Droplet Microfluidic Technology for the Early and Label-Free Isolation of Highly-Glycolytic, Activated T-Cells.

A label-free, fixation-free and passive sorting method is presented to isolate activated T-cells shortly after activation and prior to the display of activation surface markers. It uses a recently developed sorting platform dubbed "Sorting by Interfacial Tension" (SIFT) that sorts droplets based on pH. After polyclonal (anti-CD3/CD28 bead) activation and a brief incubation on chip, droplets containing activated T-cells display a lower pH than those containing naive cells due to increased glycolysis. Under specific surfactant conditions, a change in pH can lead to a concurrent increase in droplet interfacial tension. The isolation of activated T-cells on chip is hence achieved as flattened droplets are displaced as they encounter a micro-fabricated trench oriented diagonally with respect to the direction of flow. This technique leads to an enrichment of activated primary CD4+ T-cells to over 95% from an initial mixed population of naive cells and cells activated for as little as 15 min. Moreover, since the pH change is correlated to successful activation, the technique allows the isolation of T-cells with the earliest activation and highest glycolysis, an important feature for the testing of T-cell activation modulators and to determine regulators and predictors of differentiation outcomes.

Method for Passive Droplet Sorting after Photo-Tagging.

We present a method to photo-tag individual microfluidic droplets for latter selection by passive sorting. The use of a specific surfactant leads to the interfacial tension to be very sensitive to droplet pH. The photoexcitation of droplets containing a photoacid, pyranine, leads to a decrease in droplet pH. The concurrent increase in droplet interfacial tension enables the passive selection of irradiated droplets. The technique is used to select individual droplets within a droplet array as illuminated droplets remain in the wells while other droplets are eluted by the flow of the external oil. This method was used to select droplets in an array containing cells at a specific stage of apoptosis. The technique is also adaptable to continuous-flow sorting. By passing confined droplets over a microfabricated trench positioned diagonally in relation to the direction of flow, photo-tagged droplets were directed toward a different chip exit based on their lateral movement. The technique can be performed on a conventional fluorescence microscope and uncouples the observation and selection of droplets, thus enabling the selection on a large variety of signals, or based on qualitative user-defined features.

Microfluidic Platform for the Isolation of Cancer-Cell Subpopulations Based on Single-Cell Glycolysis.

High rates of glycolysis in tumors have been associated with cancer metastasis, tumor recurrence, and poor outcomes. In this light, single cells that exhibit high glycolysis are specific targets for therapy. However, the study of these cells requires efficient tools for their isolation. We use a droplet microfluidic technique developed in our lab, Sorting by Interfacial Tension (SIFT), to isolate cancer cell subpopulations based on glycolysis without the use of labels or active sorting components. By controlling the flow conditions on chip, the threshold of selection can be modified, enabling the isolation of cells with different levels of glycolysis. Hypoxia in tumors, that can be simulated with treatment with CoCl2, leads to an increase in glycolysis, and more dangerous tumors. The device was used to enrich CoCl2 treated MDA-MB 231 breast cancer cells from an untreated population. It is also used to sort K562 human chronic myelogenous leukemia cells that have either been treated or untreated with 2-deoxy-d-glucose (2DG), a pharmaceutical that targets cell metabolism. The technique provides a facile and robust way of separating cells based on elevated glycolytic activity; a biomarker associated with cancer cell malignancy.

Sorting by interfacial tension (SIFT): Label-free enzyme sorting using droplet microfluidics.

Droplet microfluidics has the ability to greatly increase the throughput of screening and sorting of enzymes by carrying reagents in picoliter droplets flowing in inert oils. It was found with the use of a specific surfactant, the interfacial tension of droplets can be very sensitive to droplet pH. This enables the sorting of droplets of different pH when confined droplets encounter a microfabricated trench. The device can be extended to sort enzymes, as a large number of enzymatic reactions lead to the production of an acidic or basic product and a concurrent change in solution pH. The progress of an enzymatic reaction is tracked from the position of a flowing train of droplets. We demonstrate the sorting of esterase isoenzymes based on their enzymatic activity. This label-free technology, that we dub droplet sorting by interfacial tension (SIFT), requires no active components and would have applications for enzyme sorting in high-throughput applications that include enzyme screening and directed evolution of enzymes.

Multiplexed Single-Cell Measurements of FDG Uptake and Lactate Release Using Droplet Microfluidics.

INTRODUCTION: Glucose utilization and lactate release are 2 important indicators of cancer metabolism. Most tumors consume glucose and release lactate at a higher rate than normal tissues due to enhanced aerobic glycolysis. However, these 2 indicators of metabolism have not previously been studied on a single-cell level, in the same cell. OBJECTIVE: To develop and characterize a novel droplet microfluidic device for multiplexed measurements of glucose uptake (via its analog 18F-fluorodeoxyglucose) and lactate release, in single live cells encapsulated in an array of water-in-oil droplets. RESULTS: Surprisingly, 18F-fluorodeoxyglucose uptake and lactate release were only marginally correlated at the single-cell level, even when assayed in a standard cell line (MDA-MB-231). While 18F-fluorodeoxyglucose-avid cells released substantial amounts of lactate, the reverse was not true, and many cells released high amounts of lactate without taking up 18F-fluorodeoxyglucose. DISCUSSION: These results confirm that cancer cells rely on multiple metabolic pathways in addition to aerobic glycolysis and that the use of these pathways is highly heterogeneous, even under controlled culture conditions. Clinically, the large cell-to-cell variability suggests that positron emission tomography measurements of 18F-fluorodeoxyglucose uptake represent metabolic flux only in an aggregate sense, not for individual cancer cells within the tumor.

Sorting by interfacial tension (SIFT): label-free selection of live cells based on single-cell metabolism.

Selection of live cells from a population is critical in many biological studies and biotechnologies. We present here a novel droplet microfluidic approach that allows for label-free and passive selection of live cells using the glycolytic activity of individual cells. It was observed that with the use of a specific surfactant utilized to stabilize droplet formation, the interfacial tension of droplets was very sensitive to pH. After incubation, cellular lactate release results in droplets containing a live cell to attain a lower pH than other droplets. This enables the sorting of droplets containing live cells when confined droplets flow over a microfabricated trench oriented diagonally with respect to the direction of flow. The technique is demonstrated with human U87 glioblastoma cells for the selection of only droplets containing a live cell while excluding either empty droplets or droplets containing a dead cell. This label-free sorting method, dubbed sorting by interfacial tension (SIFT) presents a new strategy to sort diverse cell types based on metabolic activity.

Toward a Droplet-Based Single-Cell Radiometric Assay.

Radiotracers are widely used to track molecular processes, both in vitro and in vivo, with high sensitivity and specificity. However, most radionuclide detection methods have spatial resolution inadequate for single-cell analysis. A few existing methods can extract single-cell information from radioactive decays, but the stochastic nature of the process precludes high-throughput measurement (and sorting) of single cells. In this work, we introduce a new concept for translating radioactive decays occurring stochastically within radiolabeled single-cells into an integrated, long-lasting fluorescence signal. Single cells are encapsulated in radiofluorogenic droplets containing molecular probes sensitive to byproducts of ionizing radiation (primarily reactive oxygen species, or ROS). Different probes were examined in bulk solutions, and dihydrorhodamine 123 (DHRh 123) was selected as the lead candidate due to its sensitivity and reproducibility. Fluorescence intensity of DHRh 123 in bulk increased at a rate of 54% per Gy of X-ray radiation and 15% per MBq/ml of 2-deoxy-2-[18F]-fluoro-d-glucose ([18F]FDG). Fluorescence imaging of microfluidic droplets showed the same linear response, but droplets were less sensitive overall than the bulk ROS sensor (detection limit of 3 Gy per droplet). Finally, droplets encapsulating radiolabeled cancer cells allowed, for the first time, the detection of [18F]FDG radiotracer uptake in single cells through fluorescence activation. With further improvements, we expect this technology to enable quantitative measurement and selective sorting of single cells based on the uptake of radiolabeled small molecules.

Droplet Microfluidic Platform for the Determination of Single-Cell Lactate Release.

Cancer cells release high levels of lactate that has been correlated to increased metastasis and tumor recurrence. Single-cell measurements of lactate release can identify malignant cells and help decipher metabolic cancer pathways. We present here a novel droplet microfluidic method that allows the fast and quantitative determination of lactate release in many single cells. Using passive forces, droplets encapsulated cells are positioned in an array. The single-cell lactate release rate is determined from the increase in droplet fluorescence as the lactate is enzymatically converted to a fluorescent product. The method is used to measure the cell-to-cell variance of lactate release in K562 leukemia and U87 glioblastoma cancer cell lines and under the chemical inhibition of lactate efflux. The technique can be used in the study of cancer biology, but more broadly in cell biology, to capture the full range of stochastic variations in glycolysis activity in heterogeneous cell populations in a repeatable and high-throughput manner.

Single-Cell Analysis of [18F]Fluorodeoxyglucose Uptake by Droplet Radiofluidics.

Radiolabels can be used to detect small biomolecules with high sensitivity and specificity without interfering with the biochemical activity of the labeled molecule. For instance, the radiolabeled glucose analogue, [18F]fluorodeoxyglucose (FDG), is routinely used in positron emission tomography (PET) scans for cancer diagnosis, staging, and monitoring. However, despite their widespread usage, conventional radionuclide techniques are unable to measure the variability and modulation of FDG uptake in single cells. We present here a novel microfluidic technique, dubbed droplet radiofluidics, that can measure radiotracer uptake for single cells encapsulated into an array of microdroplets. The advantages of this approach are multiple. First, droplets can be quickly and easily positioned in a predetermined pattern for optimal imaging throughput. Second, droplet encapsulation reduces cell efflux as a confounding factor, because any effluxed radionuclide is trapped in the droplet. Last, multiplexed measurements can be performed using fluorescent labels. In this new approach, intracellular radiotracers are imaged on a conventional fluorescence microscope by capturing individual flashes of visible light that are produced as individual positrons, emitted during radioactive decay, traverse a scintillator plate placed below the cells. This method is used to measure the cell-to-cell heterogeneity in the uptake of tracers such as FDG in cell lines and cultured primary cells. The capacity of the platform to perform multiplexed measurements was demonstrated by measuring differential FDG uptake in single cells subjected to different incubation conditions and expressing different types of glucose transporters. This method opens many new avenues of research in basic cell biology and human disease by capturing the full range of stochastic variations in highly heterogeneous cell populations in a repeatable and high-throughput manner.

Circulating cell membrane microparticles transfer heme to endothelial cells and trigger vasoocclusions in sickle cell disease.

Intravascular hemolysis describes the relocalization of heme and hemoglobin (Hb) from erythrocytes to plasma. We investigated the concept that erythrocyte membrane microparticles (MPs) concentrate cell-free heme in human hemolytic diseases, and that heme-laden MPs have a physiopathological impact. Up to one-third of cell-free heme in plasma from 47 patients with sickle cell disease (SCD) was sequestered in circulating MPs. Erythrocyte vesiculation in vitro produced MPs loaded with heme. In silico analysis predicted that externalized phosphatidylserine (PS) in MPs may associate with and help retain heme at the cell surface. Immunohistology identified Hb-laden MPs adherent to capillary endothelium in kidney biopsies from hyperalbuminuric SCD patients. In addition, heme-laden erythrocyte MPs adhered and transferred heme to cultured endothelial cells, inducing oxidative stress and apoptosis. In transgenic SAD mice, infusion of heme-laden MPs triggered rapid vasoocclusions in kidneys and compromised microvascular dilation ex vivo. These vascular effects were largely blocked by heme-scavenging hemopexin and by the PS antagonist annexin-a5, in vitro and in vivo. Adversely remodeled MPs carrying heme may thus be a source of oxidant stress for the endothelium, linking hemolysis to vascular injury. This pathway might provide new targets for the therapeutic preservation of vascular function in SCD.

Selective fusion of anchored droplets via changes in surfactant concentration.

We present a robust method to fuse in parallel an array of anchored droplets in a microchannel. Pairs of anchored droplets are fused by the removal of surfactant from the droplet interface by reducing the surfactant content in the flowing external oil phase. By controlling the flow of multiple oil inlets, the selective fusion of rows of droplets in a larger array is demonstrated. The technique is compatible with cells as shown with a trypan blue exclusion vitality assay. The method is easy to implement, requires no active components and is applicable to oil/water combinations where the surfactant is soluble in the external phase.

Parallel measurements of reaction kinetics using ultralow-volumes.

We present a new platform for the production and manipulation of microfluidic droplets in view of measuring the evolution of a chemical reaction. Contrary to existing approaches, our device uses gradients of confinement to produce a single drop on demand and guide it to a pre-determined location. In this way, two nanoliter drops containing different reagents can be placed in contact and merged together, in order to trigger a chemical reaction. The reaction rate is extracted from an analysis of the observed reaction-diffusion front. We show that the results obtained using this platform are in excellent agreement with stopped-flow measurements, while decreasing the sample consumption 5000 fold. We also show how the device operation can be parallelized in order to react an initial sample with a range of compounds or concentrations, on a single integrated chip. This integrated chip thus further reduces sample consumption while reducing the time required for the experimental runs from hours to minutes.

Combining rails and anchors with laser forcing for selective manipulation within 2D droplet arrays.

We demonstrate the combination of a rails and anchors microfluidic system with laser forcing to enable the creation of highly controllable 2D droplet arrays. Water droplets residing in an oil phase can be pinned to anchor holes made in the base of a microfluidic channel, enabling the creation of arrays by the appropriate patterning of such holes. The introduction of laser forcing, via laser induced thermocapillary forces to anchored droplets, enables the selective extraction of particular droplets from an array. We also demonstrate that such anchor arrays can be filled with multiple, in our case two, droplets each and that if such droplets have different chemical contents, the application of a laser at their interface triggers their merging and a chemical reaction to take place. Finally by adding guiding rails within the microfluidic structure we can selectively fill large scale arrays with monodisperse droplets with significant control over their contents. In this way we make a droplet array filled with 96 droplets containing different concentrations of fluorescent microparticles.

Rails and anchors: guiding and trapping droplet microreactors in two dimensions.

This paper presents a method to control the motion of nanolitre drops in a wide and thin microchannel, by etching fine patterns into the channel's top surface. Such control is possible for drops that are squeezed by the channel roof, by allowing them to reduce their surface energy as they enter into a local depression. The resulting gain in surface energy pulls a drop into the groove such that localized holes can be used as anchors for holding drops, while linear patterns can be used as rails to guide them along complex trajectories. An anchored drop can remain stationary indefinitely, as long as the driving flow rate is below a critical value which depends on the hole and drop sizes. By micro-fabricating holes into a grid pattern, drops can be arrayed and held in the observation field of a microscope against the mean carrier flow. Their contents can then be modulated by gas exchange with the flowing carrier oil. We demonstrate in particular how the pH or the oxygen levels within the drops can be controlled spatially and temporally, either by exposing rows of drops to two streams of oil at different gas concentrations or by periodically switching oil inputs to vary the gas concentration of drops as a function of time. Oxygen control is used to selectively deoxygenate droplets that encapsulate red blood cells from patients suffering from sickle cell disease, in order to study the polymerization of intracellular hemoglobin. Cycles of oxygenation and deoxygenation of anchored droplets induce depolymerization and polymerization of the hemoglobin, thus providing a method to simulate the cycling that takes place in physiological flows.

Sickling of red blood cells through rapid oxygen exchange in microfluidic drops.

We have developed a microfluidic approach to study the sickling of red blood cells associated with sickle cell anemia by rapidly varying the oxygen partial pressure within flowing microdroplets. By using the perfluorinated carrier oil as a sink or source of oxygen, the oxygen level within the water droplets quickly equilibrates through exchange with the surrounding oil. This provides control over the oxygen partial pressure within an aqueous drop ranging from 1 kPa to ambient partial pressure, i.e. 21 kPa. The dynamics of the oxygen exchange is characterized through fluorescence lifetime measurements of a ruthenium compound dissolved in the aqueous phase. The gas exchange is shown to occur primarily during and directly after droplet formation, in 0.1 to 0.5 s depending on the droplet diameter and speed. The controlled deoxygenation is used to trigger the polymerization of hemoglobin within sickle red blood cells, encapsulated in drops. This process is observed using polarization microscopy, which yields a robust criterion to detect polymerization based on transmitted light intensity through crossed polarizers.

Dynamic Stokes shift in green fluorescent protein variants.

Solvent reorganization around the excited state of a chromophore leads to an emission shift to longer wavelengths during the excited-state lifetime. This solvation response is absent in wild-type green fluorescent protein, and this has been attributed to rigidity in the chromophore's environment necessary to exclude nonradiative transitions to the ground state. The fluorescent protein mPlum was developed via directed evolution by selection for red emission, and we use time-resolved fluorescence to study the dynamic Stokes shift through its evolutionary history. The far-red emission of mPlum is attributed to a picosecond solvation response that is observed at all temperatures above the glass transition. This time-dependent shift in emission is not observed in its evolutionary ancestors, suggesting that selective pressure has produced a chromophore environment that allows solvent reorganization. The evolutionary pathway and structures of related fluorescent proteins suggest the role of a single residue in close proximity to the chromophore as the primary cause of the solvation response.

Ultrafast excited-state dynamics in the green fluorescent protein variant S65T/H148D. 1. Mutagenesis and structural studies.

Wild type green fluorescent protein (wt-GFP) and the variant S65T/H148D each exhibit two absorption bands, A and B, which are associated with the protonated and deprotonated chromophores, respectively. Excitation of either band leads to green emission. In wt-GFP, excitation of band A ( approximately 395 nm) leads to green emission with a rise time of 10-15 ps, due to excited-state proton transfer (ESPT) from the chromophore hydroxyl group to an acceptor. This process produces an anionic excited-state intermediate I* that subsequently emits a green photon. In the variant S65T/H148D, the A band absorbance maximum is red-shifted to approximately 415 nm, and as detailed in the accompanying papers, when the A band is excited, green fluorescence appears with a rise time shorter than the instrument time resolution ( approximately 170 fs). On the basis of the steady-state spectroscopy and high-resolution crystal structures of several variants described herein, it is proposed that in S65T/H148D, the red shift of absorption band A and the ultrafast appearance of green fluorescence upon excitation of band A are due to a very short (

Ultrafast excited-state dynamics in the green fluorescent protein variant S65T/H148D. 2. Unusual photophysical properties.

In the preceding accompanying paper [Shu, X., et al. (2007) Biochemistry 46, 12005-12013], the 1.5 A resolution crystal structure of green fluorescent protein (GFP) variant S65T/H148D is presented, and the possible consequences of an unusual short hydrogen bond (

Measurement of solvation responses at multiple sites in a globular protein.

Proteins respond to electrostatic perturbations through complex reorganizations of their charged and polar groups, as well as those of the surrounding media. These solvation responses occur both in the protein interior and on its surface, though the exact mechanisms of solvation are not well understood, in part because of limited data on the solvation responses for any given protein. Here, we characterize the solvation kinetics at sites throughout the sequence of a small globular protein, the B1 domain of streptococcal protein G (GB1), using the synthetic fluorescent amino acid Aladan. Aladan was incorporated into seven different GB1 sites, and the time-dependent Stokes shift was measured over the femtosecond to nanosecond time scales by fluorescence upconversion and time-correlated single photon counting. The seven sites range from buried within the protein core to fully solvent-exposed on the protein surface, and are located on different protein secondary structures including beta-sheets, helices, and loops. The dynamics in the protein sites were compared against the free fluorophore in buffer. All protein sites exhibited an initial, ultrafast Stokes shift on the subpicosecond time scale similar to that observed for the free fluorophore, but smaller in magnitude. As the probe is moved from the surface to more buried sites, the dynamics of the solvation response become slower, while no clear correlation between dynamics and secondary structure is observed. We suggest that restricted movements of the surrounding protein residues give rise to the observed long time dynamics and that such movements comprise a large portion of the protein's solvation response. The proper treatment of dynamic Stokes shift data when the time scale for solvation is comparable to the fluorescence lifetime is discussed.

Antibody evolution constrains conformational heterogeneity by tailoring protein dynamics.

The evolution of proteins with novel function is thought to start from precursor proteins that are conformationally heterogeneous. The corresponding genes may be duplicated and then mutated to select and optimize a specific conformation. However, testing this idea has been difficult because of the challenge of quantifying protein flexibility and conformational heterogeneity as a function of evolution. Here, we report the characterization of protein heterogeneity and dynamics as a function of evolution for the antifluorescein antibody 4-4-20. Using nonlinear laser spectroscopy, surface plasmon resonance, and molecular dynamics simulations, we demonstrate that evolution localized the Ab-combining site from a heterogeneous ensemble of conformations to a single conformation by introducing mutations that act cooperatively and over significant distances to rigidify the protein. This study demonstrates how protein dynamics may be tailored by evolution and has important implications for our understanding of how novel protein functions are evolved.