Sub-nanoliter Droplets Research Articles

Magnetic Microtweezers for High-Throughput Bioseparation in Sub-Nanoliter Droplets.

Multiomics studies at single-cell level require small volume manipulation, high throughput analysis, and multiplexed detection, characteristics that droplet microfluidics can tackle. However, the initial step of molecule bioseparation remains challenging. Here, we describe a unique magnetic device to trap and extract magnetic particles in sub-nanoliter droplets, for compartmentalisation of detection steps. Relying on electrodeposition of NiFe structures and microfluidic manipulation, the extraction of 1μm diameter magnetic particles was achieved at high throughput (20 droplets per second) with an efficiency close to 100% in 450 pL droplets. The first demonstration of its adaptability to single-cell analysis is demonstrated with the extraction of mRNA. Using a purified nucleic acid solution, this unique magnetic configuration was able to reach a RNA extraction rate of 72%. This is the first demonstration of a physical separation in droplets at high throughput at single-cell scale.

Ultrasonic investigation of the evaporation dynamics of subnanoliter droplets

With the development of ink jet printers or spotters for biological applications, the control size and homogeneity of small fluids as well as their successful deposition on various substrates has gained significant importance. We study the impingement process of such small droplets onto a solid substrate and investigate their evaporation and drying process by means of surface acoustic wave (SAW) transmission experiments. We observe a strong fluid-SAW interaction resulting in an attenuation which toward the end of the evaporation and drying process exhibits characteristic oscillations being related to temperature, SAW amplitude, and fluid properties such as viscosity and wetting angle. It is found that oscillations can only be seen in pinned, very flat droplets, while the pinning can be roughly controlled via the SAW amplitude. The frequency of those characteristic oscillations is superlinear with respect to temperature, rather proportional to the evaporation rate and can change drastically depending on the liquid used. In our experiments, isopropanol, ethanol, and water with various glycerol concentrations were loaded onto the SAW delay line. Based on the experimental results, simulations using the finite element method were performed. We find that a standing wave pattern within the droplet is responsible for the transmission oscillations. These findings might lead to new methods, e.g., sensory systems, being able to examine droplets according to their physical or chemical properties.

Controlling Droplet Impact Velocity and Droplet Volume: Key Factors to Achieving High Cell Viability in Sub-Nanoliter Droplet-based Bioprinting

Three-dimensional (3D) bioprinting systems serve as advanced manufacturing platform for the precise deposition of cells and biomaterials at pre-defined positions. Among the various bioprinting techniques, the drop-on-demand jetting approach facilitates deposition of pico/nanoliter droplets of cells and materials for study of cell-cell and cell-matrix interactions. Despite advances in the bioprinting systems, there is a poor understanding of how the viability of primary human cells within sub-nanoliter droplets is affected during the printing process. In this work, a thermal inkjet system is utilized to dispense sub-nanoliter cell-laden droplets, and two key factors – droplet impact velocity and droplet volume – are identified to have significant effect on the viability and proliferation of printed cells. An increase in the cell concentration results in slower impact velocity, which leads to higher viability of the printed cells and improves the printing outcome by mitigating droplet splashing. Furthermore, a minimum droplet volume of 20 nL per spot helps to mitigate evaporation-induced cell damage and maintain high viability of the printed cells within a printing duration of 2 min. Hence, controlling the droplet impact velocity and droplet volume in sub-nanoliter bioprinting is critical for viability and proliferation of printed human primary cells.

Droplet-Based Microfluidics Methods for Detecting Enzyme Inhibitors.

Sub-nanoliter droplets produced in microfluidic devices have gained an enormous importance for performing all kinds of biochemical assays. One of the main reasons is that the amounts of reagents employed can be reduced in approximately five orders of magnitude compared to conventional microplate assays. In this chapter, we describe how to carry out the design, fabrication, and operation of a microfluidic device that allows performing enzyme kinetics and enzyme inhibition assays in droplets. This procedure can be used effectively to screen a small size library of compounds. Then, we describe how to use this droplet microfluidic setup to screen for potential inhibitor compounds eluted from a coupled high-performance liquid chromatography (HPLC) system that separates crude natural extracts.

10,368 first dates: Microfluidic T cell matchmaking

Coencapsulation of T cells and target cells into arrays of subnanoliter droplets improves the identification of functional T cell receptors.

Optical calorimetry in microfluidic droplets.

A novel microfluidic calorimeter that measures the enthalpy change of reactions occurring in 100 μm diameter aqueous droplets in fluoropolymer oil has been developed. The aqueous reactants flow into a microfluidic droplet generation chip in separate fluidic channels, limiting contact between the streams until immediately before they form the droplet. The diffusion-driven mixing of reactants is predominantly restricted to within the droplet. The temperature change in droplets due to the heat of reaction is measured optically by recording the reflectance spectra of encapsulated thermochromic liquid crystals (TLC) that are added to one of the reactant streams. As the droplets travel through the channel, the spectral characteristics of the TLC represent the internal temperature, allowing optical measurement with a precision of ≈6 mK. The microfluidic chip and all fluids are temperature controlled, and the reaction heat within droplets raises their temperature until thermal diffusion dissipates the heat into the surrounding oil and chip walls. Position resolved optical temperature measurement of the droplets allows calculation of the heat of reaction by analyzing the droplet temperature profile over time. Channel dimensions, droplet generation rate, droplet size, reactant stream flows and oil flow rate are carefully balanced to provide rapid diffusional mixing of reactants compared to thermal diffusion, while avoiding thermal "quenching" due to contact between the droplets and the chip walls. Compared to conventional microcalorimetry, which has been used in this work to provide reference measurements, this new continuous flow droplet calorimeter has the potential to perform titrations ≈1000-fold faster while using ≈400-fold less reactants per titration.

Microfluidic screening of antibiotic susceptibility at a single-cell level shows the inoculum effect of cefotaxime on E. coli.

Measurement of antibiotic susceptibility at the level of single cells is important as it reveals the concentration of an antibiotic that leads to drug resistance in bacterial strains. To date, no solution for large-scale studies of antibiotic susceptibility at the single-cell level has been shown. Here, we present a method for production and separation of emulsions consisting of subnanoliter droplets that allows us to identify each emulsion by their spatial position in the train of emulsions without chemical barcoding. The emulsions of droplets are separated by a third immiscible phase, thus forming large compartments-tankers-each filled with an emulsion of droplet reactors. Each tanker in a train can be set under different reaction conditions for hundreds or thousands of replications of the same reaction. The tankers allow for long term incubation - needed to check for growth of bacteria under a screen of conditions. We use microfluidic tankers to analyze susceptibility to cefotaxime in ca. 1900 replications for each concentration of the antibiotic in one experiment. We test cefotaxime susceptibility for different initial concentrations of bacteria, showing the inoculum effect down to the level of single cells for more than a hundred single-cell events per tanker. Lastly, we use tankers to observe the formation of aggregates of bacteria in the presence of cefotaxime in the increasing concentration of the antibiotic. The microfluidic tankers allow for facile studies of the inoculum effect and antibiotic susceptibility, and constitute an attractive, label-free screening method for a variety of other experiments in chemistry and biology.

Analysis of ribonuclease activity in sub-nanoliter droplets by label-free fluorescence measurements.

We report the results of a label-free analysis of ribonuclease activity using droplet-based microfluidics. The ribonucleolytic activity of ribonucleases (RNases) plays a critical role in cellular functions such as development, survival, growth and differentiation. Altered ribonucleolytic activity and/or the expression level of the RNase A family are known to be associated with pancreatic, bladder, ovarian and thyroid cancers among others. For this reason, the RNase A family is a meaningful protein biomarker that can be used in the diagnosis of cancer and as a target for new drug screening. There are some successful traditional methods for analysing the RNase activity, such as radioactive label-based assay, methylene blue-based assay, gel zymography, as well as other more recently developed methods such as electrochemical assay and fluorescence resonance energy transfer (FRET). However, these methods require analytical samples with a volume ranging from microliters to milliliters, and are not suitable for high-throughput analysis. Therefore, we integrated ethidium bromide (EtBr), which intercalates the chemical itself to nucleic acid, to droplet-based microfluidics for a cost-effective, high-throughput analysis. Put simply, this method is dependent on the amount of intercalated EtBr molecules on RNA. Our assay also uses visible light that is harmless to humans, unlike previous methods that used harmful UV rays, to excite the EtBr molecules. Specifically, we monitored the ribonucleolytic activity of less than 10 nM RNase A in droplets of about 330 picoliters. Also, half the maximal inhibitory concentration (IC50) of the RNase inhibitor was successfully measured in the same volume of droplets at a frequency of 40 hertz.

Digital Droplet Multiple Displacement Amplification (ddMDA) for Whole Genome Sequencing of Limited DNA Samples.

Multiple displacement amplification (MDA) is a widely used technique for amplification of DNA from samples containing limited amounts of DNA (e.g., uncultivable microbes or clinical samples) before whole genome sequencing. Despite its advantages of high yield and fidelity, it suffers from high amplification bias and non-specific amplification when amplifying sub-nanogram of template DNA. Here, we present a microfluidic digital droplet MDA (ddMDA) technique where partitioning of the template DNA into thousands of sub-nanoliter droplets, each containing a small number of DNA fragments, greatly reduces the competition among DNA fragments for primers and polymerase thereby greatly reducing amplification bias. Consequently, the ddMDA approach enabled a more uniform coverage of amplification over the entire length of the genome, with significantly lower bias and non-specific amplification than conventional MDA. For a sample containing 0.1 pg/μL of E. coli DNA (equivalent of ~3/1000 of an E. coli genome per droplet), ddMDA achieves a 65-fold increase in coverage in de novo assembly, and more than 20-fold increase in specificity (percentage of reads mapping to E. coli) compared to the conventional tube MDA. ddMDA offers a powerful method useful for many applications including medical diagnostics, forensics, and environmental microbiology.

Microdroplet-Based Potentiometric Redox Measurements on Gold Nanoporous Electrodes.

Potentiometric redox measurements were made in subnanoliter droplets of solutions using an optically transparent nanoporous gold electrode strategically mounted on the stage of an inverted microscope. Nanoporous gold was prepared via dealloying gold leaf with concentrated nitric acid and was chemisorbed to a standard microscope coverslip with (3-mercaptopropyl)trimethoxysilane. The gold surface was further modified with 1-hexanethiol to optimize hydrophobicity of the surface to allow for redox measurements to be made in nanoscopic volumes. Time traces of the open-circuit potential (OCP) were used to construct Nernst plots to evaluate the applicability of the droplet-based potentiometric redox measurement system. Two poised one-electron transfer systems (potassium ferricyanide/ferrocyanide and ferrous/ferric ammonium sulfate) yielded Nernstian slopes of -58.5 and -60.3 mV, respectively, with regression coefficients greater than 0.99. The y-intercepts of the two agreed well to the formal potential of the two standard oxidation-reduction potential (ORP) calibrants, ZoBell's and Light's solution. The benzoquinone and hydroquinone redox couple was examined as a representative two-electron redox system; a Nernst slope of -30.8 mV was obtained. Additionally, two unpoised systems (potassium ferricyanide and ascorbic acid) were studied to evaluate the system under conditions where only one form of the redox couple is present in appreciable concentrations. Again, slopes near the Nernstian values of -59 and -29 mV, respectively, were obtained. All experiments were carried out using solution volumes between 280 and 1400 pL with injection volumes between 8 and 100 pL. The miniscule volumes allowed for extremely rapid mixing (<305 ms) as well. The small volumes and rapid mixing along with the high accuracy and sensitivity of these measurements lend support to the use of this approach in applications where time is a factor and only small volumes are available for testing.

Generation of two-dimensional concentration-gradient droplet arrays on a two-layer chip for screening of protein crystallization conditions

We developed a two-layer microfluidic chip for the generation of two-dimensional concentration-gradient droplet arrays. All of the channels, including a concentration-gradient formation channel network, an oil phase channel network, and droplet generators, were ingeniously arranged on a single polydimethylsiloxane (PDMS) substrate, which facilitates the fabrication of the chip. Ninety-six lines of subnanoliter droplets with concentration gradients can be formed on a 6-inlet PDMS chip. With the aqueous phase flow rate of 1.0 μL/min and the oil phase flow rate of 35 μL/min, relative standard deviation of the droplet sizes in the same channel was below 5.0 %, while in the 96 channels was 7.0 %. Multiparameter screening of protein crystallization conditions was demonstrated by using the two-dimensional concentration-gradient droplet arrays. Protein, precipitant, and buffer each at 16 concentrations for lysozyme crystallization were tested simultaneously in one 6-inlet chip, and five precipitants each at eight concentrations were screened at the same time in one 10-inlet chip. The concentration-gradient droplet chip proved to be a useful tool for screening crystallization conditions, characterized by low consumption, high throughput, convenience, and automatization.

Ultralocalized thermal reactions in subnanoliter droplets-in-air

Miniaturized laboratory-on-chip systems promise rapid, sensitive, and multiplexed detection of biological samples for medical diagnostics, drug discovery, and high-throughput screening. Within miniaturized laboratory-on-chips, static and dynamic droplets of fluids in different immiscible media have been used as individual vessels to perform biochemical reactions and confine the products. Approaches to perform localized heating of these individual subnanoliter droplets can allow for new applications that require parallel, time-, and space-multiplex reactions on a single integrated circuit. Our method positions droplets on an array of individual silicon microwave heaters on chip to precisely control the temperature of droplets-in-air, allowing us to perform biochemical reactions, including DNA melting and detection of single base mismatches. We also demonstrate that ssDNA probe molecules can be placed on heaters in solution, dried, and then rehydrated by ssDNA target molecules in droplets for hybridization and detection. This platform enables many applications in droplets including hybridization of low copy number DNA molecules, lysing of single cells, interrogation of ligand-receptor interactions, and rapid temperature cycling for amplification of DNA molecules.

Automated generation of libraries of nL droplets

We demonstrate an integrated system for rapid and automated generation of multiple, chemically distinct populations of ~10(3)-10(4) sub-nanoliter droplets. Generation of these 'libraries of droplets' proceeds in the following automated steps: i) generation of a sequence of micro-liter droplets of individually predetermined composition, ii) injection of these 'parental' droplets onto a chip, iii) transition from a mm- to a μm-scale of the channels and splitting each of the parental drops with a flow-focusing module into thousands of tightly monodisperse daughter drops and iv) separation of such formed homogeneous populations with plugs of a third immiscible fluid. This method is compatible both with aspiration of microliter portions of liquid from a 96-well plate with a robotic station and with automated microfluidic systems that generate (~μL) droplets of preprogrammed compositions. The system that we present bridges the techniques that provide elasticity of protocols executed on microliter droplets with the techniques for high-throughput screening of small (~pL, ~nL) droplet libraries. The method that we describe can be useful in exploiting the synergy between the ability to rapidly screen distinct chemical environments and to perform high-throughput studies of single cells or molecules and in digital droplet PCR systems.

Remarks on Microfluidic Fabrication of Nano-/subnano-liter Droplets

In the microchannel-emulsification approach for producing nano-/subnano-liter droplets, the external energy input into the droplet accounts for the variation of the droplet surface free energy during its growth on the terrace. Avoiding the demand for an infinite energy input is the mechanism responsible for the experimental observation that there is always some remaining dispersed liquid on the terrace after generation of one droplet. A better empirical relation exists for correlating the experimental data of the droplet size in the literature. Based on a balance of the surface tension and the drag force, we have also developed a simple scaling model for predicting the size of droplets fabricated in microfluidic T-junctions.

DEP actuation of emulsion jets and dispensing of sub-nanoliter emulsion droplets

Liquid Dielectrophoresis (L-DEP) has been successfully leveraged at microscopic scales and shown to provide a controllable means of on-chip precision dispensing and manipulation of sub-nanoliter single emulsion droplets. In this paper, we report on the dynamics of a DEP actuated emulsion jet prior to break-up and compare its characteristic behavior based on the lumped parameter model of Jones et al. (R. Ahmed and T. B. Jones, J. Micromech. Microeng., 2007, 17, 1052). Furthermore, features and aspects of these emulsion jets, their break-up and formation of sub-nanoliter emulsion droplets is studied in further detail. Applications of the proposed scheme in dispensing encapsulated sub-nanoliter droplets is envisioned in various fields including microTAS, on-chip handling and storage of cells and other biological samples for longer duration in controlled environments as well as solving the more general encapsulation issues in surface microfluidic devices. Scalability of the proposed scheme is shown by producing controlled sample-oil single emulsion droplets (aqueous samples in oil) in the range of 50-400 picoliters.