Department of Physical and Analytical Chemistry, University of Oviedo, Av. Julián Clavería 8, 33006 Oviedo, Spain.
Sensors (Basel). 2020 Sep 16;20(18):5287. doi: 10.3390/s20185287.
A major challenge in the development of bioanalytical methods is to achieve a rapid and robust quantification of disease biomarkers present at very low concentration levels in complex biological samples. An immunoassay platform is presented herein for ultrasensitive and fast detection of the prostate-specific antigen (PSA), a well-recognized cancer biomarker. A sandwich type immunosensor has been developed employing a detection antibody labeled with inorganic nanoparticles acting as tags for further indirect quantification of the analyte. The required high sensitivity is then achieved through a controlled gold deposition on the nanoparticle surface, carried out after completing the recognition step of the immunoassay, thus effectively amplifying the size of the nanoparticles from nm to µm range. Due to such an amplification procedure, quantification of the biomolecule could be carried out directly on the immunoassay plates using confocal microscopy for measurement of the reflected light produced by gold-enlarged nanostructures. The high specificity of the immunoassay was demonstrated with the addition of a major abundant protein in serum (albumin) at much higher concentrations. An extremely low detection limit for PSA quantification (LOD of 1.1 fg·mL PSA) has been achieved. Such excellent LOD is 2-3 orders of magnitude lower than the clinically relevant PSA levels present in biological samples (4-10 ng·mL) and even to monitor eventual recurrence after clinical treatment of a prostate tumor (0.1 ng·mL). In fact, the broad dynamic range obtained (4 orders of magnitude) would allow the PSA quantification of diverse samples at very different relevant levels.
在生物分析方法的开发中，一个主要的挑战是实现对存在于复杂生物样本中非常低浓度水平的疾病生物标志物的快速、稳健定量。本文提出了一种免疫分析平台，用于超灵敏和快速检测前列腺特异性抗原（PSA），这是一种公认的癌症生物标志物。开发了一种夹心型免疫传感器，使用标记有无机纳米粒子的检测抗体作为标签，用于进一步对分析物进行间接定量。所需的高灵敏度是通过在免疫分析完成后，在纳米粒子表面进行受控的金沉积来实现的，从而有效地将纳米粒子的尺寸从纳米级放大到微米级。由于这种放大过程，可以使用共焦显微镜直接在免疫分析板上进行生物分子的定量，通过测量金放大纳米结构产生的反射光来进行测量。通过在血清中添加浓度高得多的主要丰富蛋白（白蛋白）来证明免疫分析的高特异性。已经实现了 PSA 定量的极低检测限（PSA 的 LOD 为 1.1 fg·mL PSA）。如此优异的 LOD 比生物样本中存在的临床相关 PSA 水平（4-10 ng·mL）低 2-3 个数量级，甚至可以监测前列腺肿瘤临床治疗后的任何复发（0.1 ng·mL）。实际上，获得的宽动态范围（4 个数量级）将允许在非常不同的相关水平下对各种样本进行 PSA 定量。
