Picoliter Volumes Research Articles

An acoustofluidic picoinjector

Droplet microfluidics has emerged as a valuable technology for a multitude of chemical and biomedical applications, offering the capability to create independent microenvironments for high-throughput assays. Central to numerous droplet microfluidic applications is the picoinjection of materials into individual droplets, yet existing picoinjection methods often exhibit high power requirements, lack biocompatibility, and/or suffer from limited controllability. Here, we present an acoustofluidic picoinjector that generates acoustic pressure at the droplet interface to enable on-demand, energy-efficient, and biocompatible injection at high precision. We validate our platform by performing acid-base titrations by iteratively injecting picoliter volume reagents into droplets to induce pH transitions detectable by color change in solution. Additionally, we demonstrate the versatility of the acoustofluidic picoinjector in the synthesis of metallic nanoparticles, yielding highly monodisperse and reproducible particle morphologies compared to conventional bulk-phase techniques. By facilitating controlled delivery of reagents or biological samples with unparalleled accuracy, acoustofluidic picoinjection broadens the utility of droplet microfluidics for a myriad of applications in chemical and biological research.

Ultrahigh Throughput Evolution of Tryptophan Synthase in Droplets via an Aptamer Sensor.

Tryptophan synthase catalyzes the synthesis of a wide array of noncanonical amino acids and is an attractive target for directed evolution. Droplet microfluidics offers an ultrahigh throughput approach to directed evolution (up to 107 experiments per day), enabling the search for biocatalysts in wider regions of sequence space with reagent consumption minimized to the picoliter volume (per library member). While the majority of screening campaigns in this format on record relied on an optically active reaction product, a new assay is needed for tryptophan synthase. Tryptophan is not fluorogenic in the visible light spectrum and thus falls outside the scope of conventional droplet microfluidic readouts, which are incompatible with UV light detection at high throughput. Here, we engineer a tryptophan DNA aptamer into a sensor to quantitatively report on tryptophan production in droplets. The utility of the sensor was validated by identifying five-fold improved tryptophan synthases from ∼100,000 protein variants. More generally, this work establishes the use of DNA-aptamer sensors with a fluorogenic read-out in widening the scope of droplet microfluidic evolution.

On synergy between ultrahigh throughput screening and machine learning in biocatalyst engineering.

Protein design and directed evolution have separately contributed enormously to protein engineering. Without being mutually exclusive, the former relies on computation from first principles, while the latter is a combinatorial approach based on chance. Advances in ultrahigh throughput (uHT) screening, next generation sequencing and machine learning may create alternative routes to engineered proteins, where functional information linked to specific sequences is interpreted and extrapolated in silico. In particular, the miniaturisation of functional tests in water-in-oil emulsion droplets with picoliter volumes and their rapid generation and analysis (>1 kHz) allows screening of >107-membered libraries in a day. Subsequently, decoding the selected clones by short or long-read sequencing methods leads to large sequence-function datasets that may allow extrapolation from experimental directed evolution to further improved mutants beyond the observed hits. In this work, we explore experimental strategies for how to draw up 'fitness landscapes' in sequence space with uHT droplet microfluidics, review the current state of AI/ML in enzyme engineering and discuss how uHT datasets may be combined with AI/ML to make meaningful predictions and accelerate biocatalyst engineering.

Microfluidic 3D hepatic cultures integrated with a droplet-based bioanalysis unit

A common challenge in microfluidic cell cultures has to do with analysis of cell function without replacing a significant fraction of the culture volume and disturbing local concentration gradients of signals. To address this challenge, we developed a microfluidic cell culture device with an integrated bioanalysis unit to enable on-chip analysis of picoliter volumes of cell-conditioned media. The culture module consisted of an array of 140 microwells with a diameter of 300 m which were made low-binding to promote organization of cells into 3D spheroids. The bioanalysis module contained a droplet generator unit, 15 micromechanical valves and reservoirs loaded with reagents. Each 0.8 nL droplet contained an aliquot of conditioned media mixed with assay reagents. The use of microvalves allowed us to load enzymatic assay and immunoassay into sequentially generated droplets for detection of glucose and albumin, respectively. As a biological application of the microfluidic device, we evaluated hormonal stimulation and glucose consumption of hepatic spheroids. To mimic physiological processes occurring during feeding and fasting, hepatic spheroids were exposed to pancreatic hormones, insulin or glucagon. The droplet-based bioanalysis module was used to measure uptake or release of glucose upon hormonal stimulation. In the future, we intend to use this microfluidic device to mimic and measure pathophysiological processes associated with hepatic insulin resistance and diabetes in the context of metabolic syndrome.

Surface tension of single suspended aerosol microdroplets

The surface tension of troposphere aerosols can significantly influence their atmospheric processes and key properties, particularly on the morphology, the phase transition, the activation as cloud condensation nuclei, and the gas-particle partitioning. However, directly measuring the surface tension of single ambient aerosol is quite challenging, due to the limitations of their picolitre volumes and thermal motion. Here, we developed a dual laser tweezers Raman spectroscopy (DLT-RS) system to directly sense the surface tension of single airborne microdroplets (PM10 particles). A pair of aerosol droplets were trapped and driven to coalesce by the laser tweezers. Meanwhile, the backscattering light intensity and bright-field images during the coalescence process were recorded to characterize the aerosol surface tension. A remarkable advantage of directly sensing aerosol surface tension is that the solutes in aerosols are often supersaturated, which is common in atmospheric aerosols but almost unavailable in bulk solutions. We experimentally measured the surface tension of aerosols composed of nitrates or oxalic acid/nitrate mixture. Besides, the variation of surface tension during aerosol aging process was also explored, which brings possible implications on the surface evolution of actual ambient aerosol during their atmospheric lifetime.

Opportunities and challenges for sample preparation and enrichment in mass spectrometry for single-cell metabolomics.

Single-cell heterogeneity in metabolism, drug resistance and disease type poses the need for analytical techniques for single-cell analysis. As the metabolome provides the closest view of the status quo in the cell, studying the metabolome at single-cell resolution may unravel said heterogeneity. A challenge in single-cell metabolome analysis is that metabolites cannot be amplified, so one needs to deal with picolitre volumes and a wide range of analyte concentrations. Due to high sensitivity and resolution, MS is preferred in single-cell metabolomics. Large numbers of cells need to be analysed for proper statistics; this requires high-throughput analysis, and hence automation of the analytical workflow. Significant advances in (micro)sampling methods, CE and ion mobility spectrometry have been made, some of which have been applied in high-throughput analyses. Microfluidics has enabled an automation of cell picking and metabolite extraction; image recognition has enabled automated cell identification. Many techniques have been used for data analysis, varying from conventional techniques to novel combinations of advanced chemometric approaches. Steps have been set in making data more findable, accessible, interoperable and reusable, but significant opportunities for improvement remain. Herein, advances in single-cell analysis workflows and data analysis are discussed, and recommendations are made based on the experimental goal.

Inkjet-based bioprinting for tissue engineering

Inkjet bioprinting, a derivative of traditional inkjet technology, is gaining recognition in the fields of life sciences and tissue engineering due to its ability to produce picoliter volume droplets at high speeds in a non-contact fashion. This method has impressively evolved from enabling the production of 2-dimensional (2D) prints to complex 3-dimensional (3D) structures, and is increasingly being used in the manufacturing of electronic components. More recently, this technology has been effectively adapted for a variety of medical applications, such as cell patterning, scaffold construction, and 3D tissue fabrication. In this review, we delve into the principles and biological uses of inkjet technology. We provide an in-depth discussion on the latest developments in inkjet bioprinting, with a focus on cell patterning and 3D fabrication of tissue models, including multilayered lung, bladder, and skin. We also explore the potential of high-throughput 3D-bioprinted tissue models in toxicology and drug efficacy testing.

On-demand pitch tuning of printed chiral nematic liquid crystal droplets

Identifying facile means with which to tune the pitch of, and therefore the reflected colour from, chiral nematic liquid crystals (CLC) is of interest for many different photonics applications including optical filters, coloured displays, and mirrorless lasers. Precise control of the pitch of the helix, however, can be challenging. Here, we demonstrate the ability to tune the pitch, and consequently the reflection band, by depositing picolitre volumes of nematic LC into printed CLC droplets with a short pitch. Results are presented that demonstrate mixing of the nematic LC and CLC droplets such that the pitch elongates causing the reflection band located at blue wavelengths (430 nm) to redshift to longer wavelengths. The magnitude of the redshift can be controlled by varying the number of nematic LC droplets deposited into each CLC droplet. We consider the process of diffusion of these two separate mixtures using inkjet printing and showcase how this process of tuning the pitch can be employed to create coloured images in the form of an alphanumeric logo.

Length-scale discrepancy in the properties of epoxy resin specimens

In studies of the fibre-matrix interphase with microscale single fibre methods, the dependence of results on conversion of the thermoset resin – or degree of cure as it is often called – remains an issue. In the microbond method specifically, the curing of picolitre volume drop-on-fibre systems differs significantly from that of macroscale resin batches. The surface-to-volume ratio and vapour pressure can cause volatile components of the resin to evaporate, potentially limiting the degree of cure. Atomistic scale modelling along with experimental thermal analysis were used to understand the curing process and how it translates to resin properties, while nanoindentation was used to experimentally compare the mechanical performance of samples prepared in different length-scales. The evaporation is experimentally verified. Comparable variation in mechanical properties is shown in atomistic scale models of the epoxy network with no evaporation. The origin is in the network morphologies created by varying the curing process. Thus attributing the length-scale discrepancy solely to conversion is likely an oversimplification and understanding the network morphology from different curing conditions is also needed.

Ultrahigh-Throughput Enzyme Engineering and Discovery in In Vitro Compartments.

Novel and improved biocatalysts are increasingly sourced from libraries via experimental screening. The success of such campaigns is crucially dependent on the number of candidates tested. Water-in-oil emulsion droplets can replace the classical test tube, to provide in vitro compartments as an alternative screening format, containing genotype and phenotype and enabling a readout of function. The scale-down to micrometer droplet diameters and picoliter volumes brings about a >107-fold volume reduction compared to 96-well-plate screening. Droplets made in automated microfluidic devices can be integrated into modular workflows to set up multistep screening protocols involving various detection modes to sort >107 variants a day with kHz frequencies. The repertoire of assays available for droplet screening covers all seven enzyme commission (EC) number classes, setting the stage for widespread use of droplet microfluidics in everyday biochemical experiments. We review the practicalities of adapting droplet screening for enzyme discovery and for detailed kinetic characterization. These new ways of working will not just accelerate discovery experiments currently limited by screening capacity but profoundly change the paradigms we can probe. By interfacing the results of ultrahigh-throughput droplet screening with next-generation sequencing and deep learning, strategies for directed evolution can be implemented, examined, and evaluated.

The Microelectrode Insulator Influences Water Nanodroplet Collisions

Studying chemical reactions in very small (attoliter to picoliter) volumes is important in understanding how chemistry proceeds at all relevant scales. Stochastic electrochemistry is a powerful tool to study the dynamics of single nanodroplets, one at a time. Perhaps the most conceptually simple experiment is that of the current blockade, where the collision of an insulating particle is observed electrochemically as a stepwise decrease in current. Here, we demonstrate that nanodroplet collisions on microelectrodes are not as simple as water droplets adsorbing to the electrode to block current and that the environment immediately around the microelectrode (glass insulator) plays a pivotal role in the electrochemical collision response. We use correlated opto-electrochemical measurements to understand a variety of electrochemical responses when water nanodroplets collide with a microelectrode during the heterogeneous oxidation of decamethylferrocene in oil. The amperometric current reports not only on current blockades but also on nanodroplet coalescence events and preferential wetting to the glass around the microelectrode. Treating the glass with dichlorodimethylsilane creates a hydrophobic environment around the working electrode, and the simple current blockade response expected from the absorption of insolating nanoparticles is observed. These results highlight the importance of the environment around the working electrode for nanodroplet collision studies.

Selectable encapsulated cell quantity in droplets via label-free electrical screening and impedance-activated sorting.

Single-cell encapsulation in droplets has become a powerful tool in immunotherapy, medicine discovery, and single-cell analysis, thanks to its capability for cell confinement in picoliter volumes. However, the purity and throughput of single-cell droplets are limited by random encapsulation process, which resuts in a majority of empty and multi-cells droplets. Herein we introduce the first label-free selectable cell quantity encapsulation in droplets sorting system to overcome this problem. The system utilizes a simple and reliable electrical impedance based screening (98.9% of accuracy) integrated with biocompatible acoustic sorting to select single-cell droplets, achieving 90.3% of efficiency and up to 200​Hz of throughput, by removing multi-cells (∼60% of rejection) and empty droplets (∼90% of rejection). We demonstrate the use of the droplet sorting to improve the throughput of single-cell encapsulation by ∼9-fold compared to the conventional random encapsulation process.

Multifunctional Droplets Formed by Interfacially Self-Assembled Fluorinated Magnetic Nanoparticles for Biocompatible Single Cell Culture and Magnet-Driven Manipulation.

The ability to encapsulate and manipulate droplets with a picoliter volume of samples and reagents shows great potential for practical applications in chemistry, biology, and materials science. Magnetic control is a promising approach for droplet manipulation due to its ability for wireless control and its ease of implementation. However, it is challenged by the poor biocompatibility of magnetic materials in aqueous droplets. Moreover, current droplet technology is problematic because of the molecule leakage between droplets. In the paper, we propose multifunctional droplets with the surface coated by a layer of fluorinated magnetic nanoparticles for magnetically actuated droplet manipulation. Multifunctional droplets show excellent biocompatibility for cell culture, nonleakage of molecules, and high response to a magnetic field. We developed a strategy of coating the F-MNP@SiO2 on the outer surface of droplets instead of adding magnetic material into droplets to enable droplets with a highly magnetic response. The encapsulated bacteria and cells in droplets did not need to directly contact with the magnetic materials at the outer surface, showing high biocompatibility with living cells. These droplets can be precisely manipulated based on magnet distance, the time duration of the magnetic field, the droplet size, and the MNP composition, which well match with theoretical analysis. The precise magnetically actuated droplet manipulation shows great potential for accurate and sensitive droplet-based bioassays like single cell analysis.

Precise, wide field, and low-cost imaging and analysis of core-shell beads for digital polymerase chain reaction.

Digital droplet reactors have become a valuable tool for the analysis of single cells, organisms, or molecules by discretising reagents into picolitre or nanolitre volumes. However, DNA-based assays typically require processing of samples on the scale of tens of microlitres, with the detection of as few as one or as many as a hundred thousand fragments. Through the present work, we introduce a flow-focusing microfluidic device that produces 120 picolitre core-shell beads, which are assembled into a monolayer in a Petri dish for visualization and analysis. The bead assembly is subjected to polymerase chain reaction (PCR) amplification and fluorescence detection to digitally quantify the DNA concentration of the sample. We use a low-cost 21-megapixel digital camera and macro lens to capture wide-field fluorescence images with a 10-30 mm2 field-of-view at magnifications ranging from 5× to 2.5×. A customised Python script analysed the acquired images. Our study demonstrates the ability to perform digital PCR analysis of the entire bead assembly through end-point imaging and compare the results with those obtained through RT-qPCR.

Attomole-Level Multiplexed Detection of Neurochemicals in Picoliter Droplets by On-Chip Nanoelectrospray Ionization Coupled to Mass Spectrometry.

While droplet microfluidics is becoming an effective tool for biomedical research, sensitive detection of droplet content is still challenging, especially for multiplexed analytes compartmentalized within ultrasmall droplets down to picoliter volumes. To enable such measurements, we demonstrate a silicon-based integrated microfluidic platform for multiplexed analysis of neurochemicals in picoliter droplets via nanoelectrospray ionization (nESI)-mass spectrometry (MS). An integrated silicon microfluidic chip comprising downscaled 7 μm-radius channels, a compact T-junction for droplet generation, and an integrated nESI emitter tip is used for segmentation of analytes into picoliter compartments and their efficient delivery for subsequent MS detection. The developed system demonstrates effective detection of multiple neurochemicals encapsulated within oil-isolated plugs down to low picoliter volumes. Quantitative measurements for each neurochemical demonstrate limits of detection at the attomole level. Such results are promising for applications involving label-free and small-volume detection for monitoring a range of brain chemicals.

Monolithic Silica Microbands Enable Thin-Layer Chromatography Analysis of Single Cells.

A picoliter thin-layer chromatography (pTLC) platform was developed for analyzing extremely miniature specimens, such as assay of the contents of a single cell of 1 picoliter volume. The pTLC chip consisted of an array of microscale bands made from highly porous monolithic silica designed to accept picoliter-scale volume samples. pTLC bands were fabricated by combining sol-gel chemistry and microfabrication technology. The width (60-80 μm) and depth (13 μm) of each band is comparable to the size of single cells and acted to reduce the lateral diffusion and confine the movement of compounds along the microbands. Ultrasmall volumes (tens of pL) of model fluorescent compounds were spotted onto the microband by a piezoelectric microdispenser and successfully separated by pTLC. The separation resolution and analyte migration were dependent on the macropore size (ranging from 0.3 to 2.3 μm), which was adjustable by changing the porogen concentration during the sol-gel process. For a 0.3 μm macropore size, attomoles of analyte were detectable by fluorescence using standard microscopy methods. The separation resolution, theoretical plate number, and separation times ranged from 1.3 to 2.1, 4 to 357, and 2 to 8 min, respectively, for the chosen model biological lipids. To demonstrate the capability of pTLC for separating analytes from single mammalian cells, cells loaded with fluorescent lipophilic dyes or sphingosine kinase reporter were spotted on microbands, and the single-cell contents separated by pTLC were detected from their fluorescence. These results demonstrate the potential of pTLC for applications in many areas where miniature specimens and high-throughput parallel analyses are needed.

Efficient multi-gene expression in cell-free droplet microreactors

Cell-free transcription and translation systems promise to accelerate and simplify the engineering of proteins, biological circuits and metabolic pathways. Their encapsulation on microfluidic platforms can generate millions of cell-free reactions in picoliter volume droplets. However, current methods struggle to create DNA diversity between droplets while also reaching sufficient protein expression levels. In particular, efficient multi-gene expression has remained elusive. We here demonstrate that co-encapsulation of DNA-coated beads with a defined cell-free system allows high protein expression while also supporting genetic diversity between individual droplets. We optimize DNA loading on commercially available microbeads through direct binding as well as through the sequential coupling of up to three genes via a solid-phase Golden Gate assembly or BxB1 integrase-based recombineering. Encapsulation with an off-the-shelf microfluidics device allows for single or multiple protein expression from a single DNA-coated bead per 14 pL droplet. We envision that this approach will help to scale up and parallelize the rapid prototyping of more complex biological systems.

Accelerated protein digestion and separation with picoliter volume utilizing nanofluidics.

Single cell analyses can provide critical biological insight into cellular heterogeneity. In particular, the proteome, which governs cell functions, is much more difficult to analyze because it is principally impossible to amplify proteins compared to nucleic acids. The most promising approach to single cell proteomics is based on the liquid chromatography mass spectrometry (LC-MS) platform. However, pretreatments before MS detection have two critical issues for single cell analysis: analyte loss as a result of adsorption and artifacts due to the duration of analysis. This is a serious problem because single cells have a limited number of protein molecules and a small volume. To solve these issues, we developed an integrated nanofluidic device to manipulate samples on a femtoliter to picoliter (fL-pL) scale to achieve high-throughput analysis via suppressing analyte loss. This device can perform tryptic digestion, chromatographic separation, and non-labeled detection with high consistency. In addition, we introduced an open/close valve by physical deformation of glass on a nanometer scale to independently modify the nanochannel surfaces and control sample aliquots. The injection system equipped with this valve achieved an injection volume of 1.0 ± 0.1 pL. By using this integrated device, we found that the chromatogram of bulk-digestion for 12 hours resembled that of 15 min-digestion in the nanochannel, which indicated that these conditions reached a similar state of digestion. Therefore, an integrated device for ultra-fast protein analysis was developed on a 1 pL scale for the first time.

Droplet-Based Microfluidic Chip Design, Fabrication, and Use for Ultrahigh-Throughput DNA Analysis and Quantification.

DNA is widely used as a biomarker of contamination, infection, or disease, which has stimulated the development of a wide palette of detection and quantification methods. Even though several analytical approaches based on isothermal amplification have been proposed, DNA is still mainly detected and quantified by quantitative PCR (qPCR). However, for some analyses (e.g., in cancer research) qPCR may suffer from limitations arising from competitions between highly similar template DNAs, the presence of inhibitors, or suboptimal primer design. Nevertheless, digitalizing the analysis (i.e., individualizing DNA molecules into compartments prior to amplifying them in situ) allows to address most of these issues. By its capacity to generate and manipulate millions of highly similar picoliter volume water-in-oil droplets, microfluidics offers both the required miniaturization and parallelization capacity, and led to the introduction of digital droplet PCR (ddPCR). This chapter aims at introducing the reader to the basic principles behind ddPCR while also providing the key guidelines to fabricate, set up, and use his/her own ddPCR platform. We further provide procedures to detect and quantify DNA either purified in solution or directly from individualized cells. This approach not only gives access to DNA absolute concentration with unrivaled sensitivity, but it may also be the starting point of more complex in vitro analytical pipelines discussed at the end of the chapter.

7-Aminocoumarin-4-acetic Acid as a Fluorescent Probe for Detecting Bacterial Dipeptidyl Peptidase Activities in Water-in-Oil Droplets and in Bulk.

Droplet-based microfluidic systems are a powerful tool for biological assays with high throughput. Water-in-oil droplets (WODLs) are typically used in droplet-based microfluidic systems to culture microorganisms and perform enzyme assays. However, because of the oil surrounding the nanoliter and picoliter volumes of WODLs, availability of suitable substrates is limited. For instance, although 7-amino-4-methylcoumarin (AMC) is commonly used as a fluorescent probe of the substrate to detect peptidase activity, AMC leaks from WODLs to the oil phase due to its high hydrophobicity. Thus, AMC substrates cannot be used in droplet-based microfluidic systems with WODLs. In this study, we developed a peptidase substrate consisting of a dipeptide and 7-aminocoumarin-4-acetic acid (ACA), an AMC-derived fluorogenic compound. ACA was retained in the WODL for more than 7 days, and the dipeptidyl ACA substrate detected dipeptidyl peptidase (DPP) activity in the WODL. Compared to AMC substrates, the substrate specificity constants of DPPs for ACA substrates increased up to 4.7-fold. Fluorescence-activated droplet sorting made high-throughput screening of microorganisms based on DPP activity using the dipeptidyl ACA substrate possible. Since ACA could be applied to various substrates as a fluorescent probe, detectable microbial enzyme activities for droplet-based microfluidic systems can be largely expanded.