1. Introduction

Slab-gel electrophoresis for a long time for the separation of nucleic acids and proteins[1]–[4]. The slab-gel technique is performed in multi-lanes and can analyze samples simultaneously, compared to much more expensive capillary-based electrophoretic systems which cannot perform parallel sample analysis[5]. Despite the advantages of gel electrophoresis, the separation speed is low due to the limiting factor of Joule heating[6]. The effects of Joule heating become conspicuous when high electrical gradient are applied with high ionic concentration buffer solutions which is often the case for electrokinetic transport. Joule heating is an internal heat generation mechanism which is proportional to the externally apply potential gradient. For this reason, applying a high electrical field results in higher heat generation. Joule heating is also proportional to the electrical conductivity of the liquid hence a higher molar concentration means higher heat generation. Liquid properties such as viscosity, permittivity, electrical conductivity and thermal conductivity are strongly dependent on temperature. Strong ionic concentration leads to a strong electrical conductivity which depends strongly on temperature. In biological applications, excess temperature elevations may cause denaturing of proteins, nucleic acids and live biological samples. Large temperature excursions cause band broadening and dispersion that leads to inefficient and low quality separation for separation processes. For reproducible results and efficient separation with electrokinetic transport, effective dissipation of the heat generated by the joule heating is critical [7]–[10].

In the present study, we focus our effort on the electrophoretic component of the transport induced by applying AC fields and DC fields and the associated Joule heating effect. The AC waveforms tested for this study is the Pulsed waveform and the sawtooth waveform. When the pulsed electric fields is turned on, a constant field amplitude is applied and removed at regular time intervals leading to instant electrophoretic flow which is the migration of the charged particles at pulse start-up. When the field is removed, the electrophoretic migration halts and the electroosmotic flow which is the movement of the bulk fluid starts [6]. The sawtooth waveform on the other hand has an amplitude which is incremental and peaks at the midpoint of the period upon switching on the electric field and gradually decrease to the start point in the second half of the wave. For the sawtooth waveform, the rate of electrophoretic migration is undulates over the period of the wave. For the DC field, a constant electric field amplitude is applied for the entire duration of the applied field, therefore the electrophoretic migration of the charged particles is initiated when the DC field is started and maintained until the field is completely removed. Therefore to compute the electrophoretic mobilities of the particles in the different fields, the duty cycle of the fields become an important parameter.

2. Materials and Method

2.2 Experimental setup

The gel was prepared using 1 ml of 50X Tris-acetate-EDTA (TAE) buffer mixed with 49 ml of deionized water. Once mixed, a 0.5-gram agarose tablet was added to the solution to make a 1 % wt/vol agarose solution. The solution was thus mixed and microwaved from 30 seconds and then mixed until clear then cast in a 10 cm by 8 cm Expedon Mini-fast Electrophoresis chamber. A 1X TBE solution was used as buffer. Two dye solutions were prepared, Pyronin Y (302.12 Da) and Safranin O (350.13 Da) by adding 5 mg of the respective dye and 3 mg of sucrose to 7 ml of the previously prepared 1X TBE Buffer. The dyes have a positive N that is ionically bonded to Cl, hence the dyes have a +1 charge in aqueous solution. For tests with cartilage tissue, frozen sectioned tissue samples from whole bovine tails acquired from a local abattoir (Bringhurst Meats, Berlin, NJ, USA) were thawed, weighed and placed in the wells of the gel. Up to 7 μl of the respective dye solution was added to the wells

The chamber was connected to an Extech DCP60 (Nashua, NH, USA) power supply for DC electric field application and an AM-Systems All-In-One Stimulator (Sequim, WA, USA) for the pulsed and sawtooth fields. A field of 60 V amplitude was applied across the gel for 60 min with pictures taken at 5-min intervals. Temperature measurements were taken and recorded using a fiber-optic sensor and a temperature gun for consistency every 5 min.

3. Summary

Biological tissues, in this case a fibrocartilage, is a an extracellular matrix with a hydrated polyelectrolyte containing fixed negative charges on the proteoglycans embedded in the collagen network, with corresponding positive charges distributed in the matrix fluid. We report the electrophoretic mobility of charged molecules across fibrocartilage tissue under the influence of applied DC, square and sawtooth electric fields, taking into account the duty cycle of the pulse and sawtooth fields. We also report a gel temperature measurement during electrophoresis by using a fiber-optic temperature sensor. The results show an increase in temperature of 5 ⁰C when DC field is applied compared to about 1 ⁰C temperature rise for both square and sawtooth electric fields. Since the rate heat generation during gel electrophoresis is higher than the rate of heat dissipation, the heat is dissipated in the square and sawtooth applied fields hence the reduced temperature in the gel.

References

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