Griddient: a microfluidic array to generate reconfigurable gradients on-demand for spatial biology applications.

Biological tissues are highly organized structures where spatial-temporal gradients (e.g., nutrients, hypoxia, cytokines) modulate multiple physiological and pathological processes including inflammation, tissue regeneration, embryogenesis, and cancer progression. Current in vitro technologies struggle to capture the complexity of these transient microenvironmental gradients, do not provide dynamic control over the gradient profile, are complex and poorly suited for high throughput applications. Therefore, we have designed Griddent, a user-friendly platform with the capability of generating controllable and reversible gradients in a 3D microenvironment. Our platform consists of an array of 32 microfluidic chambers connected to a 384 well-array through a diffusion port at the bottom of each reservoir well. The diffusion ports are optimized to ensure gradient stability and facilitate manual micropipette loading. This platform is compatible with molecular and functional spatial biology as well as optical and fluorescence microscopy. In this work, we have used this platform to study cancer progression.

Microfluidic device with reconfigurable spatial temporal gradients reveals plastic astrocyte response to stroke and reperfusion.

As a leading cause of mortality and morbidity, stroke constitutes a significant global health burden. Ischemic stroke accounts for 80% of cases and occurs due to an arterial thrombus, which impedes cerebral blood flow and rapidly leads to cell death. As the most abundant cell type within the central nervous system, astrocytes play a critical role within the injured brain. We developed a novel microphysiological platform that permits the induction of spatiotemporally controlled nutrient gradients, allowing us to study astrocytic response during and after transient nutrient deprivation. Within 24 h of inducing starvation in the platform, nutrient deprivation led to multiple changes in astrocyte response, from metabolic perturbations to gene expression changes, and cell viability. Furthermore, we observed that nutrient restoration did not reverse the functional changes in astrocyte metabolism, which mirrors reperfusion injury observed in vivo. We also identified alterations in numerous glucose metabolism-associated genes, many of which remained upregulated or downregulated even after restoration of the nutrient supply. Together, these findings suggest that astrocyte activation during and after nutrient starvation induces plastic changes that may underpin persistent stroke-induced functional impairment. Overall, our innovative device presents interesting potential to be used in the development of new therapies to improve tissue repair and even cognitive recovery after stroke.

SEEMLIS: a flexible semi-automated method for enrichment of methylated DNA from low-input samples.

BACKGROUND: DNA methylation alterations have emerged as hallmarks of cancer and have been proposed as screening, prognostic, and predictive biomarkers. Traditional approaches for methylation analysis have relied on bisulfite conversion of DNA, which can damage DNA and is not suitable for targeted gene analysis in low-input samples. Here, we have adapted methyl-CpG-binding domain protein 2 (MBD2)-based DNA enrichment for use on a semi-automated exclusion-based sample preparation (ESP) platform for robust and scalable enrichment of methylated DNA from low-input samples, called SEEMLIS. RESULTS: We show that combining methylation-sensitive enzyme digestion with ESP-based MBD2 enrichment allows for single gene analysis with high sensitivity for GSTP1 in highly impure, heterogenous samples. We also show that ESP-based MBD2 enrichment coupled with targeted pre-amplification allows for analysis of multiple genes with sensitivities approaching the single cell level in pure samples for GSTP1 and RASSF1 and sensitivity down to 14 cells for these genes in highly impure samples. Finally, we demonstrate the potential clinical utility of SEEMLIS by successful detection of methylated gene signatures in circulating tumor cells (CTCs) from patients with prostate cancer with varying CTC number and sample purity. CONCLUSIONS: SEEMLIS is a robust assay for targeted DNA methylation analysis in low-input samples, with flexibility at multiple steps. We demonstrate the feasibility of this assay to analyze DNA methylation in prostate cancer cells using CTCs from patients with prostate cancer as a real-world example of a low-input analyte of clinical importance. In summary, this novel assay provides a platform for determining methylation signatures in rare cell populations with broad implications for research as well as clinical applications.

A Portable Controllable Compressive Stress Device to Monitor Human Breast Cancer Cell Protrusions at Single-Cell Resolution.

In vitro devices offer more numerous methods than in vivo models to investigate how cells respond to pressure stress and quantify those responses. Several in vitro devices have been developed to study the cell response to compression force. However, they are unable to observe morphological changes of cells in real-time. There is also a concern about cell damage during the process of harvesting cells from 3D gels. Here we report a device employing transparent, thin gel layers to clamp cells between the interfaces and applied a controllable compression force by stacking multiple layers on the top. In this approach, cells can be monitored for alteration of cellular protrusions, whose diversity has been proven to promote cancer cell dissemination, with single-cell resolution under compression force. Furthermore, p-Rac-1 and rhodamine staining on the device directly to confirm the actin filaments of lamellipodia. The method was able to fulfill real-time live-cell observation at single-cell resolution and can be readily used for versatile cell analysis. MDA-MB-231 and MCF7 breast cancer cells were utilized to demonstrate the utility of the device, and the results showed that the stimuli of compression force induce MDA-MB-231 and MCF7 to form lamellipodia and bleb protrusions, respectively. We envision the device may be used as a tool to explore mechanisms of membrane protrusion transitions and to screen drug candidates for inhibiting cancer cell protrusion plasticity for cancer therapy.

Volumeless reagent delivery: a liquid handling method for adding reagents to microscale droplets without increasing volume.

The addition of reagents for assays in digital microfluidic (DMF) systems is traditionally done by merging of droplets containing different analytes or reagents in solution. However, this process significantly increases droplet volume after each step, resulting in dilution of the analyte and reagents. Here, we report a new technique for performing reagent additions to aqueous droplets without significantly increasing the droplet's volume: volume-less reagent delivery (VRD). VRD is enabled by a physical phenomenon we call "exclusive liquid repellency" (ELR), which describes an aqueous/oil/solid 3-phase system where the aqueous phase can be fully repelled from a solid phase (contact angle ∼180°). When performing VRD, a reagent of interest in solution is deposited onto the ELR solid surface and allowed to dry. The ELR surface containing the dried reagent is then immersed under oil, followed by introduction of an aqueous droplet. By dragging the aqueous droplet over the spot of dried reagent using paramagnetic particles or via a physical sliding wall, the droplet can then recover and reconstitute the reagent with negligible increase in its total volume, returning the ELR surface to its initial liquid repellent state in the process. We demonstrate that VRD can be performed across a wide range of reagent types including sugars, proteins (antibodies), nucleic acids (DNA), antibiotics, and even complex enzyme/substrate/buffer "kit" mixtures. We believe VRD is a flexible and powerful technique which can further the development of self-contained, multi-step assays in DMF and other microfluidic systems.

Oil immersed lossless total analysis system for integrated RNA extraction and detection of SARS-CoV-2.

The COVID-19 pandemic exposed difficulties in scaling current quantitative PCR (qPCR)-based diagnostic methodologies for large-scale infectious disease testing. Bottlenecks include lengthy multi-step processes for nucleic acid extraction followed by qPCR readouts, which require costly instrumentation and infrastructure, as well as reagent and plastic consumable shortages stemming from supply chain constraints. Here we report an Oil Immersed Lossless Total Analysis System (OIL-TAS), which integrates RNA extraction and detection onto a single device that is simple, rapid, cost effective, and requires minimal supplies and infrastructure to perform. We validated the performance of OIL-TAS using contrived SARS-CoV-2 viral particle samples and clinical nasopharyngeal swab samples. OIL-TAS showed a 93% positive predictive agreement (n = 57) and 100% negative predictive agreement (n = 10) with clinical SARS-CoV-2 qPCR assays in testing clinical samples, highlighting its potential to be a faster, cheaper, and easier-to-deploy alternative for infectious disease testing.

Fluorescence "Turn-on" Lectin Sensors Fabricated by Ligand-Assisted Labeling Probes for Detecting Protein-Glycoprotein Interactions.

Elucidation of protein-protein interactions (PPIs) is often very challenging and yields complex and unclear results. Lectin-glycoprotein interactions are especially difficult to study due to the noncovalent nature of the interactions and inherently low binding affinities of proteins to glycan ligands on glycoproteins. Here, we report a "ligand-directed labeling probe (LLP)"-based approach to fabricate protein probes for elucidating protein-glycoprotein interactions. LLP was designed with dual photoactivatable groups for the introduction of an alkyne handle proximal to the carbohydrate-binding pocket of lectins, Ricinus communis agglutinin 120 (RCA120) and recombinant human Siglec-2-Fc. In proof-of-principle studies, alkynylated lectins were conjugated with a photoreactive diazirine cross-linker and an environment-sensitive fluorophore, respectively, by the bioorthogonal click reaction. The modified RCA120 or Siglec-2-Fc was used for detecting the interaction with the target glycoprotein in the solution or endogenously expressed glycoproteins on live HeLa cells. We anticipate that the fabrication of these protein probes will accelerate the discovery of novel PPIs.

Centrifugation-Assisted Immiscible Fluid Filtration for Dual-Bioanalyte Extraction.

The extraction of bioanalytes is the first step in many diagnostic and analytical assays. However, most bioanalyte extraction methods require extensive dilution-based washing processes that are not only time-consuming and laborious but can also result in significant sample loss, limiting their applications in rare sample analyses. Here, we present a method that enables the efficient extraction of multiple different bioanalytes from rare samples (down to 10 cells) without washing-centrifugation-assisted immiscible fluid filtration (CIFF). CIFF utilizes centrifugal force to drive the movement of analyte-bound glass microbeads from an aqueous sample into an immiscible hydrophobic solution to perform an efficient, simple, and nondilutive extraction. The method can be performed using conventional polymerase chain reaction (PCR) tubes with no requirement of specialized devices, columns, or instruments, making it broadly accessible and cost-effective. The CIFF process can effectively remove approximately 99.5% of the aqueous sample in one extraction with only 0.5% residual carryover, whereas a traditional "spin-down and aspirate" operation results in a higher 3.6% carryover. Another unique aspect of CIFF is its ability to perform two different solid-phase bioanalytes extractions simultaneously within a single vessel without fractionating the sample or performing serial extractions. Here we demonstrate efficient mRNA and DNA extraction from low-input samples (down to 10 cells) with slightly higher to comparable recovery compared to a traditional column-based extraction technique and the simultaneous extraction of two different proteins in the same tube using CIFF.

Automated System for Small-Population Single-Particle Processing Enabled by Exclusive Liquid Repellency.

Exclusive liquid repellency (ELR) describes an extreme wettability phenomenon in which a liquid phase droplet is completely repelled from a solid phase when exposed to a secondary immiscible liquid phase. Earlier, we developed a multi-liquid-phase open microfluidic (or underoil) system based on ELR to facilitate rare-cell culture and single-cell processing. The ELR system can allow for the handling of small volumes of liquid droplets with ultra-low sample loss and biofouling, which makes it an attractive platform for biological applications that require lossless manipulation of rare cellular samples (especially for a limited sample size in the range of a few hundred to a few thousand cells). Here, we report an automated platform using ELR microdrops for single-particle (or single-cell) isolation, identification, and retrieval. This was accomplished via the combined use of a robotic liquid handler, an automated microscopic imaging system, and real-time image-processing software for single-particle identification. The automated ELR technique enables rapid, hands-free, and robust isolation of microdrop-encapsulated rare cellular samples.

Multilayer architecture microfluidic network array for combinatorial drug testing on 3D-cultured cells.

In vitro testing of drug compounds on cell models during the drug development process represents an indispensable step in the initial screening process. Although drug testing on three-dimensional (3D) cultured cells may provide a more accurate prediction of drug efficacy, it is relatively costly and time-consuming to perform compared with conventional 2D cultures due to the thick z-axis of the 3D models. In this study, we have presented a microfluidic platform with integrated pneumatic valves for producing a thin-gel 3D cell culture-based combinatorial drug screening array (3D-µCDS array). The multilayer architecture and microfluidic layout has a smaller device footprint than a single-layer microfluidic channel arrangement, making it well suited to scaling up for high-throughput combinatorial drug screening on 3D cell model. We performed 8 × 8 combination drug screening experiments with the device using two anti-cancer drugs (doxorubicin and paclitaxel) on MDA-MB-231 and MCF-7 breast cancer cell lines for demonstration. Our results indicate that our 3D-µCDS array device allows the successful screening of multiple drug combinations while reducing the operation time and the number of sample/reagents required, making it an ideal tool for general combinatorial drug screening, as well as for applications using valuable tissues and clinical samples.

Double-exclusive liquid repellency (double-ELR): an enabling technology for rare phenotype analysis.

Double-exclusive liquid repellency (double-ELR) is an extreme wettability phenomenon in which adjacent regions selectively and completely repel immiscible liquids with different surface chemistries on a non-textured substrate (i.e., a substrate in absence of micro/nano-structures). Under double-ELR conditions, each liquid exhibits no physical contact (contact angle of 180°) with its non-preferred surface chemistry, thus enabling complete partitioning of adjacent fluidic volumes (e.g., between water and oil). This enables a new type of cell culture-based assay, where cell loss from common failure modes (e.g., biofouling from inadvertent cell adhesion, detrimental moisture loss/gain, and liquid handling dead volumes) is significantly mitigated. Importantly, the principles of double-ELR were leveraged to achieve underoil sweep patterning, a no-loss, robust and high-throughput distribution of sub-microliter volumes of aqueous media (and cells). In addition to high-efficiency distribution via sweep patterning, double-ELR can be used to construct "modular" (i.e., easily implemented and/or linked together with spatial and temporal control) higher-order architectures for in vitro imitation of physiologically relevant microenvironments that are of particular interest within the cell assay community, including multi-phenotype cultures with excellent spatial and temporal control, three-dimensional layered multi-phenotype cultures, cultures with selective mechanical cues of extracellular matrix (i.e., collagen fiber alignment), and spheroid cultures. Together, these features of double-ELR uniquely facilitate culture and high content analysis of limited cellular samples (e.g., a few hundred to a few thousand cells).

Proton-ELISA: Electrochemical immunoassay on a dual-gated ISFET array.

Here we report an electrochemical immunoassay platform called Proton-ELISA (H-ELISA) for the detection of bioanalytes. H-ELISA uniquely utilizes protons as an immunoassay detection medium, generated by the enzyme glucose oxidase (GOx) coupled with Fenton's reagent in a proton amplification reaction cascade that results in a highly amplified signal. A proton-sensitive dual-gated ion-sensitive field effect transistor (DG-ISFET) sensor was also developed for sensitive and accurate detection of the proton signal in H-ELISA. The DG-ISFET sensor comprises of a 128 × 128 array of 16,384 sensing transistors each with an individually addressable back gate to allow for a very high signal throughput and improved accuracy. We then demonstrated that the platform could detect C-reactive protein and immunoglobulin E down to concentrations of 12.5 and 125 pg/mL, respectively. We further showed that the platform is compatible with complex biological sample conditions such as human serum, suggesting that the platform is sufficiently robust for potential diagnostic applications.

Fabrication of a protein microarray by fluorous-fluorous interactions.

Fluorous-modified surfaces have emerged as a powerful tool for the immobilization of fluorous-tagged biomolecules based on their specificity and the strength of fluorous-fluorous interactions. To fabricate a fluorous-based protein microarray, we designed two strategies for site-specific modification of proteins with a fluorous tag: attaching the fluorous tag to the C-termini of expressed proteins by native chemical ligation (NCL) or to the Fc domain of antibodies through boronic acid (BA)-diol interactions. The perfluoro-tagged proteins could be easily purified by fluorous-functionalized magnetic nanoparticles (MNPs) and immobilized on a fluorous chip with minimal non-specific adsorption. Importantly, proteins immobilized on the solid support through non-covalent fluorous-fluorous interactions were sufficiently stable to withstand continuous washing. We believe that this fluorous-fluorous immobilization strategy will be a highly valuable tool in protein microarray fabrication.

Self-concentrating buoyant glass microbubbles for high sensitivity immunoassays.

Here, we report the novel application of a material with self-concentrating properties for enhancing the sensitivity of immunoassays. Termed as glass microbubbles, they are antibody functionalized buoyant hollow glass microspheres that simultaneously float and concentrate into a dense monolayer when dispensed in a liquid droplet. This self-concentrating charactaristic of the microbubbles allow for autonomous signal localization, which translates to a higher sensitivity compared to other microparticle-based immunoassays. We then demonstrated a "microbubble array" platform consisting of the glass microbubbles floating in a microfluidic liquid hemisphere array for performing multiplex immunoassays.

Towards an Endpoint Cell Motility Assay by a Microfluidic Platform.

In vitro cell motility assays are frequently used in the study of cell migration in response to anti-cancer drug treatment. Microfluidic systems represent a unique tool for the in vitro analysis of cell motility. However, they usually rely on using time-lapse microscopy to record the spatial temporal locations of the individual cells being tested. This has created a bottleneck for microfluidic systems to perform high-throughput experiments due to requirement of a costly time-lapse microscopy system. Here, we describe the development of a portable microfluidic device for endpoint analysis of cell motility. The reported device incorporates a cell alignment feature to position the seeded cells on the same initial location, so that the cells' motilities can be analyzed based on their locations at the end of the experiment after the cells have migrated. We show that the device was able to assess cancer cell motility after treatment with a migration inhibitory drug Indole-3-carbinol on MDA-MB-231 breast cancer cells, demonstrating the applicability of our device in screening anti-cancer drug compounds on cancer cells.