Electrochemiluminescence on digital microfluidics for microRNA analysis

Electrochemiluminescence (ECL) is a sensitive analytical technique with great promise for biological applications, especially when combined with microfluidics. Here, we report the first integration of ECL with digital microfluidics (DMF). ECL detectors were fabricated into the ITO-coated top plates of DMF devices, allowing for the generation of light from electrically excited luminophores in sample droplets. The new system was characterized by making electrochemical and ECL measurements of soluble mixtures of tris(phenanthroline)ruthenium(II) and tripropylamine (TPA) solutions. The system was then validated by application to an oligonucleotide hybridization assay, using magnetic particles bearing 21-mer, deoxyribose analogues of the complement to microRNA-143 (miRNA-143). The system detects single nucleotide mismatches with high specificity, and has a limit of detection of 1.5 femtomoles. The system is capable of detecting miRNA-143 in cancer cell lysates, allowing for the discrimination between the MCF-7 (less aggressive) and MDA-MB-231 (more aggressive) cell lines. We propose that DMF-ECL represents a valuable new tool in the microfluidics toolbox for a wide variety of applications

Electrochemical Impedance Immunoassay for ALS-Associated Neurofilament Protein: Matrix Effect on the Immunoplatform

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder, which has complex diagnostic steps. Electrochemical immunoassays may make the diagnosis simpler and faster. Here, we present the detection of ALS-associated neurofilament light chain (Nf-L) protein through an electrochemical impedance immunoassay on reduced graphene oxide (rGO) screen-printed electrodes. The immunoassay was developed in two different media, i.e., buffer and human serum, to compare the effect of the media on their figures of merit and calibration models. The label-free charge transfer resistance (RCT) of the immunoplatform was used as a signal response to develop the calibration models. We found that exposure of the biorecognition layer to human serum improved the impedance response of the biorecognition element with significantly lower relative error. Moreover, the calibration model obtained in the human serum environment has higher sensitivity and a better limit of detection (0.087 ng/mL) than the buffer medium (0.39 ng/mL). The analyses of the ALS patient samples show that concentrations obtained from the buffer-based regression model was higher than the serum-based model. However, a high Pearson correlation (r = 1.00) between the media suggests that concentration in one medium may be useful to predict the concentration in the other medium. Moreover, the Nf-L concentration appears to increase with age in both male and female groups, while overall higher Nf-L was found in the male group than the female group

Sequence-Independent DNA Adsorption on Few-Layered Oxygen-Functionalized Graphene Electrodes: An Electrochemical Study for Biosensing Application

DNA is strongly adsorbed on oxidized graphene surfaces in the presence of divalent cations. Here, we studied the effect of DNA adsorption on electrochemical charge transfer at few-layered, oxygen-functionalized graphene (GOx) electrodes. DNA adsorption on the inkjet-printed GOx electrodes caused amplified current response from ferro/ferricyanide redox probe at concentration range 1 aM–10 nM in differential pulse voltammetry. We studied a number of variables that may affect the current response of the interface: sequence type, conformation, concentration, length, and ionic strength. Later, we showed a proof-of-concept DNA biosensing application, which is free from chemical immobilization of the probe and sensitive at attomolar concentration regime. We propose that GOx electrodes promise a low-cost solution to fabricate a highly sensitive platform for label-free and chemisorption-free DNA biosensing

Hand-Fabricated CNT/AgNPs Electrodes using Wax-on-Plastic Platforms for Electro-Immunosensing Application

Abstract Fabrication of inexpensive and flexible electronic and electrochemical sensors is in high demand for a wide range of biochemical and biomedical applications. We explore hand fabrication of CNT modified AgNPs electrodes using wax-on-plastic platforms and their application in electrochemical immunosensing. Wax patterns were printed on polyethylene terephthalate-based substrates to laydown templates for the electrodes. Hand painting was employed to fabricate a silver conductive layer using AgNPs ink applied in the hydrophilic regions of the substrate surrounded by wax. CNT was drop cast on top of the working electrodes to improve their electrochemical signal. The device layers were characterized by scanning electron microscopy. The electrochemical performance of the hand fabricated AgNPs and CNT/AgNPs electrodes was tested using cyclic voltammetry, differential pulse voltammetry, and amperometry. The electrochemical response of CNT/AgNPs electrodes was relatively faster, higher, and more selective than unmodified AgNPs sensing electrodes. Finally, the hand-painted CNT/AgNPs electrodes were applied to detect carcinoembryonic antigen (CEA) by measuring the end-product of immunoassay performed on magnetic particles. The detection limit for CEA was found to be 0.46 ng/mL

Investigation of the Utility of Complementary Electrochemical Detection Techniques to Examine the in Vitro Affinity of Bacterial Flagellins for a Toll-Like Receptor 5 Biosensor

An initial investigation of the fabrication of a novel biosensor utilizing toll-like receptor 5 (TLR5) has been conducted. The detection assay using this sensor platform has been carried out using two complementary electrochemical techniques. The electrochemical properties of the modified bare gold surface following TLR5 immobilization were characterized. The electrochemical response to changes in the sensor film resistance and electron charge-transfer permittivity triggered by independent exposures to flagellins from Salmonella typhimurium (S. typhimurium) and Bacillus subtilis (B. subtilis) were examined and observed. The quantified film resistance data gathered using electrochemical impedance spectroscopy (EIS) over a macroscopic scale are in significant agreement with the corresponding electron charge-transfer permittivity measured locally by scanning electrochemical microscopy (SECM). Unlike other sensors that exploit pathogen recognition elements, TLR5 biosensors have the potential to carry out broad-spectrum detection of flagellated bacterial pathogens in near real time. This broad-spectrum detection platform is a significant step toward the development of fast, inexpensive clinical tools for early warning diagnoses and immediate on-site treatment