C. Drug gradient and its effect on cell metabolic activity

The potential application of concentration gradient is in determining the effect of various drugs/biomolecules on cells. We selected curcumin as the model drug to study its cytotoxic effect on HeLa cells, a commonly used model cell line for cervical cancer. First, the concentration gradient of curcumin in growth media was generated in the fabricated device. The concentration of curcumin at each outlet was determined using absorbance of the solution at 425 nm [Fig. 5(a)]. Using the MTT assay, we then tested the cytotoxicity of curcumin on HeLa cells by measuring the percent inhibition with respect to the drug collected from the device outlets [Fig. 5(b)]. As the graph shows, drug concentration coming from outlet 5 gives the IC50, i.e., inhibitory concentration at which 50% cell are viable/dead. Comparing with Fig. 5(a), the concentration of solution from outlet number 5 is found to be ∼32 μM. Therefore, IC50 of curcumin on HeLa cells is ∼32 μM. To note, our COMSOL simulation estimated that the concentration at outlet 5 is 28 μM which matches well with the experimental measurement [Fig. 4(b)]. Hence, drug concentration at different outlets can be estimated using COMSOL simulation by providing molecular weight and diffusivity of drug as input and thus eliminating the need of experimental measurement of drug concentration every time. Furthermore, to validate the device, IC50 of curcumin on HeLa was estimated using a conventional 96 well plate in which concentration gradient was generated manually by serial dilution. Using this conventional method, IC50 was found to be 34.9 ± 1.7 μM [Fig. 5(c)] which matched well with IC50 estimated from our μCGG device (32 μM). This result indicates that the μCGG can potentially be used for concentration generation in drug testing and drug screening replacing the time consuming, labor intensive, and error-prone method of creating concentration gradients manually.