Quantitative analysis of EDC‐condensed DNA on vertically aligned carbon nanofiber gene delivery arrays

Abstract

Vertically aligned carbon nanofibers (VACNFs) with immobilized DNA have been developed as a novel tool for direct physical introduction and expression of exogenous genes in mammalian cells. Immobilization of DNA base amines to the carboxylic acids on nanofibers can influence the accessibility and transcriptional activity of the DNA template, making it necessary to determine the number of accessible gene copies on nanofiber arrays. Polymerase chain reaction (PCR) and in vitro transcription (IVT) were used to investigate the transcriptional accessibility of DNA tethered to VACNFs by correlating the yields of both IVT and PCR to that of non-tethered, free DNA. Yields of the promoter region and promoter/gene region of bound DNA plasmid were high. Amplification using primers designed to cover 80% of the plasmid failed to yield any product. These results are consistent with tethered, longer DNA sequences having a higher probability of interfering with the activity of DNA and RNA polymerases. Quantitative PCR (qPCR) was used to quantify the number of accessible gene copies tethered to nanofiber arrays. Copy numbers of promoters and reporter genes were quantified and compared to non-tethered DNA controls. In subsequent reactions of the same nanofiber arrays, DNA yields decreased dramatically in the non-tethered control, while the majority of tethered DNA was retained on the arrays. This decrease could be explained by the presence of DNA which is non-tethered to all samples and released during the assay. This investigation shows the applicability of these methods for monitoring DNA immobilization techniques.

Surface characterization and functionalization of carbon nanofibers

“…1͑f͔͒, synthesized by plasma enhanced chemical vapor deposition ͑PECVD͒, 5 are highly compatible with microfabrication, thereby facilitating their incorporation as functional nanostructured components of a large variety of devices. Demonstrated CNF applications include electron field emitters, [16][17][18][19] charge and hydrogen storage media, 20,21 composite materials, 22,23 biosensors, 8,9,24,25 gene delivery arrays, [26][27][28][29] synthetic membrane structures, 30,31 electrochemical probes, 13,32,33 electrodes for neuronal interface, 34,35 and scanning probe microscopy ͑SPM͒ tips. [36][37][38] The structure and surface chemistry of the nanofibers play a crucial role in the performance characteristics of these nanofiber-based devices.…”

DNA Biosensors Based on Nanostructured Materials

“…The carbon nanofibers were found to possess an enhanced surface area, in addition to an approximately eight-fold higher amount of hybridized DNA in comparison with GCEs. Vertically aligned carbon nanofibers were also modified with DNA through the carboxylic groups on nanofibers, and used for the direct physical introduction and expression of exogenous genes in mammalian cells [133]. Transcriptional accessibility of DNA was investigated using polymerase chain reaction and in-vitro transcription.…”

Direct measurement of bending stiffness and estimation of Young's modulus of vertically aligned carbon nanofibers

“…1 Since the synthesis is catalytic, one can control the position and the diameter of individual VACNFs by patterning the catalyst, and control their length through the growth time. As a result of their synthesis process and their resemblance to carbon nanotubes (CNTs), VACNFs have been proposed to be used in a number of applications, where some have already been demonstrated including electron emitters, 2 gene delivery arrays, 3 and nanoelectromechanical systems. [4][5][6][7] CNTs are similar to VACNFs and their mechanical properties have been thoroughly studied.…”