21-01-2018

LABORATORY
EXPERIENCE
NANOBIOTECHNOLOGY

Kinetics and Thermodynamics of Au Colloid

Monolayer Self-Assembly

María Inmaculada Pérez Biedma

Università degli studi di Trieste

ABSTRACT

We describe in this article some experiments using colloidal gold nanoparticles in the
context of surface and nanomaterials chemistry. We study in it the optical properties and
stability of aqueous gold colloidal suspensions and determine the amount of protein necessary to
avoid the flocculation of colloids induced by salt.

INTRODUCTION

Some of the atoms present in the surface of materials are so important, as they
determine most of their properties and, therefore, the use we can make of them.

One approach to control the morphology of the surfaces of these materials is the use of
assembly, spherical, small and uniformly sized particles at an interface, creating a repeating
structure with a certain size.

Colloidal gold nanoparticles are a very good approximation as surface building blocks
due to the physic, chemic, and biological properties that they exhibit (inherent to their nano-
size). Their peculiar photothermic and optical properties come from the resonant oscilations of
their free electrons in the presence of light (Localized Surface Plasmon Resonance), thanks to
which the nanoparticles can radiate light or absorb light that quickly transform into heat.
Colloids gold particles are red owing to the absortion of light. The maximum absorption
absorbance in the position of surface plasmon is between 500 and 600 nm (for smaller colloids a
blue shift is expected and for larger red-shifting).

With the aim to understand the properties of these colloidal gold particles, we describe
in this article some experiments in which we study the optical properties and stability of Au
colloid self-assembly on glass microscope slides (already coated with a bifunctional
organosilane).

MATERIALS AND METHODS

 Materials
The materials used in this practice are the following:

Solutions and chemical components:

 H2O mq
 Chymotrypsin ( our protein)
 Sodium citrate
 500 mL HAuCl4 (1mM)
 NaCl (1M)

Glassware and support materials:

 1-dram glass vials

 Pipette and tips (with different dimensions)
 Small Teflon blocks

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  3 mL plastic cuvettes
 1 L Erlenmeyer flask
 Thermometrer
 Condeser
 Metal support
 Fume hood
 Boiler
 Magnetic Stirring hot-plate

(*) We should have all these materials properly cleaned but for being sure
about this, we must clean them by soaking them in 1 L 95%
CH3CH2OH,/120 mL H2O/120 g KOH or with “piranha solution” (3:1
HCl/HNO3) to be sure that there is no contaminant that could harm the
process (the most common are the organic ones).

Computer instrumentation and advanced analyzers

 UV- visible spectrophotometer

 Adequate programs
 Computer

 Methods

To explain the procedure we have followed, we differentiate two principal parts. In the
first one we prepare the gold nanoparticles and verify their correct optical properties and in the
second one we synthetize protein-coated gold nanoparticles, testing their anti-aggregation
properties.

1. Synthesis of gold nanoparticles:

1.1 First of all we boil the solution of HAuCl4 (500 mL 1mM) in a 1-L Erlenmeyer
flask, increasing the temperature until the boiling point, where we see a clear color change
(really important fact for the rest of the practice. (We have to punctuate that is really important
to use cleaned glassware in order to avoid any contaminant).
The diameter of the gold nanoparticle we want to synthetize is about 13 nm.
As we actually measure the temperature that is outside the flask, we have to increase the
temperature more than the boiling point to really heat the inside. (*) The boiling process (that
includes the synthesis of the solution) is a really long process so it was already started by the
professor.
The agitation is also very important in this process to have always the solution
homogenized so it was continuously stirred on a magnetic hot plate.

1.2 We add the reducing agent (sodium citrate, 50 mL, 38.8 mM) while it is still
stirring, playing quite attention to the rapid color transformation. The color changes from bright
yellow to blue and then to dark burgundy color. After the addition we have to wait
approximately 15 minutes to complete the reaction to AuO (mainly gold).
We have always to take into consideration the time process and wait the required time
as it is possible to have aggregates forms. (*) We used gold nanoparticles solution of other year
as we didn’t enough time.

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 After all this, we can see different colors and maybe turbidity (we saw turbidity due to
the formation of aggregates). A successful citrate-absorption on the surface would lead to a
good clearness.

1.3 Then, we perform a spectrum of these nanoparticles. For this we make a solution
more diluted to not make the absorbance be so high. With a 1:5 diluted solution (200 µL AuNP
+ 800 µL H2O mq) it is expected an absorbance of 0.7-0.9. We have to do first the blank (with
just H2O mq) and then measure the solution absorbance value. The absorption is about 500-520
nm, very similar to the permanganate one, because of its strong violet color. (The
spectrophotometer is double beam (we can measure the blank and the solution at the same time).

1.4 We add NaCl. NaCl is going to polarize the particle, which starts to aggregate
because of the colloid stabilization lost (due to the citrate). We add 5 µL of NaCl to the colloid
solution (having a concentration around 1M, as we had previously 45 µL). We have to do this
really fast in order to measure the absorption just after the adding. The color result of this step is
bluish-grey.

2. Synthesis of protein-coated nanoparticles:

2.1 We are not going to stabilize the nanoparticle by an electrostatic force, but with
steric stabilization: providing the shell that keeps the nanoparticle distant one from the other to
not precipitate and form aggregates.

2.2With this aim, we use a solution without proteins for preparing the protein-coated
colloids. We prepare 10 solutions of colloids in plastic cuvettes and then we add increasing
amounts of alpha-chymotrypsin in order to know the molar concentration needed to conclude
the stabilization. We use the same amount of particles that have to be treated in all the cuvettes
and increase the amount of proteins.

2.3. In the first cuvette there is not any protein (it is the blank) so the absorbance should
be 0. Then we increase the amount of protein in each cuvette. As we are adding just proteins, we
are changing the volumes, so we have to rectify them adding also water in order to normalize
the concentration and have the possibility to mix all them together. After adding the protein we
have to wait for a while to incubate the system.

2.4 Now we measure the absorbance of all these solutions.

2.5We add 100 µL of NaCl (1M) into the cuvettes and homogenize them a little bit.
Then we measure the new absorbance. Ideally we should measure the specters at the same time
but this is obviously not possible. This time gap makes less precise the efficiency idea of the
protein coverage.

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RESULTS

1. Synthesis of gold nanoparticles results:

The nanoparticles that we have prepared with the protocol were more or less spherical
with a 12-13 nm diameter (standard deviation around 1.5 nm). The solutions according to the
paper show a λmax at 516–518 nm, with an intense absorbance of 0.7–0.9 after dilution.

The maximum absorption of the nanoparticle war almost 0.6, actually what we expected
as the nanoparticles used were old. We obtained absorption in the red area what it is translated
into a blue color of the solution, so we could continue with the next step: addition of NaCl. This
salt shield protects the charges of the particles, allowing them to approach closely and aggregate
in large groups (typically settling to the bottom of the recipient that contains them).

When we make the addition of the NaCl and measure the absorbance with the
spectrophotometer, we observe different number results and how the peak is decreasing with
time. If we continue doing spectrums the absorbance decreases comparing with the beginning.
Comparing before and after the addition of NaCl, we realize that after 10 minutes we don’t have
almost absorbance.

2. Synthesis of protein-coated nanoparticles results:

The difference with the first part is that we have coated the nanoparticle with alpha-
chymotrypsin. Measuring the absorbance of these solutions before adding NaCl, we obtained
more or less the same absorbance for all of them. We have to expect another thing due to the
electric constant of the layer surrounding the particles as the nature of the species we have
attached affects the maximum absorption of colloids. If the dielectric constant of what we have
on the surface of the colloid changes, we have a small change in the position of the maximum
point of absorption. So we should see this effect in the graphic.

When we add NaCl we see there is not enough protein in the cuvettes to achieve the
stabilization so the nanoparticles precipitate, and we have an excess protein in the solutions
respecting to what is needed. We see the same absorbance but the point we want is that one on
the middle.

Seeing the results, there is a visible change in the two first cuvettes, where there is a
huge precipitation (no protein or 0.5 mL of protein is more or less the same). The graphic starts
with a really high absorbance an then there is a peak on the 593 nm value. There is also
absorption around 200-500 nm and a very large shoulder that it wasn’t in the colloids solution,
what means that you have aggregates. We expect the shoulder is going to disappear when we
increase the concentration so we can see the surface Plasmon band.
We also realize that the maximum now is a little bit shifted that before: from 520 nm to 593nm,
what means that the dielectric constant of the material (with which we covered the surface) has
change: the citrate has been replaced by the protein.

(*) It is important to say that the concentration of other proteins that could be present in
the solution doesn’t affect the spectrum as they absorb in other areas. Despite this, a high

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concentration (that we cannot see in the graphics) of salt could cause salting-out of the protein
and thus affects to the nanoparticle, obtaining precipitation of them too. This leads to the
formation of a fluffy pinkish precipitate made of salted-out passivated nanoparticles (due to the
high concentration of salt.

What we have talked before can happen when the concentration of salt is really high or
the proteins concentration too low. In our case the ionic strength of the medium doesn’t change
because nanoparticle is covered. On the contrary, if we had used a low amount of protein, the
nanoparticle wouldn’t have been totally covered and with the addition of NaCl could have
happened aggregation. As we see, this is a concentration-dependent effect and allows us to
understand how much protein is needed to have a complete stabilization.

Observing the following images we are going to extract some information: The blue
spectrum belongs to the solution without any protein, after the addition of NaCl. The red one is
the colloid solution that contains alpha-chymotrypsin in a concentration of 0.8 μg/mL and the
grey one is obtained from the solution with 3.2 μg /mL 0.2 μM. Looking to the graphics we can
conclude that the concentration of chymotrypsin is enough to see stabilization.

[Prot. A] Abs
[Prot. A] Vs. Abs 0,00E+00 0,22
0,5 0,051 0,35
0,4 0,205 0,42
0,3 0,512 0,39
Abs

0,2 1,024 0,37

0,1 1,536 0,37
0 2,048 0,38
0,00E+00 5,00E-01 1,00E+00 1,50E+00 4,1 0,4
[Prot. A] (μg/mL)
6,14 0,39
8,19 0,39

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 If we also see the analysis of the other solutions, we can appreciate that there is not
absorbance in the aggregate band because it remains stable. Considering all the points, we can
see a dispersion that reaches a plateau, which corresponds to an absorbance of about 0.5-0.4 nm.
From this data we can conclude that about 0.5 μg of chymotrypsin is enough to have
stabilization.

To finish we have to emphasize that we used chymotrypsin like the study protein
because it was in the laboratory. Actually it doesn’t matter the protein we use as the outcome
will be the same.

In the paper where this experiment is based on, they use a bacterial protein that binds
quite well to the Fc part of antibodies. This is so useful when we want to purify antibodies
because you can fix the protein on a support and do chromatography. You can even use
antibodies combined with nanoparticles to detect the presence of the target in the solution.

CONCLUSIONS AND NEW PERSPECTIVES

With these experiments we have learnt important concepts related to physics, kinetics,
chemistry… that are so necessary for the correct understanding of this new approach that we are
studying in the context of nanobiotechnology. We have also had the opportunity to see how the
instrumentation required (just a spectro-photometer) works, how it is its manipulation and how
we should interpret the results.

On one hand, through these experiments we have seen how is the real production in a
laboratory of a really good and useful tool that is very present today in many investigation fields
and whose application rage is broadening each day. This variability of applications is
fundamentally due to the many molecules that can be linked to its surface (mainly through thiol
groups but also through amino, phosphite, bisulfite …)

One of the largest recent studies confirms the great potential of gold nanoparticles in the
elaboration of pharmaceutical nanoporters and therapeutic macromolecules. The gold
nanoparticles are also useful in the preparation of "Intelligent Systems" that release the
therapeutic molecule encapsulated as a result of activation of an internal stimulus (release
mediated by a change of Ph) or external (release triggered by a source of Laser light).

Undoubtedly one of the most promising applications of gold nanoparticles in the field of
medicine is its use as a platform that integrates in the same system the two essential functions
for the proper treatment of cancer: diagnosis and subsequent therapy. AuNPs can be detected in
biological samples and tissues using a variety of methods: electron microscopy, darkfield,
scanning laser, optical tomography, Raman spectroscopy, X-ray image, etc.