An integrated microfluidic platform for nucleic acid testing

Abstract

This study presents a rapid and versatile low-cost sample-to-answer system for SARS-CoV-2 diagnostics. The system integrates the extraction and purification of nucleic acids, followed by amplification via either reverse transcription-quantitative polymerase chain reaction (RT-qPCR) or reverse transcription loop-mediated isothermal amplification (RT-LAMP). By meeting diverse diagnostic and reagent needs, the platform yields testing results that closely align with those of commercial RT-LAMP and RT‒qPCR systems. Notable advantages of our system include its speed and cost-effectiveness. The assay is completed within 28 min, including sample loading (5 min), ribonucleic acid (RNA) extraction (3 min), and RT-LAMP (20 min). The cost of each assay is ≈ $9.5, and this pricing is competitive against that of Food and Drug Administration (FDA)-approved commercial alternatives. Although some RNA loss during on-chip extraction is observed, the platform maintains a potential limit of detection lower than 297 copies. Portability makes the system particularly useful in environments where centralized laboratories are either unavailable or inconveniently located. Another key feature is the platform's versatility, allowing users to choose between RT‒qPCR or RT‒LAMP tests based on specific requirements.

Keywords: Chemistry; Electrical and electronic engineering; Microfluidics.

Fig. 1. Workflow of a microfluidic diagnostic system for RNA detection from sample collection to result display.

a Sample collection: A sample is collected under an outdoor tent. b Reagent setup: Test tubes filled with various reagents are used for RNA detection. c RNA preparation protocol: A flow diagram details the steps of the RNA preparation process using magnetic beads. d Sample extraction and subsequent amplification on a microfluidic chip: An illustration of the microfluidic chip shows the sample and reagents loaded into the corresponding port. e Photograph of the POCT microfluidic system: A vertical view of the microfluidic diagnostic device, highlighting its overall features with a chip in place for analysis

Fig. 2. Components of the microfluidic diagnostic system.

a System overview: The microfluidic diagnostic device featuring a stepper motor assembly and user interface with operational buttons and display. b Cross-sectional view of the temperature system: Illustration depicting the temperature system containing a thermoelectric cooler (TEC), Pt100 temperature sensor and heat sink with an electric fan. c Cross-sectional view of the optical system (left) and optical pathway schematic (right): diagram showing the LED light source passing through an excitation filter to the sample, with the emitted light collected by a photodiode (PD) after passing through an emission filter. d Software control user interface

Fig. 3. Microfluidic chip for RNA detection and schematic overview.

a Actual chip: A photograph of the microfluidic chip filled with fluorescein illuminated with blue light, thus increasing the contrast of all microfluidics channels. b Schematic diagram: The diagram illustrates the microfluidic pathway within the chip, detailing the flow (yellow dashed line) of RNA samples through various processing stages, including extraction, oil, and PCR/LAMP chambers

Fig. 4. Temperature profiling and distribution in the PCR amplification process.

a PCR protocol: Graphical representation of the PCR protocol indicating rapid heating (blue line) and cooling (red line) cycles at rates of ≈ 5.5 K·s⁻¹ and ≈ −6.7 K·s⁻¹, respectively, which are essential for efficient DNA amplification. b Infrared radiation intensity: An infrared image capturing the distribution of radiation intensity emitted from the sample area used to infer the temperature distribution across the microfluidic chip. c Melting curve analysis (MCA) and temperature mapping: 3D map derived from the MCA showing the extracted apparent melting temperature (T_M*) values across the sample area with a mean ± standard deviation of (79.02 ± 0.77) °C (mean value ± standard deviation), demonstrating uniformity in the thermal profile essential for precise nucleic acid amplification

Fig. 5. PCR amplification efficiency and standard curve analysis.

a Amplification curves generated using a commercial qPCR system showing amplification curves of DNA samples after extraction via a microfluidic chip benchmarked against original DNA samples. b Standard PCR curves extracted from the PCR amplification curves shown in (a) with an extracted Ct shift value of ≈ 4.9, indicating a sample extraction efficiency of ≈ 3.4%

Fig. 6. Comparative analysis of RT‒PCR results and melting curves from commercial qPCR machine and microfluidic chip.

a Commercial qPCR raw data: Amplification plots of N gene RNA sequences performed in triplicate on a standard qPCR machine. b MCA on a commercial machine: Post-RT‒PCR MCA showing the specific melting temperatures of the amplified products. c Derivative of melting curves: Graphical derivative representation of the melting curves highlighting the peaks corresponding to the specific melting points. d Microfluidic chip raw data: Amplification plots from RT‒PCR performed on a microfluidic chip. e Normalized chip data: RT‒PCR data from the microfluidic chip after normalization and Boltzmann fitting, revealing the amplification efficiency. f Chip MCA results: Melting curve analysis derived from the microfluidic chip’s RT‒PCR raw data indicating the melting temperature of the nucleic acids postamplification, revealing the amplification specificity

Fig. 7. RT-LAMP amplification efficiency and standard curve evaluation.

a RT-LAMP amplification curves: Real-time amplification data from RT-LAMP reactions performed on a commercial qPCR machine with five different RNA concentrations tested in triplicate, including no-template controls (NTCs). b Normalized RT-LAMP data: The raw RT-LAMP amplification data were analyzed on chips and normalized using Boltzmann function curve fitting to demonstrate the amplification progress and efficiency. c Standard RT-LAMP curves: Comparative standard curves showing the amplification efficiency of RT-LAMP assays. The left slope represents the standard curve obtained from the commercial qPCR machine with a slope of (–1.913 ± 0.053) min·dec⁻¹, and the right slope shows the standard curve derived from the microfluidic chip data with a slope of (–1.282 ± 0.077) min·dec⁻¹, both indicating the mean value and fitting error

Fig. 8. Standard curve analysis of the integrated RT-LAMP system.

Normalized Boltzmann function curve derived from raw RT-LAMP data from the chip without sample extraction and with the function of the integrated system with RNA contents of a 10⁹ copies·µl⁻¹, b 10⁷ copies·µl⁻¹, and c 10⁵ copies·µl⁻¹; each experiment was conducted in triplicate, and the results are shown as different color curves. d The standard curve (black) shows a slope of (–2.788 ± 0.098) min·dec⁻¹ (mean value ± fitting error), which is indicative of the system’s amplification capability after sample extraction. This curve is compared to the red curve with a slope of (–1.282 ± 0.077) min·dec⁻¹ from the chip without the sample extraction process, emphasizing the influence of the integrated extraction on the amplification efficiency