Amplification of Escherichia coli in a Continuous-Flow-PCR Microfluidic Chip and Its Detection with a Capillary Electrophoresis System

This protocol describes how to build a continuous-flow-polymerase chain system based on the microfluidic chip, and how to build a capillary electrophoresis system in the lab. It presents a simple way for the analysis of nucleic acids in the lab. Polymerase chain reaction is a traditional method employed for the amplification of the target gene.

However, traditional PCR is very time-consuming because of the low-temperature variation efficiency. This work proposes a continuous-flow-PCR system based on the microfluidic chip. The amplification time can be greatly reduced by running the PCR solution into a microchannel placed on heaters set at different temperatures.

As CE has many advantages, such as high resolution, high speed, and excellent reproducibility, it became a popular tool in the lab for the analysis of nucleic acids and proteins. However, most labs, especially the labs in the developing world, cannot afford this technology because of the high price of the CE instrument. Herein, we have outlined protocols for how to fabricate the CF-PCR microfluidic chip and how to build a versatile CE system in the lab.

We also demonstrate the process of amplification of Escherichia coli by the CF-PCR system, and the detection of the PCR products by the CE system. By following the procedures described in this protocol, users should be able to fabricate microfluidic chips, prepare PCR solution, build a CF-PCR system for nucleic acid amplification, and set up a simple CE system, even with limited resources, to separate DNA fragments. Heat the silicon wafer at 200 degrees Centigrade for 25 minutes to remove the moisture.

Dispense one milliliter of SU-8 2075 photoresist per inch of the wafer. Spin it on the silicon wafer using a spin coater at 500 rpm for 5 to 10 seconds with a acceleration of 100 rpm per second, and then at 2, 000 rpm for 30 seconds with a acceleration of 500 rpm per second. Soft bake it at 65 degrees Centigrade for three minutes, and 95 degrees Centigrade for 15 minutes.

Set 150 to 215 millijoules per square centimeter as the exposure energy for the photolithography machine, and engrave the designed pattern onto the photoresist with a photolithography mask. Place the silicon wafer and mask ready for exposure. Post exposure, bake the wafer at 65 degrees Centigrade for two minutes, and 95 degrees Centigrade for seven minutes.

Immerse the silicon wafer in developer solution to remove excess photoresist, and take it out when the microchannels can be seen. Then use isopropanol to rinse off the residual developer solution. Mix the polydimethylsiloxane prepolymer and curing agent at a ratio of 10 to 1.

Pour the mixed PDMS solution into the replica mold and solidify it at 80 degrees Centigrade for 60 minutes. Bond the PDMS microfluidic chip on a slide after activation using a plasma cleaner, and solidify them at 80 degrees Centigrade for 30 minutes as soon as possible. Use a vortex mixer and centrifuge to ensure the reagents are well mixed.

Prepare a centrifuge tube. First add water to the centrifuge tube. Then add the DNA template, primer, buffer, dNTP mixture, Tween 20, PVP, and finally add the DNA polymerase.

Finally, mix the solution by vortex. Prepare two PTC ceramic heaters, two solid-state relays, two temperature controllers, two temperature sensors, and a power cord. Connect the heater to the solid-state relay.

Connect the solid-state relay to the PID temperature controller. Then attach the temperature sensor probe to the bottom of the two heaters, and connect the terminal to the PID temperature controller. Finally, connect two solid-state relays in series, and connect the power cord.

3D print a slot for the two heaters and keep the heaters on the same plane. Place the microfluidic chip on the two heaters. Prepare a syringe pump and a syringe, then fix the syringe on the syringe pump.

Connect a silicone tubing with a syringe, and connect a steel needle to the top of the silicone tubing. Insert the steel needle into the inlet of the microfluidic chip. Place a pipette tip at the outlet of the chip to collect the PCR products.

Use a high-voltage power supply to generate a pulsed-field electric field. Look, this is a high-voltage power supply. These are positive and negative electrodes.

Use a mercury lamp as a light source and filter the excitation wavelength from the mercury lamp through a filter. Place the capillary on the microscope stage. Collect the fluorescence emission with the objective, and then detect it by a photomultiplier tube.

This is our microscope and this is our capillary. Now we turn on the light under dark room conditions. Excitation light is collected by the objective.

Use the self-developed LABVIEW software to control the power supply and complete the data acquisition. Preset the temperature of the heaters of the CF-PCR system at 65 degrees Centigrade and 95 degrees Centigrade. Place the microfluidic chip on the two heating blocks.

Insert the tip of the silicone tube into a centrifuge tube containing 50 microliters of the PCR solution. Pull the syringe plunger to slowly withdraw the solution. Fix the syringe on a syringe pump.

Insert the steel needle into the inlet of the microfluidic chip. Set the flow rate of the pump to 10 microliters per minute and press the start button to push the solution in the microchannel at the inlet of the microchip. Collect the PCR products at the outlet of the microfluidic chip.

Prepare a capillary with 8 centimeters total length and 6 centimeters effective length. Prepare the separation buffer by mixing 100 microliters of 1%HEC, two microliters of 100x SYBR Green, and 98 microliters of ultrapure water to get 0.5%HEC containing 1x SYBR Green. Fill the capillary with the prepared separation buffer using a vacuum pump.

Input the injection voltage and the injection time on the software interface, click the start button, and wait for the PCR products to be electrodynamically introduced into the capillary. Input the DC voltage on the software interface, click the start button, and run the electrophoresis at 100 volts per centimeter of electric field strength. Click the stop button after all DNA fragments are separated in the capillary.

Flush the capillary with sterilized water for one minute after each run. This picture represents the electropherogram of PCR products and DNA markers. We first amplified the target gene of Escherichia coli in the CF-PCR system, and the PCR solution took about 10 minutes and 30 seconds from the inlet to the outlet of the chip.

The size of the target amplicon of Escherichia coli was 544 bp. After that, we analyzed the amplified PCR products in the capillary electrophoresis system. In addition, we also performed the capillary electrophoresis of 100 bp DNA ladder under the same experimental conditions.

The size of the PCR product can be evaluated according to the electropherogram of the DNA ladder. Each experiment was performed three times for reproducibility. Data in this picture shows that the peak corresponding to PCR products of Escherichia coli was observed after separation, and the migration time of PCR products in the microfluidic chip was consistent with the one in a thermal cycler.

Both PCR and CE are two popular biotechnologies in the analysis of nucleic acids. This paper describes the amplification of Escherichia coli and the detection of the PCR products using the CF-PCR and CE systems, both built in-house. The target gene of Escherichia coli was successfully amplified within 10 minutes, and the DNA fragments smaller than 1, 500 bp were separated within eight minutes.

Also, the CF-PCR system based on the microfluidic chip and the CE system introduced in this work are easy to fabricate, and may offer a simple way for the analysis of nucleic acids in the lab. It can amplify and detect only one sample at a time, which limits its wide application. Therefore, the development of an integrated CF-PCR and CE microfluidic chip array for high-throughput detection of pathogens is still on the way.