Martinez-Perdiguero Josu, Retolaza Aritz, Bujanda Luis, Merino Santos
CIC microGUNE, Arrasate-Mondragón, Spain.
CIC microGUNE, Arrasate-Mondragón, Spain; Micro-NanoFabrication Unit, IK4-Tekniker, Eibar, Spain.
Talanta. 2014 Feb;119:492-7. doi: 10.1016/j.talanta.2013.11.063. Epub 2013 Dec 1.
A simple method for the detection of TNF-alpha protein biomarker in human serum with great sensitivity has been developed using a surface plasmon resonance biosensor. Signal amplification based on a sandwich immunoassay including gold nanoparticles was used. Detection in serum proved to be challenging due to high undesirable non-specific binding to the sensor surface stemming from the matrix nature of the sample. After optimization of the assay parameters and, in the case of serum, of a sample dilution buffer to minimize the non-specific binding, very low limits of detection were achieved: 11.6 pg/mL (211 fM) and 54.4 pg/mL (989 fM) for spiked buffer and human serum respectively. The amplification steps with high affinity biotinylated antibodies and streptavidin-fuctionalized nanoparticles greatly enhanced the signal with the advantage of additional specificity. Due to its simplicity and sensitivity, the immunoassay has proved feasible to be used for detection of low concentration biomarkers in real samples.
利用表面等离子体共振生物传感器开发了一种用于检测人血清中肿瘤坏死因子-α（TNF-α）蛋白生物标志物的简单方法，该方法具有很高的灵敏度。采用了基于包括金纳米颗粒的夹心免疫分析的信号放大技术。由于样品的基质性质导致与传感器表面的高非特异性结合，血清检测具有挑战性。在优化分析参数以及血清样品稀释缓冲液以最小化非特异性结合后，实现了非常低的检测限：加标缓冲液和人血清的检测限分别为11.6 pg/mL（211 fM）和54.4 pg/mL（989 fM）。具有高亲和力的生物素化抗体和链霉亲和素功能化纳米颗粒的放大步骤极大地增强了信号，且具有额外的特异性优势。由于其简单性和灵敏度，该免疫分析已被证明可用于检测实际样品中的低浓度生物标志物。
