Journal of Non-Crystalline Solids 285 (2001) 303±308
www.elsevier.com/locate/jnoncrysol
Aerogels as biosensors: viral particle detection by bacteria
immobilized on large pore aerogel
Mary Power a, Bouvard Hosticka a, Eric Black b, Chuck Daitch b,
Pamela Norris a,*
a
Mechanical and Aerospace Engineering, University of Virginia, 570 Edgemont Road, Charlottesville, VA 22903, USA
b
Veridian-Paci®c Sierra Research, Charlottesville, VA, USA
Abstract
A proof-of-principle study is reported in which bacteria were immobilized within macroporous, supercritically dried
silica sol±gel discs and signal induction was demonstrated by aerosolized virus particles. Escherischia coli (pET-gfp)
bacteria-doped gels were used as an aerosol collector to detect bacteriophage. The bacteriophage (105 and 108 plaque
forming units/ml) (pfu/ml) were aerosolized through the discs for 10 min, at a ¯ow rate of 1.75 l/min and aerosol
humidity of 70%. The discs were then incubated in bacterial growth media for 4 h and green ¯uorescent protein (GFP)
expression monitored. The induction of GFP indicated that both bacteriophage and bacteria survived the stressful
desiccating conditions of the aerosol challenge. Scanning confocal laser microscopic (SCLM) analysis demonstrated
that the bacteriophage contacted viable bacteria and induced expression of the GFP in 35±95% of the bacterial cells.
These ®ndings indicate that virus particles can penetrate the structure of macroporous silica gels and trigger a detectable
response in immobilized bacteria. The goal is to use microorganisms immobilized within these materials to facilitate the
detection of chemicals and organisms within the environment. Ó 2001 Elsevier Science B.V. All rights reserved.
PACS: 82.33.; 87.80
1. Introduction
Sol±gel-derived silica holds promise as a biocompatible sca€old for the immobilization of cells
in a variety of applications. The use of aerogel as a
matrix in the design of biosensors is an interesting
proposition due to several unique characteristics of
aerogels, primarily their highly porous nature,
adjustable pore size and extremely large internal
surface area. The goal is to use microorganisms
*
Corresponding author. Tel.: +1-804 924 6295; fax: +1-804
982 2037.
E-mail address: [email protected] (P. Norris).
immobilized within this material and together,
facilitate the collection of aerosols and detection of
chemicals and/or organisms within the environment.
Silica, regardless of origin, has always been a
useful matrix for the immobilization of biological
materials. Silica is both chemically and mechanically robust and biocompatible. A diculty of
most aerogel production routes for cell immobilization is the resulting small pore size. Uo et al. [1]
used pre-gelled macroporous silica matrices that
contained water-soluble polymers in order to increase the pore diameter. The polymers were removed by rinsing, yielding macroporous silica.
Saccharomyces cereviciae was loaded and upon
0022-3093/01/$ - see front matter Ó 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 0 2 2 - 3 0 9 3 ( 0 1 ) 0 0 4 7 1 - 9
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M. Power et al. / Journal of Non-Crystalline Solids 285 (2001) 303±308
incubation showed germination. Others have
shown that optimal cell growth in ceramics has
been achieved with pore diameters between one
and ®ve times the size of the cells [2,3]. It was
determined that the macroporous production
route described by Kazi et al. [4] resulted in pore
diameters in the desired range and its ability to be
colonized by Escherischia coli cells is described.
Whole-cell living bioreporters are of interest in
biosensor development since they can be used to
detect real time physical e€ects [5]. Light can be
measured non-invasively and sensitively without
disrupting the cells and this therefore enables in
situ and on-line monitoring. Green ¯uorescent
protein (GFP) is a naturally occurring protein
from the jelly®sh Aequorea victoria. This protein
has great potential as a bioreporter since it is easily
detected and requires no additional energy, other
than that required for protein expression, to emit
¯uorescence. The gene for this protein was cloned
behind a promoter that required a polymerase
enzyme produced solely by bacteria infected with
T7 bacteriophage viruses, in order to express the
protein [6]. The bacteriophage, in an aerosol
stream, were then monitored. This paper describes
the integration of macroporous supercritically
dried and re-wetted silica gels as a colonization
sca€old for bacteria, an aerosol collection device
and a biosensor system.
mother liquor. Samples were supercritically dried
using carbon dioxide to remove the ethanol and to
retain monolith structure.
The resulting macroporous gels (q ˆ
400 kg=m2 ) were machined into discs of 47 mm
diameter and 1 mm thickness. The dry discs were
soaked for 24 h in phosphate bu€ered saline (PBS;
pH 7.2) and then subjected to 20 min mild sonication in a Bransonic 2000 sonic bath in order to
replace the air in the pores with liquid. The wetted
discs were then placed into ®lter holders. Bacteria
(1 lm length) in liquid media were ¯owed
through the disc at a ¯ow rate of 0.8 ml/min for 2 h.
The discs were then stored in fresh NZY bacterial
growth media (Fisher Scienti®c) and 10 mM
MgSO4 at 30°C for 4 h, and then at 4°C until use
(for up to 4 days). Samples of these discs were also
processed for SEM.
The bacteria used in this study were E. coli
carrying a plasmid (pET-gfp)with the gene for
GFP under the control of the bacteriophage T7
polymerase promoter [6]. The presence of bacteriophage T7 polymerase is required for the expression of GFP in this system, creating a tightly
controlled system whereby green ¯uorescence is
only observed if the virus particles have come into
contact with the bacteria.
2. Experimental
The aerosol generation system consists of a
small air compressor with a HEPA ®lter and
dessicator supplying regulated air at 125 kPa to a
TSI model 3075 constant output atomizer run in
the recalculation mode. Bacteriophage CE6 virus
particles (70 nm diameter) were suspended in
PBS pH 7.0, 10 mM MgSO4 , and 2% trehalose.
The phage concentration in the liquid was 105 or
108 plaque forming units/ml (pfu/ml). The
aerosolizer was run at a ¯ow of 1.75 l/min. The
E. coli-doped aerogel discs were placed in the test
chamber of the aerosol generation system and the
¯ow was directed through 7 mm diameter apertures in fenestrated partitions housing the large
pore aerogel±aerosol collector. The aerosol humidity was run at 70%. The di€erential pressure
across the 1 mm thick discs was of the order of 15
2.1. Production of macroporous silica aerogel aerosol collectors
Macroporous silica sol±gels were fabricated by
the method of Kazi et al. [4]. Brie¯y, formamide
(45.72 g) was combined with 1.4 M nitric acid (12
g) in a 125 ml polypropylene mold. The mixture
was stirred in an ice bath until it had reached a
temperature less than 5°C. Tetramethyl orthosilicate (61.9 g) was added rapidly, and the mold was
sealed and agitated until the solution was homogeneous. The sample was then placed in a 50°C
oven for 12 h. Following gelation, the material was
removed from the mold and subsequent 1 M nitric
acid and ethanol washes were used to remove the
2.2. Aerosol generation, collection and detection of
bacteriophage virus particles
M. Power et al. / Journal of Non-Crystalline Solids 285 (2001) 303±308
kPa, dropping in 10 min runs to approximately 5
kPa as the gel dried out.
The discs were recovered from the aerosol collection device and wetted with 500 ll NZY media.
The discs were incubated for 4±8 h at 30°C. The
expression of GFP was monitored by ¯uorescence
microscopy and by scanning confocal laser microscopy (SCLM). A universal DNA stain, Syto17
(Molecular Probes, Eugene, Oregon), was used to
detect the total number of E. coli cells within the
material. The results were expressed as the ratio of
GFP expressing cells to the total cell population in
each microscopic ®eld of view. Three results were
averaged for each data point and the S.D. for all
the measurements was used for the error bars
shown. The error bars represent a measure of the
repeatability.
3. Results
The wetted macroporous silica sol±gel provided
a suitable matrix for microbial colonization. SEM
micrographs of the uncolonized gel show macropores ranging in size from approximately 10 to
100 lm (Fig. 1). The majority of the gels survived
the wetting and ¯ow procedure although cracking
did occur at the edges of the discs. Bacterial pen-
305
Fig. 1. A SEM micrograph of a macroporous silica gel disc
produced by supercritical drying examined prior to colonization
by bacteria. Macropores of the order of 10±100 lm are apparent.
etration of the wetted aerogel discs was observed
using SEM analysis. Bacteria were observed over
the entire 1 mm depth. Large numbers of cells,
often observed in clusters, accumulated on or near
the disc surface (Fig. 2(a)). However, smaller
clusters and individual cells were also observed
deep within the porous network (Fig. 2(b)).
Using SCLM the extent of colonization at different depths could be approximated. Fig. 3 is a
Fig. 2. SEM micrographs of a cross-section of a macroporous silica gel disc that had been colonized with E. coli (pET-gfp). Panel A is
the surface closest to the ¯ow and Panel B is an area near the middle of the disc. Large numbers of bacteria are evident. Arrows indicate
clusters of bacterial cells.
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M. Power et al. / Journal of Non-Crystalline Solids 285 (2001) 303±308
representative scan depicting the number of bacterial cells per ®eld of view at a single x y location
at various depths (z-axis). Depth 0 mm is the
surface in the direction of bacterial ¯ow when
colonizing and as would be expected the largest
number of bacteria were observed at this surface.
The limit of the SCLM under these conditions was
0.3 mm on either side of the disc. The disc was
turned over and examined from the reverse side at
the same x y location. Bacteria were observed at all
depths with a consistent, slight increase in numbers
at the bottom surface. Although the counts at the
di€erent x y locations on individual discs varied a
great deal, the general trend illustrated in Fig. 3
was conserved.
The collector integrity was maintained in most
of the viral aerosol challenges although occasionally the disc cracked. The cracked discs were discarded. The intact collectors were examined by
SEM following the virus aerosol challenge and a
further 4 h incubation in liquid media, which was
required for optimum GFP production. The
presence of elongated cells and dividing cells gave
a strong indication that the bacteria were still viable and growing (Fig. 4). This suggested that
mass transport of nutrients provided by the bacterial growth media and waste materials is occurring within the gel matrix. The elongated cells may
Fig. 3. A representative depth pro®le of bacterial colonization
through the thickness of a disc. The bacterial numbers are the
total number of Syto 17 stained E. coli at a single x y location
(®eld of view) as determined by SCLM. The limit of this microscope and the macroporous gel discs was 0.3 mm into the
sample. The sample was turned over and scanned at the same
x y coordinates.
be bacteria exhibiting a form of stress response,
possibly due to the overproduction of GFP.
The immobilized E. coli cells were receptive to
infection by the aerosolized virus particles. This
indicated that both the bacteria and the viruses
survived the stressful conditions they were subjected to in the aerosol. The protocol that resulted
in optimum expression of GFP by the entrapped
bacteria was a 10 min aerosol exposure to 108 pfu/
ml phage, in the aerosolized liquid, at a humidity
of 70%. This optimum exposure resulted in a large
percentage of the bacteria expressing GFP as determined by ¯uorescent microscopy. Using 97 nm
¯uorescent beads to simulate the phage it was
calculated that this would result in the deposition
of 8 104 pfu/mm2 of macroporous gel surface for
a total of 2 105 pfu (data not shown).
The virus concentration in the aerosol was
varied in an attempt to determine the microscopic
detection limits. Serial 10-fold dilutions of the
phage culture were made in PBS containing 2%
trehalose, and 10 mM MgSO4 . The maximum dilution of the phage in the ¯uid to be aerosolized
resulting in detectable expression across the entire
disc, as determined by microscopic visualization,
was 105 pfu/ml, in the liquid (Fig. 5). All further
Fig. 4. SEM micrograph of E. coli (pET-gfp) in the collector
disc after having been subjected to the 10 min aerosol stream of
viral particles. The black arrow depicts healthy looking dividing
cells and the white arrow depicts elongated cells, growing but
probably stressed.
M. Power et al. / Journal of Non-Crystalline Solids 285 (2001) 303±308
307
It was determined a 10 min aerosol collection of
this dilution would result in 2 103 pfu ¯owing
through the aerogel. At this phage concentration
(105 pfu/ml), GFP expression appeared to be induced over the entire 1 mm depth of the collector.
The ratio of GFP producing cells to the total
number of bacteria present (Syto17 stained cells)
was determined at a number of sites for three
aerosol collector discs. The SCLM data are presented in Fig. 6. At the surface of the gels, a mean
of 95% of the detectable bacteria expressed GFP
indicating that they had been infected with the
bacteriophage virus. The percentage of cells infected dropped across the 1 mm thickness with
35% of the cells expressing GFP at the surface
furthest from the aerosol.
Fig. 5. A SCLM micrograph of GFP producing E. coli (pETgfp) taken 100 lm from the surface of the disc exposed to the
aerosol. This bacteria colonized disc had been exposed to CE6
lambda bacteriophage virus particle (105 pfu/ml) for 10 min.
This dilution of phage was found to be the limit of reasonable
detection by SCLM.
studies were carried out at this maximum dilution.
The ¯uorescent bead data described above were
used to calculate the expected number of virus
particles contacting the macroporous gel surface.
Fig. 6. Ratio of the number of GFP expressing E. coli compared to the total number stained with Syto17. The values are
mean and S.D. of counts of scans from three macroporous discs
colonized with E. coli and subjected to viral aerosolization. The
limit of this microscope and the discs was 0.3 mm into the
sample. The sample was turned over and scanned at the same
x y coordinates.
4. Discussion
Cloning of the GFP protein behind the T7
polymerase promoter resulted in a system by
which GFP expression could be easily controlled.
Maintaining the T7 polymerase in the CE6 phage
gave an elegant model whereby the viral particles
were required to infect a bacterial cell in order for
GFP to be produced. This system was developed
in order to test the feasibility of using wetted
macroporous silica sol±gel-derived materials as
support matrices for living organisms to be used as
biosensors. The use of chemiluminescent and bioluminescent reporter systems to detect organic
environmental pollutants is an area that is of
growing interest [5]. There is also an interest in
developing these systems for bio-warfare agent
detection.
The macroporous gel used had pores of the
order of 10±100 lm, which is in the range that is
determined optimal for cell colonization and
growth [7]. The presence of clusters of cells and
obviously dividing and growing cells (Fig. 4) suggest that indeed transport of nutrient and waste
products was occurring into the wetted gel. Nutrient transport, including oxygen, is also required
for GFP expression con®rming that cells even deep
within the aerogel were viable. Although the pore
network within aerogels is tortuous bacteria were
observed throughout the 1 mm thickness of the
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M. Power et al. / Journal of Non-Crystalline Solids 285 (2001) 303±308
discs, indicating that there were interconnected
routes of at least 1 lm in diameter.
The viral particles in the aerosols were also able
to penetrate the entire 1mm disc thickness and
contact viable E. coli cells as evidenced by green
¯uorescence observed by SCLM (Figs. 5 and 6).
As with the bacterial colonization, more viruses
were collected near the surface of the discs. The
percentage of bacterial cells infected with phage
decreased rapidly across the aerogel ®lter with
95% infected at the surface and only 35% infected
at the opposite side (Fig. 6). This is presumably
partially because the concentration of viruses
decreased in the aerosol as they were ®ltered out
across the disc. Also, there were fewer bacteria
present further into the disc, decreasing the likelihood of contact between the two being made.
Variable distribution of bacterial cells within the
aerogel matrix and such things as clumping of
viral particles in the aerosol would contribute to
the observed variability. The increase in surface
area a€orded by these macroporous silica gels
would certainly increase the probability of
detecting smaller numbers of particles, especially if
the sensitivity of the system were increased. The
combination of using sol±gel-derived materials as
a collector and as a sensor exhibits potential as a
fast and ecient mechanism to detect chemicals or
organisms in the environment.
5. Conclusions
Aerogels are being explored as matrices for
biosensors due to a large degree of porosity and
extremely large surface area. It has been shown
that wetted macroporous silica gel can be integrated as a sca€old for bacterial growth and a
collector for viral particles. Additionally, immobilized bacterial cells can elicit a measurable response as a result of an environmental trigger.
These studies have provided the proof-of-principle
that organisms can survive within a wetted mac-
roporous silica gel subjected to aerosol exposure
and elicit a detectable physiological response to a
biological agent within the aerosol.
Although, this particular system is only valuable for testing within the laboratory, since the
detection of bacteriophage is of no economic value, this model should be transferable to other
bioreporter systems. Bacteria with speci®c biological functions could be doped into the gel. For
example, the gene for the GFP protein could be
cloned behind the promoter for the genes encoding
speci®c enzymes for biodegradation of organic
pollutants. These organisms could then be immobilized and used to detect these speci®c chemicals
in the environment as aerosols, in ground water or
in soil.
Acknowledgements
We would like to acknowledge DARPA and
Veridian±PSR for ®nancial support.
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