Report
Nanomaterials in
anomaterials constitute an
As particlesize
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emerging subdiscipline in the
I chemical and materials sciences
approaches molecular (1,2). Nanomaterials have numerous com-
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mercial and technological applications, in-
dimensions, all cluding analytical chemistry (3-6); drug
delivery; bioencapsulation; and electronic,
propertiesofa optical, and mechanical devices. In addi-
tion thisfieldposes an important funda-
I
material change, mak- mental question how do the electronic,
optical, and magnetic properties of a nano-
ing nanomaterials scopic particle differ from those of a bulk
sample of the material? This issue is
useful for particular of particular importance because all proper-
ties of a material change as the particle size
applications. approaches molecular dimensions and be-
cause it is often the unique properties of
the nanomaterial that make it useful for a
mrtirnlar annlimtinn
This Report deals with exploiting the
unique properties of nanomaterials for bio-
analytical chemistry, bioseparations, and
bioimaging. Recent advances in analytical
applications of colloidal particles will also
be discussed. Finally, the template ap-
proach for preparing nanomaterials is de-
scribed, and possible applications are re-
viewed. The objective of this Report is to
introduce analytical chemists to the numer-
ous applications of nanomaterials in chemi-
cal analysis and to encourage them to con-
sider nanomaterials in their own research
and development projects
Bioanalysis, bioseparations,
and MRI
There is no universally agreed upon defini-
tion of when a small particle qualifies as a
nanoparticle. In our research group, we con-
sider particles with at least one dimension
C h a r l e s R. M a r t i n (d) < 110 0m as s nanoparticle and particles
David T . M i t c h e l l with dimensions in the range of 100 nm
Colorado State University < d < —11 um as microparticlese
322 A Analytical Chemistry News & Features, May 1, 1998
Immunoagglutination. The use of
synthetic microparticles in bioanalysis orig-
inated in the mid-1950s, with the invention
of latex agglutination tests by Singer and
Plotz (7). These simple, ingenious tests
use suspended latex microparticles (diame-
ter ~1 um) that are chemically derivatized
with a desired antibody. The analyte is an
antigen for this antibody, and this analyte-
antigen binds to more than one antibody
molecule. In early versions of this test, a
drop of homogeneous milky-white antibody-
labeled suspension was applied to a glass
slide and mixed with a drop of the analyte
solution. The analyte chemically links
adjacent latex particles by binding to anti-
body sites on these particles resulting in
agglutination (or clumping together) of
the particles into what looks like curdled
milk
Latex agglutination tests have been de-
veloped for more than 100 analytes, includ-
ing infectious disease agents and drugs of
abuse (6). These tests are portable, simple
to use, highly selective, and fast; they can
also be quantitative. For example, ,here are
agglutination assays that relate the intensity
of light scattered by the agglutinated parti-
cles to the concentration of the analyte (6))
Particles with truly nanoscopic dimen-
sions have also been used in tests of this
type, such as the Carter-Wallace home preg-
nancy test "First Response", which uses con-
ventional micrometer-sized latex particles in
conjunction with gold nanoparticles (less
than 50-nm diameter) (6). Gold nanoparti-
cles show a characteristic visible absorption
band called the plasmon resonance absorp-
tion (8), which makes them pink. The micro-
and nanoparticles are derivatized with anti-
bodies to human chorionic gonadotrophin, a
hormone released by pregnant women.
When mixed with a urine sample containing
Analytical Chemistry
this hormone, the micro- and nanoparticles
are coagglutinated and the resulting clumps
are colored pink.
This example uses the gold nanoparti-
cle as the indicator in a conventional micro-
particle-based agglutination test. In addi-
tion, nanoparticles have been used as re-
placements for micrometer-sized particles
(4,6,9). For example, Medcalf et al. have
developed an immunoturbidimetric assay
for urine albumin, an indicator of kidney
problems. Poly(vinylnaphthalene) particles
(40-nm diameter) were coated with an
outer layer of a chloromethylstyrene poly-
mer which was used to immobilize an anti-
body raised against human albumin. Agglu-
tination in the presence of urine albumin
was detected by measuring the change in
absorbance caused by light scattering at
340 nm (9))
Using nanoparticles in such assays
offers several potential advantages. For
example, suspensions of nanoparticles do
not appreciably scatter visible light (6),
which means that the background signal
in a turbidimetric assay is lower than in
tests that use milky-white suspensions of
the microparticles (4). This results in
lower detection limits (9). In addition,
nanoparticles form more stable suspen-
sions and are therefore less susceptible to
self-agglomeration (4 9).
Analytical Chemistry News & Features, May 1, 1998 323 A
Colorimetric DNA detection. The
plasmon resonance absorption of colloidal
gold particles has recently been exploited in
a proposed DNA-detection method (10).
Mirkin and co-workers attached 13-nm-
diameter gold nanoparticles to single-stranded
oligonucleotides. The plasmon resonance
absorbance for these particles has a maxi-
mum at 520 nm, and the particles appear
red. When a linking ollgonucleotide was
added, the gold nanoparticles agglutinated,
and the color changed from red to purple.
To attach the single-stranded oligo-
nucleotides to the gold nanoparticles, a
gold sol was stirred with a solution of ter-
minally thiolated, 28 base-pair DNA. The
first 13 nucleotides served as a spacer from
the nanoparticle surface; the last 15 were
the recognition element for the target. Two
different recognition-element sequences
were used. It is important to note that
many oligomers were attached to each
gold particle (10).
The target DNA was a 30 base-pair
strand, the first 15 bases of which are com-
plementary to thefirstrecognition element,
and the remaining 15 complementary to
the second recognition element. When the
target DNA was added to the modified col-
loidal suspension, the target linked the in-
dividual colloid particles into a polymeric
network.
Because the nanoparticles were now
much closer together, the plasmon reso-
nance band shifted, and the color changed.
Although this detection method had not
yet been optimized, the detection limit was
10 fmol for the target oligonucleotide. An
additional benefit of this particular method
is that it allows visual detection, especially
if the sample is developed on a solid sup-
port such as a reversed-phase silica TLC
plate (10).
 Report
Superparamagnetic nanoparti
cles The basic concept in magnetic bio-
separations is to selectively bind the bioma-
terial of interest (e.g., a specific cell, pro-
tein, or DNA sequence) to a magnetic
particle and then separate it from its sur-
rounding matrix using a magnetic field.
Nanoparticles of Fe304 with diameters
in the 5-100 nm range are typically used
for such separations. These particles are
"superparamagnetic", meaning that they
are attracted to a magnetic field but retain
no residual magnetism after the field is
removed (11). Therefore, suspended su-
perparamagnetic particles tagged to the
biomaterial of interest can be removed
from a matrix using a magnetic field, but
they do not agglomerate (i.e., they stay
suspended) after removal of the field.
A common use of superparamagnetic
nanoparticles is for immunospecific cell
separations (11-13). Typically, the nano-
particles are dispersed within the pores of
Figure 1 . Schematic representation of cell separation using antibody-bound
superparamagnetic nanoparticles.
(a) Attachment of nanoparticles to desired cell via binding to the cell-surface antigen, (b) Separation of
nanoparticle-bound cells from unwanted cells using a column containing steel wool placed in a
magnetic field. (Adapted with permission from Ref. 12.)
324 A Analytical Chemistry News & Features, May 1, 1998
larger microparticles. In the simplest (di-
rect) method, the microparticles are coated
with a monoclonal antibody for a cell-
surface antigen. The antibody-tagged, su-
perparamagnetic microparticles are then
incubated with a solution containing the
cells of interest. The microparticles bind to
die surfaces of die desired cells, and these
cells can then be collected in a magnetic
field (13). Methods of this type have been
used to isolate or remove numerous cell
types, including lymphocytes (cells that
control immune response) and tumor cells.
In addition to loading microparticles with
superparamagnetic nanoparticles other
examples of tagging the desired biomate-
rial with individuai nanoDarticles have been
reported (12) (Figure 1)
This approach has numerous advan-
tages. Whereas bound microparticles can
affect the viability of the selected cells, nano- tendon which have a low proton density
particles supposedly do not affect cell via-
bility (12). In addition, as previously indi-
cated, nanoparticles do not affect the extent
of light scattering by the cell solution. The
disadvantage of this approach is small mag-
netic moments of individual nanoparticles;
however, this problem can be overcome by
using a high-gradient magnetic field (12).
MRI contrast agents. Superpara-
magnetic Fe304 nanoparticles are also use-
ful as magnetic resonance imaging (MRI)
contrast agents (14). MRI is essentially
proton NMR done on tissue. Protons are
excited with short pulses of radio fre-
quency radiation; the free induction decay
as they relax is measured and deconvo-
luted by means of a Fourier transform,
which provides an image of the tissue that
corresponds to proton density. Areas of
high proton density, usually in the form of
water or lipid molecules, have a strong sig-
113.l 3X1 d 3-DDetir bright Areas of bone
fit*
because of the lack of water and lipids
have a weak signal and aDDear dark
Traditionally, a major limitation of MRI
has been its inability to distinguish differ-
ences in soft tissue types (e.g., healthy
parts of the liver from diseased lesions), as
the relative proton densities can be very
similar. Other regions, such as the bowel,
are hard to image because air pockets and
fecal matter make the proton density incon-
sistent (15). Various contrast agents have
been developed to circumvent these imag-
ing problems.
Contrast agents work by changing the
strength of the MRI signal at a desired lo-
cation. For example, superparamagnetic
contrast agents change the rate at which
protons decay from their excited state to
the ground state, allowing more effective
decay through energy transfer to a neigh-
boring nucleus. As a result, regions con-
taining the superparamagnetic contrast
agent appear darker in an MRI than re-
gions without the agent. For instance,
when superparamagnetic nanoparticles are
delivered to the liver, healthy liver cells can
uptake the particles; diseased cells cannot.
Consequently the healthy regions are
darkened although the diseased regions
remain bright (16)
Superparamagnetic particles have many
advantages over other contrast agents. Un-
like agents such as perfluorochemicals,
oils, and fats, superparamagnetic particles

Figure 2. Temperature tuning of Bragg
diffraction from a 125-μm-thick
polymerized CCA film of 99-nm
polystyrene spheres embedded in a
poly(M-isopropylacrylamide) gel.
The shift of the diffraction wavelength results
from the temperature-induced volume change of
the gel, which alters the lattice spacing. Spectra
were recorded in a UV-vis/near-IR spectropho­
tometer with the sample placed normal to the
incident light beam. The inset shows the temper­
ature dependence of the diffracted wavelength
for the polymerized CCA film when the incident
light is normal to the (110) plane of the lattice.
(Adapted with permission from Ref. 22.)
are miscible with aqueous systems, which
means they can mix with material in the
bowel and be used in small volumes. Im­
miscible agents must be used in sufficient
quantity to displace intestinal matter (15).
This miscibility also allows them to be used
intravenously. Compared with other mag­
netic contrast agents (e.g., gadolinium che­
lates), they are much more potent [as
much as 50 times more effective per mole
(16)]. Another advantage is that the parti­
cles do not pass the blood-brain barrier;
thus, they are well suited for tracking
blood flow in the brain (17). A novel use of
these nanoparticles is tracking cells in vivo.
Yeh and colleagues labeled ratT-cells with
superparamagnetic Fe 3 0 4 nanoparticles
and inflamed the rat's testicles which at­
tracted the particle-attached T-cells and
caused a decrease in the MRI signal from
the testicles (18)
Other applications
Nanogold particles have been used exten­
sively as specific staining agents in biological
electron microscopy (19). The small sizes of
these particles (diameters as small as 1.4 nm)
allow them to be physically close to the
structures they stain; thus, such particles
provide high resolution. Because the nano­
particles are gold, which has a high back-
scatter coefficient, they appear bright in a
scanning electron microscope image. In con­
trast, the high density of gold makes them
appear dark in a transmission electron mi­
croscope image. Site-specific staining is ob­
tained by labeling the nanogold particles
with antibodies directed against a protein in
the region of interest. Antibody-labeled gold
nanoparticles are commercially available,
and unlabeled gold nanoparticles can also be
purchased and labeled with a specific anti­
body by the investigator.
Direct adsorption of proteins, such as
enzymes, onto bulk metal surfaces fre­
quently results in denaturation of the pro­
tein and loss of bioactivity. In contrast,
when such proteins are adsorbed to metal
nanoparticles, bioactivity is often retained
(20), such as when antibody-labeled nano­
particles are used as electron microscopy
stains, as discussed earlier. In another ex­
ample, Crumbliss and co-workers found
that they could adsorb redox enzymes to
colloidal gold with no loss of enzymatic
activity The enzyme-covered nanoparticles
were then electrodeposited onto platinum
gauze or glassv carbon to make enzyme
electrodes (20)
In addition, Natan and co-workers found
that cytochrome c retained reversible cy­
clic voltammetry when deposited onto
12-nm-diameter gold particles attached to a
conductive substrate. In contrast, if the
cytochrome c was deposited on larger sur­
face features (aggregates of the gold nano­
particles), the cyclic voltammetry became
quasireversible or irreversible, indicating
denaturation of the protein (21). These
results demonstrate another unique feature
of metal nanoparticles biocompatibility.
The repulsive electrostatic forces that
keep colloidal particles from aggregating
also cause them to form crystalline colloi­
dal arrays (CCAs) when concentrated in a
very low ionic strength liquid (22). The
periodicity of such CCAs is in the 100-
1000 nm range, so that the arrays refract
light in the visible region and thus appear
colored. Asher and co-workers found that
the periodicity of polystyrene CCAs can be
varied after incorporating them into a
swellable polymer gel. As the gel swells, ,he
Analytical Chemistry News & Features, May 1, 1998 325 A
intercolloid distance increases, causing a red
shift in the refraction maxima or color of the
CCA For example, a temperature-induced
color change could be obtained by using a
temperature-sensitive hydrogel as the CCA
matrix (22) (Figure 2))
More recently Asher and Holtz have
applied the same swellable CCA design to
chemical sensors. One such sensor used
crown ethers in the hydrogel, which bound
lead ions. The localized charge created by
the bound lead ions increased osmotic
pressure and caused the gel to swell and
the CCA to change color. A glucose-
responsive gel was also prepared. In this
case, glucose oxidase was attached to the
gel and the anionic reduced flavin that re­
sulted from oxidation of glucose caused the
swelling and color change. This
could detect concentrations of glucose as
Iowasl0 M (23)
Surfaced-enhanced Raman spectros­
copy (SERS) uses roughened metal sur­
faces to enhance the Raman scattering of
surface-adsorbed molecules, which can be
as much as 106 over the molecule's native
Raman scattering in solution. An impedi­
ment to using SERS as an analytical tech­
nique is the difficulty in reproducibly and
uniformly controlling surface roughness.
Several research groups have used self-
assembled monolayers of metal nanoparti­
cles as substrates for SERS. For example
Natan's group has studied SERS surfaces
prepared by self-assembling gold and silver
nanoparticles on glass and other sub­
strates The degree of roughness of these
surfaces is tunable bv varying the diameter
of the nanoparticles Such surfaces have
shown a high HPPTPP of reproducibilitv
tinth for
different single sur­
face and for different but identically pre
, sl]rfaces rod)
Figure 3. Schematic of a NEE.
 Report
Template-synthesized
nanomaterials
Our group and others have been exploring
a method we call "template synthesis" for
preparing nanomaterials (2,25). This
method entails synthesizing the desired
material within the pores of a membrane or
other solid. The membranes used have
cylindrical pores of uniform diameter. In
essence, we view each of these pores as a
beaker in which a particle of the desired
material is synthesized. Because of the cy-
lindrical shape of these pores a nanocylin-
der of the desired material is obtained
within each pore
Depending on the material and the chem-
istry of the pore wall, this nanocylinder may
be solid (a fibril or nanowire) or hollow (a
tubule). This is an extremely general
method for preparing nanomaterials. Nanofi-
bers and nanotubules composed of metals,
polymers, semiconductors, carbons, and
Ii+-intercalation materials have been pre-
pared (2). Nearly any chemical synthetic
method used to prepare bulk material can be
adapted so that the synthesis occurs within
the pores of such membranes.
One application entails preparing nano-
scopic electrodes. We have shown that an
electroless plating method can be used to
prepare ensembles of gold nanodisk elec-
trodes with a disk diameter as small as
10 nm. Such nanoelectrode ensembles
(NEEs) have potential applications in elec-
troanalytical chemistry because the signal-
to-background ratio (S/B) observed at the
NEE can be orders of magnitude higher
Figure 4 . Schematic of switchable
ion-selective gold nanotubule
membrane.
A negative applied potential allows passage of
cations but blocks anion transport. A positive
applied potential would allow passage of anions
but block cation transport.
326 A Analytical Chemistry News & Features, May 1, 1998
than at a conventional gold macroscopic
electrode disk, resulting in a detection limit
that can be orders of magnitude lower (3).
To understand this dramatic improve-
ment in detection limits, it is important to
consider the nature of the background
and analytical signals at the NEE. The
predominant background signal in an
electroanalytical experiment is the dou-
ble-layer charging current. Double-layer
charging occurs only at the gold surfaces.
Because only a small fraction (e.g., 0.1%)
of the NEE surface is gold, the double-
layer charging currents can be orders of
magnitude lower than at a conventional
gold macrodisk electrode of the same
geometric area (Figure 3)
The analytical signal is the faradaic cur-
rent associated with electrolysis of the ana-
lyte molecule at the surface of the elec-
trode. For our NEEs, this signal can be
identical to the current obtained at a mac-
rodisk electrode of the same geometric
area, where the entire surface is gold (3).
Hence, the faradaic current (analytical sig-
nal) at the NEE can be the same as at the
conventional gold macrodisk electrode, but
the background current is up to 3 orders of
magnitude lower. The S/B is dramatically
higher thus providing lower detection lim-
its at the NEE
Numerous other groups are also study-
ing the fundamentals of electrochemistry
and electron-transfer processes at nano-
scopic electrodes and nanometal particles.
For example, Fan and Bard have recently
shown coulombic staircase response using
electrodes of nanometer dimensions (26).
Murray et al. have also demonstrated cou-
lombic staircase response by probing a
single gold nanoparticle with a scanning
tunneling microscope tip or studying a
highly monodisperse collection of such
particles with a microelectrode (27) Other
analytical aDDlications of nanometer-sized
electrodes include Men-resolution electro-
chemical imaging and sinffle-molecule
detection (28)
The NEEs discussed above were ob-
tained by electroless plating of gold within
the pores of the template membrane for
sufficiently long times so that solid nano-
wires were produced. If plating is done for
shorter times, ensembles of nanotubules
that span the complete thickness of the
template membrane can be obtained. Gas
flux data suggest that these tubules can
have inside diameters of molecular dimen-
sions < 1 nm) (29). We have recently
shown that these gold nanotubule mem-
branes can be cation permselective, anion
permselective, or nonpermselective, de-
pending on the potential applied to them
(30) (Figure 4). This is unique to these
membranes because the nanotubules are
metal. Because of this switchability, these
membranes can be viewed as universal ion
exchangers.
Nanotubule membranes also have appli-
cations in chemical and bioseparations. For
example, we have shown that the inside
tubule diameter can be decreased until the
tubule is just large enough to accommo-
date a small molecule but too small to ac-
commodate a big molecule. Hence, these
nanotubule membranes allow for "molecu-
lar filtration", where molecules are sepa-
rated in a simple filtration-type experiment
on the basis of molecular size (29).
Template-synthesized nanotubules can
be prepared as high-density ensembles, in
which the tubes protrude from a substrate
surface like the bristles of a brush. Such
brushlike ensembles can have high surface
area, which could be useful in enzyme im-
mobilization (31). To explore this point, we
prepared brushlike ensembles of enzyme-
loaded polypyrrole tubules. Using glucose
oxidase as the enzyme we found the glu-
cose-oxidation rate to be seven times faster
for the template-synthesized bioreactor
than for a more conventional thin-film de-
sign using the components (37*) We
are currently attempting to develop biosen-
sors based on this concept
Nanomaterials have unique chemical
and physical properties that offer important
possibilities for analytical chemistry. This
field is in its infancy, and many new oppor-
tunities for nanomaterials will arise in the
coming decades.
Aspects of this work have been supported by the
Office of Naval Research, the Department of En-
ergy, and the National Science Foundation.
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389,829-32. and correspondence. SUBSCRIBE TODAY!
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1629-32. NEW! Industrial & Engineering Chemistry Research Web Ediiton. Search current
(25) Preston, C. K.; Moskovits, M.J. Phys. and back issues (through 1996) online in seconds, browse through tables of
Chem. .993,97,8495-503. contents for relevant articles, and keep important information at your fingertips
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1791-93. without sacrificing critical shelf space. View a sample issue, Frequently Asked
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Science 1997,278,655-58. ACS Member $ 87 $ 111
$76
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Science 1995,268,700-02. Nonmember $212 $244 -P 310
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1994,369, 298-301.
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Analytical Chemistry News & Features, May 1, 1998 327 A