Multiplex Single-Cell Bioprinting for Engineering of Heterogeneous Tissue Constructs with Subcellular Spatial Resolution.

Tissue development, function, and disease are largely driven by the spatial organization of individual cells and their cell-cell interactions. Precision engineered tissues with single-cell spatial resolution, therefore, have tremendous potential for next generation disease models, drug discovery, and regenerative therapeutics. Despite significant advancements in biofabrication approaches to improve feature resolution, strategies to fabricate tissues with the exact same organization of individual cells in their native cellular microenvironment have remained virtually non-existent to date. Here we report a method to spatially pattern single cells with up to eight cell phenotypes and subcellular spatial precision. As proof-of-concept we first demonstrate the ability to systematically assess the influence of cellular microenvironments on cell behavior by controllably altering the spatial arrangement of cell types in bioprinted precision cell-cell interaction arrays. We then demonstrate, for the first time, the ability to produce high-fidelity replicas of a patient's annotated cancer biopsy with subcellular resolution. The ability to replicate native cellular microenvironments marks a significant advancement for precision biofabricated in-vitro models, where heterogenous tissues can be engineered with single-cell spatial precision to advance our understanding of complex biological systems in a controlled and systematic manner.

Enhancement of dielectrophoresis-based particle collection from high conducting fluids due to partial electrode insulation.

Dielectrophoresis (DEP) is a successful method to recover nanoparticles from different types of fluid. The DEP force acting on these particles is created by an electrode microarray that produces a nonuniform electric field. To apply DEP to a highly conducting biological fluid, a protective hydrogel coating over the metal electrodes is required to create a barrier between the electrode and the fluid. This protects the electrodes, reduces the electrolysis of water, and allows the electric field to penetrate into the fluid sample. We observed that the protective hydrogel layer can separate from the electrode and form a closed domed structure and that collection of 100 nm polystyrene beads increased when this occurred. To better understand this collection increase, we used COMSOL Multiphysics software to model the electric field in the presence of the dome filled with different materials ranging from low-conducting gas to high conducting phosphate-buffered saline fluids. The results suggest that as the electrical conductivity of the material inside the dome is reduced, the whole dome acts as an insulator which increases electric field intensity at the electrode edge. This increased intensity widens the high-intensity electric field factor zone resulting in increased collection. This informs how dome formation results in increased particle collection and provides insight into how the electric field can be intensified to the increase collection of particles. These results have important applications for increasing the recovery of biologically-derived nanoparticles from undiluted physiological fluids that have high conductance, including the collection of cancer-derived extracellular vesicles from plasma for liquid biopsy applications.

On-chip dielectrophoretic recovery and detection of a lactate sensing probiotic from model human saliva.

Early detection has led to increased survival for multiple cancers; however, the 5-year survival rate of oral carcinoma (OC) has remained at 40% for the last several decades. Screening for OC is routinely done via visual examinations, followed by tissue biopsy and laboratory testing. Point-of-care testing would be a more convenient and widely available alternative for at-risk individuals. Increased lactate production is a hallmark of many head-and-neck tumors, due to the Warburg Effect, where tumor cells favor glycolysis in the place of oxidative phosphorylation. To detect excess lactate, we have modified the commensal bacterium Escherichia coli Nissle 1917 to express fluorescent reporter genes in response to extracellular lactate. Administering this commensal as a mouth wash and subsequently collecting saliva for the detection of the reporter may allow for noninvasive, early detection of cancerous lesions in at-risk individuals. Furthermore, we demonstrate a new on-chip electrokinetic technique to recover these probiotic probes from model saliva fluid to improve the detection of reporter gene activation.

Turning antibodies off and on again using a covalently tethered blocking peptide.

In their natural form, antibodies are always in an "on-state" and are capable of binding to their targets. This leads to undesirable interactions in a wide range of therapeutic, analytical, and synthetic applications. Modulating binding kinetics of antibodies to turn them from an "off-state" to an "on-state" with temporal and spatial control can address this. Here we demonstrate a method to modulate binding activity of antibodies in a predictable and reproducible way. We designed a blocking construct that uses both covalent and non-covalent interactions with the antibody. The construct consisted of a Protein L protein attached to a flexible linker ending in a blocking-peptide designed to interact with the antibody binding site. A mutant Protein L was developed to enable photo-triggered covalent crosslinking to the antibody at a specific location. The covalent bond anchored the linker and blocking peptide to the antibody light chain keeping the blocking peptide close to the antibody binding site. This effectively put the antibody into an "off-state". We demonstrate that protease-cleavable and photocleavable moieties in the tether enable controlled antibody activation to the "on-state" for anti-FLAG and cetuximab antibodies. Protein L can bind a range of antibodies used therapeutically and in research for wide applicability.

A Versatile Synthetic Pathway for Producing Mesostructured Plasmonic Nanostructures.

Highly branched gold (Au) nanostructures with sharp tips are considered excellent substrates for surface-enhanced Raman scattering (SERS)-based sensing technologies. Here, a simple synthetic route for producing Au or Au-Ag bimetallic mesostructures with multiple sharpened tips in the presence of carbon quantum dots (CQDs) is presented. The morphologies of these mesostructured plasmonic nanoparticles (MSPNs) can be controlled by adjusting the concentration of CQDs, reaction temperatures, and seed particles. The optimal molar ratio for [HAuCl4 ]/[CQDs] is found to be ≈25. At this molar ratio, the diameters of MSPNs can be tuned from 80 to 200 nm by changing the reaction temperature from 25 to 80 °C. In addition, it is found that hierarchical MSPNs consisting of multiple Au nanocrystals can be formed over the entire seed particle surface. Finally, the SERS activity of these MSPNs is examined through the detection of rhodamine 6G and methylene blue. Of the different mesostructures, the bimetallic MSPNs have the highest sensitivity with the ability to detect 10-7  m of rhodamine 6G and 10-6  m of methylene blue. The properties of these MSPN particles, made using a novel synthetic process, make them excellent candidates for SERS-based chemical sensing applications.

cyc-DEP: Cyclic immunofluorescence profiling of particles collected using dielectrophoresis.

Cancer is a highly heterogenous disease that requires precise detection tools and active surveillance methods. Liquid biopsy assays provide an agnostic way to follow the complex trajectory of cancer, providing better patient stratification tools for optimized treatment. Here, we present the development of a low-volume liquid biopsy assay called cyc-DEP (cyclic immunofluorescent imaging on dielectrophoretic chip) to profile biomarkers collected on a dielectrophoretic microfluidic chip platform. To enable on-chip cyclic imaging, we optimized a fluorophore quenching method and sequential rounds of on-chip staining with fluorescently conjugated primary antibodies. cyc-DEP allows for the quantification of a multiplex array of proteins using 25 µl of a patient plasma sample. We utilized nanoparticles from a prostate adenocarcinoma (LNCaP) cell line and a panel of six target proteins to develop our proof-of-concept technique. We then used cyc-DEP to quantify blood plasma levels of target proteins from healthy individuals, low-grade and high-grade prostate cancer patients (n = 3 each) in order to demonstrate that our platform is suitable for liquid biopsy analysis in its present form. To ensure accurate quantification of signal intensities and comparisons between different samples, we incorporated a signal intensity normalization method (fluorescent beads) and a custom signal intensity quantification algorithm that account for the distribution of signal across hundreds of collection regions on each chip. Our technique enabled a threefold improvement in multiplicity for detecting proteins associated with fluid samples, opening doors for early detection, and active surveillance through quantification of a multiplex array of biomarkers from low-volume liquid biopsies.

Theoretical and experimental analysis of negative dielectrophoresis-induced particle trajectories.

Many biomedical analysis applications require trapping and manipulating single cells and cell clusters within microfluidic devices. Dielectrophoresis (DEP) is a label-free technique that can achieve flexible cell trapping, without physical barriers, using electric field gradients created in the device by an electrode microarray. Little is known about how fluid flow forces created by the electrodes, such as thermally driven convection and electroosmosis, affect DEP-based cell capture under high conductance media conditions that simulate physiologically relevant fluids such as blood or plasma. Here, we compare theoretical trajectories of particles under the influence of negative DEP (nDEP) with observed trajectories of real particles in a high conductance buffer. We used 10-µm diameter polystyrene beads as model cells and tracked their trajectories in the DEP microfluidic chip. The theoretical nDEP trajectories were in close agreement with the observed particle behavior. This agreement indicates that the movement of the particles was highly dominated by the DEP force and that contributions from thermal- and electroosmotic-driven flows were negligible under these experimental conditions. The analysis protocol developed here offers a strategy that can be applied to future studies with different applied voltages, frequencies, conductivities, and polarization properties of the targeted particles and surrounding medium. These findings motivate further DEP device development to manipulate particle trajectories for trapping applications.

Automated fluorescence quantification of extracellular vesicles collected from blood plasma using dielectrophoresis.

Tumor-secreted exosomes and other extracellular vesicles (EVs) in circulation contain valuable biomarkers for early cancer detection and screening. We have previously demonstrated collection of cancer-derived nanoparticles (NPs) directly from whole blood and plasma with a chip-based technique that uses a microelectrode array to generate dielectrophoretic (DEP) forces. This technique enables direct recovery of NPs from whole blood and plasma. The biomarker payloads associated with collected particles can be detected and quantified with immunostaining. Accurately separating the fluorescence intensity of stained biomarkers from background (BG) levels becomes a challenge when analyzing the blood from early-stage cancer patients in which biomarker concentrations are low. To address this challenge, we developed two complementary techniques to standardize the quantification of fluorescently immunolabeled biomarkers collected and concentrated at predictable locations within microfluidic chips. The first technique was an automated algorithm for the quantitative analysis of fluorescence intensity at collection regions within the chip compared to levels at adjacent regions. The algorithm used predictable locations of particle collection within the chip geometry to differentiate regions of collection and BG. We successfully automated the identification and removal of optical artifacts from quantitative calculations. We demonstrated that the automated system performs nearly the same as a human user following a standard protocol for manual artifact removal with Pearson's r-values of 0.999 and 0.998 for two different biomarkers (n = 36 patients). We defined a usable dynamic range of fluorescence intensities corresponding to 1 to 2000 arbitrary units (a.u.). Fluorescence intensities within the dynamic range increased linearly with respect to exposure time and particle concentration. The second technique was the implementation of an internal standard to adjust levels of biomarker fluorescence based on the relative collection efficiency of the chip. Use of the internal standard reduced variability in measured biomarker levels due to differences in chip-to-chip collection efficiency, especially at low biomarker concentrations. The internal standard did not affect linear trends between fluorescence intensity and exposure time. Adjustments using the internal standard improved linear trends between fluorescence intensity and particle concentration. The optical quantification techniques described in this paper can be easily adapted for other lab-on-a-chip platforms that have predefined regions of biomarker or particle collection and that rely on fluorescence detection.

Temperature-induced liquid crystal microdroplet formation in a partially miscible liquid mixture.

Liquid-in-liquid droplets are typically generated by the partitioning of immiscible fluids, e.g. by mechanical shearing with macroscopic homogenisers or microfluidic flow focussing. In contrast, partially miscible liquids with a critical solution temperature display a temperature-dependent mixing behaviour. In this work, we demonstrate how, for a blend of methanol (MeOH) and the thermotropic liquid crystal (LC) 4-Cyano-4'-pentylbiphenyl (5CB), cooling from a miscible to an immiscible state allows the controlled formation of microdroplets. A near-room-temperature-induced phase separation leads to nucleation, growth and coalescence of mesogen-rich droplets. The size and number of the droplets is tunable on the microscopic scale by variation of temperature quench depth and cooling rate. Further cooling induces a phase transition to nematic droplets with radial configuration, well-defined sizes and stability over the course of an hour. This temperature-induced approach offers a scalable and reversible alternative to droplet formation with relevance in diagnostics, optoelectronics, materials templating and extraction processes.

Dielectrophoresis: Developments and applications from 2010 to 2020.

The 20th century has seen tremendous innovation of dielectrophoresis (DEP) technologies, with applications being developed in areas ranging from industrial processing to micro- and nanoscale biotechnology. From 2010 to present day, there have been 981 publications about DEP. Of over 2600 DEP patents held by the United States Patent and Trademark Office, 106 were filed in 2019 alone. This review focuses on DEP-based technologies and application developments between 2010 and 2020, with an aim to highlight the progress and to identify potential areas for future research. A major trend over the last 10 years has been the use of DEP techniques for biological and clinical applications. It has been used in various forms on a diverse array of biologically derived molecules and particles to manipulate and study them including proteins, exosomes, bacteria, yeast, stem cells, cancer cells, and blood cells. DEP has also been used to manipulate nano- and micron-sized particles in order to fabricate different structures. The next 10 years are likely to see the increase in DEP-related patent applications begin to result in a greater level of technology commercialization. Also during this time, innovations in DEP technology will likely be leveraged to continue the existing trend to further biological and medical-focused applications as well as applications in microfabrication. As a tool leveraged by engineering and imaginative scientific design, DEP offers unique capabilities to manipulate small particles in precise ways that can help solve problems and enable scientific inquiry that cannot be addressed using conventional methods.

Gas-Stabilizing Sub-100 nm Mesoporous Silica Nanoparticles for Ultrasound Theranostics.

Recent studies have demonstrated that gas-stabilizing particles can generate cavitating micron-sized bubbles when exposed to ultrasound, offering excellent application potential, including ultrasound imaging, drug delivery, and tumor ablation. However, the majority of the reported gas-stabilizing particles are relatively large (>200 nm), and smaller particles require high acoustic pressures to promote cavitation. Here, this paper reports the preparation of sub-100 nm gas-stabilizing nanoparticles (GSNs) that can initiate cavitation at low acoustic intensities, which can be delivered using a conventional medical ultrasound imaging system. The highly echogenic GSNs (F127-hMSN) were prepared by carefully engineering the surfaces of ∼50 nm mesoporous silica nanoparticles. It was demonstrated that the F127-hMSNs could be continuously imaged with ultrasound in buffer or biological solutions or agarose phantoms for up to 20 min. Also, the F127-hMSN can be stored in phosphate-buffered saline for at least a month with no loss in ultrasound responsiveness. The particles significantly degraded when diluted in simulated body fluids, indicating possible biodegradation of the F127-hMSNs in vivo. Furthermore, at ultrasound imaging conditions, F127-hMSNs did not cause detectable cell death, supporting the potential safety of these particles. Finally, strong cavitation activity generation by the F127-hMSNs under high-intensity focused ultrasound insonation was demonstrated and applied to effectively ablate cancer cells.

Removal of ligand-bound liposomes from cell surfaces by microbubbles exposed to ultrasound.

Gas-filled microbubbles attached to cell surfaces can interact with focused ultrasound to create microstreaming of nearby fluid. We directly observed the ultrasound/microbubble interaction and documented that under certain conditions fluorescent particles that were attached to the surface of live cells could be removed. Fluorescently labeled liposomes that were larger than 500 nm in diameter were attached to the surface of endothelial cells using cRGD targeting to αvß3 integrin. Microbubbles were attached to the surface of the cells through electrostatic interactions. Images taken before and after the ultrasound exposure were compared to document the effects on the liposomes. When exposed to ultrasound with peak negative pressure of 0.8 MPa, single microbubbles and groups of isolated microbubbles were observed to remove targeted liposomes from the cell surface. Liposomes were removed from a region on the cell surface that averaged 33.1 µm in diameter. The maximum distance between a single microbubble and a detached liposome was 34.5 µm. Single microbubbles were shown to be able to remove liposomes from over half the surface of a cell. The distance over which liposomes were removed was significantly dependent on the resting diameter of the microbubble. Clusters of adjoining microbubbles were not seen to remove liposomes. These observations demonstrate that the fluid shear forces generated by the ultrasound/microbubble interaction can remove liposomes from the surfaces of cells over distances that are greater than the diameter of the microbubble.

Drug Delivery Nanoparticles with Locally Tunable Toxicity Made Entirely from a Light-Activatable Prodrug of Doxorubicin.

PURPOSE: A major challenge facing nanoparticle-based delivery of chemotherapy agents is the natural and unavoidable accumulation of these particles in healthy tissue resulting in local toxicity and dose-limiting side effects. To address this issue, we have designed and characterized a new prodrug nanoparticle with controllable toxicity allowing a locally-delivered light trigger to convert the payload of the particle from a low to a high toxicity state. METHODS: The nanoparticles are created entirely from light-activatable prodrug molecules using a nanoprecipitation process. The prodrug is a conjugate of doxorubicin and photocleavable biotin (DOX-PCB). RESULTS: These DOX-PCB nanoparticles are 30 times less toxic to cells than doxorubicin, but can be activated to release pure therapeutic doxorubicin when exposed to 365 nm light. These nanoparticles have an average diameter of around 100 nm and achieve the maximum possible prodrug loading capacity since no support structure or coating is required to prevent loss of prodrug from the nanoparticle. CONCLUSIONS: These light activatable nanoparticles demonstrate tunable toxicity and can be used to facilitate future therapy development whereby light delivered specifically to the tumor tissue would locally convert the nanoparticles to doxorubicin while leaving nanoparticles accumulated in healthy tissue in the less toxic prodrug form.

Rapid Isolation and Detection of Exosomes and Associated Biomarkers from Plasma.

Exosomes found in the circulation are a primary source of important cancer-related RNA and protein biomarkers that are expected to lead to early detection, liquid biopsy, and point-of-care diagnostic applications. Unfortunately, due to their small size (50-150 nm) and low density, exosomes are extremely difficult to isolate from plasma. Current isolation methods are time-consuming multistep procedures that are unlikely to translate into diagnostic applications. To address this issue, we demonstrate the ability of an alternating current electrokinetic (ACE) microarray chip device to rapidly isolate and recover glioblastoma exosomes from undiluted human plasma samples. The ACE device requires a small plasma sample (30-50 µL) and is able to concentrate the exosomes into high-field regions around the ACE microelectrodes within 15 min. A simple buffer wash removes bulk plasma materials, leaving the exosomes concentrated on the microelectrodes. The entire isolation process and on-chip fluorescence analysis is completed in less than 30 min which enables subsequent on-chip immunofluorescence detection of exosomal proteins, and provides viable mRNA for RT-PCR analysis. These results demonstrate the ability of the ACE device to streamline the process for isolation and recovery of exosomes, significantly reducing the number of processing steps and time required.

Acoustic Microcannons: Toward Advanced Microballistics.

Acoustically triggered microcannons, capable of loading and firing nanobullets (Nbs), are presented as powerful microballistic tools. Hollow conically shaped microcannon structures have been synthesized electrochemically and fully loaded with nanobullets made of silica or fluorescent microspheres, and perfluorocarbon emulsions, embedded in a gel matrix stabilizer. Application of a focused ultrasound pulse leads to the spontaneous vaporization of the perfluorocarbon emulsions within the microcannon and results in the rapid ejection of the nanobullets. Such Nbs "firing" at remarkably high speeds (on the magnitude of meters per second) has been modeled theoretically and demonstrated experimentally. Arrays of microcannons anchored in a template membrane were used to demonstrate the efficient Nbs loading and the high penetration capabilities of the ejected Nbs in a tissue phantom gel. This acoustic-microcannon approach could be translated into advanced microscale ballistic tools, capable of efficient loading and firing of multiple cargoes, and offer improved accessibility to target locations and enhanced tissue penetration properties.

Dielectrophoretic recovery of DNA from plasma for the identification of chronic lymphocytic leukemia point mutations.

AIM: Circulating cell free (ccf) DNA contains information about mutations affecting chronic lymphocytic leukemia (CLL). The complexity of isolating DNA from plasma inhibits the development of point-of-care diagnostics. Here, we introduce an electrokinetic method that enables rapid recovery of DNA from plasma. MATERIALS & METHODS: ccf-DNA was isolated from 25 µl of CLL plasma using dielectrophoresis. The DNA was used for PCR amplification, sequencing and analysis. RESULTS: The ccf-DNA collected from plasma of 5 CLL patients revealed identical mutations to those previously identified by extracting DNA from CLL cells from the same patients. CONCLUSION: Rapid dielectrophoresis isolation of ccf-DNA directly from plasma provides sufficient amounts of DNA to use for identification of point mutations in genes associated with CLL progression.

Sonogenetics is a non-invasive approach to activating neurons in Caenorhabditis elegans.

A major challenge in neuroscience is to reliably activate individual neurons, particularly those in deeper brain regions. Current optogenetic approaches require invasive surgical procedures to deliver light of specific wavelengths to target cells to activate or silence them. Here, we demonstrate the use of low-pressure ultrasound as a non-invasive trigger to activate specific ultrasonically sensitized neurons in the nematode, Caenorhabditis elegans. We first show that wild-type animals are insensitive to low-pressure ultrasound and require gas-filled microbubbles to transduce the ultrasound wave. We find that neuron-specific misexpression of TRP-4, the pore-forming subunit of a mechanotransduction channel, sensitizes neurons to ultrasound stimulus, resulting in behavioural outputs. Furthermore, we use this approach to manipulate the function of sensory neurons and interneurons and identify a role for PVD sensory neurons in modifying locomotory behaviours. We suggest that this method can be broadly applied to manipulate cellular functions in vivo.

Recovery of Drug Delivery Nanoparticles from Human Plasma Using an Electrokinetic Platform Technology.

The effect of complex biological fluids on the surface and structure of nanoparticles is a rapidly expanding field of study. One of the challenges holding back this research is the difficulty of recovering therapeutic nanoparticles from biological samples due to their small size, low density, and stealth surface coatings. Here, the first demonstration of the recovery and analysis of drug delivery nanoparticles from undiluted human plasma samples through the use of a new electrokinetic platform technology is presented. The particles are recovered from plasma through a dielectrophoresis separation force that is created by innate differences in the dielectric properties between the unaltered nanoparticles and the surrounding plasma. It is shown that this can be applied to a wide range of drug delivery nanoparticles of different morphologies and materials, including low-density nanoliposomes. These recovered particles can then be analyzed using different methods including scanning electron microscopy to monitor surface and structural changes that result from plasma exposure. This new recovery technique can be broadly applied to the recovery of nanoparticles from high conductance fluids in a wide range of applications.

Optical detection of harmonic oscillations in fluorescent dye-loaded microbubbles ensonified by ultrasound.

A new optical contrast agent has been developed by exposing dye-loaded microbubbles to a rapidly-cooled thermal treatment to homogenize the dye distribution across the surface. Ultrasound causes these microbubbles to oscillate in size which changes the self-quenching efficiency of the dye molecules creating a "blinking" signal. We demonstrate for the first time that these microbubbles can reproducibly generate second, third, and even fourth harmonic fluorescence intensity modulations, in addition to the fundamental frequency of the driving ultrasound. Detecting these harmonic signals could produce a higher signal-to-noise ratio for fluorescence imaging in medical applications by allowing fundamental frequency interference and artifacts to be filtered out.

The influence of distance between microbubbles on the fluid flow produced during ultrasound exposure.

The collapse dynamics of lipid monolayer-coated microbubbles in the clinically-relevant size range under 6 µm in diameter have not been studied directly due to their small size obscuring the collapse visualization. This study investigates the influence of inter-microbubble distance on the shape of lipid debris clouds created by the collapse of the microbubble destroying the microbubble lipid monolayer. The shape was highly influenced by the fluid motion that occurred as the microbubbles collapsed. It was observed that at inter-microbubble distances smaller than 37 µm the microbubbles began to interact with one another resulting in distorted and ellipsoid-shaped debris clouds. At inter-microbubble distances less than 10 µm, significantly elongated debris clouds were observed that extended out from the original microbubble location in a single direction. These distortions show a significant distance-dependent interaction between microbubbles. It was observed that microbubbles in physical contact with one another behaved in the same manner as separate microbubbles less than 10 µm apart creating significantly elongated debris clouds. It can be hypothesized that small inter-microbubble distances influence the microbubble to collapse asymmetrically resulting in the creation of fluid jets that contribute to the formation of debris fields that are elongated in a single direction.