Our research group focuses on developing rapid and highly sensitive diagnostic technologies for non-invasive cancer diagnosis, multidimensional analysis of bacterial infections, and environmental pathogen monitoring, centered around highly integrated microfluidic chips combined with novel biochemical analysis methods.
1. Tumor Detection and Cancer Metastasis Mechanism Research
Through the analysis and detection of circulating DNA (ctDNA) in blood, early diagnosis of tumors, mutation genotyping, and disease progression monitoring can be achieved. This helps guide personalized treatment plans and assess treatment efficacy, thereby improving patient survival rates and quality of life. Research on tumor metastasis mechanisms aids in understanding the occurrence, development, and metastasis of tumors, providing a deeper theoretical foundation and new therapeutic targets for cancer treatment.
1.1 High-Density Digital PCR Chip for Tumor Liquid Biopsy
The low abundance of ctDNA in blood (ng/mL) and the high background of wild-type genes pose significant challenges for non-invasive lung cancer genotyping. To enhance the sensitivity and specificity of ctDNA mutation detection, an embedded microchamber arrangement was proposed, leading to the design of a high-density microchamber (7,000/cm²) digital PCR chip. This chip integrates 120,000 microchambers on a single chip, enabling the detection of 10-10⁷ copies/mL DNA molecules. Based on this chip, a single-molecule DNA detection technique was developed, encapsulating mutant and wild-type genes in separate microchambers, eliminating the interference of wild-type gene amplification on mutant gene detection, and reducing the detection limit of mutant genes from 1% to 0.01% compared to conventional methods. Clinical results from blood samples of lung cancer patients showed that the developed single-molecule mutation genotyping method achieved a sensitivity of 85.71% and 100% and a specificity of 94.44% and 86.96% for detecting EGFR mutations L858R and T790M resistance mutations in lung cancer, respectively.
1.2 Generation of Solidifiable Droplet Oil and Detection of Single-Cell Tumor Metastasis Protease Activity
Droplet microfluidics offer excellent flexibility and analysis throughput, showing tremendous potential in digital nucleic acid detection and high-throughput single-cell analysis. However, droplet fusion and movement make it impossible to track biochemical reactions and single-cell dynamics within individual droplets in real time. To address the issues of droplet fusion and movement, a strategy of oil-phase solidification was proposed, leading to the development of solidifiable droplet generation oil based on hydrosilylation reactions (patented as CN108543504B and CN108545692B). This oil phase was used to generate high-density static droplet arrays (18,000/cm²), achieving nearly 100% effective droplet count after PCR reactions and enabling real-time droplet digital PCR (Figures a and b). The solidifiable oil combines the high throughput and flexibility of droplet microfluidics with the stability and addressability of microchambers.
To deeply understand the relationship between single-cell MMP-9 activity and tumor metastasis, high-throughput single-cell dynamic enzyme activity analysis methods were established using solidifiable oil to generate static droplet arrays encapsulating individual tumor cells. This enabled the study of time-resolved single-cell MMP-9 activity (Figure b). The research quantitatively revealed the heterogeneity of MMP-9 expression within tumor cell populations and discovered the temporal regulation characteristics of MMP-9 activity. This enhanced the understanding of tumor metastasis mechanisms, providing new perspectives and research techniques for tumor metastasis studies
2. Lable-free pathogen isolation
Two major challenges in rapid pathogen detection are the complex separation of pathogens and the time-consuming detection of viable bacteria (culture method - 24h). Membrane filtration is the most commonly used method for label-free pathogen isolation in water samples. Due to membrane adhesion, the recovery of pathogens using membrane filtration can result in sample loss. Additionally, this method cannot remove solid particles from the sample, which may adversely affect subsequent detection. To reduce the loss rate of membrane filtration, an inert microfluidic channel capable of generating Dean vortices was introduced based on membrane filtration. This design keeps pathogens in suspension, reduces bacterial adhesion, and increases the bacterial recovery rate from 60% to 95% (Figure a).
To remove solid particles from samples, a principle of label-free, membrane-free pathogen isolation based on microfluidics was studied. By constructing a dual-layer microchannel hydrodynamic model with microstructures, the particle sorting mechanism within the channel was investigated. A particle sorting chip with dual-layer microstructures was designed, effectively separating particles larger than 5 µm (Figure b). A numerical model involving laminar flow, mass transport, and particle tracking was established, revealing the phenomenon of sheath flow interface deformation in straight channels with specific aspect ratios. This deformation causes micro-particles of different sizes to migrate directionally and aggregate within the channel (Figure c). Based on this, a sheath flow deformation microfluidic chip was designed to achieve label-free bacterial isolation in a straight channel. This method does not affect bacterial viability and can directly integrate with downstream detection.
3. Rapid Detection of Environmental Pathogens
Pathogen detection based on bacterial culture takes 24 hours, whereas rapid detection methods such as PCR and immunolabeling cannot differentiate between live and dead bacteria. Therefore, these methods cannot provide timely and reliable warnings of bacterial contamination in drinking water, leading to unnecessary economic losses. To achieve rapid detection of live bacteria, a 3D expansion strategy for microchamber arrays was first proposed. A chip with a vertically distributed microchamber array was designed, increasing the density of microchambers from 7,000/cm² in 2D arrays to 25,000/cm² in 3D arrays.
Next, the expression mechanism of the E. coli-specific metabolic enzyme glucuronidase was studied. By combining metabolic enzyme activity detection with the microarray chip, a digital live bacteria quantification technique was developed. This technique allows for the identification and quantification of live E. coli within 3.5 hours, significantly reducing the time required for standard E. coli detection. The ability to distinguish the activity status of bacteria is not achievable by nucleic acid detection and antibody labeling methods. The digital live bacteria quantification technique is not affected by water turbidity or environmental temperature and can determine the number of live bacteria in water with different pH levels. It can also assess the disinfecting effectiveness of various doses of chloramine.
4. Multidimensional Analysis of Bacterial Infections
Currently, clinical microbiology laboratories typically use a series of sequential tests to detect the presence of urinary tract infection pathogens, identify the species of pathogens, and analyze antibiotic sensitivity. This entire process takes 4-5 days and is complex to perform. This method fails to assist doctors in formulating personalized antibiotic treatment plans during a patient's first visit. Therefore, there is an urgent need for detection technology that can directly perform multidimensional analysis of pathogens in samples. This technology would be used for diagnosing bacterial infections, guiding personalized antibiotic treatments, and curbing the further development of bacterial resistance.
4. Novel Pathogen-specific Antibiotic Sensitivity Testing Markers
Phenotypic markers used for antibiotic sensitivity testing (AST) mostly lack pathogen specificity. Using these markers to directly perform AST on pathogens in samples results in data that can be influenced by commensal bacteria or bacterial concentrations, leading to inaccurate assessments of pathogen resistance. Research and discovery of novel pathogen-specific AST markers are of significant importance for achieving rapid and accurate detection of urinary pathogens. By systematically studying the response patterns of specific inducible enzyme activities in resistant and sensitive bacteria to different types of antibiotics, a correlation between specific inducible enzyme activity and antibiotic sensitivity was identified. The studies show that the inducible enzyme activity in bacteria can respond to different types of antibiotics, with response times as short as 25 minutes (see Figures a and b). AST results at varying bacterial concentrations indicate that AST test outcomes based on inducible enzyme activity are not affected by bacterial concentration (see Figure c). This feature is crucial for accurately determining the sensitivity of samples with unknown concentrations. These findings suggest that pathogen-specific inducible enzyme activity can serve as an AST marker. This discovery provides a new approach for developing direct AST technologies for pathogens in samples, facilitating rapid and accurate pathogen AST.
4. On-Site Multidimensional Analysis of Bacterial Infections
Current AST technologies are complex to operate and unsuitable for immediate on-site detection or use in primary healthcare facilities. To enable on-site AST, a ready-to-use AST chip featuring a synchronized liquid dispenser and a 3D staggered microchamber array was designed. Users only need to add the sample to the chip to initiate the AST test. This chip is driven by negative pressure, eliminating the need for external equipment to assist with sample loading and reducing AST's dependency on external devices. The reagents required for AST are pre-loaded into the 3D microchamber array, and the chip’s synchronized liquid dispenser and 3D channel network ensure that AST reagents do not leak during sample loading.
By combining specific inducible enzyme activity detection with the 3D microchamber array, an all-in-one multidimensional pathogen AST method was developed. This method allows for pathogen identification, quantification, and antibiotic sensitivity analysis within 4.5 hours, significantly shorter than the ~3 days required by traditional methods. This approach achieves rapid multidimensional bacterial infection detection with a single experiment, significantly simplifying the AST process, reducing the diagnosis time for urinary tract infections, and lowering the associated costs. It also provides a technological guarantee for promoting evidence-based antibiotic therapy.
