Tumor cell-based liquid biopsy using high-throughput microfluidic enrichment of entire leukapheresis product.

Circulating Tumor Cells (CTCs), interrogated by sampling blood from patients with cancer, contain multiple analytes, including intact RNA, high molecular weight DNA, proteins, and metabolic markers. However, the clinical utility of tumor cell-based liquid biopsy has been limited since CTCs are very rare, and current technologies cannot process the blood volumes required to isolate a sufficient number of tumor cells for in-depth assays. We previously described a high-throughput microfluidic prototype utilizing high-flow channels and amplification of cell sorting forces through magnetic lenses. Here, we apply this technology to analyze patient-derived leukapheresis products, interrogating a mean blood volume of 5.83 liters from patients with metastatic cancer, with a median of 2,799 CTCs purified per patient. Isolation of many CTCs from individual patients enables characterization of their morphological and molecular heterogeneity, including cell and nuclear size and RNA expression. It also allows robust detection of gene copy number variation, a definitive cancer marker with potential diagnostic applications. High-volume microfluidic enrichment of CTCs constitutes a new dimension in liquid biopsies.

A microfluidic transistor for automatic control of liquids.

Microfluidics have enabled notable advances in molecular biology1,2, synthetic chemistry3,4, diagnostics5,6 and tissue engineering7. However, there has long been a critical need in the field to manipulate fluids and suspended matter with the precision, modularity and scalability of electronic circuits8-10. Just as the electronic transistor enabled unprecedented advances in the automatic control of electricity on an electronic chip, a microfluidic analogue to the transistor could enable improvements in the automatic control of reagents, droplets and single cells on a microfluidic chip. Previous works on creating a microfluidic analogue to the electronic transistor11-13 did not replicate the transistor's saturation behaviour, and could not achieve proportional amplification14, which is fundamental to modern circuit design15. Here we exploit the fluidic phenomenon of flow limitation16 to develop a microfluidic element capable of proportional amplification with flow-pressure characteristics completely analogous to the current-voltage characteristics of the electronic transistor. We then use this microfluidic transistor to directly translate fundamental electronic circuits into the fluidic domain, including the amplifier, regulator, level shifter, logic gate and latch. We also combine these building blocks to create more complex fluidic controllers, such as timers and clocks. Finally, we demonstrate a particle dispenser circuit that senses single suspended particles, performs signal processing and accordingly controls the movement of each particle in a deterministic fashion without electronics. By leveraging the vast repertoire of electronic circuit design, microfluidic-transistor-based circuits enable fluidic automatic controllers to manipulate liquids and single suspended particles for lab-on-a-chip platforms.

A Microfluidic Transistor for Liquid Signal Processing.

Microfluidics have enabled significant advances in molecular biology 1-3 , synthetic chemistry 4,5 , diagnostics 6,7 , and tissue engineering 8 . However, there has long been a critical need in the field to manipulate fluids and suspended matter with the precision, modularity, and scalability of electronic circuits 9-11 . Just as the electronic transistor enabled unprecedented advances in the control of electricity on an electronic chip, a microfluidic analogue to the transistor could enable improvements in the complex, scalable control of reagents, droplets, and single cells on an autonomous microfluidic chip. Prior works on creating a microfluidic analogue to the electronic transistor 12-14 could not replicate the transistor's saturation behavior, which is crucial to perform analog amplification 15 and is fundamental to modern circuit design 16 . Here we exploit the fluidic phenomenon of flow-limitation 17 to develop a microfluidic element with flow-pressure characteristics completely analogous to the current-voltage characteristics of the electronic transistor. As this microfluidic transistor successfully replicates all of the key operating regimes of the electronic transistor (linear, cut-off and saturation), we are able to directly translate a variety of fundamental electronic circuit designs into the fluidic domain, including the amplifier, regulator, level shifter, logic gate, and latch. Finally, we demonstrate a "smart" particle dispenser that senses single suspended particles, performs liquid signal processing, and accordingly controls the movement of said particles in a purely fluidic system without electronics. By leveraging the vast repertoire of electronic circuit design, microfluidic transistor-based circuits are easy to integrate at scale, eliminate the need for external flow control, and enable uniquely complex liquid signal processing and single-particle manipulation for the next generation of chemical, biological, and clinical platforms.

Interface microstructure effects on dynamic failure behavior of layered Cu/Ta microstructures.

Structural metallic materials with interfaces of immiscible materials provide opportunities to design and tailor the microstructures for desired mechanical behavior. Metallic microstructures with plasticity contributors of the FCC and BCC phases show significant promise for damage-tolerant applications due to their enhanced strengths and thermal stability. A fundamental understanding of the dynamic failure behavior is needed to design and tailor these microstructures with desired mechanical responses under extreme environments. This study uses molecular dynamics (MD) simulations to characterize plasticity contributors for various interface microstructures and the damage evolution behavior of FCC/BCC laminate microstructures. This study uses six model Cu/Ta interface systems with different orientation relationships that are as- created, and pre-deformed to understand the modifications in the plasticity contributions and the void nucleation/evolution behavior. The results suggest that pre-existing misfit dislocations and loading orientations (perpendicular to and parallel to the interface) affect the activation of primary and secondary slip systems. The dynamic strengths are observed to correlate with the energy of the interfaces, with the strengths being highest for low-energy interfaces and lowest for high-energy interfaces. However, the presence of pre-deformation of these interface microstructures affects not only the dynamic strength of the microstructures but also the correlation with interface energy.

Isolation of circulating tumor cells.

Circulating tumor cells (CTCs) enter the vasculature from solid tumors and disseminate widely to initiate metastases. Mining the metastatic-enriched molecular signatures of CTCs before, during, and after treatment holds unique potential in personalized oncology. Their extreme rarity, however, requires isolation from large blood volumes at high yield and purity, yet they overlap leukocytes in size and other biophysical properties. Additionally, many CTCs lack EpCAM that underlies much of affinity-based capture, complicating their separation from blood. Here, we provide a comprehensive introduction of CTC isolation technology, by analyzing key separation modes and integrated isolation strategies. Attention is focused on recent progress in microfluidics, where an accelerating evolution is occurring in high-throughput sorting of cells along multiple dimensions.

Negative-Selection Enrichment of Circulating Tumor Cells from Peripheral Blood Using the Microfluidic CTC-iChip.

The ability to isolate and analyze rare circulating tumor cells (CTCs) holds the potential to increase our understanding of cancer evolution and allows monitoring of disease and therapeutic responses through a relatively non-invasive blood-based biopsy. While many methods have been described to isolate CTCs from the blood, the vast majority rely on size-based sorting or positive selection of CTCs based on surface markers, which introduces bias into the downstream product by making assumptions about these heterogenous cells. Here we describe a negative-selection protocol for enrichment of CTCs through removal of blood components including red blood cells, platelets, and white blood cells. This procedure results in a product that is amenable to downstream single-cell analytics including RNA-Seq, ATAC-Seq and DNA methylation, droplet digital PCR (ddPCR) for tumor specific transcripts, staining and extensive image analysis, and ex vivo culture of patient-derived CTCs.

Feature Blending: An Approach toward Generalized Machine Learning Models for Property Prediction.

From studying the atomic structure and chemical behavior to the discovery of new materials and investigating properties of existing materials, machine learning (ML) has been employed in realms that are arduous to probe experimentally. While numerous highly accurate models, specifically for property prediction, have been reported in the literature, there has been a lack of a generalized framework. Herein we propose a novel feature selection approach that enables the development of a unified ML model for property prediction for several classes of materials. It involves an ingenious blending of selected features from various classes of data such that the resultant feature set equips the model with global data descriptors capturing both class-specific as well as global traits. We took accurate band gaps of three distinct classes of 2D materials as our target property to develop the proposed feature blending approach. Using Gaussian process regression (GPR) with the blended features, the ML model developed here resulted in an average root-mean-squared error of 0.12 eV for unseen data belonging to any of the participating classes. The feature blending approach proposed here can be extended to additional classes of materials and also to predict other properties.

Fingerprinting shock-induced deformations via diffraction.

During the various stages of shock loading, many transient modes of deformation can activate and deactivate to affect the final state of a material. In order to fundamentally understand and optimize a shock response, researchers seek the ability to probe these modes in real-time and measure the microstructural evolutions with nanoscale resolution. Neither post-mortem analysis on recovered samples nor continuum-based methods during shock testing meet both requirements. High-speed diffraction offers a solution, but the interpretation of diffractograms suffers numerous debates and uncertainties. By atomistically simulating the shock, X-ray diffraction, and electron diffraction of three representative BCC and FCC metallic systems, we systematically isolated the characteristic fingerprints of salient deformation modes, such as dislocation slip (stacking faults), deformation twinning, and phase transformation as observed in experimental diffractograms. This study demonstrates how to use simulated diffractograms to connect the contributions from concurrent deformation modes to the evolutions of both 1D line profiles and 2D patterns for diffractograms from single crystals. Harnessing these fingerprints alongside information on local pressures and plasticity contributions facilitate the interpretation of shock experiments with cutting-edge resolution in both space and time.

Sulfur-Doped Titanium Carbide MXenes for Room-Temperature Gas Sensing.

Two-dimensional titanium carbide MXenes, Ti3C2Tx, possess high surface area coupled with metallic conductivity and potential for functionalization. These properties make them especially attractive for the highly sensitive room-temperature electrochemical detection of gas analytes. However, these extraordinary materials have not been thoroughly investigated for the detection of volatile organic compounds (VOCs), many of which hold high relevance for disease diagnostics and environmental protection. Furthermore, the insufficient interlayer spacing between MXene nanoflakes could limit their applicability and the use of heteroatoms as dopants could help overcome this challenge. Here, we report that S-doping of Ti3C2Tx MXene leads to a greater gas-sensing performance to VOCs compared to their undoped counterparts, with unique selectivity to toluene. After S-doped and pristine materials were synthesized, characterized, and used as electrode materials, the as-fabricated sensors were subjected to room-temperature dynamic impedimetric testing in the presence of VOCs with different functional groups (ethanol, hexane, toluene, and hexyl-acetate). Unique selectivity to toluene was obtained by both undoped and doped Ti3C2Tx MXenes, but an enhancement of response in the range of ∼214% at 1 ppm to ∼312% at 50 ppm (3-4 folds increase) was obtained for the sulfur-doped sensing material. A clear notable response to 500 ppb toluene was also obtained with sulfur-doped Ti3C2Tx MXene sensors along with excellent long-term stability. Our experimental measurements and density functional theory analysis offer insight into the mechanisms through which S-doping influences VOC analyte sensing capabilities of Ti3C2Tx MXenes, thus opening up future investigations on the development of high-performance room-temperature gas sensors.

Ultrahigh-throughput magnetic sorting of large blood volumes for epitope-agnostic isolation of circulating tumor cells.

Circulating tumor cell (CTC)-based liquid biopsies provide unique opportunities for cancer diagnostics, treatment selection, and response monitoring, but even with advanced microfluidic technologies for rare cell detection the very low number of CTCs in standard 10-mL peripheral blood samples limits their clinical utility. Clinical leukapheresis can concentrate mononuclear cells from almost the entire blood volume, but such large numbers and concentrations of cells are incompatible with current rare cell enrichment technologies. Here, we describe an ultrahigh-throughput microfluidic chip, LPCTC-iChip, that rapidly sorts through an entire leukapheresis product of over 6 billion nucleated cells, increasing CTC isolation capacity by two orders of magnitude (86% recovery with 105 enrichment). Using soft iron-filled channels to act as magnetic microlenses, we intensify the field gradient within sorting channels. Increasing magnetic fields applied to inertially focused streams of cells effectively deplete massive numbers of magnetically labeled leukocytes within microfluidic channels. The negative depletion of antibody-tagged leukocytes enables isolation of potentially viable CTCs without bias for expression of specific tumor epitopes, making this platform applicable to all solid tumors. Thus, the initial enrichment by routine leukapheresis of mononuclear cells from very large blood volumes, followed by rapid flow, high-gradient magnetic sorting of untagged CTCs, provides a technology for noninvasive isolation of cancer cells in sufficient numbers for multiple clinical and experimental applications.

In-flow measurement of cell-cell adhesion using oscillatory inertial microfluidics.

Multicellular clusters in circulation can exhibit a substantially different function and biomarker significance compared to individual cells. Notably, clusters of circulating tumor cells (CTCs) are much more effective initiators of metastasis than single CTCs, and correlate with worse patient prognoses. Measuring the cell-cell adhesion strength of CTC clusters is a critical step towards understanding their subsistence in the circulation and mechanism of elevated tumorigenicity. However, measuring cell-cell adhesion forces in flow is elusive using existing methods. Here, we report an oscillatory inertial microfluidics system which exerts a repeating fluidic force profile on suspended cell doublets to determine their cell-cell adhesion strength (Fs), without any biophysical modifications to the cell surface and physiological morphology. Using our system, we analyzed a large number (N > 500) of doublets from a patient-derived breast cancer CTC line. We discovered that the cell-cell adhesion strength of CTC doublets varied almost 20-fold between the weakly adhered (Fs < 28 nN) and strongly bound subpopulations (Fs > 542 nN). Our system can be used with other cancer or noncancer cells without restrictions, and may be used for rapid screening of drugs aiming to disrupt the highly-metastatic CTC clusters in circulation.

Microfluidic concentration and separation of circulating tumor cell clusters from large blood volumes.

Circulating tumor cells (CTCs) are extremely rare in the blood, yet they account for metastasis. Notably, it was reported that CTC clusters (CTCCs) can be 50-100 times more metastatic than single CTCs, making them particularly salient as a liquid biopsy target. Yet they can split apart and are even rarer, complicating their recovery. Isolation by filtration risks loss when clusters squeeze through filter pores over time, and release of captured clusters can be difficult. Deterministic lateral displacement is continuous but requires channels not much larger than clusters, leading to clogging. Spiral inertial focusing requires large blood dilution factors (or lysis). Here, we report a microfluidic chip that continuously isolates untouched CTC clusters from large volumes of minimally (or undiluted) whole blood. An array of 100 µm-wide channels first concentrates clusters in the blood, and then a similar array transfers them into a small volume of buffer. The microscope-slide-sized PDMS device isolates individually-spiked CTC clusters from >30 mL per hour of whole blood with 80% efficiency into enumeration (fluorescence imaging), and on-chip yield approaches 100% (high speed video). Median blood cell removal (in base-10 logs) is 4.2 for leukocytes, 5.5 for red blood cells, and 4.9 for platelets, leaving less than 0.01% of leukocytes alongside CTC clusters in the product. We also demonstrate that cluster configurations are preserved. Gentle, high throughput concentration and separation of circulating tumor cell clusters from large blood volumes will enable cluster-specific diagnostics and speed the generation of patient-specific CTC cluster lines.

Accelerated Data-Driven Accurate Positioning of the Band Edges of MXenes.

Functionalized MXene has emerged a promising class of two-dimensional materials having more than tens of thousands of compounds, whose uses may range from electronics to energy applications. Other than the band gap, these properties rely on the accurate position of the band edges. Hence, to synthesize MXenes for various applications, a prior knowledge of the accurate position of their band edges at an absolute scale is essential; computing these with conventional methods would take years for all the MXenes. Here, we develop a machine learning model for positioning the band edges with GW level of accuracy having a minimum root-mean-squared error of 0.12 eV. An intuitive model is proposed based on the combination of Perdew-Burke-Ernzerhof band edge and vacuum potential having a correlation of 0.93 with GW band edges. These models can be utilized to identify MXenes for a desired application in an accelerated manner.

Trapped Chromatin Fibers Damage Flowing Red Blood Cells.

Neutrophils are the most abundant white blood cells in the circulation and serve antimicrobial functions. One of their antimicrobial mechanisms involves the release of neutrophil extracellular traps (NETs), long chromatin fibers decorated with antimicrobial granular proteins that contribute to the elimination of pathogens. However, the release of NETs has also been associated with disease processes. While recent research has focused on biochemical reactions catalyzed by NETs, significantly less is known about the mechanical effect of NETs in circulation. Here, microfluidic devices and biophysical models are employed to study the consequences of the interactions between NETs trapped in channels and red blood cells (RBCs) flowing in blood over the NETs. It has been found that the RBCs can be deformed and ruptured after interactions with NETs, generating RBC fragments. Significant increases in the number of RBC fragments have also been found in the circulation of patients with conditions in which NETs have been demonstrated to be present in circulation, including sepsis and kidney transplant. Further studies will probe the potential utility of RBC fragments in the diagnostic, monitoring, and treatment of diseases associated with the presence of NETs in circulation.

Ferroelectricity, Antiferroelectricity, and Ultrathin 2D Electron/Hole Gas in Multifunctional Monolayer MXene.

The presence of ferroelectric polarization in 2D materials is extremely rare due to the effect of the surface depolarizing field. Here, we use first-principles calculations to show the largest out-of-plane polarization observed in a monolayer in functionalized MXenes (Sc2CO2). The switching of polarization in this new class of ferroelectric materials occurs through a previously unknown intermediate antiferroelectric structure, thus establishing three states for applications in low-dimensional nonvolatile memory. We show that the armchair domain interface acts as an 1D metallic nanowire separating two insulating domains. In the case of the van der Waals bilayer we observe, interestingly, the presence of an ultrathin 2D electron/hole gas (2DEG) on the top/bottom layers, respectively, due to the redistrubution of charge carriers. The 2DEG is nondegenerate due to spin-orbit coupling, thus paving the way for spin-orbitronic devices. The coexistence of ferroelectricity, antiferroelectricity, 2DEG, and spin-orbit splitting in this system suggests that such 2D polar materials possess high potential for device application in a multitude of fields ranging from nanoelectronics to photovoltaics.

Mechanistic Insight into the Chemical Exfoliation and Functionalization of Ti3C2 MXene.

MXene, a two-dimensional layer of transition metal carbides/nitrides, showed great promise for energy storage, sensing, and electronic applications. MXene are chemically exfoliated from the bulk MAX phase; however, mechanistic understanding of exfoliation and subsequent functionalization of these technologically important materials is still lacking. Here, using density-functional theory we show that exfoliation of Ti3C2 MXene proceeds via HF insertion through edges of Ti3AlC2 MAX phase. Spontaneous dissociation of HF and subsequent termination of edge Ti atoms by H/F weakens Al-MXene bonds. Consequent opening of the interlayer gap allows further insertion of HF that leads to the formation of AlF3 and H2, which eventually come out of the MAX, leaving fluorinated MXene behind. Density of state and electron localization function shows robust binding between F/OH and Ti, which makes it very difficult to obtain controlled functionalized or pristine MXene. Analysis of the calculated Gibbs free energy (ΔG) shows fully fluorinated MXene to be lowest in energy, whereas the formation of pristine MXene is thermodynamically least favorable. In the presence of water, mixed functionalized Ti3C2Fx(OH)1-x (x ranges from 0 to 1) MXene can be obtained. The ΔG values for the mixed functionalized MXenes are very close in energy, indicating the random and nonuniform functionalization of MXene. The microscopic understanding gained here unveils the challenges in exfoliation and controlling the functionalization of MXene, which is essential for its practical application.

Isolation of pristine MXene from Nb4AlC3 MAX phase: a first-principles study.

Synthesis of pristine MXene sheets from MAX phase is one of the foremost challenges in getting a complete understanding of the properties of this new technologically important 2D-material. Efforts to exfoliate Nb4AlC3 MAX phase always lead to Nb4C3 MXene sheets, which are functionalized and have several Al atoms attached. Using the first-principles calculations, we perform an intensive study on the chemical transformation of MAX phase into MXene sheets by inserting HF, alkali atoms and LiF in Nb4AlC3 MAX phase. Calculated bond-dissociation energy (BDE) shows that the presence of HF in MAX phase always results in functionalized MXene, as the binding of H with MXene is quite strong while that with F is weak. Insertion of alkali atoms does not facilitate pristine MXene isolation due to the presence of chemical bonds of almost equal strength. In contrast, weak Li-MXene and strong Li-F bonding in Nb4AlC3 with LiF ensured strong anisotropy in BDE, which will result in the dissociation of the Li-MXene bond. Ab initio molecular dynamics calculations capture these features and show that at 500-650 K, the Li-MXene bond indeed breaks leaving a pristine MXene sheet behind. The approach and insights developed here for chemical exfoliation of layered materials bonded by chemical bonds instead of van der Waals can promote their experimental realization.

Optoelectric patterning: Effect of electrode material and thickness on laser-induced AC electrothermal flow.

Rapid electrokinetic patterning (REP) is an emerging optoelectric technique that takes advantage of laser-induced AC electrothermal flow and particle-electrode interactions to trap and translate particles. The electrothermal flow in REP is driven by the temperature rise induced by the laser absorption in the thin electrode layer. In previous REP applications 350-700 nm indium tin oxide (ITO) layers have been used as electrodes. In this study, we show that ITO is an inefficient electrode choice as more than 92% of the irradiated laser on the ITO electrodes is transmitted without absorption. Using theoretical, computational, and experimental approaches, we demonstrate that for a given laser power the temperature rise is controlled by both the electrode material and its thickness. A 25-nm thick Ti electrode creates an electrothermal flow of the same speed as a 700-nm thick ITO electrode while requiring only 14% of the laser power used by ITO. These results represent an important step in the design of low-cost portable REP systems by lowering the material cost and power consumption of the system.

Dynamic optoelectric trapping and deposition of multiwalled carbon nanotubes.

In the path toward the realization of carbon nanotube (CNT)-driven electronics and sensors, the ability to precisely position CNTs at well-defined locations remains a significant roadblock. Highly complex CNT-based bottom-up structures can be synthesized if there is a method to accurately trap and place these nanotubes. In this study, we demonstrate that the rapid electrokinetic patterning (REP) technique can accomplish these tasks. By using laser-induced alternating current (AC) electrothermal flow and particle-electrode forces, REP can collect and maneuver a wide range of vertically aligned multiwalled CNTs (from a single nanotube to over 100 nanotubes) on an electrode surface. In addition, these trapped nanotubes can be electrophoretically deposited at any desired location onto the electrode surface. Apart from active control of the position of these deposited nanotubes, the number of CNTs in a REP trap can also be dynamically tuned by changing the AC frequency or by adjusting the concentration of the dispersed nanotubes. On the basis of a calculation of the stiffness of the REP trap, we found an upper limit of the manipulation speed, beyond which CNTs fall out of the REP trap. This peak manipulation speed is found to be dependent on the electrothermal flow velocity, which can be varied by changing the strength of the AC electric field.

Mapping surface tension induced menisci with application to tensiometry and refractometry.

In this work, we discuss an optical method for measuring surface tension induced menisci. The principle of measurement is based upon the change in the background pattern produced by the curvature of the meniscus acting as a lens. We measure the meniscus profile over an inclined glass plate and utilize the measured meniscus for estimation of surface tension and refractive index.