Department of Developmental BioEngineering, MIRA Institute for Biomedical Technology and Technical Medicine , University of Twente , Enschede , 7522 NB , The Netherlands.
Medical Cell Biophysics, MIRA Institute for Biomedical Technology and Technical Medicine , University of Twente , Enschede , 7522 NB , The Netherlands.
Anal Chem. 2018 Jun 5;90(11):6563-6571. doi: 10.1021/acs.analchem.8b00260. Epub 2018 May 15.
There is a large unmet need for reliable biomarker measurement systems for clinical application. Such systems should meet challenging requirements for large scale use, including a large dynamic detection range, multiplexing capacity, and both high specificity and sensitivity. More importantly, these requirements need to apply to complex biological samples, which require extensive quality control. In this paper, we present the development of an enhancement detection cascade for surface plasmon resonance imaging (SPRi). The cascade applies an antibody sandwich assay, followed by neutravidin and a gold nanoparticle enhancement for quantitative biomarker measurements in small volumes of complex fluids. We present a feasibility study both in simple buffers and in spiked equine synovial fluid with four cytokines, IL-1β, IL-6, IFN-γ, and TNF-α. Our enhancement cascade leads to an antibody dependent improvement in sensitivity up to 40 000 times, resulting in a limit of detection as low as 50 fg/mL and a dynamic detection range of more than 7 logs. Additionally, measurements at these low concentrations are highly reliable with intra- and interassay CVs between 2% and 20%. We subsequently showed this assay is suitable for multiplex measurements with good specificity and limited cross-reactivity. Moreover, we demonstrated robust detection of IL-6 and IL-1β in spiked undiluted equine synovial fluid with small variation compared to buffer controls. In addition, the availability of real time measurements provides extensive quality control opportunities, essential for clinical applications. Therefore, we consider this method is suitable for broad application in SPRi for multiplex biomarker detection in both research and clinical settings.
临床应用中需要可靠的生物标志物测量系统，但目前这一需求仍未得到满足。这些系统应满足大规模使用的挑战性要求，包括大动态检测范围、多重检测能力、高特异性和灵敏度。更重要的是，这些要求需要适用于复杂的生物样本，这需要广泛的质量控制。在本文中，我们提出了一种用于表面等离子体共振成像（SPRi）的增强检测级联的开发。级联采用抗体夹心测定法，然后使用中性亲和素和金纳米粒子增强，以在小体积的复杂流体中进行定量生物标志物测量。我们在简单的缓冲液和含有四种细胞因子（IL-1β、IL-6、IFN-γ和 TNF-α）的马关节滑液中进行了可行性研究。我们的增强级联导致了抗体依赖性的灵敏度提高了 40000 倍，从而将检测限降低至低至 50 fg/mL，动态检测范围超过 7 个对数。此外，在这些低浓度下的测量具有高度的可靠性，内和间测定 CV 在 2%至 20%之间。我们随后表明，该测定法适用于具有良好特异性和有限交叉反应性的多重测量。此外，我们还证明了该方法在未稀释的马关节滑液中检测到 IL-6 和 IL-1β 的稳健性，与缓冲液对照相比，差异较小。此外，实时测量的可用性提供了广泛的质量控制机会，这对于临床应用至关重要。因此，我们认为该方法适用于 SPRi 中广泛用于研究和临床环境中的多重生物标志物检测。
