This project develops a lab-based BD-Tau detection sensor that uses nucleic-acid aptamers together with CRISPR-Cas12a. The main experiments include molecular cloning to build the pET28a-BD-tau, pET28a-Tau and pET28a-LbCas12a plasmid, expression and purification of BD-tau, Tau and Cas12a protein in the common lab strain E. coli BL21(DE3), synthesis and annealing of various oligonucleotides and dsDNA, in-vitro transcription of crRNA, and assembly and bench-top testing of the sensor. The overall risk level is low–to–moderate: the bacterial strain used is a routine, non-pathogenic lab strain and the work does not involve environmental release or making organisms more dangerous. However, because the project involves recombinant DNA work, protein expression/purification, nucleic-acid reagents, and some chemical reagents (dyes, buffers, elution solutions), strict biosafety and chemical safety practices are required to protect people and the environment and to keep the research traceable and controlled.

1. Material and Inventory Control

Strains / vectors: This project uses E. coli DH5α, BL21(DE3), the plasmid pET28a-BD-tau, pET28a-Taua nd pET28a-LbCas12a (carries an antibiotic marker and a 6×His tag). All materials that contain recombinant plasmids must be clearly labeled and kept in controlled storage (locked fridge or −80 °C freezer). Only authorized people should access or use them.

Reagent tracking: Record the lot number, expiration date, and storage conditions for key reagents (enzymes, crRNA, reporter, biotin-ComDNA). Aliquot reagents, label the aliquots, and keep a record so you avoid repeated freeze–thaw cycles and contamination.

Gene Library: Synthesized by Genscript.,Diversity: ~1.2×10¹⁶ unique sequences.

Artificial Simulated Blood: It is composed primarily of water (up to 95% by volume) and contains important dissolved proteins (6-8%), such as serum albumin, globulins, and fibrinogen, along with glucose, clotting factors, electrolytes (Na, Ca²⁺, Mg²⁺, HCO3^-, Cl⁻, etc.), hormones, carbon dioxide, and oxygen .(Provided by Professor Lu)

2. Personal Protective Equipment

Required PPE: Lab coat, disposable gloves, and eye protection (goggles or face shield). Use chemical-resistant gloves when handling harsh chemicals. Remove outer PPE at the end of work and wash hands.

Lab conduct: No eating, drinking, applying cosmetics, or storing personal items in the lab. When you move samples or equipment, use a secondary container to prevent spills.

Waste handling: Collect disposable items (pipette tips, plates, gloves, etc.) in designated waste bins and keep them separated by category; do not mix them.

3. Key Procedure Controls

Competent cell transformation: Follow the approved SOP for CaCl₂ transformation (mixing, heat shock, recovery). Use appropriate tubes and minimize the time tubes are open. Treat post-transformation cultures and any waste from those steps as controlled biohazard material and dispose of them according to biohazard waste rules.

Expression & induction: IPTG induction and low-temperature expression should be done in a well-ventilated lab area with emergency equipment available. Make sure heating and power devices have overload protection.

Lysis / cell disruption: Sonication and high-shear methods can create aerosols. Perform these steps inside a biological safety cabinet (if appropriate) or a closed lysis system. Ensure the sonicator has proper acoustic shielding and cooling.

Nucleic acid handling: Any dsDNA, aptamer–dsDNA, or other nucleic acids that can activate Cas12a must be degraded or inactivated before disposal to prevent functional nucleic acid from entering wastewater. Perform annealing and related steps using RNase/DNase-free consumables and on clean, controlled surfaces.

4. Chemical Safety

Follow MSDS instructions: Manage acrylamide, staining dyes, solvents, glycols, strong reducing agents, and similar chemicals according to their MSDS. Keep appropriate spill kits and emergency supplies nearby.

Use lower-toxicity alternatives where possible: For example, avoid ethidium bromide (EtBr) when you can and use safer stains (e.g., SYBR family) if appropriate for your assays.

Waste liquids: Collect salt elution buffers, imidazole wash/elution fractions, staining solutions, and other chemical wastes separately and dispose of them through your institution’s chemical waste program. Do not pour these wastes down the sink.

1. Key equipment and operational requirements

Biological Safety Cabinet (BSC): Use for steps that may generate aerosols or require open handling (e.g., preparing large volumes of buffers, processing lysates). Before use, check airflow and HEPA filtration; after use, disinfect all internal surfaces routinely.

Centrifuge: Confirm balance before starting and keep the lid closed during operation. Use sealed rotors or rotor lids to reduce leakage risk. If abnormal vibration or noise occurs, stop the centrifuge immediately and inspect.

Sonicator: Install splash shields and cooling as required. Use hearing protection or other sound-dampening measures to protect operator hearing during use.

Microplate reader (plate reader): Perform regular calibration for wavelength accuracy and sensitivity. Before use, check the light source and plate positioning. Protect light-sensitive probes and samples from prolonged strong illumination that could cause degradation.

Ultra-low temperature freezers (−80 °C): Implement monitoring and alarm systems (e.g., temperature alerts by SMS/email). Aliquot critical samples to prevent loss from a single-container failure.

Autoclave: Ensure periodic maintenance and keep accurate records of sterilization cycles. Only move biological waste out of controlled areas after validated autoclave sterilization.

2. Maintenance and calibration records

Include all equipment in a maintenance schedule and keep records of maintenance dates, the person who performed the work, any issues found, and corrective actions taken. Key performance checks (e.g., centrifuge balance tests, plate reader sensitivity, BSC face velocity) should be performed at least quarterly and documented.

Equipment failure procedure: If equipment fails, immediately stop any related experiments, mark the equipment as out of service, notify the equipment administrator, and record the status of any affected samples along with instructions for follow-up handling.

Waste handling and decontamination

To prevent the release of functional nucleic acids, recombinant plasmids, or non-inactivated biological materials, all waste must be handled, inactivated, and disposed of in a compliant and fully traceable manner.

1. Biological waste

Materials such as bacterial cultures, spent media, and contaminated consumables must first be treated with an effective disinfectant (e.g., institution-approved agent such as 10% sodium hypochlorite or another validated inactivation reagent) for an appropriate contact time according to local SOPs. After chemical inactivation, materials should be autoclaved (validated cycle) and then disposed of following the institution’s biological waste procedures.

2. Nucleic-acid waste

Nucleic acids capable of activating Cas12a (e.g., dsDNA activators, aptamer–dsDNA complexes, unused crRNA templates) must be rendered non-functional prior to disposal by chemical or enzymatic degradation or by a validated high-temperature treatment. Do not pour high-concentration nucleic-acid solutions directly into the drain.

3. Chemical waste

Organic solvents, volatile wastes, wastes containing heavy metals, and staining reagents (or residues) must be collected separately according to chemical-waste categories and handled by an authorized chemical-waste disposal service in accordance with institutional procedures.

Sharps

Used single-use blades, needles, and other sharps must be placed in certified sharps containers and disposed of according to institutional policy. Sharps must never be discarded in general waste.

All participants must complete institution-mandated training in biosafety and chemical safety, as well as equipment-specific training (centrifuge, sonicator, BSC, plate reader, etc.) before working independently. Each person is also required to sign a safety commitment form. For critical procedures (such as protein purification, ultrasonic lysis, or sensor assembly), first-time operations should be carried out under the supervision of experienced personnel.

The products of this project (the magnetic bead–ComDNA–aptamer–dsDNA reagent complex, lab-prepared Cas12a protein, in-vitro transcribed crRNA, and reporter) are strictly for research use only (RUO). They have not undergone clinical validation or regulatory approval and therefore must not be used for diagnosis, treatment, or any form of clinical decision-making.

The team is committed to strictly following the safety measures outlined above within the defined experimental scope, ensuring that all activities are carried out in a compliant, controlled, and fully traceable framework. Any work beyond the scope of this safety plan (such as using new host organisms, large-scale amplification, or field deployment) will be submitted for prior institutional approval, accompanied by additional risk assessments and updated safety measures.