A miniaturized and integrated dual-channel fluorescence module for multiplex real-time PCR in the portable nucleic acid detection system

Abstract

A portable nucleic acid detection (PNAD) system based on real-time polymerase chain reaction (real-time PCR) has been developed for point-of-care testing (POCT) of infectious disease pathogens. In order to achieve "sample-in, result-out" while keeping the system compact, the hardware system integrates optical, thermal and motion control modules in a limited space for nucleic acid extraction, purification, amplification and detection. Among these hardware modules, the fluorescence module is one of the most important modules, because its performance directly affects the accuracy and sensitivity of the testing results. In this paper, a miniaturized, high-sensitivity and integrated dual-channel fluorescence module have been proposed for the homemade PNAD system. Based on the principle of confocal optical path, two group of excitation-emission optical paths of different wavelengths are integrated in a small space. In terms of circuitry, a current-light dual negative feedback light emitting diode (LED) drive circuit is applied to improve the stability of the excited light source. All optical and electronic components are integrated in a metal box of 55 mm × 45 mm × 15 mm, that helps miniaturize the detection system. Two different modules have been assembled to fit various fluorescent dyes or probes with the set of excitation and emission as follow: module 1#: 470 nm/525 nm, 570 nm/630 nm; module 2#: 520 nm/570 nm, 630 nm/690 nm. Finally, hepatitis B virus (HBV) concentration gradient detection and multiplex detection of different gene targets of SARS-CoV-2 are carried out on the PNAD system equipped with these two fluorescence modules for evaluating their performances. Compared with the commercial real-time PCR instrument, our fluorescence module has good stability and detection sensitivity.

Keywords: LED drive circuit; confocal optical path; fluorescence detection; point-of-care testing; real-time PCR.

FIGURE 1

Homemade portable nucleic acid detection (PNAD) system. (A) 3D rendering of the PNAD device. (B) Functional block diagram of the PNAD system including motion control module, thermal control module and fluorescence module to achieve “sample-in, result-out”.

FIGURE 2

The Functional block diagram of the dual-channel fluorescence module, which can be divided into two partitions of drive circuit and optical path (Take module 1# as an example).

FIGURE 3

Optical path of the dual-channel fluorescence module (Take module 1# as an example). (A) Schematic diagram of optical path: two excitation lights (blue and yellow) and two emitted lights (green and orange) forms a confocal light path with the help of four dichroic mirrors. (B) Photograph of the optical path, consistent with the schematic.

FIGURE 4

The current-light dual negative feedback LED drive circuit. (A) Working schematic block diagram of the circuit, the current-light dual negative feedback is realized by sampling the current and the light intensity of the LED. (B) Schematic of the circuit which can be divided into three partitions of current feedback, light intensity feedback and LED selection.

FIGURE 5

(A) The three-dimensional (3D) illustration of the fluorescence module in an exploded view, which includes metal shell, PCB, optical path cover, optical structure and focus lens. (B) The photograph of two fluorescence modules combined together. (C) Integration of the fluorescence module (in the red box) with a homemade portable nucleic acid detection system.

FIGURE 6

First channel optical paths (excitation/emission: 470 nm/525 nm) simulation diagram (A) 3D layout of the excitation optical path. (B) Image of the excitation optical path detector with 9.05 l m total power. (C) 3D layout of the emission optical path. (D) Image of the emission optical path detector with 5.2 × 10^-2 lm total power.

FIGURE 7

Second channel optical path (excitation/emission: 570 nm/630 nm) simulation diagram. (A) 3D layout of the excitation optical path with 2.85 l m total power. (B) Image of the excitation optical path detector. (C) 3D layout of the emission optical path. (D) Image of the emission optical path detector with 2.35 × 10^-2 lm total power.

FIGURE 8

Real-time fluorescence PCR curves (left) and their standard curves (right) for detection of HBV with concentrations ranging from 5 × 10⁶ IU/ml to 5 × 10³ IU/ml. (A) and (B): The real-time PCR curves and standard curves for Ct values from the homemade PNAD system. (C,D): The fluorescence curves and standard curves for Ct values from StepOnePlus system.

FIGURE 9

Multiplex real-time fluorescence PCR curves for detection of different targets of SARS-CoV-2. (A) Fluorescence curves of ORFlab gene, N gene and E gene of SARS-CoV-2 from StepOnePlus system. (B) Fluorescence curves of ORFlab gene, N gene, E gene and internal standard target from the homemade PNAD system.