Difference between revisions of "Team:BIT/Demonstrate/Bio-sensor"

  

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:
   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

  ii.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
   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

Figure 10. Comparison the bonding strength with AFP and complementary chain.

iv.Analysis and discussion:
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

Figure 11. Serum samples to be tested
Figure 12. Serum sample detection
Figure 13. Serum samples were added to the fluorescent plate
  

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.