A portable, shock-proof, surface-heated droplet PCR system for Escherichia coli detection

Abstract

A novel polymerase chain reaction (PCR) device was developed that uses wire-guided droplet manipulation (WDM) to guide a droplet over three different heating chambers. After PCR amplification, end-point detection is achieved using a smartphone-based fluorescence microscope. The device was tested for identification of the 16S rRNA gene V3 hypervariable region from Escherichia coli genomic DNA. The lower limit of detection was 10(3) genome copies per sample. The device is portable with smartphone-based end-point detection and provides the assay results quickly (15 min for a 30-cycle amplification) and accurately. The system is also shock and vibration resistant, due to the multiple points of contact between the droplet and the thermocouple and the Teflon film on the heater surfaces. The thermocouple also provides real-time droplet temperature feedback to ensure it reaches the set temperature before moving to the next chamber/step in PCR. The device is equipped to use either silicone oil or coconut oil. Coconut oil provides additional portability and ease of transportation by eliminating spilling because its high melting temperature means it is solid at room temperature.

Keywords: 16S rRNA; Coconut oil; Contact angle; Polymerase chain reaction; Smartphone.

Figure 1

Schematic illustrations for the device layout and its operation. (A) All the major components of the device, less the circuit. A disposable cartridge, pre-loaded with solid coconut oil at room temperature and a droplet of PCR mixture (within the oil), is connected to the device. (B) The oil melts upon initial heating and the thermocouple loop picks up the droplet (PCR mixture + sample target). The sample solution is added to the PCR mixture droplet using a pipette. (C) In one complete thermal cycle, the droplet moves from the denaturation chamber (98°C), to the annealing chamber (50°C), and then to the extension chamber (80°C). The droplet returns back to the denaturation chamber to commence another cycle. The droplet is guided across the chambers by a thermocouple loop, and it contacts on the Teflon-coated heater surfaces. A PCB heater and a surface-mounted thermocouple control the oil temperature in each chamber. The droplet stays in each chamber until the thermocouple loop detects that it has reached the desired temperature (95°C, 56°C, and 72°C, respectively). (D) A pipette dislodges the droplet upon completion of PCR thermocycling. (E) The thermocouple loop and the metal guide are moved to the extension chamber to secure room for a smartphone microscope. 1 μL of 20× SYBR Green I dye solution is added to the droplet. (F) A smartphone-based fluorescence microscope measures fluorescence. Circuit layout as seen on the breadboards. (A) There are 3 MAX31855, one for each surface-mounted thermocouple. Three JZC-11F relays, one for each heater. The temperature and PID settings are displayed on a 20×4 serial LCD (not shown). (B) The motor controller circuit, showing the AD595 used for the thermocouple loop, which measures internal droplet temperature. Also shown is the EasyDriver connected to the Arduino microcontroller and Haydon-Kerk linear stepper motor. The output of the thermocouple is displayed on another 20×4 serial LCD (not shown). There are 3 buttons, one for starting thermocycling and two for manually positioning the thermocouple loop and droplet. Images created using Fritzing software (Friends of Fritzing e.V., Berlin, Germany). T/C =temperature control.

Figure 2

(A) Graphical representation of the droplet being held steady with the thermocouple loop while simultaneously reading the internal droplet temperature for feedback to the controller. (B) The water droplet on the Teflon-coated heater surface in silicone oil immersion (top) and coconut oil immersion (bottom). The contact angle in silicone oil immersion is 154 ± 2° (n = 6), and the contact angle in coconut oil immersion is 157 ± 1° (n = 6).

Figure 3

(A) Gel electropherogram showing results from the positive control experiment using 2.6 ng of genomic DNA (equivalent to 5.2×10⁵ genomic copies) extracted from E. coli K12 and thermocycled for 30 cycles, with thermal cycle times of 30 s, on the surface-heated droplet PCR device. The genomic DNA was quantified by a Qubit 2.0 fluorimeter. The 196 bp product band is at the expected location, and there is no band observed for the no template control (NTC) sample. (B) Gel electropherogram showing the result of the dilution test. Genomic DNA in the range of 2.6 ng to 5.2 pg (5.2×10⁵ − 10³ genomic copies) was thermocycled for 30 cycles, with thermocycle times of 30 s, on the surface-heated droplet PCR device. The PCR with the lowest DNA content (5.2 pg or approximately 10³ genomic copies) produced a visible band.

Figure 4

(A) Normalized intensity is plotted against the E. coli genomic DNA content of the reaction. Green bars (left) show the average green pixel intensities of the images taken with the smartphone-based fluorescence microscope of a droplet on the device after thermocycling. Purple bars (right) show the average band intensities for the same amplifications analyzed by gel electrophoresis. All results are normalized to no target controls (NTC = no E. coli gene + PCR mixture, amplified for 30 cycles). All results are the mean of three different experiments and error bars represent standard error. Representative fluorescence images of the droplet on the device are shown above the chart for each concentration. (B) Schematic of the optical layout of the smartphone-based fluorescence microscope, which is contained within the 3D printed housing. The excitation source is a 466 nm blue LED that is filtered by a 492 ± 10 nm bandpass filter. A 500 nm dichroic shortpass filter separates the excitation light from the fluorescence emission from the droplet. The emission is further filtered by a 520 ± 10 nm bandpass filter before reaching the smartphone camera. Two lenses are used to focus the light at the position of the droplet and on the smartphone camera.

Figure 5

(A) Still images taken from a video of the vibration and impact shock tests of the surface-heated droplet PCR and the pendant droplet PCR methods. Images are shown during and after vibration and during and after impact shock (the during and after images are taken within 1 s of each other). The surface-heated droplet PCR method was able to successfully recover from vibration and impact shock and maintain droplet control. In comparison, the pendant droplet PCR method (where the droplet hangs from a syringe needle) was unable to recover from neither the vibration nor the impact shock and the droplet was dislodged from the needle tip. (B) Images taken from Supplementary Video 1 of the coconut oil melting with a 9 μL droplet of PCR mixture contained within. The images are taken at 0, 30, 90, and 120 s.