Amphiphilic Particle-Stabilized Nanoliter Droplet Reactors with a Multimodal Portable Reader for Distributive Biomarker Quantification.

Compartmentalization, leveraging microfluidics, enables highly sensitive assays, but the requirement for significant infrastructure for their design, build, and operation limits access. Multimaterial particle-based technologies thermodynamically stabilize monodisperse droplets as individual reaction compartments with simple liquid handling steps, precluding the need for expensive microfluidic equipment. Here, we further improve the accessibility of this lab on a particle technology to resource-limited settings by combining this assay system with a portable multimodal reader, thus enabling nanoliter droplet assays in an accessible platform. We show the utility of this platform in measuring N-terminal propeptide B-type natriuretic peptide (NT-proBNP), a heart failure biomarker, in complex medium and patient samples. We report a limit of detection of ∼0.05 ng/mL and a linear response between 0.2 and 2 ng/mL in spiked plasma samples. We also show that, owing to the plurality of measurements per sample, "swarm" sensing acquires better statistical quantitation with a portable reader. Monte Carlo simulations show the increasing capability of this platform to differentiate between negative and positive samples, i.e., below or above the clinical cutoff for acute heart failure (∼0.1 ng/mL), as a function of the number of particles measured. Our platform measurements correlate with gold standard ELISA measurement in cardiac patient samples, and achieve lower variation in measurement across samples compared to the standard well plate-based ELISA. Thus, we show the capabilities of a cost-effective droplet-reader system in accurately measuring biomarkers in nanoliter droplets for diseases that disproportionately affect underserved communities in resource-limited settings.

Sensitizing drug-resistant cancer cells from blood using microfluidic electroporator.

Direct assessment of patient samples holds unprecedented potential in the treatment of cancer. Circulating tumor cells (CTCs) in liquid biopsies are a rapidly evolving source of primary cells in the clinic and are ideal candidates for functional assays to uncover real-time tumor information in real-time. However, a lack of routines allowing direct and active interrogation of CTCs directly from liquid biopsy samples represents a bottleneck for the translational use of liquid biopsies in clinical settings. To address this, we present a workflow for using a microfluidic vortex-assisted electroporation system designed for the functional assessment of CTCs purified from blood. Validation of this approach was assessed through drug response assays on wild-type (HCC827 wt) and gefitinib-resistant (HCC827 GR6) non-small cell lung cancer (NSCLC) cells. HCC827 cells trapped within microscale vortices were electroporated to sequentially deliver drug agents into the cytosol. Electroporation conditions facilitating multi-agent delivery were characterized for both cell lines using an automatic single-cell image fluorescence intensity algorithm. HCC827 GR6 cells spiked into the blood to emulate drug-resistant CTCs were able to be collected with high purity, demonstrating the ability of the device to minimize background cell impact for downstream sensitive cell assays. Using our proposed workflow, drug agent combinations to restore gefitinib sensitivity reflected the anticipated cytotoxic response. Taken together, these results represent a microfluidics multi-drug screening panel workflow that can enable functional interrogation of patient CTCs in situ, thereby accelerating the clinical standardization of liquid biopsies.

Engineering Design of Concentric Amphiphilic Microparticles for Spontaneous Formation of Picoliter to Nanoliter Droplet Volumes.

Simple mixing of aqueous and oil solutions with amphiphilic particles leads to the spontaneous formation of uniform reaction volumes (dropicles) that can enable numerous applications in the analysis of biological entities (e.g., cells and molecules). Approaches to manufacture such amphiphilic particles are just starting to be investigated. Here, we investigate the tunable manufacturing of concentric amphiphilic particles, with outer hydrophobic and inner hydrophilic layers, fabricated by flowing reactive precursor streams through a 3D printed device with coaxial microfluidic channels, and curing the structured flow by UV exposure through a photomask. The dimensions of the engineered amphiphilic particles, including height, inner and outer diameters, and thicknesses of the hydrophobic and hydrophilic layers, are precisely controlled by modulating the UV exposure time, the precursor flow rate ratios, and the size of the channel in the exposure region. The particle design is systematically engineered to hold a wide range of droplet volumes, that is, from a few hundred picoliters to several nanoliters. We show that the particle size can be significantly reduced from previous reports to not only hold subnanoliter drops but the shape can also be tuned to increase the seeding density and orientation of dropicles within a well plate for imaging and analysis.

A review of biosensor technologies for blood biomarkers toward monitoring cardiovascular diseases at the point-of-care.

Cardiovascular diseases (CVDs) cause significant mortality globally. Notably, CVDs disproportionately negatively impact underserved populations, such as those that are economically disadvantaged and often located in remote regions. Devices to measure cardiac biomarkers have traditionally been focused on large instruments in a central laboratory but the development of affordable, portable devices that measure multiple cardiac biomarkers at the point-of-care (POC) are needed to improve clinical outcomes for patients, especially in underserved populations. Considering the enormity of the global CVD problem, complexity of CVDs, and the large candidate pool of biomarkers, it is of great interest to evaluate and compare biomarker performance and identify potential multiplexed panels that can be used in combination with affordable and robust biosensors at the POC toward improved patient care. This review focuses on describing the known and emerging CVD biosensing technologies for analysis of cardiac biomarkers from blood. Initially, the global burden of CVDs and the standard of care for the primary CVD categories, namely heart failure (HF) and acute coronary syndrome (ACS) including myocardial infarction (MI) are discussed. The latest United States, Canadian and European society guidelines recommended standalone, emerging, and add-on cardiac biomarkers, as well as their combinations are then described for the prognosis, diagnosis, and risk stratification of CVDs. Finally, both commercial in vitro biosensing devices and recent state-of-art techniques for detection of cardiac biomarkers are reviewed that leverage single and multiplexed panels of cardiac biomarkers with a view toward affordable, compact devices with excellent performance for POC diagnosis and monitoring.

Monodisperse drops templated by 3D-structured microparticles.

The ability to create uniform subnanoliter compartments using microfluidic control has enabled new approaches for analysis of single cells and molecules. However, specialized instruments or expertise has been required, slowing the adoption of these cutting-edge applications. Here, we show that three dimensional-structured microparticles with sculpted surface chemistries template uniformly sized aqueous drops when simply mixed with two immiscible fluid phases. In contrast to traditional emulsions, particle-templated drops of a controlled volume occupy a minimum in the interfacial energy of the system, such that a stable monodisperse state results with simple and reproducible formation conditions. We describe techniques to manufacture microscale drop-carrier particles and show that emulsions created with these particles prevent molecular exchange, concentrating reactions within the drops, laying a foundation for sensitive compartmentalized molecular and cell-based assays with minimal instrumentation.

Fabrication of 3D concentric amphiphilic microparticles to form uniform nanoliter reaction volumes for amplified affinity assays.

Reactions performed in uniform microscale volumes have enabled numerous applications in the analysis of rare entities (e.g. cells and molecules). Here, highly monodisperse aqueous droplets are formed by simply mixing microscale multi-material particles, consisting of concentric hydrophobic outer and hydrophilic inner layers, with oil and water. The particles are manufactured in batch using a 3D printed device to co-flow four concentric streams of polymer precursors which are polymerized with UV light. The cross-sectional shapes of the particles are altered by microfluidic nozzle design in the 3D printed device. Once a particle encapsulates an aqueous volume, each "dropicle" provides uniform compartmentalization and customizable shape-coding for each sample volume to enable multiplexing of uniform reactions in a scalable manner. We implement an enzymatically-amplified immunoassay using the dropicle system, yielding a detection limit of <1 pM with a dynamic range of at least 3 orders of magnitude. Multiplexing using two types of shape-coded particles was demonstrated without cross talk, laying a foundation for democratized single-entity assays.

Computational cytometer based on magnetically modulated coherent imaging and deep learning.

Detecting rare cells within blood has numerous applications in disease diagnostics. Existing rare cell detection techniques are typically hindered by their high cost and low throughput. Here, we present a computational cytometer based on magnetically modulated lensless speckle imaging, which introduces oscillatory motion to the magnetic-bead-conjugated rare cells of interest through a periodic magnetic force and uses lensless time-resolved holographic speckle imaging to rapidly detect the target cells in three dimensions (3D). In addition to using cell-specific antibodies to magnetically label target cells, detection specificity is further enhanced through a deep-learning-based classifier that is based on a densely connected pseudo-3D convolutional neural network (P3D CNN), which automatically detects rare cells of interest based on their spatio-temporal features under a controlled magnetic force. To demonstrate the performance of this technique, we built a high-throughput, compact and cost-effective prototype for detecting MCF7 cancer cells spiked in whole blood samples. Through serial dilution experiments, we quantified the limit of detection (LoD) as 10 cells per millilitre of whole blood, which could be further improved through multiplexing parallel imaging channels within the same instrument. This compact, cost-effective and high-throughput computational cytometer can potentially be used for rare cell detection and quantification in bodily fluids for a variety of biomedical applications.

Nanoplasmonic swarm biosensing using single nanoparticle colorimetry.

We demonstrate a swarm biosensing platform that detects analyte based on the change in plasmonic signal from thousands of single nanoparticles sensors, leading to increased quantitative accuracy. Following dark field microscopy, we perform computational image registration and analyses to compile the hue change from thousands of single gold nanoparticles acting as individual quantitative biosensors. This platform demonstrated a limit of detection of 10 pM with a dynamic range of at least 4 orders of magnitude in buffer solution, and the successful detection of c-reactive protein (CRP) in serum compatible with 3-tier clinical cutoffs within a 10-fold difference without the need for a blocking step. By analyzing the before-and-after status of each plasmonic sensor, our sensing scheme provides informative sensing capabilities with the flexibility to select a subset of nanoparticles with optimal performance based on their initial states. Hue comparisons within and among devices also render the platform tolerant to particle and device variation. In addition, the simplicity of the readout instrumentation based on optical imaging and the implementation of microfluidics make it promising for future adaptation into point-of-care systems.

Microscale Symmetrical Electroporator Array as a Versatile Molecular Delivery System.

Successful developments of new therapeutic strategies often rely on the ability to deliver exogenous molecules into cytosol. We have developed a versatile on-chip vortex-assisted electroporation system, engineered to conduct sequential intracellular delivery of multiple molecules into various cell types at low voltage in a dosage-controlled manner. Micro-patterned planar electrodes permit substantial reduction in operational voltages and seamless integration with an existing microfluidic technology. Equipped with real-time process visualization functionality, the system enables on-chip optimization of electroporation parameters for cells with varying properties. Moreover, the system's dosage control and multi-molecular delivery capabilities facilitate intracellular delivery of various molecules as a single agent or in combination and its utility in biological research has been demonstrated by conducting RNA interference assays. We envision the system to be a powerful tool, aiding a wide range of applications, requiring single-cell level co-administrations of multiple molecules with controlled dosages.

Direct drug cocktail analyses using microscale vortex-assisted electroporation.

Combination therapy has become one of the leading approaches for treating complex diseases because it coadministers clinically proven drugs to concurrently target multiple signaling pathways of diseased cells. Identification of synergic drug combinations at their respective effective doses without unwanted accumulative side effects is the key to success for such therapy. In this work, we demonstrate the feasibility of the vortex-assisted microfluidic electroporation system for direct drug cocktail analyses where drug substances were individually delivered into cytosols in a sequential and dosage-controlled manner. Through quantitative analyses, the synergic combinational dosage ratios of the chemotherapeutic drug and the anticancer flavonoid were identified. When integrated with high-throughput label-free rare cell purification techniques, the presented system has the potential for development of personalized medicines as the system would be capable of comprehensively assessing drug combinations directly on patients' cellular samples.

An AC electrokinetics facilitated biosensor cassette for rapid pathogen identification.

To develop a portable point-of-care system based on biosensors for common infectious diseases such as urinary tract infection, the sensing process needs to be implemented within an enclosed fluidic system. On chip sample preparation of clinical samples remains a significant obstacle to achieving robust sensor performance. Herein AC electrokinetics is applied in an electrochemical biosensor cassette to enhance molecular convection and hybridization efficiency through electrokinetics induced fluid motion and Joule heating induced temperature elevation. Using E. coli as an exemplary pathogen, we determined the optimal electrokinetic parameters for detecting bacterial 16S rRNA in the biosensor cassette based on the current output, signal-to-noise ratio, and limit of detection. In addition, a panel of six probe sets targeting common uropathogenic bacteria was demonstrated. The optimized parameters were also validated using patient-derived clinical urine samples. The effectiveness of electrokinetics for on chip sample preparation will facilitate the implementation of point-of-care diagnosis of urinary tract infection in the future.

Inducing self-rotation of cells with natural and artificial melanin in a linearly polarized alternating current electric field.

The phenomenon of self-rotation observed in naturally and artificially pigmented cells under an applied linearly polarized alternating current (non-rotating) electrical field has been investigated. The repeatable and controllable rotation speeds of the cells were quantified and their dependence on dielectrophoretic parameters such as frequency, voltage, and waveform was studied. Moreover, the rotation behavior of the pigmented cells with different melanin content was compared to quantify the correlation between self-rotation and the presence of melanin. Most importantly, macrophages, which did not originally rotate in the applied non-rotating electric field, began to exhibit self-rotation that was very similar to that of the pigmented cells, after ingesting foreign particles (e.g., synthetic melanin or latex beads). We envision the discovery presented in this paper will enable the development of a rapid, non-intrusive, and automated process to obtain the electrical conductivities and permittivities of cellular membrane and cytoplasm in the near future.

Automated rotation rate tracking of pigmented cells by a customized block-matching algorithm.

This article describes an automated rotation rate tracking algorithm for pigmented cells that undergo rotation in a dielectrophoretic (DEP) force field. In a completely automated process, we preprocess each frame of a video sequence, then analyze the sequence frame by frame using a rotating-circle template with a block-matching algorithm, and finally estimate the rotation rate of the pigmented cells using a pixel-patch correlation. The algorithm has been demonstrated to accurately calculate the DEP-induced rotation rate of the cell up to 250 rpm. Cell rotation rates in various DEP force fields (i.e., by varying the applied voltages, frequencies, and waveforms to induce different force fields) were analyzed using this automated algorithm and reported in this article. Most importantly, the algorithm is accurate even when the cells have simultaneous translational and rotational motions across the video image sequence. Also, the algorithm is capable of tracking changes in rotation speed over a long period of time (90 s) by stably analyzing a massive data set of video image frames.