Supplemental information

Biomedical Microdevices

An Integrated Microfluidic Platform for Rapid Tumor Cell Isolation, Counting and Molecular Diagnosis

Lien-Yu Hunga,Ying-Hsin Chuangb, Hsin-Tzu Kuoa, Chih-Hung Wanga,

Keng-Fu Hsuc, Cheng-Yang Chouc and Gwo-Bin Leea*

aDepartment of Power Mechanical Engineering, National Tsing Hua University,Hsinchu, Taiwan 300

bDepartment of Engineering Science, National Cheng Kung University, Tainan, Taiwan 701

cDepartment of Obstetrics and Gynecology, National Cheng Kung University, Tainan, Taiwan 701

  1. Mixing efficiency of the new moving-wall micro mixer

The optimal driving frequency of the micro incubator wasexplored by testing four different driving frequencies (0.5, 1.0, 1.5 and 2.0 Hz) of electromagnetic valves (EMVs) and their corresponding mixing indices (as shown in Supplemental Figure 1).The incubation time ranging from 5 to 10 minuteswere investigatedandthe mixing indiceswere measured accordingly. A driving frequency of 0.5 Hz showed the optimal mixing indexfor incubation of 10 minutes.

Supplemental Figure 1The mixing index under four different driving frequencies.

  1. Antibody coating testing

The labeling ratio betweenantibody and magnetic beads was measured byusing Alexa Fluor® 488 Goat Anti-Mouse IgG (Invitrogen, Life Technologies Corp., USA.), which could recognize the IgG from mouse, with flow cytometer analysis. We used six different amounts (0.5, 1.0, 2.0, 4.0, 8.0, 16.0 μg)of Ber-EP4 mAbs for 200-μL magnetic beads(4 x 108beads/mL) coating. Then Alexa Fluor® 488 Goat Anti-Mouse IgG was used for recognizing Ber-EP4 mAbs on the beads (as shown in Supplemental Figure 2(a)). After flow cytometer analysis, we compared six testing results and one beads-only sample in 1x PBS buffer as negative control (as shown in Supplemental Figure 2(b)). We did not calculate the absolute coating percentage but used relative percentage obtained from the flow cytometer analysis program with M1 (marker 1) and M2 (marker 2). While the beads were coatedwith Ber-EP4 mAbs, the fluorescence intensity would shift from M1 to M2 and the M2% would be increased,as shown in Supplemental Figure 2(c). Ata Ber-EP4 mAbsconcentration of 4.0 μg/200μL, the fluorescent signal reached saturation, thus indicating that 4.0 μg/200μL of Ber-EP4 mAbs was an optimal value for beads coating.

Supplemental Figure 2: The binding tests of Ber-EP4 antibody on magnetic beads. (a), Illustration of Alexa Fluor® 488 Goat Anti-Mouse IgG recognize the Ber-EP4 mAbs on the beads. (b), Flow cytometer analysis of beads coated with different concentration of Ber-EP4 mAbs. (c), Relative marker 2 (M2) percentages of beads coated with different concentration of Ber-EP4 mAbs.

  1. Pathological diagnosis of ascites specimens from cytospin-slides

This pathological analysisofthe cell composition of the ascites samples was from preserved frozensamples.The information may not directly reflect the composition ofthe fresh ascites fluid orthe true stage of the cancer in the patients. Supplemental Table 1 listspathological diagnosis of ovarian tumor cells in the ascites specimens.

Briefly, ascites 1 sample was analyzed tocontain about 60% TCs. The other 40% of the cells were identified as being 10% mesothelium cells and the remainderswere phagocytic cells such as macrophages and polymorphonuclear leukocytes (PMNs), which were caused by a self-protective acute inflammatory response. Furthermore, pathological diagnosis also showed that about 90% of the bead-captured cells wereTCs and there were still manyTCsthat were not captured, about 50%in the supernatant.

Ascites 2 sample was diagnosed to contain more than 90% TCs. No other cell types appeared significantly, includingonly a few cells related tothe inflammatory response. The bead-captured cells were also about90% TCs but more TCs were not captured in the supernatant, about 85%.

Ascites 3 sample was diagnosed to contain no tumor cells. Only 1% of the cells were mesothelium cells and the remainders were phagocytic cells such as macrophages and PMNs. The few captured cells were mostly macrophages. The supernatantcontained mesothelium cells and phagocytic cells.

Supplemental Table 1.Pathological diagnosisofovarian tumor cellsinthe ascites specimens

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