David Juncker

Fiber-based tissue engineering: Progress, challenges, and opportunities

Tissue engineering aims to improve the function of diseased or damaged organs by creating biological substitutes. To fabricate a functional tissue, the engineered construct should mimic the physiological environment including its structural, topographical, and mechanical properties. Moreover, the construct should facilitate nutrients and oxygen diffusion as well as removal of metabolic waste during tissue regeneration. In the last decade, fiber-based techniques such as weaving, knitting, braiding, as well as electrospinning, and direct writing have emerged as promising platforms for making 3D tissue constructs that can address the abovementioned challenges. Here, we critically review the techniques used to form cell-free and cell-laden fibers and to assemble them into scaffolds. We compare their mechanical properties, morphological features and biological activity. We discuss current challenges and future opportunities of fiber-based tissue engineering (FBTE) for use in research and clinical practice.

High-sensitivity miniaturized immunoassays for tumor necrosis factor α using microfluidic systems

We use microfluidic chips to detect the biologically important cytokine tumor necrosis factor alpha (TNF- alpha) with picomolar sensitivity using sub-microliter volumes of samples and reagents. The chips comprise a number of independent capillary systems (CSs), each of which is composed of a filling port, an appended microchannel, and a capillary pump. Each CS fills spontaneously by capillary forces and includes a self-regulating mechanism that prevents adventitious drainage of the microchannels. Thus, interactive control of the flow in each CS is easily achieved via collective control of the evaporation in all CSs by means of two Peltier elements that can independently heat and cool. Long incubation times are crucial for high sensitivity assays and can be conveniently obtained by adjusting the evaporation rate to have low flow rates of approximately 30 nL min(-1). The assay is a sandwich fluorescence immunoassay and takes place on the surface of a poly(dimethylsiloxane)(PDMS) slab placed across the microchannels. We precoat PDMS with capture antibodies (Abs), localize the capture of analyte molecules using a chip, then bind the captured analyte molecules with fluorescently-tagged detection Abs using a second chip. The assay results in a mosaic of fluorescence signals on the PDMS surface which are measured using a fluorescence scanner. We show that PDMS is a compatible material for high sensitivity fluorescence assays, provided that detection antibodies with long excitation wavelength fluorophores ( > or =580 nm) are employed. The chip design, long incubation times, proper choice of fluorophores, and optimization of the detection Ab concentration all combine to achieve high-sensitivity assays. This is exemplified by an experiment with 170 assay sites, occupying an area of approximately 0.6 mm(2) on PDMS to detect TNF-alpha in 600 nL of a dendritic cell (DC) culture medium with a sensitivity of approximately 20 pg mL(-1)(1.14 pM).

Fabricating Microarrays of Functional Proteins Using Affinity Contact Printing

Phenomena involving the binding between biomolecules are ubiquitous in biology and are essential for cell growth, signal transmission, and immune defense. In the latter system, the binding between antibody and antigen has already been exploited technologically to perform affinity purifications on columns and immunoassays on surfaces.[1] Recently, the fabrication of microarrays of proteins which require the immobilization of a large number of receptors on a surface have fueled the invention of novel patterning techniques such as pin-spotting and drop-on-demand.[2] Microarrays of proteins may find utility in proteomics, immunoassays, or for screening libraries of (bio)chemicals. It is at present not clear which patterning method will be the one best suited to pattern proteins on surfaces, but classical lithography does not seem capable of fabricating microarrays of proteins. Soft lithography[3] offers the possibility of manipulating proteins and other biomolecules by printing them from a micropatterned stamp to a surface[4] or by depositing them from a liquid using microfluidic networks ( FNs).[5] Affinity microcontact printing ( CP)[6] is a refined soft-lithographic technique that uses an elastomeric stamp made of polydimethylsiloxane (PDMS) and derivatized with binding biomolecules to extract corresponding binding partners from an impure, dilute source for placing them on a surface with spatial control. Herein, we describe, by using one particular example, how specific binding between biomolecules provides a unique opportunity to make use of self-assembly processes in technology: we propose different variants of CP to pattern surfaces with ensembles of biomolecules where the pattern on the affinity stamp ( -stamp) is not determined by its topography but by the position of various proteins covalently linked to a planar -stamp (Figure 1). This modified surface enables the simultaneous capture of different target proteins on the -stamp from a complex solution (Figure 1A). Thus, the capture step (Figure 1B) directs the assembly of an array of target molecules on the stamp (Figure 1C), which can be Figure 1. Microarrays of proteins on surfaces can be fabricated using an stamp derivatized with various capture sites that can extract target biomolecules from a complex solution and release them on a surface in a single microcontact-printing step. The -stamp can be reused for several inking and printing cycles.

Immunochromatographic Assay on Thread

Lateral-flow immunochromatographic assays are low-cost, simple-to-use, rapid tests for point-of-care screening of infectious diseases, drugs of abuse, and pregnancy. However, lateral flow assays are generally not quantitative, give a yes/no answer, and lack multiplexing. Threads have recently been proposed as a support for transporting and mixing liquids in lateral-flow immunochromatographic assays, but their use for quantitative high-sensitivity immunoassays has yet to be demonstrated. Here, we introduce the immunochromatographic assay on thread (ICAT) in a cartridge format that is suitable for multiplexing. The ICAT is a sandwich assay performed on a cotton thread knotted to a nylon fiber bundle, both of which are precoated with recognition antibodies against one target analyte. Upon sample application, the assay results become visible to the eye within a few minutes and are quantified using a flatbed scanner. Assay conditions were optimized, the binding curves for C-reactive protein (CRP) in buffer and diluted serum were established and a limit of detection of 377 pM was obtained. The possibility of multiplexing was demonstrated using three knotted threads coated with antibodies against CRP, osteopontin, and leptin proteins. The performance of the ICAT was compared with that of the paper-based and conventional assays. The results suggest that thread is a suitable support for making low-cost, sensitive, simple-to-use, and multiplexed diagnostic tests.

Antibody Colocalization Microarray: A Scalable Technology for Multiplex Protein Analysis in Complex Samples

DNA microarrays were rapidly scaled up from 256 to 6.5 million targets, and although antibody microarrays were proposed earlier, sensitive multiplex sandwich assays have only been scaled up to a few tens of targets. Cross-reactivity, arising because detection antibodies are mixed, is a known weakness of multiplex sandwich assays that is mitigated by lengthy optimization. Here, we introduce (1) vulnerability as a metric for assays. The vulnerability of multiplex sandwich assays to cross-reactivity increases quadratically with the number of targets, and together with experimental results, substantiates that scaling up of multiplex sandwich assays is unfeasible. We propose (2) a novel concept for multiplexing without mixing named antibody colocalization microarray (ACM). In ACMs, both capture and detection antibodies are physically colocalized by spotting to the same two-dimensional coordinate. Following spotting of the capture antibodies, the chip is removed from the arrayer, incubated with the sample, placed back onto the arrayer and then spotted with the detection antibodies. ACMs with up to 50 targets were produced, along with a binding curve for each protein. The ACM was validated by comparing it to ELISA and to a small-scale, conventional multiplex sandwich assay (MSA). Using ACMs, proteins in the serum of breast cancer patients and healthy controls were quantified, and six candidate biomarkers identified. Our results indicate that ACMs are sensitive, robust, and scalable.

Microfluidic quadrupole and floating concentration gradient

The concept of fluidic multipoles, in analogy to electrostatics, has long been known as a particular class of solutions of the Navier-Stokes equation in potential flows, however, experimental observations of fluidic multipoles and of their characteristics have not been reported yet. Here we present a two-dimensional microfluidic quadrupole and a theoretical analysis consistent with the experimental observations. The microfluidic quadrupole was formed by simultaneously injecting and aspirating fluids from two pairs of opposing apertures in a narrow gap formed between a microfluidic probe and a substrate. A stagnation point was formed at the center of the microfluidic quadrupole, and its position could be rapidly adjusted hydrodynamically. Following the injection of a solute through one of the poles, a stationary, tunable, and movable – i.e. “floating” – concentration gradient was formed at the stagnation point. Our results lay the foundation for future combined experimental and theoretical exploration of microfluidic planar multipoles including convective-diffusive phenomena.

Hydrogel Templates for Rapid Manufacturing of Bioactive Fibers and 3D Constructs

Hydrogel templates are formed to entrap various pre-polymers prior to their crosslinking process. Upon the completion of the crosslinking process, an independent polymer network with the same fiber geometry is formed. The hydrogel template can be removed if necessary. As the proof-of-principle, fibers from various polymers are fabricated. The fabricated hybrid polymeric fibers are bioactive and can be bioprinted or assembled using textile processes. The approach can be used for creating complex 3D constructs for various applications.

Cross-reactivity in antibody microarrays and multiplexed sandwich assays: shedding light on the dark side of multiplexing

Immunoassays are indispensable for research and clinical analysis, and following the emergence of the omics paradigm, multiplexing of immunoassays is more needed than ever. Cross-reactivity (CR) in multiplexed immunoassays has been unexpectedly difficult to mitigate, preventing scaling up of multiplexing, limiting assay performance, and resulting in inaccurate and even false results, and wrong conclusions. Here, we review CR and its consequences in single and dual antibody single-plex and multiplex assays. We establish a distinction between sample-driven and reagent-driven CR, and describe how it affects the performance of antibody microarrays. Next, we review and evaluate various platforms aimed at mitigating CR, including SOMAmers and protein fractionation-bead assays, as well as dual Ab methods including (i) conventional multiplex assays, (ii) proximity ligation assays, (iii) immuno-mass spectrometry, (iv) sequential multiplex analyte capture, (v) antibody colocalization microarrays and (vi) force discrimination assays.

Microfluidic direct writer with integrated declogging mechanism for fabricating cell-laden hydrogel constructs

Cell distribution and nutrient supply in 3D cell-laden hydrogel scaffolds are critical and should mimic the in vivo cellular environment, but been difficult to control with conventional fabrication methods. Here, we present a microfluidic direct writer (MFDW) to construct 3D cell-laden hydrogel structures with openings permitting media exchange. The MFDW comprises a monolithic microfluidic head, which delivers coaxial streams of cell-laden sodium alginate and calcium chloride solutions to form hydrogel fibers. Fiber diameter is controlled by adjusting the ratio of the volumetric flow rates. The MFDW head is mounted on a motorized stage, which is automatically controlled and moves at a speed synchronized with the speed of fiber fabrication. Head geometry, flow rates, and viscosity of the writing solutions were optimized to prevent the occurrence of curling and bulging. For continuous use, a highly reliable process is needed, which was accomplished with the integration of a declogging conduit supplying a solvent to dissolve the clogging gel. The MFDW was used for layer-by-layer fabrication of simple 3D structures with encapsulated cells. Assembly of 3D structures with distinct fibers is demonstrated by alternatively delivering two different alginate gel solutions. The MFDW head can be built rapidly and easily, and will allow 3D constructs for tissue engineering to be fabricated with multiple hydrogels and cell types.

Minimum information about a protein affinity reagent (MIAPAR)

This is a proposal developed within the community as an important first step in formalizing standards in reporting the production and properties of protein binding reagents, such as antibodies, developed and sold for the identification and detection of specific proteins present in biological samples. It defines a checklist of required information, intended for use by producers of affinity reagents, quality-control laboratories, users and databases. We envision that both commercial and freely available affinity reagents, as well as published studies using these reagents, could include a MIAPAR-compliant document describing the products properties with every available binding partner.