Picoliter-volume Droplets Research Articles

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.

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.

Droplet-assisted electrospray phase separation using an integrated silicon microfluidic platform.

We report a silicon microfluidic platform that enables monolithic integration of transparent micron-scale microfluidic channels, an on-chip segmentation of analyte flows into picoliter-volume droplets, and a nano-electrospray ionization emitter that enables spatial and temporal separation of oil and aqueous phases during electro-spray for subsequent mass spectrometry analysis.

High-speed laser speckle imaging to unravel picoliter drop-on-demand to substrate interaction.

Understanding phenomena such as evaporation and imbibition of picoliter droplets into porous substrates is crucial in printing industry to achieve a higher printing quality and print speed. After printing, the residual pigment must remain fixed at the desired location on a substrate and be of a desired volume to yield a high resolution and vibrantly printed page that has become the expectation of modern printing technology. Current research entails not only chemical composition of the ink but also how this links to the dynamics and interactions that occur between the ink and the substrate at every stage of the printed spot formation, including evaporation, wetting, and imbibition. In this paper, we present an instrument that can print on-demand picoliter volume droplets of ink onto substrates and then immediately record on evolution of the resulting dynamics when these two materials interact. This high-speed laser speckle imaging (HS-LSI) technique has been developed to monitor nanometer displacement of the drying and imbibing ink droplet at a high frame rate, up to 20000Hz, given the short timescales of these interactions. We present the design of the instrument, discuss the related challenges and the theory underlying the LSI technique, specifically how photons non-evasively probe opaque objects in a multiple scattering regime, and show how this technique can unravel the dynamics of drying and imbibition. We will finish giving a validation on the instrument and an example of its usage.

In Situ X-ray Absorption Spectroscopy and Droplet-Based Microfluidics: An Analysis of Calcium Carbonate Precipitation.

Droplet-based microfluidic systems are ideally suited for the investigation of nucleation and crystallization processes. To best leverage the features of such platforms (including exquisite time resolution and high-throughput operation), sensitive and in situ detection schemes are needed to extract real-time chemical information about all species of interest. In this regard, the extension of conventional (UV, visible, and infrared) optical detection schemes to the X-ray region of the electromagnetic spectrum is of high current interest, as techniques such as X-ray absorption spectroscopy (XAS) provide for the element-specific investigation of the local chemical environment. Accordingly, herein, we report for the first time the integration of millisecond droplet-based microfluidics with XAS. Such a platform allows for the sensitive acquisition of X-ray absorption data from picoliter-volume droplets moving at high linear velocities. Significantly, the high-temporal resolution of the droplet-based microfluidic platform enables unprecedented access to the early stages of the reaction. Using such an approach, we demonstrate in situ monitoring of calcium carbonate precipitation by extracting XAS spectra at the early time points of the reaction with a dead time as low as 10 ms. We obtain insights into the kinetics of the formation of amorphous calcium carbonate (ACC) as a first species during the crystallization process by monitoring the proportion of calcium ions converted into ACC. Within the confined and homogeneous environment of picoliter-volume droplets, the ACC content reaches 60% over the first 130 ms. More generally, the presented method offers new opportunities for the real-time monitoring of fast chemical and biological processes.

Harnessing Microfluidic Single Cell (MF-SC) and 3D “Organ-on-Chip” Assay Technologies for the Investigation of Immunotherapy and Study of Tumor Microenvironment (TME) in Antibody-Resistant Non-Hodgkin Lymphoma (NHL)

Introduction: Interactions between tumor and the TME occur through direct cell contacts and secreted factors such as cytokines and growth factors may influence behavior of tumor cells and response to therapy. Development of clinically applicable tumor models for screening therapeutics is desired to enhance our understanding of these mechanisms. We harnessed a novel MF-SC technology and an innovative 3D biomimetic tumor model to improve our understanding of the role of TME with immunotherapy and NHL tumor biology.Methods: Rituximab (R)- and obinutuzumab (O)-resistant (RR & OR) B-NHL cells (SUDHL4, SUDHL10 & Raji) were developed for evaluation of anti-CD20 resistance. Patient derived cell lines were obtained from newly diagnosed DLBCL patients. Functional response of anti-CD20 resistant & susceptible B-NHL cells were determined at SC levels in a customized MF droplet array platform. Single CD16+ NK cells were co-encapsulated with B-NHL cells at 1:1 effector-target (E:T) ratio in picoliter-volume droplets in presence/absence of R & O. Dynamic cellular interaction between E-T was monitored via live-cell time-lapse microscopy over 4-6 hrs to assess interaction profiles (eg, contact duration, frequency, and single vs serial interaction) and functional cytotoxicity. Target cell kill kinetics was assessed by loss of Calcein AM & ethidium homodimer uptake. Further, we used systems biology analyses (ie, Ingenuity Pathway Analysis (IPA) & Gene Set Enrichment Analysis (GSEA)) to identify associated biologic pathways. Additionally, the MF-SC platform was used to develop microscale 3D multicellular constructs to monitor effect of cancer-stroma interaction in a simulated TME. Heterogeneous tumor spheroids were generated using composite alginate hydrogels and varying combinations of NHL cells, fibroblasts & immune cells. Effect of TME (ie, cell composition and biological activity of immune cells) in the presence/absence of lenalidomide was evaluated based on extent of target cell survival/death (differentially labeled using CellTrace cell labeling & tracking dyes).Results: Development of anti-CD20 resistance in B-NHL cells resulted in decreased expression of CD20, loss of R binding, and decreased cytotoxic activity. Using IPA and GSEA systems biology analyses of RR and OR B-NHL cells, we observed conserved down-regulation of IFN-α/γ, TNF-α, inflammatory response, hypoxia and complement response pathways, and upregulation of MYC targets, which predicted overall immune suppression in all CD20 resistant cells. With MF-SC, we determined that NK cells induced variable duration of contacts with SUDHL10 cells irrespective of anti-CD20 resistance. While the single or multiple contacts made between NK & target cells occurred for an average of 45 minutes, addition of R or O resulted in significantly extended contact time (77 & 79 minutes; P <0.001 each) (Fig 1A). These contacts resulted in cell death with NK cells alone in 56% R-sensitive cells, which increased to 75% with addition of R (P <0.01). In RR cells, R increased NK cell-mediated target death from 37% (NK cells alone) to 52% in the presence of rituximab, but with perturbed cell-to-cell contact. The addition of O increased cell death (83%) in RR SUDHL10 cells (P <0.005) (Fig 1B). Primary DLBCL cells were also tested in this platform; there was NK-mediated cytotoxicity/death seen (68% in 80 minutes). We also developed a novel MF 3D lymphoma spheroid model using composite hydrogels optimized to host B-NHL cells (Fig 1C). In the presence of activated immune and stromal components, lenalidomide induced potent 2.5 fold increase in SUDHL10 cell death vs 3D cultures that lacked activated immune or stromal cells, which recapitulated the physiological mechanisms of TME-dependent lenalidomide drug activity.Conclusions: We identified that resistance to R & O occurs through biologically conserved mechanisms. Results in MF-SCs revealed that interactions between NHL tumor and immune cells is a highly dynamic process occurring through multiple cell contacts and that presence of R & O shift these kinetics; this was an important factor in determining cytotoxic immune activity. In addition, experiments based on reconstituted 3D NHL tumor spheroids validated the influence of TME mediating the activity of immunomodulatory drugs. This innovative MF-based 3D NHL spheroid model may be further leveraged for the discovery of immune-based therapeutic applications. [Display omitted] DisclosuresEvens:Celgene: Consultancy; Merck: Consultancy; • Spectrum Pharmaceuticals: Consultancy; Novartis: Consultancy; AbbVie: Consultancy; Seattle Genetics: Consultancy; Pharmacyclics: Consultancy; Kite Pharma: Consultancy; Millennium: Consultancy; Affimed: Consultancy; Amgen: Consultancy.

Broad-Band Spectrum, High-Sensitivity Absorbance Spectroscopy in Picoliter Volumes.

Picoliter-volume droplets within segmented flows can be probed in a rapid and efficient manner using optical detection methods. To date, however, most detection schemes for droplet content analysis have relied on the use of time-integrated fluorescence measurements. Despite its undoubted utility, the implementation of absorbance-based detectors is particularly challenging due to the reduced optical path lengths that are characteristic of microfluidic systems and deleterious scattering at droplet-oil interfaces. Unsurprisingly, efforts to develop sensitive absorbance-based detection schemes for the interrogation of rapidly moving droplets have primarily focused on ensuring adequate analytical sensitivity and, to date, have been exclusively limited to single-wavelength measurements. To address this limitation, and expand the information content associated with absorbance measurements on-chip, we herein describe a detection scheme for the extraction of broad-band absorbance spectra from pL-volume droplets with high sensitivity. The combination of a confocal optical system (that confines incident light to a reduced detection volume) and a postprocessing algorithm (that effectively removes the contribution of the carrier oil from the extracted spectra) engenders significant improvements in signal-to-noise ratios. Our system is initially calibrated by acquiring absorbance spectra from aqueous solutions of fluorescein isothiocyanate. These measurements confirm both excellent linearity over the studied range (from 0 to 100 μM) and a concentration limit of detection of 800 nM. The methodology is then used to monitor the salt-induced aggregation of gold nanoparticles with millisecond time resolution. This approach for small-volume absorbance spectroscopy allows for both high-throughput and high-information content measurements in subnanoliter volumes and will be highly desirable in a wide variety of bioanalytical applications where sensitivity and throughput are priorities.

Water-in-oil microcompartments for the study of biomimetic drug metabolism

Microcompartments in the form of water-in-oil droplets have been utilized to construct artificial cells and simulate human body environment. However, the performance of subcellular structure involved metabolism in emulsion droplets has not been explored, and the underlying mechanism is still being elucidated. In this work, drug metabolism is presented on the basis of great amounts of microcompartments formed of picoliter-volume droplets with different radius (R), using a commercial four-way valve as a droplet generator. A model substrate, phenacetin, and its metabolite, paracetamol, are quantitatively analyzed by liquid-chromatography (LC) tandem mass spectrometry (MS/MS), and the reaction kinetics is characterized. In microdroplets of varying size (R = 18, 27, 42, and 51 μm, respectively), both conversion ratio and reaction rate constant of the metabolism are influenced in different degree. For instance, the substrate conversion ratio after 60 min of incubation in R = 27 μm droplets improves from 15% to 42%, and the reaction rate constant improves nearly five-fold, compared to that in bulk phase. The influence of microcompartment size on metabolism rate is further explored by simulation using a diffusion-reaction model. The droplet-based strategy is rapid, accurate and cost-efficient, fitting especially into biomimetic metabolism studies.

Customizing droplet contents and dynamic ranges via integrated programmable picodroplet assembler

Droplet microfluidic technology is becoming increasingly useful for high-throughput and high-sensitivity detection of biological and biochemical reactions. Most current droplet devices function by passively discretizing a single sample subject to a homogeneous or random reagent/reaction condition into tens of thousands of picoliter-volume droplets for analysis. Despite their apparent advantages in speed and throughput, these droplet devices inherently lack the capability to customize the contents of droplets in order to test a single sample against multiple reagent conditions or multiple samples against multiple reagents. In order to incorporate such combinatorial capability into droplet platforms, we have developed the fully Integrated Programmable Picodroplet Assembler. Our platform is capable of generating customized picoliter-volume droplet groups from nanoliter-volume plugs which are assembled in situ on demand. By employing a combination of microvalves and flow-focusing-based discretization, our platform can be used to precisely control the content and volume of generated nanoliter-volume plugs, and thereafter the content and the effective dynamic range of picoliter-volume droplets. Furthermore, we can use a single integrated device for continuously generating, incubating, and detecting multiple distinct droplet groups. The device successfully marries the precise control and on-demand capability of microvalve-based platforms with the sensitivity and throughput of picoliter droplet platforms in a fully automated monolithic device. The device ultimately will find important applications in single-cell and single-molecule analyses.

Surface Tensions of Picoliter Droplets with Sub-Millisecond Surface Age.

Aerosols are key components of the atmosphere and play important roles in many industrial processes. Because aerosol particles have high surface-to-volume ratios, their surface properties are especially important. However, direct measurement of the surface properties of aerosol particles is challenging. In this work, we describe an approach to measure the surface tension of picoliter volume droplets with surface age <1 ms by resolving their dynamic oscillations in shape immediately after ejection from a microdroplet dispenser. Droplet shape oscillations are monitored by highly time-resolved (500 ns) stroboscopic imaging, and droplet surface tension is accurately retrieved across a wide range of droplet sizes (10-25 μm radius) and surface ages (down to ∼100 μs). The approach is validated for droplets containing sodium chloride, glutaric acid, and water, which all show no variation in surface tension with surface age. Experimental results from the microdroplet dispenser approach are compared to complementary surface tension measurements of 5-10 μm radius droplets with aged surfaces using a holographic optical tweezers approach and predictions of surface tension using a statistical thermodynamic model. These approaches combined will allow investigation of droplet surface tension across a wide range of droplet sizes, compositions, and surface ages.

Dynamic Analysis of Human Natural Killer Cell Response at Single-Cell Resolution in B-Cell Non-Hodgkin Lymphoma.

Natural killer (NK) cells are phenotypically and functionally diverse lymphocytes that recognize and kill cancer cells. The susceptibility of target cancer cells to NK cell-mediated cytotoxicity depends on the strength and balance of regulatory (activating/inhibitory) ligands expressed on target cell surface. We performed gene expression arrays to determine patterns of NK cell ligands associated with B-cell non-Hodgkin lymphoma (b-NHL). Microarray analyses revealed significant upregulation of a multitude of NK-activating and costimulatory ligands across varied b-NHL cell lines and primary lymphoma cells, including ULBP1, CD72, CD48, and SLAMF6. To correlate genetic signatures with functional anti-lymphoma activity, we developed a dynamic and quantitative cytotoxicity assay in an integrated microfluidic droplet generation and docking array. Individual NK cells and target lymphoma cells were co-encapsulated in picoliter-volume droplets to facilitate monitoring of transient cellular interactions and NK cell effector outcomes at single-cell level. We identified significant variability in NK-lymphoma cell contact duration, frequency, and subsequent cytolysis. Death of lymphoma cells undergoing single contact with NK cells occurred faster than cells that made multiple short contacts. NK cells also killed target cells in droplets via contact-independent mechanisms that partially relied on calcium-dependent processes and perforin secretion, but not on cytokines (interferon-γ or tumor necrosis factor-α). We extended this technique to characterize functional heterogeneity in cytolysis of primary cells from b-NHL patients. Tumor cells from two diffuse large B-cell lymphoma patients showed similar contact durations with NK cells; primary Burkitt lymphoma cells made longer contacts and were lysed at later times. We also tested the cytotoxic efficacy of NK-92, a continuously growing NK cell line being investigated as an antitumor therapy, using our droplet-based bioassay. NK-92 cells were found to be more efficient in killing b-NHL cells compared with primary NK cells, requiring shorter contacts for faster killing activity. Taken together, our combined genetic and microfluidic analysis demonstrate b-NHL cell sensitivity to NK cell-based cytotoxicity, which was associated with significant heterogeneity in the dynamic interaction at single-cell level.

A High-Sensitivity, Integrated Absorbance and Fluorescence Detection Scheme for Probing Picoliter-Volume Droplets in Segmented Flows.

Droplet-based microfluidic systems that incorporate flowing streams of pL-volume droplets surrounded by a continuous and immiscible carrier phase have attracted significant recent attention due to their utility in complex chemical and biological experimentation. Analysis of pL droplets, generated at kHz frequencies and moving at high linear velocities, is almost exclusively achieved using fluorescence-based detection schemes. To extend the applicability of such optical detection schemes, we herein report the development of a simple and cost-effective optofluidic platform, integrating liquid-core PDMS waveguides, that allows the accurate measurement of absorbance within individual pL-volume droplets moving within segmented flows. Using such an approach, differential measurements of "sample "and "reference" droplets can be acquired at 1 kHz and yield detection limits of 400 nM for fluorescein in water. Significantly, the presented technique enables simultaneous fluorescence and absorbance interrogation of rapidly moving droplets in a fully automated manner. Proof of principle is demonstrated through the titration and monitoring of pH gradients in real time.

Abstract 1572: Sensitive and robust targeted sequencing of pancreatic precancer and tumors using microfluidic single-molecule enrichment

Abstract Pancreatic ductal adenocarcinoma (PDAC) arises from two precursor lesions, pancreatic intraepithelial neoplasias and pancreatic cysts, such as intraductal papillary mucinous neoplasias (IPMNs). Since cyst lesions are readily detected on cross sectional imaging, early diagnosis of IPMN-associated PDAC and intervention may be feasible. However, clinical guidelines dictating which IPMNs are high-risk and require surgical resection are suboptimal. Recently, the genomic signature of IPMN-associated carcinomas has been described opening the possibility for targeted genomic analysis. Previous groups have sought to achieve this using conventional methods. However, since IPMN tissue and cyst fluid are limited in quantity and contain inhibitors of PCR, we developed a novel microfluidics-based approach to achieve sensitive and specific targeted amplification and Illumina library construction. We have optimized a novel method to enrich targeted genomic regions for next generation sequencing (NGS) platforms featuring microfluidic partitioning of the sample into uniform picoliter volume droplets containing single molecules of target DNA. All droplets contain all of the primers and PCR reagents, ensuring that every target molecule from the sample is amplified, and after endpoint PCR results in a highly uniform yield that facilitates efficient use of the NGS platform. In addition, the primers contain ‘Illumina tails’ that enable easy sample indexing and loading directly onto a MiSeq without additional library preparation. Here we detail the successful customization and use of the ThunderBolts Cancer Panel, which targets 230 commonly mutated regions in 50 cancer associated genes, with additional primers for commonly mutated genes in PDAC. Addition of the 37 PDAC-relevant primer pairs to the commercially available core panel was very straightforward and resulted in sequencing metrics similar to those of the core panel alone (100% coverage at 100x depth, mean read depth of 2500). Cyst fluid from 30 patients with IPMN resected under Sendai criteria were analyzed. We describe the mutational signature of cyst fluid and relate the presence and quantity of mutations in KRAS, GNAS and PIK3CA to the presence of invasive carcinoma and high grade dysplasia on pathology. Finally, unbiased and high resolution sequencing was obtained using this protocol with as little as 8ng of input DNA. Using picodroplet PCR technology we were able to achieve unprecedented sequencing performance on ultra-low sample inputs. Given the ease of use and customization of the panel, the same platform may be readily adapted to other applications in which samples are limited or are difficult to amplify. Future studies will utilize this platform for an expanded analysis of pancreatic cyst fluid with additional primers. Citation Format: Angelina I. Londoño-Joshi, Erica Pratt, Robert W. Cowan, Michael L. Samuels, Steve Kotsopoulos, Jeff Olson, Francis Long, Michelle A. Anderson, Diane Simeone, Andrew D. Rhim. Sensitive and robust targeted sequencing of pancreatic precancer and tumors using microfluidic single-molecule enrichment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1572. doi:10.1158/1538-7445.AM2015-1572

Abstract 4894: Targeted sequencing of myeloid cancers using single-molecule enrichment in picodroplets

Abstract A new method for enrichment of targeted genomic regions for next generation sequencing (NGS) is the use of single-molecule formatted PCR amplification, with the sample's target DNA partitioned into uniform picoliter volume droplets such that there are either zero or one target molecule in each droplet. All the droplets contain all of the primers and PCR reagents, ensuring that every target molecule from the sample is amplified, minimizing the amount of input required. Endpoint PCR using this single molecule format results in highly uniform yields that facilitate efficient use of the sequencing platform. In addition, the primers contain ‘Illumina tails’ that enable easy sample indexing and a workflow for loading directly onto a MiSeq without additional library preparation. Here we detail the use of this method with the ThunderBolts Myeloid Cancer Panel, which targets ∼550 commonly mutated regions in 49 genes implicated in the causation, prognosis, and recurrence of myeloid disorders. The panel targets include an expert-curated set of 16 full genes and 33 mutational hotspot regions, including challenging targets such as CEBPA and NOTCH1. We present results showing sample inputs as low as 10 nanograms (no pre-amplification used) provide sequencing metrics with 100% coverage at &amp;gt;100x depth (&amp;gt;95% at &amp;gt;500x depth) with a normalized mean read depth of 2500x and high uniformity (&amp;gt;90% of amplicons within 0.2x of the mean), enabling detection of low minor allele frequencies (&amp;lt;5%). The workflow is easy and rapid, with &amp;lt;2 hours hands-on time and sample-to-results in &amp;lt;48 hours. In addition, we demonstrate similar performance metrics when adding additional primer pairs (ThunderBolts PLUS), ensuring continued flexibility for researchers to use the core panel plus additional key targets as they are identified and validated. In summary, the ThunderBolts Myeloid Cancer Panel shows excellent sequencing coverage, high read depth and uniformity, and detection of low frequency mutations implicated in myeloid cancers. Starting from low input DNA samples, the workflow is easy and fast, enabling low cost targeted Illumina NGS sequencing with an expert curated panel that provides flexibility for expansion. Citation Format: Michael L. Samuels, Steve K. Kotsopoulos, Frances Long, Holly Gettler, Omo Clement, Jeff Olson. Targeted sequencing of myeloid cancers using single-molecule enrichment in picodroplets. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4894. doi:10.1158/1538-7445.AM2015-4894

A programmable droplet-based microfluidic device applied to multiparameter analysis of single microbes and microbial communities

We present a programmable droplet-based microfluidic device that combines the reconfigurable flow-routing capabilities of integrated microvalve technology with the sample compartmentalization and dispersion-free transport that is inherent to droplets. The device allows for the execution of user-defined multistep reaction protocols in 95 individually addressable nanoliter-volume storage chambers by consecutively merging programmable sequences of picoliter-volume droplets containing reagents or cells. This functionality is enabled by "flow-controlled wetting," a droplet docking and merging mechanism that exploits the physics of droplet flow through a channel to control the precise location of droplet wetting. The device also allows for automated cross-contamination-free recovery of reaction products from individual chambers into standard microfuge tubes for downstream analysis. The combined features of programmability, addressability, and selective recovery provide a general hardware platform that can be reprogrammed for multiple applications. We demonstrate this versatility by implementing multiple single-cell experiment types with this device: bacterial cell sorting and cultivation, taxonomic gene identification, and high-throughput single-cell whole genome amplification and sequencing using common laboratory strains. Finally, we apply the device to genome analysis of single cells and microbial consortia from diverse environmental samples including a marine enrichment culture, deep-sea sediments, and the human oral cavity. The resulting datasets capture genotypic properties of individual cells and illuminate known and potentially unique partnerships between microbial community members.

High-Throughput Analysis of Protein–Protein Interactions in Picoliter-Volume Droplets Using Fluorescence Polarization

Droplet-based microfluidic systems have emerged as a powerful platform for performing high-throughput biological experimentation. In addition, fluorescence polarization has been shown to be effective in reporting a diversity of bimolecular events such as protein-protein, DNA-protein, DNA-DNA, receptor-ligand, enzyme-substrate, and protein-drug interactions. Herein, we report the use of fluorescence polarization for high-throughput protein-protein interaction analysis in a droplet-based microfluidic system. To demonstrate the efficacy of the approach, we investigate the interaction between angiogenin (ANG) and antiangiogenin antibody (anti-ANG Ab) and demonstrate the efficient extraction of dissociation constants (K(D) = 10.4 ± 3.3 nM) within short time periods.

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1 In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented. Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Ultrasensitive and High-Throughput Fluorescence Analysis of Droplet Contents with Orthogonal Line Confocal Excitation

This paper describes a simple modification to traditional confocal fluorescence detection that greatly improves signal-to-noise (s/n) for the high-speed analysis of droplet streams. Rather than using the conventional epi geometry, illumination of the droplet was in the form of a line that is orthogonal to both the direction of flow and the light-collection objective. In contrast to the epi geometry where we observed high levels of scattering background from the droplets, we detected more than 10-fold less background (depending on the laser power used) when orthogonal-line-confocal illumination was used. We characterized this improvement using a standard microfluidic platform over a range of analyte concentrations and observed an improvement in limits of detection of greater than 10. Using this method, we were able to analyze picomolar concentrations of analytes contained within picoliter-volume droplets at a rate of greater than 350 droplets per second.