Experiment with colorimetric cold nanosensors - Teacher guide (age 14-18)

NANOYOU Teachers Training Kit in Nanotechnologies

Experiment C:
Colorimetric Gold
Nanosensor
Experiment Module

Written by Luisa Filipponi
Interdisciplinary Nanoscience Center (iNANO)
Aarhus University, Denmark
January 2010

Creative Commons Attribution ShareAlike 3.0 unless indicated otherwise in text or figure
captions.

This document has been created in the context of the NANOYOU project as part of 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.

NANOYOU Teachers Training Kit in Nanotechnologies‐ Experiment Module‐ Experiment C

MATERIAL INCLUDED IN THIS EXPERIMENT PACKAGE:
For teachers:

TEACHER RESOURCES FOR EXPERIMENT C

For students:

EXPERIMENT C‐STUDENT BACKGROUND READING

EXPERIMENT C‐STUDENT SYNTHESIS PROCEDURE

EXPERIMENT C‐STUDENT LABORATORY WORKSHEET

LEVEL OF EXPERIMENT: Advanced

DISCLAIMER: The experiments described in the following training kit use chemicals which need to be used accordingly to
MSDS specifications. Follow school lab safety guidelines. 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.

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The research leading to these results has received funding from the European Community's Seventh Framework Programme
(FP7/2007‐2013) under grant agreement n° 233433


TEACHER RESOURCES EXPERIMENT C: COLORIMETRIC GOLD NANO‐SENSOR
AIM: Plasmonic colorimetric nanosensors are used in nanomedicine because they allow the precise
detection of specific analytes with high sensitivity and without the need for a label. In a colloidal
plasmonic biosensor (for instance made of gold nanoparticles) the sensing event results in a change of
aggregation in the colloid, which can determine a colour change of the colloid. The aim of this
experiment is to synthesise and test a plasmonic colorimetric sensor. First the students synthesise a
colloid of gold nanoparticles and learn that the colour of the gold colloid is ruby‐red, rather than golden
as for bulk gold, so they will have direct evidence that the size of a material at the nanoscale is
fundamentally important in determining its properties. The gold colloid prepared is then tested as a
chemical colorimetric sensor: by adding a salt to the colloid; its agglomeration changes, which results in
a change in colour (from ruby‐red to blue). In the experiment the students test other solutions, such as
sugar, and observe the effect on the colloid. The last test involves the use of a protein and shows how
these can coat the nanoparticles to a point they can no longer aggregate.

FIELD OF NANOTECHNOLOGY APPLICATION: Medicine

EXTRA TEACHERS’ READING: Chapter 4 of Module 1 “Fundamental ‘Nano‐effects’” and Chapter 1 of
Module 2 “Application of Nanotechnologies: Medicine and Healthcare” in the NANOYOU Teachers
Training Kit in Nanotechnologies.

REQUIRED STUDENT PRE‐KNOWLEDGE:
‐ Electromagnetic radiation, colour and absorbance, light scattering
‐ Concept of bio‐recognition

STUDENT READING:
‐ NANOYOU Students’ background document for Experiment C

EXPECTED OUTCOME:
‐ Understanding the effect of size on the properties of a familiar material such as gold
‐ Properties of colloids; optical properties of gold nanoparticles
‐ Understanding of the use of metal colloids for sensing and in particular medical diagnostics

STUDENT ASSESSMENT:
‐NANOYOU Student laboratory worksheet Experiment C

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BACKGROUND INFORMATION
In this experiment students use a plasmonic colorimetric sensor made of nanoparticles: the gold colloid
(containing gold nanoparticles) is the sensing material, and the addition of an analyte (such as salt or
sugar) induces a change in the aggregation of the nanoparticles in the colloid, which is reflected in a
change in its colour. Here we give some background information of the properties of gold nanoparticles
and their use in medical diagnostics, and we provide some details of the synthesis of the gold colloid.

1) About gold
In this experiment the sensor is made of gold nanoparticles. Most
students will be very familiar with the colour of bulk gold and with
some of its properties, but not with its properties at the
nanoscale. Gold (Au, atomic number 79), is the most malleable
and ductile metal of all, it can be beaten to very thin sheets of
material and rolled or bent as desired. Gold creates alloys with
many metals. The colour of pure gold is metallic yellow
(“golden”), but other colours can be conferred by alloying gold
with metals such as copper and silver. For instance “rose gold” is
an alloy of gold and copper in high mass percentages (Figure 1).

Figure 1. Approximate colours of Ag‐
NOTE: Some gold alloys have a colour different from the metallic Au‐Cu alloys. (Image credit: Wiki
yellow of pure gold but this is due to the presence of two metals commons, Creative commons
in the alloy. Colloids of gold nanoparticles might have colours Attribution ShareAlike 3.0)
such as red, purple or blue, but are made only with gold.

Gold is very stable and not‐toxic, and for this reason widely used in jewellery and dentistry because is
air‐inert and is not affected by most reagents. Gold is also a good conductor of heat and electricity (due
to the fact that conduction electrons are free to move around the nucleus); it is corrosion resistant so it
is used for electrical contacts and other electronic applications. Gold has also numerous other
applications; for instance thin layers of gold (so thin as to become transparent) are applied to the
windows of large buildings to increase the reflectivity of sunlight (and reduce transmittance), so that
less air conditioning is required in summer to keep the building cool.

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2) From bulk metals to nanosized metals (“nano‐metals”)
One of the distinguishing properties of metal nanoparticles in general is their optical properties, which
are different from those of their bulk counterpart. This is due to an effect called localised surface
plasmon resonance (LSPR), which is described in Chapter 4 of Module 1 “Fundamental ‘Nano‐effects’”.
One of the consequences of the LSPR effect in metal nanoparticles is that they have very strong visible
absorption due to the resonant coherent oscillation of the plasmons. As a result, colloids of metal
nanoparticles such as gold or silver can display colours which are not found in their bulk form, like red,
purple or orange, depending on the nanoparticles’ shape, size and surrounding media. The energy of
LSPRs is sensitive to the dielectric function of the material and the surroundings and to the shape and
size of the nanoparticle. This means that if a ligand, such as a protein, attaches to the surface of the
metal nanoparticle, its LSPR energy changes. Similarly the LSPR effect is sensitive to other variations
such as the distance between the nanoparticles, which can be changed by the presence of surfactants
or ions. The fact that the LSPR depends on the dielectric environment means that the refractive index
can be used as the sensing parameter: changes in the local dielectric environment, induced by the
sensing process, are used to detect the binding of molecules in the particle nano‐environment.

3) Plasmonic colorimetric gold colloid sensor
In a plasmonic biosensor the nanoparticles can be dispersed in a medium (in which case the biosensor is
a colloidal plasmonic biosensor) or supported on a surface (surface plasmonic biosensor). Both types of
sensors exploit the fact that the sensing event changes the LSPR of the metal nanoparticles, but they use
different readout report strategies:

In a colloidal plasmonic biosensor (for instance made of gold
nanoparticles) the sensing event results in a change of aggregation
among the nanoparticles that form the colloid (Figure 2), which can
determine a colour change of the colloid. Absorption spectroscopy is
used to quantify the biosensing event. In the case of gold colloid,

which is normally red, the sensing event can result in the colloid Figure 2. Schematic representation
becoming blue. Thus metal colloids can be used as plasmonic of a colloidal plasmonic biosensor.
colorimetric biosensors. In nanomedicine this effect is used for
instance for genetic screening, where scientists look for a specific gene sequence in a sample which can
be indicative for a specific disease. How is this done? First, the sequence of bases in the target DNA is
identified. Then two sets of gold particles are prepared – one has DNA attached that binds to one end of
the target DNA, and the second set carries DNA that binds to the other end (Figure 3). The nanoparticles
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are dispersed in water. When the target DNA is added, it binds both types of nanoparticle together,
linking them together to form an aggregate. The formation of this aggregate causes a shift in the light‐
scattering spectrum from the solution, that is, a colour change in the solution that can easily be
detected. The example is illustrated in Figure 3. Since the colloid changes colour as a result of the
sensing event, this is called colorimetric sensing. Gold colloids can be used for colorimetric sensing
specifically because they have different colours depending on the environment that surrounds them.

The method is not limited to sensing DNA strands but
also other complementary biomolecules such as
antigen‐antibody systems; it is used to study the
stability of proteins.

In this experiment student will only use a qualitative
approach: the outcome of the sensing process is a
change in colour of the solution which is visible with
the naked eye (visible absorption).

Figure 3. A plasmonic colloidal nanosensor. (Image credit: reprinted with permission from Jin et al., Journal of American
Chemical Society (2003), 125 (6), 1643‐ . Copyright 2003 American Chemical Society.)

4) Does nano‐gold have other properties different from bulk gold?
Yes, it does. Nano‐gold is extremely reactive and is now being studied as a new catalyst. Nano‐gold has
been shown to be an extremely efficient catalyst in numerous pollution control studies. For example, a
company has announced an engineered nano‐gold oxidation catalyst which can reduce diesel
hydrocarbon emission 40% times more than commercially available materials. Considering that there
are over 14 million light‐duty diesel vehicles worldwide, and 2 million heavy‐duty ones, the impact of
this nanotechnology could be enormous.

5) About the gold colloid synthesis
In the present experiment students prepare a colloid of gold nanoparticles in water starting from a
solution of gold chloride hydrate and a solution of sodium citrate. This is the simplest reaction to
synthesise gold nanoparticles, which was pioneered by J. Turkevich et al. in 1951 and refined by G. Frens

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in the 1970s. The method described in this reaction is adapted from Ref 1. This synthetic method
generally produces nanoparticles 10‐20nm in size, so the end‐product is a gold colloid1. In the reaction,
the citrate acts as a weak reducing agent (reducing AuCl4‐ to Au) and as a stabiliser. A layer of citrate
anions adsorbs around each nanoparticle and prevents these from aggregating: the anions’ electrostatic
repulsion keeps the nanoparticles separated. In this state, the colloid appears ruby‐red owing to the
absorption of light by the free electron oscillations (the surface plasmon). The position of the surface
plasmon λmax for colloid gold is between 500 and 600nm depending on the particles’ size and shape, as
well as the solvent refractive index and inter‐particle distance. In this experiment λmax is around 520nm
(green) and the solution appears red. A colour wheel can be used to show the correlation between the
colour a substance absorbs and the colour it appears.

If the anion layer is removed, the nanoparticles start to approach and agglomerate. This effect can be
used to sense a certain chemical. If a strong electrolyte is added, such as NaCl, the ions of the salt shield
the negative charges on the particles, allowing them to approach and aggregate into larger and larger
clumps. The formation of agglomerates is reflected in a change of the optical spectrum and the
appearance of a second absorbance peak around 650‐750nm, causing the solution to turn deep blue. If
a high concentration of salt is added, the nanoparticles aggregate to a point at which they precipitate,
and the solution becomes clear.

Figure 4. Schematic representation of the optical changes of a colloidal gold as a solution of salt is added. (Image credit: L.
Filipponi, iNANO, Aarhus University, Creative Commons Attribution ShareAlike 3.0)

1
In a colloid 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. The particles have dimensions in the 10‐
100 nm range. Natural colloids are in the form of emulsion (milk), gel (gelatine), aerosol (fog), etc.

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Other perturbations that can be easily observed are:

‐ If a stabiliser 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
(Figure 5). In this exercise egg white is used as a very economic source of protein (mainly ovoalbumin).

Figure 5. Schematic representation showing that a protein coats the gold nanoparticles and prevents them from
aggregating as salt is added to the colloid. (Image credit: L. Filipponi, iNANO, Aarhus University, Creative Commons
Attribution ShareAlike 3.0.)

‐ If a weak or non‐electrolyte is added (e.g. sugar), the electrostatic repulsion between the gold and the
citrate ions is 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 (Figure 6).

Figure 6. Schematic
representation of the optical
changes of a colloidal gold as a
solution of sugar is added.
(Image credit: L. Filipponi,
iNANO, Aarhus University,
Creative Commons Attribution
ShareAlike 3.0)

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One peculiarity of this synthesis is the colour change that is
observed as the citrate is added to the gold chloride hydrate
solution before it reaches the final ruby‐red colour. The
reaction starts with a solution which is yellowish (solution of
HAuCl4), and after the citrate is added, the colour changes first
to clear, then light grey, dark blue‐grey, then purple, dark

purple, and finally ruby‐red. Only recently have these
intermediate colours been understood and correlated to the
formation of intermediate nanostructures before the
formation of the final gold nanoparticles. Specifically, TEM
images have shown that after few seconds of citrate addition,
Figure 7. (Left) Growth mechanism of
gold nanowires 5 to 8 nm are formed, which are responsible for nanospherical gold particles synthesised by
‐
the dark purple colour. Beyond a certain threshold, the reduction of aqueous AuCl4 by sodium
nanowires disintegrate into nanoparticles, and the solution citrate. (Right) TEM images of the dark
intermediate showing an extensive network
turns ruby‐blue. Although the chemistry and kinetics of this
of gold nanowires which were isolated from
reaction would be too advanced for class teaching, this rapid the dark purple solution. (Image credit:
and dramatic colour change during the reaction should be reprinted with permission from Pong et al.,
closely observed and this observation used to further support J. Phys. Chem. C 2007, 111, 6281‐7.
the notion that size and shape at the nanoscale are very Copyright 2003 American Chemical Society.)

important factors (Figure 7).

THIS EXERCISE IN CLASS
Depending on the class level and time available, this Exercise Module can be used in different ways. The
authors suggest:

1. Starting with a general discussion of gold, asking students what they know about gold.
‐ What are the properties of gold? As a metal? Is it a catalyst?
‐ What colour is gold?
‐ What is gold used for?
In this part teachers can also discuss other aspects of the chemistry curriculum, such as:
‐ properties of metals and metal alloys
‐ electromagnetic spectra and colour
Students should complete questions Q1 and Q2 in the Student Worksheet.

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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 (link with Chapter 1 of Module 2 “Applications of Nanotechnology:
Medicine and Healthcare”)

3. Synthesis of a gold colloid. Divide students in groups of two and ask them to follow the
synthetic procedure (students should have the “Students’ synthesis procedure for experiment C”). The
procedure is described below. Instructors should encourage students to closely monitor the colour
change of the solution during the synthesis and record their observations in the table in the student
worksheet.

MATERIAL NEEDED:
Chemicals:
‐ 0.1 g of HAuCl4*3H2O (Sigma Aldrich #G4022, 1g costs 144.50 Euro);
‐ 0.5 g of Na3C6H5O7*2H2O (Sigma Aldrich #S4641, 25g costs 24 Euro),
‐ 1 litre of distilled water
Glassware/labware:
‐ Eye protection
‐ Latex or nitrile gloves
‐ Paper towels
‐ Cylinders: 10 mL cylinder, 50 mL cylinder and 500 mL cylinder
‐ Glass pipettes: 5 mL pipette and 25 mL pipette
‐ 50mL Erlenmeyer flask or beaker
‐ 4 disposable plastic capsules for measuring solids
‐ Spatula
‐ Glass bottles: bottle 500mL, 2 small bottles 25 mL or 2 beakers of 25 mL
‐ Aluminium foil
‐ 1 laser pen
‐ 1 stirring hot plate
‐ 1 magnetic stir bar
Also needed for laser test:
‐ 1 glass of milk
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SAFETY NOTE: The experiments described in the following training kit use chemicals which need to be used according to
MSDS specifications. 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.

IMAGES TERM OF USE: Images in this experiment protocol are copyright of iNANO, Aarhus University unless specified
differently. Permission is granted to share and distribute these images under the licence Creative Commons Attribute
ShareAlike 3.0.

SPECIFIC PRECAUTIONS
Use these materials with normal chemical precautions according to MSDS. Wear eye protection and
gloves. Solids should not be inhaled and contact with skin, eyes or clothing should be avoided. Wash
thoroughly after handling. Be aware that HAuCl4*3H2O is corrosive and must be handled with caution.

DISPOSAL OF THE GOLD COLLOID
After the experiment, dispose of any gold colloidal remaining as follows: add enough NaCl solution to
the colloid to induce precipitation. Leave the solution still for at least 30 minutes (a black residue will
form as in Figure. Filter the residue on filter paper, and then dispose of it with solid normal waste.
Dispose of the clear liquid in the sink with plenty of water.

PROCEDURE (PROTOCOL FOR TEACHER):

A. Preparation of stock solutions (to be made by the
students or by the teacher depending on time). Preparation time
30 minutes. Solutions indicated below are enough for 25 students
NB. all glassware used should be clean and dry.

‐ 1.0 mM hydrogen tetrachloroaurate: Dissolve 0.1 g HAuCl4*3H2O
(orange solid) in 500 mL distilled water. Solution appears light
yellow.

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NB: This stock solution of gold(III) ions can be prepared in advance if stored in a brown bottle or in a
bottle covered well (including cap) in aluminium foil. Stored this way the solution will last a couple of
months. This 500 mL of stock solution will serve for 25 experiments.

‐ 1% trisodium citrate: Dissolve 0.5 g Na3C6H5O7*2H2O in 50 mL distilled water.
NB: this stock solution will serve for 25 experiments

‐ ~1 M NaCl: Dissolve 0.5 g of NaCl (or table salt) in 10 mL distilled water.

‐ ~ 1 M sugar: Dissolve 2 g of sugar in 10 mL distilled water.

B. Synthetic procedure of the gold colloid

1) Add 20 mL of 1.0 mM HAuCl4 stock solution to a 50 mL Erlenmeyer flask on a
stirring hot plate (turned off). The solution appears light‐yellow. Add a magnetic
stir bar inside the flask; turn the stirring on and the temperature on. Bring the
solution to the boil.

COMMENTS: After the temperature is turned on, first some bubbles are seen
forming at the bottom of the flask, then they become more and more evident. This is when the citrate
should be added, even if full bubbling is not seen. The reaction should ideally be performed under reflux
conditions, as evaporation occurs which leads to a volume loss.

2) To the boiling solution, add all at once 2 mL of a 1% solution of trisodium citrate dihydrate,
(Na3C6H5O7*2H2O). The gold colloid gradually forms as the citrate reduces the gold(III). Students should
observe the colour change as the citrate is added.



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Figure 8. Addition of the citrate. Note that immediately after the addition the solution appears clear (right image).

COMMENTS: the solution starts slightly yellow, then it becomes clear as soon as the citrate is added,
then grey, dark grey, purple, dark purple, then ruby‐red. The entire reaction (from when the hot plate is
turned on) takes about 10‐15 min. To minimise the volume loss due to evaporation, once the solution is
boiling, reduce the temperature to about 100 °C.



                                                       1 min into reaction                       2 min into reaction



                                                     3 min into reaction                           4 min into reaction



In the images above you can see the shift from deep purple to ruby‐red colour at the end of the
reaction.

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Reaction was stopped after about 6 min of total reaction (counted from the citrate addition point).
Time of reaction might vary depending on the temperature you use, or the freshness of the citrate
solution.

Appearance of final colloid:

3) Once the solution is ruby‐red, turn the hot plate off and stop the stirring. Remove the flask from the
hot plate. WARNING: the flask will be hot so use a safety glove to avoid burning your fingers! Place the
flask on a safe surface (e.g. table bench with a piece of aluminium foil)

TIP FOR TEACHER: If you decide to prepare the gold colloid in advance, you can use the NANOYOU
Video 2‐ Gold Colloid to show the synthesis to the students

CLASS DISCUSSION
After the synthesis students can test the gold colloid to understand their properties:

Let the students test the ruby‐red colloid with a laser pen (WARNING: never look straight into a laser
beam!). A scattered light path across the colloid is clearly visible. Compare the effect of shining the laser
through water or stock solutions: the light will not be scattered.

‐ Students should also analyse other familiar liquids like milk (a natural colloid), and/or other solutions
like a salt solution.

NOTE! If you test milk, note that it must be diluted (place 150 mL of water in a beaker and add 2
droplets of milk; stir and let settle for a couple of minutes). Plain milk will backscatter all the light. All
solutions (e.g., salt solution) should be made with distilled water and should let settle before testing (air
bubbles in the solution might give erroneous results).

Students should fill in questions Q3 to Q7 of the Student Worksheet

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4. Gold colloid as a plasmonic colorimetric sensor. In nanomedicine gold colloids are now
being investigated as colorimetric sensors, for instance to detect specific DNA fragments. Here we
suggest a simpler experiment. The key elements of this type of sensor are that it is very sensitive and it
does not require the use of a label.

The colloid should be tested with different solutions to see how it can be used for sensing certain
species (these tests are described in the next section):
‐ Strong ionic solutions induce a colour change from ruby‐red to blue (e.g. NaCl). Therefore it can be
used to detect the presence of electrolytes.
‐ Weak or non‐ionic solution does not induce this change (e.g. sugar).
‐ Surfactants or proteins coat the colloid to a point it is not affected by strong ionic solutions anymore
(e.g. adding a white egg solution, followed by NaCl, does not induce a colour change).
‐ 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.

MATERIAL NEEDED:
The material indicated below is intended for students working in pairs.

Chemicals:
‐ The gold colloid prepared in the synthesis (part 3), should be about 15 mL
‐ 0.5 g of NaCl (Sigma Aldrich #S7653, 250g costs about 32 Euros), fine kitchen salt can be used as
alternative
‐ 2 g of sugar
‐ 1 fresh egg
‐ 1 litre of distilled water

Glassware/labware:
‐ Eye protection
‐ Latex or nitrile gloves
‐ Paper towels
‐ Cylinders: 10 mL cylinder, 50 mL cylinder and 500 mL cylinder
‐ Glass pipettes: 5 mL pipette and 25 mL pipette, 10 Pasteur pipettes
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‐ 50 mL Erlenmeyer flask or beaker
‐ 2 disposable plastic capsules for measuring solids
‐ Spatula
‐ Glass bottles: bottle 500 mL, 2 small bottles 25 mL or 2 beakers of 25 mL
‐ 6 glass vials
‐ 1 transparent plastic container

PRECAUTIONS
Use these materials with normal chemical precautions according to MSDS. Wear eye protection and
gloves. Solids should not be inhaled and contact with skin, eyes, or clothing should be avoided. Wash
thoroughly after handling.

DISPOSAL OF THE GOLD COLLOID
After the experiment, dispose of any gold colloidal remaining as follows: add enough NaCl solution to
the colloid to induce precipitation. Leave the solution still for at least 30 minutes (a black residue will
form as in Figure 11). Filter the residue on filter paper, and then dispose of it with solid normal waste.
Dispose of the clear liquid in the sink with plenty of water.

METHOD:

1) Divide the ruby‐red gold colloid into 5 vials each containing about 3 ml

2) Use one vial as control, and the other four to perform different plasmonic colorimetric tests:

‐ TEST 1: add 6 droplets of NaCl solution: the colloid becomes blue (if it is general kitchen salt it looks
more grey‐blue). Why does this happen? In the colloid citrate ions act as stabilisers, they cover the
surface of the nanoparticles and prevent their aggregation. When a strong ionic solution (NaCl) is added,
its ions shield the charges on the particles, allowing them to approach and aggregate. The formation of
agglomerates is reflected in a change of the optical spectrum causing the solution to turn deep blue.

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Figure 11: colour change as salt is added to the gold colloid.

‐ TEST 2: add 15‐20 droplets of NaCl: the colloid becomes dark‐grey, and a black precipitate starts to
appear (figure on left). If the sample is left still for some time (30 min) the solution becomes clear and a
lot of black precipitate forms (Figure 12). Why does this happen? If a high concentration of ionic
solution (NaCl) is added, the nanoparticles aggregate to a point they precipitate, the solution becomes
clear and a black precipitate is clearly seen forming (Figure 12, left). If the vial is left to rest for about 30
minutes, aggregation continues until a big precipitate forms, and the solution appears clear (Figure 12,
right).

Figure 12. Precipitation of a gold aggregate as an excess of NaCl is added to the gold colloid.

‐ TEST 3: add 10 drops of sugar solution: the colloid doesn’t change colour and it remains ruby‐red. Why
does this happen? Sugar is anionic; therefore the citrate ions covering the gold nanoparticles remain
unperturbed.

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Figure 13. Effect of adding a solution of sugar to the gold colloid (vial on the far right). In the middle is the vial where a salt
solution was added to the gold colloid, and on the left is a control (pure gold colloid).

‐ TEST 4: Open a fresh egg and with a Pasteur take some egg white (about 1 mL or 2‐3 full Pasteurs),
place it in a empty glass vial and add one Pasteur of distilled water. Mix gently together: it will form a
foam, so let it stand a minute to reduce the foam, then take the solution from the very bottom to avoid
the foam/bubbles. Add this water/egg‐white mixture to the gold colloid: the colloid turns dark pink and
an “emulsion” is seen. Why does this happen? As the protein is added to the gold colloid, the
nanoparticles become coated with a layer of protein, which acts as a stabiliser. Nevertheless, during the
addition of the protein, a little aggregation might occur, which is reflected in a colour change (dark pink
instead of ruby‐red). More sophisticated addition methods (e.g. using stirring) might prevent this from
happening.



Figure 14. Addition of egg white to the gold colloid. Left is the control, right is the test vial.

‐TEST 5: If NaCl (6 drops) are added to the above solution (TEST 4), the colour does not change; it does
not turn blue. If more NaCl is added, the colour still doesn’t change. Why not? The protein (mainly
ovoalbumin) in the egg white acts as a surfactant, shielding the gold nanoparticles from the ions, in a
sense “protecting” the nanoparticles. The NaCl ions don’t have access to the gold nanoparticle surface
and therefore do not induce a change in their aggregation.

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egg 10
control egg
white + drops
white
10 drops NaCl
                                                 NaCl

Figure 15. Overview of the test experiments. Left is the control (only colloidal gold), then are the vials containing the colloidal
gold and the tested specie: egg white, egg white plus 10 drops of NaCl, and only 10 drops of NaCl.

After the test, students should fill in Q8 to Q15 of the student worksheet

5. Gold colloid as a medical diagnostic tool.
Students should reflect on how a gold colloid can be used as a diagnostic colorimetric tool. The general
idea is that any diagnostic process that induces a change in aggregation can (potentially) induce a visible
colour change. To quantify this change, a spectrophotometer would be needed to measure the
absorption of the solution. To be relevant in diagnosis the process must be specific, based on a
biomolecular recognition system, which can be the complementarity of DNA strands, or other processes
such as antigen‐antibody recognition.

Compared to other diagnostic tools, this one has the advantage of not requiring a label, since the
detection reaction is visible. This is an advantage because labelling is normally a long process, therefore
a label‐free detection method is faster and requires less consumables, lowering the cost of the process.

Students should fill in Q16 and Q17 of the Student Worksheet.

6. Additional topics for class teaching:
Depending on the needs and level of the class, instructors can use this experiment to discuss a number
of additional science topics:
‐ Solution vs. colloids
‐ Bulk metals and nano‐sized metals: differences in bonding, conductivity properties, etc.

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‐ Electromagnetism and properties of light (reflection, scattering, etc.)
‐ Optical spectra of a substance and optical properties of a nanomaterial
‐ Science and art: examples of ancient artefacts containing nanotechnology
‐ Proteins and their sources (e.g. egg white)

ANSWERS TO QUESTIONS
Q1. Gold alloys are made of bulk gold mixed with other metals. In this form gold alloys have different
colours (e.g. “white gold”, “pink gold”, etc.).
Q2. A metal colloid can have different colours depending on the size of the nanoparticles that form the
colloid. The metal colloid can be red, purple or blue. The colour is due to the presence of surface
plasmons.
Q3. Because there are some intermediate nanostructures that are formed during the reaction
Q4. In a colloid the substance is only dispersed (not solubilised) in the solution
Q5. Because nanoparticles scatter light, so a scattered path can be seen through the colloid
Q6. Milk, gelatine
Q7. Milk and the gold colloid
Q8. Blue
Q9. In the gold colloid, citrate ions act as stabilisers; they cover the surface of the nanoparticles and
prevent their aggregation. When a strong ionic solution (NaCl) is added, its ions shield the charges on
the particles, allowing them to approach and aggregate. The formation of agglomerates is reflected in a
change of the optical spectrum, causing the solution to turn deep blue.
Q10. The colloid aggregates so much that a black precipitate forms.
Q11. The colloid remains nearly the same colour; it shifts a bit toward pink, but it does not become blue.
The reason is that sugar is anionic, so the citrate ions covering the gold nanoparticles remain fairly
unperturbed. The fact that the colloid is pink rather than ruby‐red indicates that a small change in
aggregation has occurred.
Q12. Egg is a source of protein (ovoalbumin)
Q13. Adding egg to the colloid does not change its colour
Q14. The colloid remains red. The protein (mainly ovoalbumin) in the egg white acts as a surfactant,
shielding the gold nanoparticles from the ions, in a sense “protecting” the nanoparticles. The NaCl ions
do not have access to the gold nanoparticle surface and therefore do not induce a change in their
aggregation.
Q15. When salt is added directly to the gold colloid, the ions of the salt shield the charges on the
particles, allowing them to approach and aggregate. When a protein is added instead, it forms a shield

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around the nanoparticles, so the ions of the salt can no longer interact with the citrate around the
nanoparticles.
Q16. The fact that a change of agglomeration in colloids leads to a colorimetric change can be used to
detect a specific biomolecule. One can use the lock‐and‐key mechanisms of some biomolecules, like
antigen‐antibody. For instance one could have a gold colloid probe having gold nanoparticles
functionalised with antigen A. If we add a colloid target containing various antibodies, and a colour
change is seen, then antibody A is present in the target.
Q17. Labelling is a time‐consuming procedure, and labels are also expensive.

REFERENCES:
‐ Ref 1. Journal of Chemical Education, Vol. 81 (4), 2004;
‐ Ref 2. A more detailed description of the synthesis of colloid gold is given in: Keating et al., Journal of Chemical
Education 1999, Vol. 76 (7) pp. 949‐55.
‐ Ref 3. For the colour change as the synthesis of the gold colloid proceeds and the formation of intermediate
nanostructures, such as gold nanowires, see: Pong et al., Journal of Physical Chemistry C, 2007, Vol. 111, pp. 6281‐
7.

...................................................................................................................................
CREDIT
This exercise was partly adapted from the experiment reported in: Journal of Chemical Education, Vol. 81 (4), 2004
and from the experiment “Citrate synthesis of gold nanoparticles”, University of Whisconsi‐Madison, see:
http://www.mrsec.wisc.edu/Edetc/curriculum/index.html; A more detailed description of the synthesis of colloid
gold is given in: Keating et al., Journal of Chemical Education 1999, Vol. 76 (7) pp. 949‐55.

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Experiment with colorimetric cold nanosensors - Teacher guide (age 14-18)

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Experiment with colorimetric cold nanosensors - Teacher guide (age 14-18)