面向超低浓度生物标记物检测的磁珠富集式光微流激光酶联免疫吸附测定芯片

The rapid and accurate detection of ultra low concentration of biomarkers is critical for early diagnosis of cancer diseases. The enzyme-linked immunosorbent assay (ELISA) is a powerful tool for sensitive biomolecular detection. However, the fluorescence background increases with the decreased concentration of biomarkers, which limits the application of traditional fluorescence ELISA. Optofluidic laser ELISA overcomes the interference of fluorescence background by high intensity laser, rapid growth in laser emission, and accurate laser onset time dertermination. However, the laser onset time is long for the low concentration detection (usually 4-5 hours). We propose to develop a magnetic bead-based optofluidic laser ELISA microfluidic chip that is capable of detecting biomarkers down to sub-femto-gram concentration rapidly. The proposed magnetic bead-based optofluidic laser ELISA chip employs magnetic bead-based instead of solid-phase immunoassays. Compared to the solid-phase immunoassays optofluidic laser ELISA chip, the magnetic bead-based optofluidic laser ELISA chip has many distinct advantages. (1) Magnetic beads not only have the advantages of solid phase immunological reactions, such as high specificity, but can also reduce the detection time and improve the detection sensitivity. In addition, magnetic beads can capture more biomarkers due to the surface-to-volume ratio. Magnetic beads enrichment can increase the local concentration of fluorescent product inside a resonant cavity, which is expected to shorten the laser onset time. (2) Magnetic beads can greatly improve the specific adsorption and reduce the nonspecific adsorption due to full mixing, binding and washing. This is particularly important when we detect biomarkers of extremely low concentrations. (3) The chip can detect a variety of biomarkers by changing the type of immunomagnetic beads. The technology not only can overcome the interference of fluorescence background, but also shorten the laser onset time. The realization of the chip can further improve early detection and diagnosis of major diseases, such as cancer and AIDS, etc.

实现超低浓度生物标记物的快速准确检测对疾病的早期诊断具有非常重要的意义。酶联免疫吸附测定（ELISA）是一种进行生物分子灵敏检测的有力工具。然而背景荧光的干扰随着生物标记物浓度的降低而增强，这限制了传统荧光ELISA的应用。光微流激光ELISA利用高强度激光克服了背景荧光的干扰，但存在着产生激光阈值时间较长的缺陷。本项目提出了一种磁珠富集式光微流激光ELISA芯片，实现亚飞克浓度的生物标记物的快速检测。与固相表面固定抗体的光微流激光ELISA相比：（1）磁珠富集能够增加共振腔局域荧光产物的浓度，有望缩短产生激光的阈值时间；（2）磁珠能够通过更加充分的混合、吸附、洗脱等操作提高特异性吸附并降低非特异性吸附；（3）改变免疫磁珠能够实现多种标记物的检测，芯片能够多次使用。该技术既能够克服背景荧光的干扰，又能够缩短产生激光的阈值时间。该芯片的实现将对癌症、艾滋病等重大疾病的早期诊断带来重要提升。

超低浓度生物标记物的快速准确检测对疾病的早期诊断具有非常重要的意义。酶联免疫吸附测定（ELISA）是一种实现生物标记物的快速准确检测的非常有用的工具。传统荧光ELISA利用荧光强度作为检测信号，但背景荧光的干扰随着生物标记物浓度的降低而增强，因而限制了传统荧光ELISA的应用。光微流激光ELISA利用高强度激光克服了背景荧光的干扰，但存在着产生激光阈值时间较长的缺陷。本项目提出了一种磁珠富集式光微流激光ELISA芯片，主要完成了以下研究内容：（1）在石英基底上利用微加工技术制备了一种具有高品质因子的法布里一伯罗微腔，其品质因子达到了10^5，并探索了一种简单快速在聚苯乙烯基底上制备微结构阵列的方法，为聚合物材料基底上微腔的阵列的快速制备提供一个途径；（2）利用制备的具有微腔的微流控芯片研究了微腔对激光输出特性的影响，并利用此微腔进行了DNA错配的实验，完成了130对碱基的错配检测；（3）利用制备的具有共振微腔的微流控芯片研究了磁珠对激光性能的影响，其结果表明：磁珠的加入提高了产生激光所需的阈值能量，因此后续的的实验需要优化实验中磁珠的加入量；（4）为了提高二氧化硅磁珠对生物标记物的吸附能力，进行了玻璃表面生物改性的研究，研究结果表明：改性后的的玻璃表面具有更强的吸附生物标记物的能力；（5）完成了利用磁珠富集式光微流激光ELISA芯片检测生物标记物的初步实验。该技术既能够克服背景荧光的干扰，又能够缩短产生激光的阈值时间。该芯片的实现将对癌症、艾滋病等重大疾病的早期诊断带来进一步提升。