I. Abstract of the Paper’s Core Research

This study prepared an additive-free regenerated silk fibroin aqueous solution using silkworm cocoons as the raw material, systematically investigating the effects of pre‑treatment steps—degumming, dissolution, dialysis, and centrifugation—on the printability of silk fibroin. By integrating Optomec aerosol‑jet printing equipment, we analyzed how the sheath‑gas-to‑ink flow‑rate focusing ratio (FR) governs defects such as filament extrusion, ink pooling, and filament breakage, and established a standardized quality‑control protocol for the entire silk‑fibroin printing process. The key findings are as follows:

Critical Process Threshold System for Silk Fibroin Pretreatment

After degumming for 75 minutes, a low‑solid‑content (≤6%) silk fibroin solution can be stably atomized following 48 hours of dialysis and centrifugation at 4°C. Insufficient or excessive degumming time, as well as a concentration exceeding 10.5%, will result in premature gelation, rendering the solution completely unprintable; a corresponding troubleshooting procedure enables rapid reproduction of qualified ink.

Standardized auxiliary agent silk fibroin aerosol processing window

Ink flow rate of 23–25 sccm; a focus ratio (FR) of 3–4 represents the optimal range. If FR is too low, beam divergence and material overflow become severe; if FR exceeds 5, airflow turbulence leads to blurred lines. Excessively high ink flow can cause liquid pooling on the substrate and edge distortion.

Complete Mechanism of Defect Formation and Suppression

Low ink flow rate causes discrete particle breakage; excessive ink flow leads to droplet accumulation and pooling; overly high sheath gas induces airflow turbulence, resulting in misty overflow at the pattern edges; under optimal parameters, line edges are sharp and overflow dimensions are significantly reduced.

Long-term printing stability issues have been clarified.

Continuous printing with silk‑fibroin ink for 1–2 hours can easily lead to nozzle clogging and drift in equipment parameters; however, by using freshly prepared ink for each batch and performing standard‑shape calibration before and after printing, consistent part quality can be reliably maintained.

Multi-substrate universal forming capability

Both copper foil and polymeric flexible substrates can directly accommodate the deposition of micron‑scale silk fibroin micro‑lines, with the entire process carried out at room temperature (25°C) without the need for high‑temperature crosslinking, thereby preserving the silk fibroin’s intrinsic bioactivity in its entirety.

II. Complete Preparation Process for Regenerated Silk Fibroin Aerosols

Silk cocoon degumming: The cocoon is treated in a sodium carbonate–boiling water system for 75 minutes to achieve thorough degumming, followed by multiple rinses with deionized water and complete drying of the fibroin fibers at 80°C.

Dissolution and dialysis: Silk fibroin was dissolved in 9.3 M lithium bromide at 60°C, followed by 48-hour dialysis to remove salt ions, and low‑temperature centrifugation at 4,000 rpm to eliminate gel‑aggregated impurities.

Select qualified inks: maintain a solids content of ≤6%; discard high-concentration stock solutions outright; prepare and use as needed.

Aerosol equipment setup: 150 μm nozzle, spray distance of 3 mm, platform speed of 2 mm/s, ink flow rate of 23–25 sccm, and sheath gas ratio FR = 3–4.

At room temperature, without thermal deposition, silk fibroin micro-patterns and thin films can be directly fabricated.

Optical microscopy was used to observe line‑related flash and liquid‑pool defects, and the forming quality was compared across different gas‑flow parameters.

III. Core Advantages of Aerosol-Based Supporting Processes (Comparison of Inkjet, Spin-Coating, and Electrospinning)

Zero-Additive Green Biomanufacturing Throughout the Entire Process

No viscosity modifiers or surfactants are required; the silk fibroin film contains no extraneous impurities and does not interfere with cellular or pharmaceutical bioactivities.

Micrometer-scale, high-precision maskless patterning

CAD‑customized microfluidic channels and sensor arrays; spin coating yields only a single continuous film, and piezoelectric inkjet printing lacks sufficient precision.

Gentle deposition at room temperature, with no denaturation of silk fibroin.

The entire process is carried out at room temperature (25°C) using atomization molding, with no high-temperature or high-pressure steps, and it exhibits natural biodegradability and excellent cell‑affinity properties.

Wide adjustable spray distance, compatible with irregular-shaped bio-based substrates.

The nozzle maintains a stable working distance of 3 mm from the substrate, enabling conformal printing on curved medical dressings and flexible biofilms.

Low consumable waste and recyclable raw materials.

Localized, on-demand deposition of silk fibroin solutions via spin coating results in significant waste of precious silk raw material when applied to large areas; therefore, small‑batch biological samples offer a more cost‑effective approach.

IV. Five Core Technological Innovations of This Study

Innovation 1: A Complete Preparation Protocol for Aid-Free Regenerated Silk Fibroin Aerosol Ink

Establish a standardized process for producing atomizable silk fibroin stock solution without the addition of any polymers or surfactants, thereby eliminating impurities that could interfere with biological functions at the source.

Innovative 2‑step silk‑fibroin anti‑gelation full‑process quality‑control closed loop

The four critical thresholds—degumming, dissolution, dialysis, and concentration—are clearly defined, and a corresponding quick‑fix operating manual for gel‑related issues is provided, enabling stable batch production of inks.

Innovation 3: Focusing on the FR‑based Theory of Precise Defect Control

Quantify the matching relationship between sheath gas and ink flow rates, define the optimal processing window with no overflow or liquid accumulation, and provide direct guidance for equipment parameter tuning.

Innovation 4: A Dedicated Aerosol-Stabilized Printing Calibration Method for Bio‑Proteins

To address the inherent issues of silk fibroin—its tendency to drift and its propensity to clog the print head—we propose a batch‑by‑batch on‑demand formulation combined with pre‑ and post‑printing geometric calibration as quality‑control measures, thereby enhancing long‑term build consistency.

Innovative Low-Cost Digital Additive Manufacturing Paradigm for Bio-Microstructures

Eliminating reliance on expensive lithography and electrospinning equipment, a benchtop aerosol‑based platform enables rapid fabrication of medical micro‑patterns, lowering the barrier to biomaterials research and development.

V. Current Industry Challenges and Corresponding Solutions

Challenge 1: Regenerated silk fibroin dope is highly prone to gelation, causing nozzle clogging throughout the entire conventional printing process.

✅ Limited to a 75-minute standard degelation process, with a low solid content of ≤6% and 48-hour dialysis centrifugation, ensuring no premature gelation prior to printing.

Challenge 2: Silk fibroin printing requires the addition of additives, and impurities can compromise biocompatibility.

✅ The complete pretreatment process enhances the atomizability of the base solution without introducing any external chemical additives.

Challenge 3: There are no standardized parameters for the gas path, and multiple defects—such as material overflow, liquid accumulation, and wire breakage—occur simultaneously.

✅Established a dedicated silk‑fibroin processing window of FR = 3–4, balancing beam focusing and droplet deposition to eliminate three types of forming defects in a single pass.

Challenge 4: Long-term printing parameter drift and nozzle clogging in silk‑fibroin ink.

✅ Each batch is freshly prepared with original solution, and standard graphical calibration is performed before and after printing to ensure that continuous printing does not exceed 2 hours.

Challenge 5: Conventional techniques cannot precisely fabricate micron-scale biofunctional microarrays.

✅ Maskless direct writing of aerosols enables one-step fabrication of high-precision silk fibroin microchannels and sensor arrays.

VI. Future Diversified Industrial Application Scenarios

1 Medical Microfluidic Biochip

Silk fibroin microchannel cell culture chip, fully biodegradable and non‑cytotoxic, for in vitro organoid research.

2 Minimally Invasive Drug-Eluting Microcarriers

High-precision silk fibroin micro-patterned drug‑release films with controllable degradation enable long‑acting, targeted drug delivery.

3 Flexible Wearable Biosensing Patch

Silk‑fibroin‑based physiological signal‑sensing electrodes: skin‑friendly with no irritation, naturally biodegradable upon disposal, and a green medical consumable.

4 Biodegradable Intelligent Food Packaging

Micron‑scale silk fibroin anti‑counterfeiting and freshness‑preserving coating, replacing plastic film—environmentally friendly with no residual contamination.

5 Rapid Prototyping of Microscaffolds for Tissue Engineering

The laboratory rapidly fabricates silk fibroin microscaffolds with varying porosities, enabling high-throughput screening of material formulations for cell proliferation.

Image and Text Guide

Figure 1. RSF solution AJP process flowchart: The upper half outlines material preparation (degumming → dissolution → dialysis → centrifugation), with each gelation step triggering a feedback loop—gelation prolongs the degumming time (<90 min) and lowers the silk-to-water ratio. The lower half details material fabrication (AJP atomization → parameter tuning → printing); if pooling occurs, the degumming time is shortened (>60 min) and the silk-to-water ratio is reduced. Each batch of solution is freshly prepared from dry silk to prevent instability caused by variations in temperature, humidity, storage duration, and concentration.

Figure 2. AJP principle and operating window: (a) Ultrasonic atomization generates an aerosol; the carrier gas (N₂) transports the aerosol to the print head, while the sheath gas (N₂) is focused into a beam and deposited onto the substrate. The focusing ratio FR is defined as the sheath‑gas flow rate divided by the carrier‑gas flow rate. (b) Operating window for 48‑hour dialysis of RSF solution: carrier gas 20–27 sccm, sheath gas 20–150 sccm, FR 1–6. At the left edge (low FR/low carrier gas), discontinuous particles are observed; at the upper edge (FR > 5–6), poor focusing results in a cloud‑like appearance; at the right edge (carrier gas > 25 sccm), pooling occurs. The optimal region lies at a carrier gas flow of 23–25 sccm and an FR of 3–4.

Figure 3. Effect of FR on line morphology: (a) Optical images of line patterns under different FRs (1–4) and carrier gas flow rates (20–27 sccm); the edges within the optimal region are sharply defined. (b) Definition of line width (LW) and over‑spray (OS)—the black line represents the effective line width, while the gray area denotes the over‑spray region. (c) Variation of LW and OS with FR: at a carrier gas flow of 23 sccm, the feature size is smallest; when FR is 3–4, both OS and LW reach local minima. At a carrier gas flow of 27 sccm, for FR > 4, OS increases sharply to ~85 μm, corresponding to the pooling edge.