PCR amplification

Reagent	Volume (Unit: μL)
Mixer (2 x buffer)	25
Template (targeted genes: Siglec-1/ beta-2-Transferrin/ mCherry)	2
Primer (F)	1
Primer (R)	1
ddH2O	21

Procedure

1. Add mixer, ddH₂O, targeted genes and primers respectively (with the volume denoted above) to the 0.2 mL PCR tube using a micropipette. Label the top of the tube with “Siglec-1” which stands for Siglec-1 genes. “bTF” stands for the beta-2-Transferrin genes. “mCherry” stands for mCherry genes.

2. Put the tube into the PCR machine/thermo cycler, using the following data.

Name of Process	Temperature (Unit: ℃)	Time (Unit: s)
Denaturation	95	30
Annealing	66	30
Extention	72	120

Number of cycles: 30

 

Bacteria cultivation

Reagent	Volume (Unit: μL)
LB	4,000
Bacteria sample	20
Antibiotics (Kanamycin)	4

 

Composition of LB in 1 L ddH₂O

Reagent	Mass (Unit: g)
NaCl	10
Tryptone	10
Yeast extract	5

LB then undergoes high temperature sterilisation.

 

Agarose Gel preparation

Reagent	Mass/Volume
Agarose	1 g
TAE Buffer	100 mL

 

Gel electrophoresis

1. After the reagents above are added, use a microwave oven to heat the solution and cool the solution. Then add 5 μL of nucleic acid dye (add it before the solution solidifies).

2. Pour the gel into the mould. Insert the mould to form the wells on the gel where DNA samples can be injected. Wait until it is solidified.

3. 3 μL of Loading buffer is added to each sample of DNA. Carefully using a micropipette, add DNA marker on the first hole, and then add all the DNA sample into the gel with the corresponding order (noting down the order is recommended).

4. Start the cell. Wait for 15 minutes.

5. Using ultraviolet. Take the photo of the result.

 

Gel Extraction

1. Cut the gel with the DNA strands off.

2. Weight the mass of the gel.

3. Add buffer B2 (3 times the mass of the sampled gel).

4. Add the gel and the solution into the absorption column. Centrifuge for 30 s (8,000 x g), remove the liquid collected at the bottom.

5. Add 50 μL of wash solution to the absorption column. Centrifuge for 30 s (9,000 x g), remove the liquid collected at the bottom.

6. Repeat step 5.

7. Centrifuge the empty absorption column for one minute (9,000 x g).

8. Put the absorption column in a clean 1.5 mL centrifuge tube. Add 15 μL of elution buffer. Stand for 1 minutes at room temperature. Centrifuge for 1 minute (7,000 x g). Remove the absorption column and keep the DNA solution in the centrifuge tube. Label it with the corresponding DNA (Siglec-1/bTF-mCherry).

 

Plasmid Extraction

1. Put the absorption column in the collection tube. Then add 500 μL of buffer S (into the absorption column). Centrifuge for 1 minute (12,000 x g) and remove the liquid in the collection tube.

2. Add 1.5 mL of solution containing bacteria to the collection tube. Centrifuge for 2 minutes (8,000 x g).

3. Add 250 μL of buffer SP1 to suspend the bacteria.

4. Add 250 μL of buffer SP2 (a strong base). Shake the tube upside down gently for 10 times, then stand the tube for 2 minutes.

5. Add 350 μL of buffer SP3. Gently shake the tube upside down for 10 times.

6. Centrifuge for 5 minutes (12,000 x g). Move the supernatant into the absorption column (inserted into the collection tube). Centrifuge again for 30 s (8,000 x g). Remove the liquid in the collection tube.

7. Add 500 μL of Wash Solution. Centrifuge for 30 s (9,000 x g). Remove the liquid in the collection tube.

8. Repeat step 7 and then centrifuge the empty absorption column for one minute (9,000 x g).

9. Put the absorption column in a clean centrifuge tube (volume of 1.5 mL). Add 50 μL of Elution buffer. Stands it for one minute, and then centrifuge it for one minute (7,000 x g). Keep the solution containing DNA in the centrifuge tube and label it with “pET28a” (name of the plasmid we used).

 

Double Enzyme Digestion

Overnight enzyme digestion at 37 ℃

 

Ligation

 

Transformation

1. Carefully add the recombinant plasmids to the E.coli (DH5α/BL21) in the tube at the Super clean bench.

2. After sealing the tube. Put the tube in an ice bath for 30 minutes.

3. Put the tube on a water bath (42 ℃) for 90 s.

4. Put the tube above the ice bath for 5 minutes.

5. Add LB culture medium (add until there is a total of 1 mL of solution in the tube).

6. Put the tubes in the shaker (220 rpm, 37 ℃) for 1 hour.

7. Centrifuge the tubes (9,000 turns per minute), and then absorb 950 mL of the LB solution (at the super clean bench).

8. Carefully and gently mix the bacteria with the solution. Smear the bacteria on the growth medium.

9. Put the petri dish in the incubator (37 ℃) overnight.

 

IPTG induction

1. Add antibiotics (kanamycin), E. coli to the culture medium (with the given ratio).

2. Shake the mixture for 3-4 hours until OD₆₀₀ reaches 0.6.

3. Add IPTG to achieve a final concentration of 0.25mM and incubate overnight in a 18℃ shaker.

 

Nickel Column Purification

His-A solution composition (0.5 L, pH = 7.4)

Reagent	mass
Na2HPO4	20 mM (1.78 g)
NaCl	500 mM (14.6 g)
Imidazole	20 mM (1.02 g)

 

His-B solution composition (0.25 L, pH=7.4)

Reagent	Mass
Na2HPO4	20 mM (0.89 g)
NaCl	500 mM (7.3 g)
Imidazole	500 mM (8.5 g)

 

*The sample (proteins) need to be kept in the ice box

Procedure

1. Add 10 mL of His A solution to wash the nickel column.

2. Add 10 mL of solution (proteins), filter it. Label the 10 mL collection tube with “through.”

3. Add 10 mL His A solution, filter it. Use the 10 mL filter pipe to collect the solution and label “wash.”

4. Add 1 mL His B. Close the lids at the bottom of the nickel column. Shake the column and then collect the solution with a 2 mL EP tube.

5. Add 1 mL of His B solution again. Collect the solution filtered to the 2 mL tube (from step 4)

6. Add 5 mL of His B solution to wash the nickel column.

7. Add 5 mL of His A solution to wash the nickel column.

8. Add 10 mL of methyl alcohol solution to wash the nickel column.

 

SDS-PAGE electrophoresis

1. Insert the prepared gel (12% BT) into the electrophoresis chamber.

2. Add running buffer until the chamber is submerged.

3. Mix the protein sample and the loading buffer.

4. Add 1.5 μL of the marker to the first hole of the gel. Add 10 μL of protein sample in the following holes.

5. Set up the electrophoresis program.

Step 1: 80 V, 30 minutes

Step 2: 120V, 60 minutes

6. Start the program.

 

Coomassie Brilliant Blue staining and destaining

1. Prepare a Coomassie Brilliant Blue staining solution by mixing an appropriate volume of Coomassie Brilliant Blue dye with methanol and acetic acid and water. The final staining solution contains 40% methanol, 10% acetic acid.

2. Carefully transfer the gel into a container filled with the staining solution. Gently shake the container or use a rocking platform to ensure even staining.

3. Incubate the gel in the staining solution for 1 hours.

4. Prepare a destaining solution by mixing an appropriate volume of methanol, acetic acid, and water. The exact composition may vary based on the destaining kit used.

5. Transfer the gel into a container filled with the destaining solution.

6. Gently shake the container or use a rocking platform to facilitate destaining.

7. Replace the destaining solution every 30 minutes until satisfactory background clarity is achieved. This may take several hours or overnight.

 

Evaluation of Deletion Chamber

 

Deletion Chamber Production and Usage

Making Nickel beads for deletion chambers

Materials: Nickel beads, PBS, sTF and bTF solution at 100 ng/mL and 10 ng/mL respectively.

Steps:

1. 262.5 μL Siglec-1 (10 μg/mL) is added to solution of Nickel beads

2. Wash the Nickel beads 3 times with 1 x PBS solution. The removal of supernatant is by centrifugal of 50 x g for 30 s.

3. Total of 525 μL of overnight cultured Nickel beads were centrifuged at 50 x g for 30 s. PBS was used to wash the beads three times.

4. After the final wash, the beads were diluted in approximately 340 μL of 1x PBS solution (expected total volume of about 350 μL) and shaken well.

5. The magnetic beads were divided into 11 EP tubes, and each tube was filled with 90 μL of 1x PBS. Add 20 μL Nickel beads after thorough shaking.

A total of 11 Deletion Chambers (DC) were made, each with 110 μL Nickel beads in PBS.

 

Preparation of the solution concentration in following experiment

1. Prepared 700 μL sTF solution (1000 ng/mL, 100 ng/mL, 10 ng/mL, 1 ng/mL, 0.1 ng/mL) respectively, which will be used ELISA experiment.

2. Prepared 500 μL 100 ng/mL sTF solution and 500 μL 10 ng/mL bTF solution.

3. Discharged and mix 250 μL 100 ng/mL sTF solution with 250 μL 10 ng/mL bTF solution.

 

Test the Function of  Deletion Chamber

1. Add 190 μL of sample to the tube with 10 μL centrifuged Nickel beads, and there is totally 200 μL solution in tube.

2. Wait for 30 minutes for deletion to perform.

3. For ELISA, transfer 100 μL of solution. For the same concentration solution that used colloidal gold (AuNP) experiment, transfer the rest of 90 μL of solution.

Totally, 8 solutions are passed through Deletion Chamber (DC), which includes:

sTF: 1000 ng/mL, 100 ng/mL, 10 ng/mL, 1 ng/mL, 0.1 ng/mL

bTF: 10 ng/mL (Elisa + AuNP)

sTF: 100 ng/mL (Elisa + AuNP)

sTF + bTF: 100+10 ng/mL (Elisa + AuNP)

 

ELISA standard curve

Reagent consumables: ELISA kits, sTF, Nickel beads.

1. Samples of sTF concentration is set in maximum of 1,000 ng/mL is because the ELISA assay can only provide result in range 0 nmol/L to 1.5 nmol/L of Transferrin.

2. Diluent for sTF is exactly the same with bTF, provided by the ELISA producer.

Sample preparation for ELISA evaluation：

B-samples containing bTF; S-samples containing sTF

450 μL or 500 μL solution for sTF for ELISA and further experiments, i.e. colloidal gold experiment.

 

1. Make 1 mL of standard solution of sTF (2 mg/mL), take 250 μL of this solution, dilute it to 1,000 ng/mL to a total volume of 500 μL by adding diluent of 250 μL. Label it as S1.

2. S2 consists of 450 μL diluent and 50 μL S1 to obtain 100 ng/mL solution.

3. Repeat step 3 for making S3-S5 and then by Deletion Chamber. The solution sTF concentration is as following:

 

Tube: sTF	S1	S2	S3	S4	S5
ng/mL	0.1	1	10	100	1000

 

The dilution process for the standard curve goes as following, to obtain bTF concentration in

 

Tube: bTF	B 1	B2	B3	B4	B5	B6	B7
nmol/L	0	0.047	0.094	0.187	0.375	0.75	1.5

 

The Standard solution contains concentration of B1. Transfer 300 μL of the solution to S1. Use 150 μL from B1 to B2 and add 150 μL diluent. Repeat this procedure until B6, where the procedure is x2 dilution each time. B7 is empty, consisting solely diluent.  

 

The test samples named sTF, sTF + DC (which through the Deletion Chamber), bTF, bTF + DC

ELISA (Enzyme Linked Immunosorbent Assay)

1. Prepare the micro-plate

Plate 1:

B1	sTF
B2	bTF
B3
B4
B5
B6
B7

 

Plate 2:

B1	S1
B2	S2
B3	S3
B4	S4
B5	S5
B6
B7

 

Plate 3:

B1	bTF
B2	sTF
B3	bTF + DC
B4	sTF + DC
B5
B6
B7

 

2. Add 100 μL of solution to the well in the order stated on the diagram above. Cover the plate with adhesive membrane. Incubate it at 37 ℃ for 60 minutes. Remove the liquid and add 200 μL of wash buffer. Wait for 2 minutes, and then wash off the liquid.

3. Repeat the above step for 2 times. Invert the ELISA plate on the filter paper. Tap the plate gently to remove the remaining liquid.

4. Add 100 μL of HRP-conjugate (1x) to each well. Cover the plate with adhesive membrane. Incubate the plate at 37 ℃ for 60 minutes.

5. Remove the liquid and add 200 μL of wash buffer. Wait for 2 minutes, and then wash off the liquid. Repeat the washing step stated previously for 4 times.

6. Add 90 μL of TMB (the chromogenic substrate adopted in this experiment). Incubate the plate at 37 ℃ for 20 minutes in a darkroom.

7. Add 50 μL of stop solution to each cell to terminate the reaction. Tap the plate gently to ensure that it is mix adequately.

8. Within 5 minutes. Measure the plate at a wavelength of 450 nm.

 

Evaluation of the Test Strip with Deletion Chamber

Colloidal gold-antibody evaluation

Materials:

Linked streptavidin-gold nanoparticles (referred to as streptavidin-AuNP in the content below), 1 x PBS, 0.1 M borate buffer solution, silk protein saver, bTF solution, Tween 20, TX 100, anti-TF antibody (CSB-PA00250H0Rb) conjugated by biotin (referred to as sandwich secondary antibody below).

 

Preparation of reagents:

Adapter solution: Prepare 1 mL of adapter solution A by adding 10 mg of silk protein saver, 4 mg of Tween 20, and 50 mg of TX 100.

10 mM borate buffer solution: Prepare 10 mM borate buffer solution by dissolving 10 mL of 0.1 M borate buffer in 90 mL of ddH₂O.

Protein protectant: Prepare silk protein saver at a concentration of 50 mg/mL by dissolving 1 g in 20 mL.

 

Coupling of Colloidal Gold and Antibodies

Purpose of the experiment: preparation of results demonstrating bTF/sTF detection on NC membranes.

 

Preparing Colloidal Gold-Antibody

Materials: nanogold OD1 solution, 0.1 M borate buffer solution and 10 nM borate buffer solution (diluted 10 times with 0.1 M, approximately 5 mL), protein protective agent solution (50 mg/mL dissolved in ddH₂O, 200 μL), adapter solution (1 mL 1 x PBS, containing 50 mg/mL trehalose, 2 mg Tween 20, 5 mg protein protective agent, 10 mg Triton-X 100), 1 x PBS (1 mL), anti-TF polyclonal antibody (liquid phase) (1 mg/mL, 10 μL)

Steps:

1. 1,000 μL of streptavidin-AUNP was added to an EP tube, followed by the addition of 100 μL of borate buffer, and then 2 μL of anti-TF polyclonal antibody (1 mg/mL).

2. The mixture was incubated at room temperature for 30 minutes, and then 100 μL of protein protective agent was added.

3. The mixture was incubated at 4 ℃ for 1 hour, and then centrifuged at 13,475 g for 20 minutes at 10 ℃. Using a pipette, remove the supernatant.

4. 1 mL of 10 nM borate buffer solution was added, and the mixture was centrifuged again at 13,475 g for 20 minutes at 10 ℃.

5. Repeat step 4 for two times.

6. 550 μL of adapter solution was added to prepare 2x colloidal gold-antibody solution.

7. The working concentration was 1x (550 μL of 1 x PBS was added to obtain approximately 1,100 μL of 1 x colloidal gold-antibody solution).

 

 Further preparation of gold nanoparticles

(Performed in a 2 mL EP tube)

1. Mix 100 μL of streptavidin-AuNP, 100 μL of borate buffer, and 10 μL  (1 mg/mL) solution.

2. Incubate at 25 ℃ for 30 minutes.

3. Add 100 μg (50 mg/mL) protein saver.

4. Incubate at 4 ℃ for 60 minutes.

5. Centrifuge at 13,475 g for 20 minutes at 10 ℃.

6. Resuspend in 10 mM borate buffer solution and centrifuge again.

7. Discard the supernatant and repeat steps 4-5 for 2 times.

8. Add 550 μL of 1 x PBS and 550 μL of adapter solution.

 

Preparation of the NC membrane Test strip

1. Cut six strips of nitrocellulose (NC) membrane, each measuring 5 cm in length and 1cm in width.

2. Align and vertically add a 40 μL anti-Transferrin antibodies to each NC membrane using micropipette, 1 cm away from the right edge.

3. Wait until the Antibodies are drying.

 

Evaluation of colloidal gold test

Samples that are going to react:

sTF: 100 ng/mL

bTF: 10 ng/mL

sTF+bTF: respectively 100 ng/mL, 10 ng/mL

 

All three will have 90 μL of it untreated, 90 μL are from the three solutions that has been treated by Deletion Chamber.

 

1. Mix 50 μL colloidal gold solution, then 50 μL sample is added into the tube to make a 200 μL mixture.

2. React in room temperature for 30 minutes.

3. When waiting, drop 2 μL antibody (0.25 μg/mL) in a line ten times on the NC membrane.

4. Apply the solution to 0.1 cm to the line of antibody.

5. Wait and see whether there is an existence of color bands or not after waiting 5 minutes.

 

Fluorescence tracing

1. Prepare your sample and mount it onto a glass slide using an appropriate mounting medium. Place the prepared slide onto the stage of the fluorescence inverted microscope.

2. Turn on the microscope and adjust the light source to the desired excitation wavelength of 587 nm.

3. Adjust the focus using the coarse and fine adjustment knobs until the sample comes into view. Set the appropriate filter cube for the excitation wavelength of 587 nm.

4. Adjust the exposure time and gain settings on the camera to optimize image quality. Acquire the images by activating the camera or software capture system.

5. Switch to the emission wavelength of 610 nm by changing the appropriate filter cube.

6. Adjust the focus and image settings accordingly for the emission wavelength. Acquire the images of the NC membranes.