Abstract
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
Highlights
In order to relieve the burden on the health care system and avoid late diagnoses and deaths of patients, rapid identification and treatment of risk factors is necessary [1]
We demonstrate the rapid integration of putative cardiovascular biomarkers, mimicking autoantibodies against C-reactive protein (CRP), brain natriuretic peptide (BNP), and low-density lipoprotein (LDL), as well as the integration of cell-free mitochondrial DNA (cfmDNA) detection, into a microfluidic system through patient-oriented data collection, measurements, and data analysis
For protein detection in microfluidic chip, reagent reservoirs were filled with TBS-T buffer and 20 ng/μL of corresponding antibody (anti-CRP monoclonal antibody conjugated with Cy5 (Bioss antibodies, USA), anti-BNP monoclonal antibody conjugated with Cy5 (Bioss antibodies, USA))
Summary
In order to relieve the burden on the health care system and avoid late diagnoses and deaths of patients, rapid identification and treatment of risk factors is necessary [1]. For protein detection (mimicking autoantibodies) in microfluidic chip, reagent reservoirs were filled with TBS-T buffer and 20 ng/μL of corresponding antibody (anti-CRP monoclonal antibody conjugated with Cy5 (Bioss antibodies, USA), anti-BNP monoclonal antibody conjugated with Cy5 (Bioss antibodies, USA)). TBS-T was pumped through the flow cell to create a wet environment (see ESM, Supp Sec 2, Table S1) This was followed by a basis microbead measurement using VideoScan technology [26]. To remove the excess of antibodies in flow cell, the microbeads were washed once with TBS-T, followed by final fluorescence measurement in VideoScan technology. The biomarkers were spiked into human serum (offered from donor) in a concentration range from 0.2 to 50 ng/μL and detected with the aid of microbeads in chip assay as well as in multiwell plate. We developed the graphical user interface digilogger as R package (https:// github.com/michbur/digilogger) that eases the visual exploration of the data (see ESM, Supp Sec 10)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
