Department of Materials, Imperial College London, Exhibition Road, London SW7 2AZ, UK.
ACS Nano. 2012 Jan 24;6(1):851-7. doi: 10.1021/nn204361s. Epub 2011 Dec 20.
Nanoparticle-based labels are emerging as simpler and more sensitive alternatives to traditional fluorescent small molecules and radioactive reporters in biomarker assays. The determination of biomarker levels is a recommended clinical practice for the assessment of many diseases, and detection of multiple analytes in a single assay, known as multiplexing, can increase predictive accuracy. While multiplexed detection can also simplify assay procedures and reduce systematic variability, combining multiple assays into a single procedure can lead to complications such as substrate cross-reactivity, signal overlap, and loss of sensitivity. By combining the specificity of biomolecular interactions with the tunability of quantum dot optical properties, we have developed a detection system capable of simultaneous evaluation of the activity of two critical enzyme classes, proteases and kinases. We avoid cross-reactivity and signal overlap by synthesizing enzyme-specific peptide sequences with orthogonal terminal functionalization for attachment to quantum dots with distinct emission spectra. Enzyme activity is reported via binding of either gold nanoparticle-peptide conjugates or FRET acceptor dye-labeled antibodies, which mediate changes in quantum dot emission spectra. To the best of our knowledge, this is the first demonstration of the multiplexed sensing of the activity of two different classes of enzymes via a nanoparticle-based activity assay. Using the quantum dot-based assay described herein, we were able to detect the protease activity of urokinase-type plasminogen activator at concentrations ≥ 50 ng/mL and the kinase activity of human epidermal growth factor receptor 2 at concentrations ≥ 7.5 nM, levels that are clinically relevant for determination of breast cancer prognosis. The modular nature of this assay design allows for the detection of different classes of enzymes simultaneously and represents a generic platform for high-throughput enzyme screening in rapid disease diagnosis and drug discovery.
基于纳米粒子的标签正在成为比传统荧光小分子和放射性报告物更简单、更灵敏的生物标志物检测替代物。生物标志物水平的测定是评估许多疾病的推荐临床实践，而在单个测定中同时检测多种分析物，即多重检测，可以提高预测准确性。虽然多重检测还可以简化测定程序并减少系统变异性，但将多个测定组合成一个单一的程序可能会导致底物交叉反应、信号重叠和灵敏度降低等问题。通过将生物分子相互作用的特异性与量子点光学性质的可调谐性相结合，我们开发了一种能够同时评估两种关键酶类（蛋白酶和激酶）活性的检测系统。我们通过合成具有正交末端官能化的酶特异性肽序列来避免交叉反应和信号重叠，这些肽序列可用于与具有不同发射光谱的量子点结合。通过金纳米粒子-肽缀合物或 FRET 受体染料标记抗体的结合来报告酶活性，这介导了量子点发射光谱的变化。据我们所知，这是首次通过基于纳米粒子的活性测定来对两种不同类别的酶的活性进行多重感测的演示。使用本文所述的基于量子点的测定法，我们能够检测到尿激酶型纤溶酶原激活物的蛋白酶活性，其浓度≥50ng/mL，以及人表皮生长因子受体 2 的激酶活性，其浓度≥7.5nM，这些浓度在确定乳腺癌预后方面具有临床意义。该测定设计的模块化性质允许同时检测不同类别的酶，并且代表了用于快速疾病诊断和药物发现中的高通量酶筛选的通用平台。
