Jose L. Garcia-Cordero

LSPR Chip for Parallel, Rapid, and Sensitive Detection of Cancer Markers in Serum

Label-free biosensing based on metallic nanoparticles supporting localized surface plasmon resonances (LSPR) has recently received growing interest (Anker, J. N., et al. Nat. Mater. 2008, 7, 442-453). Besides its competitive sensitivity (Yonzon, C. R., et al. J. Am. Chem. Soc. 2004, 126, 12669-12676; Svendendahl, M., et al. Nano Lett. 2009, 9, 4428-4433) when compared to the surface plasmon resonance (SPR) approach based on extended metal films, LSPR biosensing features a high-end miniaturization potential and a significant reduction of the interrogation device bulkiness, positioning itself as a promising candidate for point-of-care diagnostic and field applications. Here, we present the first, paralleled LSPR lab-on-a-chip realization that goes well beyond the state-of-the-art, by uniting the latest advances in plasmonics, nanofabrication, microfluidics, and surface chemistry. Our system offers parallel, real-time inspection of 32 sensing sites distributed across 8 independent microfluidic channels with very high reproducibility/repeatability. This enables us to test various sensing strategies for the detection of biomolecules. In particular we demonstrate the fast detection of relevant cancer biomarkers (human alpha-feto-protein and prostate specific antigen) down to concentrations of 500 pg/mL in a complex matrix consisting of 50% human serum.

A high-throughput nanoimmunoassay chip applied to large-scale vaccine adjuvant screening

Large-scale experimentation is becoming instrumental in enabling new discoveries in systems biology and personalized medicine. We developed a multiplexed high-throughput nanoimmunoassay chip capable of quantifying four biomarkers in 384 5 nL samples, for a total of 1536 assays. Our platform, compared to conventional methods, reduces volume and reagent cost by ~1000-fold. We applied our platform in the context of systems vaccinology, to assess the synergistic production of inflammatory cytokines from dendritic cells (DCs) stimulated with 10 different adjuvants that target members of the Toll-like receptor (TLR) family. We quantified these adjuvants both alone and in all pairwise combinations, for a total of 435 conditions, revealing numerous synergistic pairs. We evaluated two synergistic interactions, MPLA + Gardiquimod and MPLA + CpG-B, in a mouse model, where we measured the same inflammatory cytokines in bronchoalveolar lavage and in blood serum at 4 different time points using our chip, and observed similar synergistic effects in vivo, demonstrating the potential of our microfluidic platform to predict agonistic immunogenicity. More generally, a high-throughput, matrix-insensitive, low sample volume technology can play an important role in the discovery of novel therapeutics and research areas requiring large-scale biomarker quantitation.

A Microfluidic Platform for High-Throughput Multiplexed Protein Quantitation

We present a high-throughput microfluidic platform capable of quantitating up to 384 biomarkers in 4 distinct samples by immunoassay. The microfluidic device contains 384 unit cells, which can be individually programmed with pairs of capture and detection antibody. Samples are quantitated in each unit cell by four independent MITOMI detection areas, allowing four samples to be analyzed in parallel for a total of 1,536 assays per device. We show that the device can be pre-assembled and stored for weeks at elevated temperature and we performed proof-of-concept experiments simultaneously quantitating IL-6, IL-1β, TNF-α, PSA, and GFP. Finally, we show that the platform can be used to identify functional antibody combinations by screening 64 antibody combinations requiring up to 384 unique assays per device.

Multiplexed surface micropatterning of proteins with a pressure-modulated microfluidic button-membrane

We report the flow-based in situ patterning of multiple proteins in microfluidic channels by simply tuning the actuation pressure of a microfluidic button-membrane. We show that a single button-membrane can pattern several concentric protein annuli, and we apply this patterning approach to a multiplexed immunoassay.

Massive Parallel Analysis of Single Cells in an Integrated Microfluidic Platform

New tools that facilitate the study of cell-to-cell variability could help uncover novel cellular regulation mechanisms. We present an integrated microfluidic platform to analyze a large number of single cells in parallel. To isolate and analyze thousands of individual cells in multiplexed conditions, our platform incorporates arrays of microwells (7 pL each) in a multilayered microfluidic device. The device allows the simultaneous loading of cells into 16 separate chambers, each containing 4640 microwells, for a total of 74u202f240 wells per device. We characterized different parameters important for the operation of the microfluidic device including flow rate, solution exchange rate in a microchamber, shear stress, and time to fill up a single microwell with molecules of different molecular weight. In general, after ∼7.5 min of cell loading our device has an 80% microwell occupancy with 1-4 cells, of which 36% of wells contained a single cell. To test the functionality of our device, we carried out a cell viability assay with adherent and nonadherent cells. We also studied the production of neutrophil extracellular traps (NETs) from single neutrophils isolated from peripheral blood, observing the existence of temporal heterogeneity in NETs production, perhaps having implications in the type of the neutrophil response to an infection or inflammation. We foresee our platform will have a variety of applications in drug discovery and cellular biology by facilitating the characterization of phenotypic differences in a monoclonal cell population.

Mechanically Induced Trapping of Molecular Interactions and Its Applications

Measuring binding affinities and association/dissociation rates of molecular interactions is important for a quantitative understanding of cellular mechanisms. Many low-throughput methods have been developed throughout the years to obtain these parameters. Acquiring data with higher accuracy and throughput is, however, necessary to characterize complex biological networks. Here, we provide an overview of a high-throughput microfluidic method based on mechanically induced trapping of molecular interactions (MITOMI). MITOMI can be used to obtain affinity constants and kinetic rates of hundreds of protein–ligand interactions in parallel. It has been used in dozens of studies to measure binding affinities of transcription factors, map protein interaction networks, identify pharmacological inhibitors, and perform high-throughput, low-cost molecular diagnostics. This article covers the technological aspects of MITOMI and its applications.

A low-cost 3-D printed stethoscope connected to a smartphone

We demonstrate the fabrication of a digital stethoscope using a 3D printer and commercial off-the-shelf electronics. A chestpiece consists of an electret microphone embedded into the drum of a 3D printed chestpiece. An electronic dongle amplifies the signal from the microphone and reduces any external noise. It also adjusts the signal to the voltages accepted by the smartphones headset jack. A graphical user interface programmed in Android displays the signals processed by the dongle. The application also saves the processed signal and sends it to a physician.We demonstrate the fabrication of a digital stethoscope using a 3D printer and commercial off-the-shelf electronics. A chestpiece consists of an electret microphone embedded into the drum of a 3D printed chestpiece. An electronic dongle amplifies the signal from the microphone and reduces any external noise. It also adjusts the signal to the voltages accepted by the smartphones headset jack. A graphical user interface programmed in Android displays the signals processed by the dongle. The application also saves the processed signal and sends it to a physician.