Preparatory work

1. Prepare Amp antibiotic
2. Prepare LB medium (solid and liquid)

Molecular cloning

1. PCR amplification
2. Agarose gel electrophoresis
3. DNA Fragment extraction
4. Homologous recombination
5. Restriction Enzyme Digestion
6. Digestion Product Purification
7. DNA Ligation
8. Transformation
9. Colony PCR
10. Plasmid Extraction

Protein expression and purification

1. Protein expression
2. Protein purification
3. SDS-PAGE and Staining
4. Protein Quantification

IgE affinity test

Cell Proliferation test

Half-life detection

Soluble Microneedles Preparation

Preparatory work
1. Prepare Amp antibiotic

Materials:

Ampicillin powder,

Sterile deionized water,

Filter sterilization unit (0.22 µm pore size).

Needle-free syringe

Equipment:

Electronic balance

Method (For 10 mL):

  1. Weighing 1 g of the ampicillin powder
  2. Dissolve in Solvent
    1. Transfer the powder to a sterile container.
    2. Add the final volume of sterile water.
    3. Mix gently until fully dissolved.
  3. Filter Sterilize
  4. Pass the solution through a 0.22 µm filter into a sterile tube. This removes bacteria and ensures sterility.
  5. Aliquot and Store
    1. Dispense into smaller aliquots (1 mL) to avoid repeated freeze-thaw cycles.
    2. Store at -20℃ for long-term storage.
2. Prepare LB medium (solid and liquid)

Materials:

Tryptone (10 g/L)

Yeast Extract (5 g/L)

NaCl (10 g/L)

Agar (for solid medium; 15 g/L)

Erlenmeyer Flask (for mixing)

Autoclave (for sterilization)

Petri dishes (for solid medium)

Equipment:

Electronic balance

Autoclave

Oven

Method:

  1. Weigh Components (For 1 L): 10 g tryptone, 5 g yeast extract, 10 g NaCl
  2. Add components to a flask with ~800 mL of water. Stir until fully dissolved (use a magnetic stirrer). Then add water to reach 1 L total volume.
  3. Transfer to an autoclavable bottle. Autoclave at 121℃ for 20 minutes.
  4. For solid LB:
    1. After autoclaving, let the medium cool to ~50℃ (warm to touch but not boiling).
    2. Add ampicillin to a final concentration of 100 μg/mL.
    3. Pour 10-15 mL per 90 mm petri dish in a sterile hood.
    4. Let plates solidify (30-60 minutes).
    5. Invert plates and store at 4℃.
Molecular cloning
1. PCR amplification

Materials:

PrimeStar Max Premix (2×)

Primers (10 μM)

Plasmid

ddH2O

Equipment: 

Thermal cycler

PCR tubes

Method:

Reagent

Volume (μL)

Procedure

PrimeStar Max Premix (2×)

25

Denaturation: 98 ℃ for 10 sec

Primer F (10 μM)

1

Annealing: Tm for 15 sec

Primer R (10 μM)

1

Extension: 72 ℃, 30 sec/kb

DNA template

1 (10 ng)

Back to step 2 (30 times)

ddH2O

22

Final elongation: 72 ℃ for 2 min

Total

up to 50

10 ℃ hold
2. Agarose gel electrophoresis

Materials:

Agarose powder

1×TAE buffer (Tris-acetate -EDTA)

DNA loading dye (6×)

DNA ladder

YeaGreen DNA stain

Equipment:

Electronic balance

Microwave

Electrophoresis equipment

UV transilluminator

Method:

  1. Prepare Gel (1%):
    1. Mix agarose 1 g powder with 100 mL TAE buffe.
    2. Heat to dissolve agarose, cool to ~50°C.
    3. Add 10 μL nucleic acid dye.
    4. Pour into gel tray with comb; let solidify.
  2. Load Samples:
    1. Mix DNA samples with loading dye.
    2. Load into gel wells, and load DNA size marker in a separate well.
  3. Run Gel:
    1. Submerge gel in TAE buffer.
    2. Apply 180 V until bands separate (About 15 min).
  4. Visualize Bands
    1. Place gel on UV transilluminator.
    2. Image bands.
3. DNA Fragment extraction

Materials:

Gel extraction kit

Ethanol

Equipment: 

Centrifuge

Water bath

Procedure:

  1. Add 96 mL ethanol into wash solution. Adjust water bath to 50 ℃.
  2. Cut the agarose gel containing the DNA fragment and put into micro-centrifuge tube.
  3. Weight the gel and add 3 times of Buffer B2. Heat in 50 ℃ water bath for 10 min.
  4. Add DNA gel solution into the spin column, 8,000 ×g for 1 min. Discard the filtrate in collection tube, then return column back to the collection tube.
  5. Add 500 μL of Wash Solution into the column, then into the spin column, 8,000 ×g for 1 min. Discard the filtrate in collection tube, then return column back to the collection tube. Repeat this step once.
  6. Centrifuge column at 8,000 ×g for 2 min.
  7. Place column into a 1.5 mL tube, then add 30 μL of ddH₂O into the column.
  8. Put in 50 ℃ dryer for 5 min.
  9. Centrifuge column at 8,000 ×g for 4 min. Store centrifuge solution in -20 °C refrigerator.
4. Homologous recombination

Materials:

ClonExpress Ultra One Step Cloning Kit

Equipment:

Thermal cycler

PCR tubes

Procedure:

Reagent

Volume (µL)

2×Clonexpress mix

5

Gene

3

pET32a Linearized vector

1

ddH2O

1

Total

10

50 ℃，10 min
5. Restriction Enzyme Digestion

Materials:

BamHI restriction enzymes

XhoI restriction enzymes

10×CutSmart buffer

Plasmid DNA

Target fragment

Nuclease-free water

Equipment: 

Thermal cycler

PCR tubes

Procedure:

Reagent

Volume (μL)

10×Cutsmart buffer

2

Plasmid DNA

3

BamHI

2

XhoI

2

ddH2O

up to 20 μL

37℃, 15 min; 65℃, 10 min
6. Digestion Product Purification

Materials:

PCR product purification kit

Ethanol

Equipment: 

Centrifuge

Water bath

Procedure:

  1. Add 5 volumes of Buffer PB directly to the digestion product
  2. Transfer the entire mixture to a spin column seated in a 2 mL collection tube.
  3. Centrifuge at 10,000 ×g for 60 seconds. Discard flow-through and reassemble column
  4. Add 700 μL Buffer PW (wash buffer containing ethanol) to the column. Centrifuge at 10,000 ×g for 60 sec. Discard flow-through.
  5. Repeat with 500 μL Buffer PW.
  6. Centrifuge the empty column at 10,000 ×g for 2 minutes to remove residual ethanol.
  7. Transfer column to a clean 1.5 mL microcentrifuge tube.
  8. Add 30 μL ddH2O directly to the center of the membrane.
  9. Incubate at room temperature for 2 minutes 
  10. Centrifuge at 10,000 ×g for 2 minutes to collect purified DNA.
  11. Using a Microspectrophotometer to Detect DNA Concentration
7. DNA Ligation

Materials:

T4 DNA Ligase

10×T4 DNA Ligase buffer

Vector and insert DNA

Nuclease-free water

Equipment:

Thermal cycler

PCR tubes

Procedure:

COMPONENT

20 μL REACTION

T4 DNA Ligase Buffer (10X)

2 μL

T4 DNA Ligase

1 μL

pET32a-V (5329 bp)

5 μL

Insert DNA (3: 1)

/

Nuclease-free water

to 20 μL
8. Transformation

Materials: 

E. coli Top10 and Origami (DE3) competent cells,

Recombination product after Gibson assembly,

Antibiotic-free LB liquid, Solid medium with antibiotics

Equipment:

Ice bath

42°C water bath

Incubator

Shaker

Procedure:

  1. Added recombination mix (10 μL) to pre-chilled competent Top10 cells, incubated on ice 30 min.
  2. Heat-shocked at 42 °C for 90 sec.
  3. Immediately placed on ice 2 min.
  4. Added 900 μL LB medium and incubated at 37 °C for 1 h.
  5. Plating: Spread 100 μL of recovered cells on LB agar + ampicillin.
  6. Incubated plates overnight at 37 °C to select for transformants.
9. Colony PCR

Materials:

PCR buffer

dNTPs

T7-pro and T7-ter primers

2×Hieff® Ultra-Rapid II HotStart PCR Master Mix

Equipment:

Thermal cycler

PCR tubes

Procedure:

Reagent

Volume (μL)

Procedure

PCR Rapid mix (2×)

5

Denaturation: 95 ℃ for 10 sec

T7-pro (10 μM)

0.4

Annealing: Tm for 15 sec

T7-ter (10 μM)

0.4

Extension: 72 ℃, 3 sec/kb

Single colony

/

Back to step 1 (20 times)

ddH2O

 4.2

Final elongation: 72 ℃ for 10 sec

Total

10

10 ℃ hold
  • After amplification, analyze 3.5 μL of the PCR reaction by agarose gel electrophoresis. The procedure is as previously described.
  • Inoculate the positive transformants in an LB culture medium containing antibiotics at 220 rpm, 37 ℃ overnight.
10. Plasmid Extraction

Materials:

DiaSpin Plasmid Mini-Preps Kit

Ethanol

Equipment: 

Centrifuge

Oven

Procedure:

(1) Culture bacterial fluid: Inoculate strains into 10 mL of liquid LB medium supplemented with 100 μg/mL Ampicillin.

(2) Place a column into a collection tube. Add 500 μL of Buffer S to column, centrifuge at 12,000 ×g for 1 min. Discard the filtrate in the collection tube, then place column back to the collection tube.

(3) Collect 10 mL of overnight bacteria culture. Centrifuge at 4,000 ×g for 10 min to pellet the bacteria. Discard the supernatant.

(4) Resuspend the bacteria pellet in 500 μL of Buffer SP1 by vortexing. Make the pellet completely resuspended.

(5) Add 500 μL of Buffer SP2 and mix by gently inverting the tube for 10 times.

(6) Add 700 μL of Buffer SP3 and mix by gently inverting the tube for 10 times.

(7) Centrifuge at 12,000 ×g for 10 min. Transfer the supernatant to the column, then centrifuge at 8,000 ×g for 1 min. Discard the filtrate in the collection tube.

(8) Add 700 μL of Wash Solution into the column, then centrifuge at 9,000 ×g for 1 min. Discard the filtrate in collection tube, then return column back to the collection tube. Repeat this step once.

(9) Place column into a 1.5 mL tube, then add 50 μL of ddH₂O into the column.

(10) Put in 50 ℃ oven for 5 min.

(11) Centrifuge column for 2 min at 9,000 ×g. Store centrifuge solution in -20 °C refrigerator.

Protein expression and purification
1. Protein expression

Materials:

Seed culture (Origami DE3 strains with recombinant plasmids)

LB liquid medium (with 100 μg/mL Amp)

1 M IPTG solution

Equipment: 

Shaker

Clean bench

Centrifuge

Procedure:

  1. Inoculated at 1: 50 ratios: 200 μL seed culture + 10 mL LB medium (with 10 μL Amp antibiotic).
  2. Incubated at 37°C with shaking (200 rpm) for 2–3 h until OD600 reached 0.4–0.6 (log phase).
  3. IPTG Induction:
    1. Reduced culture temperature to 18°C (ice bath for 10 min).
    2. Added IPTG to final concentration of 0.2 mM (2 μL 1 M IPTG + 10 mL LB medium).
    3. Induced overnight (12–16 h) at 18°C with shaking (200 rpm).
  4. Cell Pellet Collection: Centrifuged at 4,000 rpm for 10 min. Discarded supernatant; stored pellets at -80°C.
2. Protein purification

Materials:

Lysis buffer (with protease inhibitors)

Ni-NTA resin

Binding buffer (20 mM phosphate buffer, pH 7.4, 500 mM NaCl),

Wash buffer (20 mM imidazole), elution buffer (250 mM imidazole)

Native buffer (20 mM Tris-HCl, pH 8.0)

Urea

Equipment: 

Sonicator

Ice bath

Centrifuge

Procedure:

  1. Cell Lysis
  • Resuspend pellets in lysis buffer (5–10× pellet volume).
  • Sonicated on ice: 30% amplitude, 5 sec ON/OFF pulses, 10 min total.
  • Centrifuged: 12,000 rpm, 20 min, 4°C; collected supernatant.
  1. Ni-NTA Affinity Chromatography:
  • Equilibration: Washed resin with 5 column volumes of binding buffer.
  • Binding: Incubated lysate with resin on ice for 1 h with gentle agitation.
  • Wash: Centrifuged (1,000 g, 10 sec); washed twice with wash buffer.
  • Elution: Collected target protein with elution buffer.
  • Principle: His-tag binds Ni2+; imidazole competes for elution.
  1. Protein Denaturation & Renaturation:
  • Denaturation
    1. Prepare protein sample in native buffer (20 mM Tris-HCl, pH 8.0).
    2. Add ​​denaturant​​ (8 M urea) to unfold and reduce disulfide bonds.
    3. Incubate for ​​2–4 hours at room temperature​​ or ​​37°C​​ with gentle agitation.
    4. Centrifuge at ​​12,000 × g for 20 min​​ to remove insoluble aggregates.
  • ​​Renaturation by Dialysis​​
    1. Transfer denatured protein to dialysis tubing (MWCO 10 kDa).
    2. Dialyze sequentially against buffers with ​​stepwise decreasing denaturant​​ (4 M → 2 M → 0 M urea) to promote disulfide reformation.
    3. Each step requires ​​4–6 hours at 4°C​​.
3. SDS-PAGE and Staining

Materials:

12% SDS-PAGE Gel Rapid Preparation Kit

Fast Stain Solution

Equipment:

SDS-PAGE adhesive dispensing device

Procedure:

  1. Gel Casting Setup​​
    1. Clean glass plates, spacers, and comb thoroughly. Assemble the gel cassette securely to prevent leaks
  2. ​​Prepare Separating Gel (Lower Gel)​​
    1. Mix ​​2.5 mL​​ of pre-mixed Separating Gel Solution
    2. Add ​​50 μL​​ of Modified Coagulant (provided instead of TEMED)
    3. Mix gently by swirling to avoid bubble formation
    4. Immediately pour into gel cassette, leaving appropriate space for stacking gel
    5. Overlay with ​​isopropanol or water​​ to flatten the surface
    6. Polymerization occurs within ​​5-10 minutes​​
  3. ​​Prepare Stacking Gel (Upper Gel)​​
    1. After separating gel polymerization, remove the overlay liquid and dry the residual moisture
    2. Mix ​​1.0 mL​​ of pre-mixed Stacking Gel Solution
    3. Add ​​20 μL​​ of Modified Coagulant
    4. Mix gently without introducing bubbles
    5. Pour onto polymerized separating gel
    6. Insert the comb without trapping air bubbles
    7. Polymerization completes within ​​5-10 minutes​​
  4. Run the sample on the gel​​
    1. Sample preparation: Mixed 5 μL 5× SDS-PAGE loading buffer + 20 μL sample, then denatured at 98°C for 5 min
    2. Carefully remove comb after complete polymerization
    3. Rinse wells with electrophoresis buffer to remove residual acrylamide
    4. Mount gel in electrophoresis chamber and fill with running buffer
    5. Set the electrophoresis program: 150V, 1 h
  5. Staining & Destaining:
    1. Transfer gel to a tray, rinse with distilled water.
    2. Stain with Fast Stain Solution (5–30 min with gentle agitation).
    3. Destain with water until bands are clear (change water as needed).
4. Protein Quantification

Materials:

Bradford solution

BSA

PBS

Equipment:

Microplate reader

Procedure:

  • Preparation:
  1. Equilibrate all reagents to room temperature.
  2. Prepare BSA standard dilutions using the same buffer as samples (0, 10, 20, 40, 60, 80, 100, 200 μg/mL).
  • Standard Curve Setup
  1. Add 5 μL of each BSA standard or sample to a 96-well plate .
  2. Add 250 μL of Bradford solution to each well. 
  • Incubation

Incubate at room temperature for 5 minutes (avoid exceeding 1 hour).

  • Measurement
  1. Read absorbance at 595 nm using a microplate reader.
  2. Generate a standard curve by plotting absorbance vs. BSA concentration.
  3. Calculate sample protein concentration from the standard curve equation
IgE affinity test

Material:

IgE ELISA Kit (Sangon Biotech)

PBS (pH 7.2)

Blocking buffer (1% BSA in PBS)

Wash buffer (PBS containing 0.05% Tween-20)

Equipment:

Microplate reader

Procedure:

  1. Protein-Coated 96-Well Plate
    1. Dilute the allergen protein to a concentration of 10 μg/mL using PBS (pH 7.2).
    2. Add 0.1 mL of the diluted allergen protein solution to each well of an ELISA plate, and incubate overnight at 4°C.
  2. Blocking
    1. Add 100 µL of blocking buffer (1% BSA in PBS) to each well.
    2. Incubate at 37°C for 1 hour.
    3. Wash the wells to remove unbound blocking reagent.
  3. Preparation of IgE Standards
    1. Centrifuge the lyophilized IgE standard at 10,000 × g for 1 minute.
    2. Reconstitute by adding 1.0 mL of sample dilution buffer, invert gently, and allow to dissolve for 10 minutes. Mix thoroughly.
    3. Prepare a 200 ng/mL stock solution, then perform serial two-fold dilutions to obtain the following concentrations: 200, 100, 50, 25, 12.5, 6.25, 3.13, 0 ng/mL.
    4. Use 7 tubes, each preloaded with 500 µL dilution buffer. Add 500 µL from the previous tube, mix well, and continue stepwise. The final tube (0 ng/mL) serves as the blank. Add the diluted sample to the 96-well plate.
  4. IgE Binding to Allergen-Coated Wells
    1. Add 100 µL of IgE working solution (50 ng/mL) to each well. For the standard curve, use the full dilution series.
    2. Incubate at 37°C for 90 minutes.

      Note: Add samples gently to the center of the well, avoiding the walls; mix gently without bubbles. Complete sample loading within 10 minutes.

    3. Prepare biotin-labeled anti-IgE antibody by diluting the 100× stock 1:100 in SD2 buffer (calculate required volume based on 100 µL per well). Use within 30 minutes.
    4. Discard the well contents and tap dry. Add 100 µL of the biotin-labeled antibody to each well.
    5. Incubate at 37°C for 60 minutes, then wash 4 times with 350 µL wash buffer (soak 1–2 minutes per wash).
  5. Streptavidin-HRP Conjugate Binding
    1. Dilute the 100× HRP-labeled streptavidin stock 1:100 in SD3 buffer (post-centrifugation), based on 100 µL/well. Use within 30 minutes.
    2. Add 100 µL of the HRP conjugate to each well.
    3. Incubate at 37°C for 30 minutes, then wash 4 times using 300 µL per wash (30 s intervals).
  6. Color Development
    1. Add 90 µL of TMB substrate to each well (protect from light).
    2. Incubate at 37°C in the dark for ~15 minutes.
  7. Reaction Termination

    8. Add 50 µL stop solution to each well and immediately read absorbance at 450 nm using a microplate reader (within 5 minutes).

  8. Data Analysis
    1. Subtract the OD value of the blank (0 ng/mL) from all standards and sample readings.
    2. Plot the standard curve using IgE concentrations on the X-axis and corresponding OD values on the Y-axis.
    3. Fit the curve using a four-parameter logistic regression model to quantify IgE binding in unknown samples.
Cell Proliferation test

Materials:

NIH/3T3 cells

DMEM complete medium

1× DPBS

0.25% trypsin solution

T75 cell culture flasks

15 mL centrifuge tubes

hypoallergenic derivative, natural allergens, allergen fusion protein

CCK-8

Equipment:

Biosafety cabinet

Inverted microscope

CO₂ incubator (37 °C, 5% CO₂)

Centrifuge (200 ×g)

Pipettes and sterile tips

Microplate reader

Procedure:

(1) Cell Revival:

  1. Rapidly take out frozen NIH-3T3 cells from liquid nitrogen.
  2. Thaw the vial in a 37 °C water bath with gentle shaking. Do not exceed 1.5 min.
  3. In a biosafety cabinet, slowly add the 1 mL thawed cell suspension to 9 mL pre-warmed complete DMEM medium in a centrifuge tube.
  4. Mix gently by pipetting up and down.
  5. Centrifuge at 200 ×g for 5 min.
  6. Discard the supernatant carefully.
  7. Resuspend the cell pellet in 1 mL of fresh complete DMEM medium.
  8. Transfer the suspension into a T75 flask containing 9 mL complete DMEM medium.
  9. Mix gently by crosswise shaking and place the flask into a 37 °C, 5% CO₂ incubator.
  10. Replace medium with fresh complete medium on the second day, then every other day.

(2) Cell Passaging:

  1. Observe cells under an inverted microscope; proceed when ~80% confluent.
  2. In biosafety cabinet, discard old medium.
  3. Add 3 mL DPBS, gently shake, discard; repeat wash once.
  4. Add 1 mL of 0.25% trypsin along the flask wall (opposite to cells).
  5. Incubate at 37 °C, 5% CO₂ for ~2 min.
  6. Add 3 mL complete medium to stop digestion, gently shake to detach cells.
  7. Pipette up and down to disperse cell clumps.
  8. Transfer to centrifuge tube, spin at 200 ×g for 5 min, discard supernatant.
  9. Resuspend pellet in 2 mL complete medium.
  10. Add 9 mL fresh medium to a new T75 flask; seed appropriate volume of cell suspension.
  11. Mix crosswise and place in incubator (37 °C, 5% CO₂).

(3) Cell culture plate and CCK-8 treatment

  1. Observe cells under an inverted microscope; proceed when ~80% confluent.
  2. In biosafety cabinet, discard old medium. Add 3 mL DPBS, gently shake, discard; repeat wash once.
  3. Add 1 mL of 0.25% trypsin along the flask wall (opposite to cells).
  4. Incubate at 37 °C, 5% CO₂ for ~2 min.
  5. Add 3 mL complete medium to stop digestion, gently shake to detach cells.
  6. Prepare a cell suspension with a density of 1×10⁵ cells/mL using complete medium. Transfer 90 μL of the cell suspension into a 96-well plate.
  7. Preparation of drugs: Dilute each sample to 1000, 100, and 10 nM (the concentration after adding to the cells will be 100, 10, and 1 nM respectively). Add 10 μL of drug solutions at different concentrations to a 96-well cell culture plate.
  8. Add an equal volume of DPBS solution to the outer perimeter of the 96-well plate to prevent excessive evaporation.
  9. After 48h of incubation, add 10μL CCK-8 reagent to each well. Incubate at 37°C with 5% CO₂ for 30 min, then measure the absorbance at 450 nm using a microplate reader.
  10. Calculate cell viability using the following formula:

Cell viability (%) = Pc/Pb×100%

Pe represents the absorbance of the experimental group and Pb represents the absorbance of the control group.

Control group: Cells + PBS + complete medium + CCK-8

Blank group: Complete medium + CCK-8

Experimental group: Cells + drug + complete medium + CCK-8

Half-life detection

Materials​​

Purified ​​FEFKFEFK-EGFP​​ fusion protein (experimental group)

Purified ​​non-fused EGFP​​ (control group)

​​ Fetal bovine serum (FBS), sterile-filtered

PBS (pH 7.4) for dilution

​​Equipment​​:

Thermostatic incubator (37°C)

Microplate reader

Black 96-well plates (to minimize background fluorescence).

Pipettes and sterile tips.

Procedure

1. Sample Preparation:

  1. Dilute both FEFKFEFK-EGFP and non-fused EGFP to 500 µg/mL in FBS.
  2. Aliquot 200 µL of each protein-serum mixture into wells of a 96-well plate (triplicates per time point).
  3. Include blank controls-FBS

2.Incubation:

Seal the plate to prevent evaporation, and incubate at 37°C for 5 days.

3. Fluorescence Measurement:

  1. At each time point, the culture plate is briefly shaken first to prevent the precipitates from aggregating together.
  2. Measure fluorescence (excitation 488 nm, emission 507 nm).

4.Data processing

The fluorescence intensity was processed using GraphPad 8.0.2, and the half-life of the sample was predicted using the Exponential decay - one phase model.

Soluble Microneedles Preparation

Materials​​

​ PMSF microneedle molds

Sodium hyaluronate (50,000 MW)

PVA

Pigment

​​Equipment​​:

Centrifuge

Water bath

Procedure

1. Clean PMSF molds:

Place molds in a beaker containing water and briefly sonicate; remove excess moisture (using absorbent paper), then place in a 60°C oven.

2. Prepare soluble gel

  1. Weigh 0.3g sodium hyaluronate (50,000 MW) and add sequentially (3 additions total) to 1mL drug solution. After each addition, vigorously stir using a 1mL pipette tip until completely dissolved (needle layer gel).
  2. Weigh PVA and dissolve in water to prepare a 250mg/mL backing layer gel solution.
  3. Centrifuge both needle layer and backing layer gels at 3000 rpm for 3 min to degas;
  4. Transfer gel into molds using pipette tip. Cover mold cavities with non-absorbent sealing film, cap with another mold, and centrifuge in a centrifuge tube.
  5. Repeat gel addition and centrifugation as needed.
  6. Scrape off surface gel.

3. Prepare the base layer - PVA

Add an appropriate amount of backing gel solution to fill the mold completely. Dry at room temperature for 3-4 days.

4. Water Solubility Testing

(1) Immerse microneedles in pre-warmed PBS (37℃​​) in a Petri dish

(2) Maintain temperature using a water bath (37°C​​ to simulate skin conditions)

(3) Observe the dissolution of the needle tip on the light-transmitting plate.