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Over the last years, polymers have gained great attention as substrate material, because of the possibility to produce low-cost sensors in a high-throughput manner or for rapid prototyping and the wide variety of polymeric materials available with different features (like transparency, flexibility, stretchability, etc.). For almost all biosensing applications, the interaction between biomolecules (for example, antibodies, proteins or enzymes) and the employed substrate surface is highly important. In order to realize an effective biomolecule immobilization on polymers, different surface activation techniques, including chemical and physical methods, exist. Among them, plasma treatment offers an easy, fast and effective activation of the surfaces by micro/nanotexturing and generating functional groups (including carboxylic acids, amines, esters, aldehydes or hydroxyl groups). Hence, here we present a systematic and comprehensive plasma activation study of various polymeric surfaces by optimizing different parameters, including power, time, substrate temperature and gas composition. Thereby, the highest immobilization efficiency along with a homogenous biomolecule distribution is achieved with a 5-min plasma treatment under a gas composition of 50% oxygen and nitrogen, at a power of 1000 W and a substrate temperature of 80 C. These results are also confirmed by different surface characterization methods, including SEM, XPS and contact angle measurements.
An electrochemical study with three redox substances on a carbon based nanogap electrode array
(2020)
Electric cell-substrate impedance spectroscopy (ECIS) enables non-invasive and continuous read-out of electrical parameters of living tissue. The aim of the current study was to investigate the performance of interdigitated sensors with 50 μm electrode width and 50 μm inter-electrode distance made of gold, aluminium, and titanium for monitoring the barrier properties of epithelial cells in tissue culture. At first, the measurement performance of the photolithographic fabricated sensors was characterized by defined reference electrolytes. The sensors were used to monitor the electrical properties of two adherent epithelial barrier tissue models: renal proximal tubular LLC-PK1 cells, representing a normal functional transporting epithelium, and human cervical cancer-derived HeLa cells, forming non-transporting cancerous epithelial tissue. Then, the impedance spectra obtained were analysed by numerically fitting the parameters of the two different models to the measured impedance spectrum. Aluminium sensors proved to be as sensitive and consistent in repeated online-recordings for continuous cell growth and differentiation monitoring assensors made of gold, the standard electrode material. Titanium electrodes exhibited an elevated intrinsic ohmic resistance incomparison to gold reflecting its lower electric conductivity. Analysis of impedance spectra through applying models and numerical data fitting enabled the detailed investigation of the development and properties of a functional transporting epithelial tissue using either gold or aluminium sensors. The result of the data obtained, supports the consideration of aluminium and titanium sensor materials as potential alternatives to gold sensors for advanced application of ECIS spectroscopy.
Recently the use of microRNAs (miRNAs) as biomarkers for a multitude of diseases has gained substantial significance for clinical as well as point-of-care diagnostics. Amongst other challenges, however, it holds the central requirement that the concentration of a given miRNA must be evaluated within the context of other factors in order to unambiguously diagnose one specific disease. In terms of the development of diagnostic methods and devices, this implies an inevitable demand for multiplexing in order to be able to gauge the abundance of several components of interest in a patient’s sample in parallel. In this study, we design and implement different multiplexed versions of our electrochemical microfluidic biosensor by dividing its channel into subsections, creating four novel chip designs for the amplification-free and simultaneous quantification of up to eight miRNAs on the CRISPR-Biosensor X (‘X’ highlighting the multiplexing aspect of the device). We then use a one-step model assay followed by amperometric readout in combination with a 2-minute-stop-flow-protocol to explore the fluidic and mechanical characteristics and limitations of the different versions of the device. The sensor showing the best performance, is subsequently used for the Cas13a-powered proof-of-concept measurement of two miRNAs (miRNA-19b and miRNA-20a) from the miRNA-17∼92 cluster, which is dysregulated in the blood of pediatric medulloblastoma patients. Quantification of the latter, alongside simultaneous negative control measurements are accomplished on the same device. We thereby confirm the applicability of our platform to the challenge of amplification-free, parallel detection of multiple nucleic acids.