Massively parallel quantification of phenotypic heterogeneity in single-cell drug responses.

Single-cell analysis tools have made substantial advances in characterizing genomic heterogeneity; however, tools for measuring phenotypic heterogeneity have lagged due to the increased difficulty of handling live biology. Here, we report a single-cell phenotyping tool capable of measuring image-based clonal properties at scales approaching 100,000 clones per experiment. These advances are achieved by exploiting a previously unidentified flow regime in ladder microfluidic networks that, under appropriate conditions, yield a mathematically perfect cell trap. Machine learning and computer vision tools are used to control the imaging hardware and analyze the cellular phenotypic parameters within these images. Using this platform, we quantified the responses of tens of thousands of single cell­derived acute myeloid leukemia (AML) clones to targeted therapy, identifying rare resistance and morphological phenotypes at frequencies down to 0.05%. This approach can be extended to higher-level cellular architectures such as cell pairs and organoids and on-chip live-cell fluorescence assays.

Rapid capture of biomolecules from blood via stimuli-responsive elastomeric particles for acoustofluidic separation.

The detection of biomarkers in blood often requires extensive and time-consuming sample preparation to remove blood cells and concentrate the biomarker(s) of interest. We demonstrate proof-of-concept for a chip-based, acoustofluidic method that enables the rapid capture and isolation of a model protein biomarker (i.e., streptavidin) from blood for off-chip quantification. Our approach makes use of two key components - namely, soluble, thermally responsive polypeptides fused to ligands for the homogeneous capture of biomarkers from whole blood and silicone microparticles functionalized with similar, tethered, thermally responsive polypeptides. When the two components are mixed together and subjected to a mild thermal trigger, the thermally responsive moieties undergo a phase transition, causing the untethered (soluble) polypeptides to co-aggregate with the particle-bound polypeptides. The mixture is then diluted with warm buffer and injected into a microfluidic channel supporting a bulk acoustic standing wave. The biomarker-bearing particles migrate to the pressure antinodes, whereas blood cells migrate to the pressure node, leading to rapid separation with efficiencies exceeding 90% in a single pass. The biomarker-bearing particles can then be analyzed via flow cytometry, with a limit of detection of 0.75 nM for streptavidin spiked in blood plasma. Finally, by cooling the solution below the solubility temperature of the polypeptides, greater than 75% of the streptavidin is released from the microparticles, offering a unique approach for downstream analysis (e.g., sequencing or structural analysis). Overall, this methodology has promise for the detection, enrichment and analysis of some biomarkers from blood and other complex biological samples.

Injection Molded Microfluidics for Establishing High-Density Single Cell Arrays in an Open Hydrogel Format.

Here, we develop an injection molded microfluidic approach for single cell analysis by making use of (1) rapidly curing injectable hydrogels, (2) a high density microfluidic weir trap array, and (3) reversibly bonded PDMS lids that are strong enough to withstand the injection molding process, but which can be peeled off after the hydrogel sets. This approach allows for single cell patterns to be created with densities exceeding 40 cells per mm2, is amenable to high speed imaging, and creates microfluidic devices that enable efficient nutrient and gas exchange and the delivery of specific biological and chemical reagents to individual cells. We show that it is possible to organize up to 10 000 single cells in a few minutes on the device, and we developed an image analysis program to automatically analyze the single-cell capture efficiency. The results show single cell trapping rates were better than 80%. We also demonstrate that the genomic DNA of the single cells trapped in the hydrogel can be amplified via localized, multiple displacement amplification in a massively parallel format, which offers a promising strategy for analyzing single cell genomes. Finally, we show the ability to perform selective staining of individual cells with a commercial bioprinter, providing proof of concept of its ability to deliver tailored reagents to specific cells in an array for future downstream analysis. This injection molded microfluidic approach leverages the benefits of both closed and open microfluidics, allows multiday single cell cultures, direct access to the trapped cells for genotypic end point studies.

An acoustofluidic trap and transfer approach for organizing a high density single cell array.

We demonstrate a hybrid microfluidic system that combines fluidic trapping and acoustic switching to organize an array of single cells at high density. The fluidic trapping step is achieved by balancing the hydrodynamic resistances of three parallel channel segments forming a microfluidic trifurcation, the purpose of which was to capture single cells in a high-density array. Next, the cells were transferred into adjacent larger compartments by generating an array of streaming micro-vortices to move the cells to the desired streamlines in a massively parallel format. This approach can compartmentalize single cells with efficiencies of ≈67% in compartments that have diameters on the order of ∼100 um, which is an appropriate size for single cell proliferation studies and other single cell biochemical measurements.

Poly(oligo(ethylene glycol) methyl ether methacrylate) Brushes on High-κ Metal Oxide Dielectric Surfaces for Bioelectrical Environments.

Advances in electronics and life sciences have generated interest in "lab-on-a-chip" systems utilizing complementary metal oxide semiconductor (CMOS) circuitry for low-power, portable, and cost-effective biosensing platforms. Here, we present a simple and reliable approach for coating "high-κ" metal oxide dielectric materials with "non-fouling" (protein- and cell-resistant) poly(oligo(ethylene glycol) methyl ether methacrylate (POEGMA) polymer brushes as biointerfacial coatings to improve their relevance for biosensing applications utilizing advanced electronic components. By using a surface-initiated "grafting from" strategy, POEGMA films were reliably grown on each material, as confirmed by ellipsometric measurements and X-ray photoelectron spectroscopy (XPS) analysis. The electrical behavior of these POEGMA films was also studied to determine the potential impact on surrounding electronic devices, yielding information on relative permittivity and breakdown field for POEGMA in both dry and hydrated states. We show that the incorporation of POEGMA coatings significantly reduced levels of nonspecific protein adsorption compared to uncoated high-κ dielectric oxide surfaces as shown by protein resistance assays. These attributes, combined with the robust dielectric properties of POEGMA brushes on high-κ surfaces open the way to incorporate this protein and cell resistant polymer interface into CMOS devices for biomolecular detection in a complex liquid milieu.

Magnetic separation of acoustically focused cancer cells from blood for magnetographic templating and analysis.

Liquid biopsies hold enormous promise for the next generation of medical diagnoses. At the forefront of this effort, many are seeking to capture, enumerate and analyze circulating tumor cells (CTCs) as a means to prognosticate and develop individualized treatments for cancer. Capturing these rare cells, however, represents a major engineering challenge due to their low abundance, morphology and heterogeneity. A variety of microfluidic tools have been developed to isolate CTCs from drawn blood samples; however, few of these approaches offer a means to separate and analyze cells in an integrated system. We have developed a microfluidic platform comprised of three modules that offers high throughput separation of cancer cells from blood and on-chip organization of those cells for streamlined analyses. The first module uses an acoustic standing wave to rapidly align cells in a contact-free manner. The second module then separates magnetically labeled cells from unlabeled cells, offering purities exceeding 85% for cells and 90% for binary mixtures of synthetic particles. Finally, the third module contains a spatially periodic array of microwells with underlying micromagnets to capture individual cells for on-chip analyses (e.g., staining, imaging and quantification). This array is capable of capturing with accuracies exceeding 80% for magnetically labeled cells and 95% for magnetic particles. Overall, by virtue of its holistic processing of complex biological samples, this system has promise for the isolation and evaluation of rare cancer cells and can be readily extended to address a variety of applications across single cell biology and immunology.

Validation of high gradient magnetic field based drug delivery to magnetizable implants under flow.

The drug-eluting stent's increasingly frequent occurrence late stage thrombosis have created a need for new strategies for intervention in coronary artery disease. This paper demonstrates further development of our minimally invasive, targeted drug delivery system that uses induced magnetism to administer repeatable and patient specific dosages of therapeutic agents to specific sites in the human body. Our first aim is the use of magnetizable stents for the prevention and treatment of coronary restenosis; however, future applications include the targeting of tumors, vascular defects, and other localized pathologies. Future doses can be administered to the same site by intravenous injection. This implant-based drug delivery system functions by placement of a weakly magnetizable stent or implant at precise locations in the cardiovascular system, followed by the delivery of magnetically susceptible drug carriers. The stents are capable of applying high local magnetic field gradients within the body, while only exposing the body to a modest external field. The local gradients created within the blood vessel create the forces needed to attract and hold drug-containing magnetic nanoparticles at the implant site. Once these particles are captured, they are capable of delivering therapeutic agents such as antineoplastics, radioactivity, or biological cells.