I. Core Research Summary

• Three-layer integrated aerosol-forming 4×4 16-channel MEA

The bottom layer consists of silver conductive traces (thickness 5.5 ± 0.6 μm, line width 68 ± 1.4 μm); the middle layer is an NOA insulating passivation layer (thickness 33.4 ± 1.4 μm); and the top layer features PEDOT:PSS–based sensing micropillars. Electrode diameter is 50 μm, with an inter‑electrode spacing of 250 μm, along with large‑area reference electrodes on both sides, ensuring compatibility with standard data‑acquisition interfaces.

• Ultra-low electrode–interface impedance, with a substantial improvement in signal-to-noise ratio.

At 1 kHz, the impedance is only 1370 ± 30 Ω, significantly lower than that of commercial gold‑electrode MEAs (on the order of hundreds of kiloohms); the porous PEDOT structure increases the electrochemically active surface area, effectively reducing electrophysiological noise and enabling precise detection of neuronal action potentials.

• The two-week culture medium exhibits long-term stability and supports repeated assays.

PBS‑simulated culture medium was subjected to continuous electrochemical monitoring for 14 days, with negligible impedance fluctuations; after multiple cleaning and regeneration cycles, the device exhibited no significant performance degradation, enabling repeated use and substantially reducing laboratory consumables costs.

• Dual-atomization ink for layered printing requirements

Pneumatic atomization is suitable for high-viscosity silver and insulating inks, while ultrasonic atomization is ideal for low-viscosity PEDOT:PSS. Zone‑specific, localized printing exposes only the sensing electrode area, ensuring complete electrical isolation of the circuitry and minimizing inter‑channel crosstalk.

• CAD fully digital, freely customizable array

No photolithographic mask is required; the number, arrangement, and spacing of electrodes can be customized on demand, making it compatible with in vitro culture systems for diverse electrically active cells such as neurons and cardiomyocytes. Prototype iterations take only a few hours.

II. Core Working Principle of Aerosol Jet Printing and the Complete Preparation Process

(1) Complete three-layer MEA aerosol printing process flow

• Kapton flexible substrate pre‑treatment, pneumatic atomization of silver nano‑ink, printing of all conductive traces and external pads, followed by sintering and curing in a 150°C oven;
• Switch to NOA insulating‑gel pneumatic atomization for spot‑on printing that fully coats the circuitry’s insulation layer, leaving only a 50‑μm‑wide window for the sensing electrode; then use LED‑UV in‑situ curing to form an electrolyte‑blocking barrier.
• Ultrasonic atomization is used to fabricate PEDOT:PSS bio‑conductive ink, which is precisely stacked into micropillar‑based sensing layers on exposed window areas; the resulting structure undergoes low‑temperature curing at 130°C to form a biocompatible sensing interface.
• The substrate‑bonded sealing ring defines the cell culture chamber, completing the assembly of the MEA chip and enabling direct seeding of electroactive cells for electrophysiological recording.

(II) Five Core Process Advantages of Aerosol Jet Printing (Compared with Lithography and Inkjet‑Printed MEAs)

• Three-layer heterogeneous material integrated single-device fabrication

A single printer can switch between pneumatic and ultrasonic dual‑atomization modes, enabling simultaneous deposition of metallic conductive layers, insulating passivation layers, and biosensing layers. Photolithography, which traditionally requires multiple mask steps, evaporation, and etching processes, is thus significantly streamlined.

• Digital, maskless, and freely customizable electrode layout

CAD allows modification of array dimensions, channel count, and electrode spacing without the need for mold fabrication or mask production, enabling rapid iteration of custom chips tailored to neural network and cardiomyocyte cell models.

• Precise partitioned insulation, thoroughly suppresses channel crosstalk.

The aerosol‑based CNC process precisely defines the insulation layer’s printing area, exposing only the sensing sites while fully encapsulating and isolating the underlying conductive traces with NOA. This enables multi‑channel synchronous recording without signal crosstalk.

• In situ fabrication of biocompatible PEDOT micropillars

A mild room-temperature, low-temperature curing process prevents high-temperature denaturation of the conductive polymer; the porous membrane adheres to cells, addressing the cytotoxicity and high‑impedance drawbacks associated with metallic silver.

• Low-cost, reusable, and compatible with university laboratories.

Eliminates the need for costly photolithography cleanroom production lines, with low consumable waste due to on-demand ink deposition; after chip cleaning, it can be reused multiple times, significantly reducing consumable procurement costs in long-term cell culture experiments.

III. Five Key Technological Innovations

Innovation 1: A complete process for MEA fabrication featuring dual atomization and synergistic layered printing.

       Innovation 2: A Precise Window-Opening and Site-Specific Modification Strategy for the Insulating Layer

Innovative 3 PEDOT-based bio-microcolumn composite sensing interface

Innovation: A Two-Week Long-Lasting, Stable, and Reusable In Vitro Sensing Platform

Innovative Manufacturing Route for General-Purpose Flexible Substrate-Based Aqueous Sol-Gel

IV. Existing Process Challenges and Corresponding Solutions for Overcoming Them

Challenge 1: A single atomization mode cannot accommodate both high-viscosity insulating adhesives and low-viscosity conductive polymers.

Challenge 2: Direct contact between metallic silver traces and the culture medium can cause corrosion, releasing ions that are toxic to cells.

Challenge 3: Traditional planar metal electrodes have high impedance, causing the weak neuronal signals to be easily drowned out by noise.

Challenge 4: The photolithography–MEA design is fixed; switching to a different cell model requires re‑fabrication, which takes several days.

Challenge 5: Most commercial microelectrodes are suitable for only a single experiment, resulting in high costs for long-term research.

V. Future Diversified Industrial Application Scenarios

1. Basic Neuroscience Laboratory at the University

2. Cardiomyocytes / Cardiac Organ-on-a-Chip

3. High-Throughput Drug Screening Microfluidic Integrated Chip

4. In vitro monitoring platform for stem cell differentiation

5. Rapid Prototyping in Small-Scale Biological Laboratories

Figure 1. Photograph of the printed MEA and a schematic of its detailed structure; the MEA comprises contact pads for interfacing with the electronic front end.

Figure 2. Schematic illustration of the fabrication process. The first step is the printing of silver conductive traces; the second step is the deposition of a passivation layer; and the third step is the printing of electrode pads.

Figure 3. Detailed illustration of the MEA schematic. The figure shows two conductive pathways within the MEA, clearly highlighting its layered structure.

Table I: Manufacturing Parameters Required for AJP Technology

Figure 4. (a) Average profile of the coating formed by the silver wire trace (solid red line) and the NOA 81 wire trace (solid blue line). The shaded area represents the standard deviation calculated from 128 profiles acquired from four MEAs. (b) Average profile of the PEDOT:PSS coating (solid green line) and the underlying silver layer (solid red line). The shaded area indicates the standard deviation computed from 64 sensor‑electrode profiles obtained from four MEAs.

Figure 5. Distribution of measured resistance values for different conductive track lengths, with the mean and standard deviation calculated from data obtained from at least eight tracks and encompassing all four MEA types.

Figure 6. Experimental setup for electrochemical impedance spectroscopy (EIS). After filling the sensor with PBS solution, the PalmSens sensor is connected to the system, measurements are performed, and the data are transmitted to a computer for analysis.

Figure 7. Impedance magnitude as a function of frequency over 14 days (repeatability test). Error bars represent the standard deviation across two MEAs. Impedance measurements were taken between the working electrode and the reference electrode.

Figure 8. Impedance magnitude stability curves of the two sensors in PBS solution over 14 days at frequencies of 100 Hz and 1 kHz. Error bars represent the standard deviation across two MEAs.

Figure 9. Theoretical relationship of the impedance modulus as a function of frequency in the Randall circuit model, based on the electrode/electrolyte interface (used for fitting the experimental data) (see inset).

Figure 10. Nyquist plots of the real and imaginary parts of the experimental results obtained from MEA printing during a two-week stability test.