Electrospinning parameters: nitrocellulose
We examined the structural characteristics of electrospun nitrocellulose as a function of starting conditions. Samples electrospun from the 60, 80, and 100 mg/ml solutions were very similar in nature. At 60 mg/ml the bulk of the material deposited as 4–8 μm diameter beads, at 80 mg/ml foci of small diameter fibers were observed interspersed with these beads (Figure 1). The relative concentration of fibers with respect to the bead structures increased at 100 mg/ml, however, the beaded structures continued to predominate in these samples. The crenulated appearance of these beads indicates they initially form as spheres in the electrospinning (electrospray) process that contain solvent. As the solvent evaporates the beads collapse and adopt this distinctive shape [10]. At concentrations equal to or greater than 110 mg/ml the beaded structures were lost and fibers were exclusively formed in the electrospinning process (Figure 1).
Fibers electrospun from 110 mg/ml solutions were 120 nm to 1300 nm in cross sectional diameter with an average diameter of 398 nm (Figure 1). The 120 mg/ml solutions produced fibers ranging from 120 nm to 8500 nm in diameter with an average diameter of 1300 nm. The 140 mg/ml solutions produced 240 nm to 2900 nm diameter fibers with an average diameter of 725 nm. Overall, fibers in membranes prepared from the 110 mg/ml solutions were very uniform in size and, on average, were statistically smaller in diameter than fibers prepared from the 120 and 140 mg/ml solutions (P
In many solvent systems average fiber diameter varies in a predictable fashion as a function of the starting polymer concentration and the viscosity of the starting solutions. However, in this system there was not a clear relationship between these parameters (Figure 2A). Not surprising, the viscosity of nitrocellulose solutions was similar at concentrations ranging from 60 mg/ml to 100 mg/ml, the conditions that produced an electrospray and membranes composed of beads. Regression analysis of the entire data set examining the relationship between starting concentration and solution viscosity using a 1st order equation generated an R2 value of 0.677. From 100 mg/ml to 140 mg/ml solution viscosity increased markedly. Regression analysis using a linear fit model over this limited range, essentially the conditions that resulted in fiber formation, generated an R2 = 0.978 (Figure 2A). The onset of this relationship corresponded well with the onset of fiber formation in the electrospinning process. Despite this correlation, there did not appear to be a relationship between solution concentration or solution viscosity and the average fiber diameter produced during electrospinning. Regression analysis using a linear model to examine the correlation between solution concentration, over the limited range of 100–140 mg/ml, and average fiber diameter produced an R2 value of 0.328 (not shown). Plotting average fiber diameter as a function of starting solution viscosity and conducting the regression analysis with a 1st order equation generated an R2 = 0.437 (Figure 2B).
Slot blotting performance
To characterize the overall protein binding characteristics of electrospun nitrocellulose with respect to the parent starting material we conducted slot blot analysis. In these experiments membranes with fibers exhibiting an average cross-sectional diameter of less than 1 μm were prepared by electrospinning nitrocellulose from a starting concentration of 110 mg/ml. Serial dilutions of human Fn were then applied to the membranes and processed for detection. Staining and wash solutions were retained in the blotting wells during the incubation steps and were readily pulled through the parent and electrospun membranes when a vacuum was applied across the apparatus. Fn was detected on control nitrocellulose membranes across the sequence of concentrations tested (0.078 μg–80 μg) (Figure 3, Lanes A and B). The chemiluminescence signal associated with Fn bound to the electrospun membrane was several orders magnitude higher than the signal reported by the parent material (Figure 3, Lanes C and D). Control lanes that were treated with blocking buffer and incubated with primary and secondary antibodies did not exhibit detectable signal.
Electrospinning parameters: charged nylon
Charged nylon is frequently used as a solid phase substrate for protein and nucleic acid analysis. In preliminary experiments we examined the efficacy of electrospinning this material and characterized the structure of the resulting membranes. Fibers electrospun from 60 mg/ml starting suspensions were 120 nm to 1430 nm in diameter with an average diameter of 685 nm (Figure 4). At 80 mg/ml fibers were 120 nm to 3000 nm in diameter with an average of 1000 nm; at 100 mg/ml fibers were 230 nm to 6050 nm in diameter with an average of 1400 nm. The 120 mg/ml solutions produced fibers that ranged from 270 nm to 3290 nm in diameter with an average of 1400 nm; at 140 mg/ml solutions produced 370 nm to 1950 nm diameter fibers with an average of 1300 nm. Evidence of solvent induced defects and solvent welding of adjacent fibers was evident in membranes prepared from the 120 mg/ml solutions, these defects were more pronounced in the samples prepared from the 140 mg/ml solutions. Fibers produced from the 60 mg/ml solutions were smaller in diameter than all other fibers, fibers produced from the 80 mg/ml solutions were smaller than fibers produced from the 140 mg/ml solutions (Figure 4, P
Regression analysis for viscosity as a function of starting concentration using a 1st order equation generated an R2 value of 0.809, a 2nd order equation of these data produced an R2 of 0.989 (Figure 5A). A similar analysis examining the relationships between solution viscosity and average fiber diameter produced an R2 value of 0.264 for a 1st order equation (Figure 5B). These data suggest that solution viscosity, but not fiber diameter, is directly related to the starting concentration of the electrospinning solutions used to process nylon.
Western blotting
In preliminary experiments we compared and contrasted the performance of electrospun nitrocellulose and electrospun nylon with respect to one another and the parent materials. In these conventional electroblotting experiments the loft and high surface area present in membranes composed of electrospun nitrocellulose resulted in a platform that provided high sensitivity at the expense of poor band resolution (Figure 6A). Bands were ill-defined, but intensely labeled. Large sheets of this material were difficult to handle, it was soft and tended develop folds during agitation in the staining and wash buffers.
The results of experiments conducted with electrospun nylon suggest that this material provides increased protein binding capacity and increased dynamic range with respect to the parent material in Western blotting applications (Figure 6A). Band resolution was superior to the performance of the electrospun nitrocellulose membranes. In addition, electrospun nylon was more robust and remained flat during manual manipulation and agitation. In blotting experiments the control nylon membranes developed white bands in lanes loaded with the highest concentrations of Fn. This type of inverse image can occur when A) the protein binding capacity of a blotting membrane is exceeded and/or B) the antibody dilutions are too low. This artifact was absent in the electrospun nylon membranes that were processed in parallel with the parent material (Figure 6A).
To demonstrate the utility of using electrospinning to generate unique blends of material to tailor the performance of a blotting membrane we prepared composite materials. We elected to examine 2 formulations. Electrospun nylon was prepared from a starting concentration of 60 mg/ml (average fiber diameter = 685 nm) and used as a backing material for both constructs. Next, nitrocellulose was electrospun onto the nylon backing from a starting concentration of 110 mg/ml (average fiber diameter 398 nm) or 60 mg/ml (4–8 μm diameter beads). Representative SEM images of these composites are illustrated in Figure 6. This approach allowed us to alternatively test how these two very different physical forms of nitrocellulose might perform in this application.
The nylon/nitrocellulose fiber composite exhibited high signal detection but, provided low band resolution (Figure 6B). As with pure electrospun nitrocellulose we believe the higher loft of this material contributes to the poor band resolution observed in these experiments. The electrospun nylon/electrospun nitrocellulose bead composite exhibited high sensitivity while retaining band resolution (Figure 6C). We were able to clearly detect approximately 4 fold less protein on the electrospun membrane with respect to the controls (0.02 μg Fn/lane on the electrospun composite vs. 0.08 μg Fn/lane on the parent nylon) (Figure 6C).
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