Fig 2: Concept of the Ab quality verification system and fluorescence images of breast cancer cells and breast cancer tissues.(A) A schematic of the microfluidic immunostaining system and a cell array consisting of SKBR3 and MCF7. SKBR3 and MCF7 were used as positive and negative references for HER2, respectively. (B) Immunostaining results of HER2 Ab: LOT #1 in SKBR3 and MCF7 using a microfluidic system and batch incubation process. The data are mean ± s.d.; n = 3 for chip and n = 3–9 for batch. (C) HER2 Ab: LOT #2 was used as in panel B. (D) Immunostaining results of breast cancer tissues from four different patients using HER2 Abs of LOT#1 and LOT #2. (E) Surface plots based on the intensities of QD605; bottom images of panel D.

Fig 3: Standardization of three HER2 Ab immunostaining and standardized tissue staining results.Three HER2 Abs from different companies (Dako: 1.6 µg mL-1, Abcam: 0.024 µg mL-1, Thermo Fisher Scientific: 0.36 mg mL-1 [for 1.000×]) were randomly labeled as LOTs #1, #2, and, #3, respectively, to ensure anonymity. The chip-based Ab-staining process was carried out for 10 min and the batch process was carried out for 2 h. (A–C) Based on the 1.000× intensity of LOT #2, 0.750× of LOTs #1 and #3 have the most similar intensity. The data are mean ± s.d.; n = 3. Red color shows the QD605-labeled cells in tissue sections and blue color comes from the autofluorescence of tissue sections caused by the excitation of ultraviolet. (D) Sections from the same breast cancer tissues were immunostained with selected concentrations of HER2 Abs. (E) Intensity quantification result of tissue sections with HER2 Abs. The data are mean ± s.d.; n = 3–15. The error bars are based on the QD605 intensity of the imaging spot only from the epithelial region in the same tissue section.

Fig 4: Microfluidic immunostaining system for antibody (Ab) quality verification and standardization.(A) Conventional immunostaining standardization process flow. A biomarker-specific cell or tissue sample that stained with optimized intensity is stored as a reference. When a new Ab is introduced, repetitive immunostaining is performed to subjectively match the staining intensity to the reference. (B) A positive control cell block section is immunostained with linearly graded concentrations of Ab. The staining intensity of the reference is compared with those resulting from Ab incubation at various Ab concentrations to identify the best match. (C) Images of the microchannels and cell sections resulting from the concentration gradient generating network of the microfluidic immunostaining system. Fluorescein isothiocyanate (FITC) solution was introduced into the inlet with distilled water. (D) A SKBR3 cell section was stained with graded concentrations of HER2 Ab and labeled with QD605 (left) and 3,3'-diaminobenzidine (right).