1.The conjugating of GNPs-aptamer
The main measurement in our project are preparation of GNPs-aptamer and preparation of test probe &control probe. They are one of the most important parts in our test paper that used for detecting C4-HSL molecule.
Preparation of GNPs-aptamer:
GNPs-aptamer is used for detecting the C4-HSL molecule, we explored an approach to let gold nanoparticles(GNPs) successfully conjugate with aptamer, which included these processes.
1.The activation of aptamer · Adding 3 μL 100 μM SH-aptamer with 3 μL 5 mg/mL TCEP in a centrifuge tube(the volume of TCEP and GNP should be 1:1). Then the centrifuge tube was put in 4℃ for 2h.
2.Conjugation of GNP and aptamer1 ml GNP solution was mixed with 0.2 M potassium carbonate in order to change its PH value to 8.5. ·Centrifuging this solution in 10000rpm for 10 minutes, then removing 800μL supernatant. ·Shaking the GNP solution for 2 minutes. ·4μL Activated aptamer was added to the centrifuge tube that contains GNP, the final concentration of aptamer in the solution is 1 μmol/L. ·Shaking the centrifuge tube for 12h in dark environment, room temperature.
4.Removal of excess aptamer ·Centrifuging the solution in 10000rpm for 20 minutes. ·Adding 200 μL resuspension buffer which is composed of 0.01%PBS, 0.5% PEG, 5%sucrose, 0.75% Tween-20, 0.02%MgSO4, 0.05%(NH4)2SO4, and 1%OVA(1).
Preparation of test probe &control probe:
Control line in our test paper can show whether the paper is working, it is composed control probe, test line can show whether the sample contain C4-HSL, it is composed of test probe. The preparation of these two probes has the following processes.
1.Preparation of the test probe ·Mixing 35μL of 10μM DNA probe 1 with 35μL of 1mg/mL streptavidin ·Placing the solution in 4℃ for 2h ·30μL PBS buffer was added to this solution
2.Preparation of control probe ·Mixing 35μL of 10μM DNA probe 2 with 35μL of 1mg/mL streptavidin ·Placing the solution in 4℃ for 2h ·30μL PBS buffer was added to this solution
1.Since aptamer can protect GNP after conjugating with it, when 17.5μL NaCl was added gradually, the GNPs-aptamer solution it can still have red colour. (As shown in figure1,2,3,4,5,6,7,8)
Control group (aptamer was replaced be ultra-pure water）
Fig 1. Adding 4times, 8times,12times, and 17 times NaCl (from left to right respectively) for control group
Fig 2. Adding 4times, 8times,12times, and 17 times NaCl (from left to right respectively)for experiment group
2.After sample was added to the sample pad, GNPs-aptamer can cnojugate with DNA probe in control line and show red colour.
(As shown in figure 3)
(Since we added water to the sample pad, GNPs-aptamer could accumulate in control line and test line and show red colour)
2.SYBR Green Ⅰ (SG) fluorescence method protocol
We used the SYBR Green Ⅰ（SG）fluorescence method to test the function of aptamers. The principle of this method is SG acts as a DNA fluorescent dye that does not carry a fluorescent signal of its own (Abraham et al., 2018). When bound to the aptamer, it fluoresces because it is embedded in the secondary structure of the DNA. Thus when aptamer is bound to the SG dye, green fluorescence is produced when stimulated by excitation light（with the excitation Wavelength 485 nm and emission wavelength 535 nm). However, when the target small molecule is added to the system, the specific binding of the small molecule to the aptamer results in competition with the SG for the binding site, and as a result, the fluorescence value of the system decreases.
In our project, the protocol we used for the method was as follows (using C4-HSL as example):
- 1. Preparing reaction buffer(150 mM NaCl, 25 mM Tris-HCl, 25 mM KCl, 20 mM MgCl2, and pH = 7.4).
- 2. The SYBR GreenⅠ (SG) was diluted from 10000× to 100× for later use.
- 3. Preparing the 10 μM C4-HSL for later use.
- 4. Preparing 96 well plates, and added the aptamer，100× SG and C4-HSL in the well, and final volume was 100 μl with filling by buffer.
- 5. Since our experiment was gradient experiment，we set the final concentration of C4-HSL as 0,100, 200,400, 600, 800 and 1000 nM of which the 0 condition as control group. The final concentration of aptamer was 100 nM and SG was 1×. The volume of each element we add can be seen below. 3 biological duplication for every group.
- 6. The reaction took place suddenly，the fluorescence intensity can be recorded by the microplate reader with with the excitation Wavelength 485 nm and emission wavelength 535 nm.
The results of our experiment can be seen in figure 4 . It was obvious the control group (when C4-HSL was 0 nM) had a higher fluorescence intensity than the others and with the increase of C4-HSL, the fluorescence intensity decreased. So we proved that the protocol could be also used in other similar experiments.
Fig 4. The fluorescence quenching effect by C4-HSL
- Zhou, W., et al., An aptamer based lateral flow strip for on-site rapid detection of ochratoxin A in Astragalus membranaceus. J Chromatogr B Analyt Technol Biomed Life Sci, 2016. 1022: p. 102-108.
- Abraham, K.M., et al., In Vitro Selection and Characterization of a Single-Stranded DNA Aptamer Against the Herbicide Atrazine. ACS Omega, 2018. 3(10): p. 13576-13583.