Presentation Information
[3FMBS-09-KL]A Dual-Mode Detection of Cystatin C Using Aptamer- Based Rolling Circle Amplification with Nanopore and Gold Nanoparticle Assays
○chiao-min lai1 (1. Chang Gung University (Taiwan))
Keywords:
Cystatin C,nanopore,AuNP
[Purpose]Kidney diseases impose a substantial global health burden, and early diagnosis can significantly improve treatment outcomes and reduce patient suffering. Cystatin C is a reliable biomarker for chronic kidney disease (CKD) due to its biological stability and minimal interference from external physiological factors. However, existing detection methods rely heavily on specific antibodies and specialized instruments, which are costly and often require stringent storage conditions. The purpose of this study is to develop an antibody-free, cost-effective, and portable detection strategy for Cystatin C.
[Method] We developed an integrated, antibody free diagnostic workflow for Cystatin C detection that combines a gold nanoparticle (AuNP) colorimetric screening assay with a nanopore based quantitative platform. This design allows flexible implementation across different diagnostic scenarios. The core of the system is a nucleic acid aptamer with high affinity and specificity toward Cystatin C, coupled with isothermal rolling circle amplification (RCA). The assay operates through a mechanism in which the presence of Cystatin C suppresses RCA initiation. When Cystatin C is absent, the aptamer primer hybridizes with a padlock probe and triggers RCA, producing long single stranded DNA concatemers that generate frequent nanopore translocation events. In contrast, binding of Cystatin C sequesters the aptamer primer, inhibits padlock probe circularization, and leads to a pronounced reduction in nanopore signal events. To address the time and infrastructure requirements associated with nanopore analysis, an AuNP colorimetric assay is incorporated as a rapid visual screening module that requires no instrumentation. This dual platform architecture supports two practical use modes: rapid prescreening using the AuNP assay followed by precise nanopore based quantification, or independent selection of either platform depending on available resources and clinical needs.
[Results] Under optimized conditions, the nanopore platform achieved a linear detection range of 30 to 2000 ng/mL with a limit of detection of 1.08 ng/mL. The AuNP colorimetric assay showed a linear range of 30 to 1000 ng/mL and a limit of detection of 1.34 ng/mL. The assay exhibits robust signal discrimination driven by modulation of RCA activity and maintains reliable performance in protein rich sample matrices.
[Consideration] Beyond analytical sensitivity, the assay was evaluated for robustness and practical applicability in biologically relevant conditions. The antibody free architecture improves reagent stability and removes dependence on cold chain storage. The dual platform design allows flexible selection between rapid screening and quantitative analysis, depending on available resources and clinical requirements.
[Conclusion]Overall, this study establishes not merely a biosensor but a comprehensive diagnostic solution that directly addresses key limitations of current CKD testing, including high cost, antibody dependence, and limited accessibility. The modular and antibody free design provides a versatile framework for Cystatin C quantification and can be readily extended to other clinically relevant protein biomarkers through aptamer redesign, offering strong potential for point of care and decentralized diagnostic applications.
[Method] We developed an integrated, antibody free diagnostic workflow for Cystatin C detection that combines a gold nanoparticle (AuNP) colorimetric screening assay with a nanopore based quantitative platform. This design allows flexible implementation across different diagnostic scenarios. The core of the system is a nucleic acid aptamer with high affinity and specificity toward Cystatin C, coupled with isothermal rolling circle amplification (RCA). The assay operates through a mechanism in which the presence of Cystatin C suppresses RCA initiation. When Cystatin C is absent, the aptamer primer hybridizes with a padlock probe and triggers RCA, producing long single stranded DNA concatemers that generate frequent nanopore translocation events. In contrast, binding of Cystatin C sequesters the aptamer primer, inhibits padlock probe circularization, and leads to a pronounced reduction in nanopore signal events. To address the time and infrastructure requirements associated with nanopore analysis, an AuNP colorimetric assay is incorporated as a rapid visual screening module that requires no instrumentation. This dual platform architecture supports two practical use modes: rapid prescreening using the AuNP assay followed by precise nanopore based quantification, or independent selection of either platform depending on available resources and clinical needs.
[Results] Under optimized conditions, the nanopore platform achieved a linear detection range of 30 to 2000 ng/mL with a limit of detection of 1.08 ng/mL. The AuNP colorimetric assay showed a linear range of 30 to 1000 ng/mL and a limit of detection of 1.34 ng/mL. The assay exhibits robust signal discrimination driven by modulation of RCA activity and maintains reliable performance in protein rich sample matrices.
[Consideration] Beyond analytical sensitivity, the assay was evaluated for robustness and practical applicability in biologically relevant conditions. The antibody free architecture improves reagent stability and removes dependence on cold chain storage. The dual platform design allows flexible selection between rapid screening and quantitative analysis, depending on available resources and clinical requirements.
[Conclusion]Overall, this study establishes not merely a biosensor but a comprehensive diagnostic solution that directly addresses key limitations of current CKD testing, including high cost, antibody dependence, and limited accessibility. The modular and antibody free design provides a versatile framework for Cystatin C quantification and can be readily extended to other clinically relevant protein biomarkers through aptamer redesign, offering strong potential for point of care and decentralized diagnostic applications.
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