phosphodiester bonds on the outer part of the molecule. The two strands run in opposite directions and the diameter of the helix is 2.37 nm (Figure 2). There are major and minor grooves on the helical DNA surface. The paired bases are almost perpendicular to the helix axis. The size of each base pair is 0.34 nm and there are 10 base pairs per helix turn so a pitch per turn is 3.4 nm and each base pair rotates 36 degree from the adjacent one. The average molecular weight of a base pair is 650 g/mol (in sodium salt).

Figure 2 Chemical structure of DNA. Two complementary DNA strands form a double-stranded DNA (dsDNA). The pink line with phosphate and deoxyribose sugar (left) and the blue line with phosphate and deoxyribose sugar (right) show the deoxyribose-phosphate backbone. G = guanine; C = cytosine; A = adenine; T= thymine. Three hydrogen bonds are between G and C; two hydrogen bonds are between A and T.

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2.2.2 Secondary Structures of DNA 2.2.2.1 A-, B-, Z-DNA X-ray diffraction studies revealed that the conformation of DNA is variable. The double-helical DNA that Watson and Crick reported was B-DNA with 10 base pairs per turn, which is a right-handed double helix and is the most stable conformation under physiological conditions. A-DNA and Z-DNA are two variants in different conditions. Under conditions of low humidity and high salt concentration (e.g., 75%, potassium), A-DNA forms which is also right-handed DNA but shorter and wider than B-form, with 11 base pairs per helical run. Z-DNA was observed by Alexander Rich and his associates in 1979 (Wang et al., 1979) when they tried to solve the structrue of d(CG)n. In contrast with A-form and B-form, Z-form is left-handed double helix and longer and thinner with 12 base pairs in per helical run. The phosphates in the backbone take on a zigzag form. It is still uncertain that whether A-DNA exists in the cell but there is some evidence to suggest that Z-DNA plays some roles to regulate gene expression, especially for short stretches of Z-DNA containing alternating C and G at 5’end genes where regulation of transcriptional activities occurs (Devlin, 2005). The features of different comformations of DNA double helix are compared in Table 1(Berg, 2011; Brown, 2006; Nelson & Cox, 2013).

Table 1 Features of different DNA duplex comformations

Feature B-DNA A-DNA Z-DNA Type of helix right-handed right-handed left-handed

Helical diameter (nm) 2.37 2.55 1.84 Rise per base pair (nm) 0.34 0.29 0.37 Distance per complete

run (pitch) (nm) 3.4 3.2 4.5

Number of base pairs per complete turn

10 11 12

Topology of major groove

Wide, deep Narrow, deep Flat

Topology of minor groove

Narrow, shallow Broad, shallow Narrow, deep

Tilt of base pairs from perpendicular to helix

axis

1 degree 19 degree 9 degree

Glycosyl bond conformation

anti syn Anti at pyrimidine, syn at purines

Apart from the A-, B-, and Z-DNA, there are other variants in DNA conformation during the interaction between DNA with certain proteins, such as bent DNA, cruciform structure, triple-stranded DNA and four-stranded DNA (Davlin, 2005).

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2.2.2.2 Bent DNA Bent DNA forms in runs of 4 or 6 adenines separated by 10 base pairs spacer or during the interaction of DNA with certain proteins and functions as a basic molecule to carry out important biological processes such as replication, transcription and site-specific recombination. Bent DNA also acts as a signal of DNA repair to recognize base mispairing or photochemical damage. 2.2.2.3 Cruciform DNA As one type of symmetrical dsDNA sequences, inverted repeats or palindromes may serve as molecule switches of transition from replication to transcription. In inverted repeats, each DNA strand is self-complementary, which results in the base-pairing within each single-stranded DNA after denaturation of dsDNA. As a result, cruciform structure forms: cruciform DNA is important for genomic stability and basic biological regulation process in the cell (Brázda, 2011). It has been found that many proteins have specific structures for cruciform DNA binding in the cell. 2.2.2.4 Triplex and Quadruplex Forms of DNA Triplex helices can be formed by parallel binding of the third homopyrimidine strand to the homopurine strand of B-DNA through Hoogsteen hydrogen bonds, resulting in T-A-T and C+-G-C triplets, also by antiparallel binding of the third homopurine strand to the homopurine strand of B-DNA via reverse Hoogsteen hydrogen bonds to form A-A-T and G-G-C triplets. Four-stranded DNA usually occurs in G-rich region of DNA, through Hoogsteen hydrogen bonds. Four Gs interact with each other to form a guanine tetrad, and more guanine tetrads stack upon each other to form tetraplexes, or G-quadruplexes, which damages the DNA helix. The structure is stabilized by cations, especially potassium with coordination by the four O-6 oxygens in the center channel of the quadruplex. The first evidence that four-stranded DNA exists in cells was reported in 2009 (Lipps & Rhodes, 2009). In 2013, Shankar Balasubramanian and his colleagues published strong evidence that G-quadruplex does exist in the DNA of living cells (Biffi et al., 2013; Lam et al., 2013), which supports its assumptive biological functions. 2.2.3 Stability of Double-helical DNA Stability of the DNA molecule is crucial as it carries important genetic information. Generally, base-stacking and hydrogen bonds are thought to be the main forces to stabilize the duplex of DNA. In contrast to stacking forces, hydrogen bonds were better known to orient base pairing until 1963, when the importance of stacking interaction to stabilize the double duplex was verified through experiments with different reagents (Levine et al., 1963). Base-stacking,

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also termed !-! interaction, originates from hydrophobic forces and van der Waals interaction. The stacking energy for each adjacent stacked pair of nucleobases is around 2-3 times stronger than that in each hydrogen bond. Electrostatic force is another factor to effect conformation and stability of duplex DNA. The reason is that phosphodiester groups are negatively charged in physicological conditon, therefore repel each other. Cations and proteins can help to decrease repulsive forces between ionized phosphate groups (Davlin, 2005). 2.2.4 Separation of Double-helical DNA 2.2.4.1 Denaturation The hydrogen bonds between complementary bases in the centre of DNA are disrupted under certain conditons. This separation process between complementary DNA strands is termed denaturation or helix-to-coil transition or melting. A minute energy is needed to create one or more bubbles in dsDNA or in DNA-RNA complexes. Whereas these bubbles or stretches will immediately pair up at room temperature to form stable duplex structure again. At elevated temperature, the range of stretches increases and the kinetic energy overcomes the forces forming the duplex, eventually resulting in single strands. Significant changes in pH can also disrupt the double helix of DNA. Alkaline deprotonates specific positions on the DNA base to disrupt hydrogen bonds. Acid protonates hydrogen bond acceptors to block the hydrogen bond formation. Alkaline denaturation is usually used to prevent damage to the nucleosides or sugar which can happen at either low pHs or high temperature (Davlin, 2005). This technique was applied in Papers I and II. In the process of denaturation, hyperchromic effect occurs, namely the DNA absorbance of UV light at 260 nm increases. It is because the bases in duplex strands have lower ability to absorb the light than that in single strands. 2.2.5 Buffer In order to maintain certain biological processes such as polymerization, transcription, etc., maintaining a stable pH is essential. DNA structures are disrupted by significant changes in pH and enzymes function in a narrow pH range as well. Thus, there are some solutions to restrain pH changes in biological systems where the typical physical pH is around 7.4. Also, these mechanisms have been used in molecular biological studies such as denaturation, renaturation and hybridization (Paper I, II). Buffers are solutions which alleviate pH changes, and in biological systems, an important buffer is based on phosphoric acid (H3PO4), which will be deprotonated into H2PO4

-, HPO42-, and PO4

3-, respectively. H2PO4- is

proton donor and HPO42- is proton acceptor. At about pH 7.4, a mixture of equal

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concentration of H2PO4-, HPO4

2- exists, which resists the formation of either acid or base and pH change region is between about 5.9 to 7.9 (Berg, 2011). 2.3 DNA Function All the studies about how DNA functions have established the “central dogma” of molecular biology to illustrate that genetic information flows from DNA to RNA, and from RNA to proteins with exception of some viruses whose RNA stores their genetic information. In this procedure, genetic information is not only stored and transferred in DNA by precise replication, but also selectively expressed by transcription and translation. 2.3.1 DNA Replication DNA structures determine DNA functions. Uncovering the DNA double helical structure by Watson & Crick in 1953 suggested that semiconservative replication of DNA is the possbile mechanism of genetic informational transfer during cell division: “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material” (Watson & Crick, 1953). This assumption was supported by the Meselson-Stahl experiment later in 1958. Briefly, the experiment was designed in three steps to verify the hypothesis that DNA replicaiton is semiconservative replication but not conservative replication (Bloch, 1955) or dispersive replication (Delbrück, 1954). Step 1: Escherichia coli (E. coli) cells were cultured in medium with only 15N (heavy density); Step 2: To culture E. coli with 15N-DNA in the sodium with only 14N (light density), 100% DNA density of the first replication was single density between that of 15N and 14N, which excluded conservative replication. Step 3: To continue culturing the E. coli in step 2 medium. After the second replication, 50% DNA density was between that of 15N and 14N, and the other 50% DNA density was same as that of 14N. This result excluded dispersive replication and supported semiconservative replication (Micklos et al., 2003). Semiconservative replication means that in DNA replication each parental strand serves as a template to produce a daughter strand with complementary bases pairing to copy the parental genetic information, followed by the two strands winding each other. Thus in the newly generated DNA helix, one strand is “old” and the other one is “new”. Many enzymes and proteins are needed in this process. The templates of DNA replication are single-stranded DNAs so DNA helix must be unwound and separated from each other to generate two templates before replication, which is carried out by DNA helicase and topoisomerase. Then single-strand-binding proteins (SSBs) attach to single DNA strands to prevent DNA renaturation. An RNA primer is necessary to initiate DNA replication due to its free 3’ hydroxyl group (-OH). This determines the direction of DNA sythesis from 5’ to 3’. The RNA primer is made by the primase before DNA replication and replaced by DNA with the aid of DNA polymerase I after replication. Finally,

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DNA ligase connects the fragments to form a strand complementary to the template (Figure 3).

Figure 3 Schematic view of DNA semiconservative replication (See text for details) 2.3.2 Transcription In order to transfer genetic information to synthesize a variety of proteins, the copy process from DNA to RNA is also important. This process is termed transcription, which is initiated by RNA polymerases to create an RNA strand antiparallel with and complimentary to a DNA template. U in the new RNA molecule replaces T in this template. The synthesis direction is 5’ 3’. Transcription generally consists of three steps: (1) initiation, the formation of initiation complex which is made up of RNA polymerase, cofactors and a core promoter sequence in DNA, (2) elongation, the process in which the RNA polymerase complex crosses the template molecule to synthesize an RNA strand, (3) termination, which occurs when RNA polymerase complex recognizes terminator and finally releases the newly synthesized RNA strand. The transcribed products include: messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), regulatory RNAs (e.g., microRNA) and other RNA molecules. There are also some proofreading mechanisms during transcription but not as effective as in the DNA replication (Berg J et al., 2006). 2.3.3 Translation Translation is the next important step of gene expression. In this process, mRNA, tRNA and ribosomes work together to produce proteins from amino acids.

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Ribosomes, consisting of a large subunit and a small subunit, are the sites for amino acids to be assembled into protein. mRNA carries genetic information from the transcription in sequence codes and attaches to the small subunit of the ribosome. tRNA identifies specific amino acids by specific anti-codons and transfers the specific amino acids to the large subunit of the ribosome via binding its anti-codons to the code sequences of mRNA. With the ribosoms moving along the mRNA, the information in mRNA is translated into polypeptide until they meet a termination codon, when the polypeptide strand is released. 2.4 DNA Detection 2.4.1 Background In many methodologies, hybridization is the basis of DNA detection, which is a process for complementary polynucleotide strands to associate with each other. Schildkraut and his colleagues introduced this technique in 1961(Schildkraut et al., 1961). The polynucleotide strands can be homogenous or heterogenous. The technique has been applied to nucleic acid detection and quantification, e.g., genotyping, gene expression, phylogeny and genetic mapping. In this technique, a probing oligonucleotide is used to detect the complementary single-stranded DNA sequence. One of them is immobilized to a solid matrix, the other one is labeled with fluorophore or biotin then detecion of bound labels quantifies the target sequences. Hybridization technique is a basic and important tool in molecular biology (Paper I, II). 2.4.2 DNA Clone and Library Originally, the term clone refers to the process when a single cell type is reproduced to create a population of identical cells. Regarding DNA, a clone represents multiple identical copies of a particular DNA segment. This technology is called recombinant DNA technology. The goal of this technology is to amplify the target DNA, which can be used for downstream studies. This technology commonly consists of six steps: 1. To get target DNA with restriction endonucleases 2. To select a cloning vector capable to self-replication, which is treated with the same restriction endonuclease. Typically, they are plasmids or viral DNA. 3. To insert target DNA into the vector to creat recombinant DNA with DNA ligase 4. To move recombinant DNA into host cell for replication 5. To select host cells with target DNA 6. To analysis target DNA biological propertises (Nelson & Cox, 2013). A DNA library is a collection of DNA clones, including genomic DNA library and cDNA library. The goal of genomic DNA library is to produce the whole genome of an organism, and to understand genes and proteins function is the goal of cDNA library (Nelson & Cox, 2013).

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2.4.3 Polymerase Chain Reaction (PCR) 2.4.3.1 Basic Principles of PCR The first idea to replicate a short DNA fragment with enzyme and primers was described in 1971 by Kjell Kleppe (Kleppe et al., 1971). By 1983, a complete in vitro PCR technique was invented by Kary B Mullis (Bartlett et al., 2003; Mullis et al., 1986), which was mainly composed of three steps: (i) dsDNA denaturation at a high temperature, normally > 90 °C, (ii) primers annealing at a lower temperature (50-75 °C), and (iii) extension at a certain temperature (commonly 72 °C). These three steps constitute one cycle of a PCR. After the first two-three cycles, the target sequences are amplified exponentially. Therefore, after 30 cycles, billions of amplicons are produced under ideal reaction conditions. This standard PCR needs agarose gel electrophoresis to evaluate amplicon size(s) by comparing them to a DNA ladder, a known size molecular weight marker. In contrast to cell culture technique, PCR makes the detection and analysis more sensitive and robust in a short time. PCR technique became an accepted and important tool in the research field rapidly (Strachan et al., 2003). 2.4.3.2 PCR Variants Many efforts have been made to enhance the efficiency and specificity of classical PCR and this resulted in different variations of this technique. 2.4.3.2.1 Hot-start PCR Hot-start PCR is a modification of the classical PCR, that decreases unspecificity and increases specific product yields. In this system, Taq DNA polymerase is inac-tive by different methods until the reaction reaches high temperature (>95°C). For example, Platinum Taq DNA Polymerase is a hot-start polymerase where the abil-ity of the polymerase is blocked by an antibody until the initial denaturation step. However, in classical PCR, Taq DNA polymerase has some activity at low temper-ature, therefore unspecific pairing can be extended to get unspecific amplicons, which reduces the accurate target yields. 2.4.3.2.2 Nested PCR In this variant, two primers pairs are used instead of one. The products amplified from the first pair of primers become the templates of the second pair of primers, therefore the sensitivity of the nested PCR increases compared to the classical PCR. Ideally, specificity is increased at the same time because the unspecific products from the first pair of primers will not be increased by the second pair of primers; however, it depends on the design of primers. 2.4.3.3 Quantitative Real-time PCR Real-time PCR, a further development of classical PCR, was reported firstly in the early 1990s (Higuchi et al., 1992; Higuchi et al., 1993; Heid et al., 1996). Following the basic principles of classical PCR, real-time PCR integrates the techniques of thermal cycler and fluorimeter to monitor the progress of the reaction in real time

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with the use of different fluorophore(s) (Mackay et al., 2002). Compared to classical PCR, real-time PCR increases the reaction speed and reduces the risk of amplicon spread since detection and analysis are proceeded in a closed tube during an amplification, which prevents carry-over contamination of PCR products in post-PCR steps, for example, agarose gel electrophoresis. The quantitative PCR techniques are classified into two types based on the fluorescent principle: non-specific fluorophore without probe, e.g., SYBR Green I, and fluorophore labeling specific probe, e.g., TaqMan chemistry and Molecular Beacon chemistry. 2.4.4 Isothermal Amplification The amplification based on PCR needs precise thermal cycler to perform temperature changes in different steps of every run, while isothermal amplification only needs one temperature for the entire amplification reaction without thermal cycling steps, which facilitates its operation and is beneficial to on-site detection systems. Isothermal amplification was investigated as a diagnostic approach from the end of the 1980s (Blanco et al., 1989). There are various isothermal methods, such as rolling cycle amplification (RCA) (Larsson et al., 2004), helicase-dependent amplification (HDA), amongst others. RCA is an amplification technique using one primer and circular DNA. HDA uses a helicase to unwind the double stranded DNA for two primers annealing and extention via a strand displacing polymerase (Chang et al., 2012). 2.4.5 DNA Microarray DNA microarray is a high-throughput DNA detection technology based on the hybridization of DNA libraries and PCR products, which can screen many thousands, even millions of features from one or more sample(s) simultaneously (Nelson & Cox, 2013). Therefore, this technology has improved DNA identification in different fields dramatically, such as gene expression, genotype determination, SNP analysis, medical diagnosis, environment detection, forensic and even evolutionary studies. 2.4.5.1 Background With DNA hybridization technique development (e.g., Southern blot and DNA cloning), Hoheilsel et al. printed high density of clones on filters by robotics and used an imaging method to measure the parallel signals as well (Hoheilsel et al., 1994). This was thought as a start toward microarray technology. The basis of present array technology was established through the experiments of dot blots and reverse dot blots during the late 1970s and 1990s (Southern, 2001). 2.4.5.2 Principle Hybridization is the key feature of DNA microarrays, which is a technique for complementary polynucleotide strands to associate with each other. The polynucleotide strands can be homogenous or heterogenous. The technique has

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been applied in nucleic acid detection and quantification, e.g., genotyping, gene expression analysis, phylogenetic studies and genetic mapping. This thesis focuses on DNA detection. In this technique, an oligonucleotide called a probe is used to detect the complementary single-stranded DNA sequence. A probe or a ssDNA is immobilized to a solid matrix (e.g., glass or beads) while its complementary counterpart is used for detection, usually via fluorophores. After detection, the evaluation of fluorescent signal intensity quantifies the amount of target sequences (Figure 4). In the last decades, hybridization technique evolved into an important basic tool in molecular biology (Paper I, II). 2.4.5.3 Classification Based on Dimensions of Support Surfaces Hybridization between immobilized probes and target oligonucleotides happens on either a two or a three dimensional surface. 2.4.5.3.1 Two Dimensional Surfaces Many different support materials with two dimensional surfaces have been investigated for DNA detection, such as nitrocellulose and nylon membranes (Conner et al., 1983; Meinkoth, & Wahl, 1984; Saiki et al., 1989), polystyrene (Rasmussen et al., 1991), polypropylene (Matson et al., 1994), and glass (Maskos & Southern, 1992). In order to achieve a suitable support for oligonucleotide detection, several requirements have to be considered regarding support properties, such as stability and complexity, amenability to chemical modification, scattering and non-specific fluorescence background, loading capacity and cost (Guo et al., 1994). 2.4.5.3.1.1 Glass Surfaces 2.4.5.3.1.1.1 Development Compared to various other supports, glass quickly became commonly accepted as a microarray material as it met all the above needs (Guo et al., 1994; Southern, 2001). For microscopic glass slides, tens of thousands of probes can be printed in one spot for one target detection. In 1991, Maskos developed the first array with glass to analyze nucleic acid sequence (Maskos, 1991). In 1995, Schena et al. firstly reported gene expression profiling on a glass microarray (Schena et al., 1995). The first eukaryotic genome analysis on microarray was published in 1997 by Lashkari et al. (Lashkari et al., 1997). 2.4.5.3.1.1.2 Support Surface Treatment for Glass Slides For microarray techniques, support surface treatment is crucial. The goal of support surface treatment is to attach the oligonucleotides efficiently and stably by coupling chemistry termed as linker. There are several criteria for an ideal linker: the linkage should be stable, long enough to avoid steric effects from support, hydrophilic enough, and should provide specific binding to the support (Guo et al., 1994). Isothiocyanate and epoxysilane are applied to activate glass slides and provide functional groups to bind the oligonucleotide targets with their

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modifications (Guo et al., 1994; Lamture et al., 1994). Amino groups are routinely used to modify oligonucleotides to bind the functional groups of linkers. By this way, oligonucleotides can be immobilized onto isothiocyanate coated or epoxy silane-derived glass slides (Figure 4).

Figure 4 Functionalization of a solid support and the principle of microarray 1. Support surface modification. 2. Covalent binding of functional linker to surface modifiers. 3. Immobilization of molecules by covalent binding to functional groups of linkers. 4. Detection of fluorophore-targets or bead-targets via interaction between target molecules and immobilized molecules, e.g., oligonucleotide hybridization 2.4.5.3.1.1.3 5’ Amino Modifiers An amino group can be added to a 5’ terminus or 3’ terminus or internal part of DNA. Considering the steric interactions and the charges, it is necessary to convert amino groups to amino-modifiers, which are amino groups with a couple of carbon spacers (e.g., amino-C6 and amino-C12) to provide the distance between oligonucleotides, ligands and surfaces. How far the distance from an amino group to a surface is depends on the actual goals of the studies, sometimes even a longer spacer is needed than that by an amino-12C modifier (IDT). For example, in Paper I and II, a polyT spacer was integrated into each surface probe between amino-6C and oligonucleotides-NH2. This section focuses on 5’ amino modifiers. A variety of amino-modifiers can be added to 5’ terminus of oligonucleotides in the last step of oligonucleotide synthesis because synthetic direction is from 3’ to 5’.

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2.4.5.3.1.1.4 Attachment of 5’ Amino-modified Oligonucleotides An acylating reagent is needed for immobilization of amino-modified oligonucleotides, such as isothiocyanate. Carbodiimide is another common acylating agent for amino modified oligonucleotides attachment, which finally generates an amine carbonyl group. The kinetics reaction between an acylating reagent and an amine is strongly pH dependent at around pH 8.5-9.5 (Paper I, II). 5’ amino-modified oligonucleotides can also covalently attach to epoxy silane derivatized glass (Lamture et al., 1994). 2.4.5.3.1.1.5 Fabrication: Spotted or in Situ Synthesized Microarray If the probes are printed onto the glass surface, there are usually two ways to position them onto the solid array surface by a robotic printer, either by direct contact or with an ink-jet method (Goldmann et al., 2000). If the probes are synthesized in situ on the array surface directly, conventional phosphoramidite chemistry (Beaucage & Caruthers, 1981) and photolithography (Fodor et al., 1991) can be used, respectively. Both methods use protective groups through cycled reactions. In photolithographic synthesis, phosphoramidite chemistry is used with photolabile reagents. (Fodor et al., 1991). 2.4.5.3.1.2 Silicon Chips Based on the demand of high-density microarrays, Gene chip technology has been developed from the 1990s (Fodor et al., 1991) and became highly influential in this field (Affymetrix, Santa Clara, CA). In this technique, oligonucleotide probes are synthesized on the surface of quartz wafers directly by semiconductor-based photochemical synthesis. Therefore comparing with the printed probe microarrays, more than 1 million synthetic probes can be synthesized in an array around thumbnail size (Dalma-Weiszhausz, 2006; Miller & Tang, 2008). In the in situ synthesis technique, three principal steps are needed in the order of masking, optical deprotection and suitable nucleotides coupling in each synthetic cycle. The linkers with light sensitive protected groups attach to the surface of quartz wafers. Photolithographic masks are set carefully according to the probe sequences, thus the masks determine the activated positions of a gene chip in each cycle. In the exposure of UV light, linkers become deprotected and free hydroxyl groups are created for a suitable nucleotides addition, which has light protecting groups. The probes are synthesized via the repeating of the basic steps (Dalma-Weiszhausz et al., 2006; Fodor et al., 1991; Miller & Tang, 2008). 2.4.5.3.1.3 Paper Chips Paper is mainly constituted of cellulose fibers and form porous structure that offers high penetrating capacity for fluids without the involvement of pumping; also, this feature significiantly increases the surface area. This outstanding property of the paper material holds a great interest not only for academic research, but also for industrial applications as it has a huge potential commercial value for paper-based detection. Paper chips can be made of functional components in the process

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of manufacturing or be printed or coated with functional chemicals by spotting or printing after manufacturing. Many studies of different bioassays have been investigated, such as pathogen diagnosis, water and food quality monitoring (Martinez et al., 2010; Hong et al., 2008; Zhao et al., 2008; Aikio et al., 2006). 2.4.5.3.1.3.1 Paper Test Strips The objective of the paper test is to create a low-cost, easy-to-use, accurate and rapid test method. The history of paper test tracks back to the 19th century. The first paper detection of sugar and albumin was reported in 1883. Around 1920, a spot test chemistry via capillary action of filter paper was pioneered by an Austrian professor, Fritz Feigl (Voswinckel et al., 1994). That was the symbolic start of the “hour of birth of the modern test strip” as Professor Peter Voswinckel described (Voswinckel et al., 1994). Martin & Synge reported a new form of chromatography theory in 1941(Martin & Synge, 1941). The theory was exploited to separate mixtures of amino acids on a cellulose filter paper strip by Gordon, Martin & Synge in 1943; to my knowledge, this may be the first report about combination of the cellulose and this chromatography theory to separate molecules (Gordon et al., 1943). Till 1956, filter paper chromatography and electrophoresis were utilized routinely (Comer, 1956). The concept of paper or paper-like strip test was investigated in pregnancy and it was Margaret Crane who invented the first immunochromatography based pregnancy test kit in 1968 (US patent: 3579306). The studies about immunomigration by capillary action were soon initiated via antigen-antibody interaction on a porous material, e.g., filter paper, in the late 1970s and the early 1980s (Glad & Grubb, 1977; Glad & Grubb, 1978; Glad & Grubb, 1981). 2.4.5.3.1.3.2 Bioactive Filter Paper Detection The term “bioactive paper” emerged in the early 21st century with the development of paper science, biochemistry, biotechnology and microbiology and pathogen diagnostics, and was initiated by researchers in Canada with the aim of improving global public health and developing a new paper fabrication process (Pelton, 2009). Scientists explored different means to functionalize the surface of filter papers, e.g., physical adsorption and chemicals covalent modification. The functionalized filter papers enable biosensors (DNA probes, antibodies, enzymes, etc.) to bind the surface of these papers to create bioassays (e.g., pathogen detection in food and environmental sciences, Su et al., 2007). The studies about (Bio)paper-based microfluidic device were also carried out, e.g., flow channel creation (Jahanshahi-Anbuhi et al., 2012; Carrilho et al., 2009; Martinez et al., 2007) and reaction process control (Fu et al., 2010; Fu et al., 2011) and so on. In the development of bioactive paper assay, one of the biggest challenges is how to attain an immediate, sensitive and specific readout, which can be identified by naked eyes requesting no instrumentation. This challenge has been addressed in Paper II.

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2.4.5.3.2 Three Dimensional Surfaces Compared with two dimensional glass microarray or silicon chips, microbeads are ideal supports because three dimensional surfaces provide a chance to increase the concentration of oligonucleotides in the same area compared to two dimensional surfaces. There are several kinds of microbeads used nowadays. 2.4.5.3.2.1 Microbeads (Illumina): Silica Beads Three point four micron diameter Silica beads can be assembled onto different substrates to form high density bead arrays in the Illumina system: the Sentrix Array Matrix (SAM) or the Sentrix BeadChip. In SAM system, there are 96 fiber-optic bundles (1.4mm each) with 50,000 fiber-optic strands in each bundle. Every single bundle is formed into a microwell for each bead. However, in BeadChip system, microwells are created on a silicon slide via microelectromechanical technology. A single bead can be loaded in each microwell (Fan, 2005; Fan, 2006; Miller & Tang, 2009). The BeadChip can assay 1 to 24 samples at present (Illumina.com). The beads in bead arrays are randomly self-assemble to their final localization thus a “decoding” step is needed to map the bead position (Gunderson et al., 2004). ~700,000 identical capture oligonucleotides, as unique barcodes, are attached to each bead to identify every bead address (Kuhn et al., 2004). Decoding process provides quality control for intermicroarray data comparision (Bibikova et al., 2006; Miller & Tang, 2009). Bead Arrays have been used in different sides, including SNP genotying (Fan, 2003; Gunderson, 2009; Parida, 2012), gene expression profiling (Abramovitz et al., 2011; Fan et al., 2004; Kuhn et al., 2004) and DNA methylation studies (Bibikova et al., 2006; Bibikova & Fan, 2009; Triche et al., 2013). 2.4.5.3.2.2 Microbeads (Luminex): Polystyrene Beads Polystyrene beads are essentially applied in suspension bead array - a liquid phase array. The microbeads are separated based on the different ratios of their emission spectra, red (658nm emission) and infrared (712nm emission). For DNA detection in this technique, the Luminex company has developed a set of 100 fluorescent polystyrene microbeads with distinct spectral ratios of the two fluorophores. Each unique group of probes are attached to the beads with the same red-to-infrared ratio thus, in this system, 100 different samples can be assayed simultaneously in theory (xTAG; Luminex Molecular Diagnostics, Inc., Toronto, Canada). The microbeads can be detected and analyzed individually with two lasers. A 635nm laser excites the two fluorochromes and localizes beads. A 532nm laser excites report fluorochromes (phycoerythrin or PE) to analyze and quantify the hybridization. This capacity of high-throughtput detection makes the technology with microbeads attractive for clinical infectious disease detection. The first suspension bead array for infectious disease detection (Luminex, xTAG RVP) got the FDA certification in 2008 (Krunie et al., 2007; Merante et al., 2007; Miller & Tang, 2008).

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2.4.5.4 Microfluidic DNA Microarray DNA microarray technique accelerates advances of molecular biology and keeps evolving in life science due to the development of associated technologies, such as the microfluidic technology. The combination of DNA microarray and microfluidic techniques presents a method to analyze materials of minute volume/amount in a short time efficiently. Samples on picoliter level could be handled in microfluidic system (Liu et al., 2006; Wang & Li, 2011) compared with a volume of microliter level handled in DNA microarray, therefore microfluidic technique increases the target DNA concentration in microchannels. Moreover, microfluidic technique reduces the diffusion distance, therefore improves the hybridization kinetics because, in a limited time and distance, the increasing target concentration enhances hybridization chances between target DNA and probes printed on the substrate surface in the microchannels (Wang & Li, 2011). Recently, based on microfluidic technique, scientists pursue an efficient and easy parallel array hybridization on a compact disc (CD) or digital video disc (DVD) surface. This technology units microarray, microfluidic, centrifugal force and CD/ DVD, and is beneficial to develop a microfluidic DNA microarray platform even a portable lab-on-chip/DVD system (Lange et al., 2005; Madou et al., 2006; Nolte, 2009; Potyrailo et al., 2006).

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3. Specific Aims of the Studies 3.1 Paper I Activated paper surfaces for the rapid hybridization of DNA through capillary transport. Ana Catarina Araújo , Yajing Song , Joakim Lundeberg , Patrik L. Ståhl , and Harry Brumer , III. (2012). Anal Chem. 84(7): 3311-7. In pursue of a cost-effective, rapid and user-friendly candidate of microarray sup-port and establishing a forensic analysis array model to detect human and canine samples, a cellulose filter paper treatment was developed by 5 steps in this study. The 5 steps are the followings: 1. Aminated surface of cellulose filter paper 2. Functionalization of surface of cellulose filter papers with Phenylenediisothiocya-nate (PDITC). 3. Immobilization and robotic printing of synthetic oligonucleo-tides modified with amino groups by covalent bonds between amino groups and thiocyanate groups. 4. Detection of synthetic oligonucleotides/PCR products la-beled with Cy3, which are completely or partly complementary to the surface pol-ynucleotides. 5. Analysis of double strand DNA formation on the functionalized surface of filter papers with scanner. 3.2 Paper II Visual Detection of DNA on Paper Chips. Yajing Song , Gyarmati Peter , Ana Catarina Araújo , Joakim Lundeberg , Harry Brumer , III , Patrik L. Ståhl . (2013) Submitted. To simplify DNA detection further as well as lowering costs, we aimed to develop the concept of visual DNA on activated filter paper chips, which is one of the main objects in this study. Superparamagnetic beads were used to label ssDNA complementary to the printed probes on the surface of the paper chips, which was developed in Paper I. The natural brown color of the beads marked DNA detection results directly without any equipment. Moreover, for the coming commercial development, the challenge to increase the multiplexing capacity of detection on the paper chips remains the other main objective in this work.

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4 Results and Discussion 4.1 Paper I 4.1.1 Activation of Cellulose Filter Papers Cellulose is an easily available, cheap and abundant material. In pursuit of paper-based DNA detection, one important challenge is how to solve the low affinity between nucleotides and cellulose. For silicate glass slides, a general strategy is to generate amino groups on the surface of supports by aminoalkyltrialkoxysilane, such as aminopropyltriethoxysilane (APTES), followed by coupling amine-reactive functional linkers, for example, 1,4-phenylenediisothiocyanate (PDITC), which provide functional groups for the covalent immobilization of amino-modified captured targets, e.g., NH2-ssDNA (Guo, 1994). This strategy was followed in this study. Xyloglucan-NH2 (XG-NH2) aminated the surface of filter papers (Brummer H, 3rd. 2004), PDITC with two thiocyanate groups functionalized the surface of cellulose filter papers by reacting with amino groups. Synthetic NH2-oligoculeotides (ID-tag) were immobilized onto the surface of filter papers covalently by reaction between thiocyanate groups and amino groups. This work showed that PDITC was the key to functionalize the surface of cellulose. The efficiency of functionalization for PDITC in DMSO was better than that in DMF. XG-NH2 was helpful to decrease PDITC concentration to gain same intensity of fluorescence comparing to filter paper (FP) in DMSO. There was no observable intensity difference between XG-NH2-PDITC-FP group and XG-PDITC-FP group at low PDITC concentration in DMSO. 4.1.2 DNA Detection on Functionalized Papers The porous structure of cellulose filter paper produces capillary wicking ability. It drives targets to hybridize probes on surface of cellulose filter papers while paper touching the target solution. 4.1.2.1 Synthetic Oligonucleotides Fluorophore-labeled synthetic oligonucleotides complementary to the immobilized probes were used for detection. Quantitative analysis was based on measuring the fluoroscence intensity. The limit of detection in this study was proven to be ca. 0.2 pmol. 4.1.2.2 Human and Canine Samples For robotic printing, there were six features in each layout. Four of them identified four distinct PCR products, the other two were positive and negative controls. Cy3 modified 5 termini of forward primers, and ID-tag sequences were integrated into 5 termini of reverse primers where modified with biotin. After amplification and denaturation, Cy3-ssDNA-anti-ID tag was achieved to detect the immobilized ID tag sequence on the activated paper matrix. The method was specific for four different targets detection.

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4.2 Paper II 4.2.1 DNA Visualization The method in Paper I was developed parallel with this work. Superparamagnetic bead-labeled ssDNA was produced instead of Cy3-ssDNA as in paper I to base pair with the probes on the surface of cellulose filter paper chips. The color of the beads signed the readout directly without any bulk instrument, e.g., scanner, thus DNA visualization on paper chips was achieved rapidly with the capillary action of filter papers. Apart from visualizing the results by the naked eye, this assay offers quantitative measurement as well, as it was demonstrated in this study. 4.2.2 Inspiration from the Porous Structure of Cellulose Paper Chips The structure of cellulose filter paper is different from that of glass and plastic. Its porous structure endows itself a three dimensional structure, thus the surface area of filter paper is enlarged (Hong et al., 2007; Pelton, 2009), and the density of immobilized probes is increased, compared to glass and plastic. It is easier for the high density printed probes to capture target sequences when the solution with targets passes through the printed region of the paper chips rapidly by lateral flow. Meanwhile, it makes multiplex detection possible for this assay format. In this study, the average density of printed probes was increased from 2 pmol/mm2 to 3 pmol/mm2 based on the previous work ideally. 4.2.3 Multiplex Detection Enlarging feature size and increasing the immobilized probe density improve multiplex detection. In this work, in order to increase multiplexing capacity of detection on paper chips, the features per array were increased from six to ten in one array. The printing area of each feature was changed from 1mm2 to 1.7mm2 as well as increasing printed probe density. The detection was done by one, two, three and four samples. Two ways were investigated to load the detective solution containing the amplicons: the amplicons were loaded either by vertical capillary transport or by immersion. Capillary transport produced strong signals identifiable in 90 sec, while the immersion method produced weak signals in the same timeframe, supposedly because the paper pores were blocked by the solution in multiple directions. The detection was operated at room temperature. In order to ensure the accuracy of detection, there were two features in the layout to pair with the distinct moiety of each bead-ssDNA(anti-internal-probe)-anti-ID-tag target. One feature was made of synthetic oligonucleotides (ID-tag), the other one was a section of amplicon (Internal-probe). Moreover, these two probes were positioned in different row and column to reduce spatial bias. 4.2.4 Parameter Optimization The optimization of the amount of magnetic beads and amplicons, and the ideal

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printing area of filter paper region were investigated, as well as paper size and density of printed probes. 4.2.5 Future Development At present, DNA visualization was performed with magnetic beads. Ten features could be detected simultaneously; but the beads also generate background. More work can be done to increase flow rate and signal-to-noise ratio by increasing the length of filter paper chips or/and using magnetic nanobeads or golden particles or/and creating microchannels on paper chips. Nowadays, a need of an automated low-throughtput detection system has been brought up, which requests rapid, easy to use, accurate and sensitive detection and is suitable for forensic investigation and bedside clinical diagnosis (Lemieux et al., 2012). Considering visualization, sensitivity, accuracy and multiplexity, the presented filter paper chip technique could be further integrated with more advanced technologies, such as isothermal amplification (Craw & Balachandran, 2012) and microfluidic device to exploit promissing commercial applications, such as test strips or portable on-site detection device. (Wang et al., 2012; Schumacher et al., 2012).

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5 Conclusions This thesis investigates the usability of filter paper as an array surface material. A cellulose filter paper can be efficiently functionalized by XG-NH2 and PDITC in DMSO and the activated filter papers can be applied to detect DNA via fluorescence or by the naked eye with modified magnetic beads. These visualization methods show that the filter paper is a good candidate for DNA array detection. The presented technique of visual DNA detection on active filter papers has simplified DNA detection as well as reduced the associated cost; moreover, it provides the possibility to increase throughput of DNA array assays on active filter paper chips. In order to exploit the commercial potential of the presented technique, both the integration with other technologies (e.g., microfluidics devices) and the refined optimization of the parameters in this study (e.g., paper size, printing region, ways of detection) are essential. Thus, the results presented in this thesis may be helpful towards the goal to develop a portable, on-site device meeting the needs of an automated detection system (Lemieus et al., 2012).

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