ABSTRACT
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.
Subject(s)
Leukocytes , Biomarkers , HumansABSTRACT
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.
Subject(s)
Immunity , Microfluidics , Biomarkers , HumansABSTRACT
Polydimethylsiloxane (PDMS) is a common biomaterial in the fabrication of microfluidic devices due to its various beneficial properties. However, its inherent highly hydrophobic surfaces often limit PDMS-based microfluidic devices for myocardial differentiation of pluripotent stem cells. In order to improve cell interactive properties of PDMS, gelatin-dopamine was synthesized by grafting dopamine onto a gelatin backbone. The conjugation of dopamine onto gelatin was verified using proton nuclear magnetic resonance spectroscopy (1H NMR). Compared with gelatin coating, coating with gelatin-dopamine greatly reduced the water contact angle of the PDMS substrates and resulted in higher protein adsorption on the PDMS surfaces. In addition, the gelatin-dopamine coated PDMS surfaces also improved the embryonic stem cell (ESC) adhesion, proliferation and differentiation as compared to gelatin modified surfaces. The effects of gelatin-dopamine coating on the ESC-derived embryoid body (EB), myocardial differentiation on PDMS surfaces were also investigated. Compared with the PDMS surface coated with gelatin, the EB spread-out areas and myocardial differentiation efficiency greatly increased on the gelatin-dopamine coated PDMS surface. Interestingly, the lower concentration of gelatin-dopamine coating was more favorable for ESC pluripotent maintenance, while the higher concentration was more likely to support ESC myocardial differentiation. These results indicate that gelatin-dopamine is an effective coating material to improve ESC long-term culture and myocardial differentiation on PDMS substrates.
