1. Teachers Training Kit in Nanotechnologies Experiment Module
A comprehensive training kit for teachers
Experiment C
Luisa Filipponi, iNANO, Aarhus University
This document has been created in the context of the NANOYOU project. (WP4, Task 4.1) All information is provided “as is” and no guarantee or warranty is given that the information is fit for any particular purpose. The user thereof uses the information at its sole risk and liability. The document reflects solely the views of its authors. The European Commission is not liable for any use that may be made of the information contained therein.

2. Before you use this presentation
This Power Point Presentation is part of the Experiment Module of the NANOYOU Teachers Training Kit in Nanotechnologies. MATERIAL INCLUDED IN THIS EXPERIMENT C PACKAGE:
For teacher:
EXPERIMENT C-TEACHER DOCUMENT
NANOYOU VIDEO 2_GOLD COLLOID
For students*:
EXPERIMENT C-STUDENT BACKGROUND READING
EXPERIMENT C-STUDENT LABORATORY WORKSHEET
LEVEL OF EXPERIMENT: Advanced
*These documents are available for the and age group in different languages
DOCUMENTS CAN BE FOUND AT
This NANOYOU documents is distributed with Creative Commons Non-Commercial Share Alike Attribution, except where indicated differently. Please not that some images contained in this PPT are copyright protected, and to re-use them outside this document requires permission from original copyright holder. See slide 18 for details.
DISCLAIMER: The experiments described in the following training kit use chemicals which need to be used according to MSDS specifications and according to specific school safety rules. Personal protection must be taken as indicated. As with all chemicals, use precautions. Solids should not be inhaled and contact with skin, eyes or clothing should be avoided. Wash hands thoroughly after handling. Dispose as indicated. All experiments must be conducted in the presence of an educator trained for science teaching. All experiments will be carried out at your own risk. Aarhus University (iNANO) and the entire NANOYOU consortium assume no liability for damage or consequential losses sustained as a result of the carrying out of the experiments described.

3. EXPERIMENT C Age level: 11-13 and 14-18 years
Colorimetric gold nanosensor
EXPERIMENT C Age level: and years

4. Experiment C- Colorimetric gold nanosensor Applications of Nanotechnologies: Medicine1
Gold at the nanoscale has different properties than bulk gold
Nano-metals have peculiar optical proprieties due to the size confinement
Metal nanoparticles have colours that are dependent on their size and surrounding
This allows using solutions of metal nanoparticles (colloids) as sensors
Figure 1. Macroscopic and nanoscopic gold. Image credit: see slide 19

5. Experiment C- Colorimetric gold nanosensor Application of Nanotechnologies: Medicine
Metal nanoparticles have different optical properties compared to their bulk counterpart.
Generation of surface plasmons that oscillate back and forth in a synchronized way in a small space: localized surface plasmon resonance (LSPR)
When the frequency of this oscillation is the same as the frequency of the light that it generated it (i.e., the incident light), the Plasmon is said to be in resonance with the incident light.
Metal colloids have very strong visible absorption due to the resonant coherent oscillation of the plasmons
Metal nanoparticles display different colours (red, green, orange, etc.)
Figure 2. (Top) LSPR effect (2) (Bottom) Colour-size dependence in metal nanoparticles. Image credit: see slide 19

6. Experiment C- Colorimetric gold nanosensor Applications of Nanotechnologies: Medicine
The energy of LSPR's is sensitive to the dielectric function of the material and the surroundings and to the shape and size of the nanoparticle:
distance between nanoparticles in the colloid (aggregation)
attachment of a ligand on the surface of the nanoparticles
Metal colloids can be used as sensors:
In a surface plasmonic sensor, metal nanoparticles are immobilized on a surface. The sensing results in a change in refractive index which determines a change in the LSPR signal.
Figure 3. Schematic representation of a LSPR biosensors based on refractive index changes. Image credits: see slide 19

7. Experiment C- Colorimetric gold nanosensor Applications of Nanotechnologies: Medicine
Plasmonic nanosensors (cont.)
In a colloidal plasmonic biosensor (for instance made of gold nanoparticles) the sensing results in a change of aggregation among the nanoparticles that form the colloid which can determine a colour change of the colloid
( absorption spectroscopy). This is a colorimetric plasmonic nanosensor.
How it works in real nanosensors used in medicine:
Figure 4. A gold colloidal nanosensor. Image credit: see slide 19.
Colorimetric gold biosensor

8. Experiment C- Colorimetric gold nanosensor Application of Nanotechnologies: Medicine
Gold colorimetric nanosensors: what for?
Protein stability and function: search for conformation changes that are indication of disease
Genetic screening: search for a specific gene sequence in a sample which can be indicative for a specific disease
Advantages over other methods
No label required, intrinsic optical change
Works with many combinations of key-lock biomolecules
Low detection limit.

9. Experiment C- Colorimetric gold nanosensor Applications of Nanotechnologies: Medicine
Simplified experiment for school teaching (proteins, DNA etc very expensive)
Synthesis of a gold colloid in water starting from a solution of hydrogen tetrachloroaurate (HAuCl4) and a solution of trisodium citrate (Na3C6H5O7).
Gold colloid with nanoparticles 10-20nm in size.
In the reaction, the citrate acts as a weak reducing agent (reducing AuCl4- to Au) and as a stabilizer. A layer of citrate anions adsorbs around each nanoparticle and prevents these from aggregating: the anions electrostatic repulsion keeps the nanoparticle separated.
In this state, the colloid appears ruby-red.

10. Experiment C- Synthesis Protocol
Prepare stock solutions (hydrogen tetrachloroaurate (HAuCl4) and a solution of trisodium citrate (Na3C6H5O7) in water).
The solution of hydrogen tetrachloroaurate is light yellow.
Synthesize the gold colloid: heat the hydrogen tetrachloroaurate to its boiling temperature, than add quickly the citrate.Solution becomes immediately clear. Reaction is continued until the ruby-red colloid is formed.
Time of reaction from addition of citrate is about 10 min
Use Video 2_Gold Colloid if reaction is not performed in class
Before reaction
End of reaction

11. Experiment C- Details of synthesis
Immediately after the addition of citrate, the solution appears clear
After about 1 minutes some colour change is observed:
light grey» grey/blue » purple » dark purple » ruby red
Very recent studies have shown that as the reaction proceeds, some intermediate nanostructures are formed, including gold nanowires (dark purple solution).
The nanowires break down to nanoparticles
Figure 5 (Left) Growth mechanism of nanospherical gold particles (Right) TEM images of the dark intermediate showing an extensive network of gold nanowires. Image credit: see slide 19

12. How is this “nano”? This synthesis leads to a colloid.
A colloid is a chemical mixture a substance is dispersed evenly throughout another one but the particles of the dispersed substance are only suspended in the mixture, they are not completely dissolved in it.
Colloids contain nanoparticles. In this case, gold nanoparticles about 15 nm in size.
Colloids scatter light: try to shine a laser light through it. A scattered light path is observed.
Solutions do not scatter light.
Colloids exist in nature, e.g., milk (see Experiment A).
Test in the lab: use a laser pen and test your colloid. Can you see a light path?
Test also the original solutions (aurate and citrate): they do not scatter light
Test diluted milk to prove it is a natural colloid

13. Experiment C- Chemical Sensor
The gold colloid can be used as a sensor: If the anion layer is removed, the nanoparticles start to approach and agglomerate: this leads to a colour change and this effect can be used for sensing a chemical
If a strong electrolyte is added, such as NaCl, the ions of the salt shield the negative charges on the particles: the approach and aggregate This is reflected in a change of the optical spectrum and the appearance of peak around nm, causing the solution to turn deep blue.
Test in the lab: add 6 droplets of a NaCl solution to the gold colloid

14. Experiment C- Chemical Sensor
If a high concentration of salt is added, the nanoparticles aggregate to a point they precipitate, and the solution becomes clear.
Test in the lab: add excess of NaCl solution to the gold colloid

15. Experiment C- Chemical Sensor
If a stabilizer of high molecular weight is added, such as a protein or polyethylene glycol, it adsorbs to the surface of the nanoparticles with the effect of inhibiting aggregation, even at high salt concentration
Test in the lab: use egg white as a source of ovoalbumin. Add egg white to the gold colloid, followed by a NaCl solution. Try to add excess of NaCl. No aggregation occurs.
egg white + 10 drops NaCl
control
egg white
10 drops NaCl

16. Experiment C- Chemical Sensor
If a weak or non-electrolyte is added (e.g., sugar), the electrostatic repulsion between the gold and the citrate ions are not disrupted and the solution remains red. A small change in colour (e.g., from ruby red to slightly pink) can be observed which is the consequence of a very small agglomeration that occurs as the protein is added
Test in the lab: add 10 droplets of sugar solution to the gold colloid

17. Running experiment C in class
1. Start with a general discussion of gold asking the students what they know about this material.
- What are the properties of gold? As a metal? Is it a catalyst? What is gold used for?
What colour is gold? What color can gold alloys be?
2. Introduce the concept of colloids.
-What is the difference between a colloid and a solution?
- Light scattering of colloids
- Optical properties of metal colloids, and in particular of gold colloids (LSPR effect)
Applications of gold colloids (biosensors). Explain how a gold colloid can be used as a colorimetric sensor.
3. Synthesis of a gold colloid.
- Explain the synthesis, its steps and the end product. Students should confirm the presence of the colloid using a laser light pen.
- Instructors should encourage students to closely monitor the change colour of the solution during the synthesis and record their observations in the table in the student worksheet. Alternatively use the NANOYOU Video 2_ Gold Colloid.

18. Running experiment C in class (cont.)
4. Gold colloid as a plasmonic colorimetric sensor.
In nanomedicine gold colloids are investigated as colorimetric sensors. for instance to detect specific DNA fragments. Explain how id this done. Highlight that the key elements of this type of sensor are that it is very sensitive and it does not require the use of a label.
Explain that they will do a simpler experiment and use the colloid as a chemical colorimetric sensor.
The colloid should be tested with different solutions to see how it can be used for sensing certain specie : salt solution; egg white followed by salt solution; sugar solution.
Teachers can suggest testing the gold colloid to other solutions, such as vinegar (which is a mild acid) and ask the students to guess what change to expect
- Teachers can prepare a “test” solution without telling the student its content and ask the students to guess what this “test” solution is made of depending on its effect on the colour of the gold colloid.

19. Images credit
Figure 1. Dependence of colour on gold size. (Image credit: L. Filipponi, iNANO, Aarhus University, Creative Commons Attribution ShareAlike 3.0).
Figure 2 (top): Formation of plasmons in bulk metal (top) and in nanoparticles (bottom). (Image credit: D. Sutherland, iNANO, Aarhus University, Creative commons Attribution Non-Commercial ShareAlike 3.0)
Figure 2(bottom): Transmission electron micrographs and UV—Vis spectra of gold nanoparticle colloids with various geometries: (top) spheres, (middle) decahedra and (bottom) rods. (Image credit: Reprinted from: Borja Sepúlveda et al., "LSPR-based Nanobiosensors", Nano Today (2009), 4 (3), , with permission from Elsevier).
Figure 3: Schematic representation of the preparation and response of LSPR biosensors based on refractive index changes. (Image credit: Reprinted from: Borja Sepúlveda et al., “LSPR-based Nanobiosensors”, Nano Today (2009), 4 (3), , with permission from Elsevier.)
Figure 4: A plasmonic colloidal nanosensor. (Image credit: reprinted with permission from Jin et al., Journal of American Chemical Society (2003), 125 (6), Copyright 2003 American Chemical Society.)
Figure 5: (Left) Growth mechanism of nanospherical gold particles synthesised by reduction of aqueous AuCl4- by sodium citrate. (Right) TEM images of the dark intermediate showing an extensive network of gold nanowires which were isolated from the dark purple solution. (Image credit: reprinted with permission from Pong et al., J. Phys. Chem. C 2007, 111, Copyright 2003 American Chemical Society.)