Fig 1.
From digital design file to portable diagnostics device.
(a) A digital format of design file is uploaded to the internet, and downloaded by users. (b) A user uploads the digital files into 3D printer and 3D CNC milling machine for manufacturing. The 3D printer extrudes and deposits filament and the 3D CNC mill cuts and drills through material to engrave patterns and shapes. (c) This results in 3D manufactured parts for assembly. (d) The parts and off-the-shelf electronics components are assembled. (e) This assembled device can function as a portable medical diagnostic device for detecting and quantifying the pathogen in a sample through qPCR.
Fig 2.
The aluminum heating element for qPCR device.
(a) The aluminum rod has four holes for ① PCR tube insertion, ② convection cooling, ③ the excitation light path, and ④ thermistor insertion. (b) The heating element consists of an aluminum rod, a layer of polyimide film, and a coil of NiCr wire. The dimension and location of the holes are: ① ⌀ is 3.72 mm and depth is 5.46 mm, ② ⌀ is 3.72 mm and depth is 8.59 mm, ③ ⌀ is 3.56 mm and location from top is 3.70 mm, and ④ ⌀ is 1.59 mm and depth is 1.00 mm and location from top is 3.70 mm.
Fig 3.
Optical design of real-time fluorescence measurement for qPCR.
The blue LED (470 nm) emits excitation wavelength that is filtered through an excitation filter which is a band-pass filter of 455 to 495 nm. The excitation light travels through the hole in the aluminum rod and is absorbed to the sample in the PCR tube. The emitted light is collected by an avalanche photodiode through an emission filter, with a band-pass at 511 to 529 nm.
Fig 4.
Photographs of 3D printing and 3D milling of all the parts using a multitool 3D printer.
(a) The faceplate is being printed with black ABS material on a heated bed. (b) The main board is being engraved using 3D CNC mill.
Fig 5.
3D assembly of the system with exploded views.
(a) The complete device showing locations of Control assembly, Photodiode assembly, Bottom assembly, Case, and Li-Po batteries. (b) Detail of assemblies. The Control assembly contains a faceplate, a control board, a main board, and a MicroView microcontroller. This assembly provides a user interface and houses all electronic controls. The Photodiode assembly contains the photodiode and emission filter. This assembly is used to determine the target DNA concentration present in the sample. The bottom assembly contains a motor, a fan blade, spacers, and a centrifugal fan housing, forming the cooling fan system. It also contains an LED and excitation filter, establishing the light source for illuminating the sample. The cartridge is a removable assembly allowing for easy insertion of the PCR tube containing the sample. It contains the heating block, electrical contacts for connection to the main board, and a vented bottom plate allowing for the escape of warm air.
Fig 6.
The thermal simulation of the heating element’s heating and cooling cycle time.
(a) The heating time from 60°C to 95°C in various heights of heating element. The shortest (9.5 seconds) to heat is 18.5 mm in height. (b) The cooling time from 95°C to 60°C in a fan-operating condition. The shortest (23.9 seconds) to cool is 36.9 mm in height. Increasing the height of the aluminum rod increases the surface area, resulting in improved convection and the length of the cooling period is reduced. The subset graph is an expended view at 23 to 27 seconds. (c) The summary of heating and cooling time for various heights of heating element. The height with the shortest total cycling time is 18.5 mm (36.1 seconds). The heating period increases at a faster rate to the increase in height than the cooling period decreases.
Fig 7.
The temperature plot of the sample and the heating element for calibration.
The plot is used to determine proper thermocycling control thresholds. The gray curve shows the temperature of the aluminum heating block; the red curve shows the temperature inside of the 20ul sample inside of the PCR tube.
Fig 8.
The photograph of 3D manufactured qPCR device and the resulted RT-qPCR from 3D manufactured qPCR device from three separate qPCR using 3 dilutions of target virus, 2 × 107 vp/mL, 2 × 106 vp/mL, and 2 × 105 vp/mL.
(a) The dimension of the qPCR device is 12 × 7 × 6 cm3. The MicroView shows the status of amplification cycle and fluorescence reading. (b) The bottom view of the qPCR device showing cartridge bottom and an air inlet for centrifugal fan. (c) The size of the cartridge that holds PCR tube during qPCR is 7.6 × 3.4 × 2.9 cm3. (d) Measured fluorescence readings show the shift in the intensity measurements corresponding to the differing concentrations of target virus. The threshold for determining Cq is also shown is a dotted line. (e) Measured Cq for three concentrations of target DNA. The mean and standard deviation of Cq are 21.74 ± 0.39, 23.66 ± 0.70, and 25.98 ± 1.75, for 2 × 107 vp/mL, 2 × 106 vp/mL, and 2 × 105 vp/mL, respectively.
Fig 9.
Comparison of conventional qPCR machine and 3D printed device using gel electrophoresis.
Each lane included: (lane 1 and lane 6) 10-bp DNA ladders, (lane 2) negative control from conventional device, (lane 3) negative control from 3D printed device, (lane 4) 2 × 107 vp/mL sample from conventional device, and (lane 5) 2 × 107 vp/mL sample from 3D printed device.