Dynamic chemical labelling allows the measurement of the paracetamol toxicity biomarker microRNA-122 in hospital clinical laboratories

MicroRNA-122 (miR-122) is a sensitive biomarker for paracetamol-induced liver injury. This study evaluated Dynamic Chemical Labelling (DCL) combined with a clinical laboratory assay to quantify miR-122 in human serum, as a proof-of-concept for hospital use. Serum was collected from 19 healthy individuals and 19 patients with paracetamol drug-induced liver injury (DILI). miR-122 was labelled with a biotinylated SMART-Base and quantified using a multiplex, bead-based, fluorescence detection system. Median miR-122 concentration was 233.4pg/ml in DILI patients vs. 15.2pg/ml in controls. Concentrations ranged from 17.5–1376.9pg/ml (DILI) and 2.2–67.2pg/ml (controls). The Mann-Whitney U test showed significant group differences (p < 0.0001). ROC analysis yielded an AUC of 0.96, with a 29.7pg/ml cut-off providing 92.6% sensitivity and 89.4% specificity. miR-122 moderately correlated with ALT (r=0.56, p=0.03). DCL with fluorescence detection is a promising clinical tool for miR-122 quantification. Short Communication: Dynamic chemical labelling allows the measurement of the paracetamol toxicity biomarker microRNA-122 in hospital clinical laboratories Samar Alzeer 1, Antonio Marin-Romero 2,++, B´ arbara L´ opez-Longarela2, Juan J. Guardia-Monteagudo 2, F. Javier Lopez-Delgado 2, Salvatore Pernagallo 2, Juan J. Diaz-Mochon 3,4,5,6, Mavys Tabraue-Chavez2, James W. Dear1* 1 Centre for Cardiovascular Science, Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, UK 2 DESTINA Genomica S.L. Parque Tecnol´ ogico Ciencias de la Salud (PTS), Avenida de la Innovaci´ on 1, Edificio BIC, 18016, Armilla, Granada, Spain. 3 GENYO Centre for Genomics and Oncological Research, Pfizer/University of Granada/Andalusian Regio- nal Government. PTS Granada - Avenida de la Ilustraci´ on, 114, 18016, Granada, Spain. 4 Department of Medicinal & Organic Chemistry, Faculty of Pharmacy, University of Granada, Campus de Cartuja s/n, Granada, Spain. 5 Unit of Excellence in Chemistry Applied to Biomedicine and the Environment of the University of Granada, Granada, Spain. 1 Posted on 21 May 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.174782174.48540606/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary. 6 Instituto de Investigaci´ on Biosanitaria ibs.GRANADA, Granada, Spain. ++Current affiliation: Vircell S.L. Parque Tecnologico de la Salud, C/ Avicena 8, 18106 Granada. *Corresponding author E-mail address: [email protected] (J. Dear)

MicroRNA-122 (miR-122) is a sensitive biomarker for paracetamol-induced liver injury. This study evaluated Dynamic Chemical Labelling (DCL) combined with a clinical laboratory assay to quantify miR-122 in human serum, as a proof-of-concept for hospital use. Serum was collected from 19 healthy individuals and 19 patients with paracetamol drug-induced liver injury (DILI). miR-122 was labelled with a biotinylated SMART-Base and quantified using a multiplex, bead-based, fluorescence detection system. Median miR-122 concentration was 233.4pg/ml in DILI patients vs. 15.2pg/ml in controls. Concentrations ranged from 17.5–1376.9pg/ml (DILI) and 2.2–67.2pg/ml (controls). The Mann-Whitney U test showed significant group differences (p < 0.0001). ROC analysis yielded an AUC of 0.96, with a 29.7pg/ml cut-off providing 92.6% sensitivity and 89.4% specificity. miR-122 moderately correlated with ALT (r=0.56, p=0.03). DCL with fluorescence detection is a promising clinical tool for miR-122 quantification.

miR-122, paracetamol toxicity, dynamic chemical labelling, multiplex immunoassay What is already known about this subject: • Paracetamol overdose is a leading cause of acute liver failure. • Liver function biomarkers such as ALT are commonly used to assess toxicity. • miR-122 is a promising sensitive and specific biomarker for liver injury. What the study adds: • Dynamic chemical labelling enables the direct measurement of miR-122 in serum without using quan- titative PCR. • The multiplex bead-based system used for detection is available in hospital laboratories, making the assay accessible for clinical use. • The study supports the incorporation of miR-122 for assessing patients with a history of paracetamol overdose in clinical settings. 1. INTRODUCTION Paracetamol overdose is the leading cause of acute liver failure in Western countries (Arshad & Bangash, 2022; Chidiac et al., 2023). There is a need for rapid and accurate diagnosis to allow prompt initiation of the antidote N-acetylcysteine (NAC) to those patients who stand to benefit most from treatment to prevent clinically significant liver injury. The current diagnostic test for liver injury is serum alanine aminotrans- ferase (ALT) activity. Increases in ALT often lag significantly behind the onset of hepatocellular damage, potentially delaying critical interventions by hours, or even days (Dear et al., 2018; Green et al., 2010). Circulating microRNA-122 (miR-122), a highly liver-specific microRNA, has emerged as a biomarker for paracetamol-induced liver injury, demonstrating rapid and significant increases in serum within hours of toxic paracetamol exposure; importantly preceding ALT elevation (Antoine et al., 2013; Dear et al., 2018; Liu et al., 2018, Starkey Lewis et al., 2011; Wang et al., 2009;). While these, and other studies, have validated the sensitivity and specificity of miR-122, their reliance on quantitative PCR (qPCR) methods has hindered clinical translation. Although highly sensitive, qPCR requires specialized equipment, trained personnel, and relatively lengthy processing times (typically several hours), making it unsuitable for the time- critical decision-making required in acute paracetamol overdose management (Church & Watkins, 2017; Liu et al., 2018). Previous studies have demonstrated the feasibility of using Dynamic Chemical Labelling (DCL) technology for miR-122 quantification (Marin-Romero et al., 2023, Marin-Romero et al., 2021, Marin-Romero et al., 2020, Bowler et al., 2010). However, these studies utilized a highly specialized, non-IVD-certified 2 Posted on 21 May 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.174782174.48540606/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary. single-molecule array (Simoa) platform (Lopez-Longarela et al., 2020), which is not readily available in clinical laboratories. Therefore, a rapid, clinically accessible, and IVD-compatible assay for miR-122 to aid in the management of acute paracetamol overdose remains an unmet need. To address this need, we have developed a novel miR-122 immunoassay-like assay combining DCL with the Luminex xMAP platform. The Luminex xMAP technology is a bead-based multiplex immunoassay platform that is already widely used in clinical laboratories worldwide for a variety of diagnostic applications, including infectious disease testing and HLA typing (Dunbar et al., 2006). There are reportedly more than 18,000 installed Luminex xMAP platforms, of which an estimated 25-30% are in clinical laboratories. Therefore, this ’Luminex-DCL’ miR-122 assay leverages existing hospital infrastructure, eliminating the need for specialized equipment or extensive training, and offers a significantly reduced turnaround time compared to qPCR, potentially enabling results within approximately one hour. The objective of this study was to explore the diagnostic performance of the Luminex-DCL miR-122 assay for detection of paracetamol-induced liver injury in patients. We hypothesized that the serum miR-122 concentration, measured by the Luminex-DCL assay (index test), would be significantly elevated in patients with paracetamol-induced liver injury (defined by the reference standard test – ALT), compared to healthy controls. 2.METHODS 2.1 Samples A total of 38 serum samples were analysed to compare miR-122 concentration in patients diagnosed with paracetamol DILI (N = 19) and healthy subjects (N = 19). Paracetamol DILI was defined as a serum ALT activity greater than 5x the upper limit of normal (ULN) after paracetamol overdose (ULN=50 U/L). DILI samples were randomly selected from The Markers and Paracetamol Poisoning Study 2 (MAPP2, Clinical- Trials.gov identifier: NCT03497104). Ethical approval for this study was provided by London-South East Research Ethics Committee (18/LO/0894) and all patients provided written informed consent. The study established a biobank of human serum samples from patients with paracetamol overdose. MAPP2 was a prospective, observational cohort study of participants presenting to the Emergency Department at the Royal Infirmary of Edinburgh, UK. Inclusion criteria were that participants must be age 16 years and over, attending hospital with a paracetamol overdose alone or as part of a mixed overdose, and able to give in- formed consent. Healthy human serum was collected from healthy individuals on the Centre for Inflammation Research Blood Donor Register, University of Edinburgh (Ethics Reference Number: 21-EMREC-04). All samples were stored at -70degC until analysis. Before assay preparation, samples were thawed completely, vortex-mixed thoroughly, and centrifuged to ensure homogeneity and remove any debris. The processed samples were then used for downstream analysis following the assay protocol. 2.2 Index test The Liver Injury miR-122 Test (LiverACE TM Kit, LAK-96-2024) was obtained from Destina Genomica SL (Granada, Spain). This assay is based on Dynamic Chemical Labelling Technology which combines miRNAs capture probes linked to MagPlex(r) Microspheres and a biotinylated SMART-Base to selectively label duplex formation with the target miRNA sequence. Upon hybridization, the SMART-Base incorporates into the miRNA-probe duplex, allowing for specific chemical labelling. Detection was performed using a Reporter Molecule that binds to the biotin tag, generating a signal proportional to the miRNA concentration. The fluorescence signal was then analysed using the Luminex xMAP INTELLIFLEX DRSE System ( Figure 1 ). 2.3 Microsphere-based multiplexing technology The Luminex xMAP INTELLIFLEX DRSE from ThermoFisher Scientific was used in this study. This technology combines flow cytometry with microsphere-based assays, allowing for enhanced sensitivity, speci- ficity, and data precision. Data processing was refined using Luminex xMAP INTELLIFLEX DRSE software, allowing for comprehensive signal integration and normalization. The Luminex System was configured ac- cording to the assay requirements. The probe height was adjusted based on the plate used to ensure optimal 3 Posted on 21 May 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.174782174.48540606/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary. sample acquisition. In the acquisition Settings, the bead type was set to MagPlex(r), with a minimum measurement of 50 events per bead. Each sample was analysed with a sample size of 100 μL, and the gate setting was adjusted between 8,000 and 17,000 to capture the appropriate bead population. The reporter gain was set to Low PMT, with a timeout of 60 seconds to ensure adequate bead detection. The assay utilized singleplex bead sets, allowing for precise and reproducible analyte quantification. 2.4 Assay reaction The Bio-Plex Handheld Magnetic Washer (Bio-Rad, 171020100) was used during the washing steps of the assay. The procedure was conducted following the manufacturer’s instructions. Briefly, 25 μL of test solution (either serum sample or calibrator) was added to a flat-bottom 96-well plate containing 75 μL of miR-122 microsphere beads in each well. The plate was incubated on a plate shaker at 30 °C for 2 hours to allow binding of the analytes to the beads. After binding, the beads were washed three times with wash buffer. Subsequently, 50 μL of LiverAce TM Mix, containing SMART-C and a Reducing Agent in Assay Buffer, was added to each well. The plate was shaken for 1 hour at 40 °C, followed by three additional washing steps. Then, 50 μL of Streptavidin-R-Phycoerythrin (SA-PE) was added, and the plate was further incubated for 30 minutes at 30°C on a plate shaker. After a final series of three washes, the beads were resuspended in 120 μL of wash buffer and analysed on the Luminex xMAP INTELLIFLEX DRSE system. 2.5 Clinical biomarkers Blood tests were measured in the NHS Lothian Biochemistry Laboratory as described previously (Dear et al., 2018). 2.6 Statistical analysis Data were statistically analysed with GraphPad Prism 10 software (La Jolla, California)., The 95% confi- dence interval (CI) for the mean was calculated, and the interquartile range (IQR) for median values. The Mann-Whitney U tests were used for non-parametric comparisons between groups. Receiver Operating Char- acteristic (ROC) analysis was conducted to evaluate the diagnostic performance of miR-122 as a biomarker for liver injury, and the area under the curve (AUC) was calculated to determine sensitivity and specificity. Statistical significance was set at p < 0.05 for all tests. 3. RESULTS The study included a total of 38 participants, with 19 healthy controls and 19 patients with paracetamol- induced liver injury (DILI).Table 1 provides a detailed overview of the demographic and clinical character- istics of the patient cohort. The median paracetamol ingestion was 26 g (IQR: 16–46 g), and 68% of patients had co-ingested other substances, primarily ethanol or opioids. Biochemical tests were performed to assess liver function, revealing elevated ALT activity (median: 558 U/L, IQR: 306–1301) in patients with liver toxicity. Additional biochemical parameters, including alkaline phosphatase, sodium, potassium, creatinine, urea, WBC, haemoglobin, and INR, are presented in Table 1 . Figure 2A shows a calibration curve of miR-122 that was generated by plotting the fluorescence signal (MFI) against known miR-122 concentrations (pg/mL), showing a sigmoidal response. The curve was fitted using a logistic regression model to interpolate unknown sample concentrations. The standard curve demonstrated a strong correlation between fluorescence intensity and miR-122 concentration. The mean of three independent measurements per sample was used for analysis. In the patient group, the median miR-122 concentration was significantly higher (233.4 pg/mL, IQR: 74.5– 363.4) compared to the control group (15.2 pg/mL, IQR: 9.4–24). The range of miR-122 concentration varied from 2.2 to 67.2 pg/mL in controls and from 17.5 to 1376.9 pg/mL in patients. The mean miR-122 concentration was also elevated in the DILI group (317.4 ± 339.6 pg/mL) compared to controls (20.2 ± 15.9 pg/mL). Statistical analysis using the Mann-Whitney U test confirmed a significant difference between the two groups ( p < 0.0001) (Figure 2B), supporting the potential role of miR-122 as a biomarker for liver injury. 4 Posted on 21 May 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.174782174.48540606/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary. ROC analysis demonstrated that miR-122 could distinguish DILI patients from healthy controls, with an AUC of 0.96 (95% CI: 0.91–1). At a cut-off of 29.7 pg/mL sensitivity was 92.6% and specificity was 89.4% (Figure 2C ). Spearman correlation showed a moderate positive association between miR-122 and ALT (r = 0.56, 95% CI: 0.056-0,84, p = 0.031) ( Figure 2D ). 4. DISCUSSION In recent years, miR-122 has emerged as a promising biomarker for liver injury, particularly in the context of paracetamol toxicity. Multiple studies have highlighted the diagnostic and monitoring potential of circulating miRNAs in liver damage (Carreiro et al., 2020; Dear et al., 2014). In particular, miR-122 correlates with the extent of liver damage, making it a potential diagnostic tool in clinical settings (Vliegenthart et al., 2015). Our findings align with these studies, further supporting the potential of miRNAs as non-invasive biomarkers to distinguish between liver injury patients and healthy controls. Given the consistency of these findings across multiple studies, miR-122 could be a clinically valuable biomarker for liver injury, potentially offering superior diagnostic accuracy compared to traditional liver enzyme markers. Thus, miRNA-based assays hold promise for the early detection and monitoring of liver injury, especially in paracetamol overdose cases. The direct quantification of miR-122 by the novel bead-based immunoassay that uses DCL technology, offers several advantages over traditional PCR methods for measuring miR-122, particularly in clinical settings. Unlike PCR, which requires extensive sample processing, RNA extraction, and amplification steps, this assay enables direct and multiplexed quantification of miR-122, thereby reducing analysis time, procedural com- plexity, and overall costs. An important advantage is that multiplex technology is already available in many hospital laboratories, making it a feasible option for clinical translation without the need for additional in- frastructure. Moreover, multiplex assays demonstrate a broader dynamic range and enhanced reproducibility compared to PCR-based assays, making them well-suited for large-scale studies where consistency is critical (Gadsby et al., 2010; Munro et al., 2013; Pierce et al., 2016). Since microsphere-based multiplex assays do not rely on enzymatic amplification, they minimize amplification bias and variability associated with qPCR, potentially leading to more reliable and quantitative measurements. Given these advantages, the multiplex platform provides a robust, high-throughput, and cost-effective alternative for miR-122 quantification, ma- king it particularly useful for biomarker validation and clinical diagnostic applications. This proof-of-concept study has several limitations. It involved a relatively small sample size and thus requires validation in a prospective multi-centre study to confirm the findings. The time from sample application to

could be further reduced to enable more efficient clinical decision-making in acute settings. The assay needs further optimisation to function with capillary blood which would allow point of care testing. In conclusion, the Luminex-DCL miR-122 assay represents a potential advance in the diagnosis and moni- toring of liver injury as this assay can be performed using an established hospital laboratory platform and has, therefore, potential for widespread clinical adoption. Author Contributions Samar Alzeer. Writing – original draft, Methodology, Investigation, Formal Analysis, Validation.Antonio Marin-Romero : Methodology, Investigation, Formal Analysis, Validation. B´ arbara L´ opez-Longarela: Methodology, Investigation, Validation. Juan J. Guardia-Monteagudo: Resources, Investigation. Fran- cisco J. Lopez-Delgado : Resources, Investigation. Salvatore Pernagallo : Conceptualization, Metho- dology, Investigation. Juan J. D´ ıaz-Moch´ on: Conceptualization, Methodology, Writing review & editing. Mavys Tabraue-Chavez : Conceptualization, Methodology, Formal Analysis, Writing review & editing, Supervision. James W. Dear : Resources, Conceptualization, Methodology, Formal Analysis, Writing re- view & editing, Supervision. Acknowledgments J.D. acknowledges the support from the Chief Scientist’s Office Scotland via the Centre for Precision Cell Therapy for the Liver (PMAS/21/07). 5 Posted on 21 May 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.174782174.48540606/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary. Conflict of Interest statement The authors declare the following financial interests which may be considered as potential competing interests: JJDM is a founder, shareholder and Director of DESTINA Genomics Ltd. SP is a shareholder of DESTINA Genomics Ltd. DESTINA Genomica SL is a wholly owned subsidiary of DESTINA Genomics Ltd. DESTINA is interested in the exploitation of the technology developed here. Data availability statement Data available on request from the authors ORCID : Samar Alzeer: https://orcid.org/0000-0001-5119-3269 Antonio Marin-Romero: https://orcid.org/0000-0002-0589-8927 B´ arbara L´ opez Longarela: https://orcid.org/0000-0001-9043-2445 Juan J. Guardia-Monteagudo: https://orcid.org/0000-0001-5475-931X F. Javier Lopez-Delgado: https://orcid.org/0000-0002-6968-5672 Salvatore Pernagallo: https://orcid.org/0000-0002-0259-9008 Juan J. Diaz-Mochon: https://orcid.org/0000-0002-3599-1954 Mavys Tabraue Chavez: https://orcid.org/0000-0002-5161-5430 James Dear https://orcid.org/0000-0002-8630-8625

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Table 1: Descriptive statistics of paracetamol-induced liver injury (DILI) samples (N=19) used in this study Age years: Median (IQR) 30 (21-51) Gender: Male (%) 4 (21) Age: Median (IQR) 30 (21-51) Ethnicity (%) White Scottish 18 (94.7 %) White (other) 1 (5.2%) Overdose type (%) Acute overdose 8 hours 8 (42.1%) Supra-therapeutic overdose 2 (10.5%) Staggered intentional overdose (ingestion over 2 h or more) 2 (10.5%) Total paracetamol ingested in grams: Median (IQR) 26 (16.0 – 46.2) Ingestion of other drugs: Yes (%) 13 (68.4%) Anti-coagulants 0 Non-opioid analgesics 2 NSAIDs 2 Cardiovascular drugs 0 Alcohol 7 Opioids 6 SSRIs 1 Tricyclic antidepressants 1 Benzodiazepines 0 Other 1 Prothrombin (secs): Median (IQR) 14.2 (13.1-17) Alk Phos (U/L): Median (IQR) 69 (57-131) ῝ρεατινινε (μμολ/Λ): Μεδιαν (ΙΧΡ) 56 (51-76.2) Urea (mmol/L): Median (IQR) 2.9 (2.325-4.125) WBC (x109/L): Median (IQR) 8.85 (5.875-9.5) Potassium (mmol/L): Median (IQR) 3.6 (3.15-3.8) INR: Median (IQR) 1.35 (1.2-1.575) ALT (U/L): Median (IQR) 558 (306-1301) Haemoglobin (g/L): Median (IQR) 126 (114-137.25) Sodium (mmol/L): Median (IQR) 139 (137.75-141.25) Βιλιρυβιν (μμολ/Λ): Μεδιαν (ΙΧΡ) 16.5 (10-32.75) Figure 1 : Schematic representation of a miRNA detection assay using Dynamic Chemistry Labeling (DCL). (1) RNA of interest from biological samples is captured using 122 MagPlex ® beads, specifically targeting miR-122. (2) The SMART-C-Biotin reagent labels a ”blank” position in the RNA strand via DCL chemistry. (3) Biotin recognition occurs through the binding of streptavidin-phycoerythrin (SA-PE). (4) The fluorescence signal is then read using an xMAP INTELLIFLEX system for quantitative analysis. 8 Posted on 21 May 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.174782174.48540606/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary. Figure 2 : A) Standard curve for miR-122 quantificationusing microsphere-based multiplex technolo- gy. The x-axis represents miR-122 concentration (pg/mL), while the y-axis represents the median fluorescence intensity (MFI). B) Serum miR-122 concentration (pg/ml) in patients with paracetamol-induced li- ver injury (DILI) and healthy controls. Values represent the mean of three independent measurements per sample. A significant difference in miR-122 was observed between the two groups, as determined by the Mann- Whitney U test ( p < 0.0001). The bars represent the median with error bars representing the IQR. Red dotted line represents sensitivity and specificity at a cut off value of 29.7 pg/mL. C) Receiver Operating Characteristic (ROC) curve for miR-122 in distinguishing paracetamol-induced hepatotoxicity patients from healthy controls. The X-axis represents 1 - Specificity (False Positive Rate, %), and the Y- axis represents Sensitivity (True Positive Rate). The area under the curve (AUC) is 0.96 (95% CI: 0.91 to 1). D) Scatter plot depicting the correlation between circulating miR-122 concentration (pg/mL) and alanine aminotransferase (ALT) activity (U/L) in patients with paracetamol-induced liver injury . Each dot represents an individual sample. Spearman correlation analysis demonstrated a moderate positive correlation (r = 0.56, 95% CI: 0.056 to 0.84, p = 0.031). 9 Posted on 21 May 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.174782174.48540606/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary. 10 Posted on 21 May 2025 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.174782174.48540606/v1 — This is a preprint and has not been peer-reviewed. Data may be preliminary. 0 5000100001500020000 0 1000 2000 3000 4000 miR-122 (pg/ml) Median Fluorescence Intensity (MFI) 0 20 40 60 80100 0 20 40 60 80 100 100% - Specificity % Sensitivity % AUC 0.96, 95% CI: 0.9 to 1. Healthy DILI 1 10 100 1000 10000 miR-122 (pg/ml) <0.0001 Sens 93% Spec 89% 0 2000400060008000 0 200 400 600 800 1000 ALT (U/L) miR-122 (pg/ml) A B C D11