**4. Challenges and future trends**

Isolated cell culture in a confined environment for subsequent secretion detection allows good flexibility in the selection of sensing techniques, but such module-based strategy has not been achieved for real-time detection of individual cell secretion. Meanwhile, the integration of cell culture and secretion detection has shown promising results in real-time monitoring single cell secretion by physically presenting the target cells in close proximity to the sensing region. However, there are still significant challenges and difficulties ahead as indicated below.

1.Cell secretion dynamics are found associated with cell states, therefore, it is important for the sensor systems to precisely quantify the cell secretion. An obstacle for current methodology is that, the molecule secretion and molecule detection are both dynamic process occurring at the same time.

The concentrations of target molecules in solution are constantly changing, such that an equilibrium-state based calibration curves are difficult to obtain. Therefore, on one hand, novel or improved detection methods with more rapidly reached equilibrium state are required to minimize the effect of detection process. On the other hand, a comprehensive dynamic model involving secretion and detection dynamics is necessary to understand this complex process and precisely quantify the cell secretion [101, 102].

