I. Core Research Summary

• The pattern is plasma‑resistant and compatible with the entire microfluidic sealing process.

After a 30‑second oxygen plasma sealing treatment, the PEDOT:PSS thin film prepared via aerosol deposition exhibits only a 27.5% reduction in thickness, with no significant degradation of its molecular backbone. Its electrical conductivity decreases slightly but remains at 340 S/cm, and it retains its bioadhesive properties, thereby addressing the critical issue of conventional protein coatings losing activity upon exposure to plasma.

• Dual synergistic induction of directional endothelial cell alignment

PEDOT:PSS provides a substrate with high cell adhesion, and when combined with cavity‑based capillary self‑flow, it generates low‑shear‑stress fluid flow. When the alignment lines are parallel to the flow direction, over 60% of cells grow along the guidance stripes after 7 days of culture, whereas under static conditions only 31% remain within the patterned regions. In contrast, when the lines are perpendicular to the flow, cellular order is markedly reduced, confirming a dual regulatory mechanism involving both topography and fluid dynamics.

• Long-term cell‑stable culture, with adhesion resistant to fluid shear.

Under microchannel flow rates of up to 500 μL/min for 30 minutes, no extensive cell detachment was observed on the PEDOT surface; after seven consecutive days of culture, cell viability remained at 75% ± 4%, enabling the stable formation of a vascular‑mimetic monolayered endothelial tissue.

• In-situ direct writing within the aerosol chamber, eliminating the need for transfer and simplifying the fabrication process.

Unlike mold‑based and microcontact printing, which require pattern fabrication followed by alignment and bonding, the AJP nozzle can be inserted directly into the molded microcavities to write conductive routing patterns in a single step, thereby functionalizing microchannels and eliminating the need for complex alignment processes.

• Conductive microchannels integrate both cell culture and in-situ detection capabilities.

PEDOT exhibits excellent biocompatibility and electrical conductivity, and its microfluidic chamber can be directly employed for real-time transepithelial electrical resistance (TEER) monitoring, enabling the integrated platform of cell culture and electrophysiological sensing.

II. Core Operating Principle of Aerosol Spraying and Its Unique Process Advantages

(1) Complete Direct-Writing Process for Microcavities

• SU8 photolithography is used to fabricate a PDMS microfluidic substrate with a channel depth of 100 μm; the channel surface undergoes brief hydrophilic pre‑treatment with oxygen plasma.
• PEDOT:PSS is compounded with ethylene glycol and silane‑modified ink, ultrasonically dispersed, and then introduced into the aerosolization chamber.
• The nozzle is positioned 2.5 mm above the cavity floor, and a coaxial sheath gas stream focuses the aerosol plume to directly write parallel guiding lines within the closed microchannel.
• Cure in a 100°C oven for 10 minutes to remove the solvent.
• After plasma activation of the upper and lower PD layers, they are bonded to seal the microfluidic chamber, and a reservoir is connected to create a capillary‑driven microfluidic chip.

(II) Five Core Process Advantages (Compared with Microcontact Printing, Spin Coating, and Photolithography)

• In-situ direct writing within the microcavity, eliminating the need for transfer and alignment.

Traditional fabrication methods require first preparing a guiding film on a flat surface and then precisely aligning it with the microfluidic chamber, which is prone to misalignment. In contrast, an aerosol nozzle can be inserted into the formed microchannels to deposit material directly, enabling one-step fabrication without alignment errors.

• The pattern is resistant to plasma encapsulation processes.

The PEDOT conductive polymer scaffold exhibits excellent antioxidant stability, and its structural integrity and adhesive properties are preserved after plasma treatment and microfluidic sealing—features that cannot be achieved with protein-based guiding layers.

• Non-contact, gentle deposition that does not damage PDMS microstructures.

The nozzle maintains a 2.5 mm gap from the cavity bottom, eliminating mechanical compression; micron-scale microchannels and thin-walled structures remain undeformed and undamaged.

• Multi-scale microfluidic devices are flexibly programmable and compatible with a wide range of microfluidic designs.

CAD allows customization of line width, spacing, and orientation, supports parallel or perpendicular fluidic layouts, and is compatible with vascular, neural, and multi-organ microfluidic chips.

• Integrated molding of the conductive guiding layer, combining culture and sensing into one.

Conventional photolithography–based protein patterns provide only cell adhesion, whereas aerosol‑deposited PEDOT lines offer dual functionality: guiding cell growth and enabling in situ electrophysiological monitoring, thereby eliminating the need for separate fabrication of sensing electrodes.

III. Key Technological Innovations of This Study

Innovation 1: An in‑cavity direct‑write strategy for aerosols, overcoming the challenges of microfluidic sealing processes.

Innovation 2: A Plasma-Stabilized PEDOT Composite Ink System

Innovation: 3D morphology-guided + capillary fluid dual‑synergy for directional cell regulation

Innovative 4-micron ultra-thin guide film that does not interfere with fluid dynamics.

Innovation 5: A Microfluidic Platform Integrating Culture and TEER Measurement

IV. Existing Process Challenges and Corresponding Solutions for Overcoming Them

Challenge 1: Traditional protein‑targeting layers undergo complete denaturation upon exposure to oxygen plasma, leading to failure after encapsulation.

Challenge 2: Conventional printing cannot access closed microchannels; it can only be fabricated on a flat surface and then laminated, leading to significant alignment errors.

Challenge 3: External fluid pumping equipment is expensive, and experimental procedures are cumbersome.

Challenge 4: Single‑morphology induction yields only short‑term order; after 3 days of static culture, cells exhibit disordered spreading.

Challenge 5: Thick deposition lines alter the channel cross-section, disrupting the microfluidic shear environment.

V. Future Diversified Industrial Application Scenarios

1. Vascular organ-on-a-chip (cardiovascular drug screening)

2. Multi-organ串联 microfluidic platform

3. Low-Cost Microfluidic Chips for Teaching and Research

4. Implantable biosensing pre‑microfluidic unit

5. Medical In Vitro Microphysiological Testing Equipment

Figure 1. PEDOT:PSS aerosol jet‑printed patterns within a microfluidic chamber: A) Six parallel PEDOT:PSS lines with a pitch of 860 μm, uniformly distributed across the chamber and aligned along the longest dimension of the chamber (i.e., the flow direction from inlet to outlet); atomic force microscopy imaging reveals that the PEDOT:PSS lines have B) a thickness of 200 nm and C) a width of 50 μm (scale bars: A) 500 μm, B) 500 μm).

Figure 2. HUVEC culture and patterning efficiency on PEDOT: PSS–patterned PDMS devices: A) On a Matrigel‑coated PDMS substrate, HUVECs exhibit uniform distribution across the surface; B, C) On the patterned device, HUVECs preferentially adhere and proliferate along the PEDOT:PSS orientation; C) Only under capillary flow conditions do HUVECs fully proliferate along the PEDOT:PSS line direction. In the lower panel, asterisks indicate the mean calculated from nine images (bars show the standard deviation for N = 9 images across three devices), representing cell counts per unit area (256 × 2048 pixels, 160 × 1280 μm², width × height). Each image is divided into eight adjacent regions. For the PEDOT:PSS‑patterned device (B, C), only two regions correspond to the PEDOT:PSS pattern and are labeled “PEDOT” along the “transverse distance” axis; the remaining regions, lacking any pattern, are labeled “no PEDOT.” (Scale bar: 200 μm; fluorescence images: blue nuclei and green actin filaments.)

Figure 3. Effect of oxygen plasma treatment on the Raman spectra of PEDOT:PSS. A) AFM topography of untreated PEDOT:PSS (scale bar: 6 μm); B) AFM topography of PEDOT:PSS after O₂ plasma treatment (scale bar: 6 μm); C) Thickness of untreated PEDOT:PSS (97.2 ± 18.0 nm) and O₂‑plasma‑treated PEDOT:PSS (70.5 ± 4.4 nm): PSS plot (n = 9 data points from three lateral profiles; *p < 0.05); D) Root-mean-square roughness of untreated PEDOT:PSS (15.8 ± 2.7 nm) and O₂‑plasma‑treated PEDOT:PSS (10.6 ± 3.6 nm): PSS plot (bars represent standard deviations from six AFM measurements; *p < 0.05); E) Average Raman spectra of untreated (bottom green line) and O₂‑plasma‑treated PEDOT:PSS (aerosol jet printed on a glass substrate, wavenumber range: 900–1650 cm⁻¹): PSS plot (gray shaded area indicates the overlapping region of standard deviations between the treated and untreated groups; blue and green shaded areas denote regions where the standard deviations differ significantly); F) Electrical conductivity (σ) of untreated PEDOT:PSS (480 ± 226 S cm⁻¹) and O₂‑plasma‑treated PEDOT:PSS (340 ± 126 S cm⁻¹): PSS plot (bars show standard deviations from nine measurements; *p < 0.05; NS indicates no significant difference).

Figure 4. Extension of HUVECs within a patterned PEDOT:PSS‑coated PDMS device: A) Schematic of the PEDOT:PSS‑coated PDMS chamber; B) Schematic illustrating the PEDOT:PSS‑coated PDMS chamber under capillary flow (indicated by the red arrow, aligned with the patterning lines); C–E) Staining results for F‑actin (green) and nuclei (blue) on the PEDOT:PSS lines under static conditions (dotted magenta lines mark the PEDOT patterning positions; scale bar: 50 μm); F–H) Staining results for F‑actin (green) and nuclei (blue) on the PEDOT:PSS lines in a fully sealed device subjected to capillary flow (dotted magenta lines mark the PEDOT patterning positions; scale bar: 50 μm); I) Circularities of HUVECs grown on PEDOT:PSS lines within the PDMS chamber: In the open device, circularity was 0.5 ± 0.2 on day 1 and day 3, and 0.7 ± 0.1 on day 7; in the closed device, circularity was 0.4 ± 0.1 on days 1 and 3, and 0.5 ± 0.1 on day 7; J) IAR values of HUVECs grown on PEDOT:PSS lines within the PDMS chamber: In the open device, IAR was 0.3 ± 0.2 on day 7, 0.4 ± 0.2 on day 3, and 0.5 ± 0.2 on day 1; in the closed device, IAR remained constant at 0.3 ± 0.2 on days 1, 3, and 7. In panels I and J: Sample sizes for the open device were N = 105 on day 1, N = 125 on day 3, and N = 142 on day 7; for the closed device, N = 121 on day 1, N = 101 on day 3, and N = 141 on day 7; sample sizes for the PEDOT:PSS lines perpendicular to the flow direction were N = 131 on day 1, N = 139 on day 3, and N = 138 on day 7. PEDOT:PSS is abbreviated as PEDOT; *p < 0.05.

Figure 5. Alignment of HUVECs within a patterned PEDOT:PSS device: Refer to the top two rows of panels and the schematic (where the PEDOT:PSS lines are labeled as “PEDOT lines”); A–F) Polar plots of cell alignment distribution, with the 90° direction indicating the orientation of the PEDOT:PSS patterning lines. Evaluations were based on the predominant cell axis orientation. A) Polar plot of cells cultured under static conditions for 1 day (N = 105 cells); B) Polar plot of cells cultured for 3 days (N = 125 cells); C) Polar plot of cells cultured for 7 days (N = 142 cells); D) Polar plot of cells cultured under capillary flow conditions for 1 day (N = 121 cells); E) Polar plot of cells cultured for 3 days (N = 101 cells); F) Polar plot of cells cultured for 7 days (N = 141 cells).

Figure 6. Elongation and alignment of HUVECs in a PDMS microfluidic device patterned with PEDOT: PSS, with the PSS lines oriented perpendicular to the flow direction. A) Schematic illustrating the flow direction relative to the PEDOT:PSS lines (red arrows); B and C) Distribution of F‑actin and nuclei in HUVECs stained along the PEDOT:PSS lines under capillary flow conditions (scale bar: 50 μm); D) Circularities of HUVECs along the PEDOT:PSS lines under capillary flow: cell circularity parallel to the flow direction was 0.4 ± 0.1 on days 3 and 7, whereas at day 1 it was 0.5 ± 0.1; cell circularity perpendicular to the flow direction was 0.5 ± 0.2 on days 3 and 7, and 0.6 ± 0.1 at day 1 (PEDOT:PSS lines are denoted as “PEDOT”; *p < 0.05); E) IAR values of HUVECs along the PEDOT:PSS lines under capillary flow: cell IAR values parallel to the flow direction were 0.3 ± 0.2 on days 1, 3, and 7; cell IAR values perpendicular to the flow direction were 0.3 ± 0.1 on day 1, 0.4 ± 0.1 on day 3, and 0.4 ± 0.2 on day 7 (PEDOT:PSS lines are denoted as “PEDOT”; *p < 0.05); F–H) Polar plots showing the distribution of cell alignment along the PEDOT:PSS lines perpendicular to the flow direction. In all panels (D–H): sample sizes for cells aligned parallel to the flow direction were N = 121 on day 1, N = 101 on day 3, and N = 141 on day 7; sample sizes for cells aligned perpendicular to the flow direction were N = 131 on day 1, N = 139 on day 3, and N = 138 on day 7.