Hydroxypropyl Cellulose as an Adsorptive Coating Sieving Matrix for DNA Separations:  Artificial Neural Network Optimization for Microchip Analysis

Abstract

Effective DNA separations in microelectrophoretic systems are complicated by the need to passivate the surface dynamically or covalently. We describe the optimization and utilization of a novel buffer system for fast DNA separations by capillary and microchip electrophoresis without the need for any surface modification or conditioning prior to separation. At concentrations as high as 5%, hydroxypropyl cellulose (HPC) has a relatively low viscosity, allowing for microchip channel filling to be performed with ease. A MES/TRIS buffer system at pH 6.1 eliminates the need for surface preconditioning procedures due to the promotion of hydrogen bonding of HPC with the wall. An additional benefit with this buffer system is the low current observed at high fields when compared to other common DNA separation buffers. An artificial neural network (ANN) was used to model the data and to predict the optimum conditions. Utility of the ANN-optimized system for molecular diagnostic testing was demonstrated by performing microchip separations on DNA samples from patients suspected of having genetic mutations associated with Duchenne muscular dystrophy (DMD). Microchip analysis easily allowed for the patient samples positive for DMD mutations to be distinguished from patient samples negative for the disease.

Disposable polyester–toner electrophoresis microchips for DNA analysis

“…While low electroosmotic flow is key to allowing the PeT microchip to be used for DNA separation with sieving matrix without any surface pretreatment of the channel, the every same low electroosmotic flow (0.35 Â 10 À4 cm 2 V À1 s À1 ) 10 is responsible for the loss of resolution and lengthening of migration time after four injections. This feature is an advantage of this substrate material when compared to glass or PDMS chips 25 where the EOF needs to be suppressed.…”

“…Sanders et al previously demonstrated that HPC polymer solution is a low-viscosity matrix for effective separation of DNA 25 and, thus, ideal for PeT chips. Although the lamination step seals both sheets of polyester together by means of the toner layer deposited between them, the mechanical resistance is limited; therefore, the less viscous the matrix the better.…”

Surface modification in microchip electrophoresis

“…For DNA fragment analysis HEC was found to be comparable in performance to polydimethylacrylamide (PDMA), and superior to linear polyacrylamide and polyethylene oxide. In further work by the same group [49], hydroxypropylcellulose (HPC) was used as an adsorptive coating and sieving matrix for DNA separations. Compared to HEC, the use of HPC allowed for more concentrated solutions to be used without significant increase in solution viscosity.…”

Low Viscous Separation Media for Genomics and Proteomics Analysis on Microchip Electrophoresis System

“…Sanders et al 68) has used HPC with MES/TRIS buffer system (pHϭ6.1), this combination has several merits: the balanced pH (6.1) eliminates the need for surface preconditioning because it reduces the extent of the deprotonation of the silanol groups on the microchip surface 69) which cause the electro-osmotic flow (EOF) to be reduced and the hydrogen bonding of the polymer with the silica wall to increase. Also and since buffer pH is above 4 then the dsDNA will not be denatured.…”