(K-401) Microfluidics Technology for Analysis of Cell Surface Biomarkers

Mass Spectrometry (MS) has greatly helped in the studying of membrane proteins in eukaryotic cells, enabling researchers to gain insights into the complex mechanisms of cell surface biomarkers [1]. However, one persistent challenge in MS analysis of plasma membrane proteins is the much lower number of membrane proteins compared to intracellular proteins, which often leads to reduced accuracy and reliability of membrane protein detection [2].

To overcome this sensitivity limitation and improve the efficacy of MS analysis, we sought after strategies to enhance the detection of cell surface proteins while minimizing interference from intracellular components. We investigated the concept of cytoplasmic expulsion as a potential method to achieve this goal. By removing intracellular contents and isolating the membrane bound moieties, we aimed to enhance the detection of cell surface proteins by mass spectrometry.

The motivation for leveraging intracellular drug delivery methods as a basis for our approach emerged from the successful application of mechanoporation, microinjection, and hydroporation in delivering molecular compounds into cells with minimal damage to the plasma membrane. Recognizing the functionality of these techniques, we hypothesized that a similar approach could be employed “in reverse” to effectively remove the inner contents of cells, leading to improved detection of cell surface proteins by MS. In this study, we design a microfabricated device for cytoplasmic expulsion and membrane protein isolation for subsequent analysis by the electrospray ionization mass spectrometry (ESI-MS)

The hybrid device integrates mechanoporation and microinjection techniques, adopting a simplified approach in which cells are introduced at an inlet tube and propelled through a narrowing canal (Figure 1A and 1B). To achieve precise cell alignment, a microneedle situated at the channel's end gently guides cells towards its point, creating flow that leads to inertial ordering that forms a stream of individual cells (Figure 1B and 1C). This precise alignment enables the microneedle precise positioning of the cell at the end of the tapered channel to pierce and extract individual cell contents (Figure 1C).

To fabricate this device, we utilized soft photolithography on the Heidelberg MLA150 to etch channels into a photoresist layer. For optimal biocompatibility, flexibility, and transparency, Polydimethylsiloxane (PDMS) was selected as the primary material.

Each channel was designed with varying converging angles, effectively regulating the rate of cell compression observed in Figure 1C. Negative molds were created on a wafer using photoresist, and PDMS positive molds were generated by pouring PDMS over them, as illustrated in Figure 2C. After hardening, the layers forming the device (Figure 2C) were carefully assembled and bonded. Insertion of inlet tubes and micro-needles for each channel, confirmed with thorough visual analysis using a digital microscope, completed the device for performance evaluation.

The successful creation of the microfluidic device was achieved through a series of iterative improvements. Notably, adjustments were made to position the channels closer to the device edges, enabling easier microneedle insertion through the PDMS into the channel. Additionally, the introduction of a PDMS slab with guiding holes on top of the channel layer further enhanced the device's assembly. Looking ahead, the microfluidic device is now ready for testing with cells to assess its efficacy and performance, aiming to realize its potential for improving the detection of cell surface biomarkers for ESI-MS.

Throughout the device development process, specific challenges persisted, including difficulties in visualizing cells within the capillary when the microneedle were present. The biggest challenge was surmounted in visualization, i.e., developing the capability for cells became clearly visible under closer observation on a microscope slide loaded with cell suspension. The planar design was selected to avoid the light distorting curved interfaces, and PDMS was selected as the device material to minimize the refractive index mismatch between the system constituents, including water-based sample solution and glass substrate. This material and design choices ensured enhanced cell visualization within the microfluidic device.

In conclusion, the successful development of the microfluidic device, along with the insights gained from the results and discussions, establishes a solid foundation for further testing and modifications to the device. These ongoing efforts aim to propel the device's capabilities and its application in detecting cell surface biomarkers via ESI-MS.