While stability, sensitivity, and bulk reproducibility still make gold colloid an ideal detection label in rapid tests, there is more to gold than lateral flow.
Liquid components used to manufacture gold colloid.
During the past five years, gold particles and the lateral-flow tests in which they are used have become further established in point-of-care testing. The emergence of simple lateral-flow tests for many different analytes that offer simplified and accelerated testing regimes has made an impact on many industries. For example, diagnostic tests are run during clinical consultations, in which a drop of blood, urine, or saliva is used to give accurate results while patients are still present so that therapy can commence immediately. Similarly, food manufacturing companies can run quality control tests at different stages of processing without requiring trained laboratory staff or interrupting the manufacturing process. Other lateral-flow test applications in veterinary practice, food storage, and environmental monitoring require neither equipment nor training for performance or interpreting results. Such factors, combined with the speed of reaction, have made lateral-flow tests ideal for self-testing.
The quality of materials used to manufacture lateral-flow tests has been continuing to improve. Along with better test design, such improvements have led to increasing use of lateral-flow tests in which more quantitative results are needed. As the multi-billion-dollar global IVD market has continued to grow, such improvements have also led to diversity in the types of rapid and laboratory assays available. This article discusses the reasons for this diversity, and outlines some of the properties of gold that make it suitable for a variety of diagnostic applications.
Using Gold in Lateral-Flow Tests
Two types of particles are commonly used in lateral-flow tests: dyed latex and gold. Many recently developed lateral-flow tests, including the Immunocap Rapid by Phadia AB (Uppsala, Sweden) and the Singlepath and Duopath tests for foodborne pathogens by Merck KgGA (Darmstadt, Germany), have employed gold nanoparticles as the label of choice.
Figure 1. View of a lateral-flow rapid-test strip.
Particle Size. Gold nanoparticles used in lateral-flow tests are smaller than latex. The size of the pores in the test's nitrocellulose membrane is 8–10 µm, enabling many nanoparticles to be accommodated at any one time. The sensitivity of this test format is due to the mixing of particles and analytes that occurs as they pass along the length of the membrane to the capture line (see Figure 1). However, another important factor is that the small size of the particles produces dense packing at the capture line and, thus, greater visibility.
IVD manufacturers should obtain gold from reputable suppliers to ensure that compliance with stringent quality control procedures is met. Such compliance will result in particles that are uniform in size and shape, major factors in the diagnostic product remaining aggregate free and stable in storage. Gold colloid is a homogeneous solution of particles with consistent surface area and charge, satisfying two of the key criteria in protein attachment.
Particle Size Range. The range of gold particle sizes that can be used in lateral-flow tests is 5–200 nm. This size range gives the particles the versatility to be used in a number of different applications. Even the smaller particles have properties that can be used in the life sciences area.
In order to exploit the strong visual properties of larger particles in lateral-flow test applications, the uniformity of the particles is essential. Good-quality gold particles should be monodisperse and spherical, and should include less than 5% uneven shapes. Any other specifications can result in poor performance, along with wasted time and resources, while well-produced gold colloid will yield more tests per liter.
Visibility. Gold particles in the range of 20–120 nm appear bright red because they efficiently scatter green light. Even though the particles are smaller than the wavelength of incident light, the scatter occurs due to the property of surface plasmon resonance (SPR). The wavelength of visible light, which is scattered, varies with the size of the particles.
Protein Binding. Unlike covalent conjugations that are used for latex, proteins such as antibodies are passively bound to gold. A relatively simple process, such binding does not require the use of other reagents. There are three accepted modes of binding to gold particles.
Standard gold particles are surrounded by a layer of negatively charged ions. In ionic binding, positively charged entities in any protein will bind firmly to the surface. When citrate is used to produce the particles, it will bind to amino acids such as lysine.
Amino acids with high hydrophobicity (e.g., tyrosine and tryptophan) bind to surfaces such as gold by hydrophobic binding. For proteins with a high content of such amino acids (e.g., immunoglobulins), this is an important component of total binding, which explains why prolonged exposure of conjugated particles to detergent containing buffers will result in a loss of reactivity.
Gold sulphur bonding is the sharing of a lone pair of electrons by the gold and sulphur, resulting in a strong linkage. The bonding of the gold to cysteine residues within a protein is possibly the most important component in coating the antibody or antigen onto the particles. For this reason, sulphur containing buffers or preservatives such as thimerosal should be avoided.
Figure 2. Good round gold nanoparticles with a low coefficient of variation and uniform shape for optimal antibody loading and sensitivity.
Controllability. Manufacturing consistently round gold colloids for the entire usable size range is controlled via stringent quality control procedures. Many years of development experience have eliminated the problems of batch-to-batch variation and the difficulties in bulk supply (see Figure 2). The method of linkage of gold is simple and involves no reagents other than the protein, diluent buffers, and particles. With defined raw materials, IVD manufacturers can calculate the exact amount of antibody required per particle of gold to give optimal assay performance, which results in cost savings and less wasted raw materials.
Improvements in Lateral-Flow Materials
The quality of materials used in lateral-flow tests has improved, which has had a positive effect on test performance.
Particles. Techniques have been developed that can produce large volumes of consistently round gold particles in large sizes (up to 250 nm), providing a versatile label for a number of markets. For lateral-flow tests, 40-nm particles are considered to be optimal. At this size, the particle is large enough to be visible yet small enough not to create steric hindrance for proteins binding to the surface, thus optimizing the performance of the labeled material. As particles increase in size, visibility improves; initial evidence shows that using 60-nm colloid can increase the visibility of the end signal in some assays, thus potentially improving test sensitivity.
Figure 3. Size distribution comparison data, demonstrating mean diameter of 143 nm in competitor gold labeled 40 nm.
Nitrocellulose Membranes. An important feature of the membranes is the pore size that controls the surface area available for protein binding and the capillary flow rate at which samples migrate through the test strip. Gold particles should be consistent in size, as poorquality gold particles will cluster and will not flow freely through the membrane. IVD manufacturers should also confirm that the gold colloid is the specified size. Size distribution comparisons have shown that, when manufactured incorrectly, reported 40-nm gold can contain a wide range of particle sizes with mean diameters of up to 143 nm, which can lead to incorrect results (see Figures 3 and 4). Modern nitrocellulose membranes are made to be water wettable without losing protein absorption and functionality. Postproduction treatments with surfactant and specific blockers can enhance flow characteristics and remove nonspecific interactions of assay components.
Figure 4. Poor-quality 40 nm gold demonstrating nonround uneven shapes, clustering, and high coefficient of variation between particles.
Pads. Sample pads can be impregnated with chemicals that mask variations in the sample composition and improve sensitivity. Detergents, viscosity enhancers, blocking agents, and salts are commonly dried into the sample pad. This process avoids the use of complex developer/chase buffer solutions and enables a true one-step assay.
Other Membranes. Blood separation membranes now efficiently separate red blood cells and plasma in venous or capillary blood without hemolysis.1,2 Both vertical- and lateral-flow membranes are available, and can separate 10–110 ml of whole blood sample, which enables serum or plasma to be directly assayed without sample manipulation and centrifugation.
Nucleic Acid Lateral-Flow Detection
Lateral-flow tests have been developed that can detect nucleic acids in various formats. Such tests detect the products of amplification techniques such as polymerase chain reaction (PCR) and helicase-dependent amplification, an isothermal method similar to PCR in which DNA strands are separated by enzyme action instead of heating.3 This range of technologies provides detection without requiring expensive equipment.
Figure 5. (a) Detection of double-stranded PCR or tHDA product with immobilized capture antibody, antibody-conjugated gold colloid, and hapten-labeled PCR amplification primers; (b) Detection of unlabeled single-stranded nucleic acid target with immobilized capture antibody, antibody-conjugated gold, and hapten-labeled sequence-specific oligonucleotide probes; (c) Antibody-free detection with immobilized oligonucleotide capture probe and oligonucleotide-conjugated gold.
Nonsequence-Specific Detection. One approach to determine the presence or absence of nucleic acid analytes with a lateral-flow format is to use antibodies and hapten-labeled nucleic acids. In such a method, two haptens (DNP and biotin) capture and detect the analytes by binding with anti-DNP antibodies (immobilized onto the lateral-flow strip) and antibiotin antibodies (conjugated to gold nanoparticles). The haptens have the advantage of being stable in high temperatures, and they do not affect the activity of nucleic acid modifying enzymes. They are also commercially available attached to synthetic oligonucleotides. This method is suited for detecting double-stranded amplification products prepared using biotin and DNP-labeled reagents (see Figure 5a).
Sequence-Specific Detection. Many molecular diagnostic applications require sequence-specific detection. Such detection gives information on the sequence of a nucleic acid analyte in order to distinguish it from other analytes or nonspecific amplification products. Sequence specificity is achieved by using an oligonucleotide probe that will only anneal to the sequence of interest. There are a number of ways to ensure that signal generation is dependent on probe-to-target annealing by using the probe to immobilize the target or by labeling with gold nanoparticles (see Figures 5b and 5c).
Antibody-Free Nucleic Acid Lateral Flow. Eliminating the need for antibodies in a system can reduce costs and increase reproducibility, sensitivity, and specificity of detection. Antibody-free nucleic acid lateral-flow systems are based on target capture via immobilized oligonucleotide probes and detection via probes directly conjugated to gold nanoparticles (see Figure 5c).
Figure 6. Detection of multiple nucleic acid sequences with immobilized oligonucleotide capture probes and oligonucleotide-conjugated gold. Immobilized probes complementary to different sequences are striped at different positions on the nitrocellulose strip. Nucleic acid products containing each sequence are captured at each site and detected with a second, gold-conjugated probe to produce a series of lines.
Sequence-specific oligonucleotide probes can be immobilized on lateral-flow strips with methods adapted from dot blot detection. The resulting capture probes provide sensitivity and specificity comparable to antibody-based methods, and offer significant advantages in terms of multiplexing capabilities (e.g., using multiple capture lines per strip) (see Figure 6). Methods of nucleic acid conjugation to gold nanoparticles are being developed using a variety of techniques to produce stable and robust conjugates.
Other Applications for Gold
Gold colloid is also effective in rapid tests that require instruments to read the final signal. Such tests exploit the light-scattering properties of gold nanoparticles.
Although smaller than the wavelength of visible light, 20–120-nm gold nanoparticles are efficient at light scattering due to their property of SPR. SPR is the result of the interaction between the incident light of a particular wavelength and the conduction electrons present in the nanoparticles. The intensity of the light scatter is determined by the wavelength of the incident light and the size of the particles. The SPR property can be exploited as a method for detecting the particles used as a label.
Due to the property of SPR, gold nanoparticles can be used as an alternative to fluorescent labeling in microarrays. In a direct comparison of the two labels in microarrays, gold nanoparticles, as detected by resonant light scattering, produced an assay for bacterial RNA that was 50 times more sensitive than the equivalent fluorescent-labeled assay.4 Another advantage of using gold particles in such a system is that they are not photobleached.
The intensity and the maximum wavelength of light scatter are directly proportional to the size of the gold nanoparticles. By mixing populations of gold particles of different diameters, each selected for different analytes, it would be possible to develop a multiplex assay, provided that the plasmon resonant peaks of the different-sized particles can be discriminated. Such a reader would expand the range of tests that could be run in lateral-flow or other assay formats.
Use of gold nanoparticles has enabled Pointcare Technologies Inc. (Marlborough, MA) to develop a method of CD4 testing by simplifying current flow cytometric principles. The assay has been developed without different fluorescent labels and the complexity associated with their detection and measurement. This CD4 lymphocyte counting method uses colloidal gold anti-CD4 antibody conjugates that bind specifically to the surface of these lymphocytes. It scatters light in characteristic directions that distinguish these cells from other types, including CD4-expressing monocytes. The simple, small, and portable cytometry device also counts white blood cells and lymphocytes, and it is used for monitoring the treatment of HIV and AIDS patients.
Using Gold in Light-Scattering Diagnostics
There has been considerable interest in exploiting another light-scattering technique involving gold and silver nanoparticles. Surface-enhanced Raman spectroscopy (SERS) is being used in a variety of applications. Use of gold nanoparticles in SERS detection has become widespread, especially for bioanalytical applications.
SERS Process. Raman scattering refers to light scattering at a different wavelength to the incident light. Because individual substances have a unique Raman spectrum, SERS is an excellent identification tool.
However, Raman signals are characteristically weak: approximately 1 in 107 incident photons are scattered with a shift in wavelength. The signals can be enhanced by two processes. In one process, resonance Raman scattering, the laser is tuned to the absorbance of the substance of interest. The other process, surface-enhanced Raman scattering, requires the substance to be in close proximity to a metal surface, exploiting the SPR properties of suitable materials (e.g., gold or silver nanoparticles). Surface enhancement produces signal amplification of 105–106. Combining these two processes, called surface-enhanced resonance Raman scattering, is a sensitive technique that produces signal amplification of up to 1014 and is capable of single molecule detection.5
Figure 7. Use of SERS detection in sandwich immunoassay by detection of a SERS-active reporter attached to metal nanoparticles.
Using Metal Nanoparticles as SERS Substrates. The optical properties of gold and silver nanoparticles make them good surfaces or substrates for SERS detection. Metal nanoparticles are suited for solution phase assays and have the advantage of being well developed for a number of existing bioanalytical applications. Factors such as particle size, shape, and interparticle spacing are critical for signal enhancement, and only high-quality particles should be used. Developing a robust detection system is dependent on the routine production of goodquality nanoparticles that can be functionalized with biomolecules. Producing alternatives to spheres (e.g., tri- angles and rods) has particular relevance and benefit to future SERS applications.
SERS Tags. Although SERS can be used for label-free detection, it is in some cases desirable to introduce a SERS-active label, or reporter group, into the system to produce a strong, characteristic Raman signal that can be easily detected. A number of different reporters may be detected per sample, and the characteristics of SERS spectra give the potential for a higher level of multiplexing than is possible with fluorescent techniques. The reporter can be introduced as part of the nanoparticle. For example, SERS nanotags by Nanoplex Technologies Inc. (Mountain View, CA) consist of a metal nanopar-ticle covered with a layer of a SERS-active reporter molecule and coated with glass. Such particles can be used in diagnostic applications that combine SERS detection with immunoassay formats, substituting conventional particle-based detection with quantitative SERS detection of the metal-reporter complex (see Figure 7).
Figure 8. SERS detection of fluorescent-labeled nucleic acid annealed to target immobilized via thiol group (S) onto metal surface.
Homogeneous Assays. Similar SERS detection methods can also be applied to molecular diagnostics. Since nucleic acids can be labeled with a number of commercially available fluorophores, a reporter does not need to be introduced as part of the nanoparticle, but instead can be used to label the nucleic acid analyte. For example, immobilizing an oligonucleotide capture probe onto a metal surface and annealing labeled target DNA introduces the dye to the sur- face, resulting in fluorescence quenching and the production of a SERS signal (see Figure 8).6 Furthermore, using dye-labeled analytes combined with nanoparticle substrates has the advantage of a homogeneous assay format. Nucleic acids modified with amino groups and fluorophores have been used in quantitative DNA detection, following controlled aggregation of silver colloid.7 Of particular interest to clinical diagnostics is the demonstration of sample genotyping using the multiplex detection of the amplification refractory mutation system method of allele-specific PCR.8
Due to its ability to respond to increased demand for sensitivity, stability, and reliability in rapid tests, quality manufactured gold continues to be a popular label in rapid test development and manufacturing. Its size range makes it versatile for use in many different applications. When manufactured correctly, it delivers accurate results consistently, producing cost- effective and reliable rapid tests. Gold can be used to develop rapid tests in a number of ways, and can even accelerate the development of novel applications. As the market demands more-sophisticated and easy-to-use rapid tests, along with diagnostic tools to accompany specific treatments at the point-of-care, the versatility of gold will contribute to the future development of rapid assays.
Looking ahead, the attractiveness of SERS as a detection method will increase with ongoing research into areas such as substrate development and the design and synthesis of new reporters. The parallel development of instrumentation will increase its practicality for diagnostic applications. This technique is an important source of new applications for gold nanoparticles in an area that is likely to expand in the future.
|L to R: James Carney, PhD, is technical director; Helen Braven, PhD, is a senior scientist in nucleic acid research; Joanna Seal, PhD, is senior project leader of the nucleic acid research group; and Emma Whitworth, PhD, is a senior scientist in the research and development department at British BioCell International (Cardiff, UK). The authors can be reached at firstname.lastname@example.org, email@example.com, joseal@british biocell.co.uk, and firstname.lastname@example.org, respectively.|
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