Electrically Switchable Polymer Brushes for Protein Capture and Release in Biological Environments

Abstract

Interfaces functionalized with polymers are known for providing excellent resistance towards biomolecular adsorption and for their ability to bind high amounts of protein while preserving their structure. However, making an interface that switches between these two states has proven challenging and concepts to date rely on changes in the physiochemical environment, which is static in biological systems. Here we present the first interface that can be electrically switched between a high-capacity (>1 μg cm^-2 ) multilayer protein binding state and a completely non-fouling state (no detectable adsorption). Switching is possible over multiple cycles without any regeneration. Importantly, switching works even when the interface is in direct contact with biological fluids and a buffered environment. The technology offers many applications such as zero fouling on demand, patterning or separation of proteins as well as controlled release of biologics in a physiological environment, showing high potential for future drug delivery in vivo.

Keywords: Adsorption; Electrochemistry; Polymer Brushes; Proteins.

Figure 1

Interface preparation and electrochemical switching. A) Synthesis scheme for polymer brushes grafted via aryl bonds using reduction of a diazonium salt. B) States of the brush: generic hydrogen bonding state (pH≤5 at physiological ionic strength), electrostatically attracting state (if the protein has high pI) and fully repelling state (when pH is sufficiently high). C) Electrochemical QCMD data of brush switching in a pH 5.0 buffer based on proton consumption by reduction of ambient O₂. The voltage is switched on in the indicated intervals. The response is the same (but slower) when changing the bulk pH by flowing different buffers over the surface. D) Electrochemical brush switching in the opposite direction at physiological pH. Acidification occurs by oxidation of hydroquinone (5 mM). Again, the response from altering pH by buffer exchange is shown for comparison.

Figure 2

Zero fouling on demand and selective protein binding. A) QCMD data of brushes (on Pt) exposed to serum in PBS pH 7.4 (from 12 to 35 min). Crosses: Potential applied after serum proteins have bound ‐ the baseline is recovered. Circles: Potential applied before serum is introduced ‐ no binding is detected (the small step‐like response is due to the bulk viscosity change). Potential “on” means repeated sweeps from 0 to −0.5 V at 200 mV s⁻¹. B) Electrochemical tuning of adsorbed protein amount (brushes on Au). Note that the surface remains in contact with serum. More proteins are released as a stronger reductive potential is used: 25 sweeps to −0.5 V at 200 mV s⁻¹ leads to partial release (3 repeats) and 75 sweeps to −0.75 V at 200 mV s⁻¹ leads to complete release. C) Confirmation of full electrochemical release of serum proteins by SPR spectra in dry state (Au surfaces). The thicknesses are those determined using Fresnel models (solid lines), assuming a refractive index of 1.5 for the organic coating (polymer and protein). Any remaining protein amount is within the measurement uncertainty when remeasuring.

Figure 3

Protein patterning on microelectrodes. A) Microscope photo of electrodes and scanning electron microscopy image of the plasmonic nanoholes in 30 nm Au. B) The resonance in the extinction spectrum in air confirms brush synthesis and protein immobilization on the nanostructured surface. C) Superimposed fluorescence images measured from the microelectrode stripes after localized release of green‐labelled BSA and a second immobilization step of red‐labelled BSA.

Figure 4

Electrically controlled and tunable release of proteins in biological settings. A) Supporting data for the delivery strategy based on electrostatic interactions at pH 7.4 and release by native O₂ (see also Figure 2). The fluorescent intensity from electrostatically immobilized labelled avidin is measured before and after exposure to serum (30 min) at fully physiological conditions. B) QCMD data showing immobilization and tunable release of an IgG antibody (bulk pH 5.0). Cathodic potentials are applied by sweeps from 0 to −0.5 V with the number of sweeps indicated at each release event. C) Delivery strategies utilizing hydrogen bonds, tested with BSA. First, electrochemical release can be performed in serum with pH lowered to 5 as verified by fluorescence imaging. This strategy requires a locally lowered pH in the biological system. Second, spontaneous release occurs in serum at pH 7.4. This strategy requires an ongoing acidification (which is switched off for release). D) Principle of local acidification on Pt electrodes with PMAA brushes, powered by glucose and mediated by covalently bound enzymes. The interfacial pH is kept low by electrochemical oxidation of H₂O₂ produced from the glucose breakdown (no redox active species added). E) Redox activity of H₂O₂ (5 mM) during anodic sweeps on Pt surfaces functionalized with PMAA brushes. The current in the absence of H₂O₂ is shown for comparison. F) Experimental verification of brush switching based on the concept in panel D using 10 mM glucose (no redox active species introduced). The potential is always on but switches between +0.1 and +1.1 V.