Multiplex, digital nucleic acid tests have important biomedical applications, but existing methods mostly use fluorescent probes that are target-specific and difficult to optimize, limiting their widespread applications. Here, we report color-encoded, intelligent digital loop-mediated isothermal amplification (CoID-LAMP) for the coidentification of multiple nucleic acid targets. CoID-LAMP supplements different primer solutions with different dyes, generates primer droplets and sample droplets, and collectively pairs these two types of droplets in a microwell array device to perform LAMP. After imaging, the droplet colors were analyzed to decode the primer information, and the precipitate byproducts within droplets were detected to determine the target occupancy and calculate the concentrations. We first established an image analysis pipeline based on a deep learning algorithm for reliable droplet detection and validated the analytical performance in nucleic acid quantification. We then implemented CoID-LAMP using fluorescent dyes as the coding materials and established an 8-plex digital nucleic acid assay, confirming the reliable coding performance and the capability of multiplex nucleic acid quantification. We further implemented CoID-LAMP using brightfield dyes for a 4-plex assay, suggesting that the assay could be realized solely by brightfield imaging with minimal demand on the optics. Leveraging the advantages of droplet microfluidics in multiplexing and deep learning in intelligent image analysis, CoID-LAMP offers a useful tool for multiplex nucleic acid quantification.
Thimerosal (THI) is widely used as an antimicrobial preservative, but can hydrolyze to ethylmercury, causing potentially neurotoxicity. In this work, a THP-1 cell line was used to investigate the biological behavior of THI. An on-line droplet microfluidic chip system combined with time-resolved inductively coupled plasma mass spectrometry was used to quantify Hg in single THP-1 cells. The cellular uptake and elimination behaviors of THI were studied, and the toxicity of THI in terms of redox balance was discussed. The results showed that a small number of cells (<5%) exhibited a high uptake content (>200 fg/cell) for THI, and most of the cells (68.8–85.8% for different exposure groups at 25 h) exhibited a relatively low uptake content (<20 fg/cell). After stopping exposure to THI, the cells showed an elimination process for Hg, which was rapid in the first several hours and gradually slowed down. When the elimination time was 25 h, 7.4–26.3% of the cells in different exposure groups still contained a detectable amount of Hg (>2 fg/cell), indicating Hg could not be eliminated completely, which may cause cumulative toxicity to macrophages. Moreover, it was found that exposure to THI even at 50 ng/mL can cause cellular oxidative stress behavior, leading to an increase in reactive oxygen species level and a decrease in glutathione level. This trend would continue for a period of time after stopping THI exposure. With the elimination of Hg, the redox balance of cells showed a tendency to stabilize and restore, but cannot be restored to normal status, indicating a long-term chronic toxicity of THI to THP-1 cells.
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