Microfluidic On-Chip Cytotoxicity Assay

Figure 1: 3D Rendering of the on-chip assay ©ankitkumar

The whole history of urban life, in a sense,” says Richard Barnett, “is of living with parasites and trying to get rid of them.” There is an ongoing fear – the sense of cities as pregnable entities, susceptible to bacterial invaders, to infections that enter through a port and spread quickly through a population that is living at perilously close quarters. Over the last decades, these fears have flared with the SARS virus, avian flu, and swine flu, each outbreak prompting new concerns about our ability to prevent and treat disease, at a time of greatly increased drug resistance.

In times of such outbreaks, there arises a need to discover drugs, antidotes, and vaccines to control the spread of the associated pathogen as well as treat the affected ones. One of the current method of testing a potential drug in the laboratory is to perform an MTT Assay post drug-treatment for its cytotoxicity assessment. MTT assay is conventionally done on well-plates and the absorbance is measured using a standard spectrophotometer.

The MTT Cell Proliferation Assay measures the cell proliferation rate and conversely when metabolic events lead to apoptosis or necrosis, the reduction in cell viability. The MTT Reagent yields low background absorbance values in the absence of cells. For each cell type, the linear relationship between cell number and signal produced is established, thus allowing accurate quantification of changes in the rate of cell proliferation.

Figure 2: Labelled diagram of the on-chip assay ©ankitkumar

The current device (Fig. 2) is an on-chip absorbance measurement platform, which enables sensitive, yet point-of-care assays by eliminating the need of benchtop spectrophotometers. A combination of narrow spectrum Surface Mount Device (SMD) type LEDs and broad spectrum photodiodes are used for absorbance measurement and the data is processed on-chip. We have tested this approach by performing quantitative assessment of MCF-7 (breast cancer cell-line) viablility following treatment with varying concentrations of Tamoxifen- a drug known to be cytotoxic for breast cancer cells. This device was able to reproduce the viability data from a spectrophotometer, with Tamoxifen concentration varying from 0.2 uM to 20uM (Fig. 3). In addition, the device was seeded with DU145- prostate cancer cell line and cells were stained with FDA. Cells took up the stain nicely showing that they were indeed viable after 24 hours of culture (Fig. 4a,b) showing that the device itself is not cytotoxic for cells.

Figure 3a: Linearity of Output Response for Varying Concentration of Crystal Violet Dye
Figure 3b: Comparison of MTT assay measurement on-chip v/s spectrophotometer ©ankitkumar

Figure 4a: DU145 cell line growing in the device imaged after 24hrs, brightfield image.
Figure 4b: DU145 cell line growing in the device imaged after 24hrs, FDA live/dead stain.

Patent Filed: Kumar, A, Das, N, Roy, P, Satapathi, S & Indian Institute of Technology Roorkee 2019,
“Cell Cytotoxicity Assay Device and Method of Manufacturing”, IN 201911036596.