Medical Device Link.

Originally Published IVD Technology April 2004

Detection Technologies

Figure 1. Silver or gold colloidal particles with diameters of 30–120 nm efficiently scatter light in the visible spectrum. A collection of colloidal particles of which the plasmon resonant peak scattering wavelength is in the blue region of the spectrum (a). Three PRPs illuminated with white light (b).1 (Click to enlarge).

Most rapid immunochromatographic diagnostic assays use simple visual detection of colored microparticle labels to measure analyte concentration. However, as imaging and computational capabilities improve, there is a significant trend within the diagnostics industry to replace visual detection with instrument-based methods.
 
There are several instrumentation-based detection systems that are commercially available for use in home or point-of-care testing (e.g., the A1cnow Monitor by Metrika (Sunnyvale, CA), Persona by Unipath (Bedford, UK), Bio-Rad (Hercules, CA), Triage by Biosite (San Diego), and Instaquant by Provalis (Cheshire, UK)). 

The application of instrument-based detection methods has led to the increased use of traditional fluorescent or chemiluminescent microparticle labels, as well as a number of promising alternative optically detectable labels. This article presents a novel, optically detectable label based on plasmon resonant colloidal metal nanoparticles.

Optical Properties of Plasmon Resonant Particles (PRPs)

Light scattering by nanometer-sized colloidal metal particles is dominated by the incident electric-field-induced collective oscillation of the conduction electrons, known as the surface plasmon resonance. Silver or gold colloidal particles with diameters between 30 and 120 nm efficiently scatter light in the visible spectrum (see Figure 1). 

The peak light-scattering wavelength observed from a particle is generally referred to as the plasmon resonant (PR) peak. The full width at half-height of a particle PR peak centered at ~440 nm is ~40 nm (see Figure 2).1 Under the same excitation conditions, an ~80-nm-diam PRP can generate a brightness (i.e., scattering flux) equivalent to that produced by more than ~5,000,000 individual fluorescein molecules without experiencing photobleaching.^1–3

The specific color of scattered light observed (i.e., frequency band of light) is a function of the size, shape, and material properties of the particle.^{1, 4-6} The correlation of the optical scattering characteristics with the physical shape and size of the colloid, as determined by transmission electron microscopy, has demonstrated that the shape of a colloid has a strong influence on the optical scattering properties of the colloid.⁴ 

Spherical silver colloids scatter blue light (415–490 nm) preferentially, pentagons scatter green light (500–560 nm), and triangular (i.e., platelets or tetrahedrons) colloids scatter light of a longer wavelength that can reach up to 715 nm (see Figure 3). In general, the larger the size of a particle of a given shape, the farther the PR peak will shift toward the red end of the spectrum.

Significance of PRPs for Lateral-Flow Diagnostic Strips

Figure 2. The spectra of the light scattering observed from the three particles imaged in Figure 1b. The peak light scattering wavelength is generally referred to as the plasmon resonant (PR) peak. The full width at half of the height of a particle PR peak centered at ~440 nm is ~40 nm.1 (Figure courtesy National Academies of Science.) (Click to enlarge).

Many lateral-flow diagnostic strips use colloidal particles to detect the presence of an analyte. In the case of gold labels, the diameter of the particles typically ranges from 30 to 40 nm.7,8 The red color observed on the lateral-flow test-strip band is a bulk effect resulting from the scattering of light generated by the collective population of gold colloid bound to the membrane.⁸
 
Of particular interest is that gold or silver particles in this size range are plasmon resonant, and are readily viewed individually as colored light sources by darkfield observation of the scattered light (see Figure 1). The ability to optically observe individual plasmon resonant gold particles of 40–100 nm in diameter using relatively inexpensive instrumentation may permit dramatic improvements in the limits of detection of lateral-flow test-strip-based assays. 

The optimization of capture-line intensity is achieved via the contributions of many factors that, when taken together, will determine the best particle size. The important factors may include: 

• Packing constraints, which can cause a greater number of small particles to bind to a given surface area of the membrane (i.e., packing density) than large particles. 
• Steric hindrance, which accounts for the fact that smaller particles are expected to have greater mobility and accessibility to the target and the membrane.
• Electrostatic effects, including the zeta potential of particles of different size or material, which may result in electrostatic repulsion or attraction affecting both interparticle interactions and a particle’s interaction with the target molecule and the membrane. 
• Per-particle contribution to overall band intensity. For example, the signal contribution for a dyed latex particle is related to the volume of that particle. 

Figure 3. The PR peaks of individual nm-sized silver particles as a function of their shape and size.4 (Figure courtesy Journal of Chemical Physics.) (Click to enlarge).

Several of these factors that affect capture line intensity may offer insight as to why, traditionally, gold colloids of 30 and 40 nm diameter are used, whereas the optimal size for dyed latex particles is between 150 and 450 nm.⁷ Many of these factors are independent of the material (i.e., latex or metal) properties of the particles. The preference for smaller gold particles may be due to the fact that plasmon resonance is dependent on the size of the metal colloid or on interparticle interactions. 

Plasmon Resonance as a Function of Size 

A simple relationship can describe signal contribution as a function of particle size for dyed latex particles. However, the effect of PRP size on the light-scattering properties of individual metal particles is more complex. 

Dyed latex particles of different sizes will be the same color, and larger particles will be easier to see, as the total amount of dye incorporated into such particles is greater. In contrast, PRPs of different sizes will have distinct PR peaks and will scatter light of different colors (for examples of silver colloids, see Figure 3). In lateral-flow diagnostics, the peak of a small gold colloid for which the PRP peak is in the middle of the visible spectrum may be easier to see than the PR peak of a larger gold particle, which is shifted to the red end of the spectrum. 

The effect of particle size on the PR peak can be seen both for individual particles and also for bulk colloidal gold solutions (see Figures 3 and 4, respectively). Solutions of gold particles less than 2 nm in diameter are colorless; solutions of particles between 4 and 80 nm in diameter are red; and suspensions of gold particles that are larger than 80 nm in diameter are gold.⁸

Plasmon Resonance as a Function of Interparticle Spacing

Figure 4. The maximum absorption measured for colloidal gold solutions, showing a shift in wavelength as particle diameter increases. (Data courtesy BBInternational Ltd.; Cardiff, UK.) (Click to enlarge).

As already discussed, the observed color of an individual colloidal particle is related to the size of the particle. In addition, the peak scattering wavelengths (i.e., the color of the particle(s)) may be altered if the distance separating the particles is small enough to scatter the light cooperatively.⁹

For example, when the particles in a solution of 40-nm-diam gold colloids are noninteracting (i.e., separated), the color of the solution is red; however, if the particles interact strongly, then the color of the solution shifts to blue. This behavior is routinely observed during a salt titration—a colloidal gold solution is red when there is no aggregation, however after salt addition the particles aggregate and the color of the solution becomes purple or blue. Therefore, the color of a colloidal gold solution that exhibits plasmon resonance is a useful indicator of the degree of aggregation observed in that colloid. 

When smaller colloidal gold particles of between 20 and 100 nm in diameter are used in a lateral-flow assay, the observed intensity of the capture line is due, at least in part, to the size and interparticle-spacing-dependent PR properties of the particles. 

By understanding how size and interparticle interactions affect the plasmon resonance of metal particles, the full benefits of using PRPs as labels in a large number of different biological assay formats can be realized. Changing the method in which a test is read, and decreasing the density of captured PRP labels so that individual PRPs can be identified and counted, can allow for the full exploitation of the beneficial properties of PRPs in biological assays. 

Such adjustments in testing can also allow for improvements in detection sensitivity that may result from optical instrumentation detection and identification of individual PRPs.

Several applications and experiments have highlighted the potential utility of PRPs in biological diagnostic applications.10 A colorimetric DNA hybridization assay that detects the change in the spectral properties of a solution of 5'-(alkanethiol)-capped oligonucleotide-coated 13-nm-diam gold particles that aggregate in the presence of a complementary target oligonucleotide sequence has been developed.⁹ Improvements to the assay have allowed femtomoles of a target oligonucleotide that contain one base-end mismatch, deletion, or insertion to be distinguished from a fully complementary target.¹¹
 
A change in the intensity of light detected either by scattering or transmittance from a collective population of PRPs that are �30 nm in diameter has been used as a substitute for the more commonly used fluorophore label in DNA microarray-based assays.¹² An increase in sensitivity was observed when 80-nm-diam gold antibiotin antibody-coated PRP labels were compared with fluorescent labels for the detection of biotinylated DNA hybridization on commercially available high-density cDNA microarrays. 

Single-molecular-target site labeling with PRPs of 40 to 100 nm in diameter was demonstrated by the application of these individually optically detectable multicolor PRP labels for the in situ labeling of a targeted DNA site on Drosophila polytene chromosomes, and for the detection of ryanodine receptors in chicken skeletal muscle. Also, in a three-component sandwich assay, the measured signal was a count of the total number of individual particles bound to a solid substrate.¹

Figure 5. A three-component DNA sandwich assay, using a microarray format, where the measured signal (a) is an automated discrimination and counting of the total number of individual nanoparticles bound to a solid substrate. A total of 1.5 ¥ 107 template molecules were detected with a signal-to-noise ratio of 8.2. Representative darkfield images of PRP-labeled 190-µm-diameter microarray spots are shown in panels (b).13 (Click to enlarge).

More recently, 1.5 ¥ 107 template molecules were detected with a signal-to-noise ratio of 8.2 using a microarray format where the measured signal is an automated discrimination and counting of the number of individual PRPs bound to the complementary capture spot minus those bound to a noncomplementary capture spot (see Figure 5).¹³

Multicolor labeling of expressed mRNA in test and reference samples is commonly used with DNA microarray formats for the study of gene expression. Metal nanoparticles that differ in size, shape, and composition can be designed to scatter light of different wavelengths according to their distinct plasmon resonances.^1,2,4
 
Some have suggested that a multicolor analysis could be performed by using PRPs of differing colors and differing surface ligands and that the miniaturization provided by PRP labeling can be applied to the field of DNA microarrays.1 It has been reported that the imaging of light scattered by dithiane-terminated oligonucleotide-functionalized 50- and 100-nm-diam gold PRPs was used to identify two different target sequences in one solution by capture on a DNA microarray.¹⁴

Little background signal was observed in either case at the noncomplementary spot. Scattered light due to hybridized nanoparticle probes could be distinguished from background signal in the presence of as little as 1 picomole of target, which is comparable to the sensitivity of conventional fluorophore-based array detection. 

Conclusion

Colloidal metal particles have been used routinely in lateral-flow assays for many years. The imaging of colloidal metal nanoparticles using darkfield illumination is opening up exciting new areas for developers of diagnostic assays. 

For instance, any test that requires the detection of low levels of materials (e.g., viral RNA detection, which is limited by sensitivity) may be improved via the incorporation of plasmon resonant metal particles. In addition, the sensitivity of traditional techniques used to look at alternative diseases (e.g., hepatitis B) may be increased with the use of these PRP labels. 

Not only can traditional assays be made more sensitive, but also other assay formats that were limited by the sensitivity of detection techniques may be produced. 

Sheldon Schultz, PhD; David Smith, PhD; and Jack Mock at the University of California, San Diego, and Steven Oldenburg, PHD; Christine Genick, PhD; and Keith Clark at Seashell Technology (San Diego) contributed to the data presented in this article. The following sponsors provided funding support: the Richard Lounsbery Foundation, the National Human Genome Research Institute (grant #PHS HG0195901), and the National Science Foundation (grant #DBI-9876651). BBInternational Ltd. (Cardiff, UK) provided the data presented in Figure 4.

References

is technical marketing manager, diagnostics, at Whatman (Clifton, NJ)

David A. Schultz, PhD, is assistant project scientist in the physics department at the University of California, San Diego. The authors can be contacted at drkevinjones@hotmail.com  or dschultz@sdss.ucsd.edu,   respectively.

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11. JJ Storhoff et al., “One-Pot Colorimetric Differentiation of Polynucleotides with Single Base Imperfections Using Gold Nanoparticle Probes,” Journal of the American Chemical Society 120, no. 9 (1998): 1959–1964.

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