ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2018

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 1647

Magnetic Nanoparticle BasedBiosensors for Pathogen Detectionand Cancer Diagnostics

BO TIAN

ISSN 1651-6214ISBN 978-91-513-0278-2urn:nbn:se:uu:diva-346014

Dissertation presented at Uppsala University to be publicly examined in Häggsalen,Ångströmlaboratoriet, Lägerhyddsv. 1, Uppsala, Friday, 4 May 2018 at 13:15 for the degreeof Doctor of Philosophy. The examination will be conducted in English. Faculty examiner:Associate Professor Hakho Lee (Harvard University).

AbstractTian, B. 2018. Magnetic Nanoparticle Based Biosensors for Pathogen Detection andCancer Diagnostics. Digital Comprehensive Summaries of Uppsala Dissertations from theFaculty of Science and Technology 1647. 55 pp. Uppsala: Acta Universitatis Upsaliensis.ISBN 978-91-513-0278-2.

This thesis describes several magnetic nanoparticle (MNP)-based biosensing strategieswhich take advantage of different magnetic sensors, molecular tools and nanotechnologies.Proposed biosensors can be classified into three groups, i.e., immunoassay-based, molecularamplification-based, and nanoparticle assembly-based. The principal motivation is to developand optimize biosensors for out-of-lab and point-of-care testing.

Immunoassay-based biosensors described in this thesis employ antibodies as the bio-recognition element for the detection of bacteria cells/fragments or proteins. Two typicalimmunoassay formats, i.e., direct and competitive format, are studied and compared for bacteriadetection. In addition, in the protein biomarker detection, MNP chains are formed in the presenceof target analytes as well as in the external rotating magnetic field. The high shape/magneticanisotropy of the chains provides better optomagnetic performance.

Two different molecular amplification methods, i.e., rolling circle amplification (RCA) andloop-mediated isothermal amplification (LAMP), are described under the topic of molecularamplification-based biosensors. In RCA-based biosensors, DNA probe modified MNPs bind tothe amplicons after amplification. In LAMP-based biosensors, MNPs are either modified withprimers that keep growing during the amplification, or are co-precipitated with the by-product(Mg2P2O7) of the amplification.

The design of the nanoparticle assembly-based biosensors described in this thesis isbased on duplex-specific nuclease (DSN)-assisted target recycling and core-satellite magneticsuperstructures. In the presence of target microRNA, DSN cuts the DNA scaffold of the core-satellite assembly, releasing MNP satellites that can be quantified by the sensor.

Different kinds of target analytes, i.e., pathogens or cancer biomarkers, are detected at theaiming for rapid, low-cost and user-friendly diagnostics.

Keywords: Magnetic biosensors, magnetic nanoparticles, homogeneous assays, volumetricsensing

Bo Tian, Department of Engineering Sciences, Solid State Physics, Box 534, UppsalaUniversity, SE-751 21 Uppsala, Sweden.

© Bo Tian 2018

ISSN 1651-6214ISBN 978-91-513-0278-2urn:nbn:se:uu:diva-346014 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-346014)

自卑者忧 怯懦者畏 勇者克而后怕 The timid feel fear before trouble starts. The cowards feel fear as trouble happens. The bravest feel fear only after the troubles have been taken care of.

––––– Wrathion, The Black Prince

List of papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Tian, B., Bejhed, R. S., Svedlindh, P., Strömberg, M. (2016) Blu-ray optomagnetic measurement based competitive immu-noassay for Salmonella detection. Biosensors & Bioelectronics, 77:32-39

II Tian, B., Wetterskog, E., Qiu, Z., Zardán Gómez de la Torre, T., Donolato, M., Hansen, M. F., Svedlindh, P., Strömberg, M. (2017) Shape anisotropy enhanced optomagnetic measurement for prostate-specific antigen detection via magnetic chain for-mation. Biosensors & Bioelectronics, 98:285-291

III Tian, B., Zardán Gómez de la Torre, T., Donolato, M., Hansen, M. F., Svedlindh, P., Strömberg, M. (2016) Multi-scale magnet-ic nanoparticle based optomagnetic bioassay for sensitive DNA and bacteria detection. Analytical Methods, 8(25):5009-5016

IV Tian, B., Ma, J., Zardán Gómez de la Torre, T., Bálint, Á., Do-nolato, M., Hansen, M. F., Svedlindh, P., Strömberg, M. (2016) Rapid Newcastle disease virus detection based on loop-mediated isothermal amplification and optomagnetic readout. ACS Sensors, 1(10):1228-1234

V Tian, B., Qiu, Z., Ma, J., Zardán Gómez de la Torre, T., Jo-hansson, C., Svedlindh, P., Strömberg, M. (2016) Attomolar Zika virus oligonucleotide detection based on loop-mediated isothermal amplification and AC susceptometry. Biosensors & Bioelectronics, 86:420-425

VI Tian, B., Svedlindh, P., Strömberg, M., Wetterskog, E. (2017) Ferromagnetic resonance biosensor for homogeneous and vol-umetric detection of DNA. Preprint published on ChemRxiv, and submitted to ACS Sensors.

VII Tian, B., Ma, J., Qiu, Z., Zardán Gómez de la Torre, T., Donol-ato, M., Hansen, M. F., Svedlindh, P., Strömberg, M. (2017) Optomagnetic detection of microRNA based on duplex-specific nuclease-assisted target recycling and multilayer core-satellite magnetic superstructures. ACS Nano, 11(2):1798-1806

VIII Tian, B., Qiu, Z., Ma, J., Donolato, M., Hansen, M. F., Sved-lindh, P., Strömberg, M. (2018) On-particle rolling circle ampli-fication-based core-satellite magnetic superstructures for mi-croRNA detection. ACS Applied Materials & Interfaces, 10(3):2957-2964

All these above papers are published with open access.

Author’s contribution:

I All the experimental, part of the design, analysis and writing. II All the experimental, most of the design, analysis and writing. III All the experimental, part of the design, analysis and writing. IV Most of the experimental, design, analysis and writing. V Most of the experimental, design, analysis and writing. VI All the experimental, part of the design, analysis and writing. VII Most of the experimental, design, analysis and writing. VIII Most of the experimental, design, analysis and writing.

Papers not included in the thesis:

IX Bejhed, R. S.,* Tian, B.,* Eriksson, K., Brucas, R., Oscarsson, S., Strömberg, M., Svedlindh, P., Gunnarsson, K. (2015) Mag-netophoretic transport line system for rapid on-chip attomole protein detection. Langmuir, 31(37):10296-10302

X Minero, G. A. S., Nogueira, C., Rizzi, G., Tian, B., Fock, J., Donolato, M., Strömberg, M., Hansen, M. F. (2017) Sequence-specific validation of LAMP amplicons in real-time optomag-netic detection of Dengue serotype 2 synthetic DNA. Analyst, 142(18):3441-3450

*Authors contributed equally to this work.

Contents

1. Introduction ............................................................................................... 11 1.1 Aims of biosensors ............................................................................. 12 1.2 Analytes of biosensors ....................................................................... 13

1.2.1 Bacteria ....................................................................................... 13 1.2.2 Protein biomarkers ...................................................................... 14 1.2.3 Nucleic acids ............................................................................... 15 1.2.4 MicroRNAs ................................................................................ 16

1.3 Definition of limit of detection ........................................................... 16 1.4 Homogeneous and volumetric sensing ............................................... 17

2. Particles, sensors and strategies ................................................................ 19 2.1 Magnetic nanoparticles....................................................................... 19

2.1.1 Key properties ............................................................................. 19 2.1.2 Magnetic relaxation .................................................................... 20 2.1.3 Biosensing applications .............................................................. 21

2.2 Magnetic biosensors ........................................................................... 22 2.2.1 AC susceptometer ....................................................................... 23 2.2.2 Optomagnetic biosensor ............................................................. 24 2.2.3 Ferromagnetic resonance biosensor ............................................ 26

2.3 Biosensing strategies .......................................................................... 27 2.3.1 Immunoassays............................................................................. 27 2.3.2 Rotating magnetic field platform ................................................ 29 2.3.3 Rolling circle amplification ........................................................ 30 2.3.4 Loop-mediated isothermal amplification .................................... 31 2.3.5 Duplex-specific nuclease assisted target recycling ..................... 33 2.3.6 Core-satellite magnetic assemblies ............................................. 33

3. Results and discussion .............................................................................. 35 3.1 Immunoassay based bacteria/biomarker detection ............................. 35

3.1.1 Performance of immunoassays ................................................... 35 3.1.2 Immunoassay duplexing ............................................................. 36 3.1.3 Shape anisotropy enhanced immunoassays ................................ 37

3.2 Molecular amplification based DNA/RNA detection ........................ 37 3.2.1 RCA-based biosensing ................................................................ 37 3.2.2 RCA duplexing ........................................................................... 39 3.2.2 LAMP-based biosensing ............................................................. 39

3.3 Core-satellite assembly-based microRNA detection .......................... 40 3.3.1 Multilayer core-satellite superstructure based biosensing .......... 40 3.3.2 On-particle RCA enhanced core-satellite superstructures .......... 41

4. Concluding remarks .................................................................................. 42

Svensk sammanfattning ................................................................................ 43

Acknowledgements ....................................................................................... 46

References ..................................................................................................... 47

Abbreviations

AC Alternating current CFU Colony-forming unit DNA Deoxyribonucleic acid DSN Duplex-specific nuclease EPR Electron paramagnetic resonance FMR Ferromagnetic resonance GMR Giant magnetoresistance IgG Immunoglobulin G LAMP Loop-mediated isothermal amplification LOD Limit of detection MNP Magnetic nanoparticle NDV Newcastle disease virus NMR Nuclear magnetic resonance PCR Polymerase chain reaction POCT Point-of-care testing PSA Prostate-specific antigen RCA Rolling circle amplification RMF Rotating magnetic field RNA Ribonucleic acid VAM-NDA Volume-amplified magnetic nanoparticle detection assay χ Magnetic susceptibility χ' In-phase component of the magnetic susceptibility χ'' Out-of-phase component of the magnetic susceptibility τΝ Néel relaxation time τΒ Brownian relaxation time fB Brownian relaxation frequency V2' In-phase second harmonic voltage component V2'' Out-of-phase second harmonic voltage component Bres Ferromagnetic resonance field

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1. Introduction

Biosensors are analytical devices incorporating a bio-recognition element (the “bio” part) and a signal transducing element (the “sensor” part). The bio-recognition element can be a biological material (e.g., tissues, microor-ganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids and natural products), a biologically derived material (e.g., recombinant antibod-ies, engineered proteins and aptamers) or a biomimetic material (e.g., syn-thetic receptors, biomimetic catalysts, combinatorial ligands and imprinted polymers). The signal transducing element can be optical, electrochemical, thermometric, piezoelectric, magnetic or micromechanical (Turner et al., 1987). The signal transducing element can generate signals in response to the specific analyte (or group of analytes) recognized by the bio-recognition element. The signal is always digital and electronic, and is proportional to the concentration of target analyte (dose-response). Since the signal may in principle be continuous, biosensors can be configured to yield single meas-urements and the results can be transferred into the concentrations of target analyte according to the dose-response. Some biosensors do not provide digital electronic signals due to their special design. A well-known example of such a biosensor is the pregnancy test sticks, which always provide color-imetric results for qualitative naked eye judgement.

This doctoral thesis concerns magnetic nanoparticle-based magnetic or optomagnetic biosensors applied for pathogen detection and cancer diagnos-tics. Based on the strategy of signal generation, the biosensors introduced in this thesis are classified into three groups: immunoassay-based, DNA/RNA amplification-based and nanoparticle assembly-based. The principal aim of this work is to develop and optimize biosensing strategies by the combina-tion of magnetic sensors, molecular tools and nanotechnologies. Sensing method, target analyte, detection strategy and sensitivity of the biosensor in each appended paper are summarized in Table 1.

In more detail, the aims of each appended paper were as follows:

I To build a qualitative whole bacteria detection system based on immunoassay and optomagnetic readout, and to demonstrate a qualitative duplex immunoassay.

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II To investigate the effect of nanoparticle shape anisotropy in-duced by a rotating magnetic field, and to build an optomagnet-ic biosensor for a quantitative protein immunoassay.

III To build a quantitative DNA detection system based on rolling circle amplification and optomagnetic readout, and to demon-strate qualitative duplex DNA detection.

IV To build a quantitative DNA/RNA detection system based on loop-mediated isothermal amplification and optomagnetic readout.

V To build a quantitative DNA/RNA detection system based on on-particle loop-mediated isothermal amplification and AC sus-ceptometry.

VI To demonstrate a ferromagnetic resonance biosensor that can be used for homogeneous and volumetric biosensing.

VII To build a quantitative microRNA detection system based on duplex-specific nuclease-assisted target recycling and multi-layer core-satellite magnetic assemblies.

VIII To improve the biosensing performance of core-satellite assem-blies by on-particle rolling circle amplification, and to build a quantitative microRNA detection system based on the improved assemblies.

Table 1. Details of each appended paper.

Paper Sensor Analyte Strategy Sensitivity I Optomagnetic Bacteria Immunoassay 8×104 CFU/mL II Optomagnetic Protein Immunoassay 0.65 ng/mL III Optomagnetic Bacteria Immunoassay 105 CFU/mL DNA RCA 780 fM IV Optomagnetic DNA & RNA LAMP 10 aM V Susceptometric DNA LAMP 1 aM VI FMR DNA RCA 1 pM DNA LAMP 100 aM VII Optomagnetic MicroRNA DSN 4.8 fM VIII Optomagnetic MicroRNA RCA & DSN 1 fM

1.1 Aims of biosensors Biosensors have been applied to a wide variety of analytical problems in the field of, e.g., medicine, biomedical research, drug discovery, environmental research, food control, process industry, security and defence (Turner et al., 1987).

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Among the different biosensing applications, medical and clinical appli-cations are invariably foreseen as the most significant and, accordingly, most research and development have been devoted to this area (Collings and Caruso, 1997). Today, the trend is towards point-of-care testing (POCT), which is defined as the health care and self-monitoring service for patients without labour-intensive laboratory work. POCT reduces the patient turn-around times, leading to faster treatments, improved workflows and thus improving the quality of care (von Lode, 2005). POCT biosensors provide opportunities for performing tests in common settings such as the doctor’s office and the patient’s home. For such biosensors, the most representative example is the glucometer for the daily glucose testing. Implantable or in vivo devices and the real-time telemetering of body functions is another aim for medical biosensing, but there is much to be done in overcoming prob-lems of, e.g., biocompatible materials. Additionally, in foodindustry, biosen-sors are considered of high significance in four aspects: detection of contam-inants, verification of product content, monitoring raw materials conversion and product freshness (Luong et al., 1991). The aims of the biosensors pro-posed in this thesis can easily be specified according to their target analytes.

1.2 Analytes of biosensors According to the different properties utilized for designing biosensing strate-gies, target analytes in this thesis can be divided into: (a) whole or fragments of bacteria, micrometer sized; (b) protein biomarker, nanometer sized; (c) DNA or RNA, long enough for primer design and molecular amplification; and (d) microRNA, around 20 nucleotide-long and thus challenging to be detected through molecular amplification.

1.2.1 Bacteria The development of sensitive assays for identification and detection of mi-croorganisms is vital for improving diagnosis and preventing disease out-breaks. These assays also play essential roles in control programs provided by food-safety regulatory agencies and disease control and prevention agen-cies (Hood et al., 2004; Swaminathan and Feng, 1994; Virgin and Todd, 2011). Paper I deals with detection of whole or fragments of Salmonella typhimurium and Escherichia coli.

Salmonella, among all the food-borne pathogens, is one of the most fre-quent causes of food borne infectious disease, especially gastroenteritis (Majowicz et al., 2010). S. typhimurium, the top serotype associated with salmonellosis worldwide, is a Gram-negative bacteria transmitted primarily through the consumption of raw or uncooked eggs, vegetables, fruits, and poultry (Mrema et al., 2006). The presence of this organism in food can be a

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serious threat to public health worldwide (Ansari et al., 2017; Brandt et al., 2013). The routine and gold standard detection method for S. typhimurium is bacterial culture, which entails long process times (up to several days) and needs specialized laboratory infrastructure. Common amplification-based assays such as real-time polymerase chain reaction (PCR) detection (Park et al., 2013; Zheng et al., 2014) and immunoassays such as enzyme-linked immunosorbent assay (Crowley et al., 1999) are also applied for Salmonella detection but have limitations of, e.g., requiring specialized instrumentation and highly trained personnel. Therefore, there is a continuous need for a rapid, sensitive and reliable assay for S. typhimurium detection.

Escherichia coli are the most studied bacteria in biology. Significant re-search efforts have been made on improving the ability to detect E. coli in food and water supplies. The United States Food and Drug Administration has suggested to use coliforms or generic E. coli as indicators for the bacte-rial contamination level in fresh products (U. S. Food and Drug Administra-tion. Guidance for Industry: Bottled Water: Total Coliform and E. coli, Small entity compliance guide; Silver Spring, MD, 2010). Moreover, the United States Environmental Protection Agency identified E. coli as an indi-cator of the fecal contamination (U.S. Environmental Protection Agency. 40 CFR 141 Analytical Methods Approved for Compliance Monitoring under the Ground Water Rule; Washington, D.C., 2008.). The gold method to de-termine the presence of E. coli is culturing and plate counting, which is time-consuming and laborious.

1.2.2 Protein biomarkers Detection of protein biomarkers is applied as an aid for disease analysis and treatment, and has been subject to intensified interest in recent years. Bio-sensors are utilized for protein biomarker quantification in biological fluids such as serum and urine. They offer the opportunity to improve patient care through earlier and more accurate diagnosis in a convenient, non-invasive manner as well as providing a potential route towards more individually targeted treatment (Lee et al., 2008). Protein biomarker detection, i.e., quan-tification of the target protein biomarker in a mixture of a large number of bio-macromolecules with high sensitivity and specificity, is essential to achieve progress in biomarker technology. Paper II deals with the detection of native human prostate-specific antigen (30 kDa, purchased from Bio-Rad Laboratories, Kidlington, UK).

Prostate-specific antigen (PSA) is a serine protease indicator which is re-lated to many prostate diseases including prostate cancer of all grades and stages (Lilja et al., 2008). PSA, as a cancer biomarker, is widespread detect-ed both for initial diagnosis and for monitoring the response to treatment. The PSA level in blood is approximately 0.6 ng/mL for healthy adult males aged ≤ 50 years, but it can rise to 104 ng/mL for patients (Savblom et al.,

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2005). For detecting prostate cancer, the traditional threshold PSA level (in blood) is 4 ng/mL. PSA levels above 102 ng/mL have been found almost exclusively related to advanced prostate cancer (Lilja et al., 2008). Both the low threshold and the large concentration span of PSA require biosensors to have high sensitivity as well as wide detection range, which is challenging especially for POCT situations. Furthermore, analysis of a single biomarker may offer misleading diagnosis for cancer detection (Harris and Lohr, 2002), which means that the capability for multiplexing different biomarkers is important.

1.2.3 Nucleic acids Sensitive and specific detection of nucleic acids finds fast growing applica-tions in diagnostics of infectious diseases, food control, epigenetics, human identification in forensic investigations, as well as in biomedical research (Gerasimova and Kolpashchikov, 2014). Papers III-VI deal with the detec-tion of synthetic DNA sequences from Vibrio cholerae (papers III and VI), E. coli (paper III), Newcastle disease virus (paper IV) and Zika virus (papers V and VI), respectively. In addition, in paper IV, RNA virus samples were analyzed.

Vibrio cholerae, a Gram-negative, comma-shaped bacterium, is the causa-tive agent of cholera, a highly contagious and commonly fatal bacterial in-fection of the gastrointestinal tract. After infection, death can occur within hours due to hypovolemic shock or acidosis if not treated immediately (Kaper et al., 1995). The gold method for detecting cholera in environmental samples is bacterial culture, which takes approximately 8 days for a conclu-sive determination.

Newcastle disease is regarded as one of the two most significant diseases of poultry and other birds throughout the world (Aldous and Alexander, 2001). Caused by Newcastle disease virus (NDV), the Newcastle disease outbreaks can spread rapidly and have flock mortality rates up to 100% in fully susceptible chickens, resulting in severe economic losses (Aldous and Alexander, 2001; Ganar et al., 2014). The gold method for detecting NDV is virus isolation in embryonated eggs. However, molecular diagnostics meth-ods, especially real-time reverse transcription PCR, are commonly applied for NDV detection due to their reliability and short assay time (Chaka et al., 2015; Liu et al., 2016).

Zika virus, a positive-sense RNA virus, is the causative agent of the infec-tious disease Zika fever. Zika virus belongs to the family Flaviviridae, genus Flavivirus, which is related to dengue fever, yellow fever, Japanese encepha-litis and West Nile fever (Haug et al., 2016; Kuno et al., 1998). Zika fever had long been considered mild and self-limited (Petersen et al., 2016) until the outbreaks in Brazil, which has drawn the world’s attention particularly for its association with the newborns presenting microcephaly (Campos et

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al., 2015; Heukelbach et al., 2016; Rodriguez-Morales, 2015). Similar to the detection of Dengue virus, laboratory analysis of Zika virus is based mainly on the serum detection using viral RNA and/or antibody based methods (Al-Qahtani et al., 2016). Because of the high serological cross-reactivity among flaviviruses in IgM-antibody based assays, nucleic acid testing is considered more reliable (Haug et al., 2016). The current prevalent molecular detection method is reverse-transcription PCR. However, since viremia decreases over time, nucleic acid testing should be performed during the first seven days after symptom onset (Buathong et al., 2015; Haug et al., 2016), when the Zika virus loads are reported at the level of approximately 1 fM in body fluid samples (Barzon et al., 2016; Gourinat et al., 2015; Lanciotti et al., 2008); after that, reliable results can only be provided by labor-intensive serologic testing (Petersen et al., 2016).

1.2.4 MicroRNAs MicroRNAs are highly tissue-specific endogenous noncoding RNA mole-cules with lengths of approximately 19-23 nucleotides. Different combina-tions of microRNAs are expressed in different cell types and may coordi-nately regulate cell-specific target genes (Krek et al., 2005). Additionally, a considerable number of microRNAs were found to be up- or down-regulated in human cancers compared with corresponding benign tissues (Schultz et al., 2008). Recent studies indicate that microRNAs may be involved in tu-mors during the tumor development, being either under-expressed with con-sequent up-regulation of oncogenes or over-expressed with consequent down-regulation of tumor suppressor genes (Esquela-Kerscher and Slack, 2006). Therefore, microRNAs have clinical applicability for profiling tumor progression and metastasis (Calin and Croce, 2006; Esquela-Kerscher and Slack, 2006; Inui et al., 2010; Lu et al., 2005). Standard microRNA analysis strategies, e.g., Northern blotting (Lagos-Quintana et al., 2001; Valoczi et al., 2004), quantitative reverse transcription PCR (Benes and Castoldi, 2010; Chen et al., 2005; Redshaw et al., 2013) and oligonucleotide microarrays (Barad et al., 2004; Li and Ruan, 2009; Thomson et al., 2004), each have their own limitations in aspects of, e.g., poor sensitivity, labor-intensive steps, requirement of high-precision thermal cycling equipment or low speci-ficity at the 5´-end of the analyte (Baker, 2010; Shen et al., 2015).

1.3 Definition of limit of detection According to International Union of Pure and Applied Chemistry, the mini-mum single measurement result can be distinguished from a suitable blank value with a stated probability (Long and Winefordner, 1983). Note that the lowest point at which the analysis becomes possible may be different from

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the lower limit of the determinable analytical range. The limit of detection (LOD), expressed in this thesis as the concentration of the analyte, is derived from the smallest value of the signal, Xlimit, that can be detected with reason-able certainty for a given analytical strategy. The value of Xlimit is given by the equation: = +

where Xblank is the mean value of the blank measures (blank control signal), σblank is the standard deviation of the blank measures, and k is a numerical factor chosen according to the confidence level that is needed. In the thesis, the value of Xlimit is called the cutoff value, which is usually indicated by dashed horizontal lines plotted with the dose-response curves presented in chapter 3.

The value of k, in the field of biosensors, is usually chosen either to be 2 or 3, which results in a two-σ-limit or three-σ-limit, respectively. A two-σ-limit indicates data chosen randomly from a set of normally distributed data that has a 95% of probability of being within the acceptable range; while for a three-σ-limit the probability is 99.73%. In this thesis, we define the cutoff value based on the three-σ-criterion, i.e., the cutoff value is calculated as the average peak value of the blank control samples plus or minus (depending on whether the detection format is turn-on or turn-off) three standard devia-tions. Therefore, a sample with a signal value that is equal to the cutoff value is 99.73% likely to be positive.

1.4 Homogeneous and volumetric sensing Homogeneous biosensing strategies rely on signal generation within the whole sample volume (Diamandis and Christopoulos, 1996), and thus usual-ly do not need additional separation, washing or signal generation steps. The total assay time of homogeneous strategy based detection is theoretically shorter than that of the heterogeneous assay since in homogeneous assays the diffusion of both analytes and bio-recognition elements is three-dimensional (Schrittwieser et al., 2016). Therefore, homogeneous biosensors greatly ben-efit from simple “mix and measure” procedures and thus have advantages of short total assay times, a high degree of user friendliness, a low risk of con-tamination and potential for automation, which are all desirable properties for in-situ decentralized diagnostics (Foy and Parkes, 2001; Reynolds et al., 2000).

Based on the detection mechanism of signal acquisition, sensing methods can be classified into two groups, namely volumetric and surface-based sens-ing (Issadore et al., 2014). The volumetric sensors measure analytical signals coming from the entire volume of measurement, yielding simple and fast

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assays. However, compared to the surface-based sensors, the resolving pow-er of volumetric sensors is weaker, because the acquired signal is an ensem-ble average of the whole volume and it is difficult to distinguish a weak positive signal from a high level of background noise (Lee et al., 2015).

The combinations of homogenous biosensor strategies and volumetric sensing methods are typically robust, simple and rapid. Particularly, in com-bination with the large surface area and unique properties of nanoparticle labels, they are of great interest for POCT applications (Huang et al., 2017). In the papers appended in this thesis, the proposed magnetic nanoparticle-based biosensors are (or at least very close to) homogenous and volumetric.

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2. Particles, sensors and strategies

In this chapter, the vital elements on which this thesis is based are briefly described, including magnetic particles, sensors and analytical strategies.

2.1 Magnetic nanoparticles Magnetic materials are found in many daily used devices from motors to hard disks. Therefore, the urgent need for the miniaturization of these mag-netic materials can easily be understood. In addition, nanosized magnetic materials are particularly promising in medical applications due to their unique properties, e.g., superparamagnetism.

2.1.1 Key properties Magnetic moment. The very basic physical property of magnetic materials is the magnetic moment, which is to largest extent associated with the intrin-sic spin angular momentum of the electrons (Martin, 1967). Due to the posi-tive energy contribution associated with the stray field created by the aligned spin moments, bulk ferro- and ferrimagnetic materials are composed of re-gions, which are called magnetic domains. For nanosized magnetic materi-als, e.g., magnetic nanoparticles (MNPs), the material contains only one domain, and their magnetic properties are very different than those of bulk materials. In such situations, MNPs have a single domain with the magneti-zation aligned in a direction defined by the magnetic anisotropy. Moreover, the thermal energy may be large enough to change the direction of the MNP magnetization randomly, which means that the magnetic moment of an MNP may rotate randomly over time. Therefore, in the absence of an applied magnetic field, an ensemble of such MNPs displays a negligible remnant magnetic moment. This phenomenon, known as superparamagnetism, allows MNPs to forms stable colloidal suspensions that can be collected and manip-ulated by external magnetic fields without irreversible aggregation.

Magnetic susceptibility. The main parameters for bulk magnetic materi-als are composition, crystallographic structure, vacancies and defects, which together determine magnetic properties such as the magnetic susceptibility (χ). For MNPs, except for these parameters, size and shape also play an im-portant role in determining their magnetic behaviour. The magnetic aniso-

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tropic energy barrier is proportional to the product of the magnetic anisotro-py constant and the physical volume of the MNP. Therefore, in general, the smaller the MNP is, the lower the blocking temperature, i.e. the temperature where the particle moment is stable with respect to thermal fluctuations (as opposed to superparamagnetic behaviour).

Saturation magnetization. Saturation magnetization, defined as the max-imum magnetization achievable for a given magnetic material, is determined by the number of atomic magnetic dipoles per unit volume and the magnetic moment of each dipole.

Biocompatibility. For most applications, in general, MNPs with large magnetic moments are preferred since it reduces the amount of materials needed. However, in biological and medical applications, e.g., magnetic resonance imaging and magnetic hyperthermia, biocompatibility and toxicity of MNPs play more important roles. This is the reason why iron-oxide based MNPs are prevalent compared to other MNPs or even other kinds of parti-cles (e.g., quantum dots) (Sandhu et al., 2010). For intracellular and in vivo applications, the main toxicity of iron-oxide based MNPs is related to excess reactive oxygen species, which are caused by the chemical reactivity of MNPs (Kim et al., 2012; Shubayev et al., 2009). Excess reactive oxygen species have many negative effects on cells, including disrupting DNA and altering proteins, which lead to injury of the immune system and inflamma-tion (Sharifi et al., 2012; Soenen and De Cuyper, 2010).

Single- and multi-core MNPs. As mentioned above, MNPs with large magnetic moments are preferred in biosensing applications due to their strong magnetic signal which can improve the detection sensitivity (Yoon et al., 2011). However, large single-core MNPs tend to aggregate in suspension due to strong interparticle magnetic interactions. An alternative method is to use multi-core MNPs in which multiple smaller magnetic cores are embed-ded. By using this approach, the effective moment (and the size) of each MNP can be increased without losing colloidal stability. All magnetic parti-cles (nominal diameters of 56, 80, 100, 250 1000 and 5000 nm) used in this thesis were multi-core iron-oxide particles with 10-20 nm sized crystal cores embedded in a dextran or starch matrix. All magnetic particles used in this thesis were purchased from or provided by Micromod Partikeltechnologie GmbH, Rostock, Germany.

2.1.2 Magnetic relaxation In magnetic relaxation, the magnetic moments of MNPs take part in the pro-cess by which the MNPs achieve thermodynamic equilibrium. In multi-domain MNPs, the relaxation process is related to the movement of domain walls; while in single-core or multi-core superparamagnetic particles, the relaxation process is achieved by overcoming the anisotropy energy barrier (Néel relaxation), or mechanical rotation of the particle (Brownian relaxa-

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tion). For the Néel relaxation of MNPs, neglecting possible influence of magnetic interactions between crystal cores in a multi-core MNP, the relaxa-tion time, τΝ, can be estimated by the Néel-Brown model (Brown, 1963): = exp

where τ0 is the characteristic microscopic relaxation time in the order of 10-9 to 10-11 s (Svedlindh et al., 1997), K is the anisotropy energy constant of the material, V is the magnetic volume of the crystal core and kBT represents the thermal energy. On the other hand, the Brownian relaxation time, τΒ, for a MNP with a hydrodynamic volume Vh, is expressed as = 3

where η is the dynamic viscosity of the carrier liquid (Connolly and St Pierre, 2001). Since both Néel relaxation and Brownian relaxation are pre-sent, the effective relaxation time, τΕ, is given as (Chung et al., 2004) = +

Since τΝ is increasing more rapidly than τΒ with the particle size, Néel relaxa-tion dominates for the smaller MNPs while Brownian relaxation dominates for the larger ones. Note that the above models are valid only for single-core MNPs. For multi-core MNPs, number of cores, orientational distribution and relative positions of the cores also contribute to the relaxation properties.

2.1.3 Biosensing applications Due to the high surface-to-volume ratio, easy manipulation by external mag-netic fields, simplicity of bio-functionalization, and low background signals in biological samples, magnetic particles have been widely applied in chem-istry and biology (van Reenen et al., 2014). In traditional applications of magnetic particles, mainly pipette-based assays, they are utilized to simplify extraction and washing steps (Bock, 2000). In recent years, the application of magnetic particles has been extended a lot due to the fast growing nano-technology. In POCT biosensing, magnetic particles have been applied for mixing, capturing, enriching, transferring and labelling of analytes.

Mixing reaction fluids or homogenizing reagents have long been a topic in chemistry. For that purpose, methods based on magnetic particles and external magnetic fields have been utilized. In the presence of a magnetic field, magnetic particles tend to form superstructures due to the magnetic dipole-dipole forces between particles. In particular, when a rotating magnet-ic field is applied, magnetic particles in a suspension can be induced to form one-dimensional nanostructured assemblies (Vuppu et al., 2003). The mag-

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netic particle chains can be forced rotating by the field, resulting in effective mixing and accelerating reactions that may otherwise be limited by diffusion (Fermigier and Gast, 1992; Martin et al., 2009). Moreover, the angular ve-locity of the chains can help reducing the depletion layers of molecules around the particles, resulting in the increase of the reaction association con-stant (van Reenen et al., 2017). In paper II, the rotation of MNP chains was applied for biosensing.

Due to the high surface-to-volume ratio as well as the simplicity of bio-functionalization, magnetic particles, especially MNPs, are widely used for capturing, sorting, isolating and enriching targets. For specific capture, bio-recognition elements, e.g., antibodies and DNA probes, are required to func-tionalize the surface of magnetic particles. The capture rate is dependent on the total surface area of the suspended particles as well as the affinity of the bio-recognition elements. However, increasing the particle concentration is not a good choice for increasing the total surface area, since high particle concentrations can lead to (1) a higher rate of nonspecific reaction between particles and (2) a higher risk of particle aggregation.

In general, the final step of biosensing is measuring signals. If magnetic particles are utilized only as carriers, the analytes (captured on the surface of or eluted from magnetic particles) have to be further reacted with signal gen-eration probes such as enzymes and fluorescent molecules. A magnetic par-ticle itself can also serve as a label that can be detected by magnetic biosen-sors, which simplifies the strategy and shortens the total assay time. When serving as labels, MNPs are, in general, detected by their magnetic proper-ties in different magnetic sensors, which are summarized in Table 2.

Since iron-oxide MNPs have been discovered with an intrinsic peroxidase activity (Gao et al., 2007), they have also been used as labels in electro-chemical biosensors. In such applications, MNPs are utilized to catalyse oxidation of various peroxidase substrates which produce electrochemical signals when enzymatically oxidized with hydrogen peroxide (Derat and Shaik, 2006; Wei and Wang, 2008).

2.2 Magnetic biosensors Signals from MNP labels are often measured by magnetometers (Pankhurst et al., 2003). To date, different types of magnetometers have been developed as magnetic biosensors (see Table 2), among which AC susceptometers, optomagnetic biosensors and ferromagnetic resonance biosensors are fo-cused on in this thesis.

Table 2. Summary of common magnetic sensors.

Detection mode Sensor Key MNP property

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Volumetric µNMR Nuclear relaxivity AC susceptometer Brownian relaxation FMR Magnetic anisotropy Optomagnetic Brownian relaxation

Surface based GMR Magnetic moment µHall effect Magnetic moment

2.2.1 AC susceptometer Dynamics of an ensemble of MNPs can be measured by an AC susceptome-ter. In paper V, a portable commercial AC susceptometer (DynoMag®, Acreo Swedish ICT AB, Göteborg, Sweden) was used to measure the fre-quency-dependent AC susceptibility, χ. As can be seen in Figure 1a, the sensor is based on a two coil system, i.e., excitation coil and pickup coil, with a centrally positioned sample holder.

Figure 1. (a) A schematic illustration of the excitation coil and pickup coil arrange-ment in a DynoMag® AC susceptometer. For clarity, the coils are shown with a section in the middle removed to reveal the pickup coil and the sample holder. (b) Normalized magnetic AC susceptibility data (in-phase and out-of-phase components represented by solid and dashed lines, respectively) extracted from the Cole-Cole model vs frequency (τΒ=300 s and α=0.15 were used as input, where α is the Cole-Cole parameter ranging from 0 to 1, a measure of the nanoparticle size distribution width). Adapted from paper V.

The AC source, which is connected to the excitation coil, feeds a time-dependent sinusoidal current and results in a time-dependent excitation field; while the pickup coil measures the rate of magnetic flux change caused by the presence of the sample (Fornara et al., 2008; Zardán Gómez de la Torre et al., 2011). The signal obtained by the pickup coil is sent to a lock-in-amplifier, which extracts the signal amplitude at the frequency of the AC source. During the measurement, the frequency-dependent in-phase (χ') and

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out-of-phase (χ'') components of χ are recorded, as shown in Figure 1b. The out-of-phase component of χ, which means 90° phase difference with respect to the excitation field, reaches a maximum value (peak value) when the exci-tation frequency is equal to the Brownian relaxation frequency. The charac-teristic frequency for Brownian relaxation dynamics is given by = 6

which is related to the hydrodynamic volume of the relaxing entity, i.e., MNPs as well as biological objects captured by MNPs. As a result, the bind-ing and/or size increase of MNPs can be quantitatively measured by the set-up, which can be further utilized to design biosensors. Furthermore, the AC susceptometer can differentiate between MNPs of different hydrodynamic sizes based on the frequency separation between peak positions, which can be used for multiplexing.

2.2.2 Optomagnetic biosensor Optomagnetic sensors can, for instance, measure the AC magnetic field-induced modulation of the optical transmission signal from samples with suspended MNPs. Since it was first reported in 2014 by Donolato et al. (Donolato et al., 2015), a 405 nm laser-based optomagnetic readout system has been employed for the detection of several kinds of biomolecules and pathogens (Antunes et al., 2015; Mezger et al., 2015; Yang et al., 2016).

As illustrated in Figure 2, this optomagnetic sensor is typically based on an unfocused 405 nm laser source (Sony optical unit, Sony, JP) and a photo-detector (PDA36A, Thorlabs Inc., U.S.A.). The laser source provides a line-arly polarized light beam with a diameter of 2 mm. A disposable cuvette can be positioned in the beam path (the optical path is 10 mm through the sample in the cuvette), centred between a pair of electromagnets (1433428C, Murata Power Solutions Inc., U.S.A.). The AC magnetic field is applied perpendicu-lar or parallel to the laser beam with maximum AC magnetic field amplitude of approximately 2.6 mT. The distance between the electromagnets is 20 mm, and the distance between laser source and detector is 115 mm. The laser, electromagnets, cuvette, and detector are covered to avoid interference from external light sources during measurements. Signals measured by the photodetector is converted by a data acquisition unit (DAQ unit, NI USB-6341, National Instruments, U.S.A.) from analogue to digital, followed by further processing in the computer by a lock-in function.

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Figure 2. Schematic illustration of the 405 nm laser-based optomagnetic set-up. The liquid sample, contained in an optically transparent cuvette (5), is placed between two identical electromagnets (7). A 405 nm laser source (6) generates a laser beam aimed at the bottom of the cuvette. The intensity of transmitted light detected by a photo detector (4) is recorded vs time using a DAQ unit (2). The laser and electro-magnets are powered by a current source (3). A computer (1) controls the entire set-up and performs the software based lock-in detection.

The principle of the 405 nm laser-based optomagnetic readout system is based on the rotational dynamics of MNPs. MNPs employed in this study have a remnant magnetic moment, which implies that the dominating relaxa-tion mechanism upon a reversal of the magnetic field direction is a physical rotation of the particle, i.e., Brownian relaxation. The modulation of the transmitted light intensity is found in the complex second harmonic voltage component output from the photodetector = ′ + i ′′ where V2' and V2'' are the in-phase and out-of-phase signals, respectively. The modulation is measured using a lock-in amplifier as the second harmon-ic component of the voltage output from the photo detector. A typical V2'/V0 spectrum (where V0 is the simultaneously measured total transmitted light intensity) is characterized by a peak (or a valley) representing the response from suspended MNPs. The position of the peak (valley), fpeak, is related to the Brownian relaxation frequency as (Fock et al., 2017a) ≈ √33

Therefore, similar to the principle of the AC susceptometer described in section 2.2.1, the binding and/or size increase of MNPs can be quantitatively measured by the optomagnetic sensor. Additionally, the concentration of MNPs in the sample is reflected by the amplitude of the V2'/V0 spectrum at fpeak. The sign of the characteristic peaks (valleys) depends on the optical scattering properties and the measurement geometry (the AC magnetic field is applied either parallel or perpendicular to the laser beam). The sign chang-

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es of the characteristic peaks (valleys) are caused by the dependence of the optical extinction coefficient on the sizes of MNPs (Fock et al., 2017b).

In papers I, II, III, VII and VIII, quantification of target analytes was ac-complished by monitoring either the valley or peak amplitudes. In paper IV, the shift of V2'/V0 spectra were measured to quantify the size increase of MNPs.

2.2.3 Ferromagnetic resonance biosensor Ferromagnetic resonance (FMR) is a phenomenon that is related to the pre-cession of the magnetization vector of a ferro- or ferrimagnetic sample. In contrast, nuclear magnetic resonance (NMR) is related to the magnetic mo-ment of atomic nuclei. FMR has been applied in the study of magnetic ani-sotropy in single crystals (Smit and Beljers, 1955), thin films (Farle, 1998) and nanoparticles (Raikher and Stepanov, 1992; Usselman et al., 2010; Winklhofer et al., 2014). The ferromagnetic resonance field (Bres), the prin-cipal quantity of interest of FMR, is the zero-crossing of the magnetic field derivative FMR absorption spectrum. In ferromagnetic materials, Bres varies with the net magnetic (shape-, magnetocrystalline-) anisotropy of the system (Charilaou et al., 2011; Kittel, 1948). Since binding or clustering of MNPs can change the net magnetic anisotropy of the suspension, FMR sensors can possibly be used to study the binding of bio-macromolecules to MNPs by monitoring the shift of the ferromagnetic resonance field (ΔBres). This has been employed for designing a biosensor in paper VI.

In a field-swept FMR cavity measurement, microwave absorption of the sample is measured when a magnetic field is swept through the resonance of the sample. When the frequency of the microwave field matches the preces-sion frequency of the sample magnetization, absorption in FMR occurs. The output signal is the magnetic field derivative of the microwave absorption (dA/dB). In paper VI, FMR measurements were performed on a Bruker ELEXSYS-E580 EPR (electron paramagnetic resonance) spectrometer equipped with a standard X-band (9.47 GHz) cavity. The sample was trans-ferred into a quartz capillary and placed in a standard EPR tube. A sketch of the EPR cavity and magnet used for FMR sensing is shown in Figure 3a. During the measurement, the field is swept over a range of 15-8000 G and dA/dB of the sample is recorded. By using the signal obtained in the field range of 6000-8000 G, the offset is manually adjusted to 0. To extract Bres, data around the zero-crossing point is fitted using linear regression.

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Figure 3. A sketch of the EPR cavity and magnet used for FMR sensing. Black and red arrows indicate the direction of magnetic field and microwave, respectively. Adapted from paper VI.

2.3 Biosensing strategies The choice of biosensing strategies is crucial for the development of optimal biosensors. In this thesis, different strategies are introduced based on their application in papers, as summarized in Table 1.

2.3.1 Immunoassays The most prominently used bio-recognition element in biosensors is antibod-ies. Antibody-antigen reaction based sensors, which are also called immuno-sensors, have been adopted on a wide range of biosensors. Antibodies, which can also be called immunoglobulins (Ig), are proteins secreted by lympho-cytes that have extremely high affinity (dissociation constant, kd, ranges from 10-11 to 10-8 M) and selectivity for their targets (Crivianu-Gaita and Thompson, 2015). Among different kinds of Ig, IgG is the most widely uti-lized class in biosensing. IgG consists of two heavy protein chains (50 kDa each) and two light protein chains (25 kDa each) linked together by disul-phide bonds, and has a molecular weight of about 150 kDa and dimensions of 13.7 nm (width) and 8.4 nm (height) (Tan et al., 2008). Chains of IgG have both constant and variable regions. The variable region of the heavy chain is responsible for antigen binding. The variable region of the light chain contains an important part of the antigen-binding site.

In papers I-III, immuno-sensors were designed for the detection of whole bacteria or protein biomarkers. Antibodies were conjugated onto the MNPs through biotin-streptavidin (or biotin-avidin) reaction by simply mixing streptavidin (or avidin) modified MNPs with biotinylated antibodies. After incubation and washing steps using a magnetic stand, IgG-MNPs were pre-pared for further use.

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In paper I, a competitive immunoassay format was established for the de-tection of S. typhimurium cells and/or fragments, which is illustrated in Fig-ure 4. During the reaction, target cells and/or fragments, if suspended in the sample, will be captured by biotinylated IgG on the surface of the magnetic micro-particles (avidin modified 5 µm beads). The antibody-antigen reaction can sterically prevent the biotinylated IgG from binding with the subsequent-ly added detection nanoparticles (streptavidin modified MNPs). If no target is present, detection nanoparticles will attach to the magnetic micro-particles due to the streptavidin-biotin reaction, leading to a decrease of the V2'/V0 peak amplitude since the Brownian relaxation frequency of micron-sized magnetic objects is out of the detection range of the optomagnetic setup. Therefore, the V2'/V0 peak amplitude can be used for the detection of S. typhimurium in the sample. In contrast to paper I, paper III adopted a direct immunoassay format and measured the MNPs that did not bind to the cells/fragments.

Figure 4. Schematic illustration of the optomagnetic detection of S. typhimurium using a competitive immunoassay strategy. Three independent steps, i.e., capture, labelling and detection, are illustrated from left to right. Top and bottom rows repre-sent the detection of positive and blank samples, respectively. Adapted from paper I.

In paper II, antibody-antigen reactions were utilized to build a shape anisot-ropy enhanced optomagnetic biosensor, which is illustrated in Figure 5. In the presence of the rotating magnetic field (RMF, which will be specified in section 2.3.2), IgG-functionalized 250 nm MNPs form dipolar magnetic chains which are further stabilized by the multivalent antigen (target ana-lyte). After a short period of incubation, the RMF is turned off to allow dis-assembly of non-stabilized chains. In the optomagnetic measurement step, dissociated MNPs show different V2'/V0 spectra from that of MNP chains, which is utilized to quantify the multivalent antigen.

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Figure 5. Schematic illustration of the shape anisotropy enhanced optomagnetic detection strategy. Two independent steps are included, namely incubation (in RMF) and detection. Gray and red arrows indicate assays of a blank sample (the left pan-els) and of a positive sample (the right panels), respectively. Adapted from paper II.

2.3.2 Rotating magnetic field platform Magnetic field-assembled magnetic particle chains can be stabilized by the binding of multivalent molecules (Furst et al., 1998; Goubault et al., 2005). Concentrations of multivalent molecules can be quantified through the measurement of the shape anisotropy (in general, the length) of the chains, which has been utilized to design biosensors (Park et al., 2010a; Park et al., 2010b; Ranzoni et al., 2011; Vuppu et al., 2004). In these chain formation based biosensors, transmitted (or scattered) laser light is analysed to quantify the target multivalent molecules.

In paper II, a rotating in-plane magnetic field platform was used to pre-pare MNP chains during incubation of the immuno-reaction. The high shape anisotropy of the MNP chains improved the biosensor performance by offer-ing efficient mixing (via magnetic shape anisotropy) and increasing the sig-nal amplitude (via optical shape anisotropy).

The platform contained a pair of perpendicular iron-core magnetic circuits that were powered by a Hewlett Packard 6626A power supply and controlled

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by a computer. The platform could generate a homogeneous magnetic field in the central part of the platform with controlled field strength and angular rotation frequency. Formation of chains of superparamagnetic MNPs is in-fluenced by both the rotational shear force and the magnetic interaction force, which can be described by the dimensionless Mason number, Mn (Melle and Martin, 2003; Petousis et al., 2007) M ≈ 16

where ω is the angular rotation frequency of the RMF, HRMF is the magnetic field amplitude, and µ0 is the permeability of free space. Then the average number of MNPs in a stable chain can be described by (Petousis et al., 2007) ≈ M .

The binding force induced by the binding of multivalent target molecules, decreases the Mason number (Park et al., 2010a). Therefore, the average chain length is increased due to the binding force.

2.3.3 Rolling circle amplification Rolling circle amplification (RCA) is a simple isothermal enzymatic process that produces long single stranded DNA or RNA using primers, circular templates and unique DNA or RNA polymerases (e.g., Phi29 and Bst) (Kuhn et al., 2002; Lee et al., 2012; Murakami et al., 2009; Zhao et al., 2008). One major limitation for polymerase chain reaction (PCR) is the requirement for thermal cycler devices. In contrast, RCA can be performed at a constant temperature ranging from room temperature to 37°C, which makes RCA a promising amplification strategy for POCT. Traditional applications of RCA are focused on diagnostic methods by directly amplifying the target mole-cule or the template attached to the target molecule.

In papers III and VI, a method named VAM-NDA (volume-amplified magnetic nanoparticle detection assay) was applied to induce MNP aggrega-tions in the presence of target DNA (see Figure 6). In VAM-NDA, a special template, a so-called padlock probe, is designed with complementary regions to the target nucleic acid sequence. After hybridized with the target se-quence, the two ends of the padlock probe are positioned into juxtaposition, followed by a ligation reaction. Thereafter the circular padlock probe is am-plified by RCA, resulting in long single stranded products containing ap-proximately 300-1000 copies (depending on the RCA reaction time) of the sequence of the padlock probe. The micrometre-long RCA products collapse into coils in the aqueous solution. MNPs that are modified with detection probes (a short synthetic DNA sequence that is complementary to a certain sequence of the padlock probe) can bind to the RCA coils through hybridiza-

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tion, resulting in changes of their Brownian relaxation frequency as well as the net magnetic anisotropy, which can be measured by optomagnetic sen-sors and FMR sensors, respectively.

Figure 6. Schematic illustration of volume-amplified magnetic nanoparticle detec-tion assay. RCA amplicons contain repeated sequences that are identical to the loop primer (padlock probe). Adapted from paper III.

RCA products with programmable repeating sequences can be further uti-lized as scaffolds for assembling nanostructures into one-dimensional super-structures (Ali et al., 2014; Beyer et al., 2005; Deng et al., 2005; Russell et al., 2014). Moreover, RCA products can be synthetized on the particle sur-face, followed by assembling other nanostructures, resulting in programma-ble three-dimensional superstructures (Yan et al., 2015; Yan et al., 2012; Yan et al., 2010; Zhao et al., 2006; Zhu et al., 2016). In paper VIII, on-particle RCA with biotinylated DNA building blocks was used to prepare MNP core-satellite magnetic superstructures.

2.3.4 Loop-mediated isothermal amplification Loop-mediated isothermal amplification (LAMP) is an ultra-efficient iso-thermal enzymatic amplification strategy. Due to its high amplification effi-ciency, low cost and the ability of being applied to non-denatured DNA samples, LAMP holds the potential to revolutionize molecular diagnostics (Notomi et al., 2000; Tomita et al., 2008). A well-designed LAMP reaction can produce more than 109 amplicons within 1 h, but the performance of a LAMP-based assay highly depends on the readout method (Parida et al., 2008; Zhang et al., 2014). Due to the precipitation of Mg2P2O7 (a by-product of LAMP), results of LAMP reactions can be qualitatively determined by naked eye but with limited sensitivity (Zhang et al., 2014).

In paper IV, biotinylated LAMP products (LAMP using biotinylated pri-mers) were reacted with streptavidin coated MNPs that increased the hydro-dynamic size of MNPs. The size increase of MNPs, quantified by monitoring

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the shift of the V2'/V0 spectra, is related to the concentration of target mole-cules in the sample. The detection strategy is illustrated in Figure 7.

Figure 7. Schematic illustration of LAMP-optomagnetic NDV detection. Three independent assay steps (from left to right) are included as: LAMP reaction, label-ling and measurement. Adapted from paper IV.

In paper V, the LAMP reaction step and the labelling step were performed at the same time, which makes the detection strategy homogeneous and simple. Target oligonucleotides were directly mixed with streptavidin-MNPs and other LAMP reagents including biotinylated LAMP primers. Since the bio-tin-streptavidin reaction is believed to be much faster than the LAMP reac-tion, this detection strategy can be regarded as on-particle LAMP (Figure 8). In this strategy, the hydrodynamic size increase of MNPs during the LAMP reaction is caused not only by the binding of amplicons, but also by the bind-ing of Mg2P2O7 precipitation.

Figure 8. Illustration of the LAMP based susceptometry assay. Two independent assay steps, i.e., incubation and measurement, are included in the illustration from left to right. Adapted from paper V.

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In paper VI, LAMP reaction was performed with naked MNPs, resulting in co-precipitation of MNPs and Mg2P2O7. The co-precipitation changed the net magnetic anisotropy of the sample, thus being detected by the FMR sen-sor. Note that no bio-recognition element was employed in this strategy. Therefore, it is not a true biosensor and the response of the MNPs can here only be regarded as an indicator of the LAMP reaction.

2.3.5 Duplex-specific nuclease assisted target recycling In recent years, duplex-specific nuclease (DSN) has been reported for iso-thermal microRNA analysis. DSN can cleave DNA in DNA:RNA heterodu-plexes while keeping the RNA strand intact (Qiu et al., 2015). This special property means that the RNA sequence in the DNA:RNA heteroduplexes is preserved in the reaction and thus can form new heteroduplex structures with other complementary DNA sequences in the solution. This method, which is denoted DSN-assisted target recycling, has been employed in many kinds of biosensors for microRNA detection.

DSN recognizes and cuts perfect DNA:DNA duplexes no shorter than 10 bp, or perfect DNA:RNA duplexes no shorter than 15 bp (Qiu et al., 2015). It means that DSN is able to discriminate between perfectly and imperfectly matched short duplexes in case the identical part of the sequences is no long-er than 15 bp. Actually, in the experiments of this thesis, sequences that have an identical part of 16 bp can also be distinguished (but not perfectly) by DSN.

In papers VII and VIII, DSN-assisted target recycling was used with core-satellite magnetic assemblies, and the principle of detection is specified in section 2.3.6.

2.3.6 Core-satellite magnetic assemblies The special requirements of POCT diagnostics, such as ease-of-use and short assay time, are the principal motivations behind the development of biosen-sors. However, these special requirements are challenging for traditional analytical assays due to the limited capability of monofunctional-material-based biosensors (Huang and Lovell, 2017; Kelley et al., 2014; Syedmoradi et al., 2017). To meet the requirements and improve the biosensing perfor-mance, simultaneous utilization of several materials is a common strategy. Rather than using a mixture of different monofunctional materials, using a multifunctional assembly of various simple building blocks is a promising choice, which takes advantage of both the intrinsic and collective properties of the building blocks (Cheng et al., 2012; Cho et al., 2011; Kostiainen et al., 2013; Salem et al., 2003).

DNA has been widely used in nanoscience as scaffold, spacer or func-tional polymer of nano- to micron-sized assemblies. In such multifunctional

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assemblies, DNA offers unique advantages due to its controllable size, mo-lecular net charge, specific hybridization and predictable versatile enzymatic processes (Hong et al., 2017; Mucic et al., 1998; Nykypanchuk et al., 2008; Zhang et al., 2013). The DNA-assembled core-satellite superstructure offers controllable dimension, composition, functionality of the building blocks as well as the sequence of DNA, which makes it a versatile tool for POCT di-agnostics (Chou et al., 2014; He et al., 2017; Liong et al., 2014; Sebba et al., 2008; Sun et al., 2016; Wang et al., 2017; Zhao et al., 2016).

In papers VII and VIII, core-satellite magnetic assemblies were prepared and used with DSN-assisted target recycling for microRNA detection. DNA probes of target microRNA were serving as scaffold of the core-satellite superstructure, which could disassemble due to the DSN-based reaction and release MNP satellites for optomagnetic detection. The working principle is shown in the Figure 9.

Figure 9. Schematic illustration of microRNA detection based on multilayer core-satellite magnetic assemblies. The top and bottom rows show the principle of sin-gleplex and duplex detection of microRNA, respectively. Adapted from paper VII.

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3. Results and discussion

This chapter contains selected highlights from the appended papers. Meas-urement results of each detection strategy (i.e., immunoassay, RCA-based assay, LAMP-based assay, and core-satellite assembly-based assay) are shown for comparison. Dose-response curves and LODs are shown as the main results.

3.1 Immunoassay based bacteria/biomarker detection 3.1.1 Performance of immunoassays For competitive immuno-detection of S. typhimurium cells/fragments per-formed in paper I, the lowest LOD, 8×104 CFU/mL, was achieved at an IgG-modified magnetic particle concentration of 5×105 particles/mL (Figure 10a). In general, the competitive immunoassay format is less sensitive than the corresponding direct immunoassay format. However, a comparable di-rect immunoassay strategy for S. typhimurium cell/fragment (performed in paper III, see Figure 10b) has an LOD of 2×106 CFU/mL, which corrobo-rates previous results (5.6×106 CFU/mL) reported by Grossman et al. (Grossman et al., 2004), but less sensitive than the competitive detection format. Two reasons are considered for explaining this: (1) biotin groups on the surface of the IgG-magnetic particle are much more concentrated than the antigen sites on the surface of typical bacteria, and (2) the formation of immuno-magnetic aggregates shields the IgG-magnetic particles from being captured by the detection MNP.

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Figure 10. Competitive (a) and direct (b) immunoassays for the detection of S. typhimurium cells/fragments. Error bars indicate the standard deviation of three independent replicates. Adapted from paper I.

3.1.2 Immunoassay duplexing Direct and competitive immunoassays were simultaneously performed in the same sample for the detection of E. coli and S. typhimurium. MNPs of 250 nm and 100 nm sizes were used as detection labels for E. coli and S. typhi-murium, respectively. As shown in Figure 11, each spectrum has its own signature in terms of valley/peak amplitudes and positions.

Figure 11. Average V2'/V0 spectra of three independent measurements for a homo-geneous duplex immunoassay. The optomagnetic response of free IgG-250 nm MNPs and streptavidin-100 nm MNPs was characterized by a valley/peak at 18 Hz and 184 Hz, respectively. E: 107 CFU/mL E. coli; S: 107 CFU/mL S. typhimurium. 0: absence of bacteria. Adapted from paper I.

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3.1.3 Shape anisotropy enhanced immunoassays In paper II, PSA was detected in 50% serum samples using the shape anisot-ropy enhanced optomagnetic biosensor. The V2'/V0 spectra and dose-response curve of PSA samples are shown in Figure 12a and b, respectively. An LOD of 21.6 pM (0.65 ng/mL) was achieved according to the 3σ criteri-on and with an upper dynamic detection limit of 66.7 nM (2 µg/mL). The sample-to-answer time is approximately 20 min, which consists of 15 min for RMF incubation and 4.5 min for optomagnetic measurement.

Figure 12. Average V2'/V0 spectra (a) and dose-response curve (b) for PSA detec-tion using shape anisotropy enhanced optomagnetic biosensor. RMF: rotating mag-netic field. Error bars indicate the standard deviation of three independent replicates. Adapted from paper II.

3.2 Molecular amplification based DNA/RNA detection 3.2.1 RCA-based biosensing In paper III, VAM-NDA was employed for DNA detection with an LOD of 780 fM (cf. Figure 13).

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Figure 13. Peak value of V2'/V0 (normalized with respect to the blank control value) vs DNA concentration. Error bars indicate one standard deviation based on three independent measurements. Adapted from paper III.

In paper VI, VAM-NDA was employed for evaluating the FMR biosensor and for comparing with other well-studied biosensors. An LOD of 1 pM DNA coils was achieved (cf. Figure 14).

Figure 14. FMR spectra (a) and dose-response curve (b) for RCA-based homogene-ous and volumetric detection of the synthetic V. cholerae sequence. The inset in (b) shows a close-up lin-log plot of the data in panel (b). The black dotted horizontal line in (a) indicates the zero value of dA/dB. Error bars indicate the standard devia-tion of three independent replicates. Adapted from paper VI.

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3.2.2 RCA duplexing An RCA-based duplex DNA detection principle was demonstrated in paper III. In the duplex DNA detection, 100 and 250 nm MNPs were employed as detection labels to bind to RCA products originating from V. cholerae and E. coli, respectively. As shown in Figure 15, similar to the duplex immunoas-say, each spectrum has its own signature in terms of valley/peak amplitudes and positions.

Figure 15. Average V2'/V0 spectra based on three independent measurements for the RCA-based duplex DNA detection. The valley/peak at 11 Hz and 143 Hz are corre-sponding to non-bound 250 nm and 100 nm MNPs, respectively. E: 100 pM E. coli. V: 100 pM V. cholerae target sequence. 0: absence of target sequence. Adapted from paper III.

3.2.2 LAMP-based biosensing LAMP was utilized for optomagnetic detection of NDV (both synthetic DNA sequence and real RNA sample) in paper IV. A higher concentration of analyte gave longer biotinylated LAMP products, thus a denser layer of LAMP amplicons on the MNPs. The shift of the optomagnetic peak position was monitored for quantification of the analyte. An LOD of 10 aM target sequence was obtained, and the dose-response curve is shown in Figure 16 for triplicate measurements. Note that Bst 3.0 polymerase can amplify both RNA and DNA sequences under the same conditions. The results of Zika virus detection are similar and thus not shown here.

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Figure 16. Average V2'/V0 spectra (a) and dose-response curve (b) for LAMP-based optomagnetic detection of synthetic NDV target sequence. Error bars indicate the standard deviation. Adapted from paper VI.

3.3 Core-satellite assembly-based microRNA detection 3.3.1 Multilayer core-satellite superstructure based biosensing In paper VII, let-7b (a cancer related microRNA biomarker) was detected using DSN-assisted target recycling and core-satellite superstructures. The superstructure has a 1 µm magnetic bead core surrounded by 80 nm MNP satellites, DNA probes are serving as the linker/scaffold between core and satellites. After the DSN-based reaction, released MNP satellites were quan-tified by the optomagnetic sensor. As shown in Figure 17, a linear correla-tion between the V2'/V0 peak amplitude and logarithm of let-7b concentration was obtained. The LOD was calculated to be 4.8 fM.

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Figure 17. Average V2'/V0 spectra (a) and dose-response curve (b) for two-layer core-satellite superstructure based let-7b detection. Error bars indicate the standard deviation of three independent replicates. Adapted from paper VII.

3.3.2 On-particle RCA enhanced core-satellite superstructures By using RCA products as the DNA linker/scaffold between core and satel-lites, the load of MNP satellites was doubled compared with the method described in section 3.3.1. Moreover, during the DSN-based hydrolysis reac-tion, the MNP releasing speed of the RCA-based core-satellite superstruc-tures is higher than that of the DNA probe-based core-satellite superstruc-tures, which is caused by the layer-by-layer preparation strategy of the DNA probe-based core-satellite superstructures. In layer-by-layer preparation, MNP satellites were linked to the core via more than one DNA probe, which means more than one hydrolysis reaction is needed to release one single satellite.

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4. Concluding remarks

MNPs bring new aspects to out-of-lab and POCT diagnostics due to their unique properties, e.g., high surface-to-volume ratio, easy manipulation by external magnetic field, simplicity of bio-functionalization, signal stability and low background noise in biological samples. Herein, different detection strategies, sensors and nanotechnologies have been combined and studied for MNP-based biosensing. POCT related pathogens, proteins and nucleic acids were chosen as the target analytes.

For the immunoassay of Salmonella in paper I, an LOD of 8×104 CFU/mL was achieved within a total assay time of 3 h. Enhanced by the field-assembly of dipolar magnetic chains (paper II), the system achieved an LOD of 21.6 pM (0.65 ng/mL) PSA in 50% serum with a total assay time of approximately 20 min.

In papers III and VI, RCA was applied for DNA detection, resulting in LODs of 780 fM and 1 pM, respectively, within 2 h.

For LAMP-based biosensors introduced in papers IV and V, LODs of 10 aM and 1 aM were obtained in 30 min for NDV and for Zika virus, respec-tively.

At last, in paper VII, microRNA was detected with an LOD of 4.8 fM and an assay time of 70 min, which was further improved by the utilization of on-particle RCA in paper VIII.

Nearly all the proposed biosensors are designed based on homogeneous reactions as well as on volumetric sensors, which make them simple (with-out pretreatment or washing step) and rapid. In addition, rather than the sur-face-based biosensors, homogeneous and volumetric biosensors can be easi-ly adopted for in vivo clinical applications in combination with, for example, drug delivery and magnetic hyperthermia therapy. The key properties of MNPs used in these work, i.e., hydrodynamic sizes and net magnet anisotro-py, hold the potential for multiplex diagnostics.

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Svensk sammanfattning

En biosensor är en analytisk sensoranordning för att påvisa närvaron av olika sorters målmolekyler, t.ex. proteiner och bakterie- eller virus-DNA/RNA i ett prov. Provtyper kan vara t.ex. blod- och urinprover, livsmedelsprover mm. I biosensorn känns målmolekylen igen med hjälp av s.k. biomolekylära prober, t.ex. antikroppar för detektion av proteiner och DNA/RNA-sekvenser för detektion av t.ex. komplementära DNA/RNA-sekvenser. Reaktionen mellan mål- och probmolekyler skapar via en s.k. signalöverföringsmekan-ism (transduktionsmekanism) en utsignal och div. elektronik omvandlar denna signal till ett tolkningsbart svar som kan vara antingen kvalitativt (ja/nej) eller kvantitativt (hur stor koncentration). För att öka biosensorns prestanda i fråga om t.ex. känslighet kan s.k. molekylära verktyg användas vilka väldigt specifikt kan känna igen målmolekylen eller förstärka den ge-nom att t.ex. kopiera upp den med hjälp av ett enzym.

Det finns i dagsläget både ute i samhället och inom akademin ett ständigt ökande behov att utveckla nya snabba, enkla och kostnadseffektiva biosen-sorer för tillämpningar inom vitt skilda områden såsom human och veteri-närmedicin, miljösäkerhet samt livsmedelssäkerhet. För att vara attraktivt att användas i utvecklingsländer eller i andra sammanhang med begränsade ekonomiska resurser behövs biosensorerna kunna användas utanför avance-rade laboratorier (“out-of-lab”) eller i nära anslutning till patienten (“point-of-care”) eller på plats (“in-field”) av personer utan särskilda specialistkun-skaper. Under de senaste åren har en explosion av publikationer rörande enkla och kostnadseffektiva biosensormetoder baserade på olika transdukt-ionsmekanismer t.ex. optisk, akustisk, elektrokemisk och magnetisk utläs-ning skett i den vetenskapliga litteraturen. Just magnetiska biosensorer an-vänder sig av ändringar i egenskaper hos magnetiska nanopartiklar och har ett flertal unika fördelar. Eftersom de flesta biologiska prover i sig är icke-magnetiska blir bakgrundsignalen låg. Vidare kan magnetiska nanopartiklar produceras till låg kostnad och har stabila egenskaper som kan mätas med billig utrustning. Magnetiska nanopartiklar möjliggör även anrikning av målmolekylen genom att applicera ett yttre fält (bioseparation). Magnetiska biosensorer brukar indelas i två huvudkategorier, 1) ytbaserade resp. 2) vo-lymsbaserade. I ytbaserade magnetiska biosensorer används någon form av prob-funktionaliserad magnetisk sensoryta, t.ex. mikro-Hall effekt sensor eller GMR (“giant magnetoresistance”) till vilken magnetiska partiklar bin-der i närvaro av målmolekylen. I den andra kategorin görs utläsningen ho-

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mogent i hela detektionsvolymen och ett exempel på denna kategori är AC-susceptometriska biosensorer vilka mäter ändringar i den dynamiska magne-tiska responsen (komplex magnetisk volymssusceptibilitet) för magnetiska nanopartiklar till följd av att målmolekyler binder till deras yta vilket gör att partiklarnas hydrodynamiska volym ökar. Optomagnetiska biosensorer kan betraktas som en underkategori till magnetiska biosensorer som erbjuder ett homogent detektionsformat där ljus får växelverka med magnetiska nanopar-tiklar och man mäter ändringar i t.ex. polarisationsriktning eller intensiteten för ljus som gått genom provet (transmitterat ljus). Optomagnetiska biosen-sorer har fördelarna att ljusstrålen kan interagera med en stor del av provet utan att behöva vara i fysisk kontakt med provet. Vidare är instrumentering-en kostnadseffektiv. Biosensorer som baseras på s.k. ferromagnetisk reso-nans (FMR) är ytterligare en underkategori av magnetiska biosensorer som även de erbjuder ett homogent detektionsformat som utnyttjar ändringar i mikrovågsabsorption för magnetiska nanopartiklar under inverkan av ett pålagt magnetfält.

Den övergripande målsättningen med detta avhandlingsarbete har varit att med hjälp av en tvärvetenskaplig forskningsmetodik utveckla enkla, snabba och känsliga magnetiska biosensorer för två huvudapplikationer, 1) detektion av bakterier och virus relaterade till veterinärmedicin och livsmedelssäkerhet samt 2) för human cancerdiagnostik. I samtliga biosensormetoder har mag-netiska nanopartiklar av maghemit (γ-Fe2O3) använts som uppvisar s.k. Brownsk relaxation d.v.s. partiklarna roterar i ett pålagt magnetiskt AC-fält. Varje partikel består av ett kluster av mindre nanopartiklar (“multi-core”) sammanhålla stärkelse- eller dextranhölje där olika sorters biomolekylära prober enkelt kan fästas. Partiklarna är något oregelbundna till sin form och har därför en svag optisk anisotropi. Fundamenten för utläsningen i biosen-sorprinciperna har varit användandet av tre olika sensorplattformar, 1) en optomagnetisk sensorplattform som mäter modulationen av intensiteten för transmitterat blått laserljus genom ett prov med magnetiska nanopartiklar som induceras av ett pålagt svagt oscillerande magnetfält, 2) ett AC-susceptometersystem, DynoMag®, som mäter den komplexa magnetiska volymssusceptibiliteten, samt 3) ett FMR-baserat sensorsystem som mäter mikrovågsabsorption som funktion av ett pålagt DC-magnetfält. Genom att utnyttja olika molekylära verktyg för igenkänning och amplifiering av målmolekylen vilka leder till att den hydrodynamiska volymen för de mag-netiska nanopartiklarna ökar eller att partiklarna aggregerar eller att magne-tiska nanopartiklar lösgörs från ytan av mikropartiklar ändras provets opto-magnetiska, dynamiska magnetiska respons eller FMR-respons varvid kvan-tifiering av analyten kan åstadkommas.

Sammanfattningsvis, som viktigaste resultat, har följande biosensormeto-der utvecklats i denna avhandling:

1. En optomagnetisk biosensor för detektion av salmonellabakterier (fragment av bakterieceller). Detektionsprincipen som är immu-

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nomagnetisk bygger på att magnetiska mikropartiklar funktional-iserats med biotinylerade antikroppar mot salmonella varvid s.k. immunomagnetiska kluster bildas. Därefter tillsätts streptavidin-klädda magnetiska nanopartiklar vilka binder till biotingrupperna på antikropparna i en utsträckning som beror av koncentrationen sal-monellabakterier, alltså en s.k. kompetitiv immunomagnetisk detekt-ionsstrategi. Koncentrationen av fria nanopartiklar kan mätas opto-magnetiskt vilket möjliggör kvalitativ analys av mängden bakterier.

2. Hyperkänsliga optomagnetiska och AC-susceptometriska biosenso-rer för detektion av Newcastle- och Zikavirus. I dessa arbeten har en enzymatisk amplifieringsmetod som kallas LAMP (“loop-mediated isothermal amplification”) använts för igenkänning och amplifiering av virussekvenserna. LAMP-reaktionen genererar sicksackformade stora DNA-molekyler (LAMP-produkter) som binder till ytan av magnetiska nanopartiklar och därmed ökar partiklarnas hydrodyna-miska volym vilket i sin tur ändras provets AC-susceptometriska resp. optomagnetiska svar. Detta ger en kvantitativ analys av kon-centrationen bakterier.

3. FMR-baserad biosensor för detektion av RCA (“rolling circle amplifi-cation”)- och LAMP-produkter från bakteriella målsekvenser för kole-rabakterier (RCA-strategi) och Zikavirus (LAMP-strategi). RCA är en enzymatisk amplifieringsmetod som genererar stora DNA-nystan (RCA-produkter). När nanopartiklar med DNA-prober på ytan binder till nystanen skapas aggregat av partiklar vilket ändras provets FMR-respons. LAMP-reaktionen resulterar även i en biprodukt, ett magne-siumsalt, i vilket nanopartiklar kan kapslas in i vilket också skapar ag-gregat av nanopartiklar. Metoderna ger kvantitativa svar.

4. Optomagnetiska biosensorprinciper för detektion av mikro-RNA biomarkörer för cancer. Här har s.k. superstrukturer av magnetiska mikro- och nanopartiklar (“core-satellite superstructures”) använts där satellitnanopartiklar bundits till ytan av mikropartiklar via DNA-molekyler som är komplementära till målsekvensen (mikro-RNA). En annan typ av superstrukturer som använts är mikropartiklar med RCA-produkter på ytan där nanopartiklar bundits in. Ett enzym som kallas DSN (“duplex-specific nuclease”) klipper loss nanopartiklar från mikropartiklarna i närvaro av mikro-RNA vilket lösgör satellit-partiklar. Metoderna ger kvantitativa svar.

Biosensormetoderna som avhandlingsarbetet resulterat i har analystider och känsligheter jämförbara eller bättre än existerande konventionella analysme-toder och bedöms därför ha stor potential att utvecklas till kommersiella tester avsedda att användas på plats och i fält i resurssvaga delar av världen där tillgången på avancerade analyslaboratorier och specialutbildad teknisk personal är begränsad.

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Acknowledgements

First of all I would like to thank my main supervisor Mattias Strömberg for offering an opportunity to work in this amazing field and to live in this cool country. Thank you for your support and trust, based on which I enjoy max-imum freedom and can always research on what I am interested in. You nev-er require me about what should be learnt and what should be done. Sorry to say that, although you advised me one thousand times about biking for a healthy life, I keep commuting by bus. Thanks to my co-supervisor Peter Svedlindh, for your patience of teaching me physics from very basic knowledge. I would say it is a surprising, memorable and valuable experi-ence to talk and work with a physicist like you. And my apologies for never being into the equations you wrote in my papers; I just copy and paste them when needed. I would also like to express my gratitude to my co-supervisor Erik Wetterskog, your enthusiasm is really infectious. Thank you for your priceless suggestions about how to plan and continue my academic career. To Mikkel Fougt Hansen and Marco Donolato, thank you for sharing your knowledge and ideas on optomagnetic sensors. Although not been registered anywhere, I regard you as my co-supervisors.

To Zhen Qiu and Jing Ma, thank you for spending time on the experi-ments I required, and for teaching me those technologies. Thanks to all my Chinese friends in Ångström, for your tolerance for me and my vitriolic jokes. And my apologies for never serving you a party.

Thank you Sofia Kontos, it is really nice to share the office with you. You can always bring happiness (and candy) to my life. Sorry to say I ignore your invitations of going to the gym, learning Swedish, and joining your party. Many thanks go to all the people in the division of Solid State Physics for the wonderful working climate, and for fika. Thank you Bengt Götesson, the coffee machine is very important to me and my lovely biosensors.

Last but not least, Yuan, thank you for always supporting and being there for me. I love you, and your cooking skill.

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