ABSTRACT:The specific binding and uptake of protein molecules to individual hydrogel nanoparticles is measured with real-11 time single-nanoparticle surface plasmon resonance imaging (SPRI) microscopy. Nanoparticles that adsorb onto chemically 12 modified gold thin films interact with traveling surface plasmon polaritons and create individual point diffraction patterns in the 13 SPRI microscopy differential reflectivity images. The intensity of each point diffraction pattern depends on the integrated 14 refractive index of the nanoparticle; an increase in this single nanoparticle point diffraction intensity (Δ%R NP ) is observed for 15 nanoparticles that bind proteins. SPRI adsorption measurements can be used to measure an average increase in Δ%R NP that can 16 be correlated with bulk dynamic light scattering measurements. Moreover, the distribution of Δ%R NP values observed for 17 individual nanoparticles can be used to learn more about the nature of the protein−nanoparticle interaction. As a first example, 18 the binding of the lectin Concanavalin A to 180 nm N-isopropylacrylamide hydrogel nanoparticles that incorporate a small 19 percentage of mannose sugar monomer units is characterized. ■ INTRODUCTION21 Hydrogel nanoparticles (HNPs) are unique synthetic nanoma-22 terials that can incorporate various chemical functionalities 23 specifically designed to capture and release proteins, peptides, 24 or other small molecules. These capabilities have led to a 25 significant interest in the potential use of HNPs in biomedical 26 applications such as targeted drug delivery, medical diagnostics, 27 and biosensing. 1−6 For example, NIPAm-based (N-isopropyla-28 crylamide) HNPs have been utilized for detection of various 29 biomolecules, such as DNA, 7,8 proteins, 9−11 and other 30 biologically relevant small molecules. 12,13 Additionally, the 31 specific uptake of proteins into HNPs can also be used as a 32 model system for studying various biological phenomena such 33 as multivalent lectin−carbohydrate interactions. 14−20 For all of 34 these applications, it is essential that the uptake of proteins into 35 individual nanoparticles be quantitated and analyzed. For the 36 case of fluorescently labeled proteins, single nanoparticle 37 fluorescence imaging can be used to monitor affinity uptake 38 into single HNPs. 21 67 We showed that although the average HNP size (as measured 68 by DLS) did not change with melittin concentration the 69 average Δ%R NP varied linearly due to melittin uptake into the 70 HNPs. 71In this paper, we extend our use of single-nanoparticle SPRI 72 microscopy to monitor the specific adsorption and uptake of 73 proteins to individual HNPs. We have synthesized NIPAm-74 based HNPs that incorporate a small percentage of monomers 75 modified with mannose sugar units into the hydrogel polymer 76 as shown in Figure 1b. We then used SPRI microscopy to 77 monitor the interactions of the lectin Concanavalin A (Con A) 78 to these mannose-incorporated HNPs (mHNPs), shown in 79 Figure 1c. Bot...

Glycopolymers mimicking GM1 gangliosides were synthesized by incorporating multiple types of carbohydrates into the polymer backbone. The glycopolymers were immobilized onto gold surfaces, and the interactions with the cholera toxin B subunit (CTB) were analyzed using surface plasmon resonance imaging. The glycopolymer containing both galactose and neuraminic acid showed enhanced recognition of CTB. The interaction was enhanced mainly because of an improvement in the dissociation process by the binding of the neuraminic acid group in the GM1 binding pocket. This cooperativity of galactose and neuraminic acid was achieved by incorporation into the same flexible polymer backbone, and the importance of the close placement of galactose and neuraminic acid groups was revealed. These results will be valuable in medical fields and also for the development of biofunctional materials.

A two-dimensional, glycopolymer-immobilized, photonic crystal (PhC) biosensor was developed for the detection of proteins.

No abstract

Effective screening methods for the development of glycopolymers as molecular recognition materials are desirable for the discovery of novel biofunctional materials. A glycopolymer library was prepared to obtain guidelines for the design of glycopolymers for the recognition of cholera toxin B subunits (CTB). Glycopolymers with varying ratios of hydrophobic and sugar units were synthesized by reversible addition fragmentation chain transfer polymerization. N-tert-Butylacrylamide, N-phenylacrylamide, and N-cyclohexylacrylamide as hydrophobic units were copolymerized in the polymer backbone, and galactose, which contributes to CTB recognition, was introduced into the side chains by “post-click” chemistry. The thiol-terminated glycopolymers were immobilized on a gold surface. The polymer immobilization substrate was analyzed in terms of interaction with galactose recognition proteins (CTB, peanut agglutinin, and Ricinus communis agglutinin I) using surface plasmon resonance imaging. The polymers with high ratios of sugar and hydrophobic units had the strongest interactions with the CTB, which was different from the trend with peanut agglutinin and Ricinus communis agglutinin I. The binding constant of the CTB with the glycopolymer with hydrophobic units was 4.1 × 106 M–1, which was approximately eight times larger than that of the polymer without hydrophobic units. A correlation was observed between the log P value and the binding constant, indicating that the hydrophobic interaction played an important role in binding. New guidelines for the design of recognition materials were obtained by our screening method.