Ultrasensitive ELISA using enzyme-loaded nanospherical brushes as labels.

Improving the detection sensitivity of enzyme-linked immunosorbent assay (ELISA) is of utmost importance for meeting the demand of early disease diagnosis. Herein we report an ultrasensitive ELISA system using horseradish peroxidase (HRP)-loaded nanospherical poly(acrylic acid) brushes (SPAABs) as labels. HRP was covalently immobilized in SPAABs with high capacity and activity via an efficient "chemical conjugation after electrostatic entrapment" (CCEE) process, thus endowing SPAABs with high amplification capability as labels. The periphery of SPAAB-HRP was further utilized to bind a layer of antibody with high density for efficient capture of analytes owing to the three-dimensional architecture of SPAABs. Using human chorionic gonadotrophin (hCG) as a model analyte, the SPAAB-amplified system drastically boosted the detection limit of ELISA to 0.012 mIU mL(-1), a 267-fold improvement as compared to conventional ELISA systems.

Covalent immobilization of proteins on 3D poly(acrylic acid) brushes: mechanism study and a more effective and controllable process.

Polymeric brushes have emerged as a novel 3D material platform that provides great amounts of binding sites for biomolecules. This paper investigates the covalent immobilization mechanism of protein by spherical poly(acrylic acid) brushes (SPAABs) in the widely adopted N-hydroxysuccinimide/N-(3-dimethyl-aminopropyl)-N'-ethylcarbodiimide hydrochloride (NHS/EDC) process. It was discovered that electrostatic interaction plays a crucial role in the covalent immobilization of protein. Due to the existence of 3D architecture and "Donnan effect", SPAABs exhibit quite different immobilization kinetics in comparison with conventional 2D materials. Under conditions favorable to electrostatic interaction, the effect of "electrostatic interaction induced covalent binding" was observed as a result of competitive immobilization by physical adsorption and chemical binding. On the basis of the mechanism study, a new "chemical conjugation after electrostatic entrapment" (CCEE) method was developed which set the chemical and physical immobilization process apart. A more effective and well-defined covalent immobilization was achieved. And the binding capacity can be tuned in a wide range (0-4.2 mg protein/mg SPAABs) with a high level of control.

Enhancement of enzymatic activity by magnetic spherical polyelectrolyte brushes: a potential recycling strategy for enzymes.

Interactions between amyloglucosidase and magnetic spherical polyelectrolyte brushes (MSPB) were studied by turbidimetric titration, which reveals reversible and tunable behaviors of pH-dependent enzyme-SPB binding. Quantitative thermodyanmic parameters including binding affinity and stoichiometry between enzyme and SPBs were further measured by isothermal titration calorimetry (ITC). A large amount of enzyme can be loaded in MSPB without loss of MSPB stability. We demonstrated that the enzymatic activity of amyloglucosidase bound in MSPB could be greatly enhanced (catalytic reaction rate, k(bound) = 1.36k(free)) compared to free enzyme acitivity in solution. This is tremendous improvement from other carrier systems that usually lead to a significant decrease of enzymatic activity. Both the high enzyme loading capacity and the enhancement of the catalytic activity probably arise from the Coulombic interactions between the enzyme and MSPB. These findings provide a practical strategy for enhancement of enzyme activity and enzyme recycling by MSPB.

Binding between proteins and cationic spherical polyelectrolyte brushes: effect of pH, ionic strength, and stoichiometry.

Cationic spherical polyelectrolyte brushes (SPBs) were synthesized by photoemulsion polymerization, consisting of a polystyrene core with a diameter around 80 nm and a poly(2-aminoethylmethacrylate hydrochloride) (PAEMH) shell with a thickness from 10 to 50 nm densely grafted on the core surface. The binding of various proteins onto SPBs was observed by turbidimetric titration, dynamic light scattering (DLS), zeta potential, and isothermal titration calorimetry (ITC). The binding, aggregation, and releasing of proteins by SPB can be tuned by modulating pH. The pH regions of binding for bovine serum albumin (BSA), ß-lactoglobulin (BLG), and papain onto SPBs are markedly different and tunable by ionic strength and stoichiometry between protein and SPB. Binding energetics, affinity, and amount of various proteins onto cationic SPBs were determined by ITC. These findings lay the foundation for SPB applications in the protein purification and selective immobilization of different proteins, enzymes, and antibodies.

Boronic acid-functionalized spherical polymer brushes for efficient and selective enrichment of glycoproteins.

Glycoproteins are related to many biological activities and diseases, and thereby their efficient capture and enrichment for diagnostics and proteomics have emerged to exhibit great significance. However, the lack of materials with high binding capacity and selectivity is still a big obstacle for further application. Herein, we reported a facile and eco-friendly approach to fabricate spherical polymer brushes with multiple boronic acid groups. Specifically, the whole process can be divided into three steps, the polystyrene (PS) core was obtained by traditional emulsion polymerization, followed by immobilization of a home-made photoinitiator. Subsequently, boronic acid-functionalized polymer chains (PBA) were chemically grafted via photo-emulsion polymerization, leading to spherical polymer brushes (PS-PBA) with boronate affinity. The particle size, morphology, and composition of as-prepared spherical polymer brushes were systematically characterized. The characteristics of glycoproteins binding to the spherical polymer brushes under different conditions, including pH values and ionic strength, were also investigated. PS-PBA brushes possess fast binding speed (30 min) and high binding capacity for glycoprotein ovalbumin (OVA) (377.0 mg g-1) under physiological pH conditions at 25 °C, because the low steric hindrance of flexible polymeric PBA chains facilitates the interaction between boronic acid groups and glycoproteins. Moreover, the binding capacity of PS-PBA brushes for glycoprotein OVA was ∼6.7 times higher than that for non-glycoprotein bovine serum albumin (BSA), indicating the excellent selective adsorption. This study provided a facile and efficient approach for the fabrication of boronic acid-functionalized materials that will be useful in the enrichment and separation of glycoproteins for the diagnosis of diseases.

Design and preparation of bi-functionalized short-chain modified zwitterionic nanoparticles.

An ideal nanomaterial for use in the bio-medical field should have a distinctive surface capable of effectively preventing nonspecific protein adsorption and identifying target bio-molecules. Recently, the short-chain zwitterion strategy has been suggested as a simple and novel approach to create outstanding anti-fouling surfaces. In this paper, the carboxyl end group of short-chain zwitterion-coated silica nanoparticles (SiO2-ZWS) was found to be difficult to functionalize via a conventional EDC/NHS strategy due to its rapid hydrolysis side-reactions. Hence, a series of bi-functionalized silica nanoparticles (SiO2-ZWS/COOH) were designed and prepared by controlling the molar ratio of 3-aminopropyltriethoxysilane (APTES) to short-chain zwitterionic organosiloxane (ZWS) in order to achieve above goal. The synthesized SiO2-ZWS/COOH had similar excellent anti-fouling properties compared with SiO2-ZWS, even in 50% fetal bovine serum characterized by DLS and turbidimetric titration. Subsequently, SiO2-ZWS/COOH5/1 was chosen as a representative and then demonstrated higher detection signal intensity and more superior signal-to-noise ratios compare with the pure SiO2-COOH when they were used as a bio-carrier for chemiluminescence enzyme immunoassay (CLEIA). These unique bi-functionalized silica nanoparticles have many potential applications in the diagnostic and therapeutic fields. STATEMENT OF SIGNIFICANCE: Reducing nonspecific protein adsorption and enhancing the immobilized efficiency of specific bio-probes are two of the most important issues for bio-carriers, particularly for a nanoparticle based bio-carrier. Herein, we designed and prepared a bi-functional nanoparticle with anti-fouling property and bio conjugation capacity for further bioassay by improving the short-chain zwitterionic modification strategy we have proposed previously. The heterogeneous surface of this nanoparticle showed effective anti-fouling properties both in model protein solutions and fetal bovine serum (FBS). The modified nanoparticles can also be successfully functionalized with a specific antibody for CLEIA assay with a prominent bio-detection performance even in 50% FBS. In this paper, we also investigated an unexpectedly fast hydrolysis behavior of NHS-activated carboxylic groups within the pure short-chain zwitterionic molecule that led to no protein binding in the short-chain zwitterion modified nanoparticle. Our findings pave a new way for the designing of high performance bio-carriers, demonstrating their strong potential as a robust platform for diagnosis and therapy.

Antifouling performance of nano-sized spherical poly(N-hydroxyethyl acrylamide) brush.

The biomedical applications of nanoparticles are still impeded by the non-specific adsorption of proteins, cells, or others biological species in vivo/in vitro. In this work, poly(N-hydroxyethyl acrylamide) was hired to modify a solid polymer core, polystyrene (PS) nanoparticles, via surface-initiated photo-emulsion polymerization to form nano-sized spherical poly(N-hydroxyethyl acrylamide) brush (PS@PHEAA). Its antifouling ability and stability were investigated by dynamic light scattering (DLS), turbidimetric titration, and isothermal titration calorimetry (ITC). The size of PS@PHEAA was constant as a function of pH, while slightly changed with ionic strength in single protein solution. ITC data confirmed that protein was slightly adsorbed on PS@PHEAA and the ionic strength influenced the adsorption. All characterizations demonstrated that PHEAA layer reduced the interaction between nanoparticles and proteins. Thus, these nanoparticles ideal candidates for future applications in the biomedical field.

Functional short-chain zwitterion coated silica nanoparticles with antifouling property in protein solutions.

A new functional nanoparticle, consisting of a silica core onto which short-chain zwitterions are chemically connected, was successfully prepared and showed excellent antifouling performance to protein solutions. These nanoparticles (NPs) own excellent stability even in 1M NaCl solutions for at least 48 h. The interaction between these "zwitterated" NPs and proteins were investigated by dynamic light scattering (DLS), turbidimetric titration, and isothermal titration calorimetry (ITC). The results demonstrated that the zwitterated NPs had antifouling property both in single protein solutions and serum (fetal bovine serum, FBS). The zwitterated NPs also own abundant functional groups which could conjugate with biomolecules for future applications in therapeutic and diagnostic field.

ß-Lactoglobulin (BLG) binding to highly charged cationic polymer-grafted magnetic nanoparticles: effect of ionic strength.

Poly(2-(methacryloyloxy)ethyltrimethyl ammonium chloride) (PMATAC) modified magnetic nanoparticles (NPs) with a high zeta potential of ca. 50mV were synthesized by atom transfer radical polymerization (ATRP). The prepared NPs consist of a magnetic core around 13nm and a PMATAC shell around 20nm attached on the surface of magnetic nanoparticles. Thermodynamic binding parameters between ß-lactoglobulin and these polycationic NPs were investigated at different ionic strengths by high-resolution turbidimetry, dynamic light scattering (DLS), and isothermal titration calorimetry (ITC). Both turbidity and ITC show that binding affinities for BLG display a non-monotonic ionic strength dependence trend and a maximum appears at ionic strength of 50mM. Such observation should arise from the coeffects of protein charge anisotropy visualized by DelPhi electrostatic modeling and the strong electrostatic repulsion among highly charged NPs at a variety of ionic strengths.

Investigations on the interactions of proteins with polyampholyte-coated magnetite nanoparticles.

Magnetite nanoparticles have been widely used in biomedical applications, especially as contrast agents in magnetic resonance imaging. In this work, the antifouling property of polyampholyte-coating (poly(acrylic acid) (PAA)-co-3-(diethylamino)-propylamine (DEAPA)) is systematically demonstrated. Polyampholyte-coated magnetite nanoparticles (NP1) and PAA-coated magnetite nanoparticles (NP2) were synthesized to investigate their interactions with BSA and lysozyme (LYZ) by high-resolution turbidimetric titration, dynamic light scattering (DLS), and isothermal titration calorimetry (ITC) in phosphate buffer saline (PBS) buffer with pH 7.4. The abundant carboxyl groups of NP2 and polyampholyte coating of NP1 were well proven by TGA, ζ-potential, and titration methods. Turbidity change shows that NP1 have no interaction with both proteins other than NP2 having adsorption with LYZ, which was further confirmed by DLS. Besides, ITC gives the exact enthalpy change and unveils the binding stoichiometry for each interaction. All characterizations demonstrate the antifouling property of NP1 to both negatively charged protein BSA and positively charged protein LYZ. The polyampholyte-coated magnetite nanoparticles were shown to be a promising material to eliminate the strong interaction with proteins in complex medium, for example, when it is applied for MRA contrast agents with long in vivo circulation time.

A facile route to the synthesis of spherical poly(acrylic acid) brushes via RAFT polymerization for high-capacity protein immobilization.

Spherical poly(acrylic acid) brushes were prepared via a facile RAFT polymerization route from silica nanoparticles (SiO2@PAAs). A silane functionalized RAFT chain transfer agent was designed and synthesized by a one-step reaction, and then immobilized onto silica nanoparticles (SiNPs) through its R group to afford RAFT polymerization. Key structural parameters and contents of carboxyl groups of SiO2@PAAs were thoroughly characterized by transmission electron microscopy, dynamic light scattering, gel permeation chromatography, thermogravimetric analysis and conductometric titration. The SiO2@PAAs exhibit excellent dispersity, tunable brush thicknesses (14.6-68.8 nm) and abundant carboxyl groups (0.82-2.37 mmol/g). An ultra-high protein immobilization capacity (2600 µg streptavidin/mg SiO2@PAAs) was realized by virtue of its rich carboxyl groups and spherical brush structure, which opens up new possibilities for biomedical applications.