Simultaneous detection of multiple HPV DNA via bottom-well microfluidic chip within an infra-red PCR platform

Abstract

Portable Polymerase Chain Reaction (PCR) devices combined with microfluidic chips or lateral flow stripes have shown great potential in the field of point-of-need testing (PoNT) as they only require a small volume of patient sample and are capable of presenting results in a short time. However, the detection for multiple targets in this field leaves much to be desired. Herein, we introduce a novel PCR platform by integrating a bottom-well microfluidic chip with an infra-red (IR) excited temperature control method and fluorescence co-detection of three PCR products. Microfluidic chips are utilized to partition different samples into individual bottom-wells. The oil phase in the main channel contains multi-walled carbon nanotubes which were used as a heat transfer medium that absorbs energy from the IR-light-emitting diode (LED) and transfers heat to the water phase below. Cyclical rapid heating and cooling necessary for PCR are achieved by alternative power switching of the IR-LED and Universal Serial Bus (USB) mini-fan with a pulse width modulation scheme. This design of the IR-LED PCR platform is economic, compact, and fully portable, making it a promising application in the field of PoNT. The bottom-well microfluidic chip and IR-LED PCR platform were combined to fulfill a three-stage thermal cycling PCR for 40 cycles within 90 min for Human Papilloma Virus (HPV) detection. The PCR fluorescent signal was successfully captured at the end of each cycle. The technique introduced here has broad applications in nucleic acid amplification and PoNT devices.

FIG. 1.

The scheme of the bottom-well PCR microfluidic chip. (a) An overview of the bottom-well PCR microfluidic chip design. (b) An illustration of the reagent storage in the bottom-wells of the PCR microfluidic chip. (c) The final product image of the bottom-well PCR microfluidic chip.

FIG. 2.

The scheme of the IR PCR platform. (a) The physical illustration of the IR PCR platform. (b) The working principle of the IR PCR platform. (c) The demonstration of temperature control for on-chip heating, cooling, and fluorescence detection.

FIG. 3.

Flow analysis of the three dimensional water/oil phase in the microfluidic chips. (a) Scheme of sample digitization in the bottom-well microfluidic chips. The contour represents the volume of the water phase. 1 represents pure water, while 0 represents pure oil. (b) The sample retention ratio difference between the first three wells in correlation with elapsed time. (c) The influence of the well length/width on the sample retention ratio. (d) The influence of the well depth/width on the sample retention ratio. (e) The influence of inlet velocity on the sample retention ratio.

FIG. 4.

Heat transfer analysis of the three dimensional water/oil phase in microfluidic chips. (a) Scheme of the model setup in ANSYS. (b) The temperature difference of the three sections in the same well during the heat-up process. (c) The temperature distribution of the simulation diagram in the heat-up process. (d) and (e) The temperature difference of the three sections in the same well in the cool-down process without a fan (d) and the cool-down process with a fan (e). (f) The temperature distribution simulation diagram of the cool-down process.

FIG. 5.

Fluorescence detection of HPV upon the infra-red PCR platform. (a) The fluorescence detection of HPV 16 exhibits positive results. (b) The fluorescence detection of HPV 16 and 18 exhibits positive results. (c) The fluorescence curve of HPV 16 and 18 exhibits positive results. (d) The temperature ramping speed of the PCR platform. The control wells work as the negative control for PCR detection (no reverse primers were added into the control wells). Data plots in Fig. 5(c) represent Mean± S.D. from 4 detections.