Electrochemical synthesis polyaniline membrane
The polyaniline film was constructed as described in Section 2.2 after the gold electrode was cleaned with nitrogen. The result of film formation in our study was achieved using chronoamperometry method. Two potentials, 0.5 V and 0.8 V, were used for 20 seconds each. Each of the potential was stimulated for 8 times, for a total of 16 cycles, and the time setting was decided by the amperometry method which was tested before with ideal thickness. The average time of synthesis by amperometry method was 160 seconds. We doubled the overall film forming time to last for 320 seconds.
The current graph shows that a pulse current would be generated at the exactly the time of potential switch. Fig. 2a shows that the potential rise would generate a positive current, and the potential drop generates a negative current. The current intensity increased with polyaniline film formation and gradually increased to 0.01 A, which was constant from that time onwards, meaning that the film formed has good conductivity and sustainability. It can be seen in the Fig. 2b that the frequency dropped sharply at 0.8 V, while the frequency dropped at relatively slower rate at 0.5 V, appearing in a ladder shaped drop on the delta frequency graph . After forming the polyaniline film, cyclic voltammetry was used to check its conductivity.
The polyaniline film was formed as shown in Fig. 2c. Electrode surface was green in color, and appeared as a thin green membrane visible with naked eyes. After PANI film was formed on the electrode, cyclic voltammetry was used to confirm the conductivity. Fig. 2d shows that the polymer conductivity performed very well. Comparing this film with bare gold electrode and PANI generated by other method (Fig. 2e), it could be inferred that our PANI film had higher current conductivity than bare gold electrode and more stable current with negligible shift as compared to other method. This shows that using chronoamperometry to generate PANI films had good conductivity and stability, which is good enough to be used as a hippocampus gyrus biomimetic device.
Positive potential attach p-tau
In addition to the fact that proteins are generally negatively charged, phosphorylated tau are more negatively charged [26, 27]. In this part, we used positive potential as an auxiliary tool for p-tau adsorption, and compared with other proteins for natural deposition on the membrane. An additional positive potential stimulation was provided to observe whether the adsorption could be enhanced, and hence, to prove that positive potential stimulation can induce the adsorption of phosphorylated tau.
Cyclic voltammetry can be used as a preliminary test for potential adsorption of proteins [28, 29]. Therefore, we first added 1 µl of phosphorylated Tau (1 mg / ml) to the electrolytic cell and dissolved in 3 ml of ultrapure water and used cyclic voltammetry to sweep the positive potential range. These results and oxidation and reduction peak potentials generated from the solution mentioned before could be used together to establish a preliminary qualitative data for adsorption. As shown in Fig. 3a, after cyclic voltammetry sweeps the positive potential ranges from 0.1 V to 0.8 V going back and forth in this range. The range between 0.3 V to 0.6 V seem to be a potential selection choice. The current oxidation peak was between 0.5 V and 0.6 V, and there was a downward trend in the sweep from 0.6 V to 0.3 V during frequency change. So, the positive potential range was restricted from 0.3 V to 0.6 V, and this result was used to do further fixed potential adsorption tests.
After the PANI membrane was formed, four potentials were selected for fixed potential to be used for adsorbing phosphorylated Tau test. In addition, natural deposition without any additional potentials provided was included for comparison. The frequency decrease amplitude generated by different electric potentials is shown in Fig. 3b. 0.5 V adsorption effect was proven to show the highest change where the frequency dropped by 285 Hz, and the mass change per unit area of the electrode surface was about 1.95 µg/cm2. 0.3 V potential showed the second highest change, where the frequency dropped by approximately 135 Hz. This change could be calculated into a mass of approximately 0.92 µg/cm2 on the electrode surface. These results showed that these two fixed potentials were good for adsorption of phosphorylated Tau. 0.5 V was then used as the primary fixed potential for further experimentation because of its higher drop.
Based on the results of the previous part of the experiment, phosphorylated Tau adsorption due to positive potential was reproduced using 0.5 V fixed potential. After the PANI film was formed and checked for conductivity, the adsorption confirmation test was performed using low-concentration phosphorylated Tau (0.33 µg/ml), high-concentration phosphorylated Tau (1 µg/ml), and ultrapure water. These tests were performed in triplicates and averaged for comparison, and results are shown in Fig. 3c. When using ultrapure water, the frequency drop averaged at 38 Hz, which was converted into a mass change per unit area of about 0.26 µg/cm2. When using a low concentration phosphorylated Tau solution for adsorption, the average frequency drop was 283 Hz, which was converted to the mass change per unit area of about 1.93 µg/cm2. However, for the high concentration phosphorylated Tau, the average frequency drop was about 933 Hz, which was converted into the mass change per unit area of about 6.38 µg/cm2. As a result, it can be confirmed that 0.5 V seems to be the most ideal potential to be used to attach phosphorylated tau to PANI membrane, as compared to other fixed potentials, and using 0.5 V as fixed potential can certainly achieve adsorption of specific-site phosphorylated tau.
Negative potential removes p-tau and achieve dephosphorylation
In this part, we first used 3 ml of low-concentration phosphorylated Tau (0.33 µg/ml) for adsorption and desorption tests. Cyclic voltammetry was applied by screening at different voltages, Fig. 4a shows that -0.3 V~ -0.5 V has a trend of dephosphorylation in frequency change and charge effect. Thus, we applied -0.3 V, -0.4 V, and -0.5 V as fixed negative potential to test if dephosphorylation can be achieved.
After positive potential was stimulated to adsorb phosphorylated Tau on the PANI membrane, the frequency change caused by adsorption was found to be at 350 Hz, and repulsion test was performed by three negative potentials as mentioned before (Fig. 4b). In the test involving low-concentration phosphorylated Tau, the frequency change of -0.3 V has a small change of about 450 Hz is detached, which is converted to mass per unit area of about 3.08 µg/cm2. -0.5 V has the best detach effect. After the drive is detach, the frequency increases to 800 Hz, which is converted to a mass per unit area of about 5.47 µg/cm2. These results can confirm that all three potentials have repulsion effects. Among them, -0.3 V is closest to phosphate repelling potential that this study is aiming for. The concentration is increased for further testing.
In the high-concentration phosphorylated Tau test, the concentration of phosphorylated Tau was increased where 1 µg/ml of the protein was added to 3 ml of the solution and put into the device, and controlled by 0.5 V positive potential. the adsorption of phosphorylated Tau can achieve a frequency change of 1000 Hz. Identical adsorption amount was used to carry out -0.3, -0.4 V and -0.5 V fixed potential dephosphorylation tests. The results are shown in Fig. 4b. The effect of negative potential to remove phosphorylated Tau increased the most at -0.5 V, followed by -0.4 V. Frequency change at -0.3 V increased by 700 Hz, which was converted to mass per unit area for the removed p-Tau at 4.78 µg/cm2. The results were similar to those when lower concentration phosphorylated Tau were used. Compared to the other two negative potentials, -0.3 V was less effective in removing phosphorylated Tau. It also shows the ability to remove a certain quantity of proteins. However, the main point of this research is to find a negative potential that can remove phosphate, rather than the entire tau. With reference to the peak position of the cyclic voltammetry current in the previous part, it is suspected that the possible potential of dephosphorylation is -0.3 V.
Finally, in order to ensure the desorption effect and reproducibility of -0.3 V, and to confirm that the repelled protein is phosphorylated Tau or phosphoryl group, in this part, we carry out three repetitive experiments. After the PANI film was formed, 0.5 V was used as adsorption potential, and then removal by -0.3 V potential. The result can be seen in Fig. 4c. During adsorption, the phosphorylated Tau was adsorbed at a fixed potential of 0.5 V and decreased at an average frequency of 933 Hz, which was converted into a mass change per unit area of approximately 6.38 µg/cm2. During repulsion, phosphorylated Tau or only phosphate group was removed by -0.3 V and produced an average frequency increase of 793 Hz, which is converted into a mass change per unit area of approximately 5.42 µg/cm2. Compared with the standard deviation of the adsorption frequency, the frequency of the repulsion was relatively stable. This is considered to be affected by the hydrogen ion interference, but the overall performance was still stable and reproducible.
Mass spectrometry to examine the solution
After the successful repulsion test at negative potential of -0.3 V, the frequency change measured by quartz crystal microbalance showed that the material on the electrode can be driven into the solution at this negative potential. However, it cannot be ensured if the corresponding substance is phosphate or the entire phosphorylated Tau. Therefore, to investigate the repelled substance, in this part of study, the solution containing of the repelled substance was detected by high performance liquid chromatography mass spectrometer (HPLC-MS).
First, in order to determine the mass-to-charge ratio and molecular weight of phosphorylated Tau used in this study, stock solution purchased from the manufacturer was analyzed using HPLC-MS. As shown in Fig. 5a, the target peptide was separated by HPLC. After being extracted, the molecules were ionized by the ion source of mass spectrometer, and the mass analyzer separates them according to their mass-to-charge ratio to produce results. Fig. 5a shows that there are three sharp peaks, namely 562, 702 and 936. After deconvolution and back-calculation, it can be seen that these three peaks correspond to p-tau peptides used in the experiment. The m/z size was about 2806, and the signal strength was 2.5 × 106, with the detection limit of about 0.6~0.3 ppm.
In this study, in order to minimize error and to eliminate interference, deionized water which was used for solution preparation in experiments was also tested by mass spectrometer as a negative control group with no Tau added to it and the result can be seen in Fig. 5b-I. The largest signal is obtained at 705, a peak with an intensity of 1.6 × 104. However, in terms of mass spectrometer results, this signal is too small and can be neglected. In the previous part of the standard, there was no obvious signal generated at the three peak positions. It proves that negative control does not contain any substance that may interfere with the experiment, and subsequent experiments would be able to compare and confirm phosphorylated Tau with the three peaks measured by standard mass spectrometer.
In order to confirm that the phosphorylated Tau is adsorbed on the polyaniline film electrode, this part operated an additional mass spectrometry test on the waste liquid after the adsorption was completed with a positive potential of 0.5 V in the experiment to check whether the solution still contained any phosphorylated Tau or not, and the result is as shown in Fig. 5b-II. As compared to the standard product in Fig. 5a, there was no obvious phosphorylated Tau peak in the solution, and the dominant signal was the impurity peak. This can help prove that most of the phosphorylated Tau added to the solution was adsorbed. So the signal value of phosphorylated Tau cannot be detected in the solution.
The sample composition for the third part of this experiment is as shown in Supplementary Figure 1. The solution containing the substance removed by negative potential -0.3 V was used as the experimental target, and the phosphorylated Tau stock solution was diluted as the control group. Results are as shown in Fig. 5c, in both experimental group and control group, there was a main peak of 936. The signal intensity of the control group was 3.8 x 105, and the signal intensity of the experimental group was 1 × 105. The difference was about four times between the two samples, and this peak was identical to the trivalent peak results in Fig. 5a. Therefore, it can be confirmed that this peak represents phosphorylated Tau. It also showed that the positive adsorption potential and negative potential used in this study remove phosphorylated Tau were true, and the phosphorylated state can be measured using a mass spectrometer.
Beside the phosphorylated Tau signal, there are two obvious peaks at m/z 861 and 839. According to prior studies [30, 31], these two peaks can be presumed as unphosphorylated and dephosphorylated Tau respectively. After rearrangement, the results shown in Table 1 display these three peaks by their intensity and percentage. Using the overall Tau percentage, it can be found that the ratios of phosphorylated Tau detected by mass spectrometer in the two groups are similar, while the ratio of dephosphorylated Tau in the experimental group is much higher than that in the control group. The ratio of unphosphorylated Tau is relatively small. It is noteworthy that the unphosphorylated Tau of the control group is about 11 times as compared to the experimental group, while the dephosphorylated Tau of the control group is double as compared to the experimental group. The dephosphorylated Tau in the experimental group has been greatly increased, which proves that -0.3 V potential used in this study not only removes the phosphorylated Tau, but also achieves the dephosphorylation effect. This part also confirms that the 0.5 V positive potential used in this study also has the effect of adsorbing phosphorylated Tau on PANI surface, and when -0.3 V negative potential was used, it not only drove away phosphorylated Tau, but also dephosphorylated phosphorylated Tau peptide.
Table 1
Comparison of control and experimental mass spectrometry results. Phosphorylated tau, unphosphorylated tau and dephosphorylated tau intensity and ratio are compared
Peak
|
Meaning
|
Control
|
Experimental
|
Intensity
|
Percentage
|
Intensity
|
Percentage
|
936
|
[M+3H]3+
|
3.8×105
|
52%
|
1×105
|
58%
|
861
|
[M+3H]3+ - PO32-
|
2.5×105
|
34%
|
0.2×105
|
12%
|
839
|
[M+3H]3+ - H3PO4
|
1×105
|
13%
|
0.5×105
|
29%
|
* “-” means a minus sign here
* % (Percentage) is calculated by the signal intensity ratio of the three peaks
|