Heater Integrated Lab-on-a-Chip Device for Rapid HLA Alleles Amplification towards Prevention of Drug Hypersensitivity

Abstract

HLA-B*15:02 screening before administering carbamazepine is recommended to prevent life-threatening hypersensitivity. However, the unavailability of a point-of-care device impedes this screening process. Our research group previously developed a two-step HLA-B*15:02 detection technique utilizing loop-mediated isothermal amplification (LAMP) on the tube, which requires two-stage device development to translate into a portable platform. Here, we report a heater-integrated lab-on-a-chip device for the LAMP amplification, which can rapidly detect HLA-B alleles colorimetrically. A gold-patterned micro-sized heater was integrated into a 3D-printed chip, allowing microfluidic pumping, valving, and incubation. The performance of the chip was tested with color dye. Then LAMP assay was conducted with human genomic DNA samples of known HLA-B genotypes in the LAMP-chip parallel with the tube assay. The LAMP-on-chip results showed a complete match with the LAMP-on-tube assay, demonstrating the detection system's concurrence.

Keywords: lab-on-a-chip; loop-mediated isothermal amplification; microfluidics; pharmaco-genetics; point-of-care diagnostics.

Figure 1

Latex membrane-based micro-valve design in two operating states. (a) Open state. The piston requires anticlockwise rotation (90°) and pulling to change state from close to open; (i) simplified diagram indicating the floated piston on the latex membrane allowing the fluid to flow; (ii) isometric view of the valve indicating the handle for manual control; (iii) isometric view indicating the unlocked/pulled condition; (iv) isometric view (isolated) indicating the collapsed condition of the piston. (b) Closed state. The piston requires pushing and clockwise rotation (90°) to change state from open to close; (i) simplified diagram indicating the piston blocked the microchannel deforming the latex membrane; (ii) isometric view of the valve; (iii) isometric view indicating the locked/pushed condition; (iv) isometric view (isolated) indicating the expanded condition of the piston.

Figure 2

Latex membrane-based micro-pump design in two operating states. (a) Ideal/pushing state. The piston requires push action and anticlockwise rotation (90°) to change the state from pulled to pushed; (i) simplified diagram indicating the piston placed inside the air chamber by deforming the latex membrane. The chip was assembled in this state which is referred to as an ‘ideal state’; (ii) isometric view of the micro-pump indicating the handle for manual control; (iii) cross section along the x axis indicating the locked condition; (iv) isometric view (isolated) indicating the expanded condition of the piston. (b) Pulling state. The piston requires clockwise rotation (90°) and pull action to change the state from pushed to pull; (i) simplified diagram indicating the floated piston on the latex membrane; (ii) isometric view of the micro-pump; (iii) cross section along the x axis indicating the locked condition; (iv) isometric view (isolated) indicating the collapsed condition of the piston.

Figure 3

Photograph and schematic of the integrated heater and temperature sensor on a glass substrate.

Figure 4

LAMP-chip (a) photo and exploded 3D schematic. The primary function of the four 3D-printed parts: (1) supports the pistons, (2) supports the heater, (3) chamber feature, (4) inlet-outlet feature. The latex membrane supports the functionality of the micro-pump and the micro-valve. (b) Micro-pump’s push-pull state and micro-valve’s open-close state. Anticlockwise rotation and push action are required to create positive pressure (pushing state). Clockwise rotation and pulling action are required to create negative pressure (pulling state). (c) Experimental setup consists of LAMP-chip with the chip holder and control circuit board.

Figure 5

Photograph of the LAMP-chip in operation in line with the corresponding schematics. (a) Sample loading state, (b) sample transfer from loading chamber to amplification chamber, followed by the sample heating state, and (c) sample extraction state.

Figure 6

(a) Temperature profile of the micro-heater. (b) Correlation between the firmware readout and the measured thermometer by IR thermometer, where n = 5. The dots indicate the mean, and the error bars represent the standard deviation.

Figure 7

Representative photographs of the LAMP-chip and LAMP-tube after the amplification. (a) Positive control on LAMP-chip, (b) negative control on LAMP-chip, and (c) positive and negative control on LAMP-tube.