Cytokines, crucial in immune modulation, impact disease progression when their secretion is dysregulated. Existing methods for profiling cytokine secretion suffer from time-consuming and labor-intensive processes and often fail to capture the dynamic nature of immune responses. Here, iSECRETE, an integrated platform that enables synchronous cell activation, wash-free, and target-responsive protein detection for single-cell IFN-γ cytokine secretion analysis within 30 min at room temperature is presented. By incorporating a DNA proximity assay (DPA) into a multifunctional microfluidic system, one-pot homogenous cytokine signal amplification, with a limit of detection of ≈50 secreted molecules per cell is achieved. iSECRETE can robustly handle various sample types that are shown. Two distinct immune activation assay modalities are demonstrated on iSECRETE. Finally, the detection of single-cell IFN-γ secretion as an activation hallmark of chimeric antigen receptor T cells within 6 h of exposure to cancer targets is shown. iSECRETE represents the fastest single-cell sample-to-result cytokine secretion assay to date, providing a powerful tool for advancing the understanding of biological phenotypes, functions, and pathways under in vivo-like conditions.

Host immune response plays a critical role in disease manifestation and severity. In article number 2006123, Win Sen Kuan, Jongyoon Han, and co-workers present a whole blood biophysical immune response profiling tool using deterministic lateral displacement microfluidics. The speed and accuracy of immune response stratification demonstrated in an emergency department has a clear impact in enabling deployable point-of-care systems for rapid clinical triage.

Disease manifestation and severity from acute infections are often due to hyper-aggressive host immune responses which change within minutes. Current methods for early diagnosis of infections focus on detecting low abundance pathogens, which are time-consuming, of low sensitivity, and do not reflect the severity of the pathophysiology appropriately. The approach here focuses on profiling the rapidly changing host inflammatory response, which in its over-exuberant state, leads to sepsis and death. A 15-min label-free immune profiling assay from 20 µL of unprocessed blood using unconventional L and Inverse-L shaped pillars of deterministic lateral displacement microfluidic technology is developed. The hydrodynamic interactions of deformable immune cells enable simultaneous sorting and immune response profiling in whole blood. Preliminary clinical study of 85 donors in emergency department with a spectrum of immune response states from healthy to severe inflammatory response shows correlation with biophysical markers of immune cell size, deformability, distribution, and cell counts. The speed of patient stratification demonstrated here has promising impact in deployable point-of-care systems for acute infections triage, risk management, and resource allocation at emergency departments, where clinical manifestation of infection severity may not be clinically evident as compared to inpatients in the wards or intensive care units.

Enzymatic secretion of immune cells (leukocytes) plays a dominant role in host immune responses to a myriad of biological triggers, including infections, cancers, and cardiovascular diseases. Current tools to probe these leukocytes inadequately profile these vital biomarkers; the need for sample preprocessing steps of cell lysis, labeling, washing, and pipetting inevitably triggers the cells, changes its basal state, and dilutes the individual cell secretion in bulk assays. Using a fully integrated system for multiplexed profiling of native immune single-cell enzyme secretion from 50 μL of undiluted blood, we eliminate sample handling. With a total analysis time of 60 min, the integrated platform performs six tasks of leukocyte extraction, cell washing, fluorescent enzyme substrate mixing, single-cell droplet making, droplet incubation, and real-time readout for leukocyte secretion profiling of neutrophil elastase, granzyme B, and metalloproteinase. We calibrated the device, optimized the protocols, and tested the leukocyte secretion of acute heart failure (AHF) patients at admission and predischarge. This paper highlights the presence of single-cell enzymatic immune phenotypes independent of CD marker labeling, which could potentially elucidate the innate immune response states. We found that patients recovering from AHF showed a corresponding reduction in immune-cell enzymatic secretion levels and donor-specific enzymatic signatures were observed, which suggests patient-to-patient heterogeneous immune response. This platform presents opportunities to elucidate the complexities of the immune response from a single drop of blood and bridge the current technological, biological, and medical gap in understanding immune response and biological triggers.