99 results on '"Wälti, C."'
Search Results
2. Label-free electrochemical impedance biosensor to detect human interleukin-8 in serum with sub-pg/ml sensitivity
- Author
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Sharma, R., Deacon, S.E., Nowak, D., George, S.E., Szymonik, M.P., Tang, A.A.S., Tomlinson, D.C., Davies, A.G., McPherson, M.J., and Wälti, C.
- Published
- 2016
- Full Text
- View/download PDF
3. Low-temperature specific heat of the heavy electron superconductor U1-xThxBe13(x = 0, 0.033) in external magnetic fields
- Author
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Wälti, C, Felder, E, Ott, HR, Fisk, Z, and Smith, JL
- Subjects
heavy fermions ,specific heat in magnetic fields ,UBe13 ,Condensed Matter Physics ,Quantum Physics ,Macromolecular and Materials Chemistry ,General Physics - Abstract
We report on results of low-temperature specific-heat measurements of the unconventional superconductors UBe13 and U0.9669Th0.0331 Be13 in external magnetic fields up to 7 T. For T ≪ Tc, or, respectively, Tc2 for the thoriated sample, the magnetic field-induced contribution to Cp(T,B) varies linearly with temperature, which is consistent with the temperature dependence of the specific heat due to the flow of supercurrent around the vortices. © 2000 Elsevier Science B.V. All rights reserved.
- Published
- 2000
4. A COMPREHENSIVE, TWO-MONTH CIRCUIT TRAINING IN PEOPLE WITH MULTIPLE SCLEROSIS - ADHERENCE AND SATISFACTION WITH THE STUDY INTERVENTION
- Author
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Lehmann, I, Thaler, I, Verra, M, Luder, G, Damm, U, Wälti, C, Nyffeler, T, Vanbellingen, T, and Kamm, CP
- Subjects
610 Medicine & health - Abstract
Background: Exercise therapy in people with multiple sclerosis (MS) can improve strength, aerobic capacity, fatigue, balance and manual dexterity. Circuit training has been shown to be an effective exercise intervention in several neurological diseases, though it has been poorly investigated in people with MS. So far, a few small trials reported promising results, but exercise adherence and patient satisfaction have not yet been investigated. Purpose: As part of a larger randomized controlled trial this study aimed to investigate the adherence of people with MS to a structured, two-month ambulatory circuit training (MS-Fit) and to determine their overall satisfaction with the program. Methods: People with MS-related disability affecting activities of daily living (ADL) and/or quality of life (QoL) were included. The participants attended the circuit training for 2x2 hours weekly over 2 months. The circuit training was conducted in two rehabilitation centres in Switzerland and supervised by experienced physiotherapists. The program consisted of six workstations, where endurance, strength, flexibility, balance, dexterity and reaction were exercised individually. Physiotherapists evaluated and adapted quality and intensity according to the participants' performance. A training group consisted of two to six participants. Program satisfaction and adherence were evaluated using a face-validated questionnaire and attendance rate. Results: From totally 51 people with MS eligible, 37 (10 men, 27 women, mean age 52.7±10.3, median Expanded Disability Status Scale (EDSS) 3.5 (min 2.0, max 6.5) participated in the MS-fit program. Main reasons not to participate were lack of time, or family issues. All 37 participants achieved >80% (mean 90.5%±8.2) of attendance rate and sent the evaluation questionnaire back. Participants rated the program quality as good with an overall median score of 39/50 (min 26, max 50). Overall program satisfaction was median 8 (min4, max10) on a Likert scale from 0-10. 95% of participants would recommend the program to others. Moreover, participants highlighted the positive impact on social participation, peer learning opportunities and reported an increase in exercise management competences. Expectations concerning further program availability was high. Conclusion(s): The MS-Fit training program seems to be a highly appreciated form of exercise therapy with a high rate of attendance. The two-month program intensity and duration was practicable and acceptable. Furthermore, the program positively affected social participation and stimulated participants' exercise competences and learning aspects. Further research is needed to evaluate the functional, mental and physical effects of the program. Implications: The MS-Fit training program might motivate people with MS that are usually involved in one-to-one therapy settings. After analysis of the effectiveness of the circuit training program, implementation and national dispersion might be proposed to offer MS-fit access to people with MS close to their domicile.
- Published
- 2019
- Full Text
- View/download PDF
5. Direct Single-Molecule Observation of Mode and Geometry of RecA-Mediated Homology Search
- Author
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Lee, A.J., Endo, M., Hobbs, J.K., and Wälti, C.
- Abstract
Genomic integrity, when compromised by accrued DNA lesions, is maintained through efficient repair via homologous recombination. For this process the ubiquitous recombinase A (RecA), and its homologues such as the human Rad51, are of central importance, able to align and exchange homologous sequences within single-stranded and double-stranded DNA in order to swap out defective regions. Here, we directly observe the widely debated mechanism of RecA homology searching at a single-molecule level using high-speed atomic force microscopy (HS-AFM) in combination with tailored DNA origami frames to present the reaction targets in a way suitable for AFM-imaging. We show that RecA nucleoprotein filaments move along DNA substrates via short-distance facilitated diffusions, or slides, interspersed with longer-distance random moves, or hops. Importantly, from the specific interaction geometry, we find that the double-stranded substrate DNA resides in the secondary DNA binding-site within the RecA nucleoprotein filament helical groove during the homology search. This work demonstrates that tailored DNA origami, in conjunction with HS-AFM, can be employed to reveal directly conformational and geometrical information on dynamic protein-DNA interactions which was previously inaccessible at an individual single-molecule level.
- Published
- 2017
6. Cooperative RecA clustering: the key to efficient homology searching
- Author
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Lee, A.J., Sharma, R., Hobbs, J.K., and Wälti, C.
- Abstract
The mechanism by which pre-synaptic RecA nucleoprotein filaments efficiently locate sequence homology across genomic DNA remains unclear. Here, using atomic force microscopy, we directly investigate the intermediates of the RecA-mediated homologous recombination process and find it to be highly cooperative, involving multiple phases. Initially, the process is dominated by a rapid 'association' phase, where multiple filaments interact on the same dsDNA simultaneously. This cooperative nature is reconciled by the observation of localized dense clusters of pre-synaptic filaments interacting with the observed dsDNA molecules. This confinement of reactive species within the vicinity of the dsDNA, is likely to play an important role in ensuring that a high interaction rate between the nucleoprotein filaments and the dsDNA can be achieved. This is followed by a slower 'resolution' phase, where the synaptic joints either locate sequence homology and progress to a post-synaptic joint, or dissociate from the dsDNA. Surprisingly, the number of simultaneous synaptic joints decreases rapidly after saturation of the dsDNA population, suggesting a reduction in interaction activity of the RecA filaments. We find that the time-scale of this decay is in line with the time-scale of the dispersion of the RecA filament clusters, further emphasising the important role this cooperative phenomena may play in the RecA-facilitated homology search.
- Published
- 2017
7. Influence of alternating current electrokinetic forces and torque on the elongation of immobilized DNA.
- Author
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Germishuizen, W. A., Tosch, P., Middelberg, A. P. J., Wälti, C., Davies, A. G., Wirtz, R., and Pepper, M.
- Subjects
DNA ,ELECTROKINETICS ,ELECTRODES ,THIOLS ,ELECTRIC fields ,DIPOLE moments - Abstract
The authors investigate the elongation and orientation of different-sized deoxyribose nucleic acid (DNA) molecules, tethered onto gold electrodes via a terminal thiol, under the influence of high frequency ac electric fields. The DNA molecules are elongated from a random coil into an extended conformation and orientated along the electric field lines as a result of the forces acting on the molecules during the application of the ac electric fields. Elongation was observed in the frequency range 100 kHz–1 MHz, with field strengths of 0.06–1.0 MV/m. Maximum elongation for all DNA fragments tested, irrespective of size, was found for frequencies between 200 and 300 kHz. The torque acting on the induced dipole in the DNA molecules, complemented by a directional bias force, opposite in direction to the dielectrophoretic force, provides the main contribution to the elongation process. The length of elongation is limited to either half the distance between opposing electrodes or to the contour length of the DNA, whichever is shorter. Further, the authors show that the normalized length of the elongated DNA molecules is independent of the contour length of the DNA. [ABSTRACT FROM AUTHOR]
- Published
- 2005
- Full Text
- View/download PDF
8. A planar surface acoustic wave micropump for closed-loop microfluidics
- Author
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Rimsa, R., primary, Smith, A. J., additional, Wälti, C., additional, and Wood, C. D., additional
- Published
- 2017
- Full Text
- View/download PDF
9. Field-enhanced direct tunneling in ultrathin atomic-layer-deposition-grown Au−Al2O3 -Cr metal-insulator-metal structures
- Author
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Fry-Bouriaux, L., primary, Rosamond, M. C., additional, Williams, D. A., additional, Davies, A. G., additional, and Wälti, C., additional
- Published
- 2017
- Full Text
- View/download PDF
10. Solid phase synthesis of functionalised SAM-forming alkanethiol-oligoethyleneglycols
- Author
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Murray, J, Nowak, D, Pukenas, L, Azhar, R, Guillorit, M, Wälti, C, Critchley, K, Johnson, S, and Bon, RS
- Abstract
We present an efficient solid phase synthesis methodology that provides easy access to a range of functionalised long-chain alkanethiol-oligoethyleneglycols that form well-defined self-assembled monolayers on gold and are compatible with pre- or post-assembly conjugation of (bio)molecules. We demonstrate the versatility of our synthetic route by synthesising LCAT-OEGs with a range of functional moieties, including peptides, electro-active redox groups, chemical handles for post-assembly conjugation of (bio)molecules, and demonstrate the application of our LCAT-OEG monolayers in immunosensing, where they show good biocompatibility with minimal biofouling.
- Published
- 2014
11. DNA self-assembly-driven positioning of molecular components on nanopatterned surfaces
- Author
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Szymonik, M, primary, Davies, A G, additional, and Wälti, C, additional
- Published
- 2016
- Full Text
- View/download PDF
12. Barium Titanate Nanoparticles for Biomarker Applications
- Author
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Matar, O, primary, Posada, O M, additional, Hondow, N S, additional, Wälti, C, additional, Saunders, M, additional, Murray, C A, additional, Brydson, R M D, additional, Milne, S J, additional, and Brown, A P, additional
- Published
- 2015
- Full Text
- View/download PDF
13. Antibody mimetic receptor proteins for label-free biosensors
- Author
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Raina, M., primary, Sharma, R., additional, Deacon, S. E., additional, Tiede, C., additional, Tomlinson, D., additional, Davies, A. G., additional, McPherson, M. J., additional, and Wälti, C., additional
- Published
- 2015
- Full Text
- View/download PDF
14. Fabrication and characterization of gold nano-wires templated on virus-like arrays of tobacco mosaic virus coat proteins
- Author
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Wnęk, M, primary, Górzny, M Ł, additional, Ward, M B, additional, Wälti, C, additional, Davies, A G, additional, Brydson, R, additional, Evans, S D, additional, and Stockley, P G, additional
- Published
- 2012
- Full Text
- View/download PDF
15. Nanoscale programmable sequence-specific patterning of DNA scaffolds using RecA protein
- Author
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Sharma, R, primary, Davies, A G, additional, and Wälti, C, additional
- Published
- 2012
- Full Text
- View/download PDF
16. Acousto-microfluidics: Transporting microbubble and microparticle arrays in acoustic traps using surface acoustic waves
- Author
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O’Rorke, R. D., primary, Wood, C. D., additional, Wälti, C., additional, Evans, S. D., additional, Davies, A. G., additional, and Cunningham, J. E., additional
- Published
- 2012
- Full Text
- View/download PDF
17. Formation and manipulation of two-dimensional arrays of micron-scale particles in microfluidic systems by surface acoustic waves
- Author
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Wood, C. D., primary, Cunningham, J. E., additional, O’Rorke, R., additional, Wälti, C., additional, Linfield, E. H., additional, Davies, A. G., additional, and Evans, S. D., additional
- Published
- 2009
- Full Text
- View/download PDF
18. Alignment of particles in microfluidic systems using standing surface acoustic waves
- Author
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Wood, C. D., primary, Evans, S. D., additional, Cunningham, J. E., additional, O’Rorke, R., additional, Wälti, C., additional, and Davies, A. G., additional
- Published
- 2008
- Full Text
- View/download PDF
19. AC electrokinetic manipulation of DNA
- Author
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Wälti, C, primary, Germishuizen, W A, additional, Tosch, P, additional, Kaminski, C F, additional, and Davies, A G, additional
- Published
- 2006
- Full Text
- View/download PDF
20. Establishment of the ac electrokinetic elongation mechanism of DNA by three-dimensional fluorescent imaging
- Author
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Wälti, C., primary, Tosch, P., additional, Davies, A. G., additional, Germishuizen, W. A., additional, and Kaminski, C. F., additional
- Published
- 2006
- Full Text
- View/download PDF
21. Influence of the Thiol Position on the Attachment and Subsequent Hybridization of Thiolated DNA on Gold Surfaces
- Author
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Wirtz, R., primary, Wälti, C., additional, Tosch, P., additional, Pepper, M., additional, Davies, A. G., additional, Germishuizen, W. A., additional, and Middelberg, A. P. J., additional
- Published
- 2004
- Full Text
- View/download PDF
22. Low-temperature specific heat of the heavy electron superconductor U1−xThxBe13(x=0, 0.033) in external magnetic fields
- Author
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Wälti, C, primary
- Published
- 2000
- Full Text
- View/download PDF
23. Fabrication and characterization of gold nano-wires templated on virus-like arrays of tobacco mosaic virus coat proteins.
- Author
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Wnęk, M., Górzny, M. Ł., Ward, M. B., Wälti, C., Davies, A. G., Brydson, R., Evans, S. D., and Stockley, P. G.
- Subjects
NANOWIRES ,GOLD ,COAT proteins (Viruses) ,TOBACCO mosaic virus ,BIOCHEMICAL templates ,HYDROGEN-ion concentration ,SOLUTION (Chemistry) - Abstract
The rod-shaped plant virus tobacco mosaic virus (TMV) is widely used as a nano-fabrication template, and chimeric peptide expression on its major coat protein has extended its potential applications. Here we describe a simple bacterial expression system for production and rapid purification of recombinant chimeric TMV coat protein carrying C-terminal peptide tags. These proteins do not bind TMV RNA or form disks at pH 7. However, they retain the ability to self-assemble into virus-like arrays at acidic pH. C-terminal peptide tags in such arrays are exposed on the protein surface, allowing interaction with target species. We have utilized a C-terminal His-tag to create virus coat protein-templated nano-rods able to bind gold nanoparticles uniformly. These can be transformed into gold nano-wires by deposition of additional gold atoms from solution, followed by thermal annealing. The resistivity of a typical annealed wire created by this approach is significantly less than values reported for other nano-wires made using different bio-templates. This expression construct is therefore a useful additional tool for the creation of chimeric TMV-like nano-rods for bio-templating. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
24. Dielectrophoretic manipulation of surface-bound DNA.
- Author
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Germishuizen, W. A., Wälti, C., Tosch, P., Wirtz, R., Pepper, M., Davies, A. G., and Middelberg, A. P. J.
- Subjects
- *
DIELECTROPHORESIS , *DNA , *PROPERTIES of matter , *ELECTROPHORESIS , *MICROELECTRODES , *ELECTRIC fields - Abstract
Dielectrophoretic manipulation enables the positioning and orientation of DNA molecules for nanometer-scale applications. However, the dependence of the dielectrophoretic force and torque on the electric field magnitude and frequency has to be well characterised to realise fully the potential of this technique. DNA in solution is attracted to the strongest electric field gradient (i.e. the electrode edge)as a result of the dielectrophoretic force, while the dielectrophoretic torque aligns the DNA with its longest axis parallel to the electric field. In this work, the authors attached λ-DNA fragments (48 and 25 kilobases)to an array of gold microelectrodes via a terminal thiol bond and characterised the orientation and elongation as a function of electric field magnitude (0.1-0.8MV/m)and frequency (0.08-1.1MHz). Maximum elongation was observed between 200 and 500 kHz for the attached DNA. Dielectrophoresis is limited by thermal randomisation at electric fields below 0.1MV/m and by electrothermal effects above 0.7MV/m. The authors conclude that dielectrophoresis can be used to manipulate surface-immobilised DNA reproducibly. [ABSTRACT FROM AUTHOR]
- Published
- 2003
- Full Text
- View/download PDF
25. Rehabilitation in practice. The implementation of the 'International Classification of Functioning, Disability and Health' (ICF) in daily practice of neurorehabilitation: an interdisciplinary project at the Kantonsspital of Lucerne, Switzerland.
- Author
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Rentsch HP, Bucher P, Nyffeler ID, Wolf C, Hefti H, Fluri E, Wenger U, Wälti C, and Boyer I
- Published
- 2003
- Full Text
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26. High-sensitivity colorimetric detection of DNA hybridization on a gold surface with high spatial resolution
- Author
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Wirtz, R, Wälti, C, Germishuizen, W A, Pepper, M, Middelberg, A P J, and Davies, A G
- Abstract
We present a colorimetric detection method which is able to detect biotinylated oligonucleotides hybridized to thiolated complementary strands bound to a gold surface. Unlike previous colorimetric detection methods, this technique has the advantage of providing spatial information which is of higher resolution than that of an optical microscope, and can also be used on a gold surface. Bulk concentrations down to 10−9 M of biotinylated oligonucleotides were detectable representing fewer than 7000 molecules of hybridized DNA on a microarray-sized spot (
≈ 35μ m). This technique has potential for DNA detection in molecular electronics and molecular assembly applications, where a high-resolution detection on metal surfaces is required.- Published
- 2003
27. Acousto-microfluidics: Transporting microbubble and microparticle arrays in acoustic traps using surface acoustic waves.
- Author
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O'Rorke, R. D., Wood, C. D., Wälti, C., Evans, S. D., Davies, A. G., and Cunningham, J. E.
- Subjects
MICROFLUIDICS ,MICROBUBBLES ,ACOUSTIC surface waves ,MICROFLUIDIC devices ,TRANSDUCERS ,BANDWIDTHS - Abstract
We demonstrate that aqueous suspensions of microbubbles, formed into arrays using standing surface acoustic waves (SSAWs), can be transported by controlled modulation of the SSAW frequency. The array is repeatedly captured at a sequence of spatial positions along the acoustic beam path and long-range transportation is achieved by periodic cycling of the applied frequency across the transducer bandwidth. We also demonstrate that controllable alignment and transport can be achieved in a detachable microfluidic device, where the microfluidic channel, in which particle transport occurs, is separated from the piezoelectric substrate by an acoustic coupling gel. Proof-of-concept transport is first discussed using a test system of latex particles before the non-invasive manipulation technique is applied to arrays of microbubbles. We explore the role of acoustic radiation forces in the spatial control of particles by analysing the dynamics of particle manipulation by SSAWs. Our results highlight the exquisite control which we have over the position and transport of particles and we anticipate that this technique could find wide applications for the accurate and programmable, non-invasive ordering and transport of biological samples in microfluidic systems. [ABSTRACT FROM AUTHOR]
- Published
- 2012
- Full Text
- View/download PDF
28. Label-free electrochemical impedance biosensor to detect human interleukin-8 in serum with sub-pg/ml sensitivity
- Author
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Sharma R, Sarah Deacon, Nowak D, Se, George, Mp, Szymonik, Aa, Tang, Dc, Tomlinson, Ag, Davies, Mj, Mcpherson, and Wälti C
- Subjects
Inflammation ,Antibody mimetic protein ,Limit of Detection ,Point-of-care diagnostics ,Interleukin-8 ,CXCL8 ,Electric Impedance ,Label-free biosensor ,Humans ,Biosensing Techniques ,Electrochemical impedance spectroscopy ,Biomarkers ,Article - Abstract
Biosensors with high sensitivity and short time-to-result that are capable of detecting biomarkers in body fluids such as serum are an important prerequisite for early diagnostics in modern healthcare provision. Here, we report the development of an electrochemical impedance-based sensor for the detection in serum of human interleukin-8 (IL-8), a pro-angiogenic chemokine implicated in a wide range of inflammatory diseases. The sensor employs a small and robust synthetic non-antibody capture protein based on a cystatin scaffold that displays high affinity for human IL-8 with a KD of 35±10 nM and excellent ligand specificity. The change in the phase of the electrochemical impedance from the serum baseline, ∆θ(ƒ), measured at 0.1 Hz, was used as the measure for quantifying IL-8 concentration in the fluid. Optimal sensor signal was observed after 15 min incubation, and the sensor exhibited a linear response versus logarithm of IL-8 concentration from 900 fg/ml to 900 ng/ml. A detection limit of around 90 fg/ml, which is significantly lower than the basal clinical levels of 5–10 pg/ml, was observed. Our results are significant for the development of point-of-care and early diagnostics where high sensitivity and short time-to-results are essential., Highlights • A label-free electrochemical impedance-based sensor for the detection of human interleukin-8 (IL-8) in full serum was developed. • Detection limit of 90 fg/ml and time-to-result of 15 min was found. • A large dynamic range of the sensor was observed, with sensor response linear vs logarithm of IL-8 concentration from 900 fg/ml to 900 ng/ml. • The sensor employs a small and robust synthetic non-antibody capture protein, with high stability and excellent ligand specificity. • Findings are particularly relevant for the development of point-of-care and early diagnosis sensors where high sensitivity and short time-to-results are essential.
29. Field-enhanced direct tunneling in ultrathin atomic-layer-deposition-grown Au-Al2O3-Cr metal-insulator-metal structures.
- Author
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Fry-Bouriaux, L., Rosamond, M. C., Williams, D. A., Davies, A. G., and Wälti, C.
- Subjects
- *
QUANTUM tunneling , *GOLD , *METAL-insulator transitions - Abstract
Metal-insulator-metal structures based on ultrathin high-k dielectric films are underpinning a rapidly increasing number of devices and applications. Here, we report detailed electrical characterizations of asymmetric metal-insulator-metal devices featuring atomic layer deposited 2-nm-thick Al2O3 films. We find a high consistency in the current density as a function of applied electric field between devices with very different surface areas and significant asymmetries in the IV characteristics. We show by TEM that the thickness of the dielectric film and the quality of the metal-insulator interfaces are highly uniform and of high quality, respectively. In addition, we develop a model which accounts for the field enhancement due to the small sharp features on the electrode surface and show that this can very accurately describe the observed asymmetry in the current-voltage characteristic, which cannot be explained by the difference in work function alone. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
30. Multimodal nanoparticle analysis enabled by a polymer electrolyte nanopore combined with nanoimpact electrochemistry.
- Author
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Gyasi Agyemang E, Confederat S, Mohanan G, Azimzadeh Sani M, Chau C, Charnock D, Wälti C, Tschulik K, Edwards MA, and Actis P
- Abstract
Nanopores are emerging as a powerful tool for the analysis and characterization of nanoparticles at the single entity level. Here, we report that a PEG-based polymer electrolyte present inside the nanopore enables the enhanced detection of nanoparticles at low ionic strength. We develop a numerical model that recapitulates the electrical response of the glass nanopore system, revealing the response to be sensitive to the position of the polymer electrolyte interface. As proof of concept, we demonstrate the multimodal analysis of a nanoparticle sample by coupling the polymer electrolyte nanopore sensor with nanoimpact electrochemistry. This combination of techniques could deliver the multiparametric analysis of nanoparticle systems complementing electrochemical reactivity data provided by nanoimpact electrochemistry with information on size, shape and surface charge provided by nanopore measurements.
- Published
- 2024
- Full Text
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31. Photonic and electrochemical biosensors for near-patient tests-a critical comparison.
- Author
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Krauss TF, Miller L, Wälti C, and Johnson S
- Abstract
Research into diagnostic biosensors is a vibrant field that combines scientific challenge with translational opportunities; innovation in healthcare is of great societal interest and is an essential element of future healthcare provision. Photonic and electrochemical biosensors are the dominant modalities, both scientifically and commercially, yet the two scientific communities largely remain separated and siloed. It seems astute to better understand what the two fields can learn from one another so as to progress the key scientific, translational, and commercial challenges. Here, we provide an analysis of the fundamental operational characteristics of photonic and electrochemical biosensors using a classification based on energy transfer; in photonics, this separates refractive index sensors from fluorescence and vibrational spectroscopy, while in electrochemistry, it distinguishes Faradaic from non-Faradaic processes. This classification allows us to understand some of the key performance characteristics, such as the susceptibility to fouling and dependence on the clinical matrix that is being analyzed. We discuss the use of labels and the ultimate performance limits, and some of the unique advantages of photonics, such as multicolor operation and fingerprinting, and critically evaluate the requirements for translation of these technologies for clinical use. We trust that this critical review will inform future research in biosensors and support both scientific and commercial developments., Competing Interests: The author declares no conflicts of interest. No data were generated or analyzed in the present research., (© 2024 The Author(s).)
- Published
- 2024
- Full Text
- View/download PDF
32. Automated Purification of DNA Origami with SPRI Beads.
- Author
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Chau C, Mohanan G, Macaulay I, Actis P, and Wälti C
- Subjects
- Nanotechnology methods, Nanostructures chemistry, Automation, Nucleic Acid Conformation, DNA chemistry, DNA isolation & purification
- Abstract
DNA origami synthesis is a well-established technique with wide-ranging applications. In most cases, the synthesized origami must be purified to remove excess materials such as DNA oligos and other functional molecules. While several purification techniques are routinely used, all have limitations, and cannot be integrated with robotic systems. Here the use of solid-phase reversible immobilization (SPRI) beads as a scalable, high-throughput, and automatable method to purify DNA origami is demonstrated. Not only can this method remove unreacted oligos and biomolecules with yields comparable to existing methods while maintaining the high structural integrity of the origami, but it can also be integrated into an automated workflow to purify simultaneously large numbers and quantities of samples. It is envisioned that the SPRI beads purification method will improve the scalability of DNA nanostructures synthesis both for research and commercial applications., (© 2023 The Authors. Small published by Wiley‐VCH GmbH.)
- Published
- 2024
- Full Text
- View/download PDF
33. Next-Generation Nanopore Sensors Based on Conductive Pulse Sensing for Enhanced Detection of Nanoparticles.
- Author
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Confederat S, Lee S, Vang, Soulias D, Marcuccio F, Peace TI, Edwards MA, Strobbia P, Samanta D, Wälti C, and Actis P
- Subjects
- Proteins, Nanotechnology, Nanopores, Nucleic Acids, Nanoparticles
- Abstract
Nanopore sensing has been successfully used to characterize biological molecules with single-molecule resolution based on the resistive pulse sensing approach. However, its use in nanoparticle characterization has been constrained by the need to tailor the nanopore aperture size to the size of the analyte, precluding the analysis of heterogeneous samples. Additionally, nanopore sensors often require the use of high salt concentrations to improve the signal-to-noise ratio, which further limits their ability to study a wide range of nanoparticles that are unstable at high ionic strength. Here, a new paradigm in nanopore research that takes advantage of a polymer electrolyte system to comprise a conductive pulse sensing approach is presented. A finite element model is developed to explain the conductive pulse signals observed and compare these results with experiments. This system enables the analytical characterization of heterogeneous nanoparticle mixtures at low ionic strength . Furthermore, the wide applicability of the method is demonstrated by characterizing metallic nanospheres of varied sizes, plasmonic nanostars with various degrees of branching, and protein-based spherical nucleic acids with different oligonucleotide loadings. This system will complement the toolbox of nanomaterials characterization techniques to enable real-time optimization workflow for engineering a wide range of nanomaterials., (© 2023 The Authors. Small published by Wiley-VCH GmbH.)
- Published
- 2024
- Full Text
- View/download PDF
34. Therapeutic drug monitoring of immunotherapies with novel Affimer-NanoBiT sensor construct.
- Author
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Campbell E, Adamson H, Luxton T, Tiede C, Wälti C, Tomlinson DC, and Jeuken LJC
- Abstract
Concentration-therapeutic efficacy relationships have been observed for several therapeutic monoclonal antibodies (TmAb), where low circulating levels can result in ineffective treatment and high concentrations can cause adverse reactions. Rapid therapeutic drug monitoring (TDM) of TmAb drugs would provide the opportunity to adjust an individual patient's dosing regimen to improve treatment results. However, TDM for immunotherapies is currently limited to centralised testing methods with long sample-collection to result timeframes. Here, we show four point-of-care (PoC) TmAb biosensors by combining anti-idiotypic Affimer proteins and NanoBiT split luciferase technology at a molecular level to provide a platform for rapid quantification (<10 minutes) for four clinically relevant TmAb (rituximab, adalimumab, ipilimumab and trastuzumab). The rituximab sensor performed best with 4 pM limit of detection (LoD) and a quantifiable range between 8 pM-2 nM with neglectable matrix effects in serum up to 1%. After dilution of serum samples, the resulting quantifiable range for all four sensors falls within the clinically relevant range and compares favourably with the sensitivity and/or time-to-result of current ELISA standards. Further development of these sensors into a PoC test may improve treatment outcome and quality of life for patients receiving immunotherapy., Competing Interests: The authors declare the following competing financial interest(s): the Affimer reagents used in this report are owned by the University of Leeds (UoL) but licensed to Avacta Life Sciences. The UoL received royalties from Avacta Life Sciences as part of the license agreement, which is managed by the commercialization team. The authors declare no competing financial interest., (This journal is © The Royal Society of Chemistry.)
- Published
- 2023
- Full Text
- View/download PDF
35. Enzyme - Switch sensors for therapeutic drug monitoring of immunotherapies.
- Author
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Campbell E, Adamson H, Kohl D, Tiede C, Wälti C, Tomlinson DC, and Jeuken LJC
- Subjects
- Humans, Ipilimumab, Antibodies, Monoclonal therapeutic use, Trastuzumab therapeutic use, Immunotherapy, Drug Monitoring, Biosensing Techniques
- Abstract
Therapeutic monoclonal antibodies (TmAb) have emerged as effective treatments for a number of cancers and autoimmune diseases. However, large interpatient disparities in the pharmacokinetics of TmAb treatment requires close therapeutic drug monitoring (TDM) to optimise dosage for individual patients. Here we demonstrate an approach for achieving rapid, sensitive quantification of two monoclonal antibody therapies using a previously described enzyme switch sensor platform. The enzyme switch sensor consists of a β-lactamase - β-lactamase inhibitor protein (BLA-BLIP) complex with two anti-idiotype binding proteins (Affimer proteins) as recognition elements. The BLA-BLIP sensor was engineered to detect two TmAbs (trastuzumab and ipilimumab) by developing constructs incorporating novel synthetic binding reagents to each of these mAbs. Trastuzumab and ipilimumab were successfully monitored with sub nM sensitivity in up to 1% serum, thus covering the relevant therapeutic range. Despite the modular design, the BLA-BLIP sensor was unsuccessful in detecting two further TmAbs (rituximab and adalimumab), an explanation for which was explored. In conclusion, the BLA-BLIP sensors provide a rapid biosensor for TDM of trastuzumab and ipilimumab with the potential to improve therapy. The sensitivity of this platform alongside its rapid action would be suitable for bedside monitoring in a point-of-care (PoC) setting., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The Affimer reagents used in this report are owned by the University of Leeds (UoL) but licensed to Avacta Life Sciences. The UoL receive royalties from Avacta Life Sciences as part of the license agreement, which is managed by the commercialisation team. The authors declare no competing financial interest., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)
- Published
- 2023
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36. Nanopore fingerprinting of supramolecular DNA nanostructures.
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Confederat S, Sandei I, Mohanan G, Wälti C, and Actis P
- Subjects
- Nucleic Acid Conformation, DNA chemistry, Nanotechnology methods, DNA, Single-Stranded, Microscopy, Atomic Force, Nanopores, Nanostructures chemistry
- Abstract
DNA nanotechnology has paved the way for new generations of programmable nanomaterials. Utilizing the DNA origami technique, various DNA constructs can be designed, ranging from single tiles to the self-assembly of large-scale, complex, multi-tile arrays. This technique relies on the binding of hundreds of short DNA staple strands to a long single-stranded DNA scaffold that drives the folding of well-defined nanostructures. Such DNA nanostructures have enabled new applications in biosensing, drug delivery, and other multifunctional materials. In this study, we take advantage of the enhanced sensitivity of a solid-state nanopore that employs a poly-ethylene glycol enriched electrolyte to deliver real-time, non-destructive, and label-free fingerprinting of higher-order assemblies of DNA origami nanostructures with single-entity resolution. This approach enables the quantification of the assembly yields for complex DNA origami nanostructures using the nanostructure-induced equivalent charge surplus as a discriminant. We compare the assembly yield of four supramolecular DNA nanostructures obtained with the nanopore with agarose gel electrophoresis and atomic force microscopy imaging. We demonstrate that the nanopore system can provide analytical quantification of the complex supramolecular nanostructures within minutes, without any need for labeling and with single-molecule resolution. We envision that the nanopore detection platform can be applied to a range of nanomaterial designs and enable the analysis and manipulation of large DNA assemblies in real time., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
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37. A Systematic Review of the Effect of Therapeutic Drug Monitoring on Patient Health Outcomes during Treatment with Carbapenems.
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Luxton TN, King N, Wälti C, Jeuken LJC, and Sandoe JAT
- Abstract
Adjusting dosing regimens based on measurements of carbapenem levels may improve carbapenem exposure in patients. This systematic review aims to describe the effect carbapenem therapeutic drug monitoring (TDM) has on health outcomes, including the emergence of antimicrobial resistance (AMR). Four databases were searched for studies that reported health outcomes following adjustment to dosing regimens, according to measurements of carbapenem concentration. Bias in the studies was assessed with risk of bias analysis tools. Study characteristics and outcomes were tabulated and a narrative synthesis was performed. In total, 2 randomised controlled trials (RCTs), 17 non-randomised studies, and 19 clinical case studies were included. Significant variation in TDM practice was seen; consequently, a meta-analysis was unsuitable. Few studies assessed impacts on AMR. No significant improvement on health outcomes and no detrimental effects of carbapenem TDM were observed. Five cohort studies showed significant associations between achieving target concentrations and clinical success, including suppression of resistance. Studies in this review showed no obvious improvement in clinical outcomes when TDM is implemented. Optimisation and standardisation of carbapenem TDM practice are needed to improve intervention success and enable study synthesis. Further suitably powered studies of standardised TDM are required to assess the impact of TMD on clinical outcomes and AMR.
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- 2022
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38. A systematic review of the effect of therapeutic drug monitoring on patient health outcomes during treatment with penicillins.
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Luxton T, King N, Wälti C, Jeuken L, and Sandoe J
- Subjects
- Humans, Outcome Assessment, Health Care, Drug Monitoring methods, Penicillins adverse effects
- Abstract
Background: Dosing regimens guided by therapeutic drug monitoring (TDM) may be able to improve penicillin exposure in patients, which could result in improved patient health outcomes., Objectives: This systematic review aims to describe the impact penicillin TDM has on health outcomes, including antimicrobial resistance (AMR)., Methods: Studies measuring penicillins in patient samples that adjusted regimens according to the result, and reported health outcomes were selected. Study bias was assessed according to study type. Included study characteristics were tabulated and described by narrative synthesis., Results: Three randomized controlled trials (RCTs), 16 cohort studies, and 9 case studies were included. No RCTs showed statistically significant improvements in health outcomes. Five cohort studies showed improvement in at least one health outcome associated with target attainment. However, there was a high risk of bias in all studies for health outcomes. One study assessed the impact of penicillin TDM on AMR and found that improved target attainment was associated with suppression of resistance. No studies found a detrimental effect of penicillin TDM., Conclusions: There is little evidence to suggest that TDM improves health outcomes, however neither health outcomes nor impact on AMR were adequately addressed. Variations in TDM implementation meant that a meta-analysis was not suitable. Penicillin TDM needs standardization, however there is currently no clear evidence of optimal conditions. Suitably powered studies are required to resolve the ambiguity surrounding the impact of TDM on clinical outcomes, including AMR. Further, standardized protocols and concentration targets need to be identified for TDM to be implemented successfully., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)
- Published
- 2022
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39. Corrigendum to article "Micro-homology intermediates: RecA's transient sampling revealed at the single molecule level''.
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Lee AJ, Endo M, Hobbs JK, Davies AG, and Wälti C
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- 2021
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40. Micro-homology intermediates: RecA's transient sampling revealed at the single molecule level.
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Lee AJ, Endo M, Hobbs JK, Davies AG, and Wälti C
- Subjects
- DNA chemistry, Microscopy, Atomic Force, Nanostructures chemistry, Polymerization, Sequence Homology, Nucleic Acid, DNA-Binding Proteins metabolism, Escherichia coli Proteins metabolism, Homologous Recombination, Rec A Recombinases metabolism
- Abstract
Recombinase A (RecA) is central to homologous recombination. However, despite significant advances, the mechanism with which RecA is able to orchestrate a search for homology remains elusive. DNA nanostructure-augmented high-speed AFM offers the spatial and temporal resolutions required to study the RecA recombination mechanism directly and at the single molecule level. We present the direct in situ observation of RecA-orchestrated alignment of homologous DNA strands to form a stable recombination product within a supporting DNA nanostructure. We show the existence of subtle and short-lived states in the interaction landscape, which suggests that RecA transiently samples micro-homology at the single RecA monomer-level throughout the search for sequence alignment. These transient interactions form the early steps in the search for sequence homology, prior to the formation of stable pairings at >8 nucleotide seeds. The removal of sequence micro-homology results in the loss of the associated transient sampling at that location., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2021
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41. Rational design of DNA nanostructures for single molecule biosensing.
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Raveendran M, Lee AJ, Sharma R, Wälti C, and Actis P
- Subjects
- Biomarkers analysis, C-Reactive Protein analysis, Equipment Design, Humans, Limit of Detection, Nanotechnology methods, Aptamers, Nucleotide chemistry, Biosensing Techniques instrumentation, DNA chemistry, Nanostructures chemistry, Single Molecule Imaging instrumentation
- Abstract
The ability to detect low concentrations of biomarkers in patient samples is one of the cornerstones of modern healthcare. In general, biosensing approaches are based on measuring signals resulting from the interaction of a large ensemble of molecules with the sensor. Here, we report a biosensor platform using DNA origami featuring a central cavity with a target-specific DNA aptamer coupled with a nanopore read-out to enable individual biomarker detection. We show that the modulation of the ion current through the nanopore upon the DNA origami translocation strongly depends on the presence of the biomarker in the cavity. We exploit this to generate a biosensing platform with a limit of detection of 3 nM and capable of the detection of human C-reactive protein (CRP) in clinically relevant fluids. Future development of this approach may enable multiplexed biomarker detection by using ribbons of DNA origami with integrated barcoding.
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- 2020
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42. Standardized, comprehensive, hospital-based circuit training in people with multiple sclerosis: results on feasibility, adherence and satisfaction of the training intervention.
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Lehmann I, Thaler I, Luder G, Damm U, Wälti C, Steinheimer S, Verra ML, Müri RM, Nyffeler T, Vanbellingen T, and Kamm CP
- Subjects
- Activities of Daily Living, Adult, Aged, Disability Evaluation, Feasibility Studies, Female, Hospitals, Humans, Male, Middle Aged, Prospective Studies, Quality of Life, Circuit-Based Exercise methods, Circuit-Based Exercise standards, Multiple Sclerosis rehabilitation, Patient Compliance, Patient Satisfaction
- Abstract
Background: We developed a standardized, comprehensive, ambulatory, hospital-based neurorehabilitation program ("MS-Fit") to improve disability, activities of daily living and quality of life in people with multiple sclerosis (PwMS)., Aim: The aim of this study was to assess feasibility, adherence and satisfaction of the training intervention., Design: Prospective multicenter cohort study analysis., Setting: Ambulatory, hospital-based study., Population: PwMS, aged 18 to 75 years, complaining about multiple sclerosis-related disability affecting activities of daily living and/or quality of life., Methods: A standardized, ambulatory, hospital-based circuit training consisting of six workstations (aerobic exercise training, strength upper limbs, balance, manual dexterity, reactivity, strength and flexibility lower limbs) was performed two hours, twice weekly, for two months in groups of two to six participants supervised by experienced physiotherapists. Physiotherapists adapted the type and intensity of training according to the participants' individual performance using a training booklet. Program satisfaction and adherence were evaluated using a questionnaire and the attendance rate (clinicaltrials.gov Identifier: NCT02440516)., Results: Fifty-five participants started (mean age 52.82 years±10.68 standard deviation, range 29-74; 69% female; median Expanded Disability Status Scale 3.5, range 1.0-7.0) and 49 (89%) finished the training program. Main reasons to drop out during the training were lack of time, travel problems, social issues or Uthoff's phenomenon during the summer. All participants finalizing the training achieved >80% (mean 92.26%, ±7.59) attendance rate and sent back the questionnaire. Overall participant's satisfaction was high with a median of 9 points (range 4-10) on a Likert Scale from 0-10. Program quality was rated "good" with an overall median score of 39/50 points (range 26-50) and 95% of the participants would recommend the program to others., Conclusions: MS-Fit is a feasible training program with high patient satisfaction and adherence. It enables high intensity ambulatory training and can be easily reproduced due to its standardized nature., Clinical Rehabilitation Impact: MS-FIT enables a standardized ambulatory high intensity training that is easily reproducible. Participants benefit from group training and from individual adaption of the training through professional supervision.
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- 2020
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43. DNA nanostructures: A versatile lab-bench for interrogating biological reactions.
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Lee AJ and Wälti C
- Abstract
At its inception DNA nanotechnology was conceived as a tool for spatially arranging biological molecules in a programmable and deterministic way to improve their interrogation. To date, DNA nanotechnology has provided a versatile toolset of nanostructures and functional devices to augment traditional single molecule investigation approaches - including atomic force microscopy - by isolating, arranging and contextualising biological systems at the single molecule level. This review explores the state-of-the-art of DNA-based nanoscale tools employed to enhance and tune the interrogation of biological reactions, the study of spatially distributed pathways, the visualisation of enzyme interactions, the application and detection of forces to biological systems, and biosensing platforms.
- Published
- 2019
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44. Direct Single-Molecule Observation of Mode and Geometry of RecA-Mediated Homology Search.
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Lee AJ, Endo M, Hobbs JK, and Wälti C
- Subjects
- Binding Sites, DNA chemistry, Diffusion, Escherichia coli chemistry, Escherichia coli metabolism, Microscopy, Atomic Force methods, Models, Molecular, Nanostructures chemistry, Protein Binding, Protein Conformation, Rec A Recombinases chemistry, DNA metabolism, Escherichia coli enzymology, Rec A Recombinases metabolism
- Abstract
Genomic integrity, when compromised by accrued DNA lesions, is maintained through efficient repair via homologous recombination. For this process the ubiquitous recombinase A (RecA), and its homologues such as the human Rad51, are of central importance, able to align and exchange homologous sequences within single-stranded and double-stranded DNA in order to swap out defective regions. Here, we directly observe the widely debated mechanism of RecA homology searching at a single-molecule level using high-speed atomic force microscopy (HS-AFM) in combination with tailored DNA origami frames to present the reaction targets in a way suitable for AFM-imaging. We show that RecA nucleoprotein filaments move along DNA substrates via short-distance facilitated diffusions, or slides, interspersed with longer-distance random moves, or hops. Importantly, from the specific interaction geometry, we find that the double-stranded substrate DNA resides in the secondary DNA binding-site within the RecA nucleoprotein filament helical groove during the homology search. This work demonstrates that tailored DNA origami, in conjunction with HS-AFM, can be employed to reveal directly conformational and geometrical information on dynamic protein-DNA interactions which was previously inaccessible at an individual single-molecule level.
- Published
- 2018
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45. Cooperative RecA clustering: the key to efficient homology searching.
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Lee AJ, Sharma R, Hobbs JK, and Wälti C
- Subjects
- Adenosine Triphosphate metabolism, DNA chemistry, DNA genetics, Escherichia coli Proteins chemistry, Escherichia coli Proteins genetics, Microscopy, Atomic Force, Molecular Dynamics Simulation, Nucleoproteins chemistry, Nucleoproteins genetics, Nucleoproteins metabolism, Protein Binding, Protein Conformation, Rec A Recombinases chemistry, Rec A Recombinases genetics, DNA metabolism, Escherichia coli Proteins metabolism, Homologous Recombination, Rec A Recombinases metabolism
- Abstract
The mechanism by which pre-synaptic RecA nucleoprotein filaments efficiently locate sequence homology across genomic DNA remains unclear. Here, using atomic force microscopy, we directly investigate the intermediates of the RecA-mediated homologous recombination process and find it to be highly cooperative, involving multiple phases. Initially, the process is dominated by a rapid 'association' phase, where multiple filaments interact on the same dsDNA simultaneously. This cooperative nature is reconciled by the observation of localized dense clusters of pre-synaptic filaments interacting with the observed dsDNA molecules. This confinement of reactive species within the vicinity of the dsDNA, is likely to play an important role in ensuring that a high interaction rate between the nucleoprotein filaments and the dsDNA can be achieved. This is followed by a slower 'resolution' phase, where the synaptic joints either locate sequence homology and progress to a post-synaptic joint, or dissociate from the dsDNA. Surprisingly, the number of simultaneous synaptic joints decreases rapidly after saturation of the dsDNA population, suggesting a reduction in interaction activity of the RecA filaments. We find that the time-scale of this decay is in line with the time-scale of the dispersion of the RecA filament clusters, further emphasising the important role this cooperative phenomena may play in the RecA-facilitated homology search., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2017
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46. Soft Ultraviolet (UV) Photopatterning and Metallization of Self-Assembled Monolayers (SAMs) Formed from the Lipoic Acid Ester of α-Hydroxy-1-acetylpyrene: The Generality of Acid-Catalyzed Removal of Thiol-on-Gold SAMs using Soft UV Light.
- Author
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Pukenas L, Prompinit P, Nishitha B, Tate DJ, Singh NDP, Wälti C, Evans SD, and Bushby RJ
- Abstract
Under a layer of 0.1 M HCl in isopropanol, soft ultraviolet (UV) (365 nm) photolysis of the thiol-on-gold self-assembled monolayer (SAM) derived from the lipoic acid ester of α-hydroxy-1-acetylpyrene results in the expected removal of the acetylpyrene protecting group. When photolyzing through a mask, this can be used to produce a patterned surface and, at a controlled electrochemical potential, it is then possible to selectively and reversibly electrodeposit copper on the photolyzed regions. Rather surprisingly, under these photolysis conditions, there is not only the expected photodeprotection of the ester but also partial removal of the lipoic acid layer which has been formed. In further studies, it is shown that this type of acid-catalyzed photoremoval of SAM layers by soft UV is a rather general phenomenon and results in the partial removal of the thiol-on-gold SAM layers derived from other ω-thiolated carboxylic acids. However, this phenomenon is chain-length dependent. Under conditions in which there is a ∼60% reduction in the thickness of the SAM derived from dithiobutyric acid, the SAM derived from mercaptoundecanoic acid is almost unaffected. The process by which the shorter-chain SAM layers are partially removed is not fully understood because these compounds do not absorb significantly in the 365 nm region of the spectrum! Significantly, this study shows that acid catalysis photolysis of thiol-on-gold SAMs needs to be used with caution.
- Published
- 2017
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47. Rapid cell separation with minimal manipulation for autologous cell therapies.
- Author
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Smith AJ, O'Rorke RD, Kale A, Rimsa R, Tomlinson MJ, Kirkham J, Davies AG, Wälti C, and Wood CD
- Subjects
- Cell Separation instrumentation, Cells, Cultured, Dental Pulp cytology, Electrophoresis instrumentation, Electrophoresis methods, Humans, Mesenchymal Stem Cells cytology, Microfluidics instrumentation, Saccharomyces cerevisiae cytology, Sonication instrumentation, Sonication methods, Cell Separation methods, Microfluidics methods
- Abstract
The ability to isolate specific, viable cell populations from mixed ensembles with minimal manipulation and within intra-operative time would provide significant advantages for autologous, cell-based therapies in regenerative medicine. Current cell-enrichment technologies are either slow, lack specificity and/or require labelling. Thus a rapid, label-free separation technology that does not affect cell functionality, viability or phenotype is highly desirable. Here, we demonstrate separation of viable from non-viable human stromal cells using remote dielectrophoresis, in which an electric field is coupled into a microfluidic channel using shear-horizontal surface acoustic waves, producing an array of virtual electrodes within the channel. This allows high-throughput dielectrophoretic cell separation in high conductivity, physiological-like fluids, overcoming the limitations of conventional dielectrophoresis. We demonstrate viable/non-viable separation efficacy of >98% in pre-purified mesenchymal stromal cells, extracted from human dental pulp, with no adverse effects on cell viability, or on their subsequent osteogenic capabilities., Competing Interests: The authors declare no competing financial interests.
- Published
- 2017
- Full Text
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48. Enhancement of RecA-mediated self-assembly in DNA nanostructures through basepair mismatches and single-strand nicks.
- Author
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Corbett SL, Sharma R, Davies AG, and Wälti C
- Subjects
- Base Pair Mismatch genetics, DNA, Single-Stranded chemistry, Escherichia coli genetics, Kinetics, Nucleoproteins genetics, DNA chemistry, Nanostructures chemistry, Nucleoproteins chemistry, Rec A Recombinases chemistry
- Abstract
The use of DNA as a structural material for nanometre-scale construction has grown extensively over the last decades. The development of more advanced DNA-based materials would benefit from a modular approach enabling the direct assembly of additional elements onto nanostructures after fabrication. RecA-based nucleoprotein filaments encapsulating short ssDNA have been demonstrated as a tool for highly efficient and fully programmable post-hoc patterning of duplex DNA scaffold. However, the underlying assembly process is not fully understood, in particular when patterning complex DNA topologies. Here, we report the effect of basepair-mismatched regions and single-strand nicks in the double-stranded DNA scaffold on the yield of RecA-based assembly. Significant increases in assembly yield are observed upon the introduction of unpaired basepairs directly adjacent to the assembly region. However, when the unpaired regions were introduced further from the assembly site the assembly yield initially decreased as the length of the unpaired region was increased. These results suggest that an unpaired region acts as a kinetic trap for RecA-based nucleoprotein filaments, impeding the assembly mechanism. Conversely, when the unpaired region is located directly adjacent to the assembly site, it leads to an increase in efficiency of RecA patterning owing to increased breathing of the assembly site.
- Published
- 2017
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49. The influence of two-dimensional organization on peptide conformation.
- Author
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White SJ, Johnson SD, Sellick MA, Bronowska A, Stockley PG, and Wälti C
- Subjects
- Circular Dichroism, Hydrogen-Ion Concentration, Immobilized Proteins chemistry, Molecular Dynamics Simulation, Osmolar Concentration, Peptides metabolism, Protein Structure, Secondary, Protein Structure, Tertiary, Peptides chemistry
- Abstract
Molecular crowding plays a significant role in regulating molecular conformation in cellular environments. It is also likely to be important wherever high molecular densities are required, for example in surface-phase studies, in which molecular densities generally far exceed those observed in solution. Using on-surface circular dichroism (CD) spectroscopy, we have investigated the structure of a synthetic peptide assembled into a highly packed monolayer. The immobilized peptide undergoes a structural transition between α-helical and random coil conformation upon changes in pH and ionic concentration, but critically the threshold for conformational change is altered dramatically by molecular crowding within the peptide monolayer. This study highlights the often overlooked role molecular crowding plays in regulating molecular structure and function in surface-phase studies of biological molecules., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2015
- Full Text
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50. Solid phase synthesis of functionalised SAM-forming alkanethiol-oligoethyleneglycols.
- Author
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Murray J, Nowak D, Pukenas L, Azhar R, Guillorit M, Wälti C, Critchley K, Johnson S, and Bon RS
- Abstract
We present an efficient solid phase synthesis methodology that provides easy access to a range of functionalised long-chain alkanethiol-oligoethyleneglycols that form well-defined self-assembled monolayers on gold and are compatible with pre- or post-assembly conjugation of (bio)molecules. We demonstrate the versatility of our synthetic route by synthesising LCAT-OEGs with a range of functional moieties, including peptides, electro-active redox groups, chemical handles for post-assembly conjugation of (bio)molecules, and demonstrate the application of our LCAT-OEG monolayers in immunosensing, where they show good biocompatibility with minimal biofouling.
- Published
- 2014
- Full Text
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