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Sandwich immunoassays such as the enzyme-linked immunosorbent assay (ELISA) have been miniaturized and performed in a lab-on-a-chip format, but the execution of the multiple assay steps typically requires a computer or complex peripherals. Recently, an ELISA for detecting antibodies was encoded structurally in a chip thanks to the microfluidic chain reaction but the need for precise pipetting and intolerance to commonly used surfactant concentrations limited the potential for broader adoption. Here, we introduce the ELISA-on-a-chip with aliquoting functionality that obviates the need for precise pipetting, accommodates higher surfactant concentrations, includes barrier channels that delay the contact between solutions and prevent undesired mixing, and that executed a quantitative, high sensitivity assay for the SARS-CoV-2 nucleocapsid protein in 4×-diluted saliva. Upon loading the chip using disposable pipettes, capillary flow draws each reagent and the sample into a separate volumetric measuring reservoir for detection antibody (70 µL), enzyme conjugate (50 µL), substrate (80 µL), and sample (210 µL), and splits washing buffer into 4 different reservoirs of 40, 40, 60, and 20 µL. The excess volume is autonomously drained via a structurally encoded capillaric aliquoting circuit, creating aliquots with an accuracy of >93%. Next, the user click-connects the assay module, comprising a nitrocellulose membrane with immobilized capture antibodies and a capillary pump, to the chip which triggers the step-by-step, timed flow of all aliquoted solutions. A colored precipitate forming a line on a nitrocellulose strip serves as an assay readout, and upon digitization, yielded a binding curve with a limit of detection of 54 and 91 pg/mL for buffer and diluted saliva respectively, vastly outperforming rapid tests. The ELISA chip is 3D-printed, modular, adaptable to other targets and assays, and could be used to automate ELISA in the lab; or as a diagnostic test at the point of care with the convenience and form factor of rapid tests while preserving the protocol and performance of central laboratory ELISA.

Proteins are found both outside and inside of extracellular vesicles (EVs) and govern the properties and functions of EVs, while also constituting a signature of the cell of origin and of biological function and disease. Outer proteins on EVs can be directly bound by antibodies to either enrich EVs, or probe the expression of a protein on EVs, including in a combinatorial manner. However, co-profiling of inner proteins remains challenging. Here, we present the high-throughput, multiplexed analysis of extracellular vesicle inner and outer proteins (EVPio). We describe the optimization of fixation and heat-induced protein epitope retrieval for EVs, along with oligo-barcoded antibodies and branched DNA signal amplification for sensitive, multiplexed and high-throughput assays. We captured 4 subpopulations of EVs from colorectal cancer cell lines HT29 and SW403 based on EpCAM, CD9, CD63 and CD81 expression, and quantified the co-expression of 8 outer (integrins and tetraspanins) and 4 inner (heat shock, endosomal and inner leaflet) proteins. The differences in co-expression patterns were consistent with the literature and known biological function. In conclusion, EVPio analysis can simultaneously detect multiple inner and outer proteins in EVs immobilized on a surface, opening the way to extensive combinatorial protein profiles for both discovery and clinical translation.

Digital manufacturing (DM) strives for the seamless manufacture of a functional device from a digital file. DM holds great potential for microfluidics, but requirements for embedded conduits and high resolution beyond the capability of common manufacturing equipment, and microfluidic systems’ dependence on peripherals (e.g. connections, power supply, computer), have limited its adoption. Microfluidic capillaric circuits (CCs) are structurally-encoded, self-contained microfluidic systems that operate and self-fill thanks to precisely tailored hydrophilicity. CCs were heretofore hydrophilized in a plasma chamber, but which only produces transient hydrophilicity, lacks reproducibility, and limits CC design to open surface channels sealed with a tape. Here we introduce the additive DM of monolithic, fully functional and intrinsically hydrophilic CCs. CCs were 3D printed with commonly available light engine-based 3D printers using polyethylene(glycol)diacrylate-based ink co-polymerized with hydrophilic acrylic acid crosslinkers and optimized for hydrophilicity and printability. A new, robust capillary valve design and embedded conduits with circular cross-sections that prevent bubble trapping are presented, and complex interwoven circuit architectures created, and their use illustrated with an immunoassay. Finally, the need for external paper capillary pumps is eliminated by directly embedding the capillary pump in the chip as a porous gyroid structure, realizing fully functional, monolithic CCs. Thence, a computer-aided design file can be made into a CC by commonly available 3D printers in less than 30 minutes enabling low-cost, distributed, DM of fully functional ready-to-use microfluidic systems.

Chain reactions are characterized by initiation, propagation and termination, are stochastic at microscopic scales and underlie vital chemical (e.g. combustion engines), nuclear and biotechnological (e.g. polymerase chain reaction) applications.1-5 At macroscopic scales, chain reactions are deterministic and limited to applications for entertainment and art such as falling domino and Rube Goldberg machines. Appositely, the microfluidic lab-on-a-chips (also called a micro total analysis system),6,7 was envisioned as an integrated chip, akin to microelectronic integrated circuits, yet in practice remain dependent on cumbersome peripherals, connections, and a computer for automation.8-11 Capillary microfluidics integrate energy supply and flow control onto a single chip by using capillary phenomena, but programmability remains rudimentary with at most a handful (eight) operations possible.12-19 Here we introduce the microfluidic chain reaction (MCR) as the conditional, structurally programmed propagation of capillary flow events. Monolithic chips integrating a MCR are 3D printed, and powered by the free-energy of a paper pump, autonomously execute liquid handling algorithms step-by-step. With MCR, we automated (i) the sequential release of 300 aliquots across chained, interconnected chips, (ii) a protocol for SARS-CoV-2 antibodies detection in saliva, and (iii) a thrombin generation assay by continuous subsampling and analysis of coagulation-activated plasma with parallel operations including timers, iterative cycles of synchronous flow and stop-flow operations. MCRs are untethered from and unencumbered by peripherals, encode programs structurally in situ, and can form frugal, versatile, bona fide lab-on-a-chip with wide-ranging applications in liquid handling and point-of-care diagnostics.

We present the nELISA, a miniaturised, high-throughput, and high-fidelity protein profiling platform. DNA oligonucleotides are used to pre-colocalize antibody pairs on spectrally encoded microparticles and perform displacement-mediated detection while ensuring spatial separation between non-cognate antibody pairs. Read-out is performed cost-efficiently and at high-throughput using flow cytometry. We assembled an inflammatory panel of 191 targets that were multiplexed without cross-reactivity or impact to performance vs 1-plex signals, with sensitivities as low as 0.1pg/mL and measurements across the platform spanning 8 orders of magnitude. We then performed a large-scale PBMC secretome screen, with cytokines as both perturbagens and read-outs, measuring 7,392 samples and generating ∼1.5M protein datapoints in under a week, a significant advance in throughput compared to other highly multiplexed immunoassays. We uncovered 447 significant cytokine responses, including multiple putatively novel cytokine responses, that were conserved across donors and stimulation conditions. We also validated its use in phenotypic screening, and proposed applications for the nELISA in drug discovery.

This paper introduces string microfluidics (SM), which consists of stretchable strings trapping discrete microdroplets within a porous matrix, and the realization of elementary microfluidic operations such as droplet formation, transport, splitting, merging, and mixing by moving and stretching the strings. While performing these operations, SM was shown to be compatible with colorimetric biological assays. SM represents a new form of microfluidics that integrates the concepts of thread microfluidics and digital microfluidics, along with mobile or reconfigurable microfluidics where liquid transport is realized by manipulating the substrate.

Chain reactions are characterized by initiation, propagation and termination, are stochastic at microscopic scales and underlie vital chemical (e.g. combustion engines), nuclear and biotechnological (e.g. polymerase chain reaction) applications.1–5 At macroscopic scales, chain reactions are deterministic and limited to applications for entertainment and art such as falling domino and Rube Goldberg machines. Appositely, microfluidic lab-on-a-chip (also called a micro total analysis system),6,7 which were envisioned pursuant to microelectronic integrated circuits, are generally not integrated on a chip owing to an enduring dependency on cumbersome connections, peripherals, and on computers for automation.8–11 Capillary microfluidics integrate energy supply and flow control onto a single chip by using capillary phenomena, but programmability remains rudimentary with at most a handful (eight) operations possible.12–19 Here we introduce mesoscopic microfluidic chain reactions (MCRs) based on capillary phenomena for reliable programming and automation of complex liquid handling algorithms integrated in a chip. MCRs are encoded into the chip microarchitecture, 3D printed as a monolithic circuit, and deterministically propagated by the free-energy of a paper pump. With MCR, we sequentially triggered the release of 300 aliquots across chained, interconnected chips, and automated a protocol for SARS-CoV-2 antibodies detection in saliva with visual and quantitative results by cell phone imaging. We automated and miniaturized for the first time the labor-intensive thrombogram with serial and parallel operations including timers and iterative cycles of synchronous flow and stop-flow sequences. Thrombograms with normal, hemophilia-like, and anticoagulant-spiked plasma were generated. MCRs are generalizable, and both the density and number of chain reaction units are scalable. MCRs are untethered from and unencumbered by peripherals, encode programs structurally in situ, and form a frugal, versatile, bona fide lab-on-a-chip with wide-ranging applications in liquid handling and point-of-care diagnostics.

Antibody microarrays enable multiplexed protein detection with minimal reagent consumption, but they continue to be plagued by lack of reproducibility. Chemically functionalized glass slides are used as substrates, yet antibody binding spatial inhomogeneity across the slide has not been analyzed in antibody microarrays. Here, we characterize spatial bias across five commercial slides patterned with nine overlapping dense arrays (by combining three buffers and three different antibodies), and we measure signal variation for both antibody immobilization and the assay signal, generating 270 heatmaps. Spatial bias varied across models, and the coefficient of variation ranged from 4.6 to 50%, which was unexpectedly large. Next, we evaluated three layouts of spot replicates-local, random, and structured random-for their capacity to predict assay variation. Local replicates are widely used but systematically underestimate the whole-slide variation by up to seven times; structured random replicates gave the most accurate estimation. Our results highlight the risk and consequences of using local replicates: the underappreciation of spatial bias as a source of variability, poor assay reproducibility, and possible overconfidence in assay results. We recommend the detailed characterization of spatial bias for antibody microarrays and the description and use of distributed positive replicates for research and clinical applications.

A method modifying a vacuum-assisted UV micro-molding (VAUM) process is proposed for the fabrication of polymer two-level submicron high porosity membranes (2LHPM). The modified process allows for the fabrication of robust, large-area membranes over 5 × 5 cm2 with a hierarchical architecture made from a 200 nm-thick layer having submicron level pores (as small as 500 nm) supported by a 20 μm-thick layer forming a microporous structure with 10-15 μm diameter pores. The fabricated freestanding membranes are flexible and mechanically robust enough for post manipulation and filtration of cell samples. Very high white blood cell (WBC) capture efficiencies (≈97%) from healthy blood samples after red blood cell (RBC) lysis are demonstrated using a 3D-printed filter cartridge incorporated within these 2LHPM. A high release efficiency of ≈95% is also proved using the same setup. Finally, on-filter multistep immunostaining of captured cells is also shown.

Nucleic acid aptamers are single stranded DNA or RNA sequences that specifically bind a cognate ligand. In addition to their widespread use as stand-alone affinity binding reagents in analytical chemistry, aptamers have been engineered into a variety of ligand-specific biosensors, termed aptasensors. One of the most common aptasensor formats is the duplexed aptamer (DA). As defined herein, DAs are aptasensors containing two nucleic acid elements coupled via Watson-Crick base pairing: (i) an aptamer sequence, which serves as a ligand-specific receptor, and (ii) an aptamer-complementary element (ACE), such as a short DNA oligonucleotide, which is designed to hybridize to the aptamer. The ACE competes with ligand binding, such that DAs generate a signal upon ligand-dependent ACE-aptamer dehybridization. DAs possess intrinsic advantages over other aptasensor designs. For example, DA biosensing designs generalize across DNA and RNA aptamers, DAs are compatible with many readout methods, and DAs are inherently tunable on the basis of nucleic acid hybridization. However, despite their utility and popularity, DAs have not been well defined in the literature, leading to confusion over the differences between DAs and other aptasensor formats. In this review, we introduce a framework for DAs based on ACEs, and use this framework to distinguish DAs from other aptasensor formats and to categorize cis- and trans-DA designs. We then explore the ligand binding dynamics and chemical properties that underpin DA systems, which fall under conformational selection and induced fit models, and which mirror classical SN1 and SN2 models of nucleophilic substitution reactions. We further review a variety of in vitro and in vivo applications of DAs in the chemical and biological sciences, including riboswitches and riboregulators. Finally, we present future directions of DAs as ligand-responsive nucleic acids. Owing to their tractability, versatility and ease of engineering, DA biosensors bear a great potential for the development of new applications and technologies in fields ranging from analytical chemistry and mechanistic modeling to medicine and synthetic biology.

Brain implants are increasingly used to treat neurological disorders and diseases. However, the brain foreign body response (FBR) elicited by implants affects neuro-electrical transduction and long-term reliability limiting their clinical adoption. The mismatch in Young’s modulus between silicon implants (∼180 GPa) and brain tissue (∼1-30 kPa) exacerbates the FBR resulting in the development of flexible implants from polymers such as polyimide (∼1.5-2.5 GPa). However, a stiffness mismatch of at least two orders of magnitude remains. Here, we introduce (i) the first mechanically matched brain implant (MMBI) made from silicone (∼20 kPa), (ii) new microfabrication methods, and (iii) a novel dissolvable sugar shuttle to reliably implant MMBIs. MMBIs were fabricated via vacuum-assisted molding using sacrificial sugar molds and were then encased in sugar shuttles that dissolved within 2 min after insertion into rat brains. Sections of rat neocortex implanted with MMBIs, PDMS implants, and silicon implants were analyzed by immunohistochemistry 3 and 9-weeks post-implantation. MMBIs resulted in significantly higher neuronal density and lower FBR within 50 µm of the tissue-implant interface compared to PDMS and silicon implants suggesting that materials mechanically matched to brain further minimize the FBR and could contribute to better implant functionality and long-term reliability.

Common multiplex sandwich immunoassays suffer from cross-reactivity due to the mixing of detection antibodies and the combinatorial, undesired interaction between all reagents and analytes. Here we present the snap chip to perform antibody colocalization microarrays that eliminates undesirable interactions by running an array of singleplex assays realized by sequestering detection antibodies in individual nanodroplets. When detecting proteins in biological fluids, the absence of cross-reactivity allows a higher level of multiplexing, reduced background, increased sensitivity, and ensures accurate and specific results. The use of the snap chip is illustrated by measuring highly related analytes such as proteins isoforms and phospho-proteins, both particularly prone to cross-reactivity, in a single experiment. The main steps of the protocol are preparation of sample, incubation on an assay slide harboring the microarrayed capture antibodies, transfer of the microarrayed detection antibodies on their cognate spots, and measurement of the assay results by fluorescence.

Common multiplex sandwich immunoassays suffer from cross-reactivity due to the mixing of detection antibodies and the combinatorial, undesired interaction between all reagents and analytes. Here we present the snap chip to perform antibody colocalization microarrays that eliminates undesirable interactions by running an array of singleplex assays realized by sequestering detection antibodies in individual nanodroplets. When detecting proteins in biological fluids, the absence of cross-reactivity allows a higher level of multiplexing, reduced background, increased sensitivity, and ensures accurate and specific results. The use of the snap chip is illustrated by measuring highly related analytes such as proteins isoforms and phospho-proteins, both particularly prone to cross-reactivity, in a single experiment. The main steps of the protocol are preparation of sample, incubation on an assay slide harboring the microarrayed capture antibodies, transfer of the microarrayed detection antibodies on their cognate spots, and measurement of the assay results by fluorescence.

Background A major obstacle to anti-viral and -tumor cell vaccination and T cell immunotherapy is the ability to produce dendritic cells (DCs) in a suitable clinical setting. It is imperative to develop closed cell culture systems to accelerate the translation of promising DC-based cell therapy products to the clinic. The objective of this study was to investigate whether viral antigen-loaded monocyte-derived DCs (Mo-DCs) capable of eliciting specific T cell activation can be manufactured in fluorinated ethylene propylene (FEP) bags. Methods Mo-DCs were generated through a protocol applying cytokine cocktails combined with lipopolysaccharide or with a CMV viral peptide antigen in conventional tissue culture polystyrene (TCPS) or FEP culture vessels. Research-scale ( Results Monocyte differentiation into Mo-DCs led to the loss of CD14 expression with concomitant upregulation of CD80, CD83 and CD86. Significantly increased levels of IL-10 and IL-12 were observed after maturation on day 9. Antigen-pulsed Mo-DCs activated antigen-responsive CD8+ cytotoxic T cells. No significant differences in surface marker expression or tetramer-specific T cell activating potency of Mo-DCs were observed between TCPS and FEP culture vessels. Conclusions Our findings demonstrate that viral antigen-loaded Mo-DCs produced in downscaled FEP bags can elicit specific T cell responses. In view of the dire clinical need for closed system DC manufacturing, FEP bags represent an attractive option to accelerate the translation of promising emerging DC-based immunotherapies.

Bioprinting, within the emerging field of biofabrication, aims at the fabrication of functional biomimetic constructs. Different three-dimensional bioprinting techniques have been adapted to bioprint cell-laden bioinks. However, single-material bioprinting techniques oftentimes fail to reproduce the complex compositions and diversity of native tissues. Multi-material bioprinting as an emerging approach enables the fabrication of heterogeneous multi-cellular constructs that replicate their host microenvironments better than single-material approaches. Here, we briefly review bioprinting modalities, discuss how they are being adapted to multi-material bioprinting, as well as analyze their advantages and challenges, encompassing both custom-designed and commercially available technologies. The review offers a perspective of how multi-material bioprinting opens up new opportunities for tissue engineering, tissue model engineering, therapeutics development, and personalized medicine.

Immunohistochemistry (IHC) on tissue sections is widely used for quantifying the expression patterns of proteins and is part of the standard of care for cancer diagnosis and prognosis, but is limited to staining a single protein per tissue. Tissue microarray and microfluidics staining methods have emerged as powerful high throughput techniques, but they either only permit the analysis of a single protein per slide or require complex instrumentation and expertise while only staining isolated areas. Here, we introduce IHC microarrays (IHCμA) for multiplexed staining of intact tissues with preserved histological and spatial information. Droplets of a dextran solution containing antibodies were prespotted on a slide and snapped onto a preprocessed formalin-fixed, paraffin-embedded (FFPE) tissue section soaked in a polyethylene glycol solution. The antibodies are confined within the dextran droplets and locally stain the tissue below with a contrast similar to the one obtained by conventional IHC. The microarray of antibody droplets can be prespotted on a slide and stored, thus neither the preparation of the antibody solutions nor a sophisticated microarray spotter is needed. Sampling considerations with IHCμA were evaluated by taking three tissues with varying levels of cancer cells. A multiplex IHCμA with 180 spots targeting 8 cancer proteins was performed on a breast cancer tissue section to illustrate the potential of this method. This work opens the avenue of applying microarray technologies for conducting IHC on intact tissue slices and has great potential to be used in the discovery and validation of tissue biomarkers in human tumors.

Urinary tract infections (UTI) are one of the most common bacterial infections and would greatly benefit from a rapid point-of-care diagnostic test. Although significant progress has been made in developing microfluidic systems for nucleic acid and whole bacteria immunoassay tests, their practical application is limited by complex protocols, bulky peripherals, and slow operation. Here we present a microfluidic capillaric circuit (CC) optimized for rapid and automated detection of bacteria in urine. Molds for CCs were constructed using previously established design rules, then 3D-printed and replicated into poly(dimethylsiloxane). CCs autonomously and sequentially performed all liquid delivery steps required for the assay. For efficient bacteria capture, on-the-spot packing of antibody-functionalized microbeads was completed in20 s followed by autonomous sequential delivery of 100 μL of bacteria sample, biotinylated detection antibodies, fluorescent streptavidin conjugate, and wash buffer for a total volume ≈115 μL. The assay was completed in7 min. Fluorescence images of the microbead column revealed captured bacteria as bright spots that were easily counted manually or using an automated script for user-independent assay readout. The limit of detection of E. coli in synthetic urine was 1.2 × 10

Quantitative protein assays play an important role in the study of biological functions. Immunoassays and mass spectrometry are two main technologies for quantifying proteins in biological samples. The combination of immunoprecipitation (IP) with MALDI technology delivers high assay sensitivity and specificity, but the sample preparation procedure involves multiple washing and transfer steps. These steps can be performed either manually (requiring significant time and labor) or automatically (requiring the purchase of a complex liquid-handling workstation). This bottleneck has limited the widespread adoption of this technology. We present here the Bead-Extractor Assisted ready-to-use Reagent System (BEARS) technology for simplified, low cost protein and peptide immunoprecipitation combined with MALDI-MS detection. All of the reagents are stable during long-term storage and can be prepared in advance. In the BEARS technology, a magnetic-bead extractor is used to handle beads from 96 wells simultaneously. A BEARS-based method was developed for plasma renin activity (PRA) and was evaluated on fifty-three clinical samples. These experiments showed that the BEARS assay had an LOD and linear range comparable to the manual method and an automated iMALDI PRA assay, but was 4-times faster than the manual approach. The BEARS iMALDI results also correlated well with a conventional ELISA PRA assay, with a coefficient of determination of 0.98. The BEARS technology provides convenience and affordability, and extends the use of IP-based mass spectrometry technology to most research and clinical laboratories, including those in developing countries.

Hybridizing a complementary oligonucleotide to an ATP aptamer is shown to functionally regulate a newly revealed induced fit ligand-binding pathway., Duplexed aptamers (DAs) are engineered by hybridizing an aptamer-complementary element (ACE, e.g. a DNA oligonucleotide) to an aptamer; to date, ACEs have been presumed to sequester the aptamer into a non-binding duplex state, in line with a conformational selection-based model of ligand binding. Here, we uncover that DAs can actively bind a ligand from the duplex state through an ACE-regulated induced fit mechanism. Using a widely-studied ATP DNA aptamer and a solution-based equilibrium assay, DAs were found to exhibit affinities up to 1 000 000-fold higher than predicted by conformational selection alone, with different ACEs regulating the level of induced fit binding, as well as the cooperative allostery of the DA (Hill slope of 1.8 to 0.7). To validate these unexpected findings, we developed a non-equilibrium surface-based assay that only signals induced fit binding, and corroborated the results from the solution-based assay. Our findings indicate that ACEs regulate ATP DA ligand binding dynamics, opening new avenues for the study and design of ligand-responsive nucleic acids.

Surface bound guidance cues and gradients are vital for directing cellular processes during development and repair. In vivo, these cues are often presented within a soft extracellular matrix with elastic moduli E < 10 kPa, but in vitro haptotaxis experiments have been conducted primarily on hard substrates with elastic moduli in the MPa to GPa range. Here, a technique is presented for patterning haptotactic proteins with nanometer resolution on soft substrates with physiological elasticity. A new nanocontact printing process was developed that circumvented the use of plasma activation that was found to alter the mechanical properties of the substrate. A dissolvable poly(vinyl alcohol) film was first patterned by lift-off nanocontact printing, and in turn printed onto the soft substrate, followed by dissolution of the film in water. An array of 100 unique digital nanodot gradients (DNGs), consisting of millions of 200 × 200 nm2 protein nanodots, was patterned in less than 5 min with with

SummaryCirculating tumor cells (CTCs) are rare (few cells per milliliter of blood) and mostly isolated as single cell CTCs (scCTCs). CTC clusters (cCTCs), even rarer, are of growing interest, notably because of their higher metastatic potential, but very difficult to isolate. Here, we introduce gravity-based microfiltration (GµF) for facile isolation of cCTCs. We identify cluster break-up as a confounding cause, and achieve ∼85% capture efficiency. GµF from orthotopic ovarian cancer mouse models and from 10 epithelial ovarian cancer (EOC) patients uncovered cCTCs in every case, with between 2-100+ cells. cCTCs represented between 5-30% of all CTC events, and 10-80% of captured CTCs were clustered; remarkably, in two patients, more CTCs were circulating as cCTCs than scCTCs. GµF uncovered the unexpected prevalence, frequency and sometimes large size of cCTCs in EOC patients with either metastatic and localized disease, and motivates additional studies to uncover their properties and role in disease progression.

Circulating tumor cells (CTCs) have been linked to cancer progression but are difficult to isolate, as they are very rare and heterogeneous, covering a range of sizes and expressing different molecular receptors. Filtration has emerged as a simple and powerful method to enrich CTCs but only captures cells above a certain size regardless of molecular characteristics. Here, we introduce antibody-functionalized microfilters to isolate CTCs based on both size and surface receptor expression. We present a 3D printed filtration cartridge with microfabricated polymer filters with 8, 10, 12, 15, or 20 μm-diameter pores. Pristine filters were used to optimize sample dilution, rinsing protocol, flow rate, and pore size, leading to >80% for the recovery of spiked cancer cells with very low white blood cell contamination (96%. These results suggest that filtration can be optimized to target specific CTC characteristics such as size and receptor expression and that a diverse range of CTCs may be captured using particular combinations of pore size, filtration parameters, and antibody functionalization.

We recently developed capillaric circuits (CCs) – advanced capillary microfluidic devices assembled from capillary fluidic elements in a modular manner similar to the design of electric circuits (Safavieh & Juncker, Lab Chip, 2013, 13, 4180–4189). CCs choreograph liquid delivery operations according to pre-programmed capillary pressure differences with minimal user intervention. CCs were thought to require high-precision micron-scale features manufactured by conventional photolithography, which is slow and expensive. Here we present CCs manufactured rapidly and inexpensively using 3D-printed molds. Molds for CCs were fabricated with a benchtop 3D-printer, poly(dimethylsiloxane) replicas were made, and fluidic functionality was verified with aqueous solutions. We established design rules for CCs by a combination of modelling and experimentation. The functionality and reliability of trigger valves – an essential fluidic element that stops one liquid until flow is triggered by a second liquid – was tested for different geometries and different solutions. Trigger valves with geometries up to 80-fold larger than cleanroom-fabricated ones were found to function reliably. We designed retention burst valves that encode sequential liquid delivery using capillary pressure differences encoded by systematically varied heights and widths. Using an electrical circuit analogue of the CC, we established design rules to ensure strictly sequential liquid delivery. CCs autonomously delivered eight liquids in a pre-determined sequence in

Microfluidic multipoles (MFMs) have been realized experimentally and hold promise for “open-space” biological and chemical surface processing. Whereas convective flow can readily be predicted using hydraulic-electrical analogies, the design of advanced microfluidic multipole is constrained by the lack of simple, accurate models to predict mass transport within them. In this work, we introduce the complete solutions to mass transport in multipolar microfluidics based on the iterative conformal mapping of 2D advection-diffusion around a simple edge into dipoles and multipolar geometries, revealing a rich landscape of transport modes. The models are validated experimentally with a library of 3D printed devices and found in excellent agreement. Following a theory-guided design approach, we further ideate and fabricate two classes of spatiotemporally reconfigurable multipolar devices that are used for processing surfaces with time-varying reagent streams, and to realize a multistep automated immunoassay. Overall, the results set the foundations for exploring, developing, and applying open-space microfluidic multipoles., Microfluidic multipoles use arrays of sources and sinks to confine fluids and reagents without the use of physical channels. Here the authors use conformal mappings to predict both convective and diffusive transport in these flows and 3D print multipoles to automate surface-based immunoassays.

Microfluidics offer economy of reagents, rapid liquid delivery, and potential for automation of many reactions, but often require peripheral equipment for flow control. Capillary microfluidics can deliver liquids in a pre-programmed manner without peripheral equipment by exploiting surface tension effects encoded by the geometry and surface chemistry of a microchannel. Here, we review the history and progress of microchannel-based capillary microfluidics spanning over three decades. To both reflect recent experimental and conceptual progress, and distinguish from paper-based capillary microfluidics, we adopt the more recent terminology of capillaric circuits (CCs). We identify three distinct waves of development driven by microfabrication technologies starting with early implementations in industry using machining and lamination, followed by development in the context of micro total analysis systems (μTAS) and lab-on-a-chip devices using cleanroom microfabrication, and finally a third wave that arose with advances in rapid prototyping technologies. We discuss the basic physical laws governing capillary flow, deconstruct CCs into basic circuit elements including capillary pumps, stop valves, trigger valves, retention valves, and so on, and describe their operating principle and limitations. We discuss applications of CCs starting with the most common usage in automating liquid delivery steps for immunoassays, and highlight emerging applications such as DNA analysis. Finally, we highlight recent developments in rapid prototyping of CCs and the benefits offered including speed, low cost, and greater degrees of freedom in CC design. The combination of better analytical models and lower entry barriers (thanks to advances in rapid manufacturing) make CCs both a fertile research area and an increasingly capable technology for user-friendly and high-performance laboratory and diagnostic tests.

Hybrid hydrogel fibers were fabricated using hydrogel templates. On page 2146, D. Juncker, A. Khademhosseini, and co-workers show how these fibers containing different fluorescent microbeads have been assembled using textile processes, such as braiding and weaving, to create complex 3D patterns for bioengineering applications. The fabricated constructs with clinically relevant dimensions are mechanically stable.

Duplexed aptamers (DAs) are ligand-responsive constructs engineered by hybridizing an aptamer with an aptamer-complementary element (ACE, e.g., a DNA oligonucleotide). Although DAs are commonly deployed, the binding dynamics of ternary ACE-aptamer–ligand systems remain underexplored, having been conventionally described by a conformational selection framework. Here we introduce aptamer-complementary element scanning (ACE-Scan) as a method to generate comprehensive hybridization, spontaneous off-rate, and induced fit ligand-binding landscapes for entire DA families. ACE-Scan reveals induced fit in DAs engineered from small molecule- and protein-binding DNA and RNA aptamers, as well as DAs engineered from the natural add riboswitch aptamer. To validate ACE-Scan, we engineer solution-phase ATP-specific DAs from 5 ACEs with varying spontaneous and induced fit off-rates, generating aptasensors with 8-fold differences in dynamic range consistent with ACE-Scan. This work demonstrates that ACE-Scan can readily map induced fit in DAs, empowering aptamers in biosensing, synthetic biology, and DNA nanomachines., Duplexed aptamers are a common biosensor format; however, how complementary strand sequence, length, and position modulate ligand binding is not well understood. Here, the authors introduce ACE-Scan to comprehensively map binding landscapes, uncovering hotspots of enhanced binding by induced fit.

The preparation and handling of mammalian single-cell genomic DNA is limited by the complexity bottleneck inherent to performing multi-step, multi-reagent operations in a microfluidic environment. We have developed a method for benchtop preparation of high-molecular weight, intact, single-cell genomes and demonstrate the extraction of long nucleic acid molecules in a microfluidic system. Lymphoblasts are encapsulated inside of alginate microparticles using a droplet microfluidics, and cells are lysed in bulk. The purified genomes are then delivered to and imaged on a dedicated microfluidic device. High-molecular weight DNA is protected from shear and retains its original cellular identity. Using this encapsulation protocol, we were able to extract individual nucleic acid strands on the millimeter scale inside of a microfluidic channel.

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that

Antibody microarrays can detect multiple proteins simultaneously, but the need for bulky and expensive fluorescence scanners limits their adaptation in clinical settings. Here we introduce a 15-plex enzyme-mediated silver enhanced sandwich immunoassay (SENSIA) on a microarray as an economic alternative to conventional fluorescence microarray assays. We compared several gold and silver amplification schemes, optimized HRP-mediated silver amplification, and evaluated the use of flatbed scanners for microarray quantification. Using the optimized assay condition, we established binding curves for 15 proteins using both SENSIA and conventional fluorescence microarray assays and compared their limits of detection (LODs) and dynamic ranges (DRs). We found that the LODs for all proteins are in the pg/mL range, with LODs for 12 proteins below 10 pg/mL. All but two proteins (ENDO and IL4) have similar LODs (less than 10-fold difference) and all but two proteins (IL1b and MCP1) are similar in DR (less than 1.5-log difference). Furthermore, we spiked six proteins in diluted serum and measured them by both silver enhancement and fluorescence detection and found a good agreement (R(2)0.9) between the two methods, suggesting that a complex matrix such as serum has a minimal effect on the measurement. By combining enzyme-mediated silver enhancement and consumer electronics for optical detection, SENSIA presents a new opportunity for low-cost high-sensitivity multiplex immunoassays for clinical applications.

In vitro motility and laser trap assays are commonly used for molecular mechanics measurements. However, chemicals cannot be added during these measurements, because they create flows that alter the molecular mechanics. Thus, we designed a microfluidic device that allows the addition of chemicals without creating bulk flows. Biocompatibility of the components of this device was tested. A microchannel chamber was created by photolithography with the patterns transferred to polydimethylsiloxane (PDMS). The PDMS chamber was bound to a polycarbonate membrane, which itself was bound to a molecular mechanics chamber. The microchannels ensured rapid distribution of the chemicals over the membrane, whereas the membrane ensured efficient delivery to the mechanics chamber while preventing bulk flow. The biocompatibility of the materials was tested by comparing the velocity (ν(max)) of propulsion by myosin of fluorescently labeled actin filaments to that of the conventional assay; no difference in ν(max) was observed. To estimate total chemical delivery time, labeled bovine serum albumin was injected in the channel chamber and TIRF was used to determine the time to reach the assay surface (2.7 ± 0.1 s). Furthermore, the standard distance of a trapped microsphere calculated during buffer diffusion using the microfluidic device (14.9 ± 3.2 nm) was not different from that calculated using the conventional assay (15.6 ± 5.3 nm, p = 0.922). Finally, ν(max) obtained by injecting adenosine triphosphate (ATP) in the microchannel chamber (2.37 ± 0.48 μm/s) was not different from that obtained when ATP was delivered directly to the mechanics chamber (2.52 ± 0.42 μm/s, p = 0.822). This microfluidic prototype validates the design for molecular mechanics measurements.

International audience; Localized interactions are predicted to favour the evolution of cooperation amongst individuals within a population. One important factor that can localize interactions is habitat patchiness. We hypothesize that habitats with greater patchiness (greater edge-to-area ratio) can facilitate the maintenance of cooperation. This outcome is believed to be particularly relevant in pathogenic microbes that can inhabit patchy habitats such as the human respiratory tract. To test this hypothesis in a simple but spatially controlled setting, we designed a transparent microhabitat chip (MHC) with multiple patchiness treatments at the 100 micron scale. The MHC is a closed system that sustains bacterial replication and survival for up to 18 hours, and allows spatial patterns and eco-evolutionary dynamics to be observed undisturbed. Using the opportunistic pathogen Pseudomonas aeruginosa, we tracked the growth of wild-type cooperators, which produce the public good pyoverdin, in competition with mutant defectors or cheaters that use, but do not produce, pyoverdin. We found that while defectors on average outperformed cooperators in all habitats, habitat patchiness significantly alleviated the ecological pressure against cooperation due to defection, leading to coexistence. Our results confirmed that habitat-level spatial heterogeneity can be important for cooperation. The MHC enables novel experiments, allows multiple parameters to be precisely varied and studied simultaneously, and will help uncover dynamical features of spatial ecology and the evolution of pathogens.

Axonal growth cones extend during neural development in response to precise distributions of extracellular cues. Deleted in colorectal cancer (DCC), a receptor for the chemotropic guidance cue netrin-1, directs F-actin reorganization, and is essential for mammalian neural development. To elucidate how the extracellular distribution of netrin-1 influences the distribution of DCC and F-actin within axonal growth cones, we patterned nanoarrays of substrate bound netrin-1 using lift-off nanocontact printing. The distribution of DCC and F-actin in embryonic rat cortical neuron growth cones was then imaged using total internal reflection fluorescence (TIRF) microscopy. Fluorescence fluctuation analysis via image cross-correlation spectroscopy (ICCS) was applied to extract the molecular density and aggregation state of DCC and F-actin, identifying the fraction of DCC and F-actin colocalizing with the patterned netrin-1 substrate. ICCS measurement of spatially segmented images based on the substrate nanodot patterns revealed distinct molecular distributions of F-actin and DCC in regions directly overlying the nanodots compared to over the reference surface surrounding the nanodots. Quantifiable variations between the populations of DCC and F-actin on and off the nanodots reveal specific responses to the printed protein substrate. We report that nanodots of substrate-bound netrin-1 locally recruit and aggregate DCC and direct F-actin organization. These effects were blocked by tetanus toxin, consistent with netrin-1 locally recruiting DCC to the plasma membrane via a VAMP2-dependent mechanism. Our findings demonstrate the utility of segmented ICCS image analysis, combined with precisely patterned immobilized ligands, to reveal local receptor distribution and signaling within specialized subcellular compartments.

Measuring many proteins at once is of great importance to the idea of personalized medicine, in order to get a snapshot of a person's health status. We describe the antibody colocalization microarray (ACM), a variant of antibody microarrays which avoids reagent-induced cross-reactivity by printing individual detection antibodies atop their corresponding capture antibodies. We discuss experimental parameters that are critical for the success of ACM experiments, namely, the printing positional accuracy needed for the two printing rounds and the need for protecting dried spots during the second printing round. Using small sample volumes (less than 30 μL) and small quantities of reagents, up to 108 different targets can be measured in hundreds of samples with great specificity and sensitivity.

Quantitative models of Forster resonance energy transfer (FRET)-pioneered by Forster-define our understanding of FRET and underpin its widespread use. However, multicolour FRET (mFRET), which arises between multiple, stochastically distributed fluorophores, lacks a mechanistic model and remains intractable. mFRET notably arises in fluorescently barcoded microparticles, resulting in a complex, non-orthogonal fluorescence response that impedes their encoding and decoding. Here, we introduce an ensemble mFRET (emFRET) model, and apply it to guide barcoding into regimes with extreme FRET. We further introduce a facile, proportional multicolour labelling method using oligonucleotides as homogeneous linkers. A total of 580 barcodes were rapidly designed and validated using four dyes-with FRET efficiencies reaching 76%-and used for multiplexed immunoassays with cytometric readout and fully automated decoding. The emFRET model helps to expand the barcoding capacity of barcoded microparticles using common organic dyes and will benefit other applications subject to stochastic mFRET.

A vacuum assisted UV micro-molding (VAUM) process is proposed for the fabrication of freestanding and defect-free polymer membranes based on a UV-curable methacrylate polymer (MD 700). VAUM is a highly flexible and powerful method for fabricating low cost, robust, large-area membranes over 9 × 9 cm2 with pore sizes from 8 to 20 μm in diameter, 20 to 100 μm in thickness, high aspect ratio (the thickness of the polymer over the diameter of the hole is up to 15 : 1), high porosity, and a wide variety of geometrical characteristics. The fabricated freestanding membranes are flexible while mechanically robust enough for post manipulation and handling, which allows them to be cut and integrated as a plastic cartridge onto thermoplastic 3D microfluidic devices with single or double filtration stages. Very high particle capture efficiencies (≈98%) have been demonstrated in the microfluidic devices integrated with polymer membranes, even when the size of the beads is very close to the size of the pores of the microfilter. About 85% of the capture efficiency has been achieved in cancer cell trapping experiments, in which a breast cancer cell line (MDA-MB-231) spiked with phosphate-buffered saline buffer when the pore size of the filter is 8 μm and the device is operated at a flow rate of 0.1 mL min−1.