Paper-based open microfluidic platform for protein electrophoresis and immunoprobing.

Protein electrophoresis and immunoblotting are indispensable analytical tools for the characterization of proteins and posttranslational modifications in complex sample matrices. Owing to the lack of automation, commonly employed slab-gel systems suffer from high time demand, significant sample/antibody consumption, and limited reproducibility. To overcome these limitations, we developed a paper-based open microfluidic platform for electrophoretic protein separation and subsequent transfer to protein-binding membranes for immunoprobing. Electrophoresis microstructures were digitally printed into cellulose acetate membranes that provide mechanical stability while maintaining full accessibility of the microstructures for consecutive immunological analysis. As a proof-of-concept, we demonstrate separation of fluorescently labeled marker proteins in a wide molecular weight range (15-120 kDa) within only 15 min, reducing the time demand for the entire workflow (from sample preparation to immunoassay) to approximately one hour. Sample consumption was reduced 10- to 150-fold compared to slab-gel systems, owing to system miniaturization. Moreover, we successfully applied the paper-based approach to complex samples such as crude bacterial cell extracts. We envisage that this platform will find its use in protein analysis workflows for scarce and precious samples, providing a unique opportunity to extract profound immunological information from limited sample amounts in a fast fashion with minimal hands-on time.

Functional modification of fibronectin by N-terminal FXIIIa-mediated transamidation.

A straightforward strategy is presented for the site-specific incorporation of fluorophores or reactive probes into the extracellular matrix (ECM) protein fibronectin (Fn) by using the enzyme-catalyzed transamidation by activated factor XIII. Characterization by SDS-PAGE, western blotting, absorption measurements, mass spectrometry, and stepwise photobleaching for labeling quantification at the single-molecule level showed that the labeling was efficient and restricted to the N-terminal tails. The introduction of labels did not interfere with Fn fibrillogenesis, as verified by the incorporation of fluorescently labeled Fn into ECM and manually pulled Fn fibers. Site-specific incorporation of an azide was used to create a template for bioorthogonal click chemistry reactions in a second bioconjugation step, thus offering versatile modification and application possibilities in the context of matrix biology and tissue engineering.

Covalent cargo loading to molecular shuttles via copper-free "click chemistry".

An important prerequisite for molecular shuttle-based functional devices is the development of adequate linker chemistries to load and transport versatile cargoes. Copper-free "click chemistry" has not been applied before to covalently load cargo onto molecular shuttles propelled by biological motors such as kinesin. Due to the high biocompatibility and bioorthogonality of the strain-promoted azide-alkyne cycloaddition, this approach has pronounced advantages compared to previous methods.

Analyzing siRNA Concentration, Complexation and Stability in Cationic Dendriplexes by Stem-Loop Reverse Transcription-qPCR.

RNA interference (RNAi) is a powerful therapeutic approach for messenger RNA (mRNA) level regulation in human cells. RNAi can be triggered by small interfering RNAs (siRNAs) which are delivered by non-viral carriers, e.g., dendriplexes. siRNA quantification inside carriers is essential in drug delivery system development. However, current siRNA measuring methods either are not very sensitive, only semi-quantitative or not specific towards intact target siRNA sequences. We present a novel reverse transcription real-time PCR (RT-qPCR)-based application for siRNA quantification in drug formulations. It enables specific and highly sensitive quantification of released, uncomplexed target siRNA and thus also indirect assessment of siRNA stability and concentration inside dendriplexes. We show that comparison with a dilution series allows for siRNA quantification, exclusively measuring intact target sequences. The limit of detection (LOD) was 4.2 pM (±0.2 pM) and the limit of quantification (LOQ) 77.8 pM (±13.4 pM) for uncomplexed siRNA. LOD and LOQ of dendriplex samples were 31.6 pM (±0 pM) and 44.4 pM (±9.0 pM), respectively. Unspecific non-target siRNA sequences did not decrease quantification accuracy when present in samples. As an example of use, we assessed siRNA complexation inside dendriplexes with varying nitrogen-to-phosphate ratios. Further, protection of siRNA inside dendriplexes from RNase A degradation was quantitatively compared to degradation of uncomplexed siRNA. This novel application for quantification of siRNA in drug delivery systems is an important tool for the development of new siRNA-based drugs and quality checks including drug stability measurements.

Platelets drive fibronectin fibrillogenesis using integrin αIIbß3.

Platelets interact with multiple adhesion proteins during thrombogenesis, yet little is known about their ability to assemble fibronectin matrix. In vitro three-dimensional superresolution microscopy complemented by biophysical and biochemical methods revealed fundamental insights into how platelet contractility drives fibronectin fibrillogenesis. Platelets adhering to thrombus proteins (fibronectin and fibrin) versus basement membrane components (laminin and collagen IV) pull fibronectin fibrils along their apical membrane versus underneath their basal membrane, respectively. In contrast to other cell types, platelets assemble fibronectin nanofibrils using αIIbß3 rather than α5ß1 integrins. Apical fibrillogenesis correlated with a stronger activation of integrin-linked kinase, higher platelet traction forces, and a larger tension in fibrillar-like adhesions compared to basal fibrillogenesis. Our findings have potential implications for how mechanical thrombus integrity might be maintained during remodeling and vascular repair.

Rapid Detection of Pathogens in Wound Exudate via Nucleic Acid Lateral Flow Immunoassay.

The rapid detection of pathogens in infected wounds can significantly improve the clinical outcome. Wound exudate, which can be collected in a non-invasive way, offers an attractive sample material for the detection of pathogens at the point-of-care (POC). Here, we report the development of a nucleic acid lateral flow immunoassay for direct detection of isothermally amplified DNA combined with fast sample preparation. The streamlined protocol was evaluated using human wound exudate spiked with the opportunistic pathogen Pseudomonas aeruginosa that cause severe health issues upon wound colonization. A detection limit of 2.1 × 105 CFU per mL of wound fluid was achieved, and no cross-reaction with other pathogens was observed. Furthermore, we integrated an internal amplification control that excludes false negative results and, in combination with the flow control, ensures the validity of the test result. The paper-based approach with only three simple hands-on steps has a turn-around time of less than 30 min and covers the complete analytical process chain from sample to answer. This newly developed workflow for wound fluid diagnostics has tremendous potential for reliable pathogen POC testing and subsequent target-oriented therapy.

Eliminating viscosity bias in lateral flow tests.

Despite the widespread application of point-of-care lateral flow tests, the viscosity dependence of these assay results remains a significant challenge. Here, we employ centrifugal microfluidic flow control through the nitrocellulose membrane of the strip to eliminate the viscosity bias. The key feature is the balancing of the sample flow into the cassette of the lateral flow test with the air flow out of the cassette. A viscosity-independent flow rate of 3.01 ± 0.18 µl/min (±6%) is demonstrated for samples with viscosities ranging from 1.1 mPas to 24 mPas, a factor greater than 20. In a model human IgG lateral flow assay, signal-intensity shifts caused by varying the sample viscosity from 1.1 mPas to 2.3 mPas could be reduced by more than 84%.

Site-Specifically-Labeled Antibodies for Super-Resolution Microscopy Reveal In Situ Linkage Errors.

The precise spatial localization of proteins in situ by super-resolution microscopy (SRM) demands their targeted labeling. Positioning reporter molecules as close as possible to the target remains a challenge in primary cells or tissues from patients that cannot be easily genetically modified. Indirect immunolabeling introduces relatively large linkage errors, whereas site-specific and stoichiometric labeling of primary antibodies relies on elaborate chemistries. In this study, we developed a simple two-step protocol to site-specifically attach reporters such as fluorophores or DNA handles to several immunoglobulin G (IgG) antibodies from different animal species and benchmarked the performance of these conjugates for 3D STORM (stochastic optical reconstruction microscopy) and DNA-PAINT (point accumulation in nanoscale topography). Glutamine labeling was restricted to two sites per IgG and saturable by exploiting microbial transglutaminase after removal of N-linked glycans. Precision measurements of 3D microtubule labeling shell dimensions in cell lines and human platelets showed that linkage errors from primary and secondary antibodies did not add up. Monte Carlo simulations of a geometric microtubule-IgG model were in quantitative agreement with STORM results. The simulations revealed that the flexible hinge between Fab and Fc segments effectively randomized the direction of the secondary antibody, while the restricted binding orientation of the primary antibody's Fab fragment accounted for most of the systematic offset between the reporter and α-tubulin. DNA-PAINT surprisingly yielded larger linkage errors than STORM, indicating unphysiological conformations of DNA-labeled IgGs. In summary, our cost-effective protocol for generating well-characterized primary IgG conjugates offers an easy route to precise SRM measurements in arbitrary fixed samples.

One-step, wash-free, bead-based immunoassay employing bound-free phase detection.

We present a simple and fast one-step heterogeneous immunoassay, with performance characteristics that can enable easy and versatile adaptation to miniaturized, automated point-of-care systems. This novel analytical method uses magnetic and fluorescent beads as capture and detection agents respectively. Its main feature is the measurement of the fluorescent signal in the bound-free phase for (semi-)quantitative detection of analytes. Thus, no washing is required and the workflow consists only of sample and reagent supply, incubation, separation and detection. The immunoassay concept is demonstrated with C-reactive protein (CRP), a systemic inflammation marker. CRP in only 5 µL of undiluted serum was measured in the range 20-140 mg L-1 (includes clinically relevant cut-off values). The limit of detection (LOD) was 22.1 ± 6.3 mg L-1 (incubation 15 min). A CRP certified reference material was measured on five different days. Intra- and inter-assay coefficients of variation were 4.6 ± 1.9% and 5.6% respectively. To demonstrate the compatibility of the assay concept with additional matrices and concentration ranges, three oral inflammation markers, namely matrix metalloproteinases 8 and 9 (MMP-8, MMP-9) and tissue inhibitor of metalloproteinases 1 (TIMP-1), were measured in saliva in the ranges 0.47-30 ng mL-1 for MMP-8 and MMP-9, and 0.69-44 ng mL-1 for TIMP-1. LODs were 0.24 ng mL-1, 0.38 ng mL-1 and 0.39 ng mL-1 respectively (incubation 20 min). Multiplexing capacity of the assay concept was also shown with these markers. The demonstrated excellent reproducibility of the results, combined with the versatility and low complexity of the introduced immunoassay concept, make it an attractive candidate for applied analytical chemistry and automated point-of-care testing.

Flow control for lateral flow strips with centrifugal microfluidics.

Lateral flow strips (LFSs) are widely used for clinical diagnostics. The restricted flow control of the current designs is one challenge to the development of quantitative and highly sensitive LFSs. Here, we present a flow control for LFSs using centrifugal microfluidics. In contrast to previously presented implementations of lateral flow membranes into centrifugal microfluidic cartridges, we direct the flow radially outwards through the membrane. We control the flow using only the centrifugal force, thus it is independent of membrane wetting properties and permeability. The flow rate can be decreased and increased, enabling control of incubation times for a wide variety of samples. We deduced a formula as a guideline for the integration of chromatographic membranes into centrifugal microfluidic disks to ensure that all the sample liquid flows through the membrane, hence safely avoiding bypass flow around the membrane. We verified the calculated operation conditions using different membranes, different flow rates, and different sample viscosities.

Automated Pre-Analytic Processing of Whole Saliva Using Magnet-Beating for Point-of-Care Protein Biomarker Analysis.

Saliva offers many advantages for point-of-care (PoC) diagnostic applications due to non-invasive, easy, and cost-effective methods of collection. However, the complex matrix with its non-Newtonian behavior and high viscosity poses handling challenges. Several tedious and long pre-analytic steps, incompatible with PoC use, are required to liquefy and homogenize saliva samples before protein analysis can be performed. We apply magnet-beating to reduce hands-on time and to simplify sample preparation. A magnet in a chamber containing the whole saliva is actuated inside a centrifugal microfluidic cartridge by the interplay of centrifugal and magnetic forces. Rigorous mixing, which homogenizes the saliva sample, is then initiated. Consequently, fewer manual steps are required to introduce the whole saliva into the cartridge. After 4 min of magnet-beating, the processed sample can be used for protein analysis. The viscosity of whole saliva has been reduced from 10.4 to 2.3 mPa s. Immunoassay results after magnet-beating for three salivary periodontal markers (MMP-8, MMP-9, TIMP-1) showed a linear correlation with a slope of 0.99 when compared to results of reference method treated samples. Conclusively, magnet-beating has been shown to be a suitable method for the pre-analytic processing of whole saliva for fully automated PoC protein analysis.

Nanoshuttles propelled by motor proteins sequentially assemble molecular cargo in a microfluidic device.

Nanoshuttles powered by the molecular motor kinesin have the potential to capture and concentrate rare molecules from solution as well as to transport, sort and assemble them in a high-throughput manner. One long-thought-of goal has been the realisation of a molecular assembly line with nanoshuttles as workhorses. To harness them for this purpose might allow the community to engineer novel materials and nanodevices. The central milestone towards this goal is to expose nanoshuttles to a series of different molecules or building blocks and load them sequentially to build hierarchical structures, macromolecules or materials. Here, we addressed this challenge by exploiting the synergy of two so far mostly complementary techniques, nanoshuttle-mediated active transport and pressure-driven passive transport, integrated into a single microfluidic device to demonstrate the realisation of a molecular assembly line. Multiple step protocols can thus be miniaturised to a highly parallelised and autonomous working lab-on-a-chip: in each reaction chamber, analytes or building blocks are captured from solution and are then transported by nanoshuttles across fluid flow boundaries in the next chamber. Cargo can thus be assembled, modified, analysed and eventually unloaded in a procedure that requires only one step by its operator.