Takeuchi Makoto, Nagasaka Kazunori, Yoshida Mina, Kawata Yoshiko, Miyagawa Yuko, Tago Saori, Hiraike Haruko, Wada-Hiraike Osamu, Oda Katsutoshi, Osuga Yutaka, Fujii Tomoyuki, Ayabe Takuya, Kim Soo Hyeon, Fujii Teruo
Department of Obstetrics and Gynecology, Teikyo University School of Medicine, Tokyo 173-8605, Japan.
Institute of Industrial Science, University of Tokyo, Tokyo 153-8505, Japan.
Biomicrofluidics. 2019 Jul 30;13(4):044107. doi: 10.1063/1.5089796. eCollection 2019 Jul.
Several specific tests for cervical screening have been developed recently, including p16/Ki67 dual immunostaining for diagnosing high-risk human papillomavirus positive squamous intraepithelial lesion in the cervix. However, manual screening of cells in an entire glass slide is currently a standard clinical procedure for quantification and interpretation of immunocytochemical features of the cells. Here, we developed a microfluidic device containing an electroactive microwell array with barriers (EMAB) for highly efficient single-cell trapping followed by on-chip immunofluorescence analysis with minimum loss of the sample. EMAB utilizes patterned electrodes at the bottom of cell-sized microwells to trap single cells using dielectrophoresis (DEP) and cell-holding structures behind the microwells to stabilize the position of trapped cells even without DEP. Using the device, we evaluated the performance of p16/Ki67 dual immunostaining of HeLa cells on the chip. The device shows 98% cell-trapping efficiency as well as 92% cell-holding efficiency against the fixed HeLa cells, and we successfully demonstrated high-efficiency on-chip immunofluorescence analysis with minimal loss of sample. p16/Ki67 dual immunostaining using EMAB may be useful for complementary tests for cervical screening in confirming the histopathological diagnosis.
近年来已开发出几种用于宫颈癌筛查的特定检测方法，包括用于诊断宫颈高危人乳头瘤病毒阳性鳞状上皮内病变的p16/Ki67双重免疫染色。然而，目前在整个载玻片上手动筛查细胞是对细胞免疫细胞化学特征进行定量和解读的标准临床程序。在此，我们开发了一种微流控装置，其包含带有屏障的电活性微孔阵列（EMAB），用于高效捕获单细胞，随后进行芯片上的免疫荧光分析，且样本损失最小。EMAB利用细胞大小的微孔底部的图案化电极通过介电泳（DEP）捕获单细胞，并利用微孔后面的细胞固定结构即使在没有DEP的情况下也能稳定捕获细胞的位置。使用该装置，我们评估了芯片上HeLa细胞p16/Ki67双重免疫染色的性能。该装置对固定的HeLa细胞显示出98%的细胞捕获效率以及92%的细胞固定效率，并且我们成功地展示了具有最小样本损失的高效芯片上免疫荧光分析。使用EMAB进行p16/Ki67双重免疫染色可能有助于在确认组织病理学诊断时对宫颈癌筛查进行补充检测。
