Rapid single-molecule digital detection of protein biomarkers for continuous monitoring of systemic immune disorders

Abstract

Digital protein assays have great potential to advance immunodiagnostics because of their single-molecule sensitivity, high precision, and robust measurements. However, translating digital protein assays to acute clinical care has been challenging because it requires their deployment with a rapid turnaround. Herein, we present a technology platform for ultra-fast digital protein biomarker detection by employing single-molecule counting of immune-complex formation events at an early, pre-equilibrium state. This method, which we term "pre-equilibrium digital enzyme-linked immunosorbent assay" (PEdELISA), can quantify a multiplexed panel of protein biomarkers in 10 µL of serum within an unprecedented assay incubation time of 15-300 sec over a 104 dynamic range. PEdELISA allowed us to perform rapid monitoring of protein biomarkers in patients manifesting post-chimeric antigen receptor T-cell (CAR-T) therapy cytokine release syndrome (CRS), with ~30 min sample-to-answer time and a sub-pg/mL limit of detection (LOD). The rapid, sensitive, and low input volume biomarker quantification enabled by PEdELISA is broadly applicable to timely monitoring of acute disease, potentially enabling more personalized treatment.

Ultrasensitive Multiparameter Phenotyping of Rare Cells Using an Integrated Digital‐Molecular‐Counting Microfluidic Well Plate

“…A correlation between the reaction time and sensitivity of the digital immunosensors has been extensively characterized in our previous work. [ 41 ] Our digital immunosensor‐based TNF‐α assay achieves a LOD as small as 0.25 pg mL −1 , and its data correlate well with results of the gold standard ELISA. The high sensitivity and large dynamic range of the digital immunosensors permitted the assay with the number of RAW 264.7 cells ranging from 17 to 610 in each assay chamber.…”

“…The weak signal intensity could yield counting error with a conventional image processing algorithm, we therefore used a modified version of our machine learning code developed in our previous work. [ 41–43 ] We observed almost no fluorescent background (≈0.1%) in all of Aβ‐free samples whether or not LPS presented in them. The fraction of phagocytosis‐active cells steadily increased from 11.9% to 55.0% with Aβ increasing from 0.25 µм to 1 µм.…”

“…Finally, 40 µL of the QuantaRed (Qred) substrate solution was loaded to the bead channels followed by sealing and isolating the bead microwells with HFE‐7500 fluorinated oil (45 µL) for the digital counting process on a motorized microscope scanning system reported in our previous works. [ 41–43 ] The system scans the image of the bead‐filled microwell arrays on the chip right after the oil sealing step to detect the enzyme–substrate reaction activity. The motorized stage was pre‐programmed to follow the designated path to scan the entire dPP (48 cell microarrays or 48 bead arrays).…”

“…Herein, we propose an ultrasensitive multiparameter cellular phenotyping method employing digital molecular counting immunosensors [ 41–43 ] integrated into a microfluidic platform named the “digital Phenoplate (dPP).” The dPP enables analyzing cellular phenotypes associated with cytokine secretion and other cellular functions (e.g., phagocytic activity and cell death) that can be simultaneously observed by digital protein immunosensing and on‐chip image‐based cytometry. The integrated digital immunosensors preserve analyte concentration‐signal linearity even at a sub‐pg mL −1 level and achieve a limit of detection of 0.25 pg mL −1 for mouse tumor necrosis factor alpha (TNF‐α), which allows for testing fewer than 20 cells per assay.…”

Machine-Learning-Assisted Microfluidic Nanoplasmonic Digital Immunoassay for Cytokine Storm Profiling in COVID-19 Patients

“…Over the past two decades, the microfluidic immunoassay has become an emerging technology for rapid analysis of biomolecules in complex biological samples. − Microfluidics offer significant advantages in controllable fluid handling, low reagent consumption, and confined microenvironment analysis. − The integration of immunoassays to the microfluidic scale shows greatly improved analytical performance at point-of-care, such as reduced assay time, small sample volume, high throughput and multiplexity, and semi-automation. − Recent advancements in a variety of microfluidic immunoassays have demonstrated promising features for cytokine detection, including a sample-to-answer time shortened to ∼30 min, a sample volume reduced to a few μL, a throughput improved to hundreds of parallel tests, a multiplex capacity up to dozens of targets, and so on. − However, accumulating evidence suggests that the cytokine concentrations in plasma of COVID-19 patients span across a wide dynamic range (1–40,000 pg mL –1 ) with a few key inflammatory cytokines at the sub-pg mL –1 level . Current microfluidic immunoassays rely mainly on conventional signal transduction technologies based on measurement of ensemble average signals, which often require numerous captured signaling molecules to generate a detectable signal over background noise.…”