Microfluidic immunoassay platform for quantitative comparison of biomarkers on cancer tissues

Abstract

This thesis presents the development of quantitative biomarker analysis technology which can be applied to both of cells and patient tissues in a manner of highly efficient multiplexing. In particular, this development was concentrated on immunochemistry because of its importance in cancer diagnosis. For the achievement of maximized biomarker multiplexity, a microfluidic device which has bundle of microfluidic channels was used with the reversible bonding strategy. This parallel multiplexing strategy was demonstrated with simultaneous detection of eight proteins in breast cancer cells and tissues. A new method of biomarker quantification based on co-staining of loading control (LC) protein in each microchannel area was proposed to reduce staining quality variations among the specimens and to compensate the variation of cell number and volume. Also, multi-concentration antibody (Ab) incubator was developed to reproduce Ab staining quality which is important for the standardization of quantitative assay in immunohistochemistry. We demonstrated this quantum dot-based microfluidic multiple biomarker quantification (QD-MMBQ) method on cancer cells with utilization of β-actin as a LC. LC-based quantification showed the compensation effect of cell number and staining quality variation among specimens. β-actin normalization method was also demonstrated with clinical samples and enabled the quantification of multiple biomarkers. We also demonstrated that microfluidic blocking showed superior and controllable blocking effect compared to the conventional static incubation method. LC-based biomarker quantification was conducted on breast cancer tissues with cytokeratin, which is specialized LC for cancer tissues. To obtain the biomarker expressions only from the cancerous cells, the multiple channels were aligned onto the tissue area where epithelial cells are densely populated that is determined by previous cytokeratin staining. Three important breast cancer biomarkers were quantified with QD-based spectroscopic quantification system. The measured multiple biomarker expression of ten breast cancer patients was accurately correlated with the conventional scoring results which was previously determined by pathologists. This shows that our method can be compatible with conventional scoring method. Also, this method can be used for the compensation of cell number and staining quality variation among specimens as conducted in cell samples. A novel autofluorescence (AF)-removal algorithm was developed, which normalizes the reference AF spectra reconstructed from unknown AF spectra based on random sampling. For accurate quantification of QDs, we automatically and accurately removed the AF signal from 344 spots of QD-labeled tissue samples using 240 reference AF spectra. Using analytical data with ten tissue samples from breast cancer patients, the measured biomarker intensities were in good agreement with the results of conventional analyses. We developed a novel Ab quality control system by using a microfluidics technology. A microfluidic multi-concentration Ab incubator was exploited to incubate Ab at linearly diluted several concentration points of Ab. For the linear dilution of Ab, a new design of microfluidic network structure is introduced which has simple and easy design but generates stable target concentration gradient by modifying the Christmas fluidic network. By observation of the Ab labeling tendency using a microfluidic multi-concentration Ab incubator, we found out that the staining intensity of batch Ab incubation results can be reproduced by our microfluidic system at various range of concentrations. This means that our system can be used for the quality validation of Ab in real field of pathology laboratories. We also designed a special cell microarray (CMA) for the efficient use of the multi-concentration Ab incubator. CMA contains representative cell lines of breast cancer, so positive and negative control of major breast cancer biomarkers can be screened simultaneously using CMA sections. Three HER2 Abs from different Ab producing companies were validated with our system by combining a multi-concentration Ab incubator and CMA sections. After the selection of the concentration point in which resulted in the most similar intensities of three Abs staining using a microfluidic device, 2 h batch Ab incubation was processed at selected concentration points for each Ab. The results showed that similar intensities can be reproduced in both of cells and human tissues.