Precision Tumor Recognition in Clear Cell Renal Cell Carcinoma: A qHCR Platform Leveraging Tetrahedral DNA Nanostructures for Rapid Molecular Diagnosis

Abstract

Clear cell renal cell carcinoma (ccRCC) is the most prevalent and aggressive subtype of kidney cancer, demanding rapid and precise diagnostic tools to guide clinical decision-making. Current methods, such as immunohistochemistry (IHC) and frozen section analysis, face limitations in speed, sensitivity, and workflow complexity. To address these challenges, we developed a tetrahedral DNA nanostructure (TDN)-enhanced quadrivalent hybridization chain reaction (qHCR) platform targeting carbonic anhydrase IX (CAIX), a highly specific biomarker for ccRCC. This system integrates aptamer-based molecular recognition with enzyme-free signal amplification, leveraging the spatial confinement and multivalent effects of TDNs to achieve ultrasensitive detection. The qHCR platform demonstrated remarkable performance, with a reaction rate 95 times faster than traditional HCR and sustained signal stability in serum for over 36 h. In clinical validation using 40 ccRCC tissue samples, the platform enabled Precision Tumor Recognition within 25 min for frozen sections, yielding a tumor-to-normal tissue signal ratio of 3.35:1, and completed molecular profiling within 2.5 h for formalin-fixed, paraffin-embedded (FFPE) samples, showing full concordance with histopathological diagnoses. Its modular design allows seamless target switching by replacing aptamer sequences, as confirmed by successful detection of protein tyrosine kinase (PTK7) in an acute lymphoblastic leukemia model. With its cost-effectiveness ($0.15 per test), streamlined workflow, and compatibility with both intraoperative margin assessment and postoperative pathological analysis, the qHCR platform represents a transformative advancement in molecular diagnostics for ccRCC management, offering a robust solution for precision oncology and time-critical surgical decision-making.

Keywords

carbonic anhydrase IX (CAIX) / clear cell renal cell carcinoma (ccRCC) / DNA nanostructure / hybridization chain reaction / intraoperative tumor identification / molecular diagnostics

References

[1] P. C. Barata and B. I. Rini, “Treatment of Renal Cell Carcinoma: Current Status and Future Directions,” CA: A Cancer Journal for Clinicians 67 (2017): 507-524.

[2] Z. Long, C. Sun, M. Tang, et al., “Single-Cell Multiomics Analysis Reveals Regulatory Programs in Clear Cell Renal Cell Carcinoma,” Cell Discovery 8​ (2022): 68.

[3] J. J. Hsieh, M. P. Purdue, S. Signoretti, et al., “Renal Cell Carcinoma,” Nature Reviews Disease Primers 3 (2017): 17009.

[4] M. Sun, L. Marconi, T. Eisen, et al., “Adjuvant Vascular Endothelial Growth Factor-Targeted Therapy in Renal Cell Carcinoma: A Systematic Review and Pooled Analysis,” European Urology 74 (2018): 611-620.

[5] S. Buti, M. Puligandla, M. Bersanelli, et al., “Validation of a New Prognostic Model to Easily Predict Outcome in Renal Cell Carcinoma: The GRANT Score Applied to the ASSURE Trial Population,” Annals of Oncology 28 (2017): 2747-2753.

[6] B. Schatlo, J. Fandino, N. R. Smoll, et al., “Outcomes After Combined Use of Intraoperative MRI and 5-Aminolevulinic Acid in High-Grade Glioma Surgery,” Neuro-Oncology 17 (2015): 1560-1567.

[7] R. Mallick, T. M. Stevens, T. S. Winokur, et al., “Is Frozen-Section Analysis during Thyroid Operation Useful in the Era of Molecular Testing?,” Journal of the American College of Surgeons 228 (2019): 474-479.

[8] D.-Y. Hou, M.-D. Wang, X.-J. Hu, et al., “An Activated Excretion-Retarded Tumor Imaging Strategy towards Metabolic Organs,” Bioactive Materials 14 (2021): 110-119.

[9] B. Yang, L.-L. Ma, M. Qiu, et al., “A Novel Nephrometry Scoring System for Predicting Peri-Operative Outcomes of Retroperitoneal Laparoscopic Partial Nephrectomy,” Chinese Medical Journal 133 (2020): 577-582.

[10] V. A. Sahni and S. G. Silverman, “REVIEW: Biopsy of Renal Masses: When and Why,” Cancer Imaging 9 (2009): 44-55.

[11] S. G. Silverman, Y. U. Gan, K. J. Mortele, et al., “Renal Masses in the Adult Patient: The Role of Percutaneous Biopsy,” Radiology 240 (2006): 6-22.

[12] J. Dang, P. Mendez, S. Lee, et al., “Development of a Robust DNA Quality and Quantity Assessment qPCR Assay for Targeted Next-Generation Sequencing Library Preparation,” International Journal of Oncology 49 (2016): 1755-1765.

[13] A. Chatrian, R. T. Colling, L. Browning, et al., “Artificial Intelligence for Advance Requesting of Immunohistochemistry in Diagnostically Uncertain Prostate Biopsies,” Modern Pathology 34 (2021): 1780-1794.

[14] F. Pu, J. Ren, and X. Qu, “Recent Progress in Sensor Arrays Using Nucleic Acid as Sensing Elements,” Coordination Chemistry Reviews 456 (2022): 214379.

[15] E. S. Lee, B. S. Cha, Y. J. Jang, et al., “Harnessing the Potential of Aptamers in Cell-derived Vesicles for Targeting Colorectal Cancers at Pan-Dukes' stages,” International Journal of Biological Macromolecules 280 (2024): 135911.

[16] B. S. Cha, Y. J. Jang, E. S. Lee, et al., “Development of a Novel DNA Aptamer Targeting Colorectal Cancer Cell-Derived Small Extracellular Vesicles as a Potential Diagnostic and Therapeutic Agent,” Advanced Healthcare Materials 12 (2023): 2300854.

[17] M. Xuan, J. Shao, and J. Li, “Cell Membrane-Covered Nanoparticles as Biomaterials,” National Science Review 6 (2019): 551-561.

[18] M. Kimoto, Y. W. Shermane Lim, and I. Hirao, “Molecular Affinity Rulers: Systematic Evaluation of DNA Aptamers for Their Applicabilities in ELISA,” Nucleic Acids Research 47 (2019): 8362-8374.

[19] Z. Watkins, A. Karajic, T. Young, et al., “Week-Long Operation of Electrochemical Aptamer Sensors: New Insights Into Self-Assembled Monolayer Degradation Mechanisms and Solutions for Stability in Serum at Body Temperature,” ACS Sensors 8 (2023): 1119-1131.

[20] D. Kong, I. A. P. Thompson, N. Maganzini, et al., “Aptamer-Antibody Chimera Sensors for Sensitive, Rapid, and Reversible Molecular Detection in Complex Samples,” ACS Sensors 9 (2024): 1168-1177.

[21] Q. Zhou, J. Pan, L. Mo, et al., “Fluorescent on-Site Detection of Multiple Pathogens Using Smartphone-Based Portable Device With Paper-Based Isothermal Amplification Chip,” Mikrochimica Acta 189 (2022): 333.

[22] J. Liu, G. Xie, S. Lv, et al., “Recent Applications of Rolling Circle Amplification in Biosensors and DNA Nanotechnology,” Trends in Analytical Chemistry 160 (2023): 116953.

[23] B. S. Cha, E. S. Lee, J. Kim, et al., “Aptamer-Directed Tyramide Signal Amplification for Ultrasensitive Detection of Small Extracellular Vesicles for Temporally Heterogenous Colorectal Cancer,” Sensors and Actuators B: Chemical 431 (2025): 137411.

[24] R. Lin, Q. Feng, P. Li, et al., “A Hybridization-Chain-Reaction-Based Method for Amplifying Immunosignals,” Nature Methods 15 (2018): 275-278.

[25] J. Wang, D.-X. Wang, J.-Y. Ma, et al., “Three-Dimensional DNA Nanostructures to Improve the Hyperbranched Hybridization Chain Reaction,” Chemical Science 10 (2019): 9758-9767.

[26] E. S. Lee, J. Woo, J. Shin, et al., “Tetrahedral DNA Nanostructures Enhance Transcription Isothermal Amplification for Multiplex Detection of Non-Coding RNAs,” Biosensors and Bioelectronics 250 (2024): 116055.

[27] W. Wang, M. Lin, W. Wang, et al., “DNA Tetrahedral Nanostructures for the Biomedical Application and Spatial Orientation of Biomolecules,” Bioactive Materials 33 (2024): 279-310.

[28] X.-M. Zhou, S.-Y. Chen, Y.-Q. Chai, et al., “Supramolecular DNA Nanodevice Assembled via RCA and HCR Cascade Reaction on Tetrahedral DNA Nanostructure for Sensitive Detection and Intracellular Imaging of Dual-miRNAs Associated With Liver Cancer,” Analytical Chemistry 97 (2025): 686-693.

[29] M. Hu, Z. Yingyu, M. Zhang, et al., “Functionalizing Tetrahedral Framework Nucleic Acids-Based Nanostructures for Tumor in Situ Imaging and Treatment,” Colloids and Surfaces B: Biointerfaces 240 (2024): 113982.

[30] X. Wei, Z. Cai, L. Cai, et al., “Photonic Barcodes Combining Branched Hybridization Chain Reaction for Multiplex Quantification of Bladder Cancer MicroRNAs,” Advanced Materials Interfaces 9 (2022): 2102515.

[31] D.-X. Wang, J. Wang, Y.-X. Wang, et al., “DNA Nanostructure-Based Nucleic Acid Probes: Construction and Biological Applications,” Chemical Science 12 (2021): 7602-7622.

[32] H. Li, M. Han, X. Weng, et al., “DNA-Tetrahedral-Nanostructure-Based Entropy-Driven Amplifier for High-Performance Photoelectrochemical Biosensing,” ACS Nano 15 (2021): 1710-1717.

[33] P. Ji, G. Han, Y. Huang, et al., “Ultrasensitive Ratiometric Detection of Pb2+ Using DNA Tetrahedron-Mediated Hyperbranched Hybridization Chain Reaction,” Analytica Chimica Acta 1147 (2021): 170-177.

[34] J. Su, T. Liu, M. Wang, et al., “Enhanced Stability of Hairpin-Functionalized DNA Tetrahedral Nanostructures for miRNA Detection in Plasma From Ischemic Stroke Patients,” Analytica Chimica Acta 1334 (2025): 343419.

[35] C. Wang, W. Wang, Y. Xu, et al., “Tetrahedral DNA Framework-Programmed Electrochemical Biosenors With Gold Nanoparticles for Ultrasensitive Cell-Free DNA Detection,” Nanomaterials 12 (2022): 666.

[36] J. Wei, X. Gong, Q. Wang, et al., “Construction of an Autonomously Concatenated Hybridization Chain Reaction for Signal Amplification and Intracellular Imaging,” Chemical Science 9 (2018): 52-61.

[37] H. Kim, M. H. Cho, H. S. Choi, et al., “Zwitterionic Near-Infrared Fluorophore-Conjugated Epidermal Growth Factor for Fast, Real-Time, and Target-Cell-Specific Cancer Imaging,” Theranostics 9 (2019): 1085-1095.

[38] C.-P. Gui, B. Liao, C.-G. Luo, et al., “circCHST15 Is a Novel Prognostic Biomarker That Promotes Clear Cell Renal Cell Carcinoma Cell Proliferation and Metastasis Through the miR-125a-5p/EIF4EBP1 Axis,” Molecular Cancer 20 (2021): 169.

[39] M. C. Hekman, M. Rijpkema, C. H. J. Muselaers, et al., “Tumor-Targeted Dual-Modality Imaging to Improve Intraoperative Visualization of Clear Cell Renal Cell Carcinoma: A First in Man Study,” Theranostics 8 (2018): 2161-2170.

[40] N. K. Tafreshi, M. M. Bui, K. Bishop, et al., “Noninvasive Detection of Breast Cancer Lymph Node Metastasis Using Carbonic Anhydrases IX and XII Targeted Imaging Probes,” Clinical Cancer Research 18 (2012): 207-219.

[41] Y. Shao, Y. Li, J. Zhang, et al., “Involvement of Histone Deacetylation in MORC2-Mediated Down-Regulation of Carbonic Anhydrase IX,” Nucleic Acids Research 38 (2010): 2813-2824.

[42] A. Aalipour, H.-Y. Chuang, S. Murty, et al., “Engineered Immune Cells as Highly Sensitive Cancer Diagnostics,” Nature Biotechnology 37 (2019): 531-539.

[43] F. A. Gallagher, H. Sladen, M. I. Kettunen, et al., “Carbonic Anhydrase Activity Monitored in Vivo by Hyperpolarized 13C-Magnetic Resonance Spectroscopy Demonstrates Its Importance for pH Regulation in Tumors,” Cancer Research 75 (2015): 4109-4118.

[44] M.-C. Hsin, Y.-H. Hsieh, Y.-H. Hsiao, et al., “Carbonic Anhydrase IX Promotes Human Cervical Cancer Cell Motility by Regulating PFKFB4 Expression,” Cancers 13 (2021): 1174.

[45] C. Lin, S. Liang, Y. Peng, et al., “Visualized SERS Imaging of Single Molecule by Ag/Black Phosphorus Nanosheets,” Nano-Micro Letters 14 (2022): 75.

[46] L. Zhang, Y. Wu, B. Zheng, et al., “Rapid Histology of Laryngeal Squamous Cell Carcinoma With Deep-Learning Based Stimulated Raman Scattering Microscopy,” Theranostics 9 (2019): 2541-2554.

[47] X. Liu, D. Mao, Y. Song, et al., “Computer-Aided Design of Reversible Hybridization Chain Reaction (CAD-HCR) Enables Multiplexed Single-Cell Spatial Proteomics Imaging,” Science Advances 8 (2022): eabk0133.

[48] B. Koos, G. Cane, K. Grannas, et al., “Proximity-Dependent Initiation of Hybridization Chain Reaction,” Nature Communications 6 (2015): 7294.

[49] J. Zhang, Y. Huang, M. Sun, et al., “Mechanosensing View of SARS-CoV-2 Infection by a DNA Nano-Assembly,” Cell Reports Physical Science 3 (2022): 101048.

[50] GBD 2019 Diabetes Mortality Collaborators, “Diabetes Mortality and Trends Before 25 Years of Age: An Analysis of the Global Burden of Disease Study 2019,” The Lancet Diabetes & Endocrinology 10​ (2022): 177-192.

[51] H.-M. Meng, X. Zhang, Y. Lv, et al., “DNA Dendrimer: An Efficient Nanocarrier of Functional Nucleic Acids for Intracellular Molecular Sensing,” ACS Nano 8 (2014): 6171-6181.

[52] G. Lurje, W. Zhang, A. M. Schultheis, et al., “Polymorphisms in VEGF and IL-8 Predict Tumor Recurrence in Stage III Colon Cancer,” Annals of Oncology 19 (2008): 1734-1741.

[53] L. Zhu, L. Wang, Y. Liu, et al., “CAIX Aptamer-Functionalized Targeted Nanobubbles for Ultrasound Molecular Imaging of Various Tumors,” International Journal of Nanomedicine 13 (2018): 6481-6495.

RIGHTS & PERMISSIONS

2025 The Author(s). Aggregate published by SCUT, AIEI, and John Wiley & Sons Australia, Ltd.