1Department of Chemistry, Clark Atlanta University, 2Department of Physics, Clark Atlanta University, 3Department of Chemistry and Chemical Biology, Cornell University
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Doss, J., Olubi, O., Sannigrahi, B., Williams, M. D., Gadi, D., Baird, B., et al. Procedure for Fabricating Biofunctional Nanofibers. J. Vis. Exp. (67), e4135, doi:10.3791/4135 (2012).
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.
1. Synthesis of α,ω-dihydroxyl Polymer [HO-PEO-P2MS-PEO-OH]
2. Functionalization of α,ω-dihydroxyl Polymer with N-2,4-DNP-Ε-amino Caproic Acid to Obtain the Functional Polymer, CDNP-PEO-P2MS-PEO-CDNP
3. Preparation of CDNP-PEO-P2MS-PEO-CDNP/SWCNT Solution for Electrospinning
4. Electrospinning of Polymer-CNT Composite
5. Characterization of Nanofibers
6. Binding Specificity of Nanofibers with Anti-DNP IgE Protein
7. Current-Voltage Behavior of Nanofibers
8. Representative Results
The method for the synthesis of α,ω-bi[2,4-dinitrophenyl caproic][poly(ethylene oxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP) is shown in Figure 4.1 The structure of the functional polymer was confirmed by FT-IR (Figure 5) and 500 MHz 1H NMR spectroscopy (Figure 6). The FT-IR shows the complete disappearance of the -OH broad absorption around 3,500 cm-1 indicating quantitative functionalization with the CDNP group. This is also confirmed by the NMR spectrum shown in Figure 6. Using the integration of the peaks in the NMR spectrum, it was determined that the CDNP-PEO-P2MS-PEO-CDNP polymers are quantitatively functionalized.
In Figure 7, a mat of conductive nanofibers obtained by electrospinning CDNP-PEO-P2MS-PEO-CDNP /polystyrene/SWCNT from chlorobenzene is shown. Confocal images obtained showed that the protein IgE binds with the DNP on the fiber surface.3 This is an indication of the specificity of binding of electrospun DNP-polymers towards IgE antibody. The intensity of light is an indicator of the presence of IgE on the nanofibers as the protein is fluorescently tagged.
Figure 8a is an AFM (Atomic Force Microscope) image of one the nanofibers obtained by this process and Figure 8b shows the dimension of this particular nanofiber is around 150 nm in diameter. By this process fibers between 100-700 nm are obtained. At this current time it is challenging to prepare fibers with a specific dimension. This is consistent with what is observed by other groups.4 Figure 9 shows SEM images of CDNP-PEO-P2MS-PEO-CDNP /polystyrene/SWCNT nanofibers and the diameter of the nanofibers were between 200 nm to 300 nm .There are three SEM images of nanofibers shown at different magnifications. Study of the three images shows the morphologies of the fibers are linear and beaded. The overall goal is to prepare fibers which are mostly linear. Figure 10 shows the I-V plot of mats of nanofibers prepared from CDNP-PEO-P2MS-PEO-CDNP /polystyrene/SWCNT. The plot shows behavior of a resistor (Ohmic). When the antigen is bound to the nanofibers, we expect to see a change In the I-V behavior of the fiber mat as this change in resistance is a characteristic which suggests that the functional fibers have potential application as the active component in sensors for single molecule detection.
Figure 1. Polymerization reactor for synthesizing the α,ω-dihydroxyl polymer. A) The injection point for the flow UHP gas nitrogen. B.) Injection point for the solvent, monomer, and initiator. C) The reaction vessel.
Figure 2. Setup used for electrospinning using a Glassman high voltage source.
Figure 3. Setup used to measure I-V plots using a Sub-femtoamp Remote Sourcemeter (Keithley).
Figure 4. A).Synthetic approach for preparing OH-PEO-P2MS-PEO-OH polymers. B) Functionalization of α,ω-dihydroxy[poly(ethyleneoxide)-b-poly( 2-methoxystyrene)-b-poly(ethyleneoxide)].
Figure 5. FT-IR spectra of (A) OH-PEO-P2MS-PEO-OH, precursor to CDNP-PEO-P2MS-PEO-CDNP and (B) CDNP-PEO-P2MS-PEO-CDNP.
Figure 6. 500 MHz Proton NMR of CDNP-PEO-P2MS-PEO-CDNP.
Figure 7. A) Binding image of FITC- IgE with CDNP-PEO-P2MS-PEO-CDNP fibers electrospun from chlorobenzene. B) Confocal microscope image of the control (nanofibers with IgG).
Figure 8. A) AFM image of CDNP-PEO-P2MS-PEO-CDNP Fibers electrospun from chlorobenzene and B) AFM profile i.e. dimension of one fiber shown in Figure 5a.
Figure 9. SEM images of CDNP-PEO-P2MS-PEO-CDNP /polystyrene/SWCNT nanofibers.
Figure 10. I-V plot of mats of nanofibers prepared from CDNP-PEO-P2MS-PEO-CDNP /polystyrene/SWCNT.
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 which are specific to particular biomarkers of interest. Anionic living polymerization is well-established for the monomer 2-methoxystyrene.2 Electrospinning is a versatile technique in that the fiber dimensions may be easily controlled by changing the voltage and also varying concentration of the solution to be electrospun.5 The nanofibers show resistive I-V behavior and thus are promising to function as active components in biosensors i.e. the approach reported holds promise for developing biomarker detection device.6,7
The polymerization of the first monomer, 2-methoxystyrene, is 100% complete within 40 min i.e. 100% of the monomer is converted to the polymer and the second monomer polymerization is slow requiring 2 days to polymerize. That is, monomer one polymerizes faster than monomer 2. There is no unused monomer one, but end of the 2 days, there is some unused monomer but this will not contribute to the polydispersity. We have prepared homopolymers of the first monomer i.e. poly(2-methoxystyrene) and the PDI of these polymer are around 1.2 and also the block copolymers reported here is also 1.2. To the best of our knowledge, no study has been carried out that looks at the effect of PDI on the dimension of electrospun fibers but it is normally expected that low PDI contribute to better quality processed product because of reasons of chain-chain entanglements.
We used SWCNTs because of earlier work which shows that poly2-methoxystyrene is effective in wrapping around the carbon nanotube and breaking the agglomeration of the SWCNT.8 We believe this has to do with the specific size of the SWCNTs. Finally, 1% SWCNT content in the fibers results in fibers which are sufficiently electroactive for our study.
No conflicts of interest declared.
This work was supported by NSF HRD-0630456, an NSF CREST Program and NSF is DMR-0934142.
|Single walled CNTs||Sigma-Aldrich||704113|
|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|