Siyi Hu

Antigen-specific T cells constitute a low percentage of peripheral blood mononuclear cells (PBMCs) and require antigen presentation by autologous antigen-presenting cells for activation and cytokine secretion. Conventional interferon-γ release assay (IGRA) averages rare activation events beyond cellular resolution, while single-cell compartmentalization disrupts the intracellular synapses for T cell immunity. Here, we developed mSTAR (matrix-based Specific T-cell Activation Reporter), an active-matrix digital microfluidic platform that partitions microliters of PBMCs into a nanoliter-scale matrix of 16 cell microcommunities (CMCs), each retaining tens to hundreds of PBMCs in their native heterotypic composition to preserve the natural physiological immune response. In a proof of concept, mSTAR executes parallel IGRA for a tuberculosis disease model, including CMC stimulation and magnetic-bead-based immunofluorescence assay. The IGRA of the mSTAR system achieves a limit of detection of 3.70 pg/mL, yielding 16 interferon-γ quantification readouts and ratios between CMCs incubated with different stimulants (negative, positive, or specific antigen). This enables the mapping and reporting of activated antigen-specific T cells in the matrix, thereby achieving functional phenotyping of each CMC. Poisson-based modeling of the droplet matrix converts rare T-cell activation events into a nearly digital estimation, and the positive CMCs encapsulating target cells can be retrieved for further characterization without loss. By bridging functional phenotyping and molecular features without prior cell sorting or engineering, mSTAR provides a programmable immunoanalytical infrastructure for broad downstream applications such as antigen-specific immunotherapy development or T-cell receptor discovery.

High-risk human papillomavirus (HPV) genotyping is essential for risk-stratified cervical cancer screening. However, current laboratory workflows remain multistep, instrument-intensive, and difficult to scale up for high-throughput multiplex testing. Here we present an active-matrix digital microfluidic (AM-DMF) platform for high-throughput HPV genotyping, utilizing a tailing-actuated interfacial liquid mixing (TAIL-Mix) strategy to enable highly efficient nucleic acid extraction and robust downstream amplification. The AM-DMF chip features a 921,600-electrode array that uses programmable, time-sequenced electrode actuation to stretch and retract droplets. This mechanism generates TAIL-Mix, significantly enhancing mixing efficiency under low-Reynolds-number conditions. Through combined COMSOL simulations and OpenCV-based image analysis, we determined that optimal TAIL-Mix parameters (a tailing distance of 7–8 pixels; 1.5 steps s–1) achieve a homogeneous state (mixing index ≥ 0.9) within 2 s, superior to traditional schemes. This enhanced mixing proved to be critical for high-performance processing, enabling magnetic-bead DNA extraction with 75% DNA recovery while reducing the protocol time to 26 min and the sample input to 1.5 μL of sample (100-fold reduction compared with conventional protocols). By integrating TAIL-Mix-enhanced extraction, thermal cycling, and 21-plex end point fluorescence readout on a single chip, the platform successfully performs parallel 21-genotype PCR analysis for two samples. In a clinical cohort of 20 cervical samples, the AM-DMF assay shows 100% sensitivity and 100% specificity relative to qPCR. Ultimately, this TAIL-Mix-empowered AM-DMF system offers a robust solution for low-reagent consumption, high-throughput clinical diagnostics.

Despite recent advances in single-cell proteomics, a key challenge remains achieving rapid, low-background sample preparation that is sufficiently robust for complex biological samples. Here, we present AM-DMF-SCP-pro, a streamlined active-matrix digital microfluidic workflow that enables on-chip lysis and digestion in 6 nL droplets within a closed nanolitre-confined microenvironment, while reducing reagent consumption by over 100-fold. AM-DMF-SCP-pro enables reproducible single-cell proteome profiling while shortening total sample-preparation time to as little as 30 min, including a 15 min digestion step, and supports deeper, more sensitive and lower-background measurements from ultra-low peptide inputs. In murine tumors, AM-DMF-SCP-pro resolved immune and tumor compartments, identified treatment-associated tumor cell states and enabled quantitative analysis of neutrophil-associated proteomic signatures. Together, these results establish AM-DMF-SCP-pro as a practical framework for rapid single-cell proteome profiling and tumor microenvironment analysis in complex biological samples.

In this work, we developed an AM-DMF-based pyrosequencing platform for rapid and automated SNP detection, representing its first application in pharmacogenomic analysis. This approach simplifies the workflow, reduces costs, and enhances automation.

Cancer is a leading global cause of morbidity and mortality, with inter- and intra-tumor heterogeneity contributing to suboptimal therapeutic outcomes. While genomic profiling has advanced precision oncology, many tumors lack targetable mutations, underscoring the need for functional assays. Here, we present a drug sensitivity testing platform for patient-derived organoids (PDOs) based on active-matrix digital microfluidics (AM-DMF). The system comprises a microfluidic timing-control board (71% success within 4-5 tearing cycles. Evaluation of cisplatin cytotoxicity across multiple PDOs on AM-DMF chips revealed time- and dose-dependent responses, including early morphological contraction (12 h) and subsequent structural disintegration (72 h) at high concentrations. Live/dead staining confirmed membrane damage under high-dose treatment, reflected by increased red fluorescence. Quantitative analysis demonstrated consistent dose-dependent inhibition of viability across all PDOs. Notably, on-chip measurements showed response trends consistent with conventional 96-well plate assays, yet with moderately higher inhibition rates, indicating enhanced assay sensitivity. Overall, we have developed an automated, high-throughput drug-testing system for PDOs, wherein the entire workflow, including organoid culture, precise drug concentration gradient generation, multi-dose drug screening, staining, and imaging, was seamlessly integrated and performed on a single active-matrix digital microfluidics chip. Collectively, this integrated AM-DMF platform offers a systematic, scalable strategy for functional precision medicine, especially suited to scarce clinical specimens and complex multidrug regimens.

Clinical adoption of metagenomic next‑generation sequencing (mNGS) is limited by costly, labor-intensive library preparation requiring substantial DNA input. Here, we present an active‑matrix digital microfluidics library preparation platform (AM‑DMF-Lib-prep) that overcomes these barriers by automating the “sample‑in, library‑out” workflow in an enclosed, digitalized lab-on-a-chip system. The platform accommodates a wide DNA input range (1–25 ng), producing high-quality libraries with exceptional reproducibility (inter-chip R² = 0.9998) and 25.6% lower variability than conventional manual methods. It achieves ~90% reagent reduction while enhancing detection sensitivity for low-abundance pathogens by 43.7–76.7% in microbial community standard spike-in sample. Validation across 94 diverse clinical specimens confirmed high concordance with reference diagnostics. Collectively, AM‑DMF-Lib-prep establishes a precise, cost‑effective, and scalable solution with demonstrated clinical readiness, directly addressing the pre‑analytical bottleneck in routine mNGS.

Cancer is a leading cause of morbidity and mortality worldwide, characterized by substantial inter-patient and intra-tumor heterogeneity that complicates treatment efficacy. While genomic profiling has advanced precision oncology, the prevalence of tumors lacking targetable mutations highlights the need for complementary functional assays. Here, we present a drug sensitivity testing platform based on active-matrix digital microfluidics (AM-DMF) integrated with patient-derived organoids (PDOs). We optimized protocols for sample loading, droplet generation and routing, and controlled drug exposure, achieving precise concentration gradients via iterative mix-split steps. By combining AM-DMF with PDOs, we enable automated liquid handling and high-content imaging to capture heterogeneous drug responses. This approach significantly enhances the efficiency, reproducibility, and sensitivity of drug testing-particularly for limited clinical samples and complex drug combinations. The platform provides an end-to-end workflow, from sample processing to quantitative response analysis, offering a systematic and scalable strategy for precision medicine. These findings establish an automatable, integrated pipeline for functional precision oncology, especially suited to scarce specimens and multi-drug regimens.

Understanding the correlation between interdot spacing and their interactions is crucial for controlling the properties of quantum dot superlattices. In this study, the interdot spacing of the colloidal quantum dot superlattice is tuned from 1.4 to 0.5 nm by varying the self-assembly solvents and the subsequent surface ligand engineering. Reducing the spacing to 1.0 nm or below induces a secondary narrow PL peak located ∼130 meV below the conventional emission peak. The new peak is attributed to quantum resonance, evidenced by the loss of size selectivity at different excitation wavelengths and a significant red-shift at room temperature. The photoluminescence associated with the quantum resonance remains stable in oxygen-free conditions and exhibits a lifetime that is six times longer than that of the weakly coupled state. The spacing control strategy for achieving quantum resonance can be applied to develop narrow-line-width emission devices based on nanocrystal superlattices.