Report
Nanomaterials in As particlesize approaches molecular dimensions, all propertiesofa material change, making nanomaterials useful for particular applications.
anomaterials constitute an emerging subdiscipline in the I chemical and materials sciences (1,2). Nanomaterials have numerous commercial and technological applications, including analytical chemistry (3-6); drug delivery; bioencapsulation; and electronic, optical, and mechanical devices. In addition thisfieldposes an important fundamental question how do the electronic, optical, and magnetic properties of a nanoscopic particle differ from those of a bulk sample of the material? This issue is of particular importance because all properties of a material change as the particle size approaches molecular dimensions and because it is often the unique properties of I
the nanomaterial that make it useful for a mrtirnlar annlimtinn
This Report deals with exploiting the unique properties of nanomaterials for bioanalytical chemistry, bioseparations, and bioimaging. Recent advances in analytical applications of colloidal particles will also be discussed. Finally, the template approach for preparing nanomaterials is described, and possible applications are reviewed. The objective of this Report is to introduce analytical chemists to the numerous applications of nanomaterials in chemical analysis and to encourage them to consider nanomaterials in their own research and development projects Bioanalysis, bioseparations, and MRI
C h a r l e s R. M a r t i n David T . M i t c h e l l Colorado State University 322 A
There is no universally agreed upon definition of when a small particle qualifies as a nanoparticle. In our research group, we consider particles with at least one dimension (d) < 110 0m as s nanoparticle and particles with dimensions in the range of 100 nm < d < —11 um as microparticlese
Analytical Chemistry News & Features, May 1, 1998
Immunoagglutination. The use of synthetic microparticles in bioanalysis originated in the mid-1950s, with the invention of latex agglutination tests by Singer and Plotz (7). These simple, ingenious tests use suspended latex microparticles (diameter ~1 um) that are chemically derivatized with a desired antibody. The analyte is an antigen for this antibody, and this analyteantigen binds to more than one antibody molecule. In early versions of this test, a drop of homogeneous milky-white antibodylabeled 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 antibody sites on these particles resulting in agglutination (or clumping together) of the particles into what looks like curdled milk Latex agglutination tests have been developed for more than 100 analytes, including 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 particles to the concentration of the analyte (6)) Particles with truly nanoscopic dimensions have also been used in tests of this type, such as the Carter-Wallace home pregnancy test "First Response", which uses conventional micrometer-sized latex particles in conjunction with gold nanoparticles (less than 50-nm diameter) (6). Gold nanoparticles show a characteristic visible absorption band called the plasmon resonance absorption (8), which makes them pink. The microand nanoparticles are derivatized with antibodies 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 nanoparticle as the indicator in a conventional microparticle-based agglutination test. In addition, nanoparticles have been used as replacements 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 polymer which was used to immobilize an antibody 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 suspensions and are therefore less susceptible to self-agglomeration (4 9).
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-nmdiameter gold nanoparticles to single-stranded oligonucleotides. The plasmon resonance absorbance for these particles has a maximum 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 oligonucleotides to the gold nanoparticles, a gold sol was stirred with a solution of terminally 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 complementary to thefirstrecognition element, and the remaining 15 complementary to the second recognition element. When the target DNA was added to the modified colloidal suspension, the target linked the individual colloid particles into a polymeric network. Because the nanoparticles were now much closer together, the plasmon resonance 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 support such as a reversed-phase silica TLC plate (10).
Analytical Chemistry News & Features, May 1, 1998 323 A
Report Superparamagnetic nanoparti cles The basic concept in magnetic bioseparations is to selectively bind the biomaterial of interest (e.g., a specific cell, protein, or DNA sequence) to a magnetic particle and then separate it from its surrounding 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 superparamagnetic 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 nanoparticles are dispersed within the pores of
larger microparticles. In the simplest (direct) method, the microparticles are coated with a monoclonal antibody for a cellsurface antigen. The antibody-tagged, superparamagnetic 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 biomaterial with individuai nanoDarticles have been reported (12) (Figure 1)
cated, nanoparticles do not affect the extent of light scattering by the cell solution. The disadvantage of this approach is small magnetic moments of individual nanoparticles; however, this problem can be overcome by using a high-gradient magnetic field (12). MRI contrast agents. Superparamagnetic Fe304 nanoparticles are also useful as magnetic resonance imaging (MRI) contrast agents (14). MRI is essentially proton NMR done on tissue. Protons are excited with short pulses of radio frequency radiation; the free induction decay as they relax is measured and deconvoluted 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 sig113.l 3X1 d 3-DDetir bright Areas of bone
This approach has numerous advantages. Whereas bound microparticles can fit* affect the viability of the selected cells, nano- tendon which have a low proton density particles supposedly do not affect cell viabecause of the lack of water and lipids bility (12). In addition, as previously indihave a weak signal and aDDear dark Traditionally, a major limitation of MRI has been its inability to distinguish differences 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 inconsistent (15). Various contrast agents have been developed to circumvent these imaging problems. Contrast agents work by changing the strength of the MRI signal at a desired location. 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 neighboring nucleus. As a result, regions containing the superparamagnetic contrast agent appear darker in an MRI than regions 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) Figure 1 . Schematic representation of cell separation using antibody-bound Superparamagnetic particles have many superparamagnetic nanoparticles. advantages over other contrast agents. Un(a) Attachment of nanoparticles to desired cell via binding to the cell-surface antigen, (b) Separation of like agents such as perfluorochemicals, nanoparticle-bound cells from unwanted cells using a column containing steel wool placed in a oils, and fats, superparamagnetic particles magnetic field. (Adapted with permission from Ref. 12.)
324 A
Analytical Chemistry News & Features, May 1, 1998
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 backscatter 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 1001000 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
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 glucoseresponsive 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 selfassembled 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.
Analytical Chemistry News & Features, May 1, 1998 325 A
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 cylindrical shape of these pores a nanocylinder of the desired material is obtained within each pore Depending on the material and the chemistry 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. Nanofibers and nanotubules composed of metals, polymers, semiconductors, carbons, and Ii+-intercalation materials have been prepared (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 nanoscopic electrodes. We have shown that an electroless plating method can be used to prepare ensembles of gold nanodisk electrodes with a disk diameter as small as 10 nm. Such nanoelectrode ensembles (NEEs) have potential applications in electroanalytical chemistry because the signalto-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
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 improvement 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 double-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 doublelayer 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 current associated with electrolysis of the analyte molecule at the surface of the electrode. For our NEEs, this signal can be identical to the current obtained at a macrodisk electrode of the same geometric area, where the entire surface is gold (3). Hence, the faradaic current (analytical signal) 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 limits at the NEE Numerous other groups are also studying the fundamentals of electrochemistry and electron-transfer processes at nanoscopic 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 coulombic 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 electrochemical imaging and sinffle-molecule detection (28) The NEEs discussed above were obtained by electroless plating of gold within the pores of the template membrane for sufficiently long times so that solid nanowires were produced. If plating is done for shorter times, ensembles of nanotubules that span the complete thickness of the
Analytical Chemistry News & Features, May 1, 1998
template membrane can be obtained. Gas flux data suggest that these tubules can have inside diameters of molecular dimensions < 1 nm) (29). We have recently shown that these gold nanotubule membranes can be cation permselective, anion permselective, or nonpermselective, depending 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 applications 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 accommodate a small molecule but too small to accommodate a big molecule. Hence, these nanotubule membranes allow for "molecular filtration", where molecules are separated 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 immobilization (31). To explore this point, we prepared brushlike ensembles of enzymeloaded polypyrrole tubules. Using glucose oxidase as the enzyme we found the glucose-oxidation rate to be seven times faster for the template-synthesized bioreactor than for a more conventional thin-film design using the components (37*) We are currently attempting to develop biosensors 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 opportunities for nanomaterials will arise in the coming decades. Aspects of this work have been supported by the Office of Naval Research, the Department of Energy, and the National Science Foundation.
References (1) Ozin, G. A. Adv. Mater. 1192,4,612-49. (2) Martin, C. R Chem. Mater. 1996,6,1739— 46. (3) Menon, V. P.. Martin, C. R. Anal. Chem. 1995,67,1920-28.
(4) Simo, J. M.; Joven, J.; Cliville, X.; Sans, TT Clin. Chem. 1194,40,625-29. (5) Bangs, L. B./. Clin. Immunoassay 1990, 13,127-31. (6) Bangs, L. B. PureAppl. Chem. m.96, 6,6 1873-79. (7) Singer, J. M.; Plotz, C. M. Am. J. Med. 1956,21,888-96. (8) Hornyak, G. L; Patrissi, C. J.; Martin, C. R. /. Phys. Chem. B 1997,101,1548-55. (9) Medcalf, E. A.; Newman, D. J.; Gorman, E. G.; Price, C. P. Clin. Chem. 1990,36, 446-49. (10) Elghanian, R.; Storhoff, J. J.; Mucic, R C; Letsinger, R L.; Mirkin, C. A. Science 1997,277,1078-81. (11) Rye, P. D. Bio/Technology 1996,64,15557. (12) Miltenyi, S.; Muller, W.; Weichel, W.; Radbruch, A. Cytometry 1190,11,231-38. (13) Haukanes, B. I.; Kvam, C. Bio/Technology 1993,11, 60-63. (14) Hahn, P. et al. Radiology 1190,175,695700. (15) Hahn, P. et al. Radiology 1987,164,37-41. (16) Stark, D. et al. Ah. Gast. Rad. 1988,168, 297-301. (17) Forsting, M. et al. Neuroradiology gy94, 36,23-26. (18) Yeh, T.; Zhang, W.; ;ldstad, S.; Ho, CC Magn. Reson. Med. 1995,33,200-08. (19) M. Bendayan. In Practical Electron Microscopy, Hunter, E., Ed.; Cambridge University Press: New York, 1993; pp 71-92. (20) Crumbliss, A. L. et al. Biotechnol. Bioeng. 1992,40,483-90. (21) Brown, K. R; Fox, A P.; Natan, M. J./. Am. Chem. Soc. 1196,118,1154-57. (22) Weissman, J. M.; Sunkara, H. B.; Tse, A. ..; Asher, S. A Science e996,274,959-^0. (23) Holtz, J. H.; Asher, S. A Nature e197, 389,829-32. (24) Freeman, R G. et al. Science e195,267, 1629-32. (25) Preston, C. K.; Moskovits, M.J. Phys. Chem. .993,97,8495-503. (26) Fan, F. F.; Bard, A. J. Sciencce197,277, 1791-93. (27) Ingram, R. S. et all/ Am. Chem. .oc. 1997,119,9279-80. (28) Fan, F. F.; Bard, A J. Science 1995,267, 871-74. (29) Jirage, K. B.; Hulteen, J. C; Martin, C. R Science 1997,278,655-58. (30) Nishizawa, M.; Menon, V. P.; Martin, C. R Science 1995,268,700-02. (31) Parthasarathy, R.; Martin, C. R Nature 1994,369, 298-301. Charles R. Martin is professor of chemistry at Colorado State University. His research interests are nanomaterials, electrochemistry, and membrane-based chemical separations. David. T. Mitchell, a doctoral candidate at CSU, is studyingfacilitated-transport membranes and nanomaterials synthesis. Address correspondence about this article et Martin at Department of Chemistry, Colorado State University, Fort Collins, CO 80523 (
[email protected]).
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