Lehmann, W. | Search bioRxiv

bioRxiv2023

Immobilization of Lipophilic and Amphiphilic Biomarker on Hydrophobic Microbeads

BackgroundLipids 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. MethodsBased 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. ResultsBy 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. ConclusionWith the newly developed hydrophobic, dual coded and solvent stable microbeads it is possible to bind amphiphilic biomolecules directionally onto the microbead surfaces.

biochemistry↗

bioRxiv2021

A multiplex microchamber diffusion assay for the antibody-based detection of microRNAs on randomly ordered microbeads

BackgroundMicroRNAs (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. MethodsWe 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. ResultsOur 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. ConclusionThe 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.

biochemistry↗