Development of an Immunosensor of Aflatoxin B1 Based on Silica-coated Gold Nanoparticles
Abstract:
Due to the common presence of AFB1 in human diets and its toxicity, sensitive and cheap detection methods of AFB1 are needed. Many detection methods, including TLC, HPLC, ELISA, exist, but the most commonly accepted and commercially available method utilizes the intermolecular interactions between gold nanoparticles and AFB1 antibodies. However, such interactions are often weak, and gold nanoparticles tend to aggregate due to its low thermodynamic stability. Thus, by coating gold nanoparticles with a layer of silica, the thermodynamic stability of the antibody-gold nanoparticle bioconjugate can be increased. Moreover, the hydroxyl groups on the outer layer of the silica coating can connect the antibody with gold nanoparticles through strong chemical bonds. In this project, gold nanoparticles will be synthesized by using the citrate method and coated with a silica layer by using previously reported methods. Different methods of connecting the antibody and gold nanoparticle will be evaluated. More specifically, these methods include using reductive amination, michael addition, and esterification.Bibliography/Citations:
1. Aflatoxin B1. Wikipedia, the Free Encyclopedia, Wikimedia Foundation Inc., https://en.wikipedia.org/wiki/Aflatoxin_B1#cite_note-2
2. L. Zhang, Y. Mazouzi, M. Salmain, B. Liedberg, S. Boujday, Antibody-Gold Nanoparticle Bioconjugates for Biosensors: Synthesis, Characterization and Selected Applications. Biosens. Bioelectron. 165 (2020), p. 112370.
3. Immunoglobulin G. Wikipedia, the Free Encyclopedia, Wikimedia Foundation Inc., https://en.wikipedia.org/wiki/Immunoglobulin_G
4. Ö. Ertekin, Ş. Ş. Pirinçci, S. Öztürk, Monoclonal IgA antibodies for aflatoxin immunoassays. Toxins (Basel). 8, 148 (2016).
5. Y. Kobayashi et al., Synthesis of a colloid solution of silica-coated gold nanoparticles for X-ray imaging applications. J. Nanoparticle Res. 16, 1–13 (2014).
Additional Project Information
Project files
Research Plan:
Rationale
Due to the common presence of AFB1 in human diets and its toxicity, sensitive and cheap detection methods of AFB1 are needed. Many detection methods, including TLC, HPLC, ELISA, exist, but the most commonly accepted and commercially available method utilizes the intermolecular interactions between gold nanoparticles and AFB1 antibodies. However, such interactions are often weak, and gold nanoparticles tend to aggregate on their own. Thus, by coating gold nanoparticles with a layer of silica, the thermodynamic stability of the antibody-gold nanoparticle bioconjugate can be increased. Moreover, the hydroxyl groups on the outer layer of the silica coating can connect the antibody with gold nanoparticles through strong chemical bonds. In this project, gold nanoparticles will be synthesized by using the citrate method and coated with a silica layer by using previously reported methods. Different methods of connecting the antibody and gold nanoparticle will be evaluated. More specifically, these methods include using reductive amination, michael addition, and esterification. This research can make the detection of AFB1 more accurate and consistent.
Hypothesis
The sensitivity and stability of AFB1 immunosensor based on silica-coated gold nanoparticles will be better compared to AFB1 immunosensor based on normal gold nanoparticles.
Methods
- Synthesis of silica-coated gold nanoparticles
Silica-coated gold nanoparticles will be synthesized based on previously reported methods(5).
- Synthesis of antibody silica-coated gold nanoparticle bioconjugates
AFB1 antibody will be obtained commercially.
- Reductive amination method
Chloro(2-chloroethyl)dimethylsilane(figure 2), a commercially available molecule, will be used as the starting material. Ammonia will be added to the solution to substitute the chlorine connected to carbon with -NH2, which can then form an imine with the antibody, and the hydroxyl group on the outer layer of the silica coated gold nanoparticles can connect to the silicon atom.
Fig. 2. Chloro(2-chloroethyl)dimethylsilane
- Michael addition
Acryloyl chloride(figure 3) will be added to the solution of silica coated gold nanoparticles. Esterification reaction is expected to occur between the hydroxyl groups on the outer layer of the silica coated gold nanoparticles and acryloyl chloride. Primary amine groups can undergo michael addition reaction with the newly functionalized gold nanoparticle complex. Similar reactions to connect the immunoglobulin G antibody with gold nanoparticles have been reported before (2).
Fig. 3. Acryloyl chloride
- Esterification method
Succinyl chloride(figure 4) will act as the anchor between the antibody and gold nanoparticles. More specifically, due to the presence of two acyl chloride functional groups, the molecule can form an amide with the amine groups on the antibody and a ester with the hydroxyl groups on the gold nanoparticles.
Fig. 4. Succinyl chloride
- Construction of sensor
A sensor based on liquid chromatography will be constructed based on previously reported methods. The structure of the sensor is shown in figure 5 (2).
Figure. 5. Structure of the sensor
- Evaluation of the sensor
Dilute solution of AFB1 toxin(1ng/ml) will be made in the fume hood with gloves and goggles and under adult supervision. The sensor will be tested by dipping it into the solution and waiting for color change.
Safety
Gloves and goggles will be worn throughout the experiment, and an adult supervisor will be present. Reactions involving volatile organic compounds will be conducted in the fume hood. AFB1 toxin solution will be handled with great care. Aflatoxin B1 will be treated with 6% solution of hydrogen peroxide at pH 9.5 for 30 minutes at 80 °C to detoxify the toxin (6).
Reference
1. Aflatoxin B1. Wikipedia, the Free Encyclopedia, Wikimedia Foundation Inc., https://en.wikipedia.org/wiki/Aflatoxin_B1#cite_note-2
2. L. Zhang, Y. Mazouzi, M. Salmain, B. Liedberg, S. Boujday, Antibody-Gold Nanoparticle Bioconjugates for Biosensors: Synthesis, Characterization and Selected Applications. Biosens. Bioelectron. 165 (2020), p. 112370.
3. Immunoglobulin G. Wikipedia, the Free Encyclopedia, Wikimedia Foundation Inc., https://en.wikipedia.org/wiki/Immunoglobulin_G
4. Ö. Ertekin, Ş. Ş. Pirinçci, S. Öztürk, Monoclonal IgA antibodies for aflatoxin immunoassays. Toxins (Basel). 8, 148 (2016).
5. Y. Kobayashi et al., Synthesis of a colloid solution of silica-coated gold nanoparticles for X-ray imaging applications. J. Nanoparticle Res. 16, 1–13 (2014).
6. Aflatoxin B1. PubChem, National Library of Medicine, https://pubchem.ncbi.nlm.nih.gov/compound/Aflatoxin-B1#datasheet=LCSS
Questions and Answers
1. What was the major objective of your project and what was your plan to achieve it?
a. Was that goal the result of any specific situation, experience, or problem you encountered?
b. Were you trying to solve a problem, answer a question, or test a hypothesis?
The objective of this project is to build a more sensitive and stable immunosensor for AFB1. I plan is to do so by coating gold nanoparticles with silica. I'm testing the hypothesis that silica-coated gold nanoparticles are more sensitive and stable in detecting AFB1.
2. What were the major tasks you had to perform in order to complete your project?
a. For teams, describe what each member worked on.
1. Synthesize silica-coated gold nanoparticles
2. Functionalize silica-coated gold nanoparticles with AFB1 antibodies
3. What is new or novel about your project?
a. Is there some aspect of your project's objective, or how you achieved it that you haven't done before?
b. Is your project's objective, or the way you implemented it, different from anything you have seen?
c. If you believe your work to be unique in some way, what research have you done to confirm that it is?
AFB1 immunosensor based on normal gold nanoparticles have been built, but AFB1 immunosensor based on silica-coated nanoparticles, more specifically with a silica layer outside of gold nanoparticles, have not been built before. I read related literatures to make sure my project is unique in this way.
4. What was the most challenging part of completing your project?
a. What problems did you encounter, and how did you overcome them?
b. What did you learn from overcoming these problems?
I had trouble figuring out methods to connect the antibody with silica-coated gold nanoparticles. I overcome them by learning more organic chemistry and reading review articles focusing on the construction of nanoparticle immunosensors.
5. If you were going to do this project again, are there any things you would you do differently the next time?
I would start the project earlier, before the pandemic.
6. Did working on this project give you any ideas for other projects?
Yes. The experience of working on this project made me more interested in doing chemistry research in the future.
7. How did COVID-19 affect the completion of your project?
I was unable to conduct actual experiments because I had no access to my school's laboratory.