General high-capacity protein capture and tunable electrochemical release

Abstract

There is a need for improved techniques for the immobilization and controlled release of proteins. An electrochemical capture-release system (1) for repeated use, comprising a pH-responsive polymer (2) covalently bound to a structure (3) via a monolayer (4) of electrochemically insensitive aryl bonds to form a polyelectrolyte array (5), wherein the polyelectrolyte array (5) is configured to capture an entity (6), which may be a protein, vesicle, or poly(ethylene glycol)-modified compound, by non-electrostatic interactions, e.g., hydrogen bonding, when the covalently bound polymer (2) is in a neutral state, and to release the entity (6) captured by the polyelectrolyte array (5) by electrostatic repulsion when the polymer (2) is in a charged state.

Description

[Technical Field]

[0001] Technical Field

[0001] The present invention relates to an electrochemical catch-release system comprising a pH-responsive polymer covalently attached to a structure via a monolayer of electrochemically insensitive aryl bonds to form a polyelectrolyte arrangement and arranged to capture and release an entity, be it a protein, a vesicle or a poly(ethylene glycol)-modified compound, by applying an electrochemical potential to the polyelectrolyte arrangement in the presence of a redox-active species. [Background technology]

[0002] Background technology

[0002] Interfaces that are able to capture large amounts of proteins without causing denaturation are of general interest in many fields such as purification, bioanalysis or enzyme catalysis. The possibility of controllably releasing captured proteins is further explored through new lab-on-a-chip technologies (DL Huber, RP Manginell, MA Samara, BI Kim, BC Bunker, Programmed adsorption and release of proteins in a microfluidic device. Science 301, 352-354 (2003)) or drug delivery technologies (AC Anselmo, Y. Gokarn, S. Mitragotri, Non-invasive delivery strategies for biologics. Nat Rev Drug Discov 18, 19-40 (2019); E. Katz et al., Substance release triggered by biomolecular signals in bioelectronic systems. J Phys Chem Lett 6, 1340-1347 (2015); and S. Mitragotri, PA Burke, R. Langer, Overcoming the challenges in administering biopharmaceuticals: formulation and delivery strategies. Nat Rev Drug Discov 13, 655-672 (2014)).Various chemical interactions have been used to release DNA (F. Wang, D. Li, GP Li, XQ Liu, SJ Dong, Electrodissolution of inorganic ions / DNA multilayer film for tunable DNA release. Biomacromolecules 9, 2645-2652 (2008) and M. Gamella et al., DNA computing systems activated by electrochemically-triggered DNA release from a polymer-brush-modified electrode array. Electroanal 29, 398-408 (2017)), insulin (E. Honarvarfard et al., Electrochemically stimulated insulin release from a modified graphene-functionalized carbon fiber electrode. Electroanal 29, 1543-1553 (2017)), and interleukin (SM Gutowski et al., Protease-degradable PEG-maleimide coating with on-demand release of IL-1Ra to improve tissue response to neural electrodes. Biomaterials 44, 55-70 (2015)), or even whole cells (H. Zhu, J. Yan, A. Revzin, Catch and release cell sorting: Electrochemical desorption of T-cells from antibody-modified microelectrodes. Colloids and Surfaces B: Biointerfaces 64, 260-268 (2008)).Notably, proteins (especially antibodies) currently constitute most therapeutic agents (AC Anselmo, Y. Gokarn, S. Mitragotri, Non-invasive delivery strategies for biologics. Nat Rev Drug Discov 18, 19-40 (2019); and S. Mitragotri, PA Burke, R. Langer, Overcoming the challenges in administering biopharmaceuticals: formulation and delivery strategies. Nat Rev Drug Discov 13, 655-672 (2014)), and new methods are needed to achieve efficient immobilization and controlled release.

[0003] However, existing capture-release designs are often incompatible with proteins because proteins require gentle immobilization methods to preserve their structure and biological activity (K. Takasu et al., Polymer brush biointerfaces for highly sensitive biosensors that preserve the structure and function of immobilized proteins. Sensors and Actuators B: Chemical 216, 428-433 (2015)). One exception is the use of receptors to capture specific proteins, which can sometimes be combined with release via a change in the chemical environment (A. Shastri et al., An aptamer-functionalized chemomechanically modulated biomolecule catch-and-release system. Nature Chemistry 7, 447-454 (2015)). Unfortunately, such affinity-based methods place extreme demands on the receptor, for example, they must ensure that the target protein remains bound while retaining activity and being immobilized at high density. One interesting release strategy is to store molecules in microcontainers sealed with an electrochemically dissolvable thin film (ACR Grayson et al., Multi-pulse drug delivery from a resorbable polymeric microchip device. Nat Mater 2, 767-772 (2003); JT Santini, MJ Cima, R. Langer, A controlled-release microchip. Nature 397, 335-338 (1999); and Farra, R. et al., First-in-human testing of a wirelessly controlled drug delivery microchip. Sci Transl Med, 4, 122:122ra21, (2012)).While promising for many compounds, this approach has not typically been used with proteins and requires extensive microfabrication. Furthermore, existing release strategies are not tunable ("all or nothing") and are limited to single-use because the entire chemical structure is removed (F. Wang, D. Li, G. Li, X. Liu, S. Dong, Electrodissolution of inorganic ions / DNA multilayer film for tunable DNA release. Biomacromolecules 9, 2645-2652 (2008); and T. Ghaly, BE Wildt, PC Searson, Electrochemical release of fluorescently labeled thiols from patterned gold surfaces. Langmuir 26, 1420-1423 (2010)).

[0004]

[0004] Techniques for protein immobilization and controlled release are highly desirable for bioanalytical and biomedical applications. However, to date, both immobilization methods and release technologies have suffered from several limitations. It is particularly difficult to find a general concept that preserves protein structure and releases it on demand in desired amounts regardless of the environment. Therefore, there is a continuing need for improved techniques for protein immobilization and controlled release. Summary of the Invention [Problem to be solved by the invention]

[0005] Description of the Invention It is an object of the present disclosure to provide improved or at least alternative capture-release systems for capturing and releasing entities such as proteins. [Means for solving the problem]

[0006]

[0006] The invention is defined by the attached independent patent claims. Non-limiting embodiments will become apparent from the dependent claims, the accompanying drawings and the following description.

[0007]

[0007] According to a first aspect, there is provided an electrochemical capture-release system for repeated use, comprising a pH-responsive polymer covalently attached to a structure via a monolayer of electrochemically insensitive aryl bonds to form a polyelectrolyte array, the polyelectrolyte array being arranged to capture an entity which is a protein, a vesicle, or a compound modified with poly(ethylene glycol) when the covalently attached polymer is in a neutral state, and to release the entity captured in the polyelectrolyte array by electrostatic repulsion when the polymer is in a charged state, and a device for applying an electrochemical potential to the polyelectrolyte array in the presence of a redox-active species to induce switching of the polyelectrolyte array from a neutral state to a charged state or from a charged state to a neutral state.

[0008]

[0008] Thus, electrochemical capture-release systems include systems capable of performing immobilization and controlled release of entities as described herein, where the entities include, for example, proteins such as water-soluble proteins, vesicles such as water-soluble liposomes, or poly(ethylene glycol)-modified compounds.

[0009]

[0009] Furthermore, electrochemical capture-release systems can be implemented in purification, bioanalysis, enzyme catalysis, in separation techniques and analytical devices, as well as in techniques and implanted devices for the controlled release of proteins such as therapeutic antibodies from implanted devices, in bioelectrodes, lab-on-a-chip, organ-on-a-chip and drug delivery technologies.

[0010]

[0010] Furthermore, entities as described herein include intact liposomes, poly(ethylene glycol) modified compounds, biomolecule drugs, proteins useful for bioanalytical and biomedical applications, and protein drugs such as antibodies, insulin, and enzymes.

[0011]

[0011] The electrochemical capture-release system comprises the pH-responsive polymer, wherein the pH-responsive polymer may be, for example, a pH-responsive polymer containing carboxylic acid groups that have the ability to dissociate protons or take up protons to respectively increase or decrease the pH at the electrode interface, and the pH-responsive polymer is, for example, poly(acrylic acid) (PAA) or poly(methacrylic acid) (PMAA).

[0012]

[0012] Furthermore, the electrochemical capture-release system includes the above-mentioned pH-responsive polymer covalently bonded to a structure to form a polyelectrolyte array. The electrochemical capture-release system of the present invention encompasses any pH-responsive polyelectrolyte array, where the polyelectrolyte array includes, for example, a polyelectrolyte brush, film, gel, or layer covalently bonded to the electrode surface by an electrochemically insensitive aryl bond, such as, for example, a diazonium salt surface functionalization.

[0013]

[0013] Further embodiments are disclosed in which the polyelectrolyte array comprises a polyelectrolyte brush, film, gel or layer covalently bonded to the electrode surface via a monolayer of electrochemically insensitive aryl bonds formed, for example, by diazonium salt surface functionalization.

[0014] Further embodiments are disclosed in which the polyelectrolyte array includes or is a polyelectrolyte brush.

[0015]

[0015] The structure of the electrochemical capture-release system according to the present invention may be, for example, a surface, for example a planar surface, or the structure may be a nanohole array, or the structure, for example a surface, may be of a microporous or mesoporous size, allowing for a multi-scale hierarchical porous structure which may allow for a larger surface area and therefore a larger protein loading capacity.

[0016] Furthermore, the structure may be any structure capable of bonding to a diazonium salt, i.e., the structure may be carbon, silicon, or a metal, e.g., a noble metal such as Au (gold) and Pt (platinum), where the surface reacts with the diazonium salt, resulting in the formation of an aromatic organic layer covalently bonded to the surface.

[0017] The pH-responsive polymer is attached to the structure via an electrochemically insensitive bond, ie, via an aryl-containing electrochemically insensitive bond.

[0018] Electrochemical capture-release systems allow the capture of high-capacity (several μg / cm) water-soluble proteins with their secondary structure preserved. 2 ) immobilization. Furthermore, with the electrochemical capture-release system as described herein, immobilization is mediated by non-electrostatic multivalent hydrogen bonds from the carboxylic acid groups when the polyelectrolyte brush is in its protonated neutral state. The electrochemical capture-release system as described herein further allows proteins to bind in their native state.

[0019]

[0019] Furthermore, the electrochemical capture-release system described herein allows the protein to remain in its polyelectrolyte arrangement even when the electrochemical capture-release system is exposed to physiological fluids.

[0020]

[0020] Through electrochemistry, the interfacial pH of the electrochemical capture-release system of the present invention can be controlled to enable tunable capture and release. Microscale electrodes, localized delivery, and patterning can also be used with the electrochemical capture-release system described herein. In addition to proteins, intact liposomes and poly(ethylene glycol)-modified compounds can also be similarly captured and released by the electrochemical capture-release system of the present invention. Electrochemical capture-release systems such as those described herein are useful in analytical devices and drug delivery systems. Furthermore, an electrode interface is disclosed that enables non-invasive and highly efficient protein immobilization with tunable electrochemical release in biological fluids using the electrochemical capture-release system.

[0021] Furthermore, it has been shown that electrochemical capture-release systems can be used to spontaneously immobilize large amounts (multilayers) of proteins in their native state to polymer brushes when the polymer brushes are in their protonated neutral state by immobilization through non-electrostatic intramolecular attractive interactions, such as hydrogen bonding. The electrochemical capture-release system described herein allows proteins to be irreversibly bound with preserved structure and catalytic function, assuming the polymer brushes remain in their neutral state. Controllable release of captured proteins is possible through electrochemical control and, with electrochemically stable chemical anchors, via aryl-containing, electrochemically insensitive bonds, made possible with the electrochemical capture-release system described herein. The electrochemical capture-release system is also useful for liposomes and poly(ethylene glycol)-modified compounds. Furthermore, the electrode interface of the electrochemical capture-release system as described herein can be completely regenerated by releasing all bound proteins due to the pH change resulting from the electrochemical signal, and can be reused for another protein, if desired, immediately after releasing its contents without any regeneration step.

[0022] This general technique for protein capture-release, now enabled by electrochemical capture-release systems, will be useful in future bioanalytical or biomedical devices.

[0023]

[0023] Using the above electrochemical capture-release system, a new type of high-capacity protein immobilization by non-electrostatic intramolecular attractive interactions, for example, by hydrogen bonding to the neutral polyacidic brush (i.e., the polyelectrolyte array of the electrochemical capture-release system of the present invention), and subsequent release due to electrostatic repulsion induced by electrochemical control of increasing the interfacial pH, was demonstrated. Similarly, switching to protein release by electrostatic repulsion can be activated by increasing the pH of the solution to which the electrode is exposed.

[0024] Electrochemical capture-release systems allow proteins to remain bound in physiological fluids and preserve their structure. It is also possible to lower the interfacial pH to switch the surface of the polyelectrolyte array of the electrochemical capture-release system as described herein from repulsive protein binding. The key to successful electrochemical switching lies in the chemistry used to graft the polymer, i.e., the pH-responsive polymer of the electrochemical capture-release system as described herein, onto the surface, i.e., the structure of the electrochemical capture-release system as described herein. Polyelectrolyte brush switching is defined as any pH change that changes the degree of charge on the brush. The degree of charge is determined by the pK a The pH is determined by the pH of the solution to which the brush is exposed (Figure 2). a Below pK, the brushes are primarily neutral and protonated, with only a small degree of charge. a, the brush is primarily charged and the degree of charging is high. Switching can be gradual or drastic depending on the magnitude of the pH shift. As shown in Figure 2, the degree of charging is very low (<0.1) below pH 5 and very high (>0.9) above pH 7. Thus, electrochemical brush switching depends on how much the interfacial pH is displaced by the electrochemical redox reaction, which is determined by the magnitude of the applied electrochemical potential. The pK of the brush a The exact value of is subject to variation depending on the chemical identity of the brush and the composition of the solution, especially the salt concentration.

[0025] Furthermore, the pK a The surface roughness of the polyacidic brushes is varied to achieve favorable interactions with proteins and even increase protein uptake. It has been shown that high-capacity protein immobilization can be achieved even at pH 7.4 by adjusting the solution composition, e.g., low salt content. This is because the polyacidic brushes are sufficiently protonated at pH 7.4 due to the low salt concentration, and attract proteins through non-electrostatic intramolecular interactions, e.g., hydrogen bonding.

[0026]

[0026] When a relatively small voltage window (e.g., 0 to -0.5 V) is applied to a brush electrode exposed to a biological fluid containing a mixture of various proteins (e.g., serum), this results in the release of a portion of the immobilized proteins. A fraction of the released proteins has a relatively low isoelectric point pI, which is expelled due to electrostatic repulsion against the brush. The remaining fraction of proteins with a relatively high pI remains immobilized within the brush (structure). By repeating this procedure, the brush becomes enriched in proteins that exhibit a relatively high isoelectric point, which can be used as a method for isoelectric separation of proteins. Subsequent release of the purified high pI proteins is achieved by application of a larger voltage window (e.g., 0 to -0.75 V), which raises the pH at the brush interface sufficiently to release all of the captured proteins.

[0027] Although gold surfaces, i.e., the structures of the electrochemical capture-release systems as described herein, have been modified, the methods, i.e., the electrochemical capture-release systems as described herein, are applicable to any surface capable of binding diazonium salts, i.e., the structures as described herein. Similarly, although we have used planar surfaces or nanohole arrays (i.e., both are structures as described herein), the storage capacity can of course be increased even further by structures with higher effective surface areas, i.e., nanoporous structures as described herein, or by functionalization of porous conductive materials such as, but not limited to, carbon, noble metal electrodes, conductive oxides, etc. For example, we have performed polymerization of PMAA in porous carbon electrodes to achieve a binding capacity of 20 mg / cm of electrode volume, which is in the range of the binding capacity of, but not limited to, commercially available protein purification chromatography materials. 3 We achieved a protein static binding capacity in the range of 0.01 to 0.01. Further optimization of the electrochemical capture-release system as described herein can be further improved by optimizing the electrode structure and porosity in combination with full utilization of the protein binding capacity of PMAA brushes. Implementation of the electrochemical capture-release system as described herein should be straightforward in protein-focused separation techniques and analytical devices. In the long term, we envision utilizing the technology, i.e., the electrochemical capture-release system as also described herein, for the controlled release of proteins, such as therapeutic antibodies, from implanted devices.

[0028] The pH-responsive polymer can be attached to the structure via a diazonium salt, and the electrochemically insensitive bond contains an aryl.

[0029] The pH-responsive polymer may be a polyacidic polymer that contains carboxylic acid groups that act as carboxylic acid donors.

[0030]

[0030] Still further embodiments relate to electrochemical capture-release systems as described herein, wherein the pH-responsive polymer is, for example, poly(acrylic acid) (PAA) or poly(methacrylic acid) (PMAA).

[0031] The pH-responsive polymer may be any other type of polyelectrolyte, such as, but not limited to, a polybasic polyelectrolyte, e.g., poly(diethylamino)methyl methacrylate or poly(2-vinylpyridine). Alternatively, it may be any other pH-responsive polyelectrolyte capable of binding an entity, e.g., a protein, in a neutral state through non-electrostatic intramolecular protein-polyelectrolyte interactions, e.g., hydrogen bonding, and then releasing it through electrostatic repulsion, where the switch in attraction and repulsion is activated by application of an electrochemical signal that establishes a local pH gradient. Similarly, the switch to release of the protein through electrostatic repulsion can be activated by increasing the pH of the solution to which the electrode is exposed.

[0032] The entity may be a protein, such as a water-soluble protein, a vesicle, such as a water-soluble liposome, or a compound modified with poly(ethylene glycol), and / or the entity is a drug.

[0033] The structure may be a surface, for example a planar surface or an electrode surface, or the structure may be a porous material or a nanohole array.

[0034]

[0034] The structure may include or be made of carbon, a noble metal such as gold or platinum, a conductive oxide, stainless steel, or a conductive polymer.

[0035] The conductive polymer can be, for example, polythiophene, polyethyleneimine, poly(pyrrole), poly(3,4-ethylenedioxythiophene), or poly(aniline).

[0036] In one embodiment, the electrode surface is gold.

[0037] In another embodiment, the electrode surface is platinum.

[0038] In a further embodiment, the electrode surface is made of carbon.

[0039]

[0039] The electrochemical capture-release system is an entity capture system, for example a protein capture system.

[0040] A further embodiment of the present invention relates to an electrochemical capture-release system as described herein, wherein the electrochemical capture-release system is an entity release system, for example, a drug release system.

[0041]

[0041] A further embodiment of the present invention relates to an electrochemical capture-release system as described herein, wherein the electrochemical capture-release system is an entity release system, for example a cell release system, i.e., cell release via protein release.

[0042]

[0042] A further embodiment of the present invention relates to an electrochemical capture-release system as described herein in which the electrochemical capture-release system is miniaturized, for example, the dimensions of the electrochemical capture-release system or device are nanoscale, microscale or mesoscale in size.

[0043]

[0043] The redox-active species used to induce the switching of the polyelectrolyte arrangement from the charged state of the polymer to the neutral state of the polymer may be selected from hydroquinone, hydrogen peroxide, dopamine hydrochloride (DOPA), ascorbic acid, 4-aminophenethyl alcohol (tyrosol), 3,4-dihydroxyphenethylacetic acid (DOPAC), β-nicotinamide adenine dinucleotide, oxygen and reduced disodium salt hydrate (NADH).

[0044] The electrochemical capture-release system may further include an enzyme bound to a polyelectrolyte array, in which case a non-redox-active species, such as, but not limited to, glucose, which, together with dissolved oxygen, generates redox-active hydrogen peroxide by biocatalytic chemical conversion in the presence of the enzyme glucose oxidase, may be used to generate the redox-active species in the presence of a biocatalytic reaction. The enzyme may be, for example, glucose oxidase, which performs bioelectrocatalysis, in which a non-redox-active biological metabolite, such as, but not limited to, a carbohydrate, e.g., glucose, is consumed locally at the electrode interface, resulting in local acidification. The local consumption of the non-redox-active species using a surface-bound enzyme may be used to regulate the release rate of the protein or to prevent its release from the surface from the electrochemical capture and release system.

[0045]

[0045] The combined thickness of the polyelectrolyte array, e.g., polyelectrolyte brush, diazonium salt, and electrode, can be several hundred nanometers to micrometers thick. However, the dimensions of the electrochemical release system can be further reduced in addition to the lateral dimension, which is the thickness of the electrode. Therefore, further miniaturization is not limited in principle to devices based on the electrochemical capture and release systems described herein, where the electrochemical capture-release system is an entity capture system, e.g., a protein capture system. When the electrochemical capture-release system is an entity release system, e.g., a drug release system, or a protein capture system, the electrochemical capture and release system undergoes miniaturization in the interest of less invasiveness or improved functionality.

[0046]

[0046] Yet further embodiments relate to the local consumption of biologically occurring redox-active species, such as dopamine or serotonin, by applying an electrochemical potential in conjunction with an electrochemical capture and release system to maintain localized acidification of an electrode surface in a living biological system in vivo or in vitro. Local consumption of naturally occurring redox species in a biological system can be used to regulate the release rate of the protein or to prevent release from the surface until identified on demand from the electrochemical capture and release system. Similarly, through the use of biocatalysis, non-redox-active species, such as but not limited to glucose, can also be used to achieve localized pH acidification of an electrode that maintains a protein bound to the electrode.

[0047]

[0047] According to a second aspect, there is provided a protein capture system comprising the above-described electrochemical capture-release system for sensing the presence of a protein, for collecting a protein sample, or for any other bioanalytical purpose.

[0048]

[0048] According to a third aspect, there is provided a drug release system, ie, a drug delivery system for protein drugs, comprising the above-mentioned electrochemical capture-release system.

[0049]

[0049] According to a fourth aspect, there is provided a method for continuous protein repulsion by continuously applying a periodic electrochemical signal that generates a local very high pH gradient that makes the polyelectrolyte brush highly protein-repellent, i.e., anti-fouling, wherein by removing the continuous electrochemical signal, the polyelectrolyte brush electrode rapidly captures a protein sample on demand.

[0050]

[0050] According to a fifth aspect, there is provided a method for capturing and releasing an entity, which is a protein, a vesicle, a poly(ethylene glycol)-modified compound, and / or a drug, in a capture-release system, the method comprising covalently attaching a pH-responsive polymer to a structure via a monolayer of electrochemically insensitive aryl bonds to form a polyelectrolyte array; contacting a solution containing the entity with the polyelectrolyte array when the covalently attached polymer is in a neutral state, thereby enabling the polyelectrolyte array to capture the entity by non-electrostatic interactions; applying an electrochemical potential to the polyelectrolyte array in the presence of a redox-active species to induce switching of the polyelectrolyte array from the polymer-neutral state to the polymer-charged state, thereby releasing the entity from the polyelectrolyte array by electrostatic repulsion; and optionally applying an electrochemical potential to the polyelectrolyte array in the presence of a redox-active species to induce switching of the polyelectrolyte array from the polymer-charged state to the polymer-neutral state, thereby enabling the polyelectrolyte array to capture the entity by non-electrostatic interactions.

[0051]

[0051] The non-electrostatic interaction can be, for example, a hydrogen bond.

[0052] BRIEF DESCRIPTION OF THE DRAWINGS

[0052] The present invention will be explained in more detail below with reference to embodiments illustrated in the accompanying drawings. [Brief explanation of the drawings]

[0053] [Figure 1] A scheme for fabricating an electrochemically active, pH-responsive brush interface, ie, a polyelectrolyte array brush interface for an electrochemical capture-release system according to the present invention, is presented. [Figure 2]

[0054] 1 shows the determination of brush pKa by titration in SPR for polyelectrolyte brushes (PAA and PMAA), polyelectrolyte arrays of electrochemical capture-release systems according to the present invention. [Figure 3]

[0055] 1 shows the switching of electrochemical polyacidic brushes, i.e., the switching of polyelectrolyte sequences in an electrochemical capture-release system according to the present invention, monitored in a QCM. The response from changing buffer pH is also shown for comparison. [Figure 4]

[0056] Using a plasmonic nanohole array (response is linear with respect to coverage), we show monitoring of the interface of an electrochemical capture-release system according to the present invention for avidin protein immobilized on a neutral PMAA brush at pH 5 through hydrogen bonding interactions (circles), and on a charged PMAA brush at pH 8 through electrostatic attraction (crosses), followed by the complete absence of protein binding due to electrostatic repulsion at pH 11.5 (squares) (the pI of avidin is 10). [Figure 5]

[0057] Fluorescence intensity from immobilized BSA before and after exposure to serum is shown. [Figure 6]

[0058] Quantification of surface coverage of various proteins (FIB: fibrinogen, GOX: glucose oxidase, IGG: immunoglobulin, GLU: glucosidase, AVI: avidin, BSA: bovine serum albumin, NAVI: neutravidin, HRP: horseradish peroxidase, MYO: myoglobulin, LYS: lysozyme, UBI: ubiquitin, INS: insulin, INS-GLA: insulin-glargine, SERUM: 10x diluted and filtered serum) is shown. [Figure 7]

[0059] 1 shows an electrochemical QCM showing the stepwise release of IGG in real time. [Figure 8a]

[0060] An electrochemical capture and release system and various steps in a method for preparing the system are shown, and the use of such a system for capturing and releasing proteins is also described. [Figure 8b]

[0061] SPR spectra are shown during surface functionalization and before and after multiple immobilization and release cycles. [Figure 9]

[0062] Shows fluorescence of proteins immobilized on interdigitated microelectrode stripes, where patterning is achieved by localized release of green (white electrode) protein from one electrode followed by a second immobilization step adding red (black electrode) protein to an empty polymer brush electrode. [Figure 10]

[0063] Circular dichroism spectra of BSA and IGG before immobilization and after release are shown. [Figure 11]

[0064] Michaelis-Menten analysis of GOX activity before immobilization and after release is shown. [Figure 12]

[0065] Electrochemical QCM showing the collapse of the brushes due to electrochemically induced acidification. The response when a buffer solution is charged by pumping is shown for comparison. [Figure 13]

[0066] In situ electrochemical QCM demonstrates "on-demand adsorption" of proteins by applying +0.5 V in PBS containing 5 mM hydroquinone. [Figure 14]

[0067] 1 shows a scheme for the synthesis of diazonium salt 1. [Figure 15]

[0068] Figure 1 shows cyclic voltagram scans of electrochemically deposited diazonium salts compared to bare gold electrodes, and corresponding air scans by SPR comparing chemically deposited diazonium salts with electrochemically deposited diazonium salts. Only when the diazonium salts are chemically deposited are the layers thin enough for a faradaic reaction to occur, meaning that chemical deposition is required to achieve electrochemical switching of polyelectrolyte brushes, i.e., polyelectrolyte arrays according to the present invention. [Figure 16]

[0069] 1. Interaction of PMAA with liposomes at pH 5 showing highly loaded few-layer liposomes monitored by QCM and subsequent electrochemical release by an electrochemical capture-release system according to the present invention. [Figure 17]

[0070] In situ electrochemical QCM measurements are shown, in which serum (10x diluted with water and filtered, pH 7.4) was exposed to an electrochemical capture-release system, resulting in spontaneous immobilization of serum proteins (step 1). Twenty repeated CV scans with a potential window between 0 V and -0.5 V were applied at 15, 22, and 27 minutes into the experiment, resulting in partial protein release (step 2). Fifty repeated CV scans with a larger potential window from 0 V to -0.75 V were applied, resulting in complete protein release from the electrochemical capture-release system. [Figure 18]

[0071] In situ electrochemical QCM experiments are shown in which diluted serum is introduced to a PMAA brush synthesized on a platinum surface; in (A), electrochemical activation of the surface (sequential CV scan) leads to a highly non-fouling state of the surface, compared to (B), where there is no electrochemical activation but complete desorption of the bound protein is still achieved when a sufficiently large potential is applied. [Figure 19]

[0072] An in situ electrochemical QCM experiment is shown in which hydrogen peroxide is used to switch a functionalized PMAA brush on a platinum surface, and the brush rapidly switches upon application of alternating chronoamperometric potentials of +0.6 V and +0.2 V, as indicated in the frequency and dissipation signals (shown in (C)), with the corresponding current recorded as a function of time (shown in (D)). [Figure 20]

[0073] We present in situ electrochemical QCM experiments of polyelectrolyte brushes synthesized on platinum surfaces that are covalently functionalized with GOX enzyme. When positive potentials (+1.0 V and +1.2 V) are applied, the brushes switch reversibly in phosphate-buffered saline containing 10 mM glucose. DETAILED DESCRIPTION OF THE INVENTION

[0054] Detailed Description

[0074] The embodiments of the invention together with further developments described below should be considered merely as examples and are not intended in any way to limit the scope of protection provided by the claims.

[0055]

[0075] FIG. 8 illustrates an electrochemical capture-release system 1 for repeated use. System 1 includes a pH-responsive polymer 2 covalently attached to structure 3 via a monolayer 4 of electrochemically insensitive aryl bonds to form a polyelectrolyte array 5. Polyelectrolyte array 5 is configured to capture an entity 6, such as a protein, vesicle, or poly(ethylene glycol)-modified compound, through non-electrostatic intramolecular bonds, e.g., hydrogen bonds, when the covalently attached polymer 2 is in a neutral state, and to release the entity 6 captured by polyelectrolyte array 5 through electrostatic repulsion when polymer 2 is in a charged state. Device 7 is configured to apply an electrochemical potential to polyelectrolyte array 5 in the presence of a redox-active species to induce switching of polyelectrolyte array 5 from a neutral state to a charged state or from a charged state to a neutral state.

[0056] Below is described how such a system 1 can be manufactured, and the materials and chemicals required for such manufacture. Also below is described how this system can be used, and examples showing the differences in such use.

[0057] experiment material

[0076] All chemicals and proteins used were purchased from Sigma-Aldrich unless otherwise stated. HO (30%) and NHOH (28–30%) were purchased from ACROS, and HSO (98%) and ethanol (99.5%) were purchased from SOLVECO. Water was ASTM research grade type 1 ultrafiltered water (milli-Q water). The chemicals used for the synthesis of diazonium salt 1 were 4-aminophenethyl alcohol, tetrafluoroboric acid (48% solution in water), acetonitrile, tert-butyl nitrate, and diethyl ether. L-ascorbic acid was used in water to bind the diazonium salt to gold. Dichloromethane, triethylamine, and α-bromoisobutyryl bromide were used to convert the diazonium monolayer to a polymerization initiator layer. The chemicals used in the polymerization were tert-butyl acrylate, tert-butyl methacrylate, dimethyl sulfoxide, dichloromethane, methanesulfonic acid, N,N,N',N"-pentamethyldiethylenetriamine (PMDTA), CuBr2, and L-ascorbic acid. The buffers used in this study were phosphate-buffered saline (PBS) tablets (0.01 M phosphoric acid, 0.13 M NaCl, pH 7.4) or based on disodium hydrogen phosphate and NaCl titrated to a specific pH with HCl (1 M aqueous solution) or NaOH (1 M aqueous solution). PEG-succinimidyl valerate (PEG-SVA, 10,000 g / mol) was used for conjugation to BSA (Laysan Bio Inc.). The lipids phosphatidylcholine and dipalmitoylphosphatidylcholine used for the preparation of liposomes were from Avanti Polar Lipids. DNA samples (single- and double-stranded) were available from in-house collaborators. For enzyme activity assays, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and D-glucose were used.

[0058]

[0077] The proteins used in this study were avidin (AVI, ThermoFisher), bovine serum albumin (BSA), BSA-fluorescein isothiocyanate conjugate, fibrinogen (FIB) from bovine plasma, glucose oxidase (GOX) type VII G2133 from Aspergillus niger, horseradish peroxidase (HRP, ThermoFisher), insulin (INS), INS glargine, IgG antibodies from human serum, myoglobin (MYO) from equine skeletal muscle, lysozyme (LYS), neutravidin (NAVI, Pierce), and ubiquitin (UBI) from bovine erythrocytes. Human serum (from human male AB plasma) was filtered through a 40 μm hydrophilic filter and diluted 10-fold in PBS before use. Alexa Fluor TM Using the 488 and 555 labeling kits (ThermoFisher), different colors of BSA were generated to demonstrate protein patterning. The carbohydrates used to study interactions with PMAA brushes were dextran (100,000 g / mol) from Leuconostoc species and hyaluronic acid sodium salt (15,000–30,000 g / mol) from Streptococcus equi. Oxytocin acetate hydrate was also tested.

[0059]

[0078] The redox-active species used to induce the switching of polyelectrolyte arrangement from the charged state of the tested polymer to the neutral state of the polymer were hydroquinone, hydrogen peroxide, dopamine hydrochloride (DOPA), ascorbic acid, 4-aminophenethyl alcohol (tyrosol), 3,4-dihydroxyphenethylacetic acid (DOPAC), and β-nicotinamide adenine dinucleotide, reduced disodium salt hydrate (NADH).

[0060] method Diazonium salt synthesis:

[0079] The synthesis of the diazonium salt (Figure 14) required a modified literature procedure (S. Gam-Derouich et al., Aryl diazonium salt surface chemistry and ATRP for the preparation of molecularly imprinted polymer grafts on gold substrates. Surface and Interface Analysis 42, 1050-1056 (2010)). Under an inert atmosphere, 4-aminophenethyl alcohol (2.94 g, 20 mmol) and tetrafluoroboric acid (9.94 g, 113 mmol) were dissolved in acetonitrile (20 mL). In a separate flask, tert-butyl nitrate (2.269 g, 22 mmol) was dissolved in acetonitrile (12 mL). Both solutions were degassed and cooled to -20 °C with 200 mL of diethyl ether. After 20 min, the solutions were warmed to 0 °C, and the tert-butyl nitrate solution was added dropwise to the 4-aminophenethyl alcohol solution with stirring. The reaction was then stirred for an additional hour. The reaction was quenched by adding the dark yellow solution dropwise to rapidly stirring diethyl ether (200 mL). After stirring for an additional hour, the supernatant was decanted. The brown precipitate was dried to give 3.69 g of impure diazonium salt. The product was analyzed in a Varian 400 MHz NMR spectrometer to verify the purity. 1 H NMR spectra were recorded at ambient temperature. Spectra were analyzed against external TMS and referenced to the most downfield residual solvent resonance (CDCl3: δH 7.26 ppm). 1HNMR resonances were consistent with those previously reported (S. Gam-Derouich et al., Aryl diazonium salt surface chemistry and ATRP for the preparation of molecularly imprinted polymer grafts on gold substrates. Surface and Interface Analysis 42, 1050-1056 (2010)), and analysis revealed a purity of 80%.

[0061] Surface Cleaning:

[0080] Prior to surface functionalization, QCM sensor crystals (standard gold, purchased from Biolin Scientific) and SPR sensor surfaces (standard gold, purchased from BioNavis) were cleaned in piranha cleaning solution (H2SO4:H2O2, 3:1 v / v) for 10 min and then washed in a milli-Q. Next, an RCA1 cleaning (H2O:H2O2:NH4OH, 5:1:1 v / v, 75 °C for 20 min) was performed, followed by another milli-Q rinse, sonication in ethanol, and drying with N2. For microelectrodes, the piranha cleaning step was omitted to prevent surface destruction due to delamination of the porous gold film.

[0062] Surface activation:

[0081] The gold surface (QCM and SPR sensor) was placed in a septum-sealed glass vial containing diazonium salt 1 (0.301 g, 1.28 mmol), and the vial was purged with N. In a separate flask, ascorbic acid (0.028 g, 0.16 mmol) was dissolved in water (40 mL), and the solution was degassed for 1 h. The ascorbic acid solution was then transferred to a sealed vial, allowing the diazonium salt to dissolve. Using a platform shaker, the gold surface was stirred in the solution for 1 h (nitrogen bubbles appearing on the surface after 15 min indicate successful formation of a monolayer of the diazonium salt), after which it was thoroughly washed in water and then ethanol and dried. To convert the diazonium monolayer (the monolayer is shown in Figure 8a and characterized in Figures 8b and 15) to a polymerization initiator layer, the gold surface was exposed to α-bromoisobutyryl bromide (0.222 mL, 1.80 mmol) and triethylamine (0.302 mL, 2.17 mmol) in dichloromethane (20 mL) for 10 min, after which the surface was washed in ethanol and dried under N2.

[0063] Surface initiated polymerization:

[0082] PMAA polymer brushes, i.e., polyelectrolyte arrays of the present invention, were prepared using ATRP in a manner similar to published procedures (G. Ferrand-Drake del Castillo, G. Emilsson, A. Dahlin, Quantitative analysis of thickness and pH actuation of weak polyelectrolyte brushes. J Phys Chem C 122, 27516-27527 (2018)). Inhibitors were removed from the monomer tert-butyl methacrylate using an alumina column, which was then stored at -20 °C and warmed to room temperature immediately before use. The reaction was carried out using standard Schlenk line techniques under an inert atmosphere of N2. CuBr2 (0.006 g, 0.03 mmol) and PMDTA (0.052 mL, 0.246 mmol) were dissolved in dimethyl sulfoxide (20 mL) and deoxygenated by vigorous bubbling of N2 for 30 min, along with a separate flask of tert-butyl methacrylate (20 mL, 0.1231 mol). The reaction solution and monomer were then transferred via cannula to a screw-cap bottle (with a rubber septum cap) containing the initiator-prepared gold surface. The reaction was initiated by the addition of ascorbic acid (0.033 g, 0.185 mmol). The final concentrations of each component in the reaction medium were: [monomer] = 3.1 M, [CuBr2] = 0.6 mM, [PMDTA] = 6.2 mM, and [ascorbic acid] = 4.6 mM. The reaction was placed under magnetic stirring. The reaction was quenched by immersing the sample in pure ethanol. The poly(tert-butyl methacrylate) (PTBMA) brushes were then converted to PMAA by exposure to 0.2 mM methanesulfonic acid in dichloromethane (10 mL) for 15 min, followed by washing in dichloromethane and ethanol. For PAA, tert-butyl acrylate was used as the starting monomer, using the otherwise identical protocol.

[0064] Porous carbon electrode:

[0083] To evaluate the potential protein purification capabilities of the electrochemical capture and release system, a reticulated vitreous carbon electrode (Redox.Me) was used as the porous electrode. The electrode had a density of 0.05 g / cm. 3 It has a bulk density of 96.5%, a porosity of 96.5%, the number of pores is 24 pores / cm, a diameter of 20 mm and a height of 25 mm (7.85 cm). 3 ) was used. Surface activation and ATRP synthesis of PMAA were performed within a porous carbon electrode by scaling up the formulation twice with equivalent concentrations and a total reaction volume of 80 mL. Protein immobilization tests were performed by immersing the electrode in a 40 mg / mL BSA protein solution at pH 5.0 for 1 hour. The electrode was then washed and immersed in a beaker of PBS at pH 8.0 to induce release of the bound protein. The static binding capacity of the electrode was evaluated by measuring the protein concentration in the original BSA solution after immersion and the protein concentration in the beaker where the protein was released. Quantification of captured and released protein was performed using a NanoDrop (ThermoFisher Scientific).

[0065] Quartz crystal microbalance:

[0084] Measurements were performed using a Q-Sense E4 (Biolin Scientific) with a gold-coated sensor crystal. All data presented corresponded to the first or third overtone. In situ electrochemical experiments were performed using a flow cell equipped with an electrochemistry module (QEM401). A Gamry Interface 1000E potentiostat (Gamry Instruments) was connected to the electrochemical cell. For all experiments, the internal resistance of the circuit (Get Ru) and the open-circuit potential were measured to verify acceptable reference electrode performance and a properly connected circuit. The reference electrode used was a World Precision Instrument low-leakage "Dri-ref" electrode. The scan rate for CV experiments was 100 mV / s unless otherwise specified.

[0066] Surface Plasmon Resonance:

[0085] Measurements were performed in both air and water on an SPR Navi 220A instrument (BioNavis). Total internal reflection (TIR) ​​and SPR angles were recorded at three different laser wavelengths and two different flow channels. The buffer flow rate used was 20 μL / min. Electrochemical SPR measurements were performed by connecting a potentiostat (same as for the QCM) to a cell designed for this purpose (from the instrument manufacturer). Fresnel modeling (G. Ferrand-Drake del Castillo, G. Emilsson, A. Dahlin, Quantitative analysis of thickness and pH actuation of weak polyelectrolyte brushes. J Phys Chem C 122, 27516-27527 (2018)) estimated the deposited diazonium layer to have a refractive index of 1.5 (typical for organic coatings). The refractive index of the dried polymer brushes was assumed to be comparable for PAA and PMAA and set to 1.522. The refractive index of the dry protein bound to the polyelectrolyte brush was assumed to be comparable to that of the polymer. This method of SPR spectral analysis and its validity have been described and demonstrated in a previous study (G. Ferrand-Drake del Castillo et al., Enzyme immobilization in polyelectrolyte brushes: High loading and enhanced activity compared to monolayers. Langmuir 35, 3479-3489 (2019)). To obtain the surface coverage, the densities of the dry polymer and protein were assumed to be 1.22 g / cm, respectively. 3 and 1.35 g / cm 3 was set to.

[0067] Plasmonic detection using nanohole arrays:

[0086] Microscale extinction spectroscopy was performed as previously described (AB Dahlin et al., High-resolution microspectroscopy of plasmonic nanostructures for miniaturized biosensing. Analytical Chemistry 81, 6572-6580 (2009)). Briefly, the surface is imaged in transmission mode and a portion of the light is directed into the aperture of an optical fiber in the focal plane. The detection spot (typically 50 μm) depends on the fiber diameter and the objective magnification. The resolution is almost as high as for SPR (0.1 ng / cm). 2 Note that all data presented as resonance shift (nm) is obtained from plasmonic nanohole arrays, while resonance shift (degrees) is obtained from conventional angular SPR.

[0068] Fabrication of microelectrodes:

[0087] To fabricate the microscale stripe electrodes, we used a laser writer (Heidelberg Instruments DWL 2000). A photoresist (LOR3A) was spin-coated at 4000 rpm and baked on a hotplate at 180 °C for 5 minutes. A second layer of S1813 was spin-coated at 4000 rpm and baked on a hotplate at 120 °C for 2 minutes. The pattern was written using a 60 mW laser beam, and the sample was then developed in developer MF-318 for 50 seconds. Next, nanohole arrays were prepared by colloidal lithography (K. Xiong, G. Emilsson, AB Dahlin, Biosensing using plasmonic nanohole arrays with small, homogenous, and tunable aperture diameters. Analyst 141, 3803-3810 (2016)). Finally, lift-off was performed using remover RM-Rem400.

[0069] Ex-situ electrochemical desorption of proteins:

[0088] For simplicity, in some experiments, protein immobilization and release were not monitored in real time. Instead, using the kinetics from SPR and QCM measurements as a guideline, the surface was immersed in the protein solution for 30 min to ensure saturation binding. After protein loading, the surface was washed with PBS (pH 5.0) and water, then dried with N2. Desorption was performed by immersing the sample in PBS with a high pH (high enough to completely desorb the protein of interest based on SPR measurements). Alternatively, electrochemical release was performed in a beaker with a Pt cage as the counter electrode and Ag / AgCl as the reference electrode. The reference electrode was prepared by electrochemically depositing chloride ions onto a bare silver wire by applying +0.5 V in 1 M HCl for 10 min.

[0070] Fluorescence microscope:

[0089] All fluorescence measurements were performed using a Zeiss Axio Observer 7 inverted microscope equipped with an Axiocam506 camera. A 10x objective was used to image the microelectrode in air. Background fluorescence was measured on the non-functionalized glass surface area adjacent to the electrode for each dye excitation / emission filter set and subtracted from the values ​​obtained from the electrode.

[0071] Circular dichroism spectroscopy:

[0090] The polyelectrolyte brush surface was repeatedly loaded and deloaded with protein. The procedure was repeated until a sufficiently high concentration of protein was found in the collected solution. Quantification of the desorbed protein was performed using a NanoDrop (ThermoFisher Scientific). Once a detectable amount of protein was obtained, the sample was filtered through a 40 μm cellulose acetate filter and loaded onto a centrifugal column (Sartorius) with a molecular size cutoff of 40 kDa. The sample was centrifuged at 5000 rpm for 10 min to obtain approximately 100–300 μL of solution with a concentration of approximately 0.3 g / L. CD was measured using a Chirascan spectrometer (Applied Photophysics). Each spectrum was an average of 10 scans. A quartz cuvette with a path length of 0.05 cm was used. The spectra of the desorbed protein were compared with the corresponding protein solution with a concentration of 0.3 g / L in PBS at pH 5.0 and 8.0.

[0072] Activity Measurement:

[0091] The activity of GOX in bulk solution was measured using an ABTS and HRP assay. The reaction was initiated by adding GOX solution (diluted stock solution or desorbed from a PMAA brush) to a mixture of ABTS, glucose, and HRP in PBS (pH 7.4). The final assay composition was 2 mM ABTS, 1 mM glucose, 20 nM HRP, and 2 nM GOX, with a total volume of 800 μL for each measurement. To quantify the amount of GOX conversion, a standard curve was generated in which hydrogen peroxide was added in place of GOX. The absorbance was recorded at 420 nm, and the initial rate was determined by determining the slope of the absorbance increase during the first 30 s of the reaction.

[0073] Protein conjugation:

[0092] PEG conjugation of BSA was carried out by mixing 100 mg of PEG-SVA with 5 mg of BSA in PBS (pH 8.0) for 16 hours. The PEG-conjugated BSA was purified from unconjugated BSA and hydrolyzed PEG-SVA by passing the reaction solution through a 16 / 600 Superdex 200 pg size-exclusion column connected to an AKTA Start protein purification system (GE Healthcare). Analysis of the protein fraction by gel electrophoresis showed a molecular weight of over 250,000 kg / mol. Two different fluorophores were used for conjugation of fluorescent dyes to the amines of BSA: Alexa Fluor 488 and 555 (with tetrafluorophenyl or N-hydroxysuccinimide ester groups). A BSA solution (100 μL, 10 mg / mL, pH 8.5) was mixed with 100 μg of Alexa Fluor dye, and the resulting solution was inverted at 10-minute intervals for 1 hour. The reaction was terminated by the addition of PBS (pH 5.0), which lowered the pH to 5.0, and the sample was diluted to a protein concentration of 0.2 mg / mL. Fluorescent proteins were immobilized within PMAA brushes in a manner similar to that for native proteins. Conjugation of GOX to PMAA brushes was performed using the EDC / NHS protocol. First, the polymer brush surface was exposed to a mixture of EDC (4 mM) and NHS (2 mM) in MES buffer (20 mM) at pH 4.0 for 10 min. Next, the sample was washed in MES buffer and exposed to a 0.2 g / L GOX solution for 20 min. After covalent enzymatic binding, loosely bound GOX was washed away in MES buffer. To restore the pH sensitivity of the brushes, all unreacted carboxylic acids were rehydrolyzed by first briefly exposing them to phosphate buffer set at pH 8.5, followed by continuous exposure to phosphate buffer (pH 7.4) for at least 2 h. The recovery of pH sensitivity after rehydrolysis of unreacted carboxylic acids according to EDC / NHS was monitored using QCM-D by switching the solution pH between pH 5 and pH 7.4.

[0074] Vesicle preparation:

[0093] Vesicles were prepared using established methods (A. Graneli, M. Edvardsson, F. Hook, DNA-based formation of a supported, three-dimensional lipid vesicle matrix probed by QCM-D and SPR. Chemphyschem 5, 729-733 (2004)). Briefly, lipids were dried onto the inside of a glass vial by solvent evaporation, then rehydrated in buffer and extruded through a 100 nm diameter polycarbonate track-etched membrane. [Example]

[0075] Example Preparation of Poly(acrylic acid) (PAA) and Poly(methacrylic acid) (PMAA) Brushes on Gold Surfaces by Surface-Initiated Activator-Regenerated Atom Transfer Radical Polymerization (ATRP), That is, Preparation of Electrochemical Capture-Release Systems According to the Invention - Brush Characterization and Switching

[0094] We have prepared poly(acrylic acid) (PAA) and poly(methacrylic acid) (PMAA) brushes, i.e., polyelectrolyte arrays of the present invention, by surface-initiated, activator-regenerating atom transfer radical polymerization (ATRP) on gold surfaces. In other words, we have prepared an electrochemical capture-release system in accordance with the present invention. Diazonium salts (S. Gam-Derouich et al., Aryl diazonium salt surface chemistry and ATRP for the preparation of molecularly imprinted polymer grafts on gold substrates. Surf Interface Anal 42, 1050-1056 (2010)) were synthesized (Figure 14) and reduced with ascorbic acid to yield aryl monolayers covalently bound to gold (Figures 1 and 15). The monolayers were converted to ATRP initiator layers, followed by polymerization (Figure 1). The weak polyacid brushes, i.e., electrochemical capture-release systems according to the present invention, had dry thicknesses in the range of tens of nanometers as determined by surface plasmon resonance (SPR) (G. Ferrand-Drake del Castillo, G. Emilsson, A. Dahlin, Quantitative analysis of thickness and pH actuation of weak polyelectrolyte brushes. J Phys Chem C 122, 27516-27527 (2018)). The effective pK awas analyzed by titration in SPR (Figure 2). As we have previously shown (G. Ferrand-Drake del Castillo, G. Emilsson, A. Dahlin, Quantitative analysis of thickness and pH actuation of weak polyelectrolyte brushes. J Phys Chem C 122, 27516-27527 (2018)), this yields the same results as those obtained from infrared spectroscopy analysis (G. Ferrand-Drake del Castillo, G. Emilsson, A. Dahlin, Quantitative analysis of thickness and pH actuation of weak polyelectrolyte brushes. J Phys Chem C 122, 27516-27527 (2018)). The pK a values ​​are expected to be higher (compared to the same molecules in solution) (T. Wu et al., Behavior of surface-anchored poly(acrylic acid) brushes with grafting density gradients on solid substrates: 1. Experiment. Macromolecules 40, 8756-8764 (2007); and N. Schuwer, H.A. Klok, Tuning the pH sensitivity of poly(methacrylic acid) brushes. Langmuir 27, 4789-4796 (2011)), and therefore the pK aNote that the pH is higher (G. Ferrand-Drake del Castillo, G. Emilsson, A. Dahlin, Quantitative analysis of thickness and pH actuation of weak polyelectrolyte brushes. J Phys Chem C 122, 27516-27527 (2018)).

[0076]

[0095] We found that our surface functionalization protocol (shown in Figure 15) provided polymer anchors that were stable during electrochemical potential sweeps, yet still allowed for faradaic reactions. Furthermore, the brushes, i.e., our polyelectrolyte arrays, were electroresponsive to reduction potentials, as this increased the local pH through the consumption of protons and oxygen: O2+4H + +4e - → 2H2O (1), O2+2H + +2e - → H2O2(2), H2O2+2H + +2e - → 2H2O (3) (TK Tam et al., Reversible “closing” of an electrode interface functionalized with a polymer brush by an electrochemical signal. Langmuir 26, 4506-4513 (2010)).

[0077]

[0096] Electrochemical quartz crystal microbalance (OV Borisova, L. Billon, RP Richter, E. Reimhult, OV Borisov, pH- and electro-responsive properties of poly(acrylic acid) and poly(acrylic acid)-block-poly(acrylic acid-grad-styrene) brushes studied by quartz crystal microbalance with dissipation monitoring. Langmuir 31, 7684-7694 (2015)) data showed that the brushes, i.e., the electrochemical capture-release system according to the present invention, can be completely switched in their charge state by potential control. That is, the response is determined by the flowing buffer solution relative to the brush pK aThe results were the same for brushes with pHs significantly higher and lower than those shown in Figures 2 and 3. This means that application of an electrochemical potential can shift the interfacial pH between a fully charged state, in which all monomers of the polyelectrolyte within the brush are charged, and a fully neutral state, in which all monomers are neutral and protonated. Applying a weaker electrochemical signal reduces the magnitude of the pH gradient produced, leading to partial charging of the brush in proportion to the degree of charging as a function of pH shown in Figure 2. The switching was very fast (less than 1 second), even in a buffered environment, with no signs of polymer desorption. We attribute this to the highly accessible electrode surface underlying the brush—i.e., the electrochemical capture-release system according to the present invention—and the electrochemically insensitive aryl-gold bond. As expected, thiol-based anchoring resulted in polymer desorption at negative potentials (T. Ghaly, BE Wildt, PC Searson, Electrochemical release of fluorescently labeled thiols from patterned gold surfaces. Langmuir 26, 1420-1423 (2010)), and electrografted aryldiazonium layers blocked electron transfer (J. Pinson, F. Podvorica, Attachment of organic layers to conductive or semiconductive surfaces by reduction of diazonium salts. Chemical Society Reviews 34, 429-439 (2005)) (Figure 15). The brushes were significantly more difficult to switch in nitrogen-purged buffer, confirming that the effect stems from the ubiquitous O2 in aqueous solutions (approximately 0.3 mM in NTP). We observed no effect of the liquid flow rate on the switching ability.

[0078]

[0097] By applying a negative potential in an acidic pH solution, we were able to demonstrate a local increase in pH at the electrode interface. However, we also achieved switching of the polyelectrolyte brush from charged to neutral by applying a positive potential in the presence of redox species that generate protons and result in a locally acidified interface. For example, in Figure 12, electrochemically induced collapse of the polyelectrolyte brush was achieved when a positive potential (+0.5 V) was applied in the presence of 5 mM hydroquinone (HQ), which resulted in a local increase in proton concentration (a decrease in pH) according to Equation 3 (N. Fomina et al., An electrochemical platform for localized pH control on demand. Lab Chip 16, 2236-2244 (2016)). HQ → H + +Q - (3).

[0079]

[0098] The local decrease in pH can be caused by several different redox species, and we found that biologically occurring molecules, such as dopamine, can also switch the brushes, but hydroquinone reacted most readily, causing rapid acidification.

[0080]

[0099] Biological environments typically do not contain sufficiently high concentrations of quinones or redox-active neurotransmitters to be useful for electrochemically switching brushes in vivo. To extend the utility of the capture and release system to buffered, neutral pH environments, we investigated alternative routes to achieving electrochemical switching of PMAA brushes that utilize naturally abundant materials. Furthermore, we investigated the possibility of using electrode supports other than gold, which could expand the range of electrochemical reactions that can occur at the surface and possibly reduce the potential window required to achieve electrochemical switching.

[0081]

[0100] Using platinum instead of gold as the lower electrode surface resulted in altered electrochemical properties. The platinum electrode required a smaller potential magnitude (+ / - 10 mV) to switch the brush pH. In addition, the electrochemical properties of platinum allow for more electrochemical reactions to be performed; for example, hydrogen peroxide can be utilized as a redox species. For example, using a platinum surface, we were able to rapidly switch the protonation state of the brush in PBS (pH 7.4) containing 5 mM hydrogen peroxide by applying alternating potentials of +0.6 V and +0.2 V (Figure 19). At +0.6 V, hydrogen peroxide is oxidized to generate protons at the interface (the reverse of Equation (2)).

[0082]

[0101] Platinum and hydrogen peroxide for generating a pH gradient allow for the expansion of substrates beyond redox-active molecules. Biocatalytic conversions readily produce highly redox-active reaction products from less redox-active substrates. For example, GOX consumes glucose to produce hydrogen peroxide. Thus, conjugation of GOX to PMAA brushes synthesized on platinum surfaces resulted in PMAA brushes switching in neat PBS and 10 mM glucose solutions (Figure 20). Thus, GOX conjugation to platinum PMAA brush electrodes enabled electrochemical brush switching in highly buffered solutions containing physiologically relevant millimolar concentrations of glucose, strongly suggesting an electrochemically operated polyelectrolyte brush surface capable of operating in biorelevant fluids.

[0083] General high-capacity protein immobilization by electrochemical capture-release systems according to the present invention - Protein immobilization and stability in biological solutions

[0102] Next, we describe our new method for general high-capacity protein immobilization based on keeping the polyacid of the electrochemical capture-release system according to the present invention in its neutral state. aWhen the brush is in a neutral state at a pH lower than 100, there is no electrostatic repulsion between the brush and the protein, allowing the protein to diffuse into the polymer brush. Furthermore, at this pH, there is affinity between the neutral brush and the protein because the protonated carboxylic acid groups of the polyacid readily form hydrogen bonds with the surface groups of the protein. We exploited the affinity between the neutral polyacid and the protein at low pH as a mechanism for protein capture. Polyelectrolyte brushes are typically used to attach proteins via conjugation chemistry (R. Dong, S. Krishnan, BA Baird, M. Lindau, CK Ober, Patterned biofunctional poly(acrylic acid) brushes on silicon surfaces, Biomacromolecules 8, 3082-3092 (2007)), which prevents release by conventional methods. Alternatively, electrostatic interactions have been used, but to be effective, this requires low ionic strength and / or low isoelectric points (pI) and pK. aThis requires a large difference between the binding sites (J.H. Dai et al., High-capacity binding of proteins by poly(acrylic acid) brushes and their derivatives. Langmuir 22, 4274-4281 (2006); and A. Kusumo et al., High-capacity, charge-selective protein uptake by polyelectrolyte brushes. Langmuir 23, 4448-4454 (2007)). We evaluated the affinity between the brushes of the electrochemical capture-release system of the present invention and proteins using various surface-sensitive techniques at various pH values ​​while maintaining physiological ionic strength. Figure 4 shows an example of label-free nanoplasmonic detection of a highly positively charged protein (avidin, pI 10) bound to a PMAA brush, i.e., the electrochemical capture-release system of the present invention (A.B. Dahlin, Sensing applications based on plasmonic nanopores: The hole story. Analyst 140, 4748-4759 (2015)). At pH 8, the electrostatic interactions result in high binding (approximately 3000 ng / cm 2 However, at pH 5, where the brushes are approximately 90% protonated, binding is faster and reaches the same response. For proteins with more typical pI values ​​(approximately 7), such as IgG antibodies, there was no detectable binding to PMAA even at pH 6.5. However, at pH 5, binding was very high (approximately 4000 ng / cm). 2 ) remained consistent. This trend was consistent: at pH 5, we observed high binding capacity for all proteins, whereas in the pH range of 6–8, binding was efficient only for proteins with high pIs. Furthermore, as pH increased, the pI (and pK a ), we detected no protein binding at all and the bound protein desorbed. PAA brushes behaved similarly but required a lower pH to exhibit typical high-capacity binding (henceforth, only results for PMAA are presented).

[0084]

[0103] We believe that the high protein binding capacity at pH 5, i.e., the high protein binding capacity of the electrochemical capture-release system according to the present invention, is due to hydrogen bonding between the carboxylic acid donor and various acceptors on the protein, as well as the formation of intermolecular complexes between the polyacid and the hydrophilic polymer (Y. Osada, M. Sato, Thermal equilibrium of intermacromolecular complexes of polycarboxylic acids realized by cooperative hydrogen-bonding. Journal of Polymer Science Part C - Polymer Letters 14, 129-134 (1976); and K. L. Smith, A. E. Winslow, D. E. Petersen, Association reactions for Poly(alkylene oxides) and polymeric poly(carboxylic acids). Ind. Eng. Chem 51, 1361-1364 (1959)). Note that at pH 5, the secondary structure of the protein is usually preserved or at least not irreversibly altered. All proteins were irreversibly immobilized; therefore, the protein concentration during immobilization only affected the loading rate, not the final amount. Furthermore, the protein remained bound even after washing with water and exposure to physiological fluids. Figure 5 shows the fluorescence from labeled BSA in a PMAA brush, i.e., an electrochemical capture-release system according to the present invention, before and after exposure to whole serum set at pH 5, demonstrating no decrease in intensity. This also confirms that the immobilized protein cannot be displaced by other proteins, but that additional serum proteins are also immobilized if the brush has not reached its full loading capacity.

[0085]

[0104] We emphasize that our method is the opposite of previous methods for using polyelectrolyte brushes for protein immobilization: here, the polymer, in its charged state (when pH > pI), provides repulsive forces. Our results may seem contradictory to previous studies in which proteins bind to charged polymers even when they have the same net charge, for example, due to localized "patches" on the surface (X. Xu, S. Angioletti-Uberti, Y. Lu, J. Dzubiella, M. Ballauff, Interaction of proteins with polyelectrolytes: a comparison between theory and experiment. Langmuir, (2018); and AB Kayitmazer, D. Seeman, BB Minsky, PL Dubin, Y. Xu, Protein-polyelectrolyte interactions. Soft Matter 9, 2553–2583 (2013)). This is largely explained by the fact that all our measurements were performed at physiological ionic strength, where electrostatic attraction is eliminated by screening. Furthermore, brushes that are highly hydrophilic in their charged state (approximately 90% water), such as PMAA, are expected to repel proteins due to conformational entropy loss and osmotic pressure (G. Ferrand-Drake del Castillo, G. Emilsson, A. Dahlin, Quantitative analysis of thickness and pH actuation of weak polyelectrolyte brushes. J Phys Chem C 122, 27516-27527 (2018)).

[0086]

[0105] The amount of immobilized protein was quantified by fitting a Fresnel model to SPR spectra recorded in the dry state (Figure 5). This method provides high accuracy because the refractive index of proteins closely resembles that of polymers (G. Ferrand-Drake del Castillo et al., Enzyme immobilization in polyelectrolyte brushes: High loading and enhanced activity compared to monolayers. Langmuir 35, 3479-3489 (2019)). A wide range of proteins with various molecular weights (M) and pIs were tested, and in all cases, high surface coverage was obtained (Figure 6). When normalized to PMAA coverage, the protein amount correlated with M (p<0.05) but not with pI, confirming that binding is not due to electrostatic attraction. The tendency for higher M to result in higher surface coverage can be understood from a simple scaling argument: assuming that the polymer interacts with the surface of a protein with size R, the amount of polymer in contact with the protein is R. 2 and M is proportional to R 3 Therefore, in reasonable agreement with our results, the mass of immobilized protein per mass of available polymer is proportional to M 1 / 3 The remaining variation can be attributed to the varying degrees of good hydrogen bond acceptor groups on the surface of the protein. The results strongly suggest that the polymer is "wrapping around" the hydrophilic protein exterior. The lowest immobilization amount (417 ng / cm 2Note that even a thickness of 100 μm (horseradish peroxidase) still corresponds to more than a monolayer. In addition, the brushes, i.e., the polyelectrolyte arrays of the present invention, can be made thicker to accommodate even more protein. In this study, we maintained a hydration thickness equivalent to the evanescent field in SPR to enable height probing (G. Ferrand-Drake del Castillo, G. Emilsson, A. Dahlin, Quantitative analysis of thickness and pH actuation of weak polyelectrolyte brushes. J Phys Chem C 122, 27516-27527 (2018)). Protein immobilization leads to brush compression, as expected from multivalent interactions bridging several chains (J. Yu et al., Multivalent counterions diminish the lubricity of polyelectrolyte brushes. Science 360, 1434 (2018)), thereby overcoming the entropic penalty of insertion (N. Fomina et al., An electrochemical platform for localized pH control on demand. Lab Chip 16, 2236-2244 (2016)).

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[0106] The large capacity for protein immobilization on polymer brushes suggests that they could be used for protein purification applications (Jain P, Baker GL, Bruening ML. Applications of Polymer Brushes in Protein Analysis and Purification. Annu Rev Anal Chem. 2, 387-408 (2009)). In principle, the protein surface coverage of PMAA brushes on flat surfaces (Figure 6) should be translatable to equivalent surface coverage of polymer brushes in porous solid supports rather than flat surfaces. The corresponding protein binding per volume of porous solid supports functionalized with polyelectrolyte brushes could potentially be much higher. We tested this by polymerization of PMAA brushes in reticulated vitreous (glassy) carbon electrode solid supports, with minor modifications to the synthesis protocol used for flat surfaces. At 140 mg / cm of dry electrode support, 3 The weight gain of 50 mg / cm indicated successful polymerization on the entire inner surface of the electrode. The BSA protein uptake of the PMAA-functionalized electrode at pH 5 was 50 mg / cm. 3It was determined that the binding capacity was 100 mg / cm (static binding capacity). However, it is likely that the binding capacity can be increased even further by (1) optimizing the synthesis of polymer brushes within high internal surface area materials and (2) maximizing the porosity and surface area available for polymerization. In our preliminary studies, we used porous scaffolds with very high internal porosity (96.5%). This is advantageous when used in flow applications (as in most commercial protein purification devices) because it dramatically reduces the pressure drop and increases mass transport across the electrode. However, high internal porosity reduces the internal surface area. The tradeoff between porosity and surface area is most likely toward high porosity, which essentially involves optimizing the synthesis of polymer brushes within the scaffold, since the protein immobilization capacity per surface area exhibited by polymer brushes is very high (at least 100 times higher than that of conventional surfaces). In a previous study of the protein binding capacity of porous alumina membranes functionalized with poly(hydroxyethyl)acrylate (PHEA), the polymer brushes achieved a binding capacity of 150 mg / cm. 3(Sun L, Dai J, Baker GL, Bruening ML. High-capacity, protein-binding membranes based on polymer brushes grown in porous substrates. Chem Mater. 18, 4033-4039 (2006)), which should be achievable but does not limit the capacity of our porous electrode support for our electrochemical protein capture and release system. In summary, our method can be utilized for protein purification with binding capacities in the same range as conventional chromatography, while providing electrochemically induced binding and elution combined with favorable flow characteristics due to high mass transfer rates and minimal pressure drop (Gagnon P. Technology trends in antibody purification. J Chromatogr A. 1221, 57-70 (2012)). A few other compounds can also be bound in large quantities to PMAA at pH 5. One important example is poly(ethylene glycol) (PEG) and PEG-modified compounds. PEG modification of the protein did not result in its removal from the brushes. Instead, we observed binding equivalent to that of the same protein in its native state. This is predicted to result from hydrogen bonding with the ether oxygens in the PEG (Y. Osada, M. Sato, Thermal equilibrium of intermacromolecular complexes of polycarboxylic acids realized by cooperative hydrogen-bonding. Journal of Polymer Science Part C - Polymer Letters 14, 129-134 (1976)).Furthermore, liposomes can be combined in multilayers without the need for tethering (Figure 16) (CM Agrawal, KA Athanasiou, Technique to control pH in the vicinity of biodegrading PLA-PGA implants. J Biomed Mater Res 38, 105-114 (1997)).

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[0107] These results suggest potential applications in drug delivery, where PEG-modified compounds and liposome carriers are common. At the same time, no binding to other biopolymers, such as polysaccharides (e.g., hyaluronic acid), ribonucleic acids (e.g., double-stranded DNA), or short peptides (e.g., oxytocin), was detected. Thus, PMAA brushes are highly specific for proteins and a few other types of macromolecules.

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[0108] Desorption of proteins from the brush occurs when the pH of the brush increases. The mechanism behind desorption is the opposite of what occurs during protein capture. As the pH increases within the protein-loaded brush, the carboxylic acids of the polyelectrolyte brush become deprotonated, disrupting the bond between the brush and the protein. Furthermore, as the pH increases, the pK aAs the pH shifts above 100, the brush becomes negatively charged. Similarly, as the pH rises above the isoelectric point, the protein becomes net negatively charged, leading to electrostatic repulsion between the protein and the brush. The possibility of electrochemically switching the brush (Figure 3), together with the fact that proteins desorb at sufficiently high pH, ​​suggests that controlled release is possible. We first confirmed this by repeatedly applying voltammetric sweeps in a QCM (Figure 7), showing that the signal gradually returns to baseline (prior to immobilization). SPR spectra showed the same behavior (Figure 8b), and capture-release was reproducible on the same surface, demonstrating no detectable changes in storage capacity or release efficiency (up to 10 cycles tested). We also observed that all proteins tested could be fully released, although for proteins with high pIs, a negative potential must be applied for a longer period to sufficiently increase the pH. Furthermore, we demonstrated localized release and patterning using a microelectrode with a plasmonic nanohole array. Here, we also refer to AB Dahlin et al., High-resolution microspectroscopy of plasmonic nanostructures for miniaturized biosensing. Analytical Chemistry 81, 6572-6580 (2009). Figure 9 shows protein patterning on various microelectrodes by selective release and subsequent immobilization.

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[0109] To test whether the protein's higher-order structure was preserved after capture and release, we performed circular dichroism on BSA and IgG, which showed no detectable changes in secondary structure (Figure 10). In addition, Michaelis-Menten analysis of the GOX activity assay showed very little change in enzyme activity after immobilization and desorption (Figure 11). We believe the preserved bioactivity is quite remarkable, since many immobilization methods, particularly hydrophobic interactions (JN Talbert, JM Goddard, Enzymes on material surfaces. Colloids and Surfaces B: Biointerfaces 93, 8-19 (2012)), result in protein unfolding and loss of bioactivity. We believe the retained secondary structure is due to hydrogen-bonding interactions with the exterior surface of the protein, rather than its interior, consistent with the molecular weight dependence. The total salt concentration of the solution is related to the pK of the brush. a (Ferrand-Drake del Castillo G, Hailes RLN, Dahlin A. Large Changes in Protonation of Weak Polyelectrolyte Brushes with Salt Concentration - Implications for Protein Immobilization. J Phys Chem Lett. 11(13):5212-8 (2020)). In particular, pK ashifts to higher values ​​at lower salt concentrations, thus promoting protein binding to the brush at higher pH values. For serum diluted with water rather than PBS, we noted a very large amount of spontaneous protein immobilization, even at pH 7.4 (Step 1, Figure 17). In addition, we were able to obtain charge-selective separation of proteins, i.e., isoelectric focusing, based on the relative electrostatic repulsion of proteins to the brush as a function of the applied electrochemical potential. By applying continuous cyclic voltammetry scans within a small potential window (-0.5 V to 0 V) ​​during serum exposure (Step 2, Figure 17), a fraction of proteins with low pIs is released from the brush due to electrostatic repulsion to the brush. However, a fraction of proteins with high pIs remains bound to the brush due to the lack of electrostatic repulsion. When the electrochemical signal is released, new proteins bind to the then-partially empty brush, thus refilling the void volume of the brush vacated by the low pI fraction. By repeating this cycle, the brush effectively accumulates proteins with high pI from the serum solution of proteins, resulting in separation based on the proteins' isoelectric points. By changing the CV scan window to higher values ​​(Step 3, Figure 17), all of the bound proteins were released from the brush, highlighting that high pI proteins can still be removed at any time by electrochemically charging the brush to a sufficient degree. This electrochemical activation of polymer brushes for on-demand protein capture from serum may prove useful in handling proteins in bioanalytical applications. The average power consumption of the system while scanning the surface between -0.5 V and 0 V was 9 μW / cm. 2The extremely low power consumption is due to the fact that the only power required is to generate the nanoscale chemical pH gradient. This power consumption is compatible with cutting-edge biocatalytic biofuel cells and implantable technologies, which require low power (Song Y, Min J, Gao W. Wearable and Implantable Electronics: Moving toward Precision Therapy. ACS Nano. 13, 12280-12286, (2019)).

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[0110] In a dilute serum solution at pH 7.4, we noted that the polymer brush, combined with continuous cyclic voltammetry scans that maintained a high local pH, acted to create a highly protein- and biomolecule-repellent surface. Continuously scanning the PMAA brush on a platinum surface with CV scans between -0.5 V and 0 V, the brush completely resisted protein binding (Figure 18A). In comparison, without electrochemical activation prior to serum exposure (Figure 18B), a significantly larger amount of protein was immobilized on the brush (thousands of Hz and substantial dissipation changes). However, complete desorption of bound protein was achieved by applying 20 CV scans within the same potential range (-0.5 V to 0 V) ​​used in (Figure 18A).

[0092] So far, we have demonstrated on-demand protein release or prevention of protein binding by locally increasing the electrode pH (Figure 7). However, we can also locally decrease the electrode pH (Figure 12), allowing essentially any pH to be set at the electrode surface, enabling electrochemically activated capture and release of proteins from buffer solutions at any solution pH. For example, in the presence of 5 mM hydroquinone, we were able to bind proteins to polyelectrolyte brushes when a constant electrochemical potential of +0.5 V was applied (Figure 13). Despite being used in undiluted PBS (pH 7.4), the positive potential allowed for protein binding to be induced as efficiently as at pH 5. In principle, an electrochemical capture and release system consisting of a platinum electrode support, combined with functionalization of GOX, could utilize natural concentrations of glucose instead of hydroquinone to switch the brushes. Thus, the electrochemical capture and release system could function to capture or release proteins on demand in a biological environment without any additional supplementation.

[0093] conclusion

[0111] In conclusion, we have demonstrated a new type of high-capacity protein immobilization and subsequent release on polyacidic brushes, i.e., polyelectrolyte arrays of the present invention, through electrochemical control of interfacial pH, i.e., the electrochemical capture-release system of the present invention. Proteins remain bound in physiological fluids, and their structure is preserved. Surface switching is also possible due to repulsion to protein binding. The key to successful electrochemical switching lies in the chemistry used to graft the polymer onto the surface. In this work, gold, platinum, and carbon electrode surfaces were modified, but the method is applicable to any surface capable of binding diazonium salts (J. Pinson, F. Podvorica, Attachment of organic layers to conductive or semiconductive surfaces by reduction of diazonium salts. Chemical Society Reviews 34, 429-439 (2005)). Similarly, we have demonstrated that the storage capacity of the electrochemical capture and release system is compatible with current commercially available protein purification materials and may potentially be further improved by considering other structures with larger effective surface areas. We foresee several applications of this technology. Implementation should be straightforward in separation techniques and analytical devices that focus on proteins. In the long term, we envision utilizing the techniques described herein, i.e., electrochemical capture-release systems, for the controlled release of proteins, such as therapeutic antibodies, from implanted devices.

[0094] summary The electrochemical capture-release system (1) for repeated use comprises a pH-responsive polymer (2) covalently attached to a structure (3) via a monolayer (4) of electrochemically insensitive aryl bonds to form a polyelectrolyte array (5), wherein the polyelectrolyte array (5) is configured to capture an entity (6), such as a protein, vesicle, or poly(ethylene glycol)-modified compound, through non-electrostatic interactions, e.g., hydrogen bonding, when the covalently attached polymer (2) is in a neutral state, and to release the entity (6) captured by the polyelectrolyte array (5) through electrostatic repulsion when the polymer (2) is in a charged state. The system also includes a device (7) for applying an electrochemical potential to the polyelectrolyte array (5) in the presence of a redox-active species to induce switching of the polyelectrolyte array (5) from a neutral state to a charged state or from a charged state to a neutral state.

Claims

1. Covalently attached to the structure via a monolayer of electrochemically insensitive aryl bonds, A pH-responsive polymer that forms a polyelectrolyte array, the polyelectrolyte sequence is the covalently attached polymer captures an entity when in a neutral state; and When the polymer is in a charged state, electrostatic repulsion releases the entities captured in the polyelectrolyte array. a pH-responsive polymer arranged as follows: a device for applying an electrochemical potential to the polyelectrolyte array in the presence of a redox-active species to induce switching of the polyelectrolyte array from a neutral state to a charged state or from a charged state to a neutral state; Equipped with the pH-responsive polymer is a polyacidic polymer containing a carboxylic acid group; the entity is selected from fibrinogen, glucose oxidase, immunoglobulins, glucosidases, avidin, bovine serum albumin, neutravidin, horseradish peroxidase, myoglobulins, lysozyme, ubiquitin, insulin, insulin-glargine, diluted and filtered serum, and water-soluble liposomes; the redox-active species is selected from hydroquinone, hydrogen peroxide, dopamine hydrochloride (DOPA), ascorbic acid, 4-aminophenethyl alcohol (tyrosol), 3,4-dihydroxyphenethylacetic acid (DOPAC), β-nicotinamide adenine dinucleotide, oxygen, and reduced disodium salt hydrate (NADH); Electrochemical capture-release system for repeated use.

2. 2. The electrochemical capture-release system of claim 1, wherein the pH-responsive polymer is poly(methacrylic acid) (PMAA) or poly(acrylic acid) (PAA).

3. 3. The electrochemical capture-release system of claim 1, wherein the structure is a planar or electrode surface, a porous material, or a nanohole array.

4. 4. The electrochemical capture-release system of claim 1, wherein the structure comprises or is made of carbon, a noble metal, a conductive oxide, stainless steel, or a conductive polymer.

5. The electrochemical capture-release system of any one of claims 1 to 4, wherein the polyelectrolyte array comprises a polyelectrolyte brush, film, gel or layer.

6. An electrochemical capture-release system described in any one of claims 1 to 5, wherein the system is miniaturized so that the dimensions of the system are nanoscale, microscale or mesoscale in size.

7. The electrochemical capture-release system of any one of claims 1 to 6, further comprising an enzyme bound to the polyelectrolyte array.

8. A protein capture system comprising the electrochemical capture-release system according to any one of claims 1 to 7.

9. A drug release system comprising the electrochemical capture-release system according to any one of claims 1 to 7.

10. 1. A method for capturing and releasing an entity in a capture-release system, comprising: covalently attaching a pH-responsive polymer to the structure via a monolayer of electrochemically insensitive aryl bonds to form a polyelectrolyte array; contacting a solution containing the entity with the polyelectrolyte array while the covalently attached polymer is in a neutral state, allowing the polyelectrolyte array to capture the entity by non-electrostatic interactions; applying an electrochemical potential to the polyelectrolyte array in the presence of a redox active species to induce switching of the polyelectrolyte array from the neutral state of the polymer to the charged state of the polymer, thereby releasing the entity from the polyelectrolyte array by electrostatic repulsion; optionally applying an electrochemical potential to the polyelectrolyte array in the presence of a redox-active species to induce switching of the polyelectrolyte array from a charged state of the polymer to a neutral state of the polymer, thereby enabling the polyelectrolyte array to capture entities by non-electrostatic interactions; Including, the entity is selected from fibrinogen, glucose oxidase, immunoglobulins, glucosidases, avidin, bovine serum albumin, neutravidin, horseradish peroxidase, myoglobulins, lysozyme, ubiquitin, insulin, insulin-glargine, diluted and filtered serum, and water-soluble liposomes; the pH-responsive polymer is a polyacidic polymer containing a carboxylic acid group; the redox-active species is selected from hydroquinone, hydrogen peroxide, dopamine hydrochloride (DOPA), ascorbic acid, 4-aminophenethyl alcohol (tyrosol), 3,4-dihydroxyphenethylacetic acid (DOPAC), β-nicotinamide adenine dinucleotide, oxygen, and reduced disodium salt hydrate (NADH); method.

11. The method of claim 10, wherein the pH-responsive polymer is poly(methacrylic acid) (PMAA) or poly(acrylic acid) (PAA).

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