Based on this, we hypothesized that polyplexes formed at neutral pH and then acidified would release bPEI chains into the supernatant to balance the complex charge and facilitate the observed tighter packing at low pH. To test this hypothesis, we used a ninhydrin assay to detect PEI in the supernatant (Figure ). Tris-HCl buffer contains amines that can react with ninhydrin, so a 10 mM HEPES buffer (pH 7.5) was used. Fibrous bPEI-DNA samples were prepared in HEPES, washed, dried, and then transferred to fresh solutions of either 10 mM HEPES (pH 7.5) or 10 mM acetate (pH 4) buffer and equilibrated for 24 hours. The presence of bPEI in the supernatant was detected by the ninhydrin assay and quantified using a bPEI standard curve (10-200 µg/mL in HEPES buffer). As predicted, significantly higher concentrations of free bPEI chains were observed in the supernatant when equilibrated in the low pH buffer. PEI quantification revealed that 16 µg/ml, 25 µg/ml, and 54 µg/ml of PEI are released from the polymer/DNA fiber at weight ratios of 0.25, 0.5, and 1.0 at pH 4, respectively. Samples equilibrated in HEPES exhibited detectable but low levels of free bPEI in the supernatant at polymer:DNA weight ratios of 0.5 and 1.0 possibly due to adsorbed PEI chains on the fiber samples.