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Digital microfluidics (DMF) is a powerful technique for sample preparation and analysis for a broad range of biological and chemical applications. In many cases, it is desirable to carry out DMF on an open surface, such that the matrix surrounding the droplets is ambient air. However, the utility of the air-matrix DMF format has been severely limited by problems with droplet evaporation, especially when the droplet-based biochemical reactions require high temperatures for long periods of time. We present a simple solution for managing evaporation in air-matrix DMF: just-in-time replenishment of the reaction volume using droplets of solvent. We demonstrate that this solution enables DMF-mediated execution of several different biochemical reactions (RNA fragmentation, first-strand cDNA synthesis, and PCR) over a range of temperatures (4-95 °C) and incubation times (up to 1 h or more) without use of oil, humidifying chambers, or off-chip heating modules. Reaction volumes and temperatures were maintained roughly constant over the course of each experiment, such that the reaction kinetics and products generated by the air-matrix DMF device were comparable to those of conventional benchscale reactions. This simple yet effective solution for evaporation management is an important advance in developing air-matrix DMF for a wide variety of new, high-impact applications, particularly in the biomedical sciences.

Digital microfluidics (DMF) is a powerful technique for simple and precise manipulation of microscale droplets of fluid. This technique enables processing and analysis of a wide variety of samples and reagents and has proven useful in a broad range of chemical, biological, and medical applications. Handling of "real-world" samples has been a challenge, however, because typically their volumes are greater than those easily accommodated by DMF devices and contain analytes of interest at low concentration. To address this challenge, we have developed a novel "world-to-DMF" interface in which an integrated companion module drives the large-volume sample through a 10 μL droplet region on the DMF device, enabling magnet-mediated recovery of bead-bound analytes onto the device as they pass through the region. To demonstrate its utility, we use this system for extraction of RNA from human whole blood lysates (110-380 μL) and further purification in microscale volumes (5-15 μL) on the DMF device itself. Processing by the system was2-fold faster and consumed 12-fold less reagents, yet produced RNA yields and quality fully comparable to conventional preparations and supporting qRT-PCR and RNA-Seq analyses. The world-to-DMF system is designed for flexibility in accommodating different sample types and volumes, as well as for facile integration of additional modules to enable execution of more complex protocols for sample processing and analysis. As the first technology of its kind, this innovation represents an important step forward for DMF, further enhancing its utility for a wide range of applications.

Background: Dried blood spot (DBS) sampling is emerging as a valuable technique in a variety of fields, including clinical and preclinical testing of pharmaceuticals. Despite this popularity, current DBS sampling and analysis processes remain laborious and time consuming. Digital microfluidics, a microscale liquid-handling technique, characterized by the manipulation of discrete droplets on open electrode arrays, offers a potential solution to these problems. Results: We report a new digital microfluidic method for multiplexed extraction and analysis of pharmaceuticals in DBS samples. In the new method, four DBS samples are extracted in microliter-sized droplets containing internal standard, and the extract is delivered to dedicated nanoelectrospray ionization emitters for direct analysis by tandem mass spectometry and selected reaction monitoring. Conclusion: The new method allows for an order of magnitude reduction in processing time and approximately three-times reduction in extraction solvent relative to conventional techniques, while maintaining acceptable analytical performance for most drugs tested.

A microfluidic technique for combinatorial chemical synthesis of peptidomimetics has been developed. The new method is fast, automated and includes an integrated magnetic separation of inorganic catalysts from reaction products. This proof-of-concept study should lead to methods for generating libraries of compounds suitable for screening for bioactivity.

Electrospray ionization (ESI) has revolutionized mass spectrometry (MS), providing a facile method for the ionization of macromolecules for analysis by mass. The development of nanoESI-MS has further extended the utility of ESI-MS, permitting the analysis of small-volume samples with enhanced sensitivity over conventional ESI-MS. Traditional nanoESI-MS experiments use pulled-glass capillary emitters, which are expensive to purchase and require specialized instruments and training to fabricate in-house. Furthermore, these emitters suffer from problems including clogging, sample contamination, and irreproducible spray stability. Here, we report a new emitter for nanoESI-MS, made by folding small pieces of polyimide tape. In comparison with conventional pulled-glass capillary emitters, the new emitters are inexpensive and simple to make. Their low cost makes them disposable after a single use, such that sample contamination or clogging is never a problem. Emitter performance has been evaluated for diverse analytes encompassing a large mass range, including small molecules, peptides, proteins, and synthetic polymers. In all cases, the performance is similar to that of pulled-glass capillary emitters, with the advantages of low cost, ease of use, and disposability.

made with high chemoselectivity from amino acids or linear peptides, isocyanides, and amphoteric aziridine aldehydes in a one-step process. Importantly, the resulting products possess useful structural features that allow specific modification at defined positions. In our initial work, we formed serial batches of peptides using conventional macroscale synthetic techniques. The utility of this method is limited, however, without a high-throughput approach for generating focused libraries of peptide macrocycles. Such a method would be automated and would enable multistep reactions to be handled in parallel. Herein, we present a new miniaturized technique for synchronized on-chip synthesis of peptide macrocycles and related products. The most common format for miniaturized synthesis is enclosed microchannels in planar platforms. Such systems have been used for conventional organic synthesis, polymerization reactions, formation of biomolecules, such as peptides and DNA, and generation of nanoparticles and colloids. However, there are some challenges that limit the scope of their use for parallel chemical synthesis. For example, many microchannel platforms are formed from poly(dimethylsiloxane), a material that is susceptible to degradation in common organic solvents. Furthermore, control of many reagents simultaneously (a feature required to implement parallel synthetic reactions) in microchannels requires pumps, tubing, valves, and/or threedimensional channel networks that can be difficult to fabricate and operate. This has prompted researchers to develop specialized techniques to overcome this limitation. Another disadvantage associated with the microchannel format is the challenge inherent in the removal of solvents and re-dissolution of solids that are common steps in synthesis. Solid reagents and products can clog microchannels, making targeted reagent delivery difficult to control. Finally, the small volumes of samples in microchannels makes it difficult to recover them in sufficient quantities for off-line analysis techniques, such as NMR spectroscopy. In need of a platform capable of generating a) peptide macrocycles for downstream transfer onto functionalized surfaces, and b) spatially resolved macrocyclic peptide products in the solid state, we chose an alternative format for automation of synthesis, called digital microfluidics. In digital microfluidics, discrete nL to mL sized droplets of samples and reagents are controlled in parallel by applying a series of electrical potentials to an array of electrodes coated with a hydrophobic insulator (Figure 1). Digital microfluidics has become popular for biological and chemical applications, including cell culture and assays, enzyme assays, immunoassays, protein processing, clinical sample processing and analysis, and synthesis of anisotropic Scheme 1. Synthesis of peptide-based macrocycles and their structurally modified derivatives.

We present the first microfluidic method for extracting proteins from heterogeneous fluids by precipitation. The new method comprises an automated protocol for precipitation of proteins onto surfaces, rinsing the precipitates to remove impurities, and resolubilization in buffer for further analysis. The method is compatible with proteins representing a range of different physicochemical properties, as well as with complex mixtures such as fetal bovine serum and cell lysate. In all cases, the quantitative performance (measured using a fluorescent assay for % recovery) was comparable to that of conventional techniques, which are manual and require more time. Thus, this work represents an important first step in efforts to develop fully automated microfluidic methods for proteomic analyses.

The monolayer behavior of phosphatidylethanolamines bearing aliphatic chains of different length, C 16 and C 18 , and grafted with PEG chains of molecular weight 2000, DPPE-PEG2000 and DSPE-PEG2000, respectively, was studied at the air/water interface. Comparative analysis of the surface pressure–molecular area ( π – A ) isotherms, Brewster angle microscopy (BAM) and atomic force microscopy (AFM) data has provided a new insight in the nature of the high-pressure transition observed in the isotherms of both DPPE-PEG2000 and DSPE-PEG2000 monolayers. This high-pressure transition appears in the low compressibility region of the two PEG2000–phospholipid isotherms at remarkably different surface pressures, at ∼26 and ∼19 mN/m for DPPE-PEG2000 and DSPE-PEG2000, respectively. The transition has been found to originate from the monolayer collapse. As visualized by BAM and AFM, fluid DPPE-PEG2000 and DSPE-PEG2000 monolayers are unable to sustain high pressures and collapse through the formation of micellar defects in the monolayer structure as a result of 2D to 3D relaxation of an overcompressed film.

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (≥105) in milliliter-scale volumes (≥0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 μl). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

Next-generation sequencing (NGS) is emerging as a powerful tool for elucidating genetic information for a wide range of applications. Unfortunately, the surging popularity of NGS has not yet been accompanied by an improvement in automated techniques for preparing formatted sequencing libraries. To address this challenge, we have developed a prototype microfluidic system for preparing sequencer-ready DNA libraries for analysis by Illumina sequencing. Our system combines droplet-based digital microfluidic (DMF) sample handling with peripheral modules to create a fully-integrated, sample-in library-out platform. In this report, we use our automated system to prepare NGS libraries from samples of human and bacterial genomic DNA. E. coli libraries prepared on-device from 5 ng of total DNA yielded excellent sequence coverage over the entire bacterial genome, with >99% alignment to the reference genome, even genome coverage, and good quality scores. Furthermore, we produced a de novo assembly on a previously unsequenced multi-drug resistant Klebsiella pneumoniae strain BAA-2146 (KpnNDM). The new method described here is fast, robust, scalable, and automated. Our device for library preparation will assist in the integration of NGS technology into a wide variety of laboratories, including small research laboratories and clinical laboratories.

Digital microfluidics (DMF) has recently emerged as a popular technology for a wide range of applications. In DMF, nanoliter to microliter droplets containing samples and reagents can be manipulated to carry out a range of discrete fluidic operations simply by applying a series of electrical potentials to an array of patterned electrodes coated with a hydrophobic insulator. DMF is distinct from microchannel-based fluidics as it allows for precise control over multiple reagent phases (liquids and solids) in heterogeneous systems with no need for complex networks of connections, microvalves, or pumps. In this review, we discuss the most recent developments in this technology with particular attention to the potential benefits and outstanding challenges for applications in chemistry, biology, and medicine.

Dried blood spot (DBS) samples on filter paper are surging in popularity as a sampling and storage vehicle for a wide range of clinical and pharmaceutical applications. For example, a DBS sample is collected from every baby born in the province of Ontario, Canada, for quantification of approximately one hundred analytes that are used to screen for 28 conditions, including succinylacetone (SA), a marker for hepatorenal tyrosinemia. Unfortunately, the conventional methods used to evaluate DBS samples for newborn screening and other applications are tedious and slow, with limited options for automated analysis. In response to this challenge, we have developed a method to couple digital microfluidics (DMF) to nanoelectrospray ionization mass spectrometry (nESI-MS) for SA quantification in DBS samples. The new system is formed by sandwiching a pulled glass capillary emitter between the two DMF substrates such that the capillary emitter is immobilized without external seals or gaskets. Moreover, we introduce a new feedback control system that enables high-fidelity droplet manipulation across DBS samples without manual intervention. The system was validated by application to on-chip extraction, derivatization, and analysis of SA and other analytes from DBS samples, with comparable performance to gold-standard methods. We propose that the new methods described here can potentially contribute to a new generation of analytical techniques for quantifying analytes in DBS samples for a wide range of applications.

We introduce the marriage of two technologies: digital microfluidics (DMF), a technique in which droplets are manipulated by application of electrostatic forces on an array of electrodes coated by an insulator, and porous polymer monoliths (PPMs), a class of materials that is popular for use for solid-phase extraction and chromatography. In this work, circular PPM discs were formed in situ by dispensing and manipulating droplets of monomer solutions to designated spots on a DMF device followed by UV-initiated polymerization. We used PPM discs formed in this manner to develop a digital microfluidic solid-phase extraction (DMF-SPE) method, in which PPM discs are activated and equilibrated, samples are loaded, PPM discs are washed, and the samples are eluted, all using microliter droplets of samples and reagents. The new method has extraction efficiency (93%) comparable to that of pipet-based ZipTips and is compatible with preparative sample extraction and recovery for on-chip desalting, removal of surfactants, and preconcentration. We anticipate that DMF-SPE may be useful for a wide range of applications requiring preparative sample cleanup and concentration.

Estrogen is a key hormone in human reproductive physiology, controlling ovulation and secondary sexual characteristics. In addition, it plays an important role in the pathogenesis of breast cancer. Indeed, estrogen receptor antagonists and aromatase inhibitors (which block estrogen biosynthesis) are primary drugs used for treatment and prevention in at-risk populations. Despite its importance, tissue concentrations of estrogen are not routinely measured because conventional techniques require large samples of biopsies for analysis. In response to this need, we have developed a digital microfluidic method and applied it to the extraction and quantification of estrogen in 1-microliter samples of breast tissue homogenate (as would be collected with fine-needle aspiration), as well as in whole blood and serum. This method may be broadly applicable to conditions requiring frequent analysis of hormones in clinical samples (for example, infertility and cancer).

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics. Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work. Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Blood samples stored as dried blood spots (DBSs) are emerging as a useful sampling and storage vehicle for a wide range of applications. Unfortunately, the surging popularity of DBS samples has not yet been accompanied by an improvement in automated techniques for extraction and analysis. As a first step towards overcoming this challenge, we have developed a prototype microfluidic system for quantification of amino acids in dried blood spots, in which analytes are extracted, mixed with internal standards, derivatized, and reconstituted for analysis by (off-line and in-line) tandem mass spectrometry. The new method is fast, robust, precise, and most importantly, compatible with automation. We propose that the new method can potentially contribute to a new generation of analytical techniques for quantifying analytes in DBS samples for a wide range of applications.