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Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1091 Development of Electrical Readouts for Amplified Single Molecule Detection CAMILLA RUSSELL ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2015 ISSN 1651-6206 ISBN 978-91-554-9219-9 urn:nbn:se:uu:diva-247945 Dissertation presented at Uppsala University to be publicly examined in B41, BMC, Husargatan 3, Uppsala, Friday, 22 May 2015 at 13:15 for the degree of Doctor of Philosophy (Faculty of Medicine). The examination will be conducted in English. Faculty examiner: Associate professor Björn Högberg (Karolinska Institute, Department of Neuroscience). Abstract Russell, C. 2015. Development of Electrical Readouts for Amplified Single Molecule Detection. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1091. 46 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-554-9219-9. Molecular diagnostics is a fast growing field with new technologies being developed constantly. There is a demand for more sophisticated molecular tools able to detect a multitude of molecules on a single molecule level with high specificity, able to distinguish them from other similar molecules. This becomes very important for infectious diagnostics with the increasing antibiotic resistant viruses and bacteria, in gene based diagnostics and for early detection and more targeted treatments of cancer. For increased sensitivity, simplicity, speed and user friendliness, novel readouts are emerging, taking advantage of new technologies being discovered in the field of nanotechnology. This thesis, based upon four papers, examines two novel electrical readouts for amplified single molecule detection. Target probing is based upon the highly specific amplification technique rolling circle amplification (RCA). RCA enables localized amplification resulting in a long single stranded DNA molecule containing tandem repeats of the probing sequence as product. Paper I demonstrates sensitive detection of bacterial genomic DNA using a magnetic nanoparticles-based substrate-free method where as few as 50 bacteria can be detected. Paper II illustrates a new sensor concept based on the formation of conducting molecular nanowires forming a low resistance circuit. The rolling circle products are stretched to bridge an electrode gap and upon metallization the resistance drops by several orders of magnitude, resulting in an extremely high signal to noise ratio. Paper III explores a novel metallization technique, demonstrating the efficient incorporation of boranephosphonate modified nucleotides during RCA. In the presence of a silver ion solution, defined metal nanoparticles are formed along the DNA molecule with high spatial specificity. Paper IV demonstrates the ability to manipulate rolling circle products by dielectrophoresis. In the presence of a high AC electric field the rolling circle products stretch to bridge a 10 µm electrode gap. Keywords: Rolling circle amplification, metallization, electrical DNA detection, magnetic nanoparticles, dielectrophoresis, boranephosphonate modified nucleotides, DNA elongation Camilla Russell, Department of Immunology, Genetics and Pathology, Rudbecklaboratoriet, Uppsala University, SE-751 85 Uppsala, Sweden. © Camilla Russell 2015 ISSN 1651-6206 ISBN 978-91-554-9219-9 urn:nbn:se:uu:diva-247945 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-247945) To my family List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Göransson, J., Zardán Gómez de la Torre, T., Strömberg, M., Russell, C., Svedlindh, P., Strømme, M., Nilsson, M. (2010) Sensitive detection of bacterial DNA by magnetic nanoparticles. Analytical chemistry, 82(22):9138-9140 II Russell, C., Welch, K., Jarvius, J., Cai, Y., Brucas, R., Nikolajeff, F., Svedlindh, P., Nilsson, M. (2014) Gold nanowire based electrical DNA detection using rolling circle amplification. ACS Nano, 8(2):1147-1153 III Russell, C., Roy, S., Ganguly, S., Qian, X., Caruthers, M., Nilsson, M. Formation of silver nanostructures by rolling circle amplification using boranephosphonate modified nucleotides. Submitted IV Russell, C., Welch, K., Nilsson, M. Dielectrophoretic stretching of DNA amplified rolling circle products. Submitted Reprints were made with permission from the respective publishers. Related work by the author I Zardán Gómez de la Torre, T., Strömberg, M., Russell, C., Göransson, J., Nilsson, M., Svedlindh, P., Strømme, M. (2010) Investigation of immobilization of functionalized magnetic nanobeads in rolling circle amplified DNA coils. The Journal of Physical Chemistry B, 114(10):3707-3713. II Akhtar, S., Strömberg, M., Zardán Gómez de la Torre, T., Russell, C., Gunnarsson, K., Nilsson, M., Svedlindh, P., Strømme, M., Leifer, K. (2010) Real-space transmission electron microscopy investigations of attachment of functionalized magnetic nanoparticles to DNA-coils acting as a biosensor. The Journal of Physical Chemistry B, 114(41):13255-13262. Contents Introduction ................................................................................................... 11  Background ................................................................................................... 12  Detection of molecules ............................................................................. 12  Detection of nucleic acids ........................................................................ 13  Amplification methods ........................................................................ 13  Detection methods ............................................................................... 15  Rolling circle amplification ...................................................................... 16  Padlock probes ..................................................................................... 16  Ligation of padlock probes .................................................................. 17  Amplification during RCA .................................................................. 17  Circle-to-circle amplification............................................................... 18  Desirable properties for a detection method............................................. 18  Nanotechnology ....................................................................................... 20  Biocompatible magnetic particles........................................................ 20  DNA as a building block ..................................................................... 21  DNA metallization ............................................................................... 22  DNA manipulation............................................................................... 23  Biosensors ................................................................................................ 24  Electrical biosensors ............................................................................ 25  Point-of-care sensors ........................................................................... 25  Present investigations.................................................................................... 27  Paper I. Sensitive detection of bacterial DNA by magnetic nanoparticles............................................................................................. 27  Paper II. Gold nanowire based electrical DNA detection using rolling circle amplification. .................................................................................. 29  Paper III. Formation of silver nanostructures by rolling circle amplification using boranephosphonate modified nucleotides. ............... 30  Paper IV. Dielectrophoretic stretching of DNA amplified rolling circle products. ................................................................................................... 32  Perspectives .................................................................................................. 34  Acknowledgements ....................................................................................... 36  References ..................................................................................................... 39  Abbreviations 2D A AC ATP bp-dNTP C C2CA DEP DNA dNTP dsDNA ELISA G LAMP LCR LOC M / Mn+ MDA μ-TAS NA NAD+ NASBA NAT Ω PCR PEG PLA POC POC-NAT QCM qPCR R RCA RCP RNA SAM 2-dimensional Adenine Alternating current Adenosine triphosphate Boranephosphonate modified nucleotides Cytosine Circle-to-circle amplification Dielectrophoresis Deoxyribonucleic acid Deoxynucleotide triphosphate Double stranded deoxyribonucleic acid Enzyme linked immunosorbent assay Guanine Loop-mediated isothermal amplification Ligase chain reaction Lab-on-chip Metal / metal ion Multiple displacement amplification Micro total analysis system Nucleic acid Nicotinamide adenine dinucleotide (oxidized form) Nucleic acid sequence-based amplification Nucleic acid testing Ohm Polymerase chain reaction Polyethylene glycol Proximity ligation reaction Point of care Point of care nucleic acid testing Quartz crystal microbalance Real time quantitative polymerase chain reaction Reducing group Rolling circle amplification Rolling circle product Ribonucleic acid Self assembled monolayer SDA SEM SPR SQUID ssDNA T Strand displacement amplification Scanning electron microscope Surface plasmon resonance Superconducting quantum interference device Single stranded deoxyribonucleic acid Thymine Introduction Molecular diagnostics has matured as a field during the last decades resulting in a large increase in established molecular techniques and readouts. The field continues to expand with new discoveries in nanotechnology and recent advances in microfluidics and miniaturization. Nanotechnology opens for new opportunities for molecular assays with novel readout methods and novel applications that match increasingly sophisticated molecular tools. Techniques able to detect and identify molecules of interest with high specificity are still required despite the large number of molecular assays developed. This thesis describes the development of electrical readouts for amplified single molecule detection. The thesis discusses detection of molecules, more specifically nucleic acids, various molecular tools and detection methods used to detect these, with a focus on rolling circle amplification (RCA). The thesis continues with the use of nanotechnology in molecular diagnostics before demonstrating the use of rolling circle products (RCPs) in the development of electrical readouts for amplified single molecule detection. 11 Background Detection of molecules Biomolecules such as DNA, proteins and metabolites are important indicators of our health status. These molecules are biomarkers whose presence can aid in diagnosis of disease and discovery of abnormalities and permit monitoring of health. The selection of relevant biomarkers is thus of utmost importance for achieving accurate diagnosis. Not only is the inclusion of the right biomarker important but also the molecular tools used for diagnosis. The critical step in any diagnostic assay is to recognize a specific biomarker and exclude others in a complex matrix. With sensitive and highly selective molecular tools, accurate diagnosis can be achieved at an early stage of disease and thus hopefully an effective treatment can be prescribed. Proteins and nucleic acids have been extensively researched resulting in a diversity of molecular assays. The most widely used are the classic enzymelinked immunosorbent assay (ELISA) [1] for protein detection and the polymerase chain reaction (PCR) [2, 3] for nucleic acid detection. Both protein biomarkers and nucleic acids (NA) are widely used in medical diagnostics. With the available databases of genomes, sequence mutations and variations of known diseases, it is now possible to achieve large scale analysis obtaining more detailed information leading to more targeted treatments. Infectious diseases, caused by viruses or bacteria, were and still are commonly diagnosed either by microbial culturing or microscopy [4]. These methods are not very sensitive or specific and are usually slow, which can lead to delayed diagnosis [4]. Protein and NA-based (viral DNA or RNA) methods for infectious diagnostics can provide detection at an early stage and with a shorter assay time [5]. NA-based methods also enable single nucleotide variant analysis for determining antibiotic resistance, especially important with the growing number of antibiotic resistant strains of bacteria. In non-infectious diseases such as cancer or genetic disorders, NA-based diagnostics are used to detect genetic mutations or genome alterations associated with the disease. This includes circulating tumor DNA in the blood [6]. The prognosis is quite poor for some cancers due to lack of efficient diagnostic methods for early detection [7, 8]. Protein biomarkers have been and are still used for early detection, however not all cancers excrete specific proteins that can be used as biomarkers at an early stage [8]. NA-based detection of cancer biomarkers is extremely challenging due to only a few cop12 ies of NA analytes from the tumor present per ml of blood. Yet, the possibility of detecting biomarkers at low concentrations in the early stages of cancer can lead to more successful treatments [7]. Detection of nucleic acids Interest in detecting nucleic acids has rapidly increased during recent decades. Nucleic acids are difficult to detect due to their small size and usually present in low concentrations. Early nucleic acid based detection methods were typically hybridization based and performed in solution [9] or on solid support [10] using labels such as fluorophores or radioisotopes. The breakthrough in nucleic acid detection appeared with the discovery of DNA amplification, where it was shown that a low number of DNA targets could be amplified thousand fold. Amplification methods The groundbreaking technology for DNA amplification is the polymerase chain reaction (PCR), developed in the 1980s [2, 3]. There are several steps involved in PCR which are cycled for achieving several magnitudes of increase in the amount of target. PCR relies on an initial denaturing step to separate the double stranded target permitting annealing of two primers, one for each strand. Extension of the primers by polymerase copies the target strand two fold and results in two new copies from each target. These steps, requiring different temperatures, are cycled multiplying the amount of target by a factor of two after each cycle, resulting in an exponential amplification. Initially, PCR required a separate handling step for post-labelling but with the introduction of spectrofluorometric thermal cyclers it became possible to detect the formed product during amplification. Real time PCR [11] using real time labelling of fluorescent markers made it possible to achieve faster and high throughput assays. With molecular beacons [12] and TaqMan probes [13] for real time quantitative PCR (qPCR) [14] the specificity of the assay was significantly increased. PCR has revolutionized NA based diagnostics and will probably continue to be one of the main NA based techniques due to its excellent sensitivity and specificity. However, PCR is complex, needs a thermal cycler, is sensitive to contaminants, expensive and difficult to multiplex. PCR is also not easily transferred to point-of-care (POC) diagnostics. Since the development of PCR many amplification techniques has emerged such as: 13       Nucleic acid sequence-based amplification (NASBA) [15] Strand displacement amplification (SDA) [16, 17] Loop-mediated isothermal amplification (LAMP) [18] Ligase chain reaction (LCR) [19] Rolling circle amplification (RCA) [20, 21] Multiple displacement amplification (MDA) [22, 23]. Most of these techniques are isothermal removing the need for a thermal cycler as in PCR. NASBA [15] was developed as an alternative amplification method to reverse transcriptase PCR (RT-PCR), amplifying 100-250 bases long RNA targets [24]. The main advantage is the fast reaction kinetics relative to PCR without the need for a thermal cycler. SDA [25] is an isothermal amplification method resulting in exponential amplification of the target. The main limitation of this technique is its inability to amplify long target sequences [24]. LAMP, on the other hand has higher specificity, efficiency and rapidity compared to both NASAB and SDA [18]. Unlike NASAB, SDA and LAMP, LCR is a ligase dependent amplification method that amplifies the target molecule by the ligation of two complementary pairs of synthetic oligonucleotide primers, hybridized in close proximity to each other on a target DNA strand [26]. Like PCR, this method requires a thermal cycler for annealing primers to the target strand and for strand separation of the newly formed DNA fragment and target strand. The requirement of a ligation reaction provides higher specificity compared to PCR [27]. RCA is a highly specific isothermal amplification technique relying on the DNA polymerase phi29 and a ligation step [20, 21]. Unlike the previous mentioned amplification techniques, RCA allows localized amplification [28] and the synthesis of a continuous long single stranded DNA molecule. This amplification method is the technique used in this thesis and is discussed further in the coming section. Phi29 DNA polymerase is also used in isothermal MDA [23], commonly used for amplification of total genomic DNA via random primers. All of the above mentioned methods have advantages and disadvantages. The key is to choose the appropriate molecular tool for the required task. For example, large scale analysis and genotyping require highly sophisticated molecular tools able to discriminate a large set of sequences with single nucleotide variants. The detection or readout methods are almost as important as the molecular tool with the ability to aid in sensitivity, precision and multiplexability. 14 Detection methods A widely used method for nucleic acid detection is fluorescence. Fluorescence offers high sensitivity and a wide range of ready available fluorophores with various lifetimes, stability, sensitivity and excitation and emission properties. Different fluorescent probes and techniques have emerged such as TaqMan probes and molecular beacons for increased specificity. TaqMan probes are synthetic oligonucleotides containing a fluorophore in one end and a quencher in the other. As mentioned earlier, these are commonly used in qPCR where a fluorescent signal is achieved through the release of the fluorophore as the oligonucleotide is digested by the polymerase [13]. Molecular beacons are hairpin shaped synthetic oligonucleotides with a fluorophore in one end and a quencher in the other. Upon hybridization, the proximity of the quencher to the fluorophore is removed, resulting in a fluorescent signal from the fluorophore [12]. Fluorescence has further increased in sensitivity with the development of brighter and more stable probes such as quantum dots. Advances in microscope technology has further led to both simpler and more advanced instrumentation enabling easy measurements of fluorescence and imaging with nanometer resolution [29]. Despite this, fluorescence still needs advanced and expensive equipment if not to compromise in sensitivity. Other common readout methods are array-based, and with the development of microarrays [30] it is possible to produce low and high density arrays containing a few hundred up to millions of spots. These are very suitable for multiplexing and genotyping but are limited in sensitivity and specificity [31]. The labelling used in combination with arrays are typically fluorescence, silver precipitation [32] or other colorimetric techniques [33]. Electrical methods have gained a lot of research interest in recent years. There are several types of electrical readout. Many detect a change in conductance either directly related to the DNA or through a label. The majority of these are based on DNA hybridization where a change in electrochemical properties is monitored directly on an electrode [34] or nanowire [35]. The signal is generated either with or without the use of a label [34, 36]. Other electrical detection methods rely upon nanoparticle amplification. Park et. al. [37] and Urban et. al. [38] demonstrate an electrical readout based on DNA hybridization and metal enhancement. There, oligonucleotide functionalized gold nanoparticles hybridize in an electrode gap immobilized with capture probes. The gold particles are enhanced by metal deposition forming a continuous metal layer closing the circuit and giving rise to an electrical signal. The advantage of electrical detection is the ease of use, not requiring any expensive or advanced instruments. Rapid advances in nanotechnology and miniaturization also permit electrical detection to become an alternative method to fluorescence-based detection methods, rivaling in size, speed, sensitivity and specificity. With the ability to manufacture large arrays of 15 small electrodes, multiplexing and high throughput becomes possible on a portable scale [39]. Less common methods increasing in usage are surface plasmon resonance (SPR) [40] and Raman-based spectroscopy [41, 42]. SPR measures changes in the refractive index caused by the interaction of the molecule at the surface while Raman-based spectroscopy looks at the vibrational output of the molecule. Rolling circle amplification RCA was developed in 1998 [20, 21] and relies upon a hybridization and ligation event of a padlock probe prior to amplification. Figure 1. Rolling circle amplification. (I) A single stranded synthetic DNA sequence forms the padlock probe. (II) Upon recognition of a target, the padlock probe circularizes and hybridizes in a head-to-tail fashion on the target. (III) The padlock probe forms a closed circle in the presence of a ligase. (IV) Continuous amplification using phi29 DNA polymerase produces a long single stranded DNA molecule with tandem repeats of the padlock probe. Padlock probes A padlock probe is a single stranded synthetic DNA sequence comprised of two target complementary ends and a non-target complementary backbone [43]. The target complementary ends are 15-20 nucleotides long and the backbone around 40-50 nucleotides long. Upon recognition of two adjacent target regions the padlock probe becomes circularized, hybridizing in a juxtaposed position. For amplification, the circularized padlock probe needs to be closed which is achieved by the use of a DNA ligase. The specificity of 16 padlock probe is thus achieved through the requirement of two adjacent oligonucleotide hybridization events on a target strand and a DNA ligation reaction [43, 44]. Padlock probes, also known as molecular inversion probes, have further developed to achieve detection of DNA with partially known sequences for SNP genotyping [45] or in situ sequencing [46]. This is achieved through near but not completely adjacent hybridization of the two target complementary ends of the padlock probe. The resulting gap of one to a few nucleotides in length is then filled using DNA polymerase, i.e. gap filling, closing the circle and enabling amplification. Ligation of padlock probes DNA ligases seal the 3’OH and 5’PO4 ends of a nick resulting in the formation of a phosphodiester bond. When combined with padlock probes, seals the padlock probe and forms a closed circle. DNA ligases are grouped into two families discriminated by the requirement of ATP or NAD+ to drive the formation of the phosphodiester bond [47]. The ligation reaction discriminates between single nucleotide differences preventing formation of circular padlock probes when there is a mismatch [48]. This allows for multiplexing to interrogate large sets of DNA variants without cross reactivity [45]. Amplification during RCA When circularized, padlock probes are amplified in an isothermal RCA reaction catalyzed by phi29 polymerase generating long tandem-repeated singlestranded DNA molecules [20, 21]. Phi29 polymerase has a strong strand displacement activity which permits efficient amplification. The 3’ to 5’ exonuclease activity also facilitates highly accurate DNA synthesis. The rate of RCA is about 90 kb DNA per hour in a highly processive DNA polymerization reaction, making a 45 µm long single stranded product containing approximately 1000 repeats of the padlock probe. These products tend to collapse into 700 nm sized dense globular structures [49]. The backbone of the padlock probe is typically designed to contain a detection sequence and/or a restriction sequence for labelling or further amplification. The presence of these detection sites permits the generated rolling circle product (RCP) to bind roughly 700 labelled probes [50] producing a strongly fluorescent molecule with a high signal to noise ratio. These RCPs represent a single molecule making digital detection possible [51]. Alternative detection probes such as magnetic beads [52] and gold nanoparticles [53] have been used for other readout formats such as volume-amplified magnetic nanobead assays [54] and surface enhanced Raman scattering spectroscopy [55]. 17 Circle-to-circle amplification RCPs can be digested into monomers via the designed restriction sites on the padlock probes. The monomers are in turn circularized and amplified in a new RCA reaction; resulting in a circle-to-circle amplification (C2CA) [56]. Each generation of RCA results in a linear quantification. Unlike PCR C2CA is not product inhibited and can yield 100-fold higher concentration of monomer products [56]. Higher sensitivity can be achieved with the same selectivity and ability to multiplex as the padlock probes using C2CA. Figure 2. Circle-to-circle amplification. (I) Annealing of synthetic restriction oligonucleotides to RCP permits digestion by restriction enzymes resulting in (II) monomers. (III) – (IV) Circularization and ligation of monomers followed by a second rolling circle amplification step. Desirable properties for a detection method An ideal diagnostic assay should have;  Single molecule sensitivity  Exquisite specificity  Linear response  Wide dynamic range 18   High quantitative precision Multiplex format Sensitivity is important for detection of rare events and target molecules present in very low concentrations. Sensitivity of a detection method can be increased either by target amplification or signal amplification. Target based amplification occurs when the target is amplified using an amplification technique such as PCR [14] or RCA [20]. Signal based amplification occurs when the signal is directly increased in proportion to the concentration of target. This is usually achieved by a reporter molecule such as fluorescence or by an enzyme as in ELISA [1]. RCA can be used for both target amplification as in C2CA and for signal amplification resulting in a strongly fluorescent molecule. Target based amplification is considered more sensitive than signal based amplification due to increasing the amount of target rather than the signal. An important aspect is therefore to use stringent molecular tools as not to increase competing contaminants. Increasing the signal can lead to increased background noise. It is therefore important to use signal amplification techniques that do not increase the background noise. Specificity is crucial in molecular and diagnostic assays. A molecular tool should be able to distinguish a target from similar molecules in a complex matrix. This becomes increasingly more difficult when large scale analysis is required. Specificity is achieved through the use of sophisticated molecular tools. Specificity by excluding others is equally important as the specificity in selecting the right target, especially for solid phase readout methods such as electrical, SPR or quartz crystal microbalance (QCM). For these methods surface blocking is crucial, preventing adsorption of nearby contaminants and thereby avoiding interference with the signal. Concentrations of target molecules can span several orders of magnitudes, ranging from individual molecules to high abundance. It is therefore desirable to use an assay that works over a wide dynamic range, able to detect molecules in very few numbers and those in high concentration. This is usually the case in early detection of infectious diseases when the number of NA analytes is extremely low or in an advanced stage of illness when the presence of NA analytes is high. For some routine applications a simple binary answer is enough [57]. In other applications a quantitative response is required, e.g. when monitoring therapeutic response or acquiring information regarding the disease progression [58]. To achieve quantitative measurements it is preferable to have a detection method that gives a linear response to the amount of detected target. A detection method needs to have good reproducibility. For quantitative detection methods reproducibility means having high quantitative precision. 19 With increasing demands on molecular diagnostics for large scale analysis and with the need for high throughput it becomes important for detection methods to work in a multiplex format. This is possible with the use of highly specific molecular tools and the ability to manufacture platforms with nano- and microscale features. Nanotechnology The emergence of nanotechnology in life science with the introduction of gold nanoparticles [59], has taken molecular diagnostics to a new level. Molecular diagnostics has seen an increase in new materials being used both as tools and for signal amplification. Advances in manufacturing processes and the “bottom-up” approach have led to the ability to produce devices with nano- and microscale features, permitting construction of novel readouts and sophisticated Lab-on-chip (LOC) devices. Nanomaterials such as gold nanoparticles [60], carbon nanotubes [61, 62], magnetic particles [52] and quantum dots [63] are playing an important role in the uses of nanotechnology in medical diagnostics. Nanomaterials range in size from 1 nm up to 100 nm giving them a large surface to volume ratio. These have been tailored to bind biomolecules while maintaining their overall structural robustness. The most attractive feature however is the ability to modify their physical properties [64]. This gives them interesting optical and electrical properties which can improve detection of biomarkers by signal amplification and also improve performance of a sensing device [65]. The ability to change size, shape and composition of metal nanoparticles and quantum dots can produce nanomaterials with specific and narrow emission and absorption bands making these highly suitable for multiplexing [64]. Nanomaterials are also used for producing highly conductive nanowires [66]. Biocompatible magnetic particles The use of functionalized magnetic particles in molecular and diagnostic assays has increased with more readily available magnetic particles of different compositions, ranging in sizes from tens of nm up to several microns. Such particles are now extensively used due to their useful magnetic properties and biocompatibility. Applications range from concentration, target probing [67], purification and separation to use in biosensors [52], medical diagnosis and therapy [68] and drug delivery systems [69]. The use of magnetic particles introduces simplicity to the assay by its easy ability to manipulate solutions through probing, concentration and separation. 20 Paper I demonstrates the use of biocompatible magnetic nanoparticles in both probing and amplification but also in a magnetic nanoparticle-based sensor. DNA as a building block DNA is a powerful tool in nanotechnology due to its unique properties. The great attraction is its simplicity, specificity, the ability to easily synthesize desired sequences and the long term stability of the molecule. Improvements in synthesis techniques have also led to the production of more accurate sequences. DNA is a long polymer with repeating units of nucleotides. The structure of DNA is a double helix consisting of two complementary strands of oligonucleotides. Each strand consists of an alternating sugar and phosphate backbone and the four bases guanine (G), adenine (A), thymine (T) and cytosine (C). Through hydrogen bonding, G pairs only with C and A only with T, forming the double stranded DNA molecule. Despite containing only four bases, high specificity is achieved through unique sequences and base pairing to a complementary sequence. For these reasons DNA is an ideal building block, able to assemble structures with nanoscale features, and as a means for data storage [70]. Figure 3. The structure of DNA consists of a backbone of alternating sugar and phosphate groups, and four bases; adenine, thymine, cytosine and guanine. 21 DNA nanotechnology was pioneered by Nadrian Seeman with the introduction of DNA junctions and lattices [71]. DNA cubes [72] and 2D arrays [73] was soon followed by more complex structures produced by DNA origami [74, 75]. DNA nanotechnology is now moving from design and structure to focus on function [76]. DNA nanotechnology is a wide field with applications spanning from biological understanding to electronic and mechanical, with already developed uses in nanomechanics [77-79], nanoelectronics [80, 81], potential drug delivery systems [82] and tools for biological understanding such as aiding in structure analysis of biological molecules [83]. DNA itself is only slightly conductive [84] and when used in nanoelectronics, DNA is used to organize metallic nanoparticles to achieve nanowires or other 2D arrays with significantly lower resistance than the DNA itself [37]. The work in this thesis has utilized DNA as a building block for nanoelectronics, constructing 2D nanowires. DNA metallization Braun et al [85] showed in 1997 that DNA can be metallized to create an electric circuit via self-assembly and self-recognition to form a conductive silver wire. Most metallization studies have been performed on dsDNA using three different approaches, illustrated in Figure 4. Early metallization techniques were based on the first approach. The common steps for this approach are the formation of a DNA-metal complex followed by the formation of metal seeds. The metal seeds are then further enhanced by a metal deposition step leading to the formation of a continuous metal wire. The formation of the DNA-metal complex can be formed through interaction with either the backbone or base pairs. Metal ions bind to the backbone through the attraction of the negatively charged phosphate groups and the positively charged metal ions, while metal ions bind to the base pairs through the interaction of site specific metal complexes [86]. The resulting DNA-metal complexes are then reduced by a reducing agent such as sodium borohydride or hydroquinone to form the metal seeds. The disadvantage of this approach is the risk of unspecific metallization. The second approach avoids the risk of unspecific metallization by the use of reducing groups introduced on the DNA backbone. Keren et. al. [80], Burley et. al. [87] and Roy et. al. [88] report of strand specific metallization using modified phosphate groups on the backbone. Metal seeds used for further enhancement are formed along the DNA by introducing a metal ion solution. The third approach takes advantage of the ability of DNA to selfassemble. Gold or silver nanoparticles immobilized with complementary strand specific sequences can be used to assemble a network or chain of nanoparticles [89, 90]. These metal nanoparticles can then catalyze further reduction of metal in an enhancement step. 22 Formation of nanowires has been reported using silver [85], gold [66, 91] and palladium [92, 93]. Of note, for all three approaches, the enhancement step does not require the use of the same metal as the seed. Figure 4. Metallization of DNA. (I) Binding of metal ions (Mn+) through either (a) base pairing or (b) backbone. Reduction of the metal ions by reducing agents forms the corresponding metal nanoparticles. (II) Metal nanoparticles are formed along the DNA by reducing groups (R) present along the backbone. (III) Assembly of a network or chain of metal nanoparticles via self-assembly. In all three approaches, the metal nanoparticles are further enhanced by an enhancement step to form a continuous metal wire. Paper II takes advantage of the third approach while paper III explores the second approach by incorporating modified nucleotides during RCA to permit specific metallization along the resulting RCP. DNA manipulation The ability to manipulate DNA on a nano- and microscale is important in the field of nanotechnology. It enables the intrinsic assemble of DNA networks with applications in nanomechanics and nanoelectronics. DNA manipulation is also important in molecular diagnostics where it can improve detection of DNA, permit faster reaction kinetics, increase sensitivity and allow design of simple assays easily transferred to POC devices. Early DNA manipulation studies have improved our understanding of its physical and mechanical properties such as elasticity [94] and strength [95]. This was mainly achieved by the use of optical tweezers [96], AFM probes [97] and magnetic tweezers [98]. 23 A lot of DNA manipulation has focused on elongation. Bensimon et. al. [99] showed that DNA can be stretched using the receding meniscus principle. There, DNA is stretched as an air water interface is moved along a substrate leaving the tethered DNA stretched and firmly attached to the surface. Since then many techniques for DNA stretching have been developed, e.g. nanochannels [100], DNA curtains [101] and various mechanical manipulations like flow [102], magnetic [103] and optical tweezers [96]. The disadvantage of optical tweezers is the requirement of advanced equipment in the form of a laser beam set up. Flow usually requires a high force and flowrate if not used in combination with a tethered polystyrene bead or nanochannels. The flowrate close to the surface of a channel is almost zero leaving tethered DNA unaffected by the flow. Stretching in flow is dependent upon ionic strength of the solution, conformation of the DNA and the force required to stretch the DNA [104]. For single stranded DNA these parameters become much more important [104]. When using low ionic strength, stretching can be observed using less force. At low ionic strengths the electrostatic forces are more apparent while at higher ionic strength the forces involved in secondary structures dominate [104]. An easier way to manipulate DNA is through the use of the electronic properties of the DNA molecule. Electrokinetic manipulation such as dielectrophoresis (DEP) has been used for elongation [105, 106], trapping [107, 108], improving hybridization kinetics [109], specific immobilization on gold surfaces [110], separation and purification [111, 112]. Electrokinetic techniques are contact free and use the induced motion of the molecules caused by the electric field in a solution. DNA can be elongated through the motion of the molecule along the electric field gradient in a non-uniform electric field. This motion is caused by the induced dipole along the DNA in response to the alternating current (AC) electric field. Manipulation of rolling circle products in paper II is based on the receding meniscus principle; while paper IV explores manipulation of RCPs using DEP. Biosensors The goal of any developed molecular tool is to result in an application. For some this means the construction of a biosensor for molecular diagnostics. The basic building blocks of any biosensing platform are: a biorecognition element that differentiates the target molecules in the presence of other molecules, a transducing element that converts the biorecognition event into a measurable signal, and a readout platform which converts the signal into a readable form [113]. A biosensor is a small self-sufficient device able to provide analytical information using a biorecognition element [114]. Biosen24 sors can be electrical, optical or mass sensitive. This thesis will only briefly discuss electrical biosensors and the development of POC sensors. Electrical biosensors The interest in electrical DNA detection in biosensors has increased tremendously during the last few years. Many biosensors are based on electrochemical DNA chips [39], developed aiming to become simple, fast and inexpensive devices for diagnostic applications. Generally DNA biosensors rely on oligonucleotide probes immobilized on an electrode and upon hybridization of a target or change in conformation, there is a change in current [39] with or without the use of a label for signal amplification [115]. This means that the surface chemistry of the electrodes is extremely important [116].The disadvantages of these DNA biosensors are the lack of efficient hybridization of the target and unspecific adsorption which can result in false positives. Hybridization efficiency can nonetheless be improved by the use of an electric field [117]. Another type of biosensor is based upon the construction of 1D nanostructures of conducting polymer wires [118]. Electrical biosensors are extremely attractive due to their high sensitivity, small scale, capability of multiplexing and easy readout at low cost. Another branch of electrical biosensor is magnetic biosensors. Substrate based magnetic biosensors are based upon binding of a target in the presence of a magnetic field resulting in a change in electrical resistance [119]. Substratefree magnetic biosensors are based upon magnetic measurements of the Brownian relaxation time [120]. Changes of the hydrodynamic size distribution upon binding of the target can thus be monitored. Magnetic biosensors are attractive due to their high sensitivity, portability and electronic readout [121]. Point-of-care sensors POC sensor devices can be easily transported to the patient for analytical testing [122]. Common POC sensors such as the classic home pregnancy test use the lateral flow immunoassay. POC sensors are rapidly becoming more sophisticated with more integrated features such as sample preparation and complex multi-step assays. The prerequisite is the use of microfluidics to enable precise control of sample, buffer and reagent flow. Current nucleic acid testing (NAT) devices are complex and not suitable for POC [123]. The biggest challenge in making POC-NAT devices is the small sample volumes combined with the extremely low concentration of analyte. Other challenges include integration of sample collection, amplification, hybridization and detection on a microfluidic platform [124] producing LOC or micro total analysis systems (µ-TAS). The benefits of LOC and µTAS are: limited reagent use, faster sample analysis, increased sensitivity, portability, automation, low cost and user friendliness. When combined with 25 an electrical readout they become potentially very suitable for POC diagnostics in clinical settings and for developing countries. 26 Present investigations Paper I. Sensitive detection of bacterial DNA by magnetic nanoparticles. Introduction There is a need in infectious diagnostics for inexpensive readout methods with a fast assay time. This is especially important for clinics with a constrained budget. Diagnosis of infectious diseases is often based on the cultivation of bacteria which is very slow, resulting in delayed response back to the patient. Optical based methods such as PCR are fast but these require advanced and expensive equipment and are most often sensitive to contaminations. Magnetic biosensors are an alternative readout which provides high physical and chemical stability and a potentially low cost production of magnetic particles. Aim of study The aim of the study was to demonstrate detection of clinically relevant genomic DNA using a magnetic nanoparticle-based substrate-free sensor technique. This technique is based upon the Brownian relaxation principle. Magnetic nanoparticles rotate in the presence of an AC electric field and can therefore be characterized by the Brownian relaxation time. Upon binding to target molecules the hydrodynamic size of the magnetic nanoparticles increases resulting in a change in their Brownian relaxation response. This results in a decrease in the measurable Brownian relaxation frequency. The aim was to use RCPs as target molecules producing a large decrease in the Brownian relaxation frequency. 27 Brownian relaxation frequency Normalized magnetic response II Normalized magnetic response I Brownian relaxation frequency Figure 5. Schematic illustration of the normalized magnetic response of the magnetic nanoparticles to the Brownian relaxation frequency. A decrease in the amplitude of the high frequency peak for the free beads can be observed when measuring before (I) and after (II) binding to target molecules. Findings and conclusions RCPs were prepared using a solid phase-based molecular probing and amplification protocol. Micrometer sized magnetic beads were used to capture targets in an excess of padlock probes. Unbound padlock probes could then be removed to permit efficient solid phase amplification by RCA. The bound RCPs were released upon monomerization, resulting in a new generation of padlock probes. A second RCA step was then performed, ensuring an increase in the amount of 2nd generation RCPs and thereby increasing sensitivity. The presence of RCPs was monitored by the use of a superconducting quantum interference device (SQUID). Magnetic nanoparticles were functionalized with complementary strands of oligonucleotides able to hybridize to the RCPs. Each RCP was able to hybridize to several magnetic nanoparticles. Upon binding of the much larger RCP target, the hydrodynamic volume of the magnetic nanoparticles increased substantially, resulting in a large decrease in the relaxation frequency. The concentration of bacterial genomic DNA could consequently be monitored by a decrease in amplitude of the Brownian relaxation peak of the free beads. This paper demonstrates the first use of a volume-amplified magnetic nanobeads detection assay to detect clinically relevant genomic DNA. As few as 50 bacterial genomic DNA from Escherichia coli could be detected. At the time of writing of this paper, the method required advanced and expensive equipment and trained personnel for data analysis. The method was also sensitive to contaminants. Since then, the method has developed and a much simpler device can now be used [125]. The method shows large potential for automation with the further development of a miniaturized on-chip magnetic field sensor [126]. 28 Paper II. Gold nanowire based electrical DNA detection using rolling circle amplification. Introduction Current biosensors are not sensitive enough for detection of analytes present in very low concentrations e.g. in early detection of infectious diseases, motivating a demand for sensitive, inexpensive and fast biosensors suitable for point-of-care diagnostics. RCA is a highly specific amplification technique producing long single stranded DNA. The amplification technique allows localized amplification and proceeds at a constant temperature of 37C. The field of molecular diagnostics has seen an increase development of electrical readouts for NA-based diagnostics. Electrical readouts provide low cost, easy interpretation of signal and a potential to form multiplexable platforms suitable for POC. Lately DNA is being utilized for nanoelectronics with potential in molecular diagnostics. The poor conductive properties of DNA have led to a number of metallization studies, which have seen the construction of metallized DNA wires using gold, silver and palladium. Combining the specificity of RCA with the ability to form conductive wires would permit a technique and readout method suitable for POC diagnostics. The method would not suffer the same weakness of signal interference by unspecific adsorption of contaminants as other NA-based electrical readouts. It would also potentially allow increased sensitivity compared to other 2D array based metal nanoparticle enhancement methods, as one metallized RCP could potentially close the circuit and give rise to a signal. Aim of study The aim of the study was to demonstrate electrical detection of RCPs by first forcing them to stretch and bridge an electrode gap and secondly upon metallization form a low resistance circuit. Findings and conclusions Prior to forming conductive wires, the study focused on forming an optimized self-assembled monolayer (SAM). For this, a backfiller was used to control the density of the surface primer and to extend it into the solution. Two polyethylene glycol (PEG)-based backfillers were investigated, containing either a hydrophobic or hydrophilic head group. The use of a PEG backfiller not only improves the SAM but also aids stretching of the RCPs depending on the nature of the head group. A hydrophobic head group was seen to assist stretching. RCPs were stretched using the receding meniscus principle. An air/liquid interface was moved by air flow stretching the RCPs and leaving them firmly attached to the substrate. Stretching was observed through the use of fluorescently labelled complementary oligonucleotides. Oligonucleotide func29 tionalized gold nanoparticles could then be aligned along the ssDNA through hybridization. These were used as seeds for further metal enhancement using either silver or gold solutions, resulting in the formation of silver or gold nanowires. Silver enhancement caused increased background compared to gold enhancement while distinct nanowires could be seen using the latter. During metallization, the resistance was monitored every 5 minutes using a probe station. After 20 minutes of metallization the resistance of the circuit dropped noticeably, from GΩ to kΩ for silver and to Ω for gold, producing a readout with very high signal to noise ratio. Silver enhancement tends to form separate silver agglomerates producing a wire with higher resistance compared to gold. Gold is a softer metal, and fuses together to form a more homogeneous wire with lower resistance. Figure 6. SEM image of a gold nanowire produced by RCA bridging an electrode gap and closing the circuit. Scale bar is 1µm. The paper also demonstrates an electrical signal upon detection of specific target DNA sequence in the form of Escherichia coli genomic DNA. The method shows potentials having demonstrated a nearly 10 orders of magnitude in signal to noise ratio. A large signal to noise ratio is an essential feature for achieving a highly sensitive assay. The hope is to utilize this potential to achieve a sensitive and specific biosensor suitable for POC. Paper III. Formation of silver nanostructures by rolling circle amplification using boranephosphonate modified nucleotides. Introduction RCA is a strongly emerging molecular tool for molecular diagnostics using various readouts e.g. fluorescence, optical, magnetic and electrical. Combining metallization with RCA would allow multi-modal detection including optical and electrical and for some applications this would be an advantage. Roy et. al. [127] has previously reported on a novel metallization technique able to form silver nanoassemblies with high spatial resolution. This technique uses boranephosphonate modified nucleotides (bp-dNTPs) as reducing agents for metallization. One of the oxygen atoms on the phosphate is replaced by a borane group. Metal ions such as silver, gold and platinum are 30 thus reduced by the borane group to their corresponding metal nanoparticle. Other metallization techniques such as metal ions bound to the backbone of DNA reduced by reducing agents, or hybridized functionalized nanoparticles have shown limitations in spatial resolution and in interparticle repulsion. Combining a specific metallization technique with RCA would allow efficient construction of more homogeneous gold nanowires produced via RCA. Aim of study The aim of the study was to explore the incorporation of boranephosphonate modified nucleotides into RCPs using phi29 polymerase and investigate the metallization of the resulting RCPs containing boranephosphonate internucleotide linkages. Findings and conclusions The rate of RCA using bp-dNTPs was determined using two independent methods. The first method was an indirect method where fluorescence digitally quantified the amount of RCPs formed. RCPs tend to collapse into globular shaped molecules that when labelled with fluorescence appear as bright dots. These can thus be counted when passing a laser in a microfluidic channel. The second method was a direct method using real time monitoring of RCA with SYBR Green II, an intercalator dye which only fluoresces upon binding to ssDNA. The amount of fluorescence can therefore be monitored using a real time PCR machine as the amount of bound dye increases with the growing RCPs. The RCA rates obtained for the four modified nucleotides using both methods showed similar outcomes. A slight decrease in rate was seen for G, T and A and a slight increase was seen for C resulting in an average of a 1.4fold decrease in incorporation rate. The paper also demonstrates enzymatic synthesis of an all boranephosphonate internucleotide linked long DNA molecule. The RCA rates were monitored using both methods when more than one natural dNTP was exchanged for bp-dNTPs. The results show a decrease in amplification rate as the number of exchanged nucleotides increase. The rates were however not estimated as these are also dependent on the sequence. To note is the formation of an all boranephosphonate linked long DNA molecule (>100 bases long) when all four natural dNTPs were exchanged. Metallization using a silver nitrate solution of RCPs when all four natural nucleotides were exchanged for bp-dNTPs saw the formation of well-defined silver nanoparticles along the DNA molecule. Similar metallization was observed when three natural nucleotides were exchanged. In contrast, when only two nucleotides were exchanged large silver nanoparticles with an irregular distribution were formed. Previous results have shown that metallization requires the linkage of several bp-dNTPs in a row. The large, irregularly distributed silver nanoparticles are a result of the coiling of RCPs allowing 31 close proximity of several bp-dNTP and thus creating “hot spots” for metal deposition. Combining RCA with metallization using bp-dNTPs has potentials in nanotechnology and for sensor techniques based on RCA. Depending on usage, the formation of small regular shaped metal nanoparticles or large irregular distributed nanoparticles might be desired. The sequence of the padlock probe can also be tailored to allow incorporation of several bpdNTPs in a row when only one or two natural nucleotides are exchanged allowing for formation of small defined nanoparticles. Paper IV. Dielectrophoretic stretching of DNA amplified rolling circle products. Introduction The ability to manipulate molecules on a nano- and microscale has great usage in nanotechnology. This would allow increased specificity in molecular diagnostics and also aid in more fundamental studies for increased biological understanding. Contact-free methods such as DEP and other electrokinetic techniques are attractive manipulation methods due to their simplicity and ease of implementation. DEP results in movement of a molecule in a non-uniform field due to the dielectric properties of the molecule. DEP has previously shown the ability to manipulate DNA by separation, purification, trapping and stretching. Aim of study The aim of the study was to stretch tethered RCPs across an electrode gap using dielectrophoresis and to investigate the frequency and electric field required to achieve complete bridging. Two designs of electrodes were used, one sawtooth shaped electrode and one interdigitated electrode. Findings and conclusions The study demonstrates that stretching of tethered RCPs is possible using DEP. Sterile filtered MilliQ treated water was used to provide a low conductivity medium, necessary for DEP. The low conductivity medium also prevents oxidation of the adhesive titanium layer used for adhering the gold electrodes to the substrate. The RCPs were seen to elongate from 1 kHz, while complete bridging was seen at 100 kHz. During RCA, the RCPs where labelled with fluorescently labelled complementary strands of DNA, making them partially double stranded. The RCPs were therefore more flexible than dsDNA with less overall charge, but more rigid than ssDNA. A higher electric field than reported for dsDNA was thus required for elongation of the RCPs. 32 At low frequencies (<10 kHz), a high electric field caused migration of RCPs. Increasing the electric field further, caused electrolysis and consequently removal of the RCPs. A high electric field was possible at high frequencies without causing migration or electrolysis. At 100 kHz complete bridging was seen using an electric field of 1.4 MV/m. Increasing the electric field at this frequency led to more efficient stretching with more RCPs closing the gap. Electric field simulations using the software COMSOL illustrates a nonuniform electric field at the tips of the sawtooth shaped electrode while a larger area of high electric field could be achieved using an interdigitated electrode. Elongation of RCPs was possible for both designs using DEP, although a larger amount of RCPs were stretched using the interdigitated electrode. Figure 7. Electric field simulations of (Ia) a sawtooth shaped electrode and (IIa) an interdigitated electrode. Elongation of RCPs tethered to (Ib) a sawtooth shaped electrode and (IIb) an interdigitated electrode visualized by fluorescently labelled oligonucleotides. The ability to manipulate RCPs through DEP has large potentials. If taken further to include capturing of target on a multiplex array by DEP it should be possible to realize a simple yet sensitive detection method. 33 Perspectives The main focus of this thesis has been the development of a simple electrical readout by forming conductive wires from RCPs. The thesis has demonstrated proof of concept and illustrated alternative metallization and stretching techniques. The critical steps in the assay are; (1) the efficient binding of target to generate RCPs, (2) efficient stretching of RCPs to bridge the electrode gap and (3) efficient, specific and homogeneous metallization of the RCPs. Currently, the assay is relatively complex with many different steps. The metallization technique used in Paper II can form wires with incomplete metallization indicating that the hybridization of the gold label is inefficient. Paper III illustrates a simpler metallization technique with high spatial resolution. This technique has the potential to form more homogeneous metal wires which is required if to advance the readout to produce more quantitative measurements. The disadvantage of bp-dNTPs is the increased hydrophobicity resulting in less efficient stretching of the RCPs. This can be overcome using a polar aprotic solvent. Metallization of stretched RCPs using this technique still remains to be evaluated. Inefficient stretching, observed in Paper II, and the desire to simplify stretching with consistent reproducible results led to the investigation of an alternative technique seen in Paper IV. This demonstrates a very simple contact free method to stretch RCPs. As yet, this method has only been used to stretch the RCPs but it also has the potential to trap targets to allow fast and efficient hybridization. The method of electrical detection of conductive wires produced by RCA has just passed the stage of initial proof of concept while many possibilities lie ahead. Further studies in the use of electrokinetic manipulation and in the design of the electrodes to allow stretching of all generated RCPs are needed. The realization, to achieve a simple detection method with high sensitivity and specificity, should be possible when combining metallization by the use of bp-dNTPs with electrokinetic manipulation in more steps than stretching. The assay can be further improved by optimizing substrate and design of electrodes. The simple setup can easily be transferred to a microfluidic platform to allow further simplicity and ease of handling. For proof of concept the focus has been on nucleic acid detection. Protein detection is also possible using the same readout method. Antibody recognition elements can be used in combination with RCA for signal amplification. Combining antibody recognition elements in the form of proximity ligation 34 probes with RCA produces the proximity ligation assays (PLA) [128, 129], while combining a DNA labelled antibody recognition element with RCA produces the immuno-RCA assay [130]. The thesis has also shown the use of an alternative electrical readout in the form of a magnetic nanoparticle-based sensor. This method offers a robust portable system and multiplexing is possible to some degree due to beads of different sizes display peaks at different Browninan relaxation frequency. However, it is important to use magnetic nanobeads with narrow size distribution. 35 Acknowledgements First I would like to acknowledge the Uppsala Berzelii Technology Centre for Neurodiagnostics, financed by the Swedish Governmental Agency for Innovation Systems, the Swedish Research Council, and Uppsala University for funding me during my PhD. Then I would like to express my gratitude to a number of people who have been important throughout my PhD, starting with my main supervisor Mats Nilsson. Thank you for allowing me to do my PhD in your group, for all the support when I got stuck, your enthusiasm and always coming up with new ideas when something isn’t working. My co-supervisor Jonas Jarvius; for introducing me to “DNA threads”, your never ending enthusiasm and helping me with practical solutions for my set up. I would also like to thank my opponent Björn Högberg for being my opponent. The examining committee: Mikael Karlsson, Valerio Beni and Masood Kamali-Moghaddan for taking your time to examine my work. Elin Ekberg for helping me with all the admin stuff, Christina Magnusson for your kindness and support, and Johanna Herö for fun conversations and for taking over the responsibility of all the chemicals. Everyone in the lab: Karin for all the good conversations and good times when we shared office, Linda also for all the good times when we shared office and your enthusiasm for exercise, Christina for all the advice and discussion we had on baking, David for always having answers to my molecular biology questions and for being a good office friend, Liza for your positive energy that you bring to the lab every day, Agata for all the interesting fun conversations and coming back to the lab, Lotta for all the conversation in the lab and during lunch, Elin F for encouraging me to start running again, Lotte for all the help in the lab, Caroline for interesting discussions, Gaëlle for your expertise in microscopy, Tonge for letting me borrow your slides when I have run out, Felipe for your fun humor that you bring to the lab, Rasel for all the nice interesting conversations, Andries for being a fellow Berzelii member, Johan O for all the help with my computer and other computers, Erik for helping me with all the Berzelli reports, Joakim for interesting discussions, Axel for always knowing something about every36 thing, Lei for your discussions about RCA, Junhong for your kindness and having answers to my questions about thesis production and defense, Peter and Johan B for good conversations at lunch, Ola for leading the in situ PLA group and Masood (again) for leading the solid-phase PLA group. I would also like to thank all the newcomers and students in the lab, especially Megha and Lu for helping out in making the lab a fun work environment. Everyone in the Stockholm lab: Elin L for always helping out in organizing things in the lab and organizing the move to Stockholm, Marco for all the help in teaching the microscope course with me, Annika for your endless support and kindness and also all the help with microfluidics, Anja for your expertise in conjugating magnetic nanoparticles, Di for all the good conversations during our Berzelii PhD forum, Pavan for the good collaboration and all the interesting discussions, Jessica for the fun when we went fishing together during the retreat, Tagrid for all the nice conversations, Thomas for your expertise in microscopy, Amel for being a good roommate during the retreat, Ivan for all your help with the membranes even though we could not use your syringe set up, Malte for your inspiring enthusiasm to everything regarding science, Tomasz for good discussions during meetings, Xiaoyan for all the help with Cellprofiler and image analysis of my metal nanoparticles. I would also like to thank all the newcomers in the lab and all the students who help in creating a nice work place when I come and visit. Old group members: Björn for the fun you brought into the lab and all the good conversations, Carl-Magnus for all the interesting discussions during lunch, Rachel for teaching me how to use the array printer, Rongqin for all the help when we started out as PhD students, Jenny Göransson for all the good conversations and teaching me RCA when I was new, Anna Engström for all your support and the fun collaboration in the TB project, Maria Hammond for help with antibody conjugation, Monica for all the microfluidic discussions, Lucy for giving me some E. coli genomic DNA when I needed some for my paper. I would also like to thank all the other old group members that were in the lab when I started for making the lab a fun working place, and a special thanks to Lena Spångberg and Carla Annik for taking care of everything in the lab. I would also like to thank Anne-Li Lind for all the good times in the Berzelii centre. My students that I have had over the years; Swapneel, Nushrat and Hanqian. I would also like to express my gratitude to all my collaborators that I have had during my time as a PhD student. Most of all Ken Welch for all your 37 help with my projects and being an invaluable collaborator, Teresa and Mattias for introducing me to magnetic biosensors and all the good discussions, Rebecca and Klas Gunnarson for all the work on magnetic tweezers, Fredrik Nikolajeff for helping me throughout my PhD with my projects and introducing me to new collaborators, Maria Strömme for your work in nanotechnology, Shubh for giving excellent feedback on my manuscript and synthesizing bp-dNTPs for me, Rimantas for all your help with the gold electrodes, Yixiao Cai for all your SEM help and good discussions, Jonas Hansson for making an excellent microfluidic chip for me and finally Maoxiang (Alora) for the interesting work you do with your 3D chips and all the good times we have had in the lab. 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(Prior to January, 2005, the series was published under the title “Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine”.) Distribution: publications.uu.se urn:nbn:se:uu:diva-247945 ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2015