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.

Background Lipids and amphiphilic molecules are ubiquitous and play a central role in cell signalling, cell membrane structure, and lipid transport in the human body. However, they also appear in many diseases such as atherosclerosis, cardiovascular diseases, infections, inflammatory diseases, cancer, and autoimmune diseases. Thus, it is necessary to have detection systems for lipids and amphiphilic molecules. Microbeads can be one of these systems for the simultaneous detection of different lipophilic biomarkers. Methods Based on the fundamentals of microbead development, novel hydrophobic microbeads were produced. These not only have a hydrophobic surface, but are also fluorescently encoded and organic solvent resistant. The challenge after the development of the hydrophobic microbeads was to immobilise the amphiphilic molecules, in this study phospholipids, on the microbead surface in an oriented direction. After successful immobilisation of the biomarkers, a suitable antibody based detection assay was established. Results By passive adsorption, the phospholipids cardiolipin, phosphatidylethanolamine and phosphatidylcholine could be bound to the microbead surface. With the application of the enzymes phospholipase A2 and phospholipase C, the directional binding of the phospholipids to the microbead surface was demonstrated. The detection of directional binding indicated the need for the hydrophobic surface. Microbeads with no hydrophobic surface bound the phospholipids non-directionally (with the hydrophilic head) and were thus no longer reactively accessible for detection. Conclusion With the newly developed hydrophobic, dual coded and solvent stable microbeads it is possible to bind amphiphilic biomolecules directionally onto the microbead surfaces.

For improving aptamer-ligand binding we have developed a screening system that defines optimal binding buffer composition. Using multiplex assays, one buffer system is needed which guarantees the specific binding of all aptamers. We investigated nine peer-reviewed DNA aptamers. Non-specific binding of aptamers is an obstacle. To address this, we investigated 16 proteins as specificity controls bound covalently to encoded microbeads in a multiplex assay. Increasing the NaCl concentration decreased the binding for all aptamers. Changing pH values by one unit higher or lower did not influence the aptamer binding significantly. However, pH < 5 led to non-specific binding for all aptamers. The Pf LDH-aptamer selected in the absence of divalent cations exhibited doubling of its binding signal by the addition of Ca 2+ and Mg 2+ . We confirmed Ca 2+ and Mg 2+ dependency of the aptamers for streptavidin and thrombin by observing a 90% and 50% binding decrease, respectively. We also achieved a doubling of binding for the streptavidin aptamer when replacing Ca 2+ and Mg 2+ by Mn 2+ . A buffer suitable for all aptamers can have considerable variations in pH or ionic strength, but divalent cations (Ca 2+ , Mg 2+ , Mn 2+ ) are essential.

Background: Laboratory diagnosis of Lyme borreliosis refers to some methods with known limitations. Molecular diagnostics using specific nucleic acid probes may overcome some of these limitations. Methods: We describe the novel reporter fluorescence real-time polymerase chain reaction (PCR) probe system LoopTag for detection of Borrelia species. Advantages of the LoopTag system include having cheap conventional fluorescence dyes, easy primer design, no restrictions for PCR product lengths, robustness, high sequence specificity, applicability for multiplex real-time PCRs, melting curve analysis (single nucleotide polymorphism analysis) over a large temperature range, high sensitivity, and easy adaptation of conventional PCRs. Results: Using the LoopTag probe system we were able to detect all nine tested European species belonging to the Borrelia burgdorferi (sensu lato) complex and differentiated them from relapsing fever Borrelia species. As few as 10 copies of Borrelia in one PCR reaction were detectable. Conclusion: We established a novel multiplex probe real-time PCR system, designated LoopTag, that is simple, robust, and incorporates melting curve analysis for the detection and in the differentiation of European species belonging to the Borrelia burgdorferi s.l. complex.

One of the most commonly used bonds between two biomolecules is the bond between biotin and streptavidin (SA) or streptavidin homologues (SAHs). A high dissociation constant and the consequent high-temperature stability even allows for its use in nucleic acid detection under polymerase chain reaction (PCR) conditions. There are a number of SAHs available, and for assay design, it is of great interest to determine as to which SAH will perform the best under assay conditions. Although there are numerous single studies on the characterization of SAHs in solution or selected solid phases, there is no systematic study comparing different SAHs for biomolecule-binding, hybridization, and PCR assays on solid phases. We compared streptavidin, core streptavidin, traptavidin, core traptavidin, neutravidin, and monomeric streptavidin on the surface of microbeads (10-15 mu m in diameter) and designed multiplex microbead-based experiments and analyzed simultaneously the binding of biotinylated oligonucleotides and the hybridization of oligonucleotides to complementary capture probes. We also bound comparably large DNA origamis to capture probes on the microbead surface. We used a real-time fluorescence microscopy imaging platform, with which it is possible to subject samples to a programmable time and temperature profile and to record binding processes on the microbead surface depending on the time and temperature. With the exception of core traptavidin and monomeric streptavidin, all other SA/SAHs were suitable for our investigations. We found hybridization efficiencies close to 100% for streptavidin, core streptavidin, traptavidin, and neutravidin. These could all be considered equally suitable for hybridization, PCR applications, and melting point analysis. The SA/SAH-biotin bond was temperature sensitive when the oligonucleotide was mono-biotinylated, with traptavidin being the most stable followed by streptavidin and neutravidin. Mono-biotinylated oligonucleotides can be used in experiments with temperatures up to 70 degrees C. When oligonucleotides were bis-biotinylated, all SA/SAH-biotin bonds had similar temperature stability under PCR conditions, even if they comprised a streptavidin variant with slower biotin dissociation and increased mechanostability.

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.

Amplification curves from quantitative Real-Time PCR experiments typically exhibit a sigmoidal shape. They can roughly be divided into a ground or baseline phase, an exponential amplification phase, a linear phase and finally a plateau phase, where in the latter, the PCR product concentration no longer increases. Nevertheless, in some cases the plateau phase displays a negative trend, e.g. in hydrolysis probe assays. This cycle-to-cycle fluorescence decrease is commonly referred to in the literature as the hook effect. Other detection chemistries also exhibit this negative trend, however the underlying molecular mechanisms are different. In this study we present two approaches to automatically detect hook effect-like curvatures based on linear (hookreg) and nonlinear regression (hookregNL). As the hook effect is typical for qPCR data, both algorithms can be employed for the automated identification of regular structured qPCR curves. Therefore, our algorithms streamline quality control, but can also be used for assay optimization or machine learning.

Typing and classification of Escherichia coli (E. coli) according to cell wall components, like polysaccharides, is routinely done by serotyping. Given the presence of 188 known O-antigens, this process is complex. The authors present a proof-of-concept planar microbead array for multiplexed O-serotyping. Ten clinically relevant E. coli serotypes associated with high risk for diarrhea in humans were examined (O26, O55, O78, O118, O124, O127, O128, O142, O145 and O157). Antisera were assigned to specific microbead populations, which can be differentiated by size and fluorescence color. Automatted image processing and data analysis were conducted by a microscopic interpretation platform. Homogenous antiserum coating of the microbeads was demonstrated by an intra-population CV that ranges from 3.3 to 6.3% and by an inter-population CV of 9.5%. Typical detections limits are in the range from 0.31 to 0.71 refMFI. Significantly elevated fluorescence signals revealed that E. coli of a certain serogroup bound specifically to microbeads with the matching antiserum (p < 0.001). In our perception, the method represents a viable diagnostic tool for automated multiplex serotyping of E. coli. It enables simultaneous and high-throughput screening for different O-antigens by a simple staining and binding protocol.

Real-time quantitative PCR (qPCR) is a well-established method for the precise quantification of nucleic acids. However, there is an ongoing development of novel device concepts for qPCRs, hybridization and melting curve analysis. This includes lab-on-chip systems or the recently published microbead-based VideoScan platform. Besides dedicated hardware an easy to use software for data analysis is also a requirement during developmental processes. Experimental instruments usually collect data of different formats and quality. Analysis software must therefore be able to preprocess data of different origins. Preprocessing includes data import, removal of missing values, selection of data ranges, outlier removal, detection of background signal, data transformation and smoothing /normalization of data. Subsequently, the software must be able to analyze and quantify both, the amplification data and melting curve data. To automate this process we developed a set of plugins for the open source software RKWard. RKWard is a GUI and an Integrated Development Environment (IDE) for statistical analysis with R which provides tools to manage and edit different types of data objects. Statistical procedures and plots were implemented using the easily extendable RKWard plugin architecture based on ECMAScript, XML, and R. The plugins are enhanced by a set of functions for data preprocessing. Core features rely on the excellent qpcR package for qPCR quantification and algorithms from the MBmca package for melting curve analysis. RKWard provides all tools of R for an interactive data analysis and presentation, a dedicated output file and an output window for documenting the results and figures. The software aims to accelerate the development process and to enable easier collaboration between colleagues in the hardware and wetware disciplines.

The need for new highly sensitive, cost-efficient, fast and robust nucleic acid detection and quantification technologies is a driving force. PCR, especially quantitative PCR (qPCR), is the method of choice in diagnostics and life-sciences. The digital PCR (dPCR) provides a new technology to measure absolute quantities of nucleic acids without the need for calibration curves. This review gives details on the dPCR technology and available platforms. We discuss the platform differences, common features as well as their advantages and disadvantages.

Microbead-based technologies represent elegant and versatile approaches for highly parallelized quantitative multiparameter assays. They also form the basis of various techniques for detection and quantification of nucleic acids and proteins. Nucleic acid-based methods include hybridization assays, solid-phase PCR, sequencing, and trapping assays. Microbead assays have been improved in the past decades and are now important tools in routine and point-of-care diagnostics as well as in life science. Its advances include low costs, low workload, high speed and high-throughput automation. The potential of microbead-based assays therefore is apparent, and commercial applications can be found in the detection and discrimination of single nucleotide polymorphism, of pathogens, and in trapping assays. This review provides an overview on microbead-based platforms for biosensing with a main focus on nucleic acid detection (including amplification strategies and on selected probe systems using fluorescent labeling). Specific sections cover chemical properties of microbeads, the coupling of targets onto solid surfaces, microbead probe systems (mainly oligonucleotide probes), microbead detection schemes (with subsections on suspension arrays, microfluidic devices, and immobilized microbeads), quantification of nucleic acids, PCR in solution and the detection of amplicons, and methods for solid-phase amplification. We discuss selected trends such as microbead-coupled amplification, heterogeneous and homogenous DNA hybridization assays, real-time assays, melting curve analysis, and digital microbead assays. We finally discuss the relevance and trends of the methods in terms of high-level multiplexed analysis and their potential in diagnosis and personalized medicine. Contains 211 references. Figure ᅟ

PCR is a simplistic and robust laboratory technology for nucleic acid detection. However, for research and diagnostics processing multiple targets within one reaction in an automatic fashion is a demanded feature. Combining two multiplex read out technologies, such as microarray and microbeads, the VideoScan platform was designed. This microscope imaging technology enables an automatable high throughput multiplex measurement of genetic material from biological and patient samples.

The analysis of different biomolecules is of prime importance for life science research and medical diagnostics. Due to the discovery of new molecules and new emerging bioanalytical problems, there is an ongoing demand for a technology platform that provides a broad range of assays with a user-friendly flexibility and rapid adaptability to new applications. Here we describe a highly versatile microscopy platform, VideoScan, for the rapid and simultaneous analysis of various assay formats based on fluorescence microscopic detection. The technological design is equally suitable for assays in solution, microbead-based assays and cell pattern recognition. The multiplex real-time capability for tracking of changes under dynamic heating conditions makes it a useful tool for PCR applications and nucleic acid hybridization, enabling kinetic data acquisition impossible to obtain by other technologies using endpoint detection. The paper discusses the technological principle of the platform regarding data acquisition and processing. Microbead-based and solution applications for the detection of diverse biomolecules, including antigens, antibodies, peptides, oligonucleotides and amplicons in small reaction volumes, are presented together with a high-content detection of autoimmune antibodies using a HEp-2 cell assay. Its adaptiveness and versatility gives VideoScan a competitive edge over other bioanalytical technologies.

Intestinal colonization is influenced by the ability of the bacterium to inhabit a niche, which is based on the expression of colonization factors. Escherichia coli carries a broad range of virulence-associated genes (VAGs) which contribute to intestinal (inVAGs) and extraintestinal (exVAGs) infection. Moreover, initial evidence indicates that inVAGs and exVAGs support intestinal colonization. We developed new screening tools to genotypically and phenotypically characterize E. coli isolates originating in humans, domestic pigs, and 17 wild mammal and avian species. We analyzed 317 isolates for the occurrence of 44 VAGs using a novel multiplex PCR microbead assay (MPMA) and for adhesion to four epithelial cell lines using a new adhesion assay. We correlated data for the definition of new adhesion genes. inVAGs were identified only sporadically, particularly in roe deer (Capreolus capreolus) and the European hedgehog ( Erinaceus europaeus). The prevalence of exVAGs depended on isolation from a specific host. Human uropathogenic E. coli isolates carried exVAGs with the highest prevalence, followed by badger (Meles meles) and roe deer isolates. Adhesion was found to be very diverse. Adhesion was specific to cells, host, and tissue, though it was also unspecific. Occurrence of the following VAGs was associated with a higher rate of adhesion to one or more cell lines: afa-dra, daaD, tsh, vat, ibeA, fyuA, mat, sfa-foc, malX, pic, irp2, and papC. In summary, we established new screening methods which enabled us to characterize large numbers of E. coli isolates. We defined reservoirs for potential pathogenic E. coli. We also identified a very broad range of colonization strategies and defined potential new adhesion genes.

Background: The human cytomegalovirus (HCMV) and the human herpesvirus 6 (HHV6) are widely distributed in the human population. The variants A and B of HHV6 are closely related to each other and cannot be distinguished by common serological methods like enzyme-linked immunosorbent assay (ELISA) or immunofluorescence test (IFT). The aim of this study was to develop a microwell-adapted blot system for specificity detection of human cytomegalovirus and human herpesvirus 6A and 6B (HHV6A, HHV6B) that combines the advantages of ELISA (automation and multiplex detection) and immunoblotting (antigen-specific antibody detection with high specificity).Methods: Ten HCMV, five HHV6A and five HHV6B antigens were expressed as fusion proteins and tested with sera of children (n=30), of healthy young adults (n=30) and of older adults (n=30) in a newly developed microblot system.Results: Sensitivity and specificity of HCMV and HHV6 microblots were comparable to commercially available ELISA, IFT and to line assay tests. The advantage of the HHV6 microblot is the possibility of distinguishing between HHV6A-monovalent sera, HHV6B-monovalent sera and HHV6A/B-polyvalent sera. Most sera of children younger than 2 years showed only HHV6B antigen positivity, while most sera of adults and children aged over 2 years reacted with HHV6A and B proteins, although predominance for HHV6B was observed.Conclusions: The authors were able to detect HCMV positive sera and to distinguish between HHV6A-monovalent sera, HHV6B-monovalent sera and HHVA/B-polyvalent sera with the new developed microblot system. Predominance of HHV6B was observed in sera of children and adults.

Sera of tumor patients frequently contain autoantibodies to tumor associated antigens. Here we describe a miniaturized immunoblot platform allowing us to screen sera of patients for the presence of autoantibodies to ten autoantigens in parallel.