Simultaneous detection and quantification of DNA and protein biomarkers in spectrum of cardiovascular diseases in a microfluidic microbead chip
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.
KeywordsMicrofluidic Multiplex Microbeads Cardiovascular disease Biomarker Autoantibodies
In order to relieve the burden on the health care system and thus avoid late diagnoses and deaths of patients, rapid identification and treatment of risk factors is necessary . Biomarkers are indicator tools to assess physiological, pathogenic, or pharmacological processes.
They are used to monitor or detect diseases . Prerequisites for biomarkers are high specificity, easy accessibility for high diagnostic sensitivity, high stability, and long plasma half-life . For example, cardiovascular diseases (CVD) are one of the major reasons of mortality and morbidity worldwide caused by genetic predisposition, lifestyle, high misdiagnosis, and lack of clearly defined risk assessment criteria [3, 4, 5]. Immunoassays belong to the most import type of analytical methods in clinical environments and a large number of biomarkers have been investigated in CVD over the last 10 years [6, 7]. This includes C-reactive protein (CRP), brain natriuretic peptide (BNP), and low-density lipoprotein (LDL). However, there are new DNA-based biomarkers such as cell-free mitochondrial DNA (cfmDNA) that indicate systemic inflammation . High concentrations of cfmDNA lead to cytokine production in monocytes and thus to an induced inflammatory reaction. This reaction may be involved in age-related CVD diseases such as heart failure, atherosclerosis, and ischemic heart disease [8, 9].
In recent years, there has been an ongoing discussion about the influence of autoantibodies on the pathology of inflammatory cardiovascular disease . Several studies suggest that autoantibodies play a role in the development of cardiovascular disease, regardless of whether the person is affected by autoimmune disease or not [12, 13, 14]. In systemic lupus erythematosus (SLE), a multiorganic inflammatory disorder, CRP levels can be very low despite inflammatory activity. However, autoantibodies against CRP occur, which may be a link to acute coronary syndrome and thus a link between SLE and an increased risk of CVD [15, 16, 17]. Another autoantibody involved in SLE is anti-oxidized LDL (oxLDL). Increased detection of anti-oxLDL indicates atherosclerosis and may also serve as a marker for cardiovascular disease [18, 19, 20].
To detect several biomarkers simultaneously for a quick diagnostic statement, a combination of microfluidic and microbead technology is proposed. In comparison to conventional analytical methods, offer these technologies advantages in terms of multiplexing, time reduction, the use of smaller reaction quantities, and automation [7, 21]. The application of microbeads is suitable for the observation of the health condition of patients by a fast, multiparametric, economical, and sensitive sample analysis. The integration of microbeads in microfluidic devices brings further advantages such as reaction optimization. This can be achieved by volume reduction, meaning the use of small reaction volumes in the pico- and microliter range. Furthermore, high surface-to-volume ratios increase sensitivity; low local temperature fluctuations enable higher assay reproducibility and reduced incubation time . In addition, microfluidic integration poses to potential for point-of-care testing by running an assay completely automatically in self-contained cartridges without human interference.
Biomolecules application in microbead-based microfluidic chip. CRP, BNP, and LDL served as model targets to mimic autoantibody reactions
Term and sequences
Monoclonal antibody (IgG) against CRP, Cy5 conjugated
Protein-based cardiovascular biomarker immunofluorescence dilution 1:50–1:200
Brain natriuretic peptide
Monoclonal antibody (IgG) against BNP, Cy5 conjugated
Protein-based cardiovascular biomarker immunofluorescence dilution 1:50–1:200
Polyclonal antibody (IgG) against LDL, Cy5 conjugated
Protein-based cardiovascular biomarker immunofluorescence dilution 1:50–1:200
Cell-free mitochondrial DNA 5′-TGG GAG TGG GAG GGG AAA ATA ATG TGT TAG TTG GGG GGT GAC TGT TAA AAG TGC ATA CCG CCA AAA GAT AAA ATT TGA AAT CTG GTT AGG CTG GTG TTA GGG TTC TTT GTT TTT GGG GTT TGG CAG AGA TGT GTT TAA GTG CTG TGG CCA-3′
Capture probe 5′-(T)20ATCTCTGCCAAACCCC-3′3′biotinylated detection probe 5′-TTGGCGGTATGCACTT-3′, 5′Cy5 conjugated
DNA-based cardiovascular biomarker
Capture probe 5′-(T)20ACWCCTACGGGWGGCWGC-3′, 3′biotinylated
EUB338 5′-GCWGCCWCCCGTAGGWGT-3′, 5′Cy5 conjugated 
Unrelated internal control from fluorescence in situ Hybridization experiments
Human papilloma virus 72 5′-CATCTGTTGGTTTAATGAGCTT-3′, 3′Cy5 conjugated, 5′biotinylated
Detection probe 5′-AAGCTCATTAAACCAACAGATG-3′, 3′biotinylated
Unrelated internal control
Assignment of biomarkers to microbeads
Covalent coupling of biomarkers on microbeads
Independently, proteins CRP (antibodies-online GmbH, Germany), BNP (Bachem, Switzerland), LDL (Biozol, Germany), and streptavidin (internally produced according to ) were covalently bound to microbeads (Table 2) using a crosslinker 1-ethyl-3- (3- dimethylaminopropyl) carbodiimid hydrochloride (EDC, Karl Roth, Germany). Streptavidin was used for the immobilization of biotinylated DNA probes. Per population 105 microbeads were washed first with 200 μL 2-(N-morpholino)ethanosulfonic acid 0.1 M (MES, pH 4.6, Karl Roth, Germany). Afterwards, carboxyl groups were activated by shaking for 20 min at 28 °C and 1200 rpm with EDC solution (25 mg/mL, dissolved in MES). Followed by washing step with 0.05 × phosphate-buffered saline (PBS) and incubated by shaking (3 h at 28 °C 1200 rpm) with corresponding protein (300 μg/μL, dissolved in 0.05 × PBS). Microbeads were washed three times with 200 μL tris-buffered saline containing 0.1 % Tween (TBS-T).
Coupling of capture probes on microbeads
The biotinylated capture probes for cfmDNA (cfmDNA CP, biomers.net GmbH, Germany) EUB338 (unrelated internal control, biomers.net GmbH, Germany) and HPV72 (unrelated internal control, biomers.net GmbH, Germany) were independently coupled to streptavidin coated microbeads (MB4, MB6, and MB7) at 50 nM concentration (1 h at 28 °C and 1200 rpm). Afterwards the microbeads were washed three times with 200 μL TBS-T to remove unbound capture probes (see ESM, Supp Sec 1, Fig. S1).
All coated microbead populations (MB1–MB7) were pooled in equal parts. A mixture of approximately 2000 microbeads was immobilized with immobilization solution (Medipan GmbH, Germany) on flow cell surface on microfluidic chip and dried over night at RT. The multiwell plate does not have to be prepared.
Protein assay—mimicking autoantibody testing
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)). First, 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 . Then, antibody solution was pumped through flow cell and was incubated for 1 h at RT in the dark. 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. For protein detection in the multiwell plate, the assay was performed in the same order as in the chip. However, the microbead mix was incubated with the corresponding antibody for 1 h at RT and 1200 rpm in the thermomixer and then washed three times with TBS-T. One washing step consisted of a centrifugation step (1 min at 10,000 rpm), the aspiration of the supernatant and the subsequent addition of 200 μL TBS-T. The microbeads were then transferred to a multiwell plate and measured using VideoScan technology. For dilution series, the assay was performed with different concentrations (0.2 ng/μL to 100 ng/μL) of the corresponding antibody.
In addition, 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.
DNA assay for cfmDNA testing
Reagent reservoirs of microfluidic chip were filled with TBS-T, cfmDNA (10 ng/μL, Biotez Berlin Buch GmbH, Germany) and detection probe for cfmDNA (cfmDNA DP, 50 nM, Cy5 conjugated, Biomers.net GmbH, Germany). As described in subsection before (“Protein assay—mimicking autoantibody testing”), the method was equally performed until step of basis measurement. cfmDNA solution was pumped through flow cell and incubated for 1 h at RT in the dark. To remove the excess of cfmDNA in flow cell, the microbeads were washed once with TBS-T. Afterwards, the cfmDNA DP solution was pumped through flow cell and incubated for 1 h at RT in the dark. After washing with TBS-T, the fluorescence signal was measured in VideoScan technology. In the multiwell plate, the assay was performed in the same order as in the chip. The incubation steps with the cfmDNA to be detected and cfmDNA DP were carried out for 1 h at RT and 1200 rpm in the thermomixer, interrupted by washing with TBS-T three times each.
The microbead suspension was then transferred to a multiwell plate and measured using VideoScan technology. For dilution series, the assay was performed with different concentrations (0.5 to 100 ng/μL) of the cfmDNA.
In addition, the cfmDNA was spiked into human serum (offered from donor) in a concentration range from 0.05 to 50 ng/μL and detected with the aid of microbeads in chip assay as well as in multiwell plate.
Microfluidic chips for kinetic experiments were prepared as described before. The fluorescence signal was measured every 3 min during 1 h incubation of antibody solution in protein assay and cfmDNA DP incubation in DNA assay.
Images were acquired using the VideoScan platform and the image data analyzed using the FastFluoScan software (see  section 2.1–2.2). The proper linear range of fluorescence intensity was determined by an adaptive algorithm. Details can be found in the ESM (Supp Sec 5.) All raw data (shown in ESM in files Raw_data.xlsx and Serum_Data.xlsx) were analyzed with the R statistical  computing environment. Non-linear kinetic curve data were fitted using the drc package  and plotted with the 95 % confidence interval (see ESM, Supp Sec 6–9). The biomolecular interaction is reported as refMFI (referenced fluorescence intensity, see  for details). Precision medicine yields a large amounts of data that surpasses human ability to understand it . Therefore, there is a growing need of a dedicated software streamlining getting the gist of out the data. For this project, 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).
Results and discussion
Conventional detection methods of biomarkers are single detection by immunoassay (membrane or chip based) or qPCR [31, 32]. Growing attention is paid to the role of autoantibodies in CVD [11, 33]. We have developed a model system, to simultaneously measure and quantify protein and DNA biomarkers in a microfluidic microbead chip. Experiments have been carried out to demonstrate the functionality of our model, including the optimization of the system through a suitable buffer system, an applicable immobilization method, and the analysis of biomarkers in human serum.
Optimization of microbead immobilization and assay environment
Optimal assay conditions in a microfluidic chip are based on an optimized microbead immobilization. Three options (method A, immobilization by drying; method B, poly-L-lysine (PLL); method C, immobilization solution (Medipan GmbH)) were tested. The end point was the lowest microbead loss after all washing steps. The immobilization of the microbeads by method A showed the highest loss of microbeads with 37 %. Method B had the lowest microbead loss of 6 %. The immobilization of microbeads with PLL was described as the most effective method in . However, unspecific binding of oligonucleotides to the coated surface was observed. Thus, method B was discarded. Microbead immobilization with method C resulted in ~ 25 % microbead loss. No unspecific binding was observed. Thus, method C was further used to immobilize the microbeads on chip surface.
Next, we optimized the buffer system (see ESM, Supp Sec 3, Fig. S2). Four different buffer systems (PBS, PBS-T, TBS, and TBS-T) were used. All biomarkers were tested in single measurements in each buffer. Both PBS-T and TBS-T were found to be suitable for further use. Using PBS and TBS without the surfactant and spreading agent Tween 20 (Polysorbate 20), only low signals were obtained or microbeads aggregated in the flow cell. Finally, TBS-T was selected as buffer system for further experiments, since the use of phosphate-buffered systems can lead to unspecific binding .
Proof of concept for the detection of biomarkers in microfluidic microbead chip system
Fluorescence intensity determination as a function of time
Simultaneous detection of protein and DNA-based biomarker in a microfluidic microbead chip
Comparison of microfluidic microbead chip and conventional multiwell plate
Comparison of microfluidic microbead chip and multiwell plate
Microfluidic microbead chip
Low background signal
Small reaction volumes
Few washing steps
High surface-to-volume ratios
Expensive (small scale)
Time-consuming preparation (not automatized)
Low throughput (higher hands-on-time)
High background signal
Many (manual) washing steps
A model system for the simultaneous detection of protein and DNA-based cardiovascular biomarkers has been successfully developed. For example, it is possible to use this system for autoantibody detection as well as for antibody titer determination. The detection of biomarkers in a microfluidic microbead chip has advantages over measurements in multiwell format, such as low background signals, small reaction volumes, and fast reaction. We have chosen a simple microfluidic structure and a low chamber containing randomly ordered microbeads. We never observed channel blockage as described by others . We found the system is easy to assemble and can therefore be adapted to new tests. In order to make the system more usable, further optimizations have to be carried out. For the readout of the image data, we used the FastFluoScan software of the VideoScan system. There are alternative open source bioimage informatics software packages  and smartphone-based readout  that could be used. These include improving the reproducibility of cfmDNA detection and reducing the overall cost of a chip (including beads, biomarkers, and detection molecules). In addition, a secondary antibody or aptamers  for signal amplification could be integrated into the system for further experiments and the application of patient sample material with tested autoantibodies is also required. More biomarkers should be integrated into the system. Our technology theoretically allows the use of up to 18 different microbead populations.
We thank Kurt J. G. Schmailzl for the concept of the digilog project (ISBN 978-3-00-062150-5) and his continous support.
The authors received funding from the Brandenburg Ministry of Sciences, Research and Cultural Affairs (MWFK; Grant No. 06-GeCa:H228-05/002/004, digilog—Gesundheitscampus Brandenburg).
Compliance with ethical standards
Conflict of interest
Werner Lehmann has a management role in and is a shareholder of attomol GmbH. This company is a diagnostic manufacturer. Joerg Nestler is managing director and shareholder of BiFlow Systems GmbH. All other authors declare that they have no competing financial and nonfinancial interests.
The studies on blood material (serum) have been granted by the ethics committee of the Brandenburg University of Technology (BTU) Cottbus-Senftenberg, Cottbus, Germany, (Ethikkommissionssatzung BTU, document number EK2018—3) and were conducted in accordance with the Helsinki Declaration of 1964 (revised 2008). The blood donor gave a written informed consent.
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