Nanomaterials in Analytical Chemistry

As particle size approaches molecular dimensions, all properties of a material change, making nanomaterials useful for particular applications.

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

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)  100 nm as a nanoparticle and particles with dimensions in the range of 100 nm < d < ~10 m as microparticles.

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 m) 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 antibody sites on these particles, resulting in agglutination (or clumping together) of the particles into what looks like curdled milk (6).

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, there 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 micro- and nanoparticles are derivatized with antibodies to human chorionic gonadotrophin, a hormone released by pregnant women. When mixed with a urine sample containing 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. Agglutination 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-nm-diameter 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 oligonucleotide 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 the first recognition 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).

Superparamagnetic nanoparticles. 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 Fe₃O₄ 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 cell-surface antigen. The antibody-tagged, superparamagnetic microparticles are then incubated with a solution containing the cells of interest. The microparticles bind to the surfaces of the 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 individual nanoparticles have been reported (12) (Figure 1).

Figure 1. Schematic representation of cell separation using antibody-bound superparamagnetic nanoparticles.

This approach has numerous advantages. Whereas bound microparticles can affect the viability of the selected cells, nanoparticles supposedly do not affect cell viability (12). In addition, as previously indicated, 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 Fe₃O₄ 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 signal and appear bright. Areas of bone or tendon, which have a low proton density because of the lack of water and lipids, have a weak signal and appear 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).

Superparamagnetic particles have many advantages over other contrast agents. Unlike agents such as perfluorochemicals, oils, and fats, superparamagnetic particles are miscible with aqueous systems, which means they can mix with material in the bowel and be used in small volumes. Immiscible agents must be used in sufficient quantity to displace intestinal matter (15). This miscibility also allows them to be used intravenously. Compared with other magnetic contrast agents (e.g., gadolinium chelates), they are much more potent [as much as 50 times more effective per mole (16)]. Another advantage is that the particles 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 rat T-cells with superparamagnetic Fe₃O₄ nanoparticles and inflamed the rat's testicles, which attracted the particle-attached T-cells and caused a decrease in the MRI signal from the testicles (18).

Other applications

Nanogold particles have been used extensively 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 nanoparticles are gold, which has a high backscatter coefficient, they appear bright in a scanning electron microscope image. In contrast, the high density of gold makes them appear dark in a transmission electron microscope image. Site-specific staining is obtained 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 antibody by the investigator.

Direct adsorption of proteins, such as enzymes, onto bulk metal surfaces frequently results in denaturation of the protein and loss of bioactivity. In contrast, when such proteins are adsorbed to metal nanoparticles, bioactivity is often retained (20), such as when antibody-labeled nanoparticles are used as electron microscopy stains, as discussed earlier. In another example, 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 glassy carbon to make enzyme electrodes (20).

In addition, Natan and co-workers found that cytochrome c retained reversible cyclic voltammetry when deposited onto 12-nm-diameter gold particles attached to a conductive substrate. In contrast, if the cytochrome c was deposited on larger surface features (aggregates of the gold nanoparticles), 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 colloidal 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, the 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).

Figure 2. Temperature tuning of Bragg diffraction from a 125-m-thick polymerized CCA film of 99-nm polystyrene spheres embedded in a poly(N-isopropylacrylamide) gel.

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 resulted from oxidation of glucose caused the swelling and color change. This sensor could detect concentrations of glucose as low as 10^-12 M (23).

Surfaced-enhanced Raman spectroscopy (SERS) uses roughened metal surfaces 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 impediment to using SERS as an analytical technique is the difficulty in reproducibly and uniformly controlling surface roughness. Several research groups have used self-assembled monolayers of metal nanoparticles 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 substrates. The degree of roughness of these surfaces is tunable by varying the diameter of the nanoparticles. Such surfaces have shown a high degree of reproducibility, both for different locations on a single surface and for different--but identically prepared--surfaces (24).

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 Li⁺-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 signal-to-background ratio (S/B) observed at the NEE can be orders of magnitude higher 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 double-layer charging currents can be orders of magnitude lower than at a conventional gold macrodisk electrode of the same geometric area (Figure 3).

Figure 3. Schematic of a NEE.

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 applications of nanometer-sized electrodes include high-resolution electrochemical imaging and single-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 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.

Figure 4. Schematic of switchable ion-selective gold nanotubule membrane.

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 enzyme-loaded 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 same components (31). 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.

References

 (1) Ozin, G. A. Adv. Mater. 1992, 4, 612-49.

 (2) Martin, C. R. Chem. Mater. 1996, 8, 1739-46.

 (3) Menon, V. P.; Martin, C. R. Anal. Chem. 1995, 67, 1920-28.

 (4) Simo, J. M.; Joven, J.; Cliville, X.; Sans, T. Clin. Chem. 1994, 40, 625-29.

 (5) Bangs, L. B. J. Clin. Immunoassay 1990, 13, 127-31.

 (6) Bangs, L. B. Pure Appl. Chem. 1996, 68, 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. J. 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, 14, 155-57.

(12) Miltenyi, S.; Muller, W.; Weichel, W.; Radbruch, A. Cytometry 1990, 11, 231-38.

(13) Haukanes, B. I.; Kvam, C. Bio/Technology 1993, 11, 60-63.

(14) Hahn, P. et al. Radiology 1990, 175, 695-700.

(15) Hahn, P. et al. Radiology 1987, 164, 37-41.

(16) Stark, D. et al. Ab. Gast. Rad. 1988, 168, 297-301.

(17) Forsting, M. et al. Neuroradiology 1994, 36, 23-26.

(18) Yeh, T.; Zhang, W.; Ildstad, S.; Ho, C. 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. J. Am. Chem. Soc. 1996, 118, 1154-57.

(22) Weissman, J. M.; Sunkara, H. B.; Tse, A. S.; Asher, S. A. Science 1996, 274, 959-60.

(23) Holtz, J. H.; Asher, S. A. Nature 1997, 389, 829-32.

(24) Freeman, R. G. et al. Science 1995, 267, 1629-32.

(25) Preston, C. K.; Moskovits, M. J. Phys. Chem. 1993, 97, 8495-503.

(26) Fan, F. F.; Bard, A. J. Science 1997, 277, 1791-93.

(27) Ingram, R. S. et al. J. Am. Chem. Soc. 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 studying facilitated-transport membranes and nanomaterials synthesis. Address correspondence about this article to Martin at Department of Chemistry, Colorado State University, Fort Collins, CO 80523 (crmartin@lamar.colostate.edu).