Measuring Protein Binding to Individual Hydrogel Nanoparticles with Single-Nanoparticle Surface Plasmon Resonance Imaging Microscopy

Abstract

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...

Label-free surface-sensitive photonic microscopy with high spatial resolution using azimuthal rotation illumination

“…This single nanosphere is consistent with all previously reported works in that it produces a wave-like pattern with parabolic tails on the image (Fig. 2B) acquired using conventional SPRM (SPRM-SDI) ( 7 , 8 , 13 , 15 , 18 ). Both theoretical ( 23 , 24 ) and experimental ( 18 , 25 , 26 ) investigations have indicated that this wave-like pattern is due to the scattered SPs generated by the nanosphere.…”

“…When compared with the surface-sensitive microscopy techniques of single-molecule TIRFM, SPRM offers the advantageous capability to monitor individual binding events continuously without the need to account for photo bleaching or blinking of the fluorophores. Therefore, in recent years, SPRM has found numerous applications in the study of biological targets, including cells ( 12 ), bacteria ( 13 ), viruses ( 7 ), DNA molecules ( 14 ), and proteins ( 15 ); the local electrochemical reactions of heterogeneous surfaces ( 16 , 17 ); and the catalytic reactions of nanomaterials ( 18 , 19 ). However, SPRM has obvious disadvantages that prevent the general use of this interesting technique.…”

Characterizing Single Polymeric and Protein Nanoparticles with Surface Plasmon Resonance Imaging Measurements

“… 41 , 47 In addition to nanoparticle-counting measurements, changes in the intensity of the average single-nanoparticle SPRI response (⟨Δ% R NP ⟩) have been used to quantitate the bioaffinity uptake of polypeptides and proteins by hydrogel nanoparticles. 42 , 43 …”

Detection and Digital Resolution Counting of Nanoparticles with Optical Resonators and Applications in Biosensing

“…A variety of nanoscale particles, such as metallic nanoparticles [129], dielectric nanoparticles [130,131], protein nanoparticles [132,133] and single DNA molecules [134,135] have been observed with this system. In addition, orthogonal and complementary measurement techniques, such as electrochemistry [129,136,137] and local thermal measurement [138], have also be incorporated into the system.…”