Institute for Molecular Bioscience, The University of Queensland, 306 Carmody Road, Brisbane, 4072 QLD, Australia.
Institute for Molecular Bioscience, The University of Queensland, 306 Carmody Road, Brisbane, 4072 QLD, Australia; School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, 4072 QLD, Australia.
Biosens Bioelectron. 2015 May 15;67:769-74. doi: 10.1016/j.bios.2014.09.048. Epub 2014 Sep 28.
Nanoparticles used in biosensor applications often fail when deployed directly in complex biological fluids. This is due to surface fouling and interference from the large concentration of non-specific binders (proteins, lipids, nucleic acids and saccharides) in the matrix. We systematically investigate four orthogonal approaches for decorating nanoparticle surfaces with affinity probes and evaluate their performance in buffer and serum. Carbodiimide coupling, cooper-mediated 'click' coupling, copper-free click coupling and thiol-maleimide coupling were quantitatively controlled during the fabrication process. Analyte mediated aggregation of fluorescent reporters and paramagnetic nanoparticle in a sandwich immunoassay was then used to probe assay sensitivity and specificity using an early biomarker of dengue fever, NS-1, as an exemplar and clinically relevant analyte. The type of surface functionalization played a vital role in assay performance in buffer versus serum at the assay sensitivity limit (3 ng/mL in serum) and over the linearity of response of the assay's dynamic range. There was a 10 fold increase on the dynamic range of the detection of NS1 comparing copper free click coupling to carbodiimide coupling, one of the most common approaches for nanoparticle functionalization. By tuning their size, we could carefully monitor the evolution of nanoparticle populations by flow cytometer and discriminate between unbound and fluorescent nanoparticles. This subtle control on each assay component resulted in more than a 10-fold reduction in fluorescence background and improved the sensitivity of almost two orders of magnitude compared to endpoint measurements.
用于生物传感器应用的纳米粒子在直接部署到复杂的生物流体中时经常失效。这是由于表面污垢和基质中大量非特异性结合物（蛋白质、脂质、核酸和糖）的干扰。我们系统地研究了在纳米粒子表面修饰亲和探针的四种正交方法，并在缓冲液和血清中评估了它们的性能。在制造过程中，对碳二亚胺偶联、协同介导的“点击”偶联、无铜点击偶联和巯基-马来酰亚胺偶联进行了定量控制。然后，使用荧光报告物和顺磁纳米粒子的分析物介导的聚集来探测夹心免疫测定的分析灵敏度和特异性，以登革热的早期生物标志物 NS-1 为例，并使用临床相关分析物。在检测灵敏度极限（血清中 3ng/mL）和测定线性范围内，表面功能化的类型在缓冲液与血清中的测定性能中起着至关重要的作用。与最常用的纳米粒子功能化方法之一碳二亚胺偶联相比，无铜点击偶联使 NS1 的检测动态范围增加了 10 倍。通过调整它们的大小，我们可以通过流式细胞仪仔细监测纳米粒子群体的演变，并区分未结合的和荧光的纳米粒子。对每个测定组件的这种细微控制导致荧光背景降低了 10 倍以上，与终点测量相比，灵敏度提高了近两个数量级。
