Amplification-free electrochemical biosensor detection of circulating microRNA to identify drug-induced liver injury

Circulating microRNA detection using electrochemical biosensor for rapid diagnosis of liver disease in dogs

“…Screen-printed gold electrodes (DRP-C223BT) were purchased from Metrohm DropSens (Oviedo, Spain) and functionalised with PNA probes following the protocol developed in our earlier studies. 23,24,41 In short, electrodes were electrochemically cleaned using cyclic voltammetry (0–1.6 V potential range, 100 mV s −1 scan rate and 10 cycles) in 100 mM sulfuric acid aqueous solution. The cleaned gold working electrode was promptly incubated with a ternary MCH/HDT/PNA probe layer, comprising 6 μM PNA probe, 100 μM MCH, 200 μM HDT and 5 mM TCEP (reducing reagent), and kept in a humid chamber for 16 h for probe immobilisation.…”

“…EIS measurements (see Scheme 1A, frequency range 0.3 Hz–100 kHz, AC signal 10 mV rms at the measured open circuit potential using measurement buffer) were carried out in an EIS measurement buffer (10 mM sodium phosphate, pH 7, 20 mM sodium chloride and 0.2 mM potassium ferri/ferrocyanide redox mediator) and by using a flow-based sample cycling set-up as developed in our previous study. 24 This flow-based system (Scheme 1B) was contained with sample reservoirs maintained in a heat block (Starlab, N2400-4002) to control the temperature, a multichannel dispenser peristaltic pump (ISMATEC, ISM930C) to regulate the flow rate, enclosed flow cells with functionalised electrodes placed on a hot plate for temperature-controlled hybridisation measurements, and flow cells connected with the potentiostat and display system. The total volume required to run this closed-loop sample cycling setup was 600 μL.…”

“…In a subsequent publication, we optimised the biosensor assay in a continuous flow-based system using size-matched probe. 24 The flow-based system indeed enhanced the assay performance by facilitating the fast transport of target molecules to the immobilised probes on the electrode surface. Following thorough characterisation of flow rate and temperature to achieve higher detection sensitivity and specificity, the optimised flow conditions showed a detection limit of 50 pM for miR-122 with the size-matched probe.…”

“…For point-of-care detection of microRNA, we previously developed electrochemical biosensors and used them for diagnostic test development for drug-induced liver injury (DILI) in humans by detecting miR-122 in clinical samples. 23,24 Our biosensor assay uses screen-printed electrodes functionalised with sequence-specific peptide nucleic acid (PNA) probes, enabling direct and amplification-free detection of the miR-122 target using electrochemical impedance spectroscopy (EIS), making it suitable for point-of-care applications. A comprehensive investigation of fundamental parameters influencing sequence-specific microRNA binding was performed by altering the probe length, orientation of probe immobilisation, target overhangs, and degrees of base overhang on the electrode surface.…”

The Evolution of Circulating Biomarkers for Use in Acetaminophen/Paracetamol-Induced Liver Injury in Humans: A Scoping Review

“…While there are concerns about the biological variation in miR-122 [136] as well as technical challenges with current measurement methods (e.g., PCR), new analytical approaches are in development that may help to overcome some of the relevant problems [178][179][180]. In addition, an alternative approach may be to measure the levels of proteins encoded by genes that are miR-122 targets.…”