| 1. |
- Svirelis, Justas, 1993, et al.
(author)
-
Stable trapping of multiple proteins at physiological conditions using nanoscale chambers with macromolecular gates
- 2023
-
In: Nature Communications. - 2041-1723 .- 2041-1723. ; 14:1
-
Journal article (peer-reviewed)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.
|
|
| 2. |
- Andersson, John, 1993, et al.
(author)
-
Pore performance: artificial nanoscale constructs that mimic the biomolecular transport of the nuclear pore complex
- 2022
-
In: Nanoscale Advances. - : Royal Society of Chemistry (RSC). - 2516-0230. ; 4:23, s. 4925-4937
-
Research review (peer-reviewed)abstract
- The nuclear pore complex is a nanoscale assembly that achieves shuttle-cargo transport of biomolecules: a certain cargo molecule can only pass the barrier if it is attached to a shuttle molecule. In this review we summarize the most important efforts aiming to reproduce this feature in artificial settings. This can be achieved by solid state nanopores that have been functionalized with the most important proteins found in the biological system. Alternatively, the nanopores are chemically modified with synthetic polymers. However, only a few studies have demonstrated a shuttle-cargo transport mechanism and due to cargo leakage, the selectivity is not comparable to that of the biological system. Other recent approaches are based on DNA origami, though biomolecule transport has not yet been studied with these. The highest selectivity has been achieved with macroscopic gels, but they are yet to be scaled down to nano-dimensions. It is concluded that although several interesting studies exist, we are still far from achieving selective and efficient artificial shuttle-cargo transport of biomolecules. Besides being of fundamental interest, such a system could be potentially useful in bioanalytical devices.
|
|
| 3. |
|
|
| 4. |
- Andersson, John, 1993, et al.
(author)
-
Surface plasmon resonance sensing with thin films of palladium and platinum - quantitative and real-time analysis
- 2022
-
In: Physical Chemistry Chemical Physics. - : Royal Society of Chemistry (RSC). - 1463-9084 .- 1463-9076. ; 24:7, s. 4588-4594
-
Journal article (peer-reviewed)abstract
- Surface plasmon resonance (SPR) is a highly useful technique in biology and is gradually becoming useful also for materials science. However, measurements to date have been performed almost exclusively on gold, which limits the possibility to probe chemical modifications of other metals. In this work we show that 20 nm Pd and Pt films work "fairly well" for quantitative SPR sensing of organic films despite the high light absorption. In the interval between total reflection and the SPR angle, high intensity changes occur when a film is formed on the surface. Fresnel models accurately describe the full angular spectra and our data analysis provides good resolution of surface coverage in air (a few ng cm(-2)). Overall, the Pd sensors behave quite similarly to 50 nm gold in terms of sensitivity and field extension, although the noise level in real-time measurements is similar to 5 times higher. The Pt sensors exhibit a longer extension of the evanescent field and similar to 10 times higher noise compared to gold. Yet, formation of organic layers a few nm in thickness can still be monitored in real-time. As a model system, we use thiolated poly(ethylene glycol) to make Pd and Pt protein repelling. Our findings show how SPR can be used for studying chemical modifications of two metals that are important in several contexts, for instance within heterogeneous catalysis. We emphasize the advantages of simple sample preparation and accurate quantitative analysis in the planar geometry by Fresnel models.
|
|
| 5. |
- Svirelis, Justas, 1993, et al.
(author)
-
Accurate Correction of the "bulk Response" in Surface Plasmon Resonance Sensing Provides New Insights on Interactions Involving Lysozyme and Poly(ethylene glycol)
- 2022
-
In: ACS Sensors. - : American Chemical Society (ACS). - 2379-3694. ; 7:4, s. 1175-1182
-
Journal article (peer-reviewed)abstract
- Surface plasmon resonance is a very well-established surface sensitive technique for label-free analysis of biomolecular interactions, generating thousands of publications each year. An inconvenient effect that complicates interpretation of SPR results is the "bulk response"from molecules in solution, which generate signals without really binding to the surface. Here we present a physical model for determining the bulk response contribution and verify its accuracy. Our method does not require a reference channel or a separate surface region. We show that proper subtraction of the bulk response reveals an interaction between poly(ethylene glycol) brushes and the protein lysozyme at physiological conditions. Importantly, we also show that the bulk response correction method implemented in commercial instruments is not generally accurate. Using our method, the equilibrium affinity between polymer and protein is determined to be KD = 200 μM. One reason for the weak affinity is that the interaction is relatively short-lived (1/koff < 30 s). Furthermore, we show that the bulk response correction also reveals the dynamics of self-interactions between lysozyme molecules on surfaces. Besides providing new insights on important biomolecular interactions, our method can be widely applied to improve the accuracy of SPR data generated by instruments worldwide.
|
|
| 6. |
- Andersson, John, 1993, et al.
(author)
-
Control of Polymer Brush Morphology, Rheology, and Protein Repulsion by Hydrogen Bond Complexation
- 2021
-
In: Langmuir. - : American Chemical Society (ACS). - 1520-5827 .- 0743-7463. ; 37:16, s. 4943-4952
-
Journal article (peer-reviewed)abstract
- Polymer brushes are widely used to alter the properties of interfaces. In particular, poly(ethylene glycol) (PEG) and similar polymers can make surfaces inert toward biomolecular adsorption. Neutral hydrophilic brushes are normally considered to have static properties at a given temperature. As an example, PEG is not responsive to pH or ionic strength. Here we show that, by simply introducing a polymeric acid such as poly(methacrylic acid) (PMAA), the highly hydrated brush barrier can change its properties entirely. This is caused by multivalent hydrogen bonds in an extremely pH-sensitive process. Remarkably, it is sufficient to reduce the pH to 5 for complexation to occur at the interface, which is two units higher than in the corresponding bulk systems. Below this critical pH, PMAA starts to bind to PEG in large amounts (comparable to the PEG amount), causing the brush to gradually compact and dehydrate. The brush also undergoes major rheology changes, from viscoelastic to rigid. Furthermore, the protein repelling ability of PEG is lost after reaching a threshold in the amount of PMAA bound. The changes in brush properties are tunable and become more pronounced when more PMAA is bound. The initial brush state is fully recovered when releasing PMAA by returning to physiological pH. Our findings are relevant for many applications involving functional interfaces, such as capture-release of biomolecules.
|
|
| 7. |
- Andersson, John, 1993
(author)
-
Functional polymer brush coatings for nanoscale devices
- 2022
-
Doctoral thesis (other academic/artistic)abstract
- Nanobiotechnology is an interdisciplinary field that has garnered considerable attention for offering exciting new opportunities of studying and manipulating biomolecules at the nanoscale. This prospect bears large potential benefits in the field of medicine and the whole life science sector in general. Fabrication of different nanostructure devices that can handle liquids at the scale of biomolecules, such as nanochannels or nanopores, provide a good basis within nanobiotechnology. However, the materials of nanostructures tend to not interact with complex biomolecules in ways that are sufficiently specific or controlled. This issue can be avoided by functionalising the surface of nanostructures with different organic coatings, and polymer brushes have shown a diverse range of functionality in this regard. This thesis summarises efforts towards designing functional polymer brush coatings for nanoscale devices. Surface sensitive techniques are used to characterise the grafting of dense poly(ethylene glycol) brushes to various noble metals and silicon dioxide. The new functionalisation protocol for polymer brushes on silicon dioxide provides excellent biofunctionality and is demonstrated to be compatible with two different nanostructures. The specific hydrogen-bond mediated interaction between a poly(ethylene glycol) brush and poly(methacrylic acid) in solution at low pH is shown to make the polymer brush reversibly stimuli-responsive. Preliminary results further demonstrate how this interaction can be controlled electrochemically and indicates its suitability as a macromolecular gating mechanism for nanosized openings. Finally, characterisation and fabrication of plasmonic nanopore arrays with separately functionalisable compartments using electron beam lithography techniques is presented.
|
|
| 8. |
- Andersson, John, 1993
(author)
-
Interpolymer complexation of a polymer brush
- 2020
-
Licentiate thesis (other academic/artistic)abstract
- Controllable macromolecular gating between nanoscopic compartments is of high interest for single molecule studies of biological macromolecules. By definition, a good macromolecular gate should completely stop biomolecules, such as proteins, from crossing between compartments in its closed state while letting them pass in its open state. Polymer brushes of poly(ethylene glycol) have been proven excellent barriers for proteins in previous work, but are limited in terms of stimuli-responsive behaviour needed for macromolecular gating. In this thesis work, the pH reversible interpolymer complexation between a poly(ethylene glycol) brush and poly(methacrylic acid) was investigated as a potential macromolecular gating mechanism. Conclusions were based on the evaluation of the resulting surface complex using three surface sensitive characterisation techniques. Upon complexing at low pH, the polymer layer was found to adopt a shrunken state with significant behavioural changes, while completely reversing back to its initial state after a neutral pH had been introduced. This pH reversible interaction show great promise as a pH controlled macromolecular gating mechanism and calls for further studies with suitable nanostructures. To this end, the fabrication and properties of a new solid-state nanopore device is also presented, together with the direction needed for future work towards a new macromolecular gating system.
|
|
| 9. |
- Andersson, John, 1993, et al.
(author)
-
Polymer Brushes on Silica Nanostructures Prepared by Aminopropylsilatrane Click Chemistry: Superior Antifouling and Biofunctionality
- 2023
-
In: ACS Applied Materials & Interfaces. - : American Chemical Society (ACS). - 1944-8252 .- 1944-8244. ; 15:7, s. 10228-10239
-
Journal article (peer-reviewed)abstract
- In nanobiotechnology, the importance of controlling interactions between biological molecules and surfaces is paramount. In recent years, many devices based on nanostructured silicon materials have been presented, such as nanopores and nanochannels. However, there is still a clear lack of simple, reliable, and efficient protocols for preventing and controlling biomolecule adsorption in such structures. In this work, we show a simple method for passivation or selective biofunctionalization of silica, without the need for polymerization reactions or vapor-phase deposition. The surface is simply exposed stepwise to three different chemicals over the course of ∼1 h. First, the use of aminopropylsilatrane is used to create a monolayer of amines, yielding more uniform layers than conventional silanization protocols. Second, a cross-linker layer and click chemistry are used to make the surface reactive toward thiols. In the third step, thick and dense poly(ethylene glycol) brushes are prepared by a grafting-to approach. The modified surfaces are shown to be superior to existing options for silica modification, exhibiting ultralow fouling (a few ng/cm2) after exposure to crude serum. In addition, by including a fraction of biotinylated polymer end groups, the surface can be functionalized further. We show that avidin can be detected label-free from a serum solution with a selectivity (compared to nonspecific binding) of more than 98% without the need for a reference channel. Furthermore, we show that our method can passivate the interior of 150 nm × 100 nm nanochannels in silica, showing complete elimination of adsorption of a sticky fluorescent protein. Additionally, our method is shown to be compatible with modifications of solid-state nanopores in 20 nm thin silicon nitride membranes and reduces the noise in the ion current. We consider these findings highly important for the broad field of nanobiotechnology, and we believe that our method will be very useful for a great variety of surface-based sensors and analytical devices.
|
|
| 10. |
|
|
