Department of Chemical and Biomedical Engineering, University of Missouri, Columbia, MO 65211, USA.
Department of Electrical and Computer Engineering, University of Denver, Denver, CO 80210, USA.
Int J Mol Sci. 2024 Aug 7;25(16):8603. doi: 10.3390/ijms25168603.
Sensitive, accurate, and early detection of biomarkers is essential for prompt response to medical decisions for saving lives. Some infectious diseases are deadly even in small quantities and require early detection for patients and public health. The scarcity of these biomarkers necessitates signal amplification before diagnosis. Recently, we demonstrated single-molecule-level detection of tuberculosis biomarker, lipoarabinomannan, from patient urine using silver plasmonic gratings with thin plasma-activated alumina. While powerful, biomarker binding density was limited by the surface density of plasma-activated carbonyl groups, that degraded quickly, resulting in immediate use requirement after plasma activation. Therefore, development of stable high density binding surfaces such as high binding polystyrene is essential to improving shelf-life, reducing binding protocol complexity, and expanding to a wider range of applications. However, any layers topping the plasmonic grating must be ultra-thin (<10 nm) for the plasmonic enhancement of adjacent signals. Furthermore, fabricating thin polystyrene layers over alumina is nontrivial because of poor adhesion between polystyrene and alumina. Herein, we present the development of a stable, ultra-thin polystyrene layer on the gratings, which demonstrated 63.8 times brighter fluorescence compared to commercial polystyrene wellplates. Spike protein was examined for COVID-19 demonstrating the single-molecule counting capability of the hybrid polystyrene-plasmonic gratings.
灵敏、准确和早期检测生物标志物对于及时做出医疗决策以挽救生命至关重要。一些传染病即使数量很少也可能致命，因此需要对患者和公众健康进行早期检测。这些生物标志物的稀缺性需要在诊断前进行信号放大。最近，我们使用带有薄等离子体激活氧化铝的银等离子体光栅，从患者尿液中检测到结核生物标志物脂阿拉伯甘露聚糖，实现了单分子水平的检测。虽然这种方法很强大，但由于等离子体激活的羰基基团的表面密度有限，且这些基团很快降解，因此在等离子体激活后立即需要使用，导致生物标志物的结合密度受到限制。因此，开发稳定的高密度结合表面，如高结合聚苯乙烯，对于提高保质期、降低结合方案的复杂性以及扩展到更广泛的应用范围至关重要。然而，任何位于等离子体光栅顶部的层都必须非常薄（<10nm），以增强相邻信号的等离子体增强。此外，由于聚苯乙烯和氧化铝之间的附着力差，在氧化铝上制造薄的聚苯乙烯层并非易事。在此，我们提出了在光栅上开发稳定的超薄聚苯乙烯层的方法，与商业聚苯乙烯微孔板相比，该方法的荧光强度提高了 63.8 倍。我们还检测了冠状病毒 spike 蛋白，展示了杂交聚苯乙烯-等离子体光栅的单分子计数能力。
