2025-09-01 理化学研究所,東京都健康長寿医療センター
図1 バイオマーカー酵素の1分子デジタルSERS計数法の仕組み
(A)マイクロチップ内における酵素反応生成物のSERS信号発生メカニズム
微小試験管の底面には銀ナノ粒子の凝集体が固定化されている。生成物が銀ナノ粒子の表面に吸着すると、その表面増強効果によって、生成物由来のSERS信号が検出される。
(B)酵素(アセチルコリンエステラーゼ)を標的とした1分子デジタルSERS計数法の実施例
<関連情報>
- https://www.riken.jp/press/2025/20250902_1/index.html
- https://www.pnas.org/doi/10.1073/pnas.2510559122
単一酵素バイオマーカーのデジタルSERS生体分析 Digital SERS bioanalysis of single-enzyme biomarkers
Jun Ando, Kazue Murai, Tomoe Michiyuki, +7 , and Rikiya Watanabe
Proceedings of the National Academy of Sciences Published:September 2, 2025
DOI:https://doi.org/10.1073/pnas.2510559122
Significance
Single-enzyme reactions in digital bioanalysis have been observed using bright fluorogenic substrates encapsulated in microchambers, but their broad emission spectrum has limited molecular selectivity and multiplexing capability. Raman scattering spectroscopy provides molecular fingerprints with sharp and distinct peaks, but low sensitivity has hindered its application to digital bioanalysis. Here, we developed plasmonically active, uniform microchamber arrays, enabling highly sensitive, molecular-selective, and multiplexed detection of single-enzyme reactions using surface-enhanced Raman scattering (SERS) spectroscopy. SERS-based digital bioanalysis enabled precise quantification of acetylcholinesterase in cerebrospinal fluid, demonstrating its clinical potential for type-specific dementia diagnosis based on minute enzyme-level differences.
Abstract
Digital bioanalysis enables highly sensitive detection of biomolecules at the single-molecule level, making it a widely used technique in biomedical research. However, conventional approaches typically rely on fluorescence detection of single-enzyme reactions, which limits molecular selectivity and the ability to analyze multiple targets simultaneously. To address these limitations, we developed a digital bioanalysis platform based on surface-enhanced Raman scattering spectroscopy and microchamber arrays decorated with silver nanoparticles. This platform achieves a million-fold amplification of Raman signals from products generated by single-enzyme reactions, enabling precise digital counting of enzyme biomarkers with high molecular selectivity and multiplexing capability. We applied this platform to detect and distinguish two closely related enzyme biomarkers, acetylcholinesterase (AChE) and butyrylcholinesterase. By leveraging the sharp and distinct Raman spectral signatures of the reaction products, the platform achieved multiplexed biomarker quantification with femtomolar-level sensitivity. As a proof-of-concept, the platform successfully quantified AChE in human cerebrospinal fluid within 8.5 min, highlighting its potential utility in clinical diagnostics, particularly for differentiating types of dementia based on subtle differences in enzyme levels. Hence, this study presents a valuable alternative to fluorescence-based digital bioanalysis by offering enhanced molecular selectivity and multiplexing capability. Its application extends the scope of digital bioanalysis and broadens its capacity to quantify multiple biomarkers in complex biological samples with high precision and efficiency.
