# Hollow Disc and Sphere-Shaped Particles from  Red Blood Cell Templates

Authors: Preston B. Landon; Jose J. Gutierrez; Sara A. Alvarado; Sujatha Peela; Srinivasan Ramachandran; Fernando Teran Arce; Ratnesh Lal
Journal: Research Letters in Physical Chemistry
 (2008)

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## Abstract

Colloidal gold particles with uniform size distributions were fabricated utilizing human red blood cells (RBCs) as templates. The gold shells were charged with a metal chelating agent to prevent flocculation. The procedure described here allows control over the shape of the colloidal particles. Thus, it was possible to fabricate discs and spheres by controlling the osmotic pressure.


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## Body

## 1. Introduction

Colloidal particles with specific
shape and function [1, 2]  are currently a subject of intense investigation for
the development of pigments [3], microcapsules [3, 4], guided drug carriers
[5, 6], synthetic opals [7], transparent metals [8], photonic crystals [7, 8],
thin-film transistors, organic light emitting diodes [9], solar cells [10, 11],
and tunable lasers [12].Large populations of nonspherical
colloidal particles with uniform size distributions have many potential
applications. Nonetheless, there are only a few techniques for the creation of
nonspherical particles since their formation has been proven difficult. In
recent years there has been a considerable effort in the formation of colloidal
nanorods of various materials [13, 14]. Another example of nonspherical
particles is the formation of oval-shaped polystyrene by applying mechanical
force to spherical particles in the solid phase [15].On the one hand, it is difficult to
create uniform populations of nonspherical colloids in the laboratory. On the
other hand, nature has been extremely efficient at this task. Moreover, unique
shapes of colloidal particles are found throughout nature.Human blood cells have a biconcave disc shape with an average diameter of
8.1μm. They are roughly 2 μm thick at
the outer edges and 1-μm thick in their center [16]. In a typical person, red blood cells (RBCs) have a coefficient of variation of approximately 11% [16]. Thus, the size distribution in this colloidal population can be considered relatively
uniform. The outer surface of a red blood cell is composed of a phospholipid
bilayer membrane [16] similar to that of most eukaryotic cells. However, unlike
most cells, RBCs do not divide or reproduce themselves. They are fabricated as
individual particles elsewhere within the body [16] and are, thus, truly
colloidal in nature. The colloidal nature of RBCs and their unique shape
motivated us to fabricate metallic colloidal particles using them as templates.
Several potential commercial applications are envisioned  [1–10].


## 2. Experimental Section

### 2.1. Materials

All chemicals were obtained from
Aldrich Chemical Company and were used as received unless otherwise noted. Gold
chloride was prepared as described elsewhere [17]. RBCs were obtained using a
diabetic finger lancet. A boric acid and sodium borate isotonic contact lens
saline solution was used in all experiments. Samples were centrifuged using a
Hamilton Bell Van Guard V6500, which is a fixed speed blood centrifuge. All
solutions were prepared with deionized water with a resistivity below 17 M Ω cm.


### 2.2. Colloidal Discs

Colloidal gold discs were prepared at room temperature as follows. Three
drops of blood were dispersed in 1.5 mL of saline solution in 2 mL centrifuge
tubes. 100μL of 0.06 M aqueous sodium citrate (used as anticoagulant) followed
by 100 μL of 0.006 M aqueous gold chloride were added at once. The mixture was
shaken by hand and centrifuged for 60 seconds. The supernatant was decanted and
the remaining sediment was quickly redispersed in pure saline solution. The
coating process (i.e., addition of aqueous AuCl3) was repeated two
additional times. The gold coated RBCs were centrifuged and redispersed several
times in 400 μL of 0.035 M aqueous 16-mercaptohexadecanoic acid for the purpose
of purification.


### 2.3. Colloidal Gold Spheres

Six drops of blood were dispersed in 1.5 ml of saline solution. 120μL of .06 M aqueous sodium citrate
followed by 400 μL of 0.006 M aqueous gold chloride were added at once. After
addition of gold chloride, the sample was immediately shaken and centrifuged
for 60 seconds. The supernatant was decanted and the remaining sediment was
quickly redispersed in pure saline solution.
The coating process was repeated one additional time. The sediment was
redispersed in 1.5 mL of saline solution and 300 μL of aqueous gold chloride. On
the last coating cycle, the gold chloride remained in solution one minute before
the addition of 400 μL of 0.035 M aqueous 16-mercaptohexadecanoic acid and 50 μL
of 7.5 M aqueous sodium hydroxide. The particles were centrifuged and
redispersed in deionized water containing 50 μL of aqueous sodium hydroxide.
The spheres were centrifuged and redispersed several times in deionized water
to purify the samples with 400 μL of the aqueous 16-mercaptohexadecanoic acid. The
spheres were then centrifuged and redispersed two additional times and were allowed
to sediment for 7 minutes before removing all the supernatant with a pipette,
leaving only a small amount of sediment at the bottom. The purified spheres
were redispersed in water and were allowed to selfassemble by sedimentation.


### 2.4. Sintering of the Metal Layers

The metal-coated discs were thermally
sintered in a vacuum tube furnace as described: the gold-coated cells were
heated at 80°C in air for 4 hours and then placed under vacuum. Once under
vacuum, the temperature was increased by 10°C per hour for 4 hours. Temperature
was held at 120°C for 5 hours and then raised to 500°C, at a rate of 50°C per
hour. The samples were kept at 500°C for 10 hours and then cooled to room
temperature at a rate of 50°C per hour.


## 2.1. Materials

All chemicals were obtained from
Aldrich Chemical Company and were used as received unless otherwise noted. Gold
chloride was prepared as described elsewhere [17]. RBCs were obtained using a
diabetic finger lancet. A boric acid and sodium borate isotonic contact lens
saline solution was used in all experiments. Samples were centrifuged using a
Hamilton Bell Van Guard V6500, which is a fixed speed blood centrifuge. All
solutions were prepared with deionized water with a resistivity below 17 M Ω cm.


## 2.2. Colloidal Discs

Colloidal gold discs were prepared at room temperature as follows. Three
drops of blood were dispersed in 1.5 mL of saline solution in 2 mL centrifuge
tubes. 100μL of 0.06 M aqueous sodium citrate (used as anticoagulant) followed
by 100 μL of 0.006 M aqueous gold chloride were added at once. The mixture was
shaken by hand and centrifuged for 60 seconds. The supernatant was decanted and
the remaining sediment was quickly redispersed in pure saline solution. The
coating process (i.e., addition of aqueous AuCl3) was repeated two
additional times. The gold coated RBCs were centrifuged and redispersed several
times in 400 μL of 0.035 M aqueous 16-mercaptohexadecanoic acid for the purpose
of purification.


## 2.3. Colloidal Gold Spheres

Six drops of blood were dispersed in 1.5 ml of saline solution. 120μL of .06 M aqueous sodium citrate
followed by 400 μL of 0.006 M aqueous gold chloride were added at once. After
addition of gold chloride, the sample was immediately shaken and centrifuged
for 60 seconds. The supernatant was decanted and the remaining sediment was
quickly redispersed in pure saline solution.
The coating process was repeated one additional time. The sediment was
redispersed in 1.5 mL of saline solution and 300 μL of aqueous gold chloride. On
the last coating cycle, the gold chloride remained in solution one minute before
the addition of 400 μL of 0.035 M aqueous 16-mercaptohexadecanoic acid and 50 μL
of 7.5 M aqueous sodium hydroxide. The particles were centrifuged and
redispersed in deionized water containing 50 μL of aqueous sodium hydroxide.
The spheres were centrifuged and redispersed several times in deionized water
to purify the samples with 400 μL of the aqueous 16-mercaptohexadecanoic acid. The
spheres were then centrifuged and redispersed two additional times and were allowed
to sediment for 7 minutes before removing all the supernatant with a pipette,
leaving only a small amount of sediment at the bottom. The purified spheres
were redispersed in water and were allowed to selfassemble by sedimentation.


## 2.4. Sintering of the Metal Layers

The metal-coated discs were thermally
sintered in a vacuum tube furnace as described: the gold-coated cells were
heated at 80°C in air for 4 hours and then placed under vacuum. Once under
vacuum, the temperature was increased by 10°C per hour for 4 hours. Temperature
was held at 120°C for 5 hours and then raised to 500°C, at a rate of 50°C per
hour. The samples were kept at 500°C for 10 hours and then cooled to room
temperature at a rate of 50°C per hour.


## 3. Results and Discussion

### 3.1. Self Assembly and Sintering

Colloidal metal disc-shaped
particles were self-assembled by sedimentation and then slowly dried in air (Figure1(a)). Once the particles were dried, they were slowly sintered as described in
the experimental section (Figure 1(b)).(a) Colloidal gold discs self-assembled on a silicon substrate prior to sintering. (b) A hollow colloidal gold replica of a red blood cell on a silicon substrate after sintering at 500°C.
(a)(b)Gold chloride is
converted to metallic gold and chlorine gas at temperatures above 350°C [17]. The
sintering process should therefore result in the thermal conversion of virtually
all of the remaining gold chloride to metallic gold. This was verified by energy dispersive X-ray (EDX) (Table 1). Accordingly, chlorine was present in a ratio far below its
composition in gold chloride.  This was
evident in both the atomic and weight composition.Table 1
Elemental composition of gold
chloride-coated red blood cells as determined by energy dispersive X-ray spectroscopy (EDX) after sintering.
ElementWeight%Atomic%Au40.078.86Cl3.344.11When particles with thick
metal shells were sintered, the shells fused at their interfaces causing widening
between the necking of the shells (Figure2(a)).  This suggests that there was excess gold
chloride remaining in the metal shells before sintering allowing them to neck
together during the thermal process, which was significantly below 1064°C, the melting
temperature of gold.  Roughening of the
particles’ surface was observed after sintering (Figure 2(a)). Interestingly,
the gold-coated particles that were sintered have dodecahedral-shaped nodules
visible on the surface of the particles, which were not present prior to
sintering (Figure 2(b)). It would be interesting to investigate the formation
and evolution of these dodecahedral aggregates on the particles surface during
the sintering process, while the gold chloride converts to metallic gold.(a) Gold discs with a thick layer of gold prior to sintering at 500°C, (b) a 300–400 nm thick
layer of gold deposited on the blood cells from repeated coatings of gold prior
to sintering. The arrows indicate excess colloidal gold, (c) an SEM image of a
red blood cell that bursts because the gold shell was too thin to contain it.
(a)(b)(c)


### 3.2. Properties of the Gold Shells

The coating process
described in the experimental section outlines the optimum procedure to create the
thinnest shell necessary to stabilize the particles. This allowed us to dry the
samples and obtain SEM images without bursting (Figure2(c)) or collapsing (Figure 3(a)) the shells. The thickness of this gold layer was found to be
approximately 30–120 nm thick.
This was determined by examining the remains of particles in the SEM that were
shattered by thermal shock (Figure 3(a)).(a) Collapsed gold-coated RBC
disc. (b) A thermally shattered hollow gold disc with a 50–120 nm thick layer of gold. (c) A doublet formed from two thinly coated spheres.
(a)(b)(c)Once the cells were encapsulated
with a layer of gold, they could be further coated by placing them in solutions
that had much higher concentrations of gold chloride. The particles could also
be left in these plating solutions for any desired amount of time since they
were completely coated and indifferent to osmotic changes in solution (Figure2(b)).
This allowed the formation of much thicker gold shells and it resulted in reproducible
control of the thickness of the gold shells.


### 3.3. Red Blood Cells

The cell membrane of an
RBC is permeable to water, but only relatively permeable to ions [16]. Thus,
the volume of the individual cells can quickly change due to flux of water
across the membrane arising from a change in the osmotic pressure across the
cells membrane [16, 18]. When the osmotic pressure drives water out of the
interior of the cell, the volume of the cell contracts and the cell is said to
be crenated. When the osmotic pressure drives water into the cell, its volume
expands [19]. This allowed us to modify the shape.The membrane of a red
blood cell is relatively elastic and the volume of the cell can increase from 30%
to 40% before bursting in a process called hemolysis [16, 19, 20]. When hemolysis
occurred in our samples, it was evident because the samples turned blood red.
This red color did not sediment when centrifuged during the coating process and
was a very convenient indicator of how the coating process was proceeding.
After hemolysis, the membrane of a red blood cell will often reclose or remain
stable. These blood cells are called ghosts. Many interesting ghosts were found
in our samples and were prevalent when the layer of gold was very thin or
coated using a process where the gold layer was deposited too slowly.Under normal
physiological conditions the osmotic pressure across an RBC membrane is about 3.4 times stronger than the atmospheric pressure [19]. The osmotic sensitivity of
the cells makes the coating process a challenging time-dependent task that is
both pH and electrolyte sensitive. The coating process appears to be a time-sensitive
one and only attempts using a quick coating process resulted in reproducible
work. A failed coating attempt usually resulted in hemolysis of virtually all
of the cells. The speed of the coating process was controlled by the
concentration of gold chloride, which also affected the osmotic pressure. The
increased osmotic pressure (resulting from excess solution) allowed the
formation of spherical gold-coated RBCs (Figure 3(c)).


### 3.4. Colloidal Stabilization and Mechanical Properties

In order to stabilize the
colloidal particles, their surfaces needed to be electrostatically charged.
This was achieved using the following multistep process. First, the particles were
charged using 16-mercaptohexadecanoic acid (a metal chelating agent) [21]. Addition
of the chelating agent resulted in the removal of small unwanted gold particles
(Figure 2(b)) during the rinsing process described in the experimental section.
Thus, they remained dispersed after centrifuging and were decanted during the purification
procedure. This also greatly reduced the number of doublet spheres found in the
samples (Figure 3(b)). This means that the surfaces of the gold-coated cells must
remain relatively malleable after the coating process. This malleability was observed
in some of the SEM images of the gold-coated cells prior to sintering (Figure 4(a)).
For comparison, an SEM image of dried and uncoated red blood cells is included
as a control (Figure 4(c)).(a) A previous doublet formed
from two gold spheres that had been forced apart. The arrows indicate where the
two spheres were joined. (b) A wide-field SEM image of colloidal disc-shaped particles. (c) An SEM image of dried untreated RBCs on a silicon substrate.
(a)(b)(c)


## 3.1. Self Assembly and Sintering

Colloidal metal disc-shaped
particles were self-assembled by sedimentation and then slowly dried in air (Figure1(a)). Once the particles were dried, they were slowly sintered as described in
the experimental section (Figure 1(b)).(a) Colloidal gold discs self-assembled on a silicon substrate prior to sintering. (b) A hollow colloidal gold replica of a red blood cell on a silicon substrate after sintering at 500°C.
(a)(b)Gold chloride is
converted to metallic gold and chlorine gas at temperatures above 350°C [17]. The
sintering process should therefore result in the thermal conversion of virtually
all of the remaining gold chloride to metallic gold. This was verified by energy dispersive X-ray (EDX) (Table 1). Accordingly, chlorine was present in a ratio far below its
composition in gold chloride.  This was
evident in both the atomic and weight composition.Table 1
Elemental composition of gold
chloride-coated red blood cells as determined by energy dispersive X-ray spectroscopy (EDX) after sintering.
ElementWeight%Atomic%Au40.078.86Cl3.344.11When particles with thick
metal shells were sintered, the shells fused at their interfaces causing widening
between the necking of the shells (Figure2(a)).  This suggests that there was excess gold
chloride remaining in the metal shells before sintering allowing them to neck
together during the thermal process, which was significantly below 1064°C, the melting
temperature of gold.  Roughening of the
particles’ surface was observed after sintering (Figure 2(a)). Interestingly,
the gold-coated particles that were sintered have dodecahedral-shaped nodules
visible on the surface of the particles, which were not present prior to
sintering (Figure 2(b)). It would be interesting to investigate the formation
and evolution of these dodecahedral aggregates on the particles surface during
the sintering process, while the gold chloride converts to metallic gold.(a) Gold discs with a thick layer of gold prior to sintering at 500°C, (b) a 300–400 nm thick
layer of gold deposited on the blood cells from repeated coatings of gold prior
to sintering. The arrows indicate excess colloidal gold, (c) an SEM image of a
red blood cell that bursts because the gold shell was too thin to contain it.
(a)(b)(c)


## 3.2. Properties of the Gold Shells

The coating process
described in the experimental section outlines the optimum procedure to create the
thinnest shell necessary to stabilize the particles. This allowed us to dry the
samples and obtain SEM images without bursting (Figure2(c)) or collapsing (Figure 3(a)) the shells. The thickness of this gold layer was found to be
approximately 30–120 nm thick.
This was determined by examining the remains of particles in the SEM that were
shattered by thermal shock (Figure 3(a)).(a) Collapsed gold-coated RBC
disc. (b) A thermally shattered hollow gold disc with a 50–120 nm thick layer of gold. (c) A doublet formed from two thinly coated spheres.
(a)(b)(c)Once the cells were encapsulated
with a layer of gold, they could be further coated by placing them in solutions
that had much higher concentrations of gold chloride. The particles could also
be left in these plating solutions for any desired amount of time since they
were completely coated and indifferent to osmotic changes in solution (Figure2(b)).
This allowed the formation of much thicker gold shells and it resulted in reproducible
control of the thickness of the gold shells.


## 3.3. Red Blood Cells

The cell membrane of an
RBC is permeable to water, but only relatively permeable to ions [16]. Thus,
the volume of the individual cells can quickly change due to flux of water
across the membrane arising from a change in the osmotic pressure across the
cells membrane [16, 18]. When the osmotic pressure drives water out of the
interior of the cell, the volume of the cell contracts and the cell is said to
be crenated. When the osmotic pressure drives water into the cell, its volume
expands [19]. This allowed us to modify the shape.The membrane of a red
blood cell is relatively elastic and the volume of the cell can increase from 30%
to 40% before bursting in a process called hemolysis [16, 19, 20]. When hemolysis
occurred in our samples, it was evident because the samples turned blood red.
This red color did not sediment when centrifuged during the coating process and
was a very convenient indicator of how the coating process was proceeding.
After hemolysis, the membrane of a red blood cell will often reclose or remain
stable. These blood cells are called ghosts. Many interesting ghosts were found
in our samples and were prevalent when the layer of gold was very thin or
coated using a process where the gold layer was deposited too slowly.Under normal
physiological conditions the osmotic pressure across an RBC membrane is about 3.4 times stronger than the atmospheric pressure [19]. The osmotic sensitivity of
the cells makes the coating process a challenging time-dependent task that is
both pH and electrolyte sensitive. The coating process appears to be a time-sensitive
one and only attempts using a quick coating process resulted in reproducible
work. A failed coating attempt usually resulted in hemolysis of virtually all
of the cells. The speed of the coating process was controlled by the
concentration of gold chloride, which also affected the osmotic pressure. The
increased osmotic pressure (resulting from excess solution) allowed the
formation of spherical gold-coated RBCs (Figure 3(c)).


## 3.4. Colloidal Stabilization and Mechanical Properties

In order to stabilize the
colloidal particles, their surfaces needed to be electrostatically charged.
This was achieved using the following multistep process. First, the particles were
charged using 16-mercaptohexadecanoic acid (a metal chelating agent) [21]. Addition
of the chelating agent resulted in the removal of small unwanted gold particles
(Figure 2(b)) during the rinsing process described in the experimental section.
Thus, they remained dispersed after centrifuging and were decanted during the purification
procedure. This also greatly reduced the number of doublet spheres found in the
samples (Figure 3(b)). This means that the surfaces of the gold-coated cells must
remain relatively malleable after the coating process. This malleability was observed
in some of the SEM images of the gold-coated cells prior to sintering (Figure 4(a)).
For comparison, an SEM image of dried and uncoated red blood cells is included
as a control (Figure 4(c)).(a) A previous doublet formed
from two gold spheres that had been forced apart. The arrows indicate where the
two spheres were joined. (b) A wide-field SEM image of colloidal disc-shaped particles. (c) An SEM image of dried untreated RBCs on a silicon substrate.
(a)(b)(c)


## 4. Conclusion

Hollow gold colloidal disc and sphere-shaped shells were
fabricated by encapsulating human red blood cell templates in gold chloride
shells. The thickness of the gold shell and the shape of the resulting
particles were found to be controllable during the encapsulating process.
Coated red blood cells were electrostatically charged, self-assembled and the
residual gold chloride was converted to metallic gold by sintering under vacuum
at 500°C.



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*Source: 726285-2008-04-29.xml*