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Magnetic Nanoparticle Based Biosensors for Pathogen Detection and Cancer Diagnostics
Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Solid State Physics.ORCID iD: 0000-0002-5249-4415
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes several magnetic nanoparticle (MNP)-based biosensing strategies which take advantage of different magnetic sensors, molecular tools and nanotechnologies. Proposed biosensors can be classified into three groups, i.e., immunoassay-based, molecular amplification-based, and nanoparticle assembly-based. The principal motivation is to develop and 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 typical immunoassay formats, i.e., direct and competitive format, are studied and compared for bacteria detection. In addition, in the protein biomarker detection, MNP chains are formed in the presence of target analytes as well as in the external rotating magnetic field. The high shape/magnetic anisotropy of the chains provides better optomagnetic performance.

Two different molecular amplification methods, i.e., rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP), are described under the topic of molecular amplification-based biosensors. In RCA-based biosensors, DNA probe modified MNPs bind to the amplicons after amplification. In LAMP-based biosensors, MNPs are either modified with primers 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 is based on duplex-specific nuclease (DSN)-assisted target recycling and core-satellite magnetic superstructures. 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 the aiming for rapid, low-cost and user-friendly diagnostics.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. , p. 55
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1647
Keywords [en]
Magnetic biosensors, magnetic nanoparticles, homogeneous assays, volumetric sensing
National Category
Other Engineering and Technologies not elsewhere specified
Research subject
Engineering Science with specialization in Solid State Physics
Identifiers
URN: urn:nbn:se:uu:diva-346014ISBN: 978-91-513-0278-2 (print)OAI: oai:DiVA.org:uu-346014DiVA, id: diva2:1190177
Public defence
2018-05-04, Häggsalen, Ångströmlaboratoriet, Lägerhyddsv. 1, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2018-04-13 Created: 2018-03-13 Last updated: 2018-04-24
List of papers
1. Blu-ray optomagnetic measurement based competitive immunoassay for Salmonella detection
Open this publication in new window or tab >>Blu-ray optomagnetic measurement based competitive immunoassay for Salmonella detection
2016 (English)In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 77, p. 32-39Article in journal (Refereed) Published
Abstract [en]

A turn-on competitive immunoassay using a low-cost Blu-ray optomagnetic setup and two differently sized magnetic particles (micron-sized particles acting as capture particles and nano-sized particles acting as detection particles) is here presented. For Salmonella detection, a limit of detection of 8 x 10(4) CFU/mL is achieved within a total assay time of 3 h. The combination of a competitive strategy and an optomagnetic setup not only enables a turn-on read-out format, but also results in a sensitivity limit about a factor of 20 times lower than of volumetric magnetic stray field detection device based immunoassays. The improvement of sensitivity is enabled by the formation of immuno-magnetic aggregates providing steric hindrance protecting the interior binding sites from interaction with the magnetic nanoparticle labels. The formation of immuno-magnetic aggregates is confirmed by fluorescence microscopy. The system exhibits no visible cross-reaction with other common pathogenic bacteria, even at concentrations as high as 10 CFU/mL. Furthermore, we present results when using the setup for a qualitative and homogeneous biplex immunoassay of Escherichia coli and Salmonella typhimurium.

Keywords
Biosensors; Immunoassays; Magnetic particles; Magneto-optics
National Category
Engineering and Technology
Research subject
Engineering Science with specialization in Solid State Physics
Identifiers
urn:nbn:se:uu:diva-264720 (URN)10.1016/j.bios.2015.08.070 (DOI)000366766900006 ()
Funder
Swedish Research Council Formas, 221-2012-444
Available from: 2015-10-15 Created: 2015-10-15 Last updated: 2019-04-24Bibliographically approved
2. Shape anisotropy enhanced optomagnetic measurement for prostate-specific antigen detection via magnetic chain formation
Open this publication in new window or tab >>Shape anisotropy enhanced optomagnetic measurement for prostate-specific antigen detection via magnetic chain formation
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2017 (Swedish)In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 98, p. 285-291Article in journal (Refereed) Published
Abstract [en]

We demonstrate a homogeneous biosensor for the detection of multivalent targets by combination of magnetic nanoparticle (MNP) chains and a low-cost 405 nm laser-based optomagnetic system. The MNP chains are assembled in a rotating magnetic field and stabilized by multivalent target molecules. The number of chains remaining in zero field is proportional to the target concentration, and can be quantified by optomagnetic measurements. The shape anisotropy of the MNP chains enhances the biosensor system in terms of providing efficient mixing, reduction of depletion effects (via magnetic shape anisotropy), and directly increasing the optomagnetic signal (via optical shape anisotropy). We achieve a limit of detection (LOD) of 5.5 pM (0.82 ng/mL) for the detection of a model multivalent molecule, biotinylated anti-streptavidin, in PBS. For the measurements of prostate-specific antigen (PSA) in 50% serum using the proposed method, we achieve an LOD of 21.6 pM (0.65 ng/mL) and a dynamic detection range up to 66.7 nM (2 µg/mL) with a sample-to-result time of approximately 20 min. The performance for PSA detection therefore well meets the clinical requirements in terms of LOD (the threshold PSA level in blood is 4 ng/mL) and detection range (PSA levels span from < 0.1–104 ng/mL in blood), thus showing great promise for routine PSA diagnostics and for other in-situ applications.

Keywords
Magnetic nanoparticles, Magnetic chains, Rotating magnetic field, Brownian relaxation, Optomagnetic biosensor, Prostate-specific antigen
National Category
Nano Technology
Research subject
Engineering Science with specialization in Solid State Physics
Identifiers
urn:nbn:se:uu:diva-330181 (URN)10.1016/j.bios.2017.06.062 (DOI)000407538300039 ()28689114 (PubMedID)
Funder
Swedish Research Council Formas, 221-2012-444Swedish Research Council Formas, 221-2014-574Swedish Research Council Formas, 2011-1692EU, FP7, Seventh Framework Programme, Grant No. 604448-NanoMag
Available from: 2017-09-27 Created: 2017-09-27 Last updated: 2019-04-24Bibliographically approved
3. Multi-scale magnetic nanoparticle based optomagnetic bioassay for sensitive DNA and bacteria detection
Open this publication in new window or tab >>Multi-scale magnetic nanoparticle based optomagnetic bioassay for sensitive DNA and bacteria detection
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2016 (English)In: Analytical Methods, ISSN 1759-9660, E-ISSN 1759-9679, Vol. 8, no 25, p. 5009-5016Article in journal (Refereed) Published
Abstract [en]

Benefiting from their rapid readout, highly flexible devices and low-cost portable systems, optomagnetic biosensors have drawn increased attention in recent years as bioassay technologies for small molecules, biomarkers, DNA, and bacteria. Herein, an optomagnetic bioassay strategy suitable for point-of-care diagnostics, utilizing functionalized magnetic nanoparticles (100 nm) with Brownian relaxation behavior is optimized in order to obtain higher detection sensitivity for DNA molecules and bacteria. Presence of target DNA sequences or bacteria changes the dynamic behavior of the magnetic nanoparticles (binding to the target) and thus the optomagnetic response of the sample, which is measured by an optomagnetic setup including a 405 nm laser and a photodetector. The limit of detection is mainly set by the lowest measurable concentration of magnetic nanoparticles. Herein, as new results compared to previous work, we systematically optimize the concentration of 100 nm magnetic nanoparticles to increase the assay sensitivity and lower the limit of detection. To enable biplex detection, we perform this optimization in the presence of larger 250 nm magnetic nanoparticles that do not interact with the target. We show that the optimization and lowering of the 100 nm magnetic nanoparticle concentration result in a limit of detection of 780 fM of DNA coils formed by rolling circle amplification (size of about 1 mu m) and 10(5) CFU per mL Salmonella (for immunoassay). These values are 15 times lower than those reported previously for this readout principle. Finally, we show that the 250 nm magnetic nanoparticles can serve as a second detection label for qualitative biplex detection of DNA coils formed by rolling circle amplification from V. cholerae and E. coli DNA coils using 100 nm and 250 nm magnetic detection nanoparticles, respectively.

National Category
Bioprocess Technology
Identifiers
urn:nbn:se:uu:diva-300098 (URN)10.1039/c6ay00721j (DOI)000378941500005 ()
Funder
Swedish Research Council Formas, 221-2012-444 221-2014-574 2011-1692Swedish Foundation for Strategic Research
Available from: 2016-08-02 Created: 2016-08-02 Last updated: 2019-04-24Bibliographically approved
4. Rapid Newcastle Disease Virus Detection based on Loop-Mediated Isothermal Amplification and Optomagnetic Readout
Open this publication in new window or tab >>Rapid Newcastle Disease Virus Detection based on Loop-Mediated Isothermal Amplification and Optomagnetic Readout
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2016 (English)In: ACS Sensors, ISSN 2379-3694, Vol. 1, no 10, p. 1228-1234Article in journal (Refereed) Published
Abstract [en]

Rapid and sensitive diagnostic methods based on isothermal amplification are ideal substitutes for PCR in out-of-lab settings. However, there are bottlenecks in terms of establishing low-cost and user-friendly readout methods for isothermal amplification schemes. Combining the high amplification efficiency of loop-mediated isothermal amplification (LAMP) with an optomagnetic' nanoparticle-based readout system, we demonstrate ultrasensitive and rapid detection of Newcastle disease virus RNA. Biotinylated amplicons of LAMP and reverse transcription LAMP (RT-LAMP) bind to streptavidin-coated magnetic nanoparticles (MNPs) resulting in a dramatical increase in the hydrodynamic size of the MNPs. This increase was measured by an optomagnetic readout system and provided quantitative information on the amount of LAMP target sequence. Our assay resulted in a limit of detection of 10 aM of target sequence with a total assay time of 30 min. The assay has also been tested on clinical samples (vaccine and tissue specimens) with a performance comparable to real-time RT-PCR By changing the LAMP primers, this strategy can serve as a general method for the detection of other DNA/RNA targets with high specificity and sensitivity.

Keywords
out-of-lab diagnostics, Newcastle disease virus, loop-mediated isothermal amplification, optomagnetic bioassay, magnetic nanoparticles
National Category
Medical Laboratory and Measurements Technologies Physical Sciences
Research subject
Engineering Science with specialization in Solid State Physics
Identifiers
urn:nbn:se:uu:diva-305174 (URN)10.1021/acssensors.6b00379 (DOI)000386747600012 ()
Funder
Swedish Research Council Formas, 221-2012-444Swedish Research Council Formas, 221-2014-574Swedish Research Council Formas, 2011-1692
Available from: 2016-10-12 Created: 2016-10-12 Last updated: 2019-04-24Bibliographically approved
5. Attomolar Zika virus oligonucleotide detection based on loop-mediated isothermal amplification and AC susceptometry
Open this publication in new window or tab >>Attomolar Zika virus oligonucleotide detection based on loop-mediated isothermal amplification and AC susceptometry
Show others...
2016 (English)In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 86, p. 420-425Article in journal (Refereed) Published
Abstract [en]

Because of the serological cross-reactivity among the flaviviruses, molecular detection methods, such as reverse-transcription polymerase chain reaction (RT-PCR), play an important role in the recent Zika outbreak. However, due to the limited sensitivity, the detection window of RT-PCR for Zika viremia is only about one week after symptom onset. By combining loop-mediated isothermal amplification (LAMP) and AC susceptometry, we demonstrate a rapid and homogeneous detection system for the Zika virus oligonucleotide. Streptavidin-magnetic nanoparticles (streptavidin-MNPs) are premixed with LAMP reagents including the analyte and biotinylated primers, and their hydrodynamic volumes are dramatically increased after a successful LAMP reaction. Analyzed by a portable AC susceptometer, the changes of the hydrodynamic volume are probed as Brownian relaxation frequency shifts, which can be used to quantify the Zika virus oligonucleotide. The proposed detection system can recognize 1 aM synthetic Zika virus oligonucleotide in 20% serum with a total assay time of 27 min, which can hopefully widen the detection window for Zika viremia and is therefore promising in worldwide Zika fever control.

Keywords
Zika virus, Loop-mediated isothermal amplification, Magnetic nanoparticles, Brownian relaxation, AC susceptometer
National Category
Biophysics Medical Biotechnology
Identifiers
urn:nbn:se:uu:diva-307258 (URN)10.1016/j.bios.2016.06.085 (DOI)000384853300057 ()
Funder
Swedish Research Council Formas, 221-2012-444 221-2014-574 2011-1692
Available from: 2016-11-14 Created: 2016-11-11 Last updated: 2019-04-24Bibliographically approved
6. Ferromagnetic Resonance Biosensor for Homogeneous and Volumetric Detection of DNA
Open this publication in new window or tab >>Ferromagnetic Resonance Biosensor for Homogeneous and Volumetric Detection of DNA
Show others...
2018 (English)In: ACS Sensors, ISSN 2379-3694, Vol. 3, no 6, p. 1093-1101Article in journal (Refereed) Published
Abstract [en]

The ability to detect and analyze the state ofmagnetic labels with high sensitivity is of crucial importance fordeveloping magnetic biosensors. In this work, we demonstrate, forthefirst time, a ferromagnetic resonance (FMR) basedhomogeneous and volumetric biosensor for magnetic labeldetection. Two different isothermal amplification methods, i.e.,rolling circle amplification (RCA) and loop-mediated isothermalamplification (LAMP), are adopted and combined with a standardelectron paramagnetic resonance (EPR) spectrometer for FMRbiosensing. For the RCA-based FMR biosensor, binding of RCAproducts of a syntheticVibrio choleraetarget DNA sequence givesrise to the formation of aggregates of magnetic nanoparticles.Immobilization of nanoparticles within the aggregates leads to adecrease of the net anisotropy of the system and a concomitant increase of the resonancefield. A limit of detection of 1 pM isobtained with a linear detection range between 7.8 and 250 pM. For the LAMP-based sensing, a synthetic Zika virus targetoligonucleotide is amplified and detected in 20% serum samples. Immobilization of magnetic nanoparticles is induced by theircoprecipitation with Mg2P2O7(a byproduct of LAMP) and provides a detection sensitivity of 100 aM. The fast measurement,high sensitivity, and miniaturization potential of the proposed FMR biosensing technology makes it a promising candidate fordesigning future point-of-care devices.

National Category
Condensed Matter Physics Engineering and Technology
Research subject
Engineering Science with specialization in Solid State Physics
Identifiers
urn:nbn:se:uu:diva-345595 (URN)10.1021/acssensors.8b00048 (DOI)000436525800005 ()
Funder
Swedish Research Council Formas, 221-2012-444Swedish Research Council Formas, 2011-1692EU, FP7, Seventh Framework Programme, FP7-NMP-601118
Available from: 2018-03-09 Created: 2018-03-09 Last updated: 2019-04-24Bibliographically approved
7. Optomagnetic Detection of MicroRNA Based on Duplex-Specific Nuclease-Assisted Target Recycling and Multilayer Core-Satellite Magnetic Superstructures
Open this publication in new window or tab >>Optomagnetic Detection of MicroRNA Based on Duplex-Specific Nuclease-Assisted Target Recycling and Multilayer Core-Satellite Magnetic Superstructures
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2017 (English)In: ACS Nano, ISSN 1936-0851, E-ISSN 1936-086X, Vol. 11, no 2, p. 1798-1806Article in journal (Refereed) Published
Abstract [en]

Superstructural assembly of magnetic nanoparticles enables approaches to biosensing by combining specially tailored properties of superstructures and the particular advantages associated with a magnetic or optomagnetic read-out such as low background signal, easy manipulation, cost-efficiency, and potential for bioresponsive multiplexing. Herein, we demonstrate a sensitive and rapid miRNA detection method based on optomagnetic read-out, duplex-specific nuclease (DSN)-assisted target recycling, and the use of multilayer core-satellite magnetic superstructures. Triggered by the presence of target miRNA and DSN-assisted target recycling, the core-satellite magnetic superstructures release their "satellites" to the suspension, which subsequently can be quantified accurately in a lowcost and user-friendly optomagnetic setup. Target miRNAs are preserved in the cleaving reaction and can thereby trigger more cleavage and release of "satellites". For singleplex detection of let-7b, a linear detection range between 10 fM and 10 nM was observed, and a detection limit of 4.8 fM was obtained within a total assay time of 70 min. Multiplexing was achieved by releasing nanoparticles of different sizes in the presence of different miRNAs. The proposed method also has the advantages of single-nucleotide mismatch discrimination and the ability of quantification in a clinical sample matrix, thus holding great promise for miRNA routine multiplex diagnostics.

Place, publisher, year, edition, pages
AMER CHEMICAL SOC, 2017
Keywords
miRNA detection, duplex-specific nuclease, magnetic nanoparticles, core-satellite superstructures, optomagnetic bioassay
National Category
Biomaterials Science Nano Technology
Research subject
Engineering Science with specialization in Nanotechnology and Functional Materials
Identifiers
urn:nbn:se:uu:diva-319101 (URN)10.1021/acsnano.6b07763 (DOI)000395357300073 ()28177611 (PubMedID)
Funder
Swedish Research Council Formas, 221-2012-444 221-2014-574 2011-1692
Available from: 2017-04-03 Created: 2017-04-03 Last updated: 2019-04-24Bibliographically approved
8. On-Particle Rolling Circle Amplification-Based Core-Satellite Magnetic Superstructures for MicroRNA Detection
Open this publication in new window or tab >>On-Particle Rolling Circle Amplification-Based Core-Satellite Magnetic Superstructures for MicroRNA Detection
Show others...
2018 (English)In: ACS Applied Materials and Interfaces, ISSN 1944-8244, E-ISSN 1944-8252, Vol. 10, no 3, p. 2957-2964Article in journal (Refereed) Published
Abstract [en]

Benefiting from the specially tailored properties of the building blocks as well as of the scaffolds, DNA-assembled core satellite superstructures have gained increasing interest-in drug delivery, imaging, and biosensing. The load of satellites plays,,a vital role in core satellite superstructures, and it determines the signal intensity in response to a biological/physical stimulation/actuation. Herein, for the first time, we utilize on-particle rolling circle amplification (RCA) to prepare rapidly responsive-core satellite magnetic superstructures With a high load of magnetic nanoparticle (MNP) Satellites. Combined with duplex-specific nuclease-assisted target recycling) the proposed magnetic superstructures hold great promise in sensitive and rapid microRNA detection. The long single-stranded DNA produced by RCA serving as the scaffold of the core satellite superstructure can be hydrolyzed by duplex-Specific nuclease in the presence of target microRNA, resulting in a release of MNPs that can be quantified in an optomagnetic sensor. The proposed biosensor has a-simple mix separate measure strategy. For let-7b detection, the proposed biosensor offers a wide linear detection range of approximately 5 orders of magnitude with a detection sensitivity of 1 fM. Moreover, it has the capability to discriminate single-nucleotide mismatches and to detect let-7b in cell extracts and serum, thus showing considerable potential for clinical applications.

National Category
Engineering and Technology
Research subject
Engineering Science with specialization in Solid State Physics
Identifiers
urn:nbn:se:uu:diva-345594 (URN)10.1021/acsami.7b16293 (DOI)000423496500087 ()29266917 (PubMedID)
Funder
Swedish Research Council Formas, 221-2012-444
Available from: 2018-03-09 Created: 2018-03-09 Last updated: 2019-04-24Bibliographically approved

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