Printed Antifouling Electrodes for Biosensing Applications.

Biosensors based on miniaturized, functional electrodes are of high potential for various biosensing applications, especially at the point-of-care setting among others. However, the sensor performance of such electrochemical devices is still strongly limited, especially due to surface fouling in complex sample fluids, such as blood serum. Electrode coatings based on conductive nanomaterials embedded in antifouling matrices offer a promising strategy to overcome this limitation. However, known composite coatings require long (typically >24 h) and complex fabrication processes, which pose a strong barrier for cost-effective mass manufacturing and successful commercialization. Here, we describe a novel polymer/carbon nanotube (CNT) composite coating that can be produced from an ink containing a photoreactive and antifouling copolymer as well as conductive CNTs using fast and highly scalable printing processes. Coatings were prepared on screen-printed electrodes and characterized using cyclic voltammetry (CV) and protein fouling experiments. The coatings offered an electroactive surface area (EASA) comparable to uncoated screen-printed electrodes and retained >90% of initial EASA after 1 h of exposure to concentrated bovine serum albumin solution, while uncoated electrodes decreased to <20% of initial EASA after the same treatment. Utilizing the universal crosslinking reaction of the polymer coating, antibodies against the inflammatory biomarker C-reactive protein (CRP) were photochemically immobilized on the electrodes. Functionalized electrodes were fabricated in <2 h and were successfully used to quantify nanogram-range concentrations of CRP spiked in undiluted human blood serum using a sandwich-immunoassay with electrochemical read-out, demonstrating the high potential of the platform for biosensing applications.

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.

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.

Traditional and Digital Biomarkers: Two Worlds Apart?

The identification and application of biomarkers in the clinical and medical fields has an enormous impact on society. The increase of digital devices and the rise in popularity of health-related mobile apps has produced a new trove of biomarkers in large, diverse, and complex data. However, the unclear definition of digital biomarkers, population groups, and their intersection with traditional biomarkers hinders their discovery and validation. We have identified current issues in the field of digital biomarkers and put forth suggestions to address them during the DayOne Workshop with participants from academia and industry. We have found similarities and differences between traditional and digital biomarkers in order to synchronize semantics, define unique features, review current regulatory procedures, and describe novel applications that enable precision medicine.

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.

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.

Functional Cryogel Microstructures Prepared by Light-Induced Cross-Linking of a Photoreactive Copolymer.

A novel, highly efficient method for the preparation of functional, microstructured and surface-attached cryogels is described. Photoinduced C,H-insertion reactions are used to generate cryogels in a single, rapid photo-cross-linking process. To this end, solutions containing both a photoreactive copolymer and the (bio)molecules to be immobilized are placed on a polymeric substrate followed by freezing and a short UV exposure. This strategy combines photolithography and cryogel formation allowing for a simultaneous generation and (bio)functionalization of cryogels in a single reaction step. To demonstrate the potential of the generated materials for bioanalytical applications, we successfully prepared DNA and protein cryogel microarrays.