TY - JOUR
T1 - Amplification-free electrochemical biosensor detection of circulating microRNA to identify drug-induced liver injury
AU - Roychoudhury, Appan
AU - Dear, James W
AU - Kersaudy-Kerhoas, Maiwenn
AU - Bachmann, Till T
N1 - Funding Information:
DILI is one of the key challenges for the drug development. It can lead to early market removal of new drugs after launch, resulting huge financial losses for the pharmaceutical companies. A problem in this context is the lack of reliable diagnostic and prognostic biomarkers, making it difficult to identify patients who are at risk. MicroRNA122 (miR-122) is significantly raised in the circulation of DILI patients, and can be detected earlier than conventional clinical liver biomarkers e.g., alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (Starkey Lewis et al. 2011, 2012). ALT and AST activity increases 12–16 h after paracetamol overdose, whereas miR-122 can diagnose liver injury within 4 h (Vliegenthart et al., 2015b). While ALT is the gold standard biomarker for hepatocyte injury, it lacks tissue specificity, and can cause false positives, reducing the confidence in DILI diagnosis (Starkey Lewis et al., 2011). In contrast, miR-122 is distinctly and abundantly expressed in the liver, representing 70% of the total hepatocyte miRNA hepatic complement (∼40,000 copies per hepatocyte) (de Rie et al., 2017). During liver injury, miR-122 is released from necrotic hepatocytes, causing high miR-122 concentrations in the circulation (Wang et al., 2009). After paracetamol overdose and liver injury, the miR-122 concentration can rise up to 100 to 10,000 fold higher than the healthy concentration (Antoine et al., 2013; Dear et al., 2014; Starkey Lewis et al., 2011). In vitro, miR-122 has been demonstrated as a biomarker of cellular toxicity during the development of new drugs (Kia et al., 2015). The US Food and Drug Administration (FDA) has provided formal regulatory support for the miR-122 to be utilised as an exploratory DILI biomarker in the clinical trials.Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), sulfuric acid (H2SO4), monosodium phosphate (NaH2PO4), disodium phosphate (Na2HPO4), sodium chloride (NaCl), potassium ferricyanide [K3Fe(CN)6] and potassium ferrocyanide [K4Fe(CN)6] were purchased from Sigma-Aldrich (Gillingham, UK). 6-Mercapto-1-hexanol (MCH) and 1,6-hexanedithiol (HDT) were procured from ProChimia Surfaces (Gdynia, Poland). All of the other chemicals were of analytical grade unless otherwise stated. All of the aqueous solutions were made with deionised water (resistivity >18 MΩ cm) from a Millipore MilliQ water purification system (Bedford, MA, USA). The specific and non-specific peptide nucleic acid (PNA) single-stranded probes for miR-122 target sequence were ordered via Cambridge Research Biochemicals (Cleveland, UK) and obtained from Panagene (Daejeon, South Korea). Probes (>95% HPLC purified) were synthesized with a linker consisting of three ethylene glycol units (abbreviated as AEEEA) and a terminal thiol group on either N-end (equivalent to 5′-end of DNA) or C-end (equivalent to 3′-end of DNA) of the PNA for self-assembled monolayer (SAM) formation on gold electrode surface. The PNA stock solutions were prepared in 50% (v/v) dimethylformamide (DMF) aqueous solution and kept at −20 °C during storage. The complementary and nearly or non-complementary RNA target sequences were obtained from Metabion (Martinsried, Germany). Stock solutions of the RNA targets were prepared in nuclease-free deionised (DI) water and stored at −80 °C. The sequence and structural details of PNA probes and RNA targets are provided in Table S1 of the Supporting Information.The presence of PNA probes on the electrode surface following immobilisation was confirmed using atomic force microscopy (AFM), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX) and cyclic voltammetry (CV). The AFM micrograph of the bare screen-printed gold working electrode (Fig. 1A) taken in a 10 × 10 μm2 scan area revealed a rough surface with porous morphology due to the presence of screen printed gold microparticles. The bare electrode showed a mean roughness (Ra) of 448.7 nm, a root mean square roughness (Rq) of 589.8 nm, and a roughness factor of 1.719. The electrode surface appeared less porous after probe immobilisation (Fig. 1B), and the roughness parameters increased, with Ra = 460 nm, Rq = 613 nm and roughness factor 1.792. The line profiles obtained from AFM surface profile analysis (see Fig. S1) demonstrate an increase in baseline height following the probe immobilisation, confirming less porous surface morphology of the electrode after modification with the probes. For further comparison, SEM, EDX and CV studies were conducted and the results are shown in Fig. 1C–F, and explained in Supporting Information.In present study, commercially available screen-printed gold electrodes were functionalised with specific PNA probe molecules to develop a disposable biosensor for miR-122 detection. The bare electrodes have a rough surface morphology, as observed in AFM and SEM studies, which is to be expected given that the surface was formed by screen printing of gold particles suspended in an ink (Butterworth et al., 2019). Following probe immobilisation, the increasing roughness parameters, the deposition of an additional layer with a less porous electrode surface morphology, and the higher presence of carbon as observed from the AFM, SEM and EDX studies each independently support the existence of a PNA probe layer on electrode surface. Earlier AFM studies also noted the rising roughness parameters upon DNA probe immobilisation on electrode surface (Lee et al., 2014). As supported by previous literature, the increase in peak-to-peak potential separation, reduction in peak current amplitudes and a lower surface concentration of ionic species from the CV studies indicate the presence of PNA probes on the electrode interface, which interrupt the reversible redox reaction and the facile electron transfer between electrolyte and electrode (Steel et al., 1998). The ratio of oxidation and reduction peak current is less than 1 in the probe-modified electrode, confirming the quasi-reversible behaviour of the prepared electrode, which we anticipate resulting from the biological layer of the probe molecules.The authors acknowledge financial support from the Rosetrees Trust (Ref. no. CF1\100010). We thank Dr. Holger Schulze for useful discussion and the Bioimaging and Cryo FIB-SEM facilities at the University of Edinburgh for the AFM and SEM measurements, respectively. Author JD acknowledges the support from the Chief Scientist's Office Scotland via the Centre for Precision Cell Therapy for the Liver (PMAS/21/07).
Funding Information:
The authors acknowledge financial support from the Rosetrees Trust (Ref. no. CF1\100010 ). We thank Dr. Holger Schulze for useful discussion and the Bioimaging and Cryo FIB-SEM facilities at the University of Edinburgh for the AFM and SEM measurements, respectively. Author JD acknowledges the support from the Chief Scientist's Office Scotland via the Centre for Precision Cell Therapy for the Liver ( PMAS/21/07 ).
Publisher Copyright:
© 2023 Elsevier B.V.
PY - 2023/4/5
Y1 - 2023/4/5
N2 - Drug-induced liver injury (DILI) is a major challenge in clinical medicine and drug development. There is a need for rapid diagnostic tests, ideally at point-of-care. MicroRNA 122 (miR-122) is an early biomarker for DILI which is reported to increase in the blood before standard-of-care markers such as alanine aminotransferase activity. We developed an electrochemical biosensor for diagnosis of DILI by detecting miR-122 from clinical samples. We used electrochemical impedance spectroscopy (EIS) for direct, amplification free detection of miR-122 with screen-printed electrodes functionalised with sequence specific peptide nucleic acid (PNA) probes. We studied the probe functionalisation using atomic force microscopy and performed elemental and electrochemical characterisations. To enhance the assay performance and minimise sample volume requirements, we designed and characterised a closed-loop microfluidic system. We presented the EIS assay's specificity for wild-type miR-122 over non-complementary and single nucleotide mismatch targets. We successfully demonstrated a detection limit of 50 pM for miR-122. Assay performance could be extended to real samples; it displayed high selectivity for liver (miR-122 high) comparing to kidney (miR-122 low) derived samples extracted from murine tissue. Finally, we successfully performed an evaluation with 26 clinical samples. Using EIS, DILI patients were distinguished from healthy controls with a ROC-AUC of 0.77, a comparable performance to qPCR detection of miR-122 (ROC-AUC: 0.83). In conclusion, direct, amplification free detection of miR-122 using EIS was achievable at clinically relevant concentrations and in clinical samples. Future work will focus on realising a full sample-to-answer system which can be deployed for point-of-care testing.
AB - Drug-induced liver injury (DILI) is a major challenge in clinical medicine and drug development. There is a need for rapid diagnostic tests, ideally at point-of-care. MicroRNA 122 (miR-122) is an early biomarker for DILI which is reported to increase in the blood before standard-of-care markers such as alanine aminotransferase activity. We developed an electrochemical biosensor for diagnosis of DILI by detecting miR-122 from clinical samples. We used electrochemical impedance spectroscopy (EIS) for direct, amplification free detection of miR-122 with screen-printed electrodes functionalised with sequence specific peptide nucleic acid (PNA) probes. We studied the probe functionalisation using atomic force microscopy and performed elemental and electrochemical characterisations. To enhance the assay performance and minimise sample volume requirements, we designed and characterised a closed-loop microfluidic system. We presented the EIS assay's specificity for wild-type miR-122 over non-complementary and single nucleotide mismatch targets. We successfully demonstrated a detection limit of 50 pM for miR-122. Assay performance could be extended to real samples; it displayed high selectivity for liver (miR-122 high) comparing to kidney (miR-122 low) derived samples extracted from murine tissue. Finally, we successfully performed an evaluation with 26 clinical samples. Using EIS, DILI patients were distinguished from healthy controls with a ROC-AUC of 0.77, a comparable performance to qPCR detection of miR-122 (ROC-AUC: 0.83). In conclusion, direct, amplification free detection of miR-122 using EIS was achievable at clinically relevant concentrations and in clinical samples. Future work will focus on realising a full sample-to-answer system which can be deployed for point-of-care testing.
KW - Drug-induced liver injury
KW - microRNA detection
KW - Electrochemical impedance spectroscopy
KW - Point-of-care diagnostics
KW - Continuous-flow measurements
U2 - 10.1016/j.bios.2023.115298
DO - 10.1016/j.bios.2023.115298
M3 - Article
SN - 0956-5663
VL - 231
JO - Biosensors and Bioelectronics
JF - Biosensors and Bioelectronics
M1 - 115298
ER -