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Optical heterogeneous bioassay for the detection of the inflammatory biomarker suPAR

机译:光学异质生物测定法检测炎性生物标志物suPAR

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Soluble urokinase plasminogen activator receptor (suPAR) is an inflammatory protein present in blood and a marker of disease presence, severity and prognosis. A heterogeneous sandwich assay is proposed for quantifying suPAR by employing a capture antibody from rat and a biotinylated detection antibody from mouse. Optical detection was achieved by a successive exposure of the biotinylated sandwich to streptavidin labelled with ATTO647N. The heterogeneous assay was implemented on a multichannel polymethylmetacrylate (PMMA) optical biochip, potentially capable of the simultaneous detection of more than one analyte. Capture antibody was immobilized on the PMMA surface of the microfluidic channel and the assay was performed with the following protocol: ⅰ) surface blocking with BSA, ⅱ) incubation with suPAR or PBS, ⅲ) incubation with biotinylated suPAR detection Ab and iv) incubation with streptavidin-ATTO647N. Promising preliminary results were obtained with this protocol. Moreover, an improved optical setup is proposed which avoids the mechanical scanning of the chip and consequently the in-series fluorescence excitation and read out, allowing the simultaneous measurement of the fluorescence on all the channels of the microfluidic chip.
机译:可溶性尿激酶纤溶酶原激活剂受体(suPAR)是血液中存在的一种炎症蛋白,是疾病存在,严重程度和预后的标志。提出了一种异质夹心测定法,其通过使用来自大鼠的捕获抗体和来自小鼠的生物素化的检测抗体来定量suPAR。通过将生物素化的三明治连续暴露于用ATTO647N标记的抗生蛋白链菌素来实现光学检测。异质分析是在多通道聚甲基丙烯酸甲酯(PMMA)光学生物芯片上实施的,可能能够同时检测一种以上的分析物。将捕获抗体固定在微流体通道的PMMA表面上,并按以下规程进行测定:ⅰ)用BSA阻断表面,ⅱ)用suPAR或PBS孵育,ⅲ)用生物素化的suPAR检测抗体孵育Ab和iv)用链霉亲和素-ATTO647N。该协议获得了有希望的初步结果。此外,提出了一种改进的光学装置,其避免了芯片的机械扫描,因此避免了串联荧光激发和读出,从而允许同时测量微流控芯片所有通道上的荧光。

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