The rapid and simultaneous detection of DNA and protein biomarkers is necessary to detect the outbreak of a disease or to monitor a disease. For example, cardiovascular diseases are a major cause of adult mortality worldwide. We have developed a rapidly adaptable platform to assess biomarkers using a microfluidic technology. Our model mimics autoantibodies against three proteins, C-reactive protein (CRP), brain natriuretic peptide (BNP), and low-density lipoprotein (LDL). Cell-free mitochondrial DNA (cfmDNA) and DNA controls are detected via fluorescence probes. The biomarkers are covalently bound on the surface of size- (11–15 μm) and dual-color encoded microbeads and immobilized as planar layer in a microfluidic chip flow cell. Binding events of target molecules were analyzed by fluorescence measurements with a fully automatized fluorescence microscope (end-point and real-time) developed in house. The model system was optimized for buffers and immobilization strategies of the microbeads to enable the simultaneous detection of protein and DNA biomarkers. All prime target molecules (anti-CRP, anti-BNP, anti-LDL, cfmDNA) and the controls were successfully detected both in independent reactions and simultaneously. In addition, the biomarkers could also be detected in spiked human serum in a similar way as in the optimized buffer system. The detection limit specified by the manufacturer is reduced by at least a factor of five for each biomarker as a result of the antibody detection and kinetic experiments indicate that nearly 50 % of the fluorescence intensity is achieved within 7 min. For rapid data inspection, we have developed the open source software digilogger, which can be applied for data evaluation and visualization. Graphical abstract Electronic supplementary materialThe online version of this article (10.1007/s00216-019-02199-x) contains supplementary material, which is available to authorized users.

BACKGROUND: The 3D printing is relevant as a manufacturing technology of functional models for forensic, pharmaceutical and bioanalytical applications such as drug delivery systems, sample preparation and point-of-care tests. OBJECTIVE: Melting behavior and autofluorescence of materials are decisive for optimal printing and applicability of the product which are influenced by varying unknown additives. METHODS: We have produced devices for bioanalytical applications from commercially available thermoplastic polymers using a melt-layer process. We characterized them by differential scanning calorimetry, fluorescence spectroscopy and functional assays (DNA capture assay, model for cell adhesion, bacterial adhesion and biofilm formation test). RESULTS: From 14 tested colored, transparent and black materials we found only deep black acrylonitrile-butadiene-styrene (ABS) and some black polylactic acid (PLA) useable for fluorescence-based assays, with low autofluorescence only in the short-wave range of 300-400 nm. PLA was suitable for standard bioanalytical purposes due to a glass transition temperature of approximately 60 • C, resistance to common laboratory chemicals and easy print processing. For temperature-critical methods, such as hybridization reactions up to 90 • C, ABS was better suited. CONCLUSIONS: Autofluorescence was not a disadvantage per se but can also be used as a reference signal in assays. The rapid development of individual protocols for sample processing and analysis required the availability of a material with consistent quality over time. For fluorescence-based assays, the use of commercial standard materials did not seem to meet this requirement.

Background: MicroRNAs (miRNAs) are small, conserved, noncoding RNAs regulating gene expression that functions in RNA silencing and post-transcriptional regulation of gene expression. Altered miRNA profiles have been implicated in many human diseases, and due to their circulating abilities, they have excited great interest in their use as clinical biomarkers. The development of innovative methods for miRNA detection has become of high scientific and clinical interest. Methods: We developed a diffusion-driven microbead assay and combined it with an antibody-based miRNA detection. The diffusion process was carried out in two different approaches a) co-diffusion of miRNA and antibodies (termed diffusion approach I, DAI) and b) diffusion of miRNA in an antibody-saturated environment (DAII). In both approaches, neutravidin-coated microbeads were loaded with specific biotinylated DNA capture probes, which targets either miR-21-5p, miR-30a-3p or miR-93-5p. The miRNAs were time- and dose-dependently detected in a diffusion microchamber by primary anti-DNA:RNA hybrid and fluorescence-labeled secondary antibodies using our in-house developed inverse fluorescence microscope imaging platform VideoScan. Results: Our assay offers the advantage that several target molecules can be detected simultaneously and in real-time in one reaction environment (multiplex), without any amplification steps. We recorded the diffusion process over a period of 24 h and found that the reaction was almost completed after 2 h. The specificity of the assay was 96.7 % for DAI and 92.3 % for DAII. The detection limits were in a concentration range of 0.03-0.43 nM for DAI and 0.14-1.09 nM for DAII, depending on the miRNA. Conclusion: The miRNAs are successively exposed to the capture probe-loaded randomly ordered microbeads (p value of CSR 0.23-0.96), which leads to microbeads that become saturated with the target molecules first in front rows. Non-bonded miRNAs continue to diffuse further and can therefore subsequently bind to the microbeads with free binding sites. Our detection principle differs from other microbead assays, in which all microbeads are simultaneously mixed with the sample solution, so that all target molecules bind equally distributed to the microbeads, resulting in an averaged signal intensity.

Quantitative analysis of a target molecule in a microbead-based fluorescent assay requires a specific labeling procedure. For nucleic acid analysis the hybridization with florescent labeled oligonucleotides is the most common method. However, disadvantages are the necessity for direct labeling of probes and the sensitivity to detect low amounts of target molecules. In this study we established an alternative detection method for biomolecules on microbeads, the tyramide signal amplification (TSA). Hereby, biomolecules are detected by enzymatically activated and fluorophore-conjugated tyramides that bind to specific protein residues. This method has proven to be a versatile and robust enzyme amplification technique for sensitive immunohistochemical detection. Now, we present the feasibility of the TSA procedure to detect hybridized biotinylated oligonucleotide probes bound to protein coated microbead surfaces TSA was performed using fluorescent, sizeencoded and streptavidin coated microbeads that were loaded with dual-biotinylated DNA capture probes, prepared from polymerase chain reaction. Beside streptavidin alone for surface coating of those microbeads, we applied different quantities of streptavidin in combination with bovine serum albumin, immunoglobulin G or Protein G/A, to check for positive effects on the resulting signal intensities through specific binding of tyramide molecules. For this method, streptavidin turned out as appropriate protein for the surface binding, without the need for further molecules. In comparison to a standard detection with common streptavidin-fluorophore-conjugates TSA showed its advantage in the detection of low probe amounts down to a concentration of 3.3•10 −4 ng/L.

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