To ensure ease of use and full automation, the microfluidic cartridge was designed to perform on-chip nucleic acid lysis and LAMP amplification. The illustration of the microfluidic cartridge was displayed in Fig. 1a, and the LAMP reagent was preloaded into the reaction chambers of the cartridge as the lyophilized beads. The overall design and the description of the cartridge were displayed in Fig. 1b. The cartridge consists of a top cover, a reagent storage rotor, a base plate, a waterproof debubbler membrane, and two pistons. The base plate contains multiple microchannels and four independent reaction chambers, which enabled the detection for up to 4 targets within a single cartridge. The reagent storage rotor comprises three chambers: a lysis chamber (A), a sample loading chamber (B), and a mixing chamber (C). Rotation of the reagent rotor is driven by a motor, which in turn activates pistons to control liquid movement between chambers, enabling nucleic acid lysis, mixing, and eventual transfer to the amplification chambers.
The overall cartridge functions and fluidic control process were displayed in Fig. 1c. After sample collection using a nasopharyngeal or oropharyngeal swab, the swab was broken off and inserted directly into the sample-loading chamber (Step 1). The chamber was then sealed by closing the cap integrated into the top cover. The flat-head piston pushed the lysis reagent from the lysis chamber into the sample-loading chamber via lysis buffer transfer microchannel, initiating nucleic acid extraction (Step 2 to Step 3). As the rotor continues to turn, the ball-head piston lifted under the guidance of a grooved cam structure, drawing the lysed sample into the mixing chamber (Step 4). Forward and reverse rotation of the rotor allowed the ball-head piston to perform repeated up-and-down strokes, ensuring thorough mixing (Step 5 to Step 6). Finally, continued rotation directed the mixed solution into an inlet channel, which leaded the liquid into the reaction chambers (Step 7). There, the lyophilized reagents were rehydrated, and the cartridge ready for amplification and fluorescence-based detection (Step 8).
The FA-RMP supports reverse-transcription LAMP (RT-LAMP) reactions within a consumable microfluidic chip, and integrates temperature control and fluorescence signal detection into a fully automated workflow. The system is capable of simultaneously processing up to four individual detection channels enabling high-throughput diagnosis.
As depicted in Fig. 2a, the platform integrates three subsystems: (i) microfluidic cartridge, (ii) a temperature-control stack, and (iii) a mobile fluorescence-detection head. After the user introduces the lysed sample into the chip inlet, a stepper motor drives the transmission component to rotate the upper groove of the microfluidic chip, thereby actuating a piston that sequentially allocates the sample to distinct chambers for nucleic-acid extraction and mixing and ultimately to the reaction chamber. The chip is clamped between upper and lower heater blocks to maintain isothermal amplification at 65 °C. The optical head is mounted on a linear rail and is moved beneath the chip by a second stepper motor to sequentially scan all wells, producing real-time fluorescence curves. Control software loaded on the system executes automated management of the entire process.
Accurate, uniform, and rapidly adjustable temperature is indispensable for isothermal LAMP assays. The system adopts a sandwich-type temperature-control module: during the heating phase, two polyimide-foil heaters clamp the chip's amplification chamber between thermally conductive plates, while an NTC thermistor probe continuously acquires temperature data and feeds it to the control unit, providing feedback to a PID controller until the designated RT-LAMP temperature is reached. As the result shown in Fig. 2b, the system can maintain a constant temperature of 65 °C for 30 min at least. Real-time signal acquisition is accomplished by a compact optical head carried on a stepper-driven linear rail, which sequentially scans the four reaction regions. A dilution series of fluorescent sodium solution from 0.3125 to 5 μM was analyzed to evaluate the performance of intensity measurement of the optical module, and the result shown in Fig. 2c exhibited the linear correlation between the concentrations and fluorescence (R = 0.9820). The result displayed in Fig. 2d indicated that the fluorescence intensities of each chamber and channel was no significant difference, which contained the 1 μM of fluorescent sodium. The above results strongly proved the platform demonstrated in this study could support the needs of the pathogens detection.
Using the selected primers, the concentration of the primers in the LAMP reaction was optimized. The primers were evaluated by comparing the Tp value and the non-specific amplification signal of non-template control group. As the results shown in Fig. 3a, 60-80 μM of the inner primers (FIP/BIP) would lead to nonspecific signals due to the high-concentration primers form double-stranded structures and then bound by the fluorescence dye non-specifically. The 20-40 μM of the primers with no significant differences in values of Tp and 20 μM was selected to minimize the risk of non-specific amplification while preserving efficient amplification performance. Likewise, outer primers (F3/B3) were optimal at 5 μM per test to prevent the risk of non-specific amplification under high concentration of primers as Fig. 3b, and loop primers at 10 μM per test as Fig. 3c.
Mg plays a critical role in regulating polymerase activity and optimizes the concentration of Mg emerges as a viable strategy to enhance the performance of LAMP assays. As shown in Fig. 3d, different concentrations of Mg (from 2 mM to 8 mM) were added in the reaction mixture, and the Tp values were obtained. The groups added with 8 mM and 6 mM Mg exhibited the nonspecific signals, which may be attributed to the high concentration of Mg causing incorrect binding between primer and template. The optimal Mg concentration was defined as the concentration that still achieved a rapid amplification of the positive sample without yielding any amplification in the negative control and 4 mM was finally chosen as the optimal concentration.
To address the application requirements, the LAMP mixture was turned as lyophilized beads with advantages in terms of transportation, long-term storage stability, and compatibility. The efficiency of the LAMP lyophilized beads was validated by incubation at 65 °C for 30 min and the result was shown in Fig. 4. The standard cultures with MP DNA were pre-mixed with nucleic acid release solution in 10 min at room temperature and then detected as the sample. The amplification curves of Positive 1 (LAMP mixture) and Positive 2 (lyophilized beads) with MP DNA were standard S profiles, which indicated the LAMP lyophilized beads can realize the amplification and detection of MP accurately. The amplification curve of the group with positive 3 (lyophilized beads on chip) was same with the positive 2, indicating that the LAMP lyophilized beads could be utilized to detect the MP DNA released on-chip or off-chip.
To determine the limit of detection (LoD) of FA-RMP, each sample was serially diluted to different concentrations (5000-20 copies μL) and evaluated in 3 independent replicates per level on microfluidic cartridges under the optimized LAMP conditions. The platform reliably detected down to 50 copies μL of MP culture samples (Fig. 5a). Linear regression of logarithm of MP concentration versus Tp yielded R = 0.9528 (Fig. 5b), indicating quantitative potential within the range 5000-50 copies μL.
The detection specificity is crucial to evaluate the specificity of the FA-RMP. The screening primers of MP were preloaded and a variety of pathogens standard cultures including M. pneumoniae, Influenza A virus (Flu A), Influenza B virus (Flu B), Respiratory syncytial virus, Ureaplasma urealyticum, Chlamydia pneumoniae, Staphylococcus aureus, Legionella pneumophila, Streptococcus pneumoniae were chosen to test the specificity analysis of system and the intensity of the fluorescence in 30 min were obtained. The results shown in Fig. 5c indicate that the primer sets for M. pneumonia could only amplify the target pathogen and generate strong fluorescence signal intensity. There was no cross-reaction with other templates, which proved that the FA-RMP have great specificity.
The diagnostic procedure of the FA-RMP follows a streamlined sequence as shown in Fig. 6a. A nasopharyngeal or oropharyngeal swab is first collected from the individual. The swab tip is placed into the sample-loading chamber of a disposable microfluidic chip after breaking off the shaft, and the lid is closed to seal the chamber. The chip is then inserted into the benchtop instrument, where proper positioning is ensured by a mechanical interlock. The analysis is initiated by a single click within the dedicated software on the connected computer. The overall process of the FA-RMP for "samples-in, results-out" with microfluidic cartridge was shown in Fig. 6b, and the FA-RMP enabled automatically detection processes on-chip including nucleic acid release, isothermal amplification, and real-time fluorescence detection within ~30 min. The developed platform automatically operated by the software on the computer is shown in Fig. 6c.
A total of 18 nasopharyngeal samples (healthy controls, n = 4; MP infected individuals, n = 12; Flu A infected individual, n = 1; Flu B infected individual, n = 1) were acquired from Peking University Third Hospital. The study protocol was reviewed and approved by the Peking University Third Hospital medical science research ethics committee (IRB approval number: 00006761-M2022102). To demonstrate the practicality and the consistency, the collected throat swabs of were detected by the developed platform (FA-RMP) and clinical standard device (7500 real time PCR, Thermo Fisher Scientific.). The result shown in Fig. 7 displayed the Tp value of the clinical samples under the two platforms. The reaction condition for qPCR was were configured following the the Mycoplasma pneumoniae detection kit (Sansure Biotech INC., China): initial denaturation at 94 °C for 5 min; 45 cycles of denaturation at 94 °C for 15 s, annealing at 57 °C for 30 s (with fluorescence acquisition). It also defined that the Tp value of sample ≤35 cycles were classified as positive and the results of all clinical samples based on qPCR were consistent with expectations. The FA-RMP is considered capable of identifying positive samples within 30 min, with Tp values greater than 30 uniformly classified as negative results. For the 12 positive samples detected by FA-RMP, all Tp values were below 20; negative samples exhibited no amplification trend. The testing results between FA-RMP and clinical standard device were 100%, which fully proved the clinical practicability of the FA-RMP.
The clinical samples with Flu A, Flu B and MP were used to verify the ability of the developed platform for pathogens multiplexed detection. The microfluidic cartridges were prepared with sealing the microchambers and loading the nucleic acid release reagent and LAMP lyophilized beads. The LAMP primers for Flu A and Flu B were listed in the Table S1. The results displayed in Figs. S1, S2 proved the developed LAMP assays could be used to amplify M1 gene of Flu A and NA gene of Flu B with strong specificity.
After establishing the optimized conditions with the MP assay, we evaluated the ability of the system to perform simultaneous detection of multiple respiratory pathogens. The clinical samples were pretreated with the nucleic acid release reagent and then added into the microfluidic cartridge. As the results displayed in Fig. 8, We confirmed that known influenza A, influenza B, and MP samples were each detected specifically in the appropriate chamber with no cross-signal. This demonstrates the assay's applicability to clinical specimens and its ability to multiplex targets on the chip.