1. |
- Aliakbarinodehi, Nima, 1986, et al.
(författare)
-
Interaction Kinetics of Individual mRNA-Containing Lipid Nanoparticles with an Endosomal Membrane Mimic: Dependence on pH, Protein Corona Formation, and Lipoprotein Depletion
- 2022
-
Ingår i: ACS Nano. - : American Chemical Society (ACS). - 1936-086X .- 1936-0851. ; 16:12, s. 20163-20173
-
Tidskriftsartikel (refereegranskat)abstract
- Lipid nanoparticles (LNPs) have emerged as potent carriers for mRNA delivery, but several challenges remain before this approach can offer broad clinical translation of mRNA therapeutics. To improve their efficacy, a better understanding is required regarding how LNPs are trapped and processed at the anionic endosomal membrane prior to mRNA release. We used surface-sensitive fluorescence microscopy with single LNP resolution to investigate the pH dependency of the binding kinetics of ionizable lipid-containing LNPs to a supported endosomal model membrane. A sharp increase of LNP binding was observed when the pH was lowered from 6 to 5, accompanied by stepwise large-scale LNP disintegration. For LNPs preincubated in serum, protein corona formation shifted the onset of LNP binding and subsequent disintegration to lower pH, an effect that was less pronounced for lipoprotein-depleted serum. The LNP binding to the endosomal membrane mimic was observed to eventually become severely limited by suppression of the driving force for the formation of multivalent bonds during LNP attachment or, more specifically, by charge neutralization of anionic lipids in the model membrane due to their association with cationic lipids from earlier attached LNPs upon their disintegration. Cell uptake experiments demonstrated marginal differences in LNP uptake in untreated and lipoprotein-depleted serum, whereas lipoprotein-depleted serum increased mRNA-controlled protein (eGFP) production substantially. This complies with model membrane data and suggests that protein corona formation on the surface of the LNPs influences the nature of the interaction between LNPs and endosomal membranes.
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2. |
- Emilsson, Gustav, 1989, et al.
(författare)
-
Polymer brushes in solid-state nanopores form an impenetrable entropic barrier for proteins
- 2018
-
Ingår i: Nanoscale. - : Royal Society of Chemistry (RSC). - 2040-3372 .- 2040-3364. ; 10:10, s. 4663-4669
-
Tidskriftsartikel (refereegranskat)abstract
- Polymer brushes are widely used to prevent the adsorption of proteins, but the mechanisms by which they operate have remained heavily debated for many decades. We show conclusive evidence that a polymer brush can be a remarkably strong kinetic barrier towards proteins by using poly(ethylene glycol) grafted to the sidewalls of pores in 30 nm thin gold films. Despite consisting of about 90% water, the free coils seal apertures up to 100 nm entirely with respect to serum protein translocation, as monitored label-free through the plasmonic activity of the nanopores. The conclusions are further supported by atomic force microscopy and fluorescence microscopy. A theoretical model indicates that the brush undergoes a morphology transition to a sealing state when the ratio between the extension and the radius of curvature is approximately 0.8. The brush-sealed pores represent a new type of ultrathin filter with potential applications in bioanalytical systems.
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3. |
- Ferrand-Drake Del Castillo, Gustav, 1990, et al.
(författare)
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Quantitative Analysis of Thickness and pH Actuation of Weak Polyelectrolyte Brushes
- 2018
-
Ingår i: Journal of Physical Chemistry C. - : American Chemical Society (ACS). - 1932-7447 .- 1932-7455. ; 122:48, s. 27516-27527
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Tidskriftsartikel (refereegranskat)abstract
- Polymer brushes are widely used as surface coatings for various inert, functional, or responsive interfaces. If the polymer can alter its protonation state (a polyelectrolyte (PE)), the brush can switch between a collapsed and swollen state with pH, which enables applications such as nanoscale actuators. However, changes in brush height as the polymer alters its charge state are not straightforward to measure accurately. Here, we show how surface plasmon resonance can be used to determine the thickness of PE brushes both in their charged and neutral states. We use different methods to measure the heights of brushes consisting of poly(acrylic acid) and the polybasic poly(2-(diethylamino)ethyl methacrylate), both prepared by atom transfer radical polymerization. We find polymers in solution that can act as refractive index probes, which do not interact with the grafted polyelectrolytes, thus providing an "exclusion height" of the brush. Importantly, the angular reflection spectrum can be used to directly identify if a probe is indeed noninteracting. Furthermore, using different noninteracting probes results in small but significant changes (∼10%) in the exclusion height as long as the probe is reasonably large (approximately >2 kg/mol). These differences cannot be attributed to probe charge. Data from multiple brushes show that the relative height increase (at physiological ionic strength), i.e., the "collapse ratio" upon charging due to pH alterations, increases with the absolute brush height. In addition, we show that the plasmonic response to the pH switching of the polyelectrolyte brush is opposite to the response of hydrophilic polymer brushes collapsing at the lower critical solution temperature. This phenomenon is explained by an increase in refractometric constant upon charging. Our study shows that surface plasmon resonance is an excellent tool for characterizing polyelectrolyte brushes and provides useful insights into pH actuation not easily obtained by other methods.
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4. |
- Malekian, Bita, 1986, et al.
(författare)
-
Detecting Selective Protein Binding Inside Plasmonic Nanopores: Toward a Mimic of the Nuclear Pore Complex
- 2018
-
Ingår i: Frontiers in Chemistry. - : Frontiers Media SA. - 2296-2646. ; 6:December 2018
-
Tidskriftsartikel (refereegranskat)abstract
- Biosensors based on plasmonic nanostructures offer label-free and real-time monitoring of biomolecular interactions. However, so do many other surface sensitive techniques with equal or better resolution in terms of surface coverage. Yet, plasmonic nanostructures offer unique possibilities to study effects associated with nanoscale geometry. In this work we use plasmonic nanopores with double gold films and detect binding of proteins inside them. By thiol and trietoxysilane chemistry, receptors are selectively positioned on the silicon nitride interior walls. Larger (similar to 150 nm) nanopores are used detect binding of averaged sized proteins (similar to 60 kg/mol) with high signal to noise (>100). Further, we fabricate pores that approach the size of the nuclear pore complex (diameter down to 50 nm) and graft disordered phenylalanine-glycine nucleoporin domains to the walls, followed by titration of karyopherin beta 1 transport receptors. The interactions are shown to occur with similar affinity as determined by conventional surface plasmon resonance on planar surfaces. Our work illustrates another unique application of plasmonic nanostructures, namely the possibility to mimic the geometry of a biological nanomachine with integrated optical sensing capabilities.
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5. |
- Acimovic, Srdjan, 1982, et al.
(författare)
-
Antibody−antigen interaction dynamics revealed by analysis of single-molecule equilibrium fluctuations on individual plasmonic nanoparticle biosensors
- 2018
-
Ingår i: ACS Nano. - : American Chemical Society (ACS). - 1936-086X .- 1936-0851. ; 12:10, s. 9958-9965
-
Tidskriftsartikel (refereegranskat)abstract
- Antibody−antigen interactions are complex events central to immune response, in vivo and in vitro diagnostics, and development of therapeutic substances. We developed an ultrastable single-molecule localized surface plasmon resonance (LSPR) sensing platform optimized for studying antibody−antigen interaction kinetics over very long time scales. The setup allowed us to perform equilibrium fluctuations analysis of the PEG/anti-PEG interaction. By time and frequency domain analysis, we demonstrate that reversible adsorption of monovalently bound anti-PEG antibodies is the dominant factor affecting the LSPR fluctuations. The results suggest that equilibrium fluctuation analysis can be an alternative to established methods for determination of interaction rates. In particular, the methodology is suited to analyze molecular systems whose properties change during the initial interaction phases, for example, due to mass transport limitations or, as demonstrated here, because the effective association rate constant varies with surface concentration of adsorbed molecules.
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6. |
- Acimovic, Srdjan, 1982, et al.
(författare)
-
Superior LSPR substrates based on electromagnetic decoupling for on-a-chip high-throughput label-free biosensing
- 2017
-
Ingår i: Light: Science and Applications. - : Springer Science and Business Media LLC. - 2047-7538 .- 2095-5545. ; 6:8, s. e17042-
-
Tidskriftsartikel (refereegranskat)abstract
- Localized surface plasmon resonance (LSPR) biosensing based on supported metal nanoparticles offers unparalleled possibilities for high-end miniaturization, multiplexing and high-throughput label-free molecular interaction analysis in real time when integrated within an opto-fluidic environment. However, such LSPR-sensing devices typically contain extremely large regions of dielectric materials that are open to molecular adsorption, which must be carefully blocked to avoid compromising the device readings. To address this issue, we made the support essentially invisible to the LSPR by carefully removing the dielectric material overlapping with the localized plasmonic fields through optimized wet-etching. The resulting LSPR substrate, which consists of gold nanodisks centered on narrow SiO2 pillars, exhibits markedly reduced vulnerability to nonspecific substrate adsorption, thus allowing, in an ideal case, the implementation of thicker and more efficient passivation layers. We demonstrate that this approach is effective and fully compatible with state-of-the-art multiplexed real-time biosensing technology and thus represents the ideal substrate design for high-throughput label-free biosensing systems with minimal sample consumption.
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7. |
- Aliakbarinodehi, Nima, 1986, et al.
(författare)
-
Time-Resolved Inspection of Ionizable Lipid-Facilitated Lipid Nanoparticle Disintegration and Cargo Release at an Early Endosomal Membrane Mimic
- 2024
-
Ingår i: ACS Nano. - 1936-086X .- 1936-0851. ; In Press
-
Tidskriftsartikel (refereegranskat)abstract
- Advances in lipid nanoparticle (LNP) design have contributed notably to the emergence of the current clinically approved mRNA-based vaccines and are of high relevance for delivering mRNA to combat diseases where therapeutic alternatives are sparse. LNP-assisted mRNA delivery utilizes ionizable lipid-mediated cargo translocation across the endosomal membrane driven by the acidification of the endosomal environment. However, this process occurs at a low efficiency, a few percent at the best. Utilizing surface-sensitive fluorescence microscopy with a single LNP and mRNA resolution, we have investigated pH-controlled interactions between individual LNPs and a planar anionic supported lipid bilayer (SLB) formed on nanoporous silica, mimicking the electrostatic conditions of the early endosomal membrane. For LNPs with an average diameter of 140 nm, fusion with the anionic SLB preferentially occurred when the pH was reduced from 6.6 to 6.0. Furthermore, there was a delay in the onset of LNP fusion after the pH drop, and upon fusion, a significant fraction (>70%) of mRNA was released into the acidic solution representing the endosomal lumen, while a fraction of mRNA remained bound to the SLB even after reversing the pH to neutral cytosolic conditions. Finally, a comparison of the fusion efficiency of two LNP formulations with different surface concentrations of gel-forming lipids correlated with differences in the protein translation efficiency previously observed in human primary cell transfection studies. Together, these findings emphasize the relevance of biophysical investigations of ionizable lipid-containing LNP-assisted mRNA delivery mechanisms while potentially also offering means to optimize the design of LNPs with enhanced endosomal escape capabilities.
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8. |
- Emilsson, Gustav, 1989, et al.
(författare)
-
Gating Protein Transport in Solid State Nanopores by Single Molecule Recognition
- 2018
-
Ingår i: ACS Central Science. - : American Chemical Society (ACS). - 2374-7951 .- 2374-7943. ; 4:8, s. 1007-1014
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Tidskriftsartikel (refereegranskat)abstract
- Control of molecular translocation through nanoscale apertures is of great interest for DNA sequencing, biomolecular filters, and new platforms for single molecule analysis. However, methods for controlling the permeability of nanopores are very limited. Here, we show how nanopores functionalized with poly(ethylene glycol) brushes, which fully prevent protein translocation, can be reversibly gated to an "open" state by binding of single IgG antibodies that disrupt the macromolecular barrier. On the basis of surface plasmon resonance data we propose a two-state model describing the antibody-polymer interaction kinetics. Reversibly (weakly) bound antibodies decrease the protein exclusion height while irreversibly (strongly) bound antibodies do not. Our results are further supported by fluorescence readout from pore arrays and high-speed atomic force microscopy on single pores. This type of dynamic barrier control on the nanoscale provides new possibilities for biomolecular separation and analysis.
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9. |
- Emilsson, Gustav, 1989, et al.
(författare)
-
Identifying bacteria using DNA binding maps
- 2013
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Ingår i: 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013; Freiburg; Germany; 27 October 2013 through 31 October 2013. - 9781632666246 ; 1, s. 473-475
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Konferensbidrag (refereegranskat)abstract
- We have developed an assay, based on nanofluidic channels and fluorescence microscopy, for optical mapping of DNA based on competitive binding between two molecules - one fluorescent and one sequence selective. From the experimental data we can extract binding constants for the two competing DNA binders, which may be subsequently used to calculate a theoretical reference map of any DNA with known sequence. The goal is to create a method for fast identification of bacteria from single DNA molecules without the need for additional cultivation or amplification. We here demonstrate a proof-of-principle experiment on phage DNA and furthermore show that the method can be used to distinguish between two strains of E. coli DNA and to map pieces of DNA onto the full genome.
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10. |
- Emilsson, Gustav, 1989, et al.
(författare)
-
Strongly Stretched Protein Resistant Poly(ethylene glycol) Brushes Prepared by Grafting-To
- 2015
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Ingår i: ACS Applied Materials & Interfaces. - : American Chemical Society (ACS). - 1944-8252 .- 1944-8244. ; 7:14, s. 7505-7515
-
Tidskriftsartikel (refereegranskat)abstract
- We present a new grafting-to method for resistant non-fouling poly(ethylene glycol) brushes, which is based on grafting of polymers with reactive end groups in 0.9 M Na2SO4 at room temperature. The grafting process, the resulting brushes, and the resistance toward biomolecular adsorption are investigated by surface plasmon resonance, quartz crystal microbalance, and atomic force microscopy. We determine both grafting density and thickness independently and use narrow molecular weight distributions which result in well-defined brushes. High density (e.g., 0.4 coils per nm(2) for 10 kDa) and thick (40 nm for 20 kDa) brushes are readily achieved that suppress adsorption from complete serum (10x dilution, exposure for 50 min) by up to 99% on gold (down to 4 ng/cm(2) protein coverage). The brushes outperform oligo(ethylene glycol) monolayers prepared on the same surfaces and analyzed in the same manner. The brush heights are in agreement with calculations based on a simple model similar to the de Gennes strongly stretched brush, where the height is proportional to molecular weight. This result has so far generally been considered to be possible only for brushes prepared by grafting-from. Our results are consistent with the theory that the brushes act as kinetic barriers rather than efficient prevention of adsorption at equilibrium. We suggest that the free energy barrier for passing the brush depends on both monomer concentration and thickness. The extraordinary simplicity of the method and good inert properties of the brushes should make our results widely applicable in biointerface science.
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11. |
- Emilsson, Gustav, 1989, et al.
(författare)
-
Surface plasmon resonance methodology for monitoring polymerization kinetics and morphology changes of brushes-evaluated with poly(N-isopropylacrylamide)
- 2017
-
Ingår i: Applied Surface Science. - : Elsevier BV. - 0169-4332. ; 396, s. 384-392
-
Tidskriftsartikel (refereegranskat)abstract
- Polymerization from surfaces and the resulting “brushes” have many uses in the development of novel materials and functional interfaces. However, it is difficult to accurately monitor the polymerization rate, which limits the use of polymer brushes in applications where control of thickness is desirable. We present a new methodology based on angular surface plasmon resonance (SPR) which provides real-time measurements of the thickness evolution during atom transfer radical polymerization, using poly(N-isopropylacrylamide) as an example. Our data analysis shows that the growth is linear with a rate of ?20 nm/min in a water/methanol mixture up to ?100 nm after which chain termination gradually reduces the growth rate. Further, we introduce an improved method in SPR which makes it possible to determine changes in brush height and refractive index during switching of responsive polymers. The ratio between heights in the coil to globule transition at 32 °C in water was found to be almost 5, independent of the initial absolute height up to ?200 nm, in agreement with theory. Complementary quartz crystal microbalance and atomic force microscopy data confirm the accuracy of our results. With the methodology presented here the established SPR technique can be used for quantitative characterization of surface-initiated polymerization and responsive polymer brushes.
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12. |
- Emilsson, Gustav, 1989, et al.
(författare)
-
The In Vivo Fate of Polycatecholamine Coated Nanoparticles Is Determined by a Fibrinogen Enriched Protein Corona
- 2023
-
Ingår i: ACS Nano. - 1936-086X .- 1936-0851. ; 17:24, s. 24725-24742
-
Tidskriftsartikel (refereegranskat)abstract
- Polycatecholamine coatings have attracted significant attention in the past 10 years owing to their ability to functionalize a wide range of materials. Here we apply the use of such coatings to drug nanocrystals, made from a poorly soluble drug compound, to postfunctionalize the nanocrystal surface with the aim of providing steric stabilization and extending their circulation time after intravenous injection. We show that both polydopamine and polynorepinephrine can be used to successfully modify drug nanocrystals and subsequently incorporate end-functionalized PEG to the surface. Even though high grafting densities of PEG were achieved, we observed rapid clearance and increased liver uptake for polycatecholamine functionalized drug nanocrystals. Using both surface sensitive model systems and protein corona profiling, we determine that the rapid clearance was correlated with an increase in adsorption of proteins involved in coagulation to the polycatecholamine surface, with fibrinogen being the most abundant. Further analysis of the most abundant proteins revealed a significant increase in thiol-rich proteins on polycatecholamine coated surfaces. The observed interaction with coagulation proteins highlights one of the current challenges using polycatecholamines for drug delivery but might also provide insights to the growing use of these materials in hemostatic applications.
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13. |
- Emilsson, Gustav, 1989
(författare)
-
Towards Functional Nanopores Using Polymers
- 2016
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In nature there are several examples of nanometer sized gateways able to control the passage of molecules and other substances with remarkable precision. This has inspired research in trying to create nanoscale openings which resemble such gateways. To gain functionalities similar to what is available in nature it is necessary to functionalize such mimics with soft materials. One candidate for such functionalization is polymers, as these posses a lot of the characteristics desirable to mimic biological systems. This thesis is focused on how polymers behave at interfaces and how polymer surfaces can be prepared and characterized. Different approaches to functionalize surfaces with two kinds of polymers is described: poly(ethylene glycol), which e.g. can be used to reduce non-specific adsorption of serum by up to 97 %, and poly(N-isopropylacrylamide) which can be used to create thermoresponsive surfaces with tunable thickness. The approaches involve "grafting to", when polymers are attached to a surface using material specific end-groups, and "grafting from" where a radical polymerization scheme is used to grow the polymer directly from the surface. The formed brushes are characterized using different methods, such as surface plasmon resonance and quartz crystal microbalance with dissipation, and the basis of these methods are presented. In particular new ways to use surface plasmon resonance for height determination is utilized and discussed.The protocols described in this thesis is intended for implementation with plasmonic nanostructures, in particular nanopores, to create different kinds of passive and active nanoscale gates with uses in bioseparation and biosensing
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14. |
- Ferhan, Abdul Rahim, et al.
(författare)
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Nanoplasmonic Sensing Architectures for Decoding Membrane Curvature-Dependent Biomacromolecular Interactions
- 2018
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 90:12, s. 7458-7466
-
Tidskriftsartikel (refereegranskat)abstract
- Nanoplasmonic sensors have emerged as a promising measurement approach to track biomacromolecular interactions involving lipid membrane interfaces. By taking advantage of nanoscale fabrication capabilities, it is possible to design sensing platforms with various architectural configurations. Such capabilities open the door to fabricating lipid membrane-coated nanoplasmonic sensors with varying degrees of membrane curvature in order to understand how biomacromolecular interaction processes are influenced by membrane curvature. Herein, we employed an indirect nanoplasmonic sensing approach to characterize the fabrication of supported lipid bilayers (SLBs) on silica-coated nanowell and nanodisk sensing platforms and to investigate how membrane curvature influences membrane-peptide interactions by evaluating the corresponding measurement responses from different spectral signatures that are sensitive to specific regions of the sensor geometries. SLBs were prepared by the vesicle fusion method, as monitored in real-time by nanoplasmonic sensing measurements and further characterized by fluorescence recovery after photobleaching (FRAP) experiments. By resolving different spectral signatures in the nanoplasmonic sensing measurements, it was determined that peptide binding induces membrane disruption at positively curved membrane regions, while peptide binding without subsequent disruption was observed at planar and negatively curved regions. These findings are consistent with the peptide's known preference to selectively form pores in positively curved membranes, providing validation to the nanoplasmonic sensing approach and highlighting how the integration of nanoplasmonic sensors with different nanoscale architectures can be utilized to study the influence of membrane curvature on biomacromolecular interaction processes.
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15. |
- Junesch, Juliane, 1987, et al.
(författare)
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Location-specific nanoplasmonic sensing of biomolecular binding to lipid membranes with negative curvature
- 2015
-
Ingår i: Nanoscale. - : Royal Society of Chemistry (RSC). - 2040-3372 .- 2040-3364. ; 7:37, s. 15080-15085
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Tidskriftsartikel (refereegranskat)abstract
- The biochemical processes of cell membranes are sensitive to the geometry of the lipid bilayer. We show how plasmonic "nanowells" provide label-free real-time analysis of molecules on membranes with detection of preferential binding at negative curvature. It is demonstrated that norovirus accumulate in invaginations due to multivalent interactions with glycosphingolipids.
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16. |
- Malekian, Bita, 1986, et al.
(författare)
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Fabrication and Characterization of Plasmonic Nanopores with Cavities in the Solid Support
- 2017
-
Ingår i: Sensors. - : MDPI AG. - 1424-8220. ; 17:6, s. Article no. 1444 -
-
Tidskriftsartikel (refereegranskat)abstract
- Plasmonic nanostructures are widely used for various sensing applications by monitoring changes in refractive index through optical spectroscopy or as substrates for surface enhanced Raman spectroscopy. However, in most practical situations conventional surface plasmon resonance is preferred for biomolecular interaction analysis because of its high resolution in surface coverage and the simple single-material planar interface. Still, plasmonic nanostructures may find unique sensing applications, for instance when the nanoscale geometry itself is of interest. This calls for new methods to prepare nanoscale particles and cavities with controllable dimensions and curvature. In this work, we present two types of plasmonic nanopores where the solid support underneath a nanohole array has been etched, thereby creating cavities denoted as 'nanowells' or 'nanocaves' depending on the degree of anisotropy (dry or wet etch). The refractometric sensitivity is shown to be enhanced upon removing the solid support because of an increased probing volume and a shift of the asymmetric plasmonic field towards the liquid side of the finite gold film. Furthermore, the structures exhibit different spectral changes upon binding inside the cavities compared to the gold surface, which means that the structures can be used for location-specific detection. Other sensing applications are also suggested.
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17. |
- Malekian, Bita, 1986, et al.
(författare)
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Optical properties of plasmonic nanopore arrays prepared by electron beam and colloidal lithography
- 2019
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Ingår i: Nanoscale Advances. - : Royal Society of Chemistry (RSC). - 2516-0230. ; 1:11, s. 4282-4289
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Tidskriftsartikel (refereegranskat)abstract
- Solid state nanopores are central structures for many applications. To date, much effort has been spent on controlled fabrication of single nanopores, while relatively little work has focused on large scale fabrication of arrays of nanopores. In this work we show wafer-scale fabrication of plasmonic nanopores in 50 nm thick silicon nitride membranes with one or two 30 nm gold films, using electron beam lithography with a negative resist or a new version of colloidal lithography. Both approaches offer good control of pore diameter (even below 100 nm) and with high yield (>90%) of intact membranes. Colloidal lithography has the advantage of parallel patterning without expensive equipment. Despite its serial nature, electron beam lithography provides high throughput and can make arbitrary array patterns. Importantly, both methods prevent metal from ending up on the membrane pore sidewalls. The new fabrication methods make it possible to compare the optical properties of structurally identical plasmonic nanopore arrays with either long-range order (e-beam) or short-range order (colloidal). The resonance features in the extinction spectrum are very similar for both structures when the pitch is the same as the characteristic spacing in the self-assembled colloidal pattern. Long-range ordering slightly enhances the magnitude of the extinction maximum and blueshift the transmission maximum by tens of nm. Upon reducing the diameter in long-range ordered arrays, the resonance is reduced in magnitude and the transmission maximum is further blue shifted, just like for short-range ordered arrays. These effects are well explained by interpreting the spectra as Fano interference between the grating-type excitation of propagating surface plasmons and the broad transmission via individual pores in the metal film. Furthermore, we find that only the short-range ordered arrays scatter light, which we attribute to the highly limited effective period in the short-range ordered system and the corresponding lack of coherent suppression of scattering by interference effects.
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18. |
- Nilsson, Adam, et al.
(författare)
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Competitive binding-based optical DNA mapping for fast identification of bacteria - multi-ligand transfer matrix theory and experimental applications on Escherichia coli.
- 2014
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 1362-4962 .- 0305-1048. ; 42:15, s. 118-118
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Tidskriftsartikel (refereegranskat)abstract
- We demonstrate a single DNA molecule optical mapping assay able to resolve a specific Escherichia coli strain from other strains. The assay is based on competitive binding of the fluorescent dye YOYO-1 and the AT-specific antibiotic netropsin. The optical map is visualized by stretching the DNA molecules in nanofluidic channels. We optimize the experimental conditions to obtain reproducible barcodes containing as much information as possible. We implement a multi-ligand transfer matrix method for calculating theoretical barcodes from known DNA sequences. Our method extends previous theoretical approaches for competitive binding of two types of ligands to many types of ligands and introduces a recursive approach that allows long barcodes to be calculated with standard computer floating point formats. The identification of a specific E. coli strain (CCUG 10979) is based on mapping of 50-160 kilobasepair experimental DNA fragments onto the theoretical genome using the developed theory. Our identification protocol introduces two theoretical constructs: a P-value for a best experiment-theory match and an information score threshold. The developed methods provide a novel optical mapping toolbox for identification of bacterial species and strains. The protocol does not require cultivation of bacteria or DNA amplification, which allows for ultra-fast identification of bacterial pathogens.
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19. |
- Nyberg, Lena, 1979, et al.
(författare)
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Optical mapping of single DNA molecules in nanochannels: A novel method for identification and characterization of antibiotic resistance
- 2015
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Ingår i: 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014. - 9780979806476 ; , s. 1045-1047
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Konferensbidrag (refereegranskat)abstract
- The use, and overuse, of antibiotics has during the last decade led to a dramatic increase in antibiotic resistance and there is a crying need for novel methods for fast identification of antibiotic resistance genes. We here demonstrate how our previously developed assay for optical mapping of DNA in nanochannels can be used for characterization of resistance genes located on plasmid DNA from bacteria. The assay requires extremely small sample volumes and does neither rely on PCR, nor culturing of bacteria, which greatly reduces the time for analysis.
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20. |
- Nyberg, Lena, 1979, et al.
(författare)
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Rapid identification of intact bacterial resistance plasmids via optical mapping of single DNA molecules
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 6
-
Tidskriftsartikel (refereegranskat)abstract
- The rapid spread of antibiotic resistance - currently one of the greatest threats to human health according to WHO - is to a large extent enabled by plasmid-mediated horizontal transfer of resistance genes. Rapid identification and characterization of plasmids is thus important both for individual clinical outcomes and for epidemiological monitoring of antibiotic resistance. Toward this aim, we have developed an optical DNA mapping procedure where individual intact plasmids are elongated within nanofluidic channels and visualized through fluorescence microscopy, yielding barcodes that reflect the underlying sequence. The assay rapidly identifies plasmids through statistical comparisons with barcodes based on publicly available sequence repositories and also enables detection of structural variations. Since the assay yields holistic sequence information for individual intact plasmids, it is an ideal complement to next generation sequencing efforts which involve reassembly of sequence reads from fragmented DNA molecules. The assay should be applicable in microbiology labs around the world in applications ranging from fundamental plasmid biology to clinical epidemiology and diagnostics.
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21. |
- Rupert, Deborah, 1986, et al.
(författare)
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Dual-Wavelength Surface Plasmon Resonance for Determining the Size and Concentration of Sub-Populations of Extracellular Vesicles
- 2016
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 88:20, s. 9980-9988
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Tidskriftsartikel (refereegranskat)abstract
- Accurate concentration determination of subpopulations of extracellular vesicles (EVs), such as exosomes, is of importance both in the context of understanding their fundamental biological role and of potentially using them as disease biomarkers. In principle, this can be achieved by measuring the rate of diffusion-limited mass uptake to a sensor surface modified with a receptor designed to only bind the subpopulation of interest. However, a significant error is introduced if the targeted EV subpopulation has a size, and thus hydrodynamic diffusion coefficient, that differs from the mean size and diffusion coefficient of the whole EV population and/or if the EVs become deformed upon binding to the surface. We here demonstrate a new approach to determine the mean size (or effective film thickness) of bound nanoparticles, in general, and EV subpopulation carrying a marker of interest, in particular. The method is based on operating surface plasmon resonance simultaneously at two wavelengths with different sensing depths and using the ratio of the corresponding responses to extract the particle size on the surface. By estimating in this way the degree of deformation of adsorbed EVs, we markedly improved their bulk concentration determination and showed that EVs carrying the exosomal marker CD63 correspond to not more than around 10% of the EV sample.
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22. |
- Schoch, Rafael L., et al.
(författare)
-
Protein exclusion is preserved by temperature sensitive PEG brushes
- 2017
-
Ingår i: Polymer. - : Elsevier BV. - 0032-3861. ; 132, s. 362-367
-
Tidskriftsartikel (refereegranskat)abstract
- Poly(ethylene glycol) (PEG) is widely used in biotechnology-related applications yet its temperature-dependent functionality is not well understood. Here, we use bovine serum albumin (BSA) monomers and cross-linked dimers to directly probe the height of strongly stretched PEG brushes using surface plasmon resonance (SPR) in aqueous solution. Our results show that PEG brush height follows a smooth decrease as a function of increasing temperature commencing near room temperature. Measurements obtained by BSA monomers and dimers are comparable and suggest that BSA effectively probes the leading edge of the brush with minimal penetration into its interior being supported by SPR reflectivity calculations. Further, the BSA-PEG interaction remains largely inert over the entire temperature range. Overall, PEG brushes undergo a smooth conformational transition while fully preserving its protein excluding properties far from the lower critical solution temperature.
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23. |
- Svirelis, Justas, 1993, et al.
(författare)
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Stable trapping of multiple proteins at physiological conditions using nanoscale chambers with macromolecular gates
- 2023
-
Ingår i: Nature Communications. - 2041-1723 .- 2041-1723. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- The possibility to detect and analyze single or few biological molecules is very important for understanding interactions and reaction mechanisms. Ideally, the molecules should be confined to a nanoscale volume so that the observation time by optical methods can be extended. However, it has proven difficult to develop reliable, non-invasive trapping techniques for biomolecules under physiological conditions. Here we present a platform for long-term tether-free (solution phase) trapping of proteins without exposing them to any field gradient forces. We show that a responsive polymer brush can make solid state nanopores switch between a fully open and a fully closed state with respect to proteins, while always allowing the passage of solvent, ions and small molecules. This makes it possible to trap a very high number of proteins (500-1000) inside nanoscale chambers as small as one attoliter, reaching concentrations up to 60 gL−1. Our method is fully compatible with parallelization by imaging arrays of nanochambers. Additionally, we show that enzymatic cascade reactions can be performed with multiple native enzymes under full nanoscale confinement and steady supply of reactants. This platform will greatly extend the possibilities to optically analyze interactions involving multiple proteins, such as the dynamics of oligomerization events.
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24. |
- Xiong, Kunli, 1987, et al.
(författare)
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Biosensing using plasmonic nanohole arrays with small, homogenous and tunable aperture diameters
- 2016
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Ingår i: The Analyst. - : Royal Society of Chemistry (RSC). - 0003-2654 .- 1364-5528. ; 141:12, s. 3803-3810
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Tidskriftsartikel (refereegranskat)abstract
- Plasmonic nanohole arrays are widely used for optical label-free molecular detection. An important factor for many applications is the diameter of the apertures. So far nanohole arrays with controllable diameters below 100 nm have not been demonstrated and it has not been systematically investigated how the diameter influences the optical properties. In this work we fine-tune the diameter in short range ordered nanohole arrays down to 50 nm. The experimental far field spectra show how the wavelength of maximum extinction remains unaffected while the transmission maximum blue shifts with smaller diameters. The near field is visualized by numerical simulations, showing a homogenous enhancement throughout the cylindrical void at the transmission maximum for diameters between 50 and 100 nm. For diameters below 50 nm plasmon excitation is no longer possible experimentally or by simulations. Further, we investigate the refractive index sensing capabilities of the smaller holes. As the diameter was reduced, the sensitivity in terms of resonance shift with bulk liquid refractive index was found to be unaltered. However, for the transmission maximum the sensitivity becomes more strongly localized to the hole interior. By directing molecular binding to the bottom of the holes we demonstrate how smaller holes enhance the sensitivity in terms of signal per molecule. A real-time detection limit well below one protein per nanohole is demonstrated. The smaller plasmonic nanoholes should be suitable for studies of molecules confined in small volumes and as mimics of biological nanopores.
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25. |
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26. |
- Xiong, Kunli, 1987, et al.
(författare)
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Switchable Plasmonic Metasurfaces with High Chromaticity Containing only Abundant Metals
- 2017
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Ingår i: Nano Letters. - : American Chemical Society (ACS). - 1530-6984 .- 1530-6992. ; 17:11, s. 7033-7039
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Tidskriftsartikel (refereegranskat)abstract
- Plasmonic color generation offers several advantages but is also limited by the cost and availability of noble metals like gold. In this work, we present color-tunable metasurfaces with high chromaticity and reflectivity consisting of an aluminum mirror, a dielectric spacer, and a plasmonic nanohole array in copper. Copper is shown to be an excellent alternative to gold when properly protected from oxidation and makes it possible to generate a wide RGB gamut covering 27% of the standard RGB. By patterning the metasurfaces into microscale pixel triplets, color photos can be well reproduced with high resolution over wafer-sized areas. Further, we demonstrate active modulation of the reflected intensity using an electrochromic conductive polymer deposited on top of the nanostructures by screen printing. This technology opens up for ultrathin and flexible reflective displays in full color, that is, plasmonic electronic paper, compatible with large-scale sustainable production.
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