Procedure for Fabricating Biofunctional Nanofibers

Summary

An efficient approach for preparing nanofibers decorated with functional groups capable of specifically interacting with proteins is described. The approach first requires the preparation of a polymer functionalized with the appropriate functional group. The functional polymer is fabricated into nanofibers by electrospinning. The effectiveness of the binding of the nanofibers with a protein is studied by confocal microscopy.

Abstract

Electrospinning is an effective processing method for preparing nanofibers decorated with functional groups. Nanofibers decorated with functional groups may be utilized to study material-biomarker interactions i.e. act as biosensors with potential as single molecule detectors. We have developed an effective approach for preparing functional polymers where the functionality has the capacity of specifically binding with a model protein. In our model system, the functional group is 2,4-dinitrophenyl (DNP) and the protein is anti-DNP IgE (Immunoglobulin E). The functional polymer, α,ω-bi[2,4-dinitrophenyl caproic][poly(ethylene oxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP), is prepared by anionic living polymerization. The difunctional initiator utilized in the polymerization was prepared by electron transfer reaction of α-methylstyrene and potassium (mirror) metal. The 2-methoxystyrene monomer was added first to the initiator, followed by the addition of the second monomer, ethylene oxide, and finally the living polymer was terminated by methanol. The α,ω-dihydroxyl polymer [HO-PEO-P2MS-PEO-OH] was reacted with N-2,4-DNP-∈-amino caproic acid, by DCC coupling, resulting in the formation of α,ω-bi[2,4-dinitrophenylcaproic][poly(ethyleneoxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP). The polymers were characterized by FT-IR, ¹H NMR and Gel Permeation Chromatography (GPC). The molecular weight distributions of the polymers were narrow (1.1-1.2) and polymers with molecular weights greater than 50,000 was used in this study. The polymers were yellow powders and soluble in tetrahydrofuran. A water soluble CDNP-PEO-P2MS-PEO-CDNP/ DMEG (dimethoxyethylene glycol) complex binds and achieves steady state binding with solution IgE within a few seconds. Higher molecular weight (water insoluble i.e. around 50,000) CDNP-PEO-P2MS-PEO-CDNP polymers, containing 1% single wall carbon nanotubes (SWCNT) were processed into electroactive nanofibers (100 nm to 500 nm in diameter) on silicon substrate. Fluorescence spectroscopy shows that anti-DNP IgE interacts with the nanofibers by binding with the DNP functional groups decorating the fibers. These observations suggest that appropriately functionalized nanofibers hold promise for developing biomarker detection device.

Protocol

1. Synthesis of α,ω-dihydroxyl Polymer [HO-PEO-P2MS-PEO-OH] Assemble polymerization reactor as shown in Figure 1. The reactor for this experiment consist of a 100 ml round bottom 2-neck flask having a standard taper outer joint (Chemglass), two flow control adapters with stopcocks (Chemglass), and one Teflon stirring rod. Adapter A (Figure 1) was used to keep Ultra High Purity(UHP) Nitrogen flowing through the system in order to prevent air and moisture from enter…

Discussion

In this report, we have presented a powerful approach for preparing biofunctional nanofibers. The nanofibers are decorated to a functional group which is specific to a model protein. The procedure and approach reported in this communication is general in nature and may be used to prepare nanofibers decorated with any functional group desired. The anionic living polymerization is powerful method to synthesize controlled polymer structures covalently connected to any number of interesting functional or functional groups wh…

Materials

Name of the reagent	Company	Catalogue number
Sodium Metal	Sigma-Aldrich	282065
Benzophenone	Sigma-Aldrich	239852
2-methoxystyrene	Sigma-Aldrich	563064
Tetrahydrofuran	Sigma-Aldrich	178810
Chlorobenzene	Sigma-Aldrich	319996
Single walled CNTs	Sigma-Aldrich	704113
Polystyrene	Sigma-Aldrich	81416
Silicon Wafers	Silicon Quest Int’l	720200
Zeiss FESEM	Carl Zeiss Inc.	Ultra 60
Probestation with Bausch & Lomb MicroZoom II High Performance Microscope	Bausch and Lomb
Leica Scanning Confocal System	Leica Microsystems	TCS SP2
Sub-femtoamp Remote Sourcemeter	Keithley Instruments	6430
Autoranging Digital Multimeter	Keithley Instruments	175A
Syringe Pump	Chemyx Inc.	Fusion 200
Zeiss Optical Microscope	Carl Zeiss Inc.	Zeiss/Axiotech