2017.igem.org/Team:BIT/demonstrate/biosensor

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Part 1:The specific binding of AFP to AP-273

The main experimental material:

Purified AFP protein, HRP-labeled Streptavidin, Aptamer AP273, BSA block buffer (Bovine Serum Albumin solution), PBS buffer , TMB Single-Component Substrate solution

Clinical significance:

Experimental principle:The protein(AFP) was incubated on a 96-wells plate at 37 ℃ for 2 h ,then remove the excess protein that is not fixed on the 96-wells plate, and then injected the aptamer(AP273) into the correspond orifice plate and incubated at 37 ℃for 2 h, continue to wash out the aptamer which does not bind to the AFP, at next step we injected HRP- streptavidin into the plate, HRP -streptavidin binds to the aptamer at 37 ℃ for 1 h.

Then wash away excess HRP -streptavidin, add TMB in the dark for color reaction.Finally, the absorbance of TMB color reaction was measured by microplate reader.

Detection equipment: microplate reader

Main steps of our Experiment:
① coated: take 20μL (10mg / ml) Purified protein(AFP), then take 1980μL coated buffer, 100μL per well to add in 96-wells plate, Incubate in a 37 ℃ incubator for 2 hours. ② emptied and dry the residual liquid, and washed the 96-wells plate twice with 300μl of Washing buffer. ③ Added 300μL (5 mg / ml) of BSA block Buffers to each well and allowed to stand at 4 ℃for 1 hour ④ discard the liquid, wash the plate with 300μl washing-buffer 3 times, during the last time after washing empty the liquid and pat dry ⑤ The aptamer was arranged into diluted solution of 0.2,0.4,0.6,0.8,1.0μmol/L in concentration, 100 μl of the appropriate concentration of the aptmer was added to 96-wells .The blank group was added with TE buffer, and the above substances were incubated at 37 ℃for 2h. ⑥ discard the liquid, wash the plate with 300μl washing-buffer 3 times, during the last time after washing empty the liquid and pat dry. ⑦ Added 100 μl 1: 2000 diluted HRP- streptavidin to the 96-wellss and incubated at 37 ° C for 1.5 h. ⑧ discard the liquid,wash the plate with 300 μl of washing buffer three times, then soak with 300 μl of rinse buffer for 5 min,then empty the liquid and pat dry,and then wash the plate with 300μl washing buffer 2 times,during the last time after washing empty the liquid and pat dry. ⑨ Added 10μL of TMB solution to 96-wells. After 20 mins of color development in the dark, added 50 μL stop solution to terminate the reaction immediately. After the reaction was stopped, read the absorbance value at 450 nm.

Part 3: The deprotection of Boc-Lysine

Methouds:

In this part we choose TEA(Trifluoroacetate) to realize the deprotection of Boc-Lysine Tert-Butoxycarbonylaminocaproic acid (boc-lys, 4g) was added to a mixed solution of trifluoroacetic acid (5 ml) and dichloromethane (10 ml), and the reaction was terminated after stirring for 1 hour under a room temperature. And then evaporate impurities under rotation conditions. The remaining material was dissolved in an appropriate amount of ethyl acetate (5 ml), washed with 5% Na2CO3 (25-30 ml) solution to pH 8-9, And then we removing the solvent by rotary evaporation to get the lysine.

Analysis and discussion:

The data "345.2062" in Figure 1 is the mass ratio of boc-lysine before the reaction. Figure 2 shows the mass spectrum of the product after "THF deprotection" reaction. It can be seen that the peak of "345.2062" in figure1 disappears, and instead appears a strong peak“145.0922 " in figure 2(that is, the product contains lysine), indicating that the deprotection experiment was successful, we get the deprotection of lysine.

Part 4: Linear relationship between AFP and lysine

Methouds:

1. Resuspend the in the vial (i.e. vortex for >30 sec, or tilt and rotate for 5 min). 2. Transfer the desired volume of Dynabeads to a tube. 3. Add an equal volume of Washing buffer, or at least 1 m L, and mix (vortex for 5 sec, or keep on a roller for at least 5 min). 4. Place the tube on a magnet for 1 min and discard the supernatant. 5. Remove the tube from the magnet and resuspend the washed Dynabeads in the same volume of washing buffer as the initial volume of Dynabeads taken from the vial (step 2). Immobilize Nucleic Acids: 1. Resuspend beads in 2X B&W Buffer to a final concentration of 5 µg/µL (twice original volume). 2. To immobilize, add an equal volume of the biotinylated AP273 in distilled water to dilute the Na Cl concentration in the 2 B&W Buffer from 2 M to 1 M for optimal binding. 3. Incubate for 15 min at room temperature using gentle rotation. 4. Separate the biotinylated AP273 coated beads with a magnet for 2–3 min. 5. Wash 2–3 times with a 1X B&W Buffer. 6.Resuspend to the desired concentration. Binding is now complete., suitable for downstream applications. Combination of Apt and Fluorescent Complementary Chain: 1. Take 10 μl of the above-mentioned washed magnetic beads to add to 12 1.5 ml centrifuge tubes 2. Add 120 μl of 1 μmol / L of the fluorescent complementary strand and 40 μl of 1 μmol / L of AP273 to each of the centrifuge tubes 3. Heated the mixed solution from room temperature to 90 ℃and then cooled to room temperature. At this time, the fluorescent complementary strand is bonded to the aptamer by hydrogen bonding. 4. Place the mixed solution on the magnetic frame for 5 min, discard the supernatant, and repeatedly wash on the magnetic frame to remove the unbound complementary 5. And then added the concentration of 2,4,6,8μg / ml of AFP solution to 12 centrifuge tubes .incubated for 2 hours at 37 ℃ , place every tube in the magnetic frame for 5min, absorb 100μl of supernatant from every tube. 6. Measured the fluorescence value at an absorption wavelength of 492 nm and an emission wavelength of 518 nm.

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