Diagnosis of Endometriosis: Dual-Amplification Strategy Driven by Copper Nanoclusters

Endometriosis is a prevalent gynecologic disorder associated with infertility and increased cancer risk, necessitating the development of sensitive and reliable diagnostic methods. Circulating microRNAs (miRNAs) have emerged as promising noninvasive biomarkers for early disease detection. However, low abundance and sequence similarity among miRNA family members hinder accurate detection. Herein, we first conducted differential expression analysis of publicly available miRNA-sequencing data sets to identify potential diagnostic biomarkers for endometriosis, from which miR-199a-5p was selected as a representative target. Building on this selection, we subsequently developed a highly sensitive and specific fluorescent biosensing platform for miR-199a-5p detection by integrating poly(thymine) (polyT) DNA-templated copper nanoclusters (CuNCs) with a dual isothermal amplification strategy. The biosensing system utilizes a 3′-phosphorylated, biotinylated hairpin DNA probe immobilized on streptavidin-coated magnetic beads. Upon hybridization with the target miR-199a-5p, duplex-specific nuclease (DSN) mediates selective cleavage, enabling target recycling and simultaneously generating a 3′-hydroxyl terminus. This newly exposed terminus subsequently serves as a primer for terminal deoxynucleotidyl transferase (TdT)-catalyzed polyT elongation. The resulting polyT sequence functions as an effective scaffold for the in situ formation of copper nanoclusters (CuNCs), thus producing a label-free fluorescence signal within 2 h. In this design, magnetic beads not only facilitate efficient separation from serum matrices but also enhance reaction efficiency through surface-initiated enzymatic polymerization. As a result, the sensing platform exhibits excellent specificity, including reliable discrimination of single-base mismatches, and maintains robust performance in complex biological samples. This integrated platform, combining bioinformatic prescreening with a CuNCs-based sensing strategy, offers rapid, cost-effective, and label-free detection of miRNA, showing promise for early diagnosis and clinical monitoring of endometriosis. This publication is licensed under License Summary* You are free to share(copy and redistribute) this article in any medium or format and to adapt(remix, transform, and build upon) the material for any purpose, even commercially within the parameters below: Creative Commons (CC): This is a Creative Commons license. Attribution (BY): Credit must be given to the creator. *Disclaimer This summary highlights only some of the key features and terms of the actual license. It is not a license and has no legal value. Carefully review the actual license before using these materials. License Summary* You are free to share(copy and redistribute) this article in any medium or format and to adapt(remix, transform, and build upon) the material for any purpose, even commercially within the parameters below: Creative Commons (CC): This is a Creative Commons license. Attribution (BY): Credit must be given to the creator. *Disclaimer This summary highlights only some of the key features and terms of the actual license. It is not a license and has no legal value. Carefully review the actual license before using these materials. License Summary* You are free to share(copy and redistribute) this article in any medium or format and to adapt(remix, transform, and build upon) the material for any purpose, even commercially within the parameters below: Creative Commons (CC): This is a Creative Commons license. Attribution (BY): Credit must be given to the creator. *Disclaimer This summary highlights only some of the key features and terms of the actual license. It is not a license and has no legal value. Carefully review the actual license before using these materials.

Experimental Section Materials, Chemicals, and Apparatus MiRNA Differential Expression Analysis Hairpin Probe Design and Optimization Specificity of Hairpin Probe Melting Curve Analysis of the Hairpin Probe and the Hairpin Probe/Target Duplex Preparation and Characterization of the Conjugated hDNA on MB (MB-hDNA) Synthesis and Characterization of Copper Nanoclusters (CuNCs) Validation of DSN Reaction on Magnetic Beads Optimization of the Reaction Conditions of TdT-Mediated Polymerization Procedure for the Detection of miR-199a-5p Detection of miR-199a-5p in Serum Samples from Endometriosis Patients and Controls MiR-199a-5p Quantification Using TaqMan MicroRNA Assays

and Discussion Identification of Dysregulated MiRNAs in Endometriosis Assay Principle The “Balloon-like” Probe hDNA Design Characterization of the hDNA-Conjugated Magnetic Beads (MB-hDNA) Characterization and Optimization of the Synthesis Conditions of CuNCs Feasibility Investigation of the Biosensing Platform Assay Optimization Assay Performance Detection of miR-199a-5p in Serum Samples Using the DSN-TdT Dual Mode MB-hDNA Assay

Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.6c00532. Listing the sequences of oligonucleotides (Table S1) and design (Table S3) used in this study, detection workflow summary (Table S2), comparison of dynamic light scattering results for MB-hDNA and bare MB (Table S4), calculation of probe density (Table S5), comparison of various biosensor platforms (Table S6), clinical characteristics of patients with endometriosis or without endometriosis (Table S7) and comparison between the proposed biosensor and the clinical standard method (Table S8); supplementary figures demonstrating heatmap of differentially expressed miRNAs in two data sets (Figure S1 and S2), rational design of probes (Figure S3) and characterization (Figure S4), characterization of MB-hDNA (Figure S5 and S6), characterization of fluorescent polyT-templated CuNCs (Figure S7), optimization of the synthesis conditions for polyT-templated CuNCs (Figure S8), the effects of reaction buffer composition on the property of polyT-templated CuNCs (Figure S9), fluorescence stability of polyT-templated CuNCs (Figure S10), feasibility assessment of the biosensing platform (Figure S11), characterization of TdT-mediated extension in free solution at different reaction times (Figure S12), assay optimization (Figure S13), characterization of hairpin probes immobilized at different probe density (Figure S14) and containing different thymidine linkers (Figure S15) in the DSN-assisted amplification reaction on magnetic beads, intra-and inter- day reproducibility of the dual mode MB-hDNA assay (Figure S16), evaluation of matrix effects using a spiked-in sample in different biological media (Figure S17), and quantitative analyses of the. serum samples from individuals with endometriosis and healthy controls (Figure S18) (PDF) Terms & Conditions Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html. Acknowledgments The authors gratefully acknowledge the financial support from the Ministry of Education of Taiwan (Higher Education Sprout Project, 113L895204 and 114L893404) and the National Science and Technology Council under Grant Nos. 109-2113-M-002-007-MY3 and 112-2113-M-002-026-MY3. Thanks also go to Ms. Fu-Mei Lin, Yu-Ling Liang and Tzu-Yuan Wang for numerous help in project administration.

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Supporting Information Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.6c00532. Listing the sequences of oligonucleotides (Table S1) and design (Table S3) used in this study, detection workflow summary (Table S2), comparison of dynamic light scattering results for MB-hDNA and bare MB (Table S4), calculation of probe density (Table S5), comparison of various biosensor platforms (Table S6), clinical characteristics of patients with endometriosis or without endometriosis (Table S7) and comparison between the proposed biosensor and the clinical standard method (Table S8); supplementary figures demonstrating heatmap of differentially expressed miRNAs in two data sets (Figure S1 and S2), rational design of probes (Figure S3) and characterization (Figure S4), characterization of MB-hDNA (Figure S5 and S6), characterization of fluorescent polyT-templated CuNCs (Figure S7), optimization of the synthesis conditions for polyT-templated CuNCs (Figure S8), the effects of reaction buffer composition on the property of polyT-templated CuNCs (Figure S9), fluorescence stability of polyT-templated CuNCs (Figure S10), feasibility assessment of the biosensing platform (Figure S11), characterization of TdT-mediated extension in free solution at different reaction times (Figure S12), assay optimization (Figure S13), characterization of hairpin probes immobilized at different probe density (Figure S14) and containing different thymidine linkers (Figure S15) in the DSN-assisted amplification reaction on magnetic beads, intra-and inter- day reproducibility of the dual mode MB-hDNA assay (Figure S16), evaluation of matrix effects using a spiked-in sample in different biological media (Figure S17), and quantitative analyses of the. serum samples from individuals with endometriosis and healthy controls (Figure S18) (PDF) Terms & Conditions Most electronic Supporting Information files are available without a subscription to ACS Web Editions. 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