(J-380) A CMOS-Based Integrated Circuit for Rapid and Real-Time Differentiation of Macrophage Activation State

Macrophages, a vital component of the innate immune response, play a crucial role in defending against infections and injuries. Upon stimulation, they polarize into two distinct phenotypes: M1, releasing pro-inflammatory cytokines to combat foreign microbes, cancer cells, and debris, and M2, releasing pro-healing cytokines to promote cell growth. However, pathogens can exploit the M2 phenotype for their long-term colonization, highlighting the importance of understanding macrophage phenotype changes upon stimulation for unraveling host-microbe interactions, pathology, and drug efficacy.

Currently, common assays used to confirm macrophage polarization, such as ELISA, qPCR, and flow cytometry, require binding samples with cytokine-specific reagents and hours of reaction time. Therefore, there is a pressing need for a cost-effective and labor-efficient assay capable of providing rapid indications of macrophage phenotypes.

To address this challenge, we have developed a novel CMOS-based integrated circuit employing capacitance sensors to detect the total dielectric properties of proteins in the media, which are dependent on macrophage metabolism. Our setup demonstrates the capability to accurately differentiate macrophage activation states with high efficiency. Moreover, we propose a real-time cell culture setup that can continuously track cell activation, allowing dynamic monitoring of macrophage phenotype changes.

Our CMOS-based integrated circuit presents an advancement in the study of macrophage biology and immune responses, giving new approaches for understanding host-pathogen interactions and improving drug development.

1. Integrated Capacitance Sensing Chip:

Our integrated capacitance sensing chip is designed to detect macrophage activations by monitoring changes in the dielectric properties of the culture media. The sensor employs a ring oscillator that exhibits increased vibration as media concentration rises, resulting in a corresponding decrease in capacitance readout. When changes occur in the media content, either caused by cell proliferation or activation, variations in protein and ion concentrations are digitized into frequency changes.

2. Hardware Setup:

The experiments are conducted within a standard incubation environment set at 37°C with 5% CO2. A microsystem is mounted on a printed circuit board, housing the CMOS chip at its center for media protein detection. Murine RAW 264.7 cells are cultivated on a diffusion membrane with a pore size of 0.4 um, effectively blocking the cells while enabling secreted factors to reach the CMOS sensor beneath. To minimize evaporation, the wells containing the cells and media are securely capped.

For signal transduction, the PCB  is connected to a motherboard that includes a microcontroller and additional ancillary circuits dedicated to capacitance data collection and system control.

3. Cell Culture and analysis:

Macrophages are cultured in DMEM with 10% FBS over a period of 48 hours. To induce macrophage polarization, stimulants such as IFN-gamma and IL-4 are added at the beginning of each trial.

After the incubation, the cell media are rapidly frozen at -70°C to facilitate subsequent secretome analysis. Furthermore, cells are harvested to confirm their polarization state.

Result
1. General protein assay on chip

Bovine serum albumin (BSA) is commonly used as a protein concentration standard in biology assays. A titration of BSA recording on chip shows that increasing in protein concentration results in decrease in capacitance reading. So far from preliminary results, our limit of detection is < 100 ug/ml.

2. Capacitance Sensing and Secretome Analysis

The integrated capacitance sensing chip recorded a significant deviation in capacitance readings for M1-polarized cells, followed by M2-polarized cells. In comparison, the untreated DMEM (cell culture media) displayed the highest capacitance reading, indicating the absence of secretome factors.

3. Confirmation of Polarization:

The observed differences in capacitance readings were further verified through the BCA assay, a general protein test. The M1-polarized group showed the highest protein production, followed by the M2-polarized group, then the untreated group.

These results demonstrate the successful induction of M1 and M2 macrophage polarization and highlight the sensitivity of our capacitance sensing chip in detecting changes in the secretome composition associated with different macrophage activation states.

4. Real time cell polarization on chip

To monitor macrophage polarization in real-time, cells were cultured on a membrane above the CMOS chip for 48 hours while recording capacitance signals. The IFN-gamma treated group displayed a notable downward slope at time = 30 hr, indicating an increase in protein secretion, consistent with M1 polarization. The delay in CMOS detection from time = 0 hr, when the stimulant was added, is attributed to the time required for protein diffusion, which takes several hours to occur.

Conclusions

In conclusion, our CMOS chip proved to be an effective tool for differentiating between M1, M2, and the original macrophage phenotypes. Through real-time capacitance signal recordings, we were able to track and distinguish the distinct polarization states of macrophages.

Discussion

Our future work will focus on being target-specific. By incorporating antibodies that selectively bind to key biomarkers associated with distinct macrophage phenotypes, we seek to improve the chip's ability to confidently predict cell phenotype changes. This targeted approach will enable us to distinguish between various macrophage activation states with heightened accuracy and sensitivity.