TEAM/BIT/BS1
Demonstrate
Part 1:The specific binding of AFP to AP-273
Part 2:The binding of the complementary strand to lysine (Lys)
Part 3: The deprotection of Boc-Lysine
Part 4: Linear relationship between AFP and lysine
Part 1：The specific binding of AFP to AP-273:
i.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
ii.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.
iii.Detection equipment:
microplate reader
iv. 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.
v.Test results:
vi.Analysis and discussion:
The figure above is the experimental results obtained by TMB colorimetric method.we can see When the amount of AFP is constant (or excess), the OD value increases with the increase of Apt concentration. From the fitting degree of the graph, we know that this two substances Concentration has a linear relationship to some degree . This data validates the binding special ability between AFP and aptamer AP273.
Part 2：The binding of the complementary strand to lysine (Lys):
ii.Main steps of our Experiment:
i.Experimental principle:
With the existence of one molecules contains an amine and the other contains a carboxylate group, amino and carboxyl groups condensates can be formed using EDC.N-substituted carbodiimides can react with carboxylic acids to form highly reactive, o-acylisourea intermediates.This active species then can react with sulfo-NHS to form the carbonyl compound with ester functional group ,which can continue reacting with a nucleophile such as a primary amine to form an amide bond. Sulfo-NHS esters are hydrophilic reactive groups that couple rapidly with amines on target molecules.
Figure 3. EDC-mediated principle
1.Dissolve 1 mg L-lysine in 1ml of activation buffer(0.1M MES,0.5M NaCl,pH6.0),at a concentration of 1mg/ml.
2.Add coupled reagent EDC 0.4mg and sulfo-NHS 0.6mg to the solution in step 1,at a concentration of 0,4mg/ml of EDC and 0.6mg/ml of sulfo-NHS.
3.Shake up and react for 15min in dark at room temperature.
4.Add 1.4ul β-mercaptoethanol to the reaction solution.Mix and incubate for 10min at room temperature to neutralize extra EDC.
5.Add 10 ul amino-modified complementary strand(100uM) to coupling buffer(phosphate buffer 100mM sodium phosphate,150mM NaCl,pH 7.2)
6.Adjust the pH of the activation buffer over 7.0.
7.Mix the activation buffer with the coupling buffer,shake up and react for 2 hours in dark at room temperature.
8.Use ultrafiltration to concentrate and purify the solution after the end of the reaction.
9.Using mass spectrometer and infrared detector for testing.
iii.Test results:
vi.Analysis and discussion:
Figure 1 shows the mass spectrum of pure complementary strand samples. It can be seen from the figure that the nucleo plasmic relation of the complementary strand is between 882.5255-908.5209, and Figure 2 is the result of the product after coupling. It can be found that the nucleo plasmic relation of the BOC-Lys is 373.2497, the complementary strand is 901.5145 and the coupling product is 1766.0513. In order to ensure the accuracy of the experimental results, infrared spectroscopy detection was additionally used. The results shown in Figure 3. The absorption peak was appeared at 1650 cm-1 showing the existence of amide bond. The result confirms that lysine is indeed coupled successfully with the complementary strand.
Part 3：The deprotection of Boc-Lysine:
ii.Analysis and discussion: Part 4：Linear relationship between AFP and lysine iv.Analysis and discussion:
i.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.
Figure 7. The Mass Spectrum Result of Boc-Lys(Boc)-OH
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.
i.Preparation of the beads:
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).
ii.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.
iii.Combination of Apt and Fluorescent Complementary Chain：
1Take 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.
iii.Test results：
i.Serum sample test results
From the results we can see that with the increase of AFP content, the fluorescence intensity of the supernatant also increased, and in a certain range has a good linear relationship.
Extended
ii.Substituted lysine with lysine peptide
The idea of replacing AFP with a single lysine has been achieved to some extent, but the experimental results show that if only one to one ratio is replaced, 0-3μg / ml has a linear relationship. In order to be able to reduce the detection limit, the project intended to use 10 lysine-formed lysine polypeptide chain instead of a single lysine coupled to the complementary chain. The advantages are as follows: 1.In theory, the detection limit can be reduced to one-tenth of the previous.
2. Trypsin acting on the lysine polypeptide chain is much more efficient than the usual amino and carboxyl groups formed by the amide bond .
iii.Coupling the carboxyl group with the amino group by coupling the mercapto group with the amino group
In the course of the experiment, we found that in the process of deprotection of TFA, a part of the lysine with a successful ligation resulted in the breakage of the amide bond, which resulted in a decrease in the coupling product and an increase in the cost of the whole experiment and production. This condition is not very stable. Therefore, the project is intended to use mercapto and amino coupling, can enhance the coupling.The stability of the fruit, and the lysine connection is more simple.
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