Enhancing protein delivery for tissue regeneration: Development of AGR2-loaded hydrogels with controlled release properties.

The growth factor Anterior Gradient 2 (AGR2) has been shown to have an effective role in tissue regeneration, but remained largely unexplored in localized tissue engineering applications. Alginate beads have been proven as safe carriers for protein encapsulation, but they suffer from fragility and uncontrolled protein release. For such alginate systems, little is known about how changes in concentrations and ion-crosslinking affect protein release and accumulation in 3-D matrices. To address these questions, an engineered interpenetrating polymer network (IPN) has been used to synthesize a novel hybrid system consisting of AGR2 loaded beads composed of calcium-crosslinked sodium alginate (SA) and carboxymethyl cellulose (CMC). These beads are embedded in films consisting of SA and polyvinyl alcohol (PVA), using a simple ion gelation technique. We assess protein release kinetics and accumulation within the hybrid system by varying polymer concentrations and cross-linking parameters. The IPN hybrid system maintains controlled release over two weeks, without an initial burst period. Through this approach efficicnt delivery of AGR2 is achieved which in turn effectively mediates cell migration and proliferation, resulting in excellent cell viability and complete wound closure. The described release system opens new perspectives in tissue engineering.

Particle ID: A Multiplexed Hydrogel Bead Platform for Biomedical Applications.

We present a new platform based on hydrogel beads for multiplex analysis that can be fabricated, barcoded, and functionalized in a single step using a simple microfluidic assembly and a photo-cross-linking process. The beads are generated in a two-phase flow fluidic system and photo-cross-linking of the polymer in the aqueous phase by C,H insertion cross-linking (CHic). The size and shape of the hydrogel particles can be controlled over a wide range by fluidic parameters. During the fabrication of the beads, they are barcoded by using physical and optical barcoding strategies. Magnetic beads and fluorescent particles, which allow identification of the production batch number, are added simultaneously as desired, resulting in complex, multifunctional beads in a one-step reaction. As an example of biofunctionalization, Borrelia antigens were immobilized on the beads. Serum samples that originated from infected and non-infected patients could be clearly distinguished, and the sensitivity was as good as or even better than ELISA, the state of the art in clinical diagnostics. The ease of the one-step production process and the wide range of barcoding parameters offer strong advantages for multiplexed analytics in the life sciences and medical diagnostics.

Controlling Fluorescent Readout in Paper-based Analytical Devices.

Paper is an ideal candidate for the development of new disposable diagnostic devices because it is a low-cost material, allows transport of the liquid on the device by capillary action, and is environmentally friendly. Today, colorimetric analysis is most often used as a detection method for rapid tests (test strips or lateral flow devices) but usually gives only qualitative results and is limited by a relatively high detection threshold. Here, we describe studies using fluorescence as a readout tool for paper-based diagnostics. We study how the optical readout is affected by light transmission, scattering, and fluorescence as a function of paper characteristics such as thickness (grammage), water content, autofluorescence, and paper type/composition. We show that paper-based fluorescence analysis allows better optical readout compared to that of nitrocellulose, which is currently the material of choice in colorimetric assays. To reduce the loss of analyte molecules (e.g., proteins) due to adsorption to the paper surface, we coat the paper fibers with a protein-repellent hydrogel. For this purpose, we use hydrophilic copolymers consisting of N,N-dimethyl acrylamide and a benzophenone-based cross-linker, which are photochemically transformed into a fiber-attached polymer hydrogel on the paper fiber surfaces in situ. We show that the combination of fluorescence detection and the use of a protein-repellent coating enables sensitive paper-based analysis. Finally, the success of the strategy is demonstrated by using a simple LFD application as an example.

An advanced and efficient asymmetric PCR method for microarray applications.

The sensitivity of a PCR based biochip assay relies on the efficiency of PCR amplicons in binding to the microarray spots. The essential factor determining the sensitivity is the amount of single stranded (ss) amplicons available for biochip hybridization. Asymmetric PCR can generate ss-amplicons depending on the ratio of primers used in the amplification process, but this process is often inefficient. We report a novel variant of PCR called the Asymmetric Exponential and Linear Amplification (AELA) which can overcome these issues and generate large amounts of single stranded amplicons. AELA-PCR introduces an amplification strategy that makes use of both exponential and linear amplification of the target nucleic acid. This is done by specifically designed primers and choice of adequate thermal profiles. In conventional PCR with a classical thermal profile, these specifically designed primers will work normally and contribute to an exponential increase of amplicons. A designed sequence extension of one of the primers and a very specific thermal profile, will result in a situation that the extended primer will be the only functional one for amplification, resulting in a linear phase of the amplification process. That is why during this step only one of the two strands of the target is amplified linearly and no longer exponentially. The result of the whole process is an amplification product enriched very strongly in one of the two single strands of the target. These adaptions in PCR are particularly favorable where the generation of ss-DNA/RNA is required. We demonstrate the higher biochip sensitivity of AELA-PCR compared to conventional amplification methods with an example of the Staphylococcus aureus detection on a DNA oligonucleotide microarray.

Single-Color Barcoding for Multiplexed Hydrogel Bead-Based Immunoassays.

Current developments in precision medicine require the simultaneous detection of an increasing number of biomarkers in heterogeneous, complex solutions, such as blood samples. To meet this need, immunoassays on barcoded hydrogel beads have been proposed, although the encoding and decoding of these barcodes is usually complex and/or resource-intensive. Herein, an efficient method for the fabrication of barcoded, functionalized hydrogel beads is presented. The hydrogel beads are generated using droplet-based microfluidics in combination with photochemically induced C-H insertion reactions, allowing photo-crosslinking, (bio-) functionalization, and barcode integration to be performed in a single step. The generated functionalized beads carry single-color barcodes consisting of green-fluorescent particles of different sizes and concentrations, allowing simple and simultaneous readout with a standard plate reader. As a test example, the performance of barcoded hydrogel beads (3 × 3 matrix) functionalized with capture molecules of interest (e.g., antigens) is investigated for the detection of Lyme-disease-specific antibodies in patient sera. The described barcoding strategy for hydrogel beads does not interfere with the bioanalytical process and captivates by its simplicity and versatility, making it an attractive candidate for multiplex bioanalytical processes.

Reducing Unspecific Protein Adsorption in Microfluidic Papers Using Fiber-Attached Polymer Hydrogels.

Microfluidic paper combines pump-free water transport at low cost with a high degree of sustainability, as well as good availability of the paper-forming cellulosic material, thus making it an attractive candidate for point-of-care (POC) analytics and diagnostics. Although a number of interesting demonstrators for such paper devices have been reported to date, a number of challenges still exist, which limit a successful transfer into marketable applications. A strong limitation in this respect is the (unspecific) adsorption of protein analytes to the paper fibers during the lateral flow assay. This interaction may significantly reduce the amount of analyte that reaches the detection zone of the microfluidic paper-based analytical device (µPAD), thereby reducing its overall sensitivity. Here, we introduce a novel approach on reducing the nonspecific adsorption of proteins to lab-made paper sheets for the use in µPADs. To this, cotton linter fibers in lab-formed additive-free paper sheets are modified with a surrounding thin hydrogel layer generated from photo-crosslinked, benzophenone functionalized copolymers based on poly-(oligo-ethylene glycol methacrylate) (POEGMA) and poly-dimethyl acrylamide (PDMAA). This, as we show in tests similar to lateral flow assays, significantly reduces unspecific binding of model proteins. Furthermore, by evaporating the transport fluid during the microfluidic run at the end of the paper strip through local heating, model proteins can almost quantitatively be accumulated in that zone. The possibility of complete, almost quantitative protein transport in a µPAD opens up new opportunities to significantly improve the signal-to-noise (S/N) ratio of paper-based lateral flow assays.

Cryogel Monoliths for Analyte Enrichment by Capture and Release.

A platform based on cryogel monoliths in small capillaries, which allows very strong enrichment of an analyte through a capture and release process, is described. For their preparation, a photoreactive copolymer solution containing capture molecules of interest is filled into a capillary, frozen in, and then photochemically transformed into cryogel monoliths through C,H-insertion cross-linking reactions. As a test example, the platform is used for the preconcentration of dopamine from bovine serum albumin and urine samples through capture and release processes. During capture from a large volume and release into a smaller volume, the platform shows recovery rates up to 97% and allows up to a roughly 630-fold enrichment of the concentration of the analyte. The presented platform could be used as a disposable device for the purification and enrichment of a variety of cis-diol-containing samples.

Linear Cryogel Arrays: On the Fast Track for Borreliosis Detection.

The detection of IgG/IgM antibodies is a crucial tool for the diagnosis of infectious diseases as they give specific information such as the stage of infection or when it approximately occurred. In this work, a linear cryogel array (LCA) technology is described for the detection of IgG and IgM antibodies, indicative of a borreliosis infection in human sera. The LCA consists of a transparent capillary filled with functionalized cryogel compartments. For the generation of these cryogel arrays, solutions containing a photo-copolymer and the appropriate antigens are sucked into a surface-modified glass capillary. The solution compartments are separated from each other through air pockets. After freezing the solutions, a photo-induced cross-linking process is performed, through which the solutions are transformed into cryogel compartments, covalently attached to the capillary walls. We show that the LCA technology allows the simultaneous detection of IgG and IgM antibodies via a sandwich immunoassay in sera from Borrelia-infected patients within 1 h for sample sizes of only 12 µL. A study with sera from 42 patients conducted with the LCAs and referenced - depending on the source of the sera - to a commercial line immunoassay and a chemiluminescent immunoassay, which are currently widely used for Lyme disease screening, demonstrates the diagnostic potential of the approach.

Diazo-Based Copolymers for the Wet Strength Improvement of Paper Based on Thermally Induced CH-Insertion Cross-Linking.

We present an alternative to commonly used, but from an environmental point of view, problematic wet strength agents, which are usually added to paper to prevent a loss of mechanical stability and finally disintegrate when they get into contact with water. To this end, diazoester-containing copolymers are generated, which are coated onto paper and by heating to 110-160 °C for short periods of time become activated and form carbene intermediates, which undergo a CH-insertion cross-linking reaction. The process leads to a simultaneous cross-linking of the polymer and its attachment to the cellulose substrate. The immobilization process of copolymers consisting of a hydrophilic matrix based on N,N-dimethylacrylamide and a diazoester-based comonomer to a cellulose model surface and to laboratory-engineered, fibrous paper substrates is investigated as a function of time, temperature, and cross-linker composition. The distribution of the polymer in the fiber network is studied using confocal fluorescence microscopy. Finally, the tensile properties of modified wet and dry eucalyptus sulfate papers are measured to demonstrate the strong effect of the thermally cross-linked copolymers on the wet strength of paper substrates. Initial experiments show that the tensile indices of the modified and wetted paper samples are up to 50 times higher compared to the values measured for unmodified samples. When dry and wet papers coated with the above-described wetting agents are compared, relative wet strengths of over 30% are observed.

"CHicable" and "Clickable" Copolymers for Network Formation and Surface Modification.

In this study, we present the generation of novel, multifunctional polymer networks through a combination of C,H-insertion cross-linking (CHic) and click chemistry. To this, copolymers consisting of hydrophilic N,N-dimethylacrylamide as matrix component and repeat units containing azide moieties, as well as benzophenone or anthraquinone groups, are generated. The benzophenone or anthraquinone groups allow photo-cross-linking, surface attachment or covalent immobilization of adjacent (bio)molecules through CHic reactions. The azide moieties either can react with available alkynes through conventional click reactions or can be activated to form nitrenes, which can also undergo CHic reactions. By choosing appropriate reaction conditions, the same polymer can be used to follow very different reaction paths, opening up a plethora of choices for the generation of functional polymer networks. In the exemplary presented case ("CHic-Click"), irradiation of the copolymers with UV-A light (λirr = 365 nm) leads to cross-linking (network formation) and surface attachment simultaneously. The azide units remain intact during this cross-linking step, and alkyne-modified (bio)molecules can be bound through click reactions. Biofunctionalization of the polymer network with alkynylated streptavidin, followed by application of biotin-conjugated antibody and a model analyte, highlights the potential of these surface architectures as a toolbox which can be adapted for diverse bioanalytical applications.

Breaking the Interface: Efficient Extraction of Magnetic Beads from Nanoliter Droplets for Automated Sequential Immunoassays.

Droplet-based microfluidic systems offer a high potential for miniaturization and automation. Therefore, they are becoming an increasingly important tool in analytical chemistry, biosciences, and medicine. Heterogeneous assays commonly utilize magnetic beads as a solid phase. However, the sensitivity of state of the art microfluidic systems is limited by the high bead concentrations required for efficient extraction across the water-oil interface. Furthermore, current systems suffer from a lack of technical solutions for sequential measurements of multiple samples, limiting their throughput and capacity for automation. Taking advantage of the different wetting properties of hydrophilic and hydrophobic areas in the channels, we improve the extraction efficiency of magnetic beads from aqueous nanoliter-sized droplets by 2 orders of magnitude to the low µg/mL range. Furthermore, the introduction of a switchable magnetic trap enables repetitive capture and release of magnetic particles for sequential analysis of multiple samples, enhancing the throughput. In comparison to conventional ELISA-based sandwich immunoassays on microtiter plates, our microfluidic setup offers a 25-50-fold reduction of sample and reagent consumption with up to 50 technical replicates per sample. The enhanced sensitivity and throughput of this system open avenues for the development of automated detection of biomolecules at the nanoliter scale.

Hydrogel based protein biochip for parallel detection of biomarkers for diagnosis of a Systemic Inflammatory Response Syndrome (SIRS) in human serum.

The Systemic Inflammatory Response Syndrome (SIRS), a sepsis related inflammatory state, is a self-defense mechanism against specific and nonspecific stimuli. The six most extensively studied inflammatory biomarkers for the clinical diagnosis of SIRS are interleukin 4 (hIL-4), interleukin 6 (hIL-6), interleukin 10 (hIL-10), tumor necrosis factor alpha (hTNF-α), interferon gamma (hIFN-γ) and procalcitonin (hPCT). These biomarkers are naturally present (but usually only at low concentration) in SIRS infected patients [1, 2] and thus the development of a highly sensitive detection method is of major clinical interest. However, the existing analytical techniques are lacking in required analytical sensitivity and parallel determination of these biomarkers. We developed a fast, easy and cost-efficient protein microarray biochip where the capture molecules are attached on hydrogel spots, enabling SIRS diagnosis by parallel detection of these six clinically relevant biomarkers with a sample volume of 25 µl. With our hydrogel based protein microarray biochip we achieved a limit of detection for hIL-4 of 75.2 pg/ml, for hIL-6 of 45.1 pg/ml, for hIL-10 of 71.5 pg/ml, for hTNF-α of 56.7 pg/ml, for IFN-γ of 46.4 pg/ml and for hPCT of 1.1 ng/ml in spiked human serum demonstrating sufficient sensitivity for clinical usage. Additionally, we demonstrated successful detection of two relevant SIRS biomarkers in clinical patient samples with a turnaround time of the complete analysis from sample-to-answer in less than 200 minutes.

Biophysical Insights on the Enrichment of Cancer Cells from Whole Blood by (Affinity) Filtration.

Circulating tumor cells (CTCs) play a key role during the metastatic process of human cancers and their reliable detection and characterization could enable new and effective ways of cancer diagnosis, monitoring and treatment. However, due to their ultralow concentration in patient blood, the CTCs must first be enriched before such analysis can be performed. Classical microfiltration is an important and widely used method for the mechanical enrichment of CTCs. This method exploits that CTCs are generally larger than the accompanying blood cells, however, does not differentiate the cells in other ways. In an affinity filtration, selectivity is added by functionalizing the membrane with specific antibodies against a CTC-characteristic surface protein such as the epithelial cell adhesion molecule (EpCAM). A common shortcoming of both filtration approaches is that there is still a poor understanding of the enrichment process and the systems developed so far are frequently operated under non-optimized conditions. To address this, systematic filtration experiments are performed in this work using the EpCAM+ cell line MCF-7 as CTC-model and standard track-etched membranes modified with or without antibodies against EpCAM. The influences of the key filtration parameters time and applied pressure are studied and it is found that in all cases the extent of cell recovery is limited by a lysis process which occurs on the membrane surface. Counterintuitively, it is found that filtration at rather high pressures is advantageous to ensure high recovery rates. To describe the pressure-induced lysis process a biophysical model is developed. This model allows the determination of optimum filtration conditions to achieve both high cancer cell recovery and large blood sample throughput. It is demonstrated that this way practically 100% of spiked cancer cells can be recovered from milliliters of undiluted whole blood within seconds.

In Vitro Effects of a Novel Coating Agent on Bacterial Biofilm Development on Ureteral Stents.

OBJECTIVES: Ureteral stenting is a widely used method for noninvasive urinary drainage in ureteral obstruction. However, biofilm development due to transient bacteriuria can cause severe complications such as incrustation with subsequent obstruction as well as recurrent urinary tract infection. Apart from local ailment such as dysuria, this increases both stent replacement frequency and incidence of complications. In this work, we investigated in vitro the bacterial adhesion to a surface-attached and cross-linked poly(N,N-dimethylacrylamide) (PDMAA) hydrogel network, which is known for its nonfouling and protein-repellent characteristics. MATERIALS AND METHODS: To mimic the conditions encountered in vivo, PDMAA-coated and uncoated cyclic olefin polymer (COP) slides as well as polyurethane (PU)-coated glass slides were incubated in sterile human urine for 48 hours. Colonization was then simulated by adding known uropathogens, cultivated from clinical urine samples (such as Escherichia coli). After further incubation for 24 and 48 hours, slides were washed, and the remaining adherent bacteria were solubilized by ultrasound. CFUs were counted after plating and incubation for 48 hours of the resulting solution. RESULTS: PDMAA reduced adherent E. coli about fivefold on coated PU glass slides as well as in PDMAA-coated COP slides. With adherent Enterococcus faecalis and Klebsiella pneumoniae there was a tendency to decreased biofilm formation, but the difference was not statistically significant. CONCLUSIONS: PDMAA reduces surface adherence of the most common uropathogen significantly. Assessment of clinical relevance and of the effect on further uropathogens needs further experimental and clinical evaluations. German Clinical Trial Register ID: DRKS00013264 (approved WHO primary register).

Blocking-Free and Substrate-Independent Serological Microarray Immunoassays.

In bioanalytical applications, many coating strategies have been established for so-called "blocking" of the surfaces. However, most of the procedures developed so far require additional processing steps for surface blocking and small variations in the blocking efficiency result in increased background noise, which lowers the overall sensitivity of an assay. In this study, we demonstrate the preparation of a bioanalytical surface with a thin film of a photo-cross-linkable copolymer that is transformed photochemically into a surface-attached hydrogel network. The presented coating is directly applicable to various plastic substrates that are used for bioassays without the need for any prior surface modification. Such a strategy allows facile one-step immobilization of biomolecules for bioanalysis and protein-repellent properties for avoiding unspecific adsorption of analyte molecules during the assay. The protein adsorption behavior of the hydrogel-coated and blank surfaces is measured by SPR with human serum and physisorption of labeled detection antibodies. We show that the hydrogel surfaces used lower unspecific background signals and background noise and thus increase the sensitivities of the microarray immunoassays.

One-Step Photochemical Generation of Biofunctionalized Hydrogel Particles via Two-Phase Flow.

Biofunctional hydrogel particles have become increasingly popular in medical diagnostics; however, their generation is time-consuming and typically requires several process steps. We report on a new method for the simple, fast, and reproducible one-step generation of monodisperse hydrogel particles equipped with biofunctional molecules such as proteins or DNA. Key to the approach is the simultaneous photo cross-linking of the polymer chains and covalent binding of proteins or DNA through a C,H insertion reaction inside aqueous plug compartments that are produced via microfluidics. The strong performance in biological binding assays of the functionalized particles is demonstrated.

Geometrically enhanced sensor surfaces for the selective capture of cell-like particles in a laminar flow field.

Medical wires inserted into the blood stream of patients offer an attractive perspective to capture rare cells such as circulating tumor cells in vivo. A major challenge in such systems is to achieve an efficient interaction of the desired cells with the sensing surface and avoid those cells that simply flow by the wire without any contact while floating in a laminar flow field at some small distance to the sensor surface. We describe a new strategy to increase the interaction of cells or cell-like particles to such wire-shaped sensor surfaces both from an experimental and a theoretical point of view. For model experiments, we use cell-like particles that are flowing past the profile wire in a blood-like liquid stream. In the fluid dynamics simulations, this sensor is inserted into small capillaries. The influence of geometry and orientation of the wire with respect to the surrounding capillary onto the capture behavior is studied. Parameters, such as wire diameter, profile shape, wire torsion, and orientation of it with respect to the liquid stream, induce in some cases quite strong crossflows. These crossflows enhance the contact probability compared to a straight line wire of the same length by factors of up to about 80. A general model connecting the wire geometry with the crossflow intensity and the particle capture behavior is developed. Particle capture experiments demonstrate that the identified geometric factors can improve the capture of cell-like particles in laminar fluid flows and enhance the performance of such cell sensors.

Surface-attached hydrogel coatings via C,H-insertion crosslinking for biomedical and bioanalytical applications (Review).

A novel method for the generation of surface-attached hydrogel coatings and their use in biomedical applications is discussed. This short review concentrates on surface architectures that are prepared from prepolymers carrying reactive groups suitable for crosslinking via C,H insertion reactions [C,H insertion crosslinking (CHic)]. Upon photochemical or thermal activation these groups do not only induce the crosslinking of the system, but also connect the forming gel to the surface as long as the surface itself consists of an organic material. C,H groups as the reaction partner are available in abundance at practically all types of organic surfaces such as biomaterials or polymers, rendering the technique almost universally applicable. Surface-attached gels prepared this way show unique swelling properties due to the confinement of the chains, as the obtained essentially two-dimensional gels can only swell in one dimension. This anisotropic swelling does not permit penetration of the layers by macromolecules so that the surfaces become bioinert, i.e., are strongly protein and cell repellent. It is discussed how this property can be used to control the interaction of surfaces with biological species ranging from the level of biomolecules to living cells. A combination of the CHic chemistry and microstructuring techniques opens further avenues for the study of the behavior of cells to the generation of novel bioanalytical devices.

Preparation of Linear Cryogel Arrays as a Microfluidic Platform for Immunochromatographic Assays.

We describe a new microfluidic platform to perform immunochromatographic assays. The platform consists of a linear assembly of small, porous cryogel monoliths functionalized with various biomolecules. The cryogels are anchored in an optically transparent capillary, which serves as the microfluidic carrier. This assembly enables fluid flow by capillary action and simple optical detection. Using an in situ preparation method, individual compartments are generated from small plugs of polymer solutions that are transformed into small individually functionalized cryogel monoliths through a photoinduced cross-linking reaction. In the same reaction step, the monoliths are firmly anchored to the surface of the capillary. As proof-of-concept, a prototype platform is successfully used for the detection of the inflammatory marker interleukin 6 via a sandwich immunoassay. We observe excellent assay performance metrics that include high sensitivity, good linearity, and low variation. We also demonstrate fluid transport solely by passive means, which is a critical attribute for point-of-care diagnostics.