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Under the condition of primer ratio of 20:1, asymmetric PCR products were obtained. Electrophoresis was performed at 130V for 30min to obtain the test gel pattern. (Fig 1)
Except for marker, the first four channels are NA chains （none aptamer chain）and the last four channels are AP (chain with aptamer)chains. The length of NA double strand is 206bp, while the length of AP double strand is 139bp. The position of double chain is undoubtedly correct, but the movement of single chain can not be judged by marker alone. To make sure we get the right single chain, we used the same asymmetric PCR method to obtain complementary chains of NA and AP ssDNA. We put the NA chain and its complementary chain together, 98 ℃ for 5min, and cooled naturally in a water bath to get recombinant double chains. So was the AP chain. We performed an electrophoresis to compare the original NA and AP double strands and the recombinant double strands. (Fig 2)
The left two channels are original AP and NA chains, the next two channels are recombinant chains. From the figure we can find that the two NA chains are at the same position, the two AP chains as well. It proves that the single strands obtained by asymmetric PCR are correct.
Positive normal template
Before the formal experiment, in order to test the resolution ratios that the RPA kit can achieve, we carried out RPA experiment with the positive normal templates. (Fig 3)
When the concentration of positive normal templates is 0.4pM, the curve can be significantly different from that of no template chains. Besides, the RPA kit can detect a difference of 0.3pM. The resolution required for our experiment is within the range that the kit can achieve, which indicates that our experiment can be continued.
We use fluorescence quantitative PCR instrument to detect the real-time fluorescence signal of our system. Every 20 seconds the signal strength will be recorded. According to our design, the template can be amplified by enzymes in the absence of aptamer. In order to verify the correctness of this design, we selected different concentrations of aptamer-free template for RPA amplification and got the fluorescence intensity curves with time change. (Fig 4)
The concentrations of template chains are 10-8, 10-9, 10-10, 10-11, 10-12 and 10-13 mol/L. The lowest curve represents blank control group. It can be seen from the figure that except for the 10-12 and 10-13 groups, the fluorescence intensity of template chains with different concentrations is obviously different, and the signal is positively correlated with chain concentration. The two nearly overlapping curves indicate that fluorescence signal intensity cannot show significant difference when the concentration of template chain is too low, so we only selected the data with concentration of 10-12mol/L for analysis in these two groups. All other curves are above the blank control group, confirming the validity of those data.
According to our design, the probe can combine with complete template chain obtained from RPA. At the same reaction time, the higher the concentration of template chains is, the more sites the probe can bind, which leads to a higher signal. Our image fully illustrates that the vacant part of template chain will be filled, and complete AP complementary chain will be obtained under the action of chain 1 primers and enzymes, so that the binding site of chain 2 primer will appear. In other words, the image shows that the template can amplify without block and the difference in fluorescence intensity is consistent with our hypothesis.
To confirm that the aptamer can bind to the single-strand portion of template and block the amplification of template chain in the absence of tau, we conducted a comparative experiment with different concentrations of aptamers and without aptamers. (Fig 5)
The concentrations of the template chains we chose are still from 10-8 to 10-13 mol/L. In the groups with aptamers, we added twice the template concentration of aptamers to ensure more template bind aptamers. The gray curve is aptamer-free while the red curve is with aptamer, and the blue curve is blank group. We can easily make a distinction between the curves representing aptamer and aptamer-free. At any concentration, the curves representing aptamer-free is above the curves representing aptamer, while the blank control group is the lowest in the figures. Both curves are above the blank control group, proving that our data are valid. It means that the aptamer can successfully bind to the template strand, and the block can inhibit the DNA polymerase from completing the template strand. The amplification reaction is blocked and reflected in the fluorescence signal intensity.
Those figures above preliminarily prove the feasibility of our experiment. According to experiment data, when the concentration of template is 10-10mol/L, the comparison between the presence and absence of aptamers is obvious enough, and there is no need to select template chain with a higher concentration. Therefore, further experiments on Tau protein were carried out under the condition of template chain of 10-10 mol/L. In addition, it is proved that the experiment can achieve good results under the condition of two-equivalent aptamer, so the two-equivalent aptamer was also selected in the subsequent experiments.
According to our experimental design, when tau protein is present, it will bind to aptamer with block so the aptamer will release from the template chain. Then RPA reaction begins. Therefore, we added different concentrations of tau protein into the reaction system for experiments (Fig 6).
The experimental results shows that the fluorescence intensity will increase as the tau protein concentration grows. This suggests that tau protein can bind to aptamer with blocks and take the aptamer and blocks away from the NA and AP chains. It can be seen from the figure that when the concentration of tau protein is between 4pg/ μL and 6pg/ μL, the fluorescence intensity of the reaction takes a leap. We suspect that this is because tau protein may bind to the aptamer sequence on the template chain, resulting in the change of nucleic acid conformation. This will promote the opening of hydrogen bonds, which to some extent plays a role in unzipping the DNA. This phenomenon is especially obvious when the concentration of tau protein is high enough.
To verify that our design can be used in a real blood test, we replaced water with a serum buffer. Under the same condition, we got the figure of different concentrations of tau protein and fluorescence intensity. (Fig 7)
From top to bottom, the concentration of tau proteins represented by the curves decreases sequentially. The fluorescence intensity at 20 min into the reaction was plotted against the concentration, and a good linear relationship was obtained. This result shows that our design is not just a theory. The device is not limited to aqueous laboratory solutions, but can be used in actual blood tests. In the actual test we can use the standard curve (Fig 8) for quantitative determination of proteins. This is important for the early detection of Alzheimer's disease because it allows for the detection of blood markers on a large scale with extremely fast response times and precise measurement accuracy.
Compared with ELISA
The concentration of tau protein was detected by ELISA kit and we obtained the relation between the absorbance of the system and the concentrations. (Fig 9)
The liner relationship between absorbance and protein concentration is excellent, indicating that ELISA kit can accurately determine the concentration of tau protein, and its detection limit can reach at least 7.81pg/ mL, which is beyond our reach at present. However, it takes a long time about 3-4h with high requirements for the operator and every test costs a lot. Our method has the advantages of rapid detection, simple operation and low expense. According to our experiment results, good effects can be achieved after about 20 minutes of the reaction. Significant differences can be shown in the groups with different concentrations of tau protein added. Compared with ELISA kit, the two biggest advantages of our device are simple operations and short reaction time.
At present, our experiment is limited by the affinity between tau protein and aptamer, so the minimum concentration of tau protein that can be measured by our device is limited, and the accuracy of the experiment will also be affected. Currently, the aptamer sequence we use are obtained from the literature, and the affinity with tau protein is not the highest value that the aptamers can reach today. Besides，the head and tail of the aptamer we are using are complementary, which easily leads to dimerization and even polymerization of the aptamer, affecting the experiment results. So this aptamer is not the best choice. If we can get new aptamers with a higher affinity through screening, the minimum protein concentration we can measure will continue to decrease and that we can achieve higher detection accuracy.