Gold-nanoparticle aptamer conjugates
1st iteration
Design: In the beginning we had no experience in working with Lateral-Flow-Assays(LFAs). We oriented ourselves on the papers of Shijia Wu et al.1 and Guodong Liu et al. 2 to get an idea of what we needed to do and on how to design and build our test.
Build: After getting everything we needed, it was time to start the first experiment. We started with the preparation of the gold nanoparticle (AuNPs)-aptamer-conjugates and the verification of the thiol-gold binding with a gel electrophoresis.
Test: In this experiment we already faced the first problem: the gold nanoparticle-solution turned from red to blue, which indicates that they have aggregated. When gold nanoparticles aggregate this is irreversible and they become unusable for our application.
Learn: After some research we found two possible causes for this aggregation. First, the NaCl or its specific concentration in the PBS buffer could have caused the AuNPs to aggregate. We learned that using a buffer with high ionic strength, like PBS, with citrate stabilized gold nanoparticles, can lead to aggregation. The aggregation occurs due to shield charging of the nanoparticles3. Second, the protocol itself could have some issues that we are not aware of. They synthesized their gold nanoparticles themselves, which might make a difference. Therefore we first needed to figure out what went wrong.
2nd iteration
Design: To get to the bottom of which of the problems was the most likely one, we first wanted to figure out whether the buffer was causing the aggregation. And if it was the cause we wanted to know if it caused aggregation due to the NaCl concentration or something else in it.
Build: For that we prepared five different buffers with different concentrations of NaCl, PBS and sodium phosphate buffer, which does not contain NaCl.
Test: When we resuspended the AuNP-aptamer-solution in the five different probes, all five turned blue once again and were no longer usable for us.
Learn: This result showed us that the solutions we came up with did not fix our problem. We therefore came to the conclusion that if the aggregation was caused by the buffer, it was not related to the concentration.
3rd iteration
Design: We then thought that maybe the citrate buffer our gold nanoparticles were delivered in might make them less stable. In order to test this hypothesis we tried our protocol again but this time we rebuffered the AuNPs before use.
Build: Four samples were set up. We washed the AuNPs and resuspended them in 1) milliQ water, 2) PB (phosphate buffer) and 3) PBS. Sample 4) was a control sample where the AuNPs stayed in the citrate buffer they came in. Additionally, we made two more samples and slowly added only 0.1 mM of PBS at a time until we reached our needed PBS concentration. This was done to test if adding the buffer more slowly, giving the AuNPs more time to adapt, could prevent them from aggregating
Test: When we rebuffered the AuNPs in different buffers, we had our first successful results. Only the sample that got resuspended in PBS turned blue. The others 2.- 4. stayed red.
Figure 1: from top to bottom: PBS, PB, H2O, citrate
In the additional experiment, in every step of adding small amounts of PBS, the AuNPs stayed red until the last time, when we added 10 mM PBS.
Learn: We concluded from this experiment that changing the buffer of the AuNPs was possible but PBS was not suitable for this. In the second experiment we could see that the pace and concentration at which the PBS is added can affect whether the AuNPs aggregate or not.
4th iteration
Design: To test, if the newly generated samples stayed red during the further process, we used the PB and the citrate buffered sample from the previous experiment. We then tested if the AuNps could form conjugates with the aptamers. This was done via gel electrophoresis. Due to the red color of the AuNps no additional dyes are needed. If conjugates were formed we expected the samples to run more slowly than the control AuNPs, as they effectively increase in size.
Build: The further processing included an incubation step in which the aptamers should bind to the AuNps and two washing steps at the end of which the AuNps were resuspended in water. To see if there truly was conjugation between the AuNps and the aptamers, a 2% agarose gel was prepared in 0.5xTBE and run at 100 V for 90 min. 50% sucrose was added to each sample to increase their density so they would sink into the gel's pockets.
Test: The originally phosphate buffered sample turned blue during the washing steps. The originally citrate buffered sample and a control sample with AuNPs ran through the gel. Surprisingly, they passed through the gel very slowly, but at the same speed.
Figure 2: 1. lane: originally phosphate buffered AuNP-aptamer-conjugates; 2. lane: originally citrate buffered AuNP-aptamer-conjugates; 3. lane: control AuNPs
Learn: These results could either mean that there was no conjugation in the citrate buffered sample or the gel was too dense to separate the sample and the control.
5th iteration - success
Design: Due to the inconclusive results from the prior experiment, we decided to try out a different agarose gel, this time with 1% agarose, and to use the originally citrate buffered AuNPs-aptamer-conjugates.
Build: The 1% agarose gel was placed in 0,5xTBE and run at 80 V for 90 min.
Test: Finally, the band of the AuNP-conjugates ran slower than the control AuNPs, which indicates that the conjugates have formed.
Figure 3: 1. lane: originally citrate buffered AuNP-aptamer-conjugates; 2. lane: control AuNPs
Learn: From this experiment we learned that in order for the gold nanoparticles to not aggregate and bind to the aptamers, we could not change their buffer to anything but water. Furthermore, from now on we will exclude PBS and the included NaCl from our experiments with AuNPs, since this always leads to the aggregation of the AuNPs.
Side note: Unfortunately, we were not able to recreate this again, so we had to walk through another Engineering cycle.
6th iteration
Design: Having researched our problem one more time, we found another protocol that stated that heating the aptamers could help4.
Build: 100 µM aptamers were heated to 90 °C and were allowed to cool down slowly to room temperature. 99 µl of these aptamers were mixed with a spatula tip SDS reacted for 30 min. at room temperature. 60 µl AuNPs and 10 µl aptamer-SDS-mix were combined and left to stand for 16 h at room temperature.
Test: The new AuNP-aptamer conjugates stayed red again and we ran them again on a 1% agarose gel to check if the conjugation worked. Unexpectedly the new conjugates ran faster than the control AuNP.
Figure 4: left: new AuNPs-aptamer conjugates; right: control AuNPs
Learn: We doubt that the conjugation worked properly, since in all the papers we read, it is stated that the conjugates should run slower. We suppose that maybe due to the bound DNA, the AuNP-aptamer-conjugates have more negative charge than the unmodified AuNPs and therefore run faster than expected.