A 3D Microfluidic Chip for Electrochemical Detection of Hydrolysed Nucleic Bases by a Modified Glassy Carbon Electrode

Modification of carbon materials, especially graphene-based materials, has wide applications in electrochemical detection such as electrochemical lab-on-chip devices. A glassy carbon electrode (GCE) modified with chemically alternated graphene oxide was used as a working electrode (glassy carbon modified by graphene oxide with sulphur containing compounds and Nafion) for detection of nucleobases in hydrolysed samples (HCl pH = 2.9, 100 °C, 1 h, neutralization by NaOH). It was found out that modification, especially with trithiocyanuric acid, increased the sensitivity of detection in comparison with pure GCE. All processes were finally implemented in a microfluidic chip formed with a 3D printer by fused deposition modelling technology. As a material for chip fabrication, acrylonitrile butadiene styrene was chosen because of its mechanical and chemical stability. The chip contained the one chamber for the hydrolysis of the nucleic acid and another for the electrochemical detection by the modified GCE. This chamber was fabricated to allow for replacement of the GCE.

Basic Systems Operations The whole system was controlled by the unit with a program allowing elementary changes in the detection procedure. The main features were sample volume, HCl volume, NaOH volume, electrolyte volume and technical parameters such as hydrolysed period (TIME_H), temperature hydrolysis (TEMP) and area of tubes between hydrolysed and detection part (TRANSPORT). Before the detection process, tubes from hydrolysis solutions and electrolyte had to be immersed deep enough. The working unmodified electrode was inserted.
1. Magnetic valve A was opened and syringe pump1 sucked a small amount of sample to get the liquid to the end of tubing. Magnetic valve A was closed. 2. Magnetic valve B was opened and syringe pump1 sucked a small amount of sample to get the liquid to the end of tubing. Magnetic valve B was closed. 3. Magnetic valve C was opened and syringe pump1 sucked a small amount of sample to get the liquid to the end of tubing. Magnetic valve C was closed.

OPEN ACCESS
4. Servo 2 with valve 2 switched to position B and syringe pump 2 sucked a small volume of electrolyte to get the liquid to the end of tubing. 5. Servo 2 with valve 2 was switched to position C and syringe pump 2 pushed the liquid out. 6. Servo 2 with valve 2 was switched to position A. 7. Servo 3 with valve 3 was switched to position B, servo 1 with valve 1 was switched to position B and syringe pump 1 sucked rinse fluid to the maximal volume of the syringe pump. 8. Servo 1 with valve 1 was switched to position A and syringe pump 1 pushed rinse fluid out. 9. Servo 2 with valve 2 was switched to position C and syringe pump 1 sucked the volume back for washing of hydrolysis cell. 10. Syringe pump pushed liquid out back to zero volume of the syringe pump after 5 seconds . 11. Servo 3 with valve 3 was switched to position A, servo 1 with valve 1 was switched to position B and syringe pump 1 sucked in air to the maximal volume of the syringe pump. 12. Servo 1 with valve 1 was switched to position A and syringe pump 1 pushed rinse fluid out to the detection cell. Pump 3 sucked rinse fluid out to waste.

Detection Procedure
The initial conditions for the start of the detection process were: all pumps were in zero positions, all servos with valves were in position A, magnetic valves were closed, all solutions were sucked into the tubes and remaining tubes were washed and emptied. The main electrode was modified and inserted.
1. Magnetic valve A was opened and the syringe pump sucked in a defined volume of sample. 2. Magnetic valve A was closed, servo 1 with valve was switched to the position B and the syringe pump went to zero position. 3. Servo 1 with valve 1 was switched to position A, magnetic valve B was opened and pump 1 sucked in a defined amount of HCl. 4. Magnetic valve B was closed, servo 1 with valve 1 was switched to the position B and syringe pump 1 went to zero position. 5. Servo 1 with valve 1 was switched to position A, magnetic valve C was opened and syringe pump 1 sucked in a defined amount of NaOH. 6. Magnetic valve C was closed, servo1 with valve 1 was switched on to position B and syringe pump 1 went to zero position. 7. Control unit regulated temperature by sensors IT100 and heater sensors according to variable TEMP. 8. Twenty seconds before the end of hydrolysis servo 2 with valve 2 was switched to the position B and syringe pump 2 sucked in a defined volume of electrolyte. 9. Servo 2 with valve 2 was switched to the position C and syringe pump 2 pushed the liquid out. 10. Servo 2 with valve 2 was switched to the position A. 11. During hydrolysis servo 1 with valve 1 was switched to the position B and syringe pump sucked in volume due to variable TRANSPORT. 12. Immediately after that control unit detected that the time period TIME_H is over, servo 1 with valve 1 was switched to the position A and the syringe pump pushed the sample out to the detection part. 13. Control unit gave an order for detection to the special unit which carried out the detection.