Safety

To ensure a safe and professional working environment, the following safety practices must be strictly observed at all times during laboratory sessions:

• Supervision
  Never work alone in the laboratory without prior approval and knowledge of the instructor. All activities must be conducted under proper supervision.
• Behavior and hygiene
  Maintain a professional attitude. No running, joking, or eating in the lab. Avoid touching your face during experiments, and rinse immediately if exposed to hazardous materials.
• Personal protective equipment (PPE)
  Wear a lab coat and closed-toe shoes. Use gloves and goggles when required, and avoid touching personal items or door handles while wearing gloves.
• Equipment use
  Do not operate any laboratory equipment without proper instruction and authorization. Immediately report any damaged, malfunctioning, or broken equipment to the instructor.
• Workspace organization
  Keep laboratory benches clear of unnecessary items such as backpacks, personal belongings, and unrelated books. Only essential materials for the experiment should be present on the work surface.

E. coli operation
The E. coli strains DH5α and BL21 applied in our experiment for cloning and protein expression belong to the WHO-defined Risk Group 1, which do not cause serious human or animal diseases.

1. Operating bacterium-related experiments in the biosafety cabinet.
2. All biowaste would be sterilized by steam sterilization.
3. Report all accidents and any broken equipment to the instructor.
4. Use small trays to carry all sharp objects. When handling, point their tips down and away from other people.
5. Lab benches should be kept free of extraneous items while conducting experiments.

HEK293T operation
According to ATCC, HEK293T cells are recommended to be handled at Biosafety Level 2 (BSL-2), which corresponds to Risk Group 2 (RG2) classification under local biosafety regulations. HEK293T applied in our experiment for protein expression.

1. Conducting HEK293T in biosafety level 2 lab.
2. Don’t release the cell out of the lab.
3. Operating cell-related experiments in the biosafety cabinet.
4. All biowaste would be sterilized by steam sterilization.
5. Report all accidents and any broken equipment to the instructor.
6. Use small trays to carry all sharp objects. When handling, point their tips down and away from other people.
7. Lab benches should be kept free of extraneous items while conducting experiments.

Ethidium bromide (EtBr)
EtBr is applied in agarose gel electrophoresis. EtBr is a potential mutagen that will intercalate double-stranded DNA and RNA. Inhalation of excessive EtBr is even deadly. We set up a separate EtBr-only zone for EtBr-related experiments and always equipped with gloves when conducting experiments in this zone. The used EtBr will be destroyed by sunlight exposure for two days.

Phenylmethylsulfonyl fluoride (PMSF)
PMSF is used to inhibit the serine protease function to prevent protein degradation. PMSF is a corrosive and irritant liquid to skin. To prevent hazards caused by PMSF, we wear gloves during PMSF-related experiments to refrain from any possible contact with PMSF.

Acetic acid
Acetic acid is used as a solvent for preparing chitosan solutions and has a pungent, irritating odor. High concentrations can severely irritate the eyes, skin, and respiratory tract, while even dilute solutions require careful handling to avoid splashing or prolonged skin contact. Proper protection, including lab coat, goggles, and gloves, is essential, and any accidental exposure should be rinsed immediately with plenty of water.

Sodium hydroxide (NaOH)
Sodium hydroxide is used in the cross-linking of hydrogels and is highly corrosive. Inhalation of concentrated fumes can damage the respiratory tract, while ingestion causes severe injury to the esophagus and stomach, often with intense pain and vomiting. To minimize these risks, personnel should wear lab coats, safety goggles, and solutions must be handled carefully to prevent splashing or aerosol formation.

Patch utilization

• Environmental safety
  The materials used in our product, including collagen, chitosan, and the sRAGE-CBD fusion protein, are biodegradable and commonly used in biomedical applications. These components naturally break down over time and are not expected to accumulate in the environment.
• Application safety
  The product should only be applied under the guidance of a licensed physician or according to the provided instructions. To ensure protein stability and maintain efficacy, the patch should be stored and used in shaded areas, avoiding direct sunlight or high temperatures that may induce protein denaturation. Users with known allergies to collagen or chitosan should perform a skin sensitivity test prior to application to prevent potential adverse reactions.

Device operation

• Local network access
  Flask application is accessible to all devices on the same Wi-Fi network. While you are currently using a password-protected Wi-Fi network, anyone with the password can access the website. This could be a problem if the network is shared with untrusted individuals or if the password is leaked. A potential solution is to configure the Flask app to permit access only from a designated IP address, rather than making it available to all devices on the network.
• Unencrypted website traffic
  Since Flask website is not using HTTPS, all data transmitted between the Raspberry Pi and the connected device is unencrypted. This means that if an attacker is on the same network, they can use network sniffing tools to intercept and view the data.
• Local API and data security
  The APIs and potentially some of the captured images are stored directly on the Raspberry Pi. If the Raspberry Pi is compromised (e.g., through an exposed port or a malicious script), an attacker could gain access to these files. This could lead to a breach of your private data, with images being stolen or even manipulated.
• Outdated software and vulnerabilities
  Over time, the Raspberry Pi's operating system and the software(like Flask) will have known vulnerabilities that are discovered and patched. If you don't keep your system updated, these unpatched vulnerabilities can be exploited by attackers to gain unauthorized access. To mitigate this, you should regularly update your Raspberry Pi's operating system and all installed software. Use commands like “sudo apt update” and “sudo apt upgrade” frequently to apply the latest security patches. This is a fundamental practice for maintaining a secure system and protecting against known exploits.