Team:DUT China A/Measurement
Principle
Gold nanoparticles (Au NPs) covered by Citrate ion are charged negatively and they repel each other, remaining dispersive in the solution. After adding NaCl into this solution system, the double electrode layer of the Au NPs are destroyed and they tend to being coagulated, resulting the color of the solution changes from red to blue-purple, and the spectrum of the solution shifts red. The absorbance of 620nm obviously increase. However, short ssDNA are readily adsorbed onto the surface of AuNPs without any modification and that such ssDNA-treated AuNPs are more stable than untreated ones. Adding aptamers into the system, the citrate root of nano gold surface is replaced. AuNPs can still remain negatively charged instead of being coagulated even if there is NaCl due to the non-specific interaction between DNA side chain phosphate groups and Au NPs, keeping the solution color red. When the determinand is added in to the solution, the adaptor configuration changes and falls off the surface of the Au NPs because there is affinity between the determinand and the aptamers. AuNPs is going to coagulate. The changing concentrations of determinand lead to the changing AuNPs solution spectra. Adopting this method, the binding of the aptamer to determinand can be tested and the determinand in the solution can be easily and quickly detected.
Protocol & Expected Result
2.1. Preparation for Au NPs
Add 125mL HAuCl(1mM) water solution to 250mL round bottom flask. Heat it until boils then quickly add 12.5 mL (38.8mM) sodium citrate solution into this system. Heat for 10min, remove the heating sleeve, stir for 15min. Cool to room temperature and obtain Au NPs with particle size of about 13nm.2.2. Exploration on the least concentration of NaCl to precipitate a certain concentration of Au NPs
Fabricate Au NPs systems with different concentration of NaCl in 96 well plates and measure their absorbance ratios at wavelengths of 520 nm and 620 nm, the trend line of A620/A520 should be like Figure.1 & Figure.2. It reaches a plateau when the concentration of NaCl comes to a certain concentration (concenrtaion 1). We can assume that NaCl at concentration 1 is able to precipitate all the Au NPs in this system.In the following experiments, we will make NaCl at concentration 1 in every system.
2.3. Explorations on the least concentration of the aptamer of determinand to protect all the Au NPs from being precipitated
Once there are extra aptamers in the solution system, they will bond to the determinand while the Au NPs are still totally protected, which will not lead to a change on A620/A520. We don’t want this happen. Therefore, we must ensure that the aptamers at the working concentration are not excessive.Fabricate Au NPs systems with different concentration of the aptamer of determinand in 96 well plates when the NaCl is at concentration 1 and measure their absorbance ratios at wavelengths of 520 nm and 620 nm, the trend line of A620/A520 should be like Figure.3. It is likely to just reach a plateau when the concentration of the aptamer of determinand comes to another certain concentration (concenration 2). We can assume that the aptamer of determinand at this amount are just enough to protect all the Au NPs at 150 nM.
In the following experiment, we will make the aptamer of determinand at concentration 2 in the system.
2.4. Characterization of Aptamers
As the principle indicates, once the A620/A520 declines with the increasing concentration of determinand, it can be proved that our aptamers can bind to determinand efficiently.Incubate 150 nM of AuNP and 0.20μM of he aptamer of determinand for 30min at 37℃, with system volume being 200 μL. Add different concentrations of determinand into the system with the final concentration of determinand between 0 and 0.45 μM.
Add 5μL, 3M of NaCl solution, making its final concentration at 0.075 M. All these steps are done in 96-well plates. Measure the absorbance at 620nm and 520nm by uv-vis spectrometer, and the changes of A 620 / A 520 are recorded and analyzed, the trend line of A620/A520 should be like Figure.4.
