1. Summer Project Report, 19 October 2016
Summer Project Report
Science without Borders through Nanoengineering Materials at Chemical – Bio Interfaces
for Mechanical Repair of Concrete and Human Bone
Professor Paul Sermon
Iman Al-Timimi
Tenório Itiro Fukushima Feliciano da Silva
Student Number: 1406170
1. INTRODUCTION – Biomimetic Material
After 1950’s, when Otto Schimdt related biology with technology transfers as biomimetic (Vicent,
2006), this area became an important field for researchers for the fact that these kind of materials
provides innovative solutions for the design of a new generation of bio inspired functional materials
( [2] , website). Natural materials display a wealth of structures and fulfil a variety of functions.
Hierarquial structuring is one of the keys to providing multifunctionality and to adapting to varying
needs of an organism. (Paris, 2010).
Organic syntheses were the initial realm of biomimetic chemistry (Breslow, 1972) but its impact has
progressed to bioinorganic interfaces with benefits to materials design. With evolution many living
plant and animal species have constructed species-specific bio composite structures (Sarikaya,
2003) that have nano-architectures whose green processing is the envy of materials scientists
(Mann, 2008). In Biomimetic Material, researchers develop and replicate these nature’s three-
dimensional self-assembled biotemplates. Over the years, nature compounds will be gradually
replaced by a systematic approach involving the study of natural tissues in materials laboratories,
the application of engineering principles to the further development of bio-inspired ideas and the
generation of specific databases.

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2. LABORATORY METHODOLOGY
The experimental procedure was realised at Bragg Building, Wolfson Centre, Brunel University as a
complement of researches developed by Iman Al-Timimi.
First of all, spores from mushroom (PMS), usually found in markets, were collected. The mushrooms
were left in a glass container for some days and the deposited spores were harvested.
Secondly, solutions of calcium nitrate tetra-hydrate Ca(NO3)2 and ammonium phosphate dibasic
(NH4)2HPO4, were prepared in four different concentrations: 1M, 0.1M, 0.01M and 1mM, resulting in
eight solutions.
The pHs of each solution are shown in table 1.
Table 1. pH of solutions in different concentrations
Concentration
pH
Calcium Nitrate Tetra-hydrate
Ca(NO3)2
Ammonium Phosphate dibasic
(NH4)2HPO4
1M 6.43 8.21
0.1M 6.79 8.12
0.01M 6.87 8.12
0.001M 6.60 8.10
2.5 mg of PMS were inserted in 2ml of each solution and let them react, resulting on eight samples.
The eight samples (solutions + PMS) in addition to the sample with PMS in water were analysed in
optical microscopy and some of them in Scanning electron microscopy with energy dispersive X-ray
spectroscopy (SEM-EDX). Experimental Techniques Centre (ETC) provided these equipment.
Spores were placed in calcium nitrate solution and after ammonium phosphate solution in order to
produce Hydroxyapatite with the reaction between Ca2+
and PO4
3+
in the following proportion
according to the stoichiometric equation below:
10 Ca(NO3)2 + 6 (NH4)2HPO4 → Ca10(PO4)6(OH)2 + 18H2O (1)
For this, three samples were prepared varying concentration of each solution.
• Sample 1: Spores were left in 0.1M of calcium nitrate solution and after 0.107M of
ammonium phosphate dibasic solution.
• Sample 2: Spores were left in 0.01M of calcium nitrate solution and after 0.0107M of
ammonium phosphate dibasic solution.

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• Sample 3: Spores were left in 0.001M of calcium nitrate solution and after 0.00107M of
ammonium phosphate dibasic solution.
Finally, to analyse the aggregation in a 2-dimmension plan, the sample 3 were placed between 2
parallel glass plates and images were taken from optical microscopy.
3. DISCUSSION OF RESULTS
3.1. Influence of solutions in PMS dimension
Spores from each sample with different concentrations of calcium nitrate solution were removed in
order to analyse the influence of this solution in the spores’ dimension. A hundred spores from each
sample were measure in length and width dimension and average and standard deviation were
calculated. Table 2 shows the results.
Table 2. Spores Dimension under Ca2+
influence
Concentration of
Ca2+
Length
(µm)
Width
(µm)
1 M 6.7 ± 0.5 4.9 ± 0.3
0.1 M 6.8 ± 0.5 4.9 ± 0.3
0.01 M 6.6 ± 0.5 4.8 ± 0.3
0.001 M 6.6 ± 0.5 4.8 ± 0.3
Plotting this values in one graph (1), there is:
Graph 1. Length x Width of Spores under Ca2+
influence

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According to the graph and the table it is possible to notice that the influence of Ca2+
ions in
mushroom spores dimensions is not clear once the average of width and the error bars of one
samples enclose length of other one. In other words, statistically all the four samples have the
same width and length measure.
In addition, the dimensions of spores under PO4
3+
in concentration of 0.001M were also measured.
Table 3. Length x Width of Spores under PO4
3+
influence
Lenght. (µm) Width (µm)
7.6 ± 0.6 5.5 ± 0.4
The dimensions under ammonium phosphate are around 10% higher compared under calcium
nitrate influence.
3.2. Optical Microscopy images
The images from optical microscopy were taken in 40x zoom configuration.
Spores in water are shown in figure 1.
Figure 1. Spores in water
Then, images from spores under different concentrations of calcium nitrate dibasic were taken.

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Figure 2. Spores under calcium nitrate influence in different concentrations: (1) 0.001M (2) 0.01M
(3) 0.1M (4) 1M
For ammonium phosphate:

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Figure 3. Spores under ammonium phosphate influence in different concentrations: (1) 0.001M
(2) 0.01M (3) 0.1M (4) 1M
Lastly, spores under hydroxyapatite influence (Samples 1, 2 and 3):

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Figure 4. Spores under hydroxyapatite influence in different proportions: (1) Sample 3
(2) Sample 2 (3) Sample 1
3.3. SEM - EDX
Spores in 1mM of calcium nitrate solution were analysed in SEM.
Figure 5. Spores under 1mM solution of calcium nitrate (SEM-EDX)

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The areas 1, 2, 3, 4 and 5 were used in order to know the percentage amounts of each element
(atomic and weight percentages). The average values of areas 1, 2, 3 and 4 provided by the
software are shown in the table bellow.
Table 4. The average percentage of elements in spore having seen calcium nitrate 1mM solution
(area 1, 2, 3 and 4)
Element Weight % Atomic %
Carbon (C K) 50.96 69.88
Gold (Au M) 22.21 1.86
Oxygen (O K) 14.41 14.84
Nitrogen (N K) 10.82 12.73
Calcium (Ca K) 0.73 0.30
Potassium (K K) 0.57 0.24
Phosphorus (P K) 0.19 0.10
Sulphur (S K) 0.09 0.04
Silica (Si K) 0.02 0.01
The highest values are carbon and gold for the fact that carbon tape was used and the sample was
coated with gold. The next elements, oxygen, nitrogen and calcium came from the dissociation of
calcium nitrate.
The percentages in background, area 5, are shown in table 5.
Table 5. The average percentage of elements in background (area 5)
Element Weight % Atomic %
Carbom (C K) 73.60 92.24
Gold (AuM) 18.47 1.41
Oxygen (O K) 5.14 4.83
Nitrogen (N K) 0.49 0.52
Calcium (CaK) 0.86 0.32
Potassium (K K) 0.82 0.32
Phosphurus (P K) 0.02 0.01
Sulfur (S K) 0.21 0.1
Silica (SiK) 0.14 0.07

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In this region, there are no spores, just background was analysed, and therefore the predominance of
carbon is notice (carbon tape).
In addition, the samples 1, 2 and 3 were also analysed in SEM – EDX.
Figure 6. Sample 1 (higher concentration) 5000X (section: 20kX)
The spores were not washed with water before the analysis, resulting in the accumulation of
hydroxyapatite between spores. However in the maximized area it is possible to notice a little
chamber that could be caused by the crack up of a spore.
Figure 7. Sample 2 (10mM solution used) 5000X

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In Figure 7, it is not possible to notice any alteration in the spore surface due to the excessive
accumulation of hydroxyapatite. Wash may have removed the excess of hydroxyapatite.
Figure 8. Sample 3 (lower concentration) 20000X
Sample 3 is the one that has lower concentrations of calcium nitrate and ammonium phosphate. This
reveals a layer accumulation of hydroxyapatite on spores surface, just located accumulations are
seen around the surface.
3.4. Spores Translucency
Using the software Image SMX (version 1.97) the translucency of spores was measured, in other
words, the spores darkness was analysed in order to compare the difference influence on them.
Due to the fact that the software just analyses images files (optical microscopy images in case), all of
them were taken in the same day, at same conditions (Brightness: 70%, Saturation: 1.40, Gamma:
0.70, Brightness: 8.2) in order to avoid errors.
The table bellow shows the results provided by the software.

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Table 6. Spores Translucency in different influences
Influency of
Translucency degree
Max. Min.
Calcium Nitrate 1mM 185 10
Calcium Nitrate 0.01M 190 30
Ammonium Phosphate 1mM 170 20
Ammonium Phosphate 0.01M 160 20
Hydroxyapatite (1mM) 170 0
Hydroxyapatite (0.01M) 170 0
Water 175 10
Obs.: The higher the degree is, the darker the spore is.
The maximum values are related to region around the spore border whereas the minimum values are
related to central region of spores.
According to the table above, Calcium nitrate causes on spores to be about 10% darker than in water
or other solutions, which practically have the same values. However, 10% is not considered a
substantially amount and can the included in experimental errors.
3.5. Aggregation
The method used to analyse the aggregation in a 2D plan was to drop spores in hydroxyapatite
solution (1mM) in a narrow gap between 2 glass plates. As the plates are very close to each other, the
surface tension of them is as considerable as gravity force, causing spores to aggregate in the plate’s
surface.
Figure 9. Aggregation of spores in hydroxyapatite 1mM (10x)

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In this image it is not possible to see many details, just islands of spores. Higher zoom configuration is
required to notice further details, however the maximization 40k and 100k of microscopy were not
working properly.
4. CONCLUSIONS
Mushroom spores are quite similar when immersed in calcium nitrate tetra hydrate solution and
ammonium phosphate dibasic solution in different concentrations. Furthermore the translucency of
them remains almost unchanged by Ca2+
and PO4
3-
influence.
Hydroxyapatite forms on the surface of the mineral form of calcium moves to the surface of spore.
Between two spores or among them, hydroxyapatite creates a connection, causing in some cases the
aggregation of them.
5. ACKNOWLEDGEMENT
I would like to express my sincere gratitude to professor Paul Sermon for being my tutor in the
Summer Project and for giving me the opportunity to learn a little bit about biomimetic materials.
Besides my tutor, I would like to thank Iman Al-Timimi for all given support and all knowledge shared,
being like my second tutor over the last 3 months.

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6. REFERENCES
[1] J.F.V.Vincent, O.A.Bogatyreva, N.R.Bogatyrev, A.Bowyer and A-K.PahlInterface3,471-
482,(2006)
[2] https://www.beilstein-journals.org/bjnano/single/articleFullText.htm?vt=f&publicId=2190-4286-2-
16&sn=3&tpn=0&bpn=singleSeries, accessed 9 July 2015.
[3] O. Paris, Ingo Burgert and Peter Fratzl (2010). Biomimetics and Biotemplating of Natural
Materials. MRS Bulletin, 35, pp 219-225. doi:10.1557/mrs2010.655.
[4] R.Breslow, Chem.Soc.Rev.1,553,(1972).
[5] M.Sarikaya, C.Tamerler, A.K.Y.Jen, K.Schulten and F.BaneyxNature Materials 2,577,(2003).
[6] A.B.Mann, R.R.Naik, H.C.DeLong, K.H.Sandhge, J.Mater.Res.2008,23,3137.