Layer-by-Layer Deposition with Polymers Containing Nitrilotriacetate

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Layer-by-layer Deposition with Polymers Containing Nitrilotriacetate, A Convenient Route to Fabricate Metal- and Protein-Binding Films Salinda Wijeratne, Weijing Liu, Jinlan Dong, Wenjing Ning, Nishanka Dilini Ratnayake, Kevin D Walker, and Merlin L. Bruening ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b00896 • Publication Date (Web): 04 Apr 2016 Downloaded from http://pubs.acs.org on April 12, 2016

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ACS Applied Materials & Interfaces

Layer-by-layer

Deposition

with

Polymers

Containing Nitrilotriacetate, A Convenient Route to Fabricate Metal- and Protein-Binding Films Salinda Wijeratnea, Weijing Liua, Jinlan Dong, Wenjing Ning, Nishanka Dilini Ratnayake, Kevin D. Walker and Merlin L. Bruening* Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA a

These authors contributed equally to this work.

*Corresponding author. Email: [email protected] Phone: +1 517 355-9715 ext. 237 Fax: +1 517 353-1793

KEYWORDS:

Layer-by-layer

deposition,

His-tagged

protein,

polyelectrolytes, NTA

ACS Paragon Plus Environment

membrane

adsorbers,


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Abstract

This paper describes a convenient synthesis of nitrilotriacetate (NTA)-containing polymers and subsequent layer-by-layer adsorption of these polymers on flat surfaces and in membrane pores. The resulting films form NTA-metal-ion complexes and capture 2 to 3 mmol of metal ions per mL of film. Moreover, these coatings bind multilayers of polyhistidine-tagged proteins through association with NTA-metal-ion complexes. Inclusion of acrylic acid repeat units in NTAcontaining copolymers promotes swelling to increase protein binding in films on Au-coated wafers. Adsorption of NTA-containing films in porous nylon membranes gives materials that capture ~46 mg of His-tagged ubiquitin per mL. However, the binding capacity decreases with the protein molecular weight. Due to the high affinity of NTA for metal ions, the modified membranes show modest leaching of Ni2+ in binding and rinsing buffers. Adsorption of NTA-containing polymers is a simple method to create metal- and protein-binding films and may, with future enhancement of stability, facilitate development of disposable membranes that rapidly purify tagged proteins.

Introduction

Films that bind metal ions are attractive for applications ranging from water remediation1-3 to metal-affinity chromatography of peptides4 and proteins.5 Deposition of such coatings on beads or in porous supports enables high-capacity capture of metal ions and biomolecules, and film formation in membrane pores facilitates rapid analyte capture because of low radial diffusion distances and convective flow.6-11 Anchored metal-ion complexes are also important for binding proteins in microarrays and sensors.12-15 In most cases, increased protein binding will lower detection limits in microarrays and increase output in affinity-based purification. Thus, these applications will benefit from high-capacity coatings.


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Traditional methods for covalently immobilizing metal-ion-binding ligands yield only a monolayer of ligand,12-17 which limits capacity. In contrast, polymer coatings may contain many multilayers of ligands that potentially capture multilayers of protein.4 Common approaches to controlled formation of polymer films on flat surfaces, beads and in membrane pores include synthesis of polymer brushes18-23 and layer-by-layer (LBL) polyelectrolyte adsorption,5,24-26 and the latter technique is attractive for its simplicity. Several groups examined metal-ion binding in LBL polyelectrolyte films,5,27,28 but most studies employed weak-binding ligands such as the carboxylic acid groups of poly(acrylic acid) (PAA). Multilayer films containing PAA and branched polyethyleneimine (BPEI) or quaternized poly-4-vinylpyridine bind Co2+ and/or Cu2+ ions through coordination to amine or acid groups.28,29 Additionally, post-deposition functionalization of PAA/protonated poly(allylamine) (PAH) or PAA/BPEI films with nitrilotriacetate (NTA) yields coatings with a high affinity for a number of metal ions,5 but the derivatization process is expensive and inefficient. To simplify the immobilization of metal-ion-binding ligands in thin films, we began developing relatively inexpensive ligand-containing polymers for LBL adsorption.30,31 Until now, these polymers contained iminodiacetate (IDA) ligands, and multilayer polyelectrolyte films formed using these polymers bind large amounts of metal ions (Cu2+ binding capacity of ~2.5 mmol per mL of film)30 and proteins (60±6 mg of His-tagged ubiquitin per mL of membrane).31 However, IDA binds metal ions less strongly than does NTA, which contains an extra carboxylate group for metal-ion complexation. As an example, the equilibrium constant for the reaction of Cu2+ with ligand to form the Cu2+-ligand complex is 3 orders of magnitude less for IDA than for NTA.32 Thus, in metal-affinity adsorption IDA will allow significantly more metal-ion leaching than NTA.



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Figure 1. Representation of the assembly of (PAH/PNTA-X)n films on Au-coated substrates modified with a monolayer of 3-mercaptopropionic acid (MPA), and metal-ion and protein binding to these films. Polymers are much more intermingled in the actual film structure. This

study

reports

a

convenient

synthesis

of

poly(2,2-(5-acrylamido-1-

carboxypentylazanediyl) diacetic acid) [PNTA-100 or PNTA], an NTA-containing polymer, and incorporation of this polymer into polyelectrolyte multilayers to capture metal ions as well as proteins that bind to these immobilized ions (Figure 1). We also examine whether copolymers with both NTA ligands and acrylic acid promote polymer swelling to increase protein binding to NTA-metal-ion complexes. Direct adsorption of NTA-containing polymers to construct proteinbinding films is more convenient and should be less expensive than post-deposition functionalization of coatings by reaction with an NTA derivative.5 Membranes modified with LBL films containing PNTA-100 capture as much as 46 mg of His-tagged protein per mL of membrane,


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and coatings prepared with copolymers that contain acrylic acid bind multilayers of proteins despite a relatively low density of NTA groups.

Experimental Section Materials. Poly(allylamine hydrochloride) (PAH, Mw=120,000–210,000, Alfa-Aesar), branched polyethyleneimine (BPEI, Mw = 25,000, Sigma-Aldrich), and poly(acrylic acid) (PAA, Mw = 90,000, 25% aqueous solution, Polysciences) were employed for LBL deposition. Hydroxylated nylon membranes (LoProdyne® LP, Pall, 1.2 μm pore size, 110 μm thick) were cut into 25 mmdiameter discs prior to use. The supporting information and the results and discussion section describe synthesis and characterization of PNTA-100 and the copolymers poly(ε-acryloyl-Llysine-44-co-acrylic acid-56) [PLys-44] and poly(ε-acryloyl-L-lysine-19-co-acrylic acid-81) [PLys-19].

N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide

hydrochloride

(EDC),

N-

hydroxysuccinimide (NHS), Nα, Nα-bis(carboxymethyl)-L-lysine hydrate (aminobutyl NTA), 3mercaptopropionic acid (MPA, 99%), L-lysine monohydrochloride (98%), acryloyl chloride (97%, contains

Layer-by-Layer Deposition with Polymers Containing Nitrilotriacetate

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