Thermoresponsive and Photocrosslinkable Poly(2-alkyl-2-oxazoline) Toolbox - Customizable Ultralow-Fouling Hydrogel Coatings for Blood Plasma Environments.

This study focuses on developing surface coatings with excellent antifouling properties, crucial for applications in the medical, biological, and technical fields, for materials and devices in direct contact with living tissues and bodily fluids such as blood. This approach combines thermoresponsive poly(2-alkyl-2-oxazoline)s, known for their inherent protein-repellent characteristics, with established antifouling motifs based on betaines. The polymer framework is constructed from various monomer types, including a novel benzophenone-modified 2-oxazoline for photocrosslinking and an azide-functionalized 2-oxazoline, allowing subsequent modification with alkyne-substituted antifouling motifs through copper(I)-catalyzed azide-alkyne cycloaddition. From these polymers surface-attached networks are created on benzophenone-modified gold substrates via photocrosslinking, resulting in hydrogel coatings with several micrometers thickness when swollen with aqueous media. Given that poly(2-alkyl-2-oxazoline)s can exhibit a lower critical solution temperature in water, their temperature-dependent solubility is compared to the swelling behavior of the surface-attached hydrogels upon thermal stimulation. The antifouling performance of these hydrogel coatings in contact with human blood plasma is further evaluated by surface plasmon resonance and optical waveguide spectroscopy. All surfaces demonstrate extremely low retention of blood plasma components, even with undiluted plasma. Notably, hydrogel layers with sulfobetaine moieties allow efficient penetration by plasma components, which can then be easily removed by rinsing with buffer.

Responsive Hydrogel Binding Matrix for Dual Signal Amplification in Fluorescence Affinity Biosensors and Peptide Microarrays.

A combined approach to signal enhancement in fluorescence affinity biosensors and assays is reported. It is based on the compaction of specifically captured target molecules at the sensor surface followed by optical probing with a tightly confined surface plasmon (SP) field. This concept is utilized by using a thermoresponsive hydrogel (HG) binding matrix that is prepared from a terpolymer derived from poly(N-isopropylacrylamide) (pNIPAAm) and attached to a metallic sensor surface. Epi-illumination fluorescence and SP-enhanced total internal reflection fluorescence readouts of affinity binding events are performed to spatially interrogate the fluorescent signal in the direction parallel and perpendicular to the sensor surface. The pNIPAAm-based HG binding matrix is arranged in arrays of sensing spots and employed for the specific detection of human IgG antibodies against the Epstein-Barr virus (EBV). The detection is performed in diluted human plasma or with isolated human IgG by using a set of peptide ligands mapping the epitope of the EBV nuclear antigen. Alkyne-terminated peptides were covalently coupled to the pNIPAAm-based HG carrying azide moieties. Importantly, using such low-molecular-weight ligands allowed preserving the thermoresponsive properties of the pNIPAAm-based architecture, which was not possible for amine coupling of regular antibodies that have a higher molecular weight.

A new ultralow fouling surface for the analysis of human plasma samples with surface plasmon resonance.

Surface plasmon resonance (SPR) has been widely used to detect a variety of biomolecular systems, but only a small fraction of applications report on the analysis of patients' samples. A critical barrier to the full implementation of SPR technology in molecular diagnostics currently exists for its potential application to analyze blood plasma or serum samples. Such capability is mostly hindered by the non-specific adsorption of interfering species present in the biological sample at the functional interface of the biosensor, often referred to as fouling. Suitable polymeric layers having a thickness ranging from 15 and about 70 nm are usually deposited on the active surface of biosensors to introduce antifouling properties. A similar approach is not fully adequate for SPR detection where the exponential decay of the evanescent plasmonic field limits the thickness of the layer beyond the SPR metallic sensor surface for which a sensitive detection can be obtained. Here, a triethylene glycol (PEG(3))-pentrimer carboxybetaine system is proposed to fabricate a new surface coating bearing excellent antifouling properties with a thickness of less than 2 nm, thus compatible with sensitive SPR detection. The high variability of experimental conditions described in the literature for the quantitative assessment of the antifouling performances of surface layers moved us to compare the superior antifouling capacity of the new pentrimeric system with that of 4-aminophenylphosphorylcholine, PEG-carboxybetaine and sulfobetaine-modified surface layers, respectively, using undiluted and diluted pooled human plasma samples. The use of the new coating for the immunologic SPRI biosensing of human arginase 1 in plasma is also presented.

UV-Laser Interference Lithography for Local Functionalization of Plasmonic Nanostructures with Responsive Hydrogel.

A novel approach to local functionalization of plasmonic hotspots at gold nanoparticles with biofunctional moieties is reported. It relies on photocrosslinking and attachment of a responsive hydrogel binding matrix by the use of a UV interference field. A thermoresponsive poly(N-isopropylacrylamide)-based (pNIPAAm) hydrogel with photocrosslinkable benzophenone groups and carboxylic groups for its postmodification was employed. UV-laser interference lithography with a phase mask configuration allowed for the generation of a high-contrast interference field that was used for the recording of periodic arrays of pNIPAAm-based hydrogel features with the size as small as 170 nm. These hydrogel arrays were overlaid and attached on the top of periodic arrays of gold nanoparticles, exhibiting a diameter of 130 nm and employed as a three-dimensional binding matrix in a plasmonic biosensor. Such a hybrid material was postmodified with ligand biomolecules and utilized for plasmon-enhanced fluorescence readout of an immunoassay. Additional enhancement of the fluorescence sensor signal by the collapse of the responsive hydrogel binding matrix that compacts the target analyte at the plasmonic hotspot is demonstrated.

Free-standing hydrogel-particle composite membrane with dynamically controlled permeability.

The preparation and investigation of a free-standing membrane made from a composite of thermoresponsive poly(N-isopropylacrylamide) (pNIPAAm) and polystyrene nanoparticles (PS NP) with temperature-controlled permeability is reported. The method exploits the light-induced crosslinking of the photo-reactive pNIPAAm-based polymer and mechanical reinforcement of the membrane structure by the polystyrene nanoparticles. About micrometer thick layers were either directly attached to a gold surface or prepared as free-standing layers spanning over arrays of microfluidic channels with a width of about hundred microns by using template stripping. Diffusion of liquid medium, low molecular weight molecules, and large molecular weight proteins contained in blood through the composite membrane was observed with combined surface plasmon resonance (SPR) and optical waveguide spectroscopy (OWS). The swelling ratio, permeability, and nonspecific sorption to these composite membranes were investigated by SPR and OWS as a function of molecular weight of analyte, loading of PS NP in the composite film, and temperature. The authors show successful preparation of a defect-free membrane structure that acts as a thermoresponsive filter with nanoscale pores spanning over an area of several square millimeters. This membrane can be reversibly switched to block or allow the diffusion of low mass molecules to the sensor surface by temperature-triggered swelling and collapsing of the hydrogel component. Blocking of diffusion and low unspecific sorption of proteins contained in blood serum is observed. These features make this platform interesting for potential future applications in continuous monitoring biosensors for the analysis of low molecular weight drug analytes or for advanced cell-on-chip microfluidic studies.

Tunable Plasmonic Nanohole Arrays Actuated by a Thermoresponsive Hydrogel Cushion.

New plasmonic structure with actively tunable optical characteristics based on thermoresponsive hydrogel is reported. It consists of a thin, template-stripped Au film with arrays of nanoholes that is tethered to a transparent support by a cross-linked poly(N-isopropylacrylamide) (pNIPAAm)-based polymer network. Upon a contact of the porous Au surface with an aqueous environment, a rapid flow of water through the pores enables swelling and collapsing of the underlying pNIPAAm network. The swelling and collapsing could be triggered by small temperature changes around the lower critical solution temperature (LCST) of the hydrogel. The process is reversible, and it is associated with strong refractive index changes of Δn ∼ 0.1, which characteristically alters the spectrum of surface plasmon modes supported by the porous Au film. This approach can offer new attractive means for optical biosensors with flow-through architecture and actively tunable plasmonic transmission optical filters.

Reversibly tunable plasmonic bandgap by responsive hydrogel grating.

Reversible actuating of surface plasmon propagation by responsive hydrogel grating is reported. Thermo-responsive poly(N-isopropylacrylamide)-based (pNIPAAm) hydrogel nanostructure was designed and tethered to a gold surface in order to switch on and off Bragg scattering of surface plasmons which is associated with an occurrence of a bandgap in their dispersion relation. pNIPAAm-based grating with a period around 280 nm was prepared by using photo-crosslinkable terpolymer and laser interference lithography and it was brought in contact with water. The temperature induced swelling and collapse of pNIPAAm hydrogel grating strongly modulates its refractive index (Δn~0.1) which leads to the reversible opening and closing of a plasmonic bandgap. The experiments demonstrate partial opening of a bandgap with the width of 12 nm at wavelength around 800 nm where SPR exhibited the spectral width of about 75 nm.