Point of Care Testing and Use Of Gold In Rapid Assay Tests For The Future Including Lateral Flow Tests From BBInternational

Topics Covered

Background

Point of Care Tests

What Makes Gold so Suitable for LFTs?

Particle Size

Range of Particle Sizes

Visibility

Binding to Proteins

Controllability

Continuing Improvements in Lateral Flow Materials

Particles

Nitrocellulose Membranes

Pads

Membranes

Background

Superior stability, sensitivity, and bulk reproducibility still make gold colloid an excellent detection label in rapid tests, but there is more to gold than lateral flow.

In the five years since BBInternational last wrote an evaluation of the place of gold in rapid testing, both the particles and the lateral flow tests which utilise them have become further established at the forefront of point of care testing.

There have been continuing improvements in the quality of material used in manufacture, which, along with better test design has led to the increasing use of lateral flow tests (LFTs) where a more quantitative result is required. As the multi-billion dollar global in-vitro diagnostic market has continued to grow, these improvements have led to diversity in the types of rapid and laboratory assays that are available. Here we discuss the reasons for this, and outline some of the properties of gold which make it suitable for the wide variety of diagnostic applications.

Point of Care Tests

The emergence of simple LFTs for many different analytes now offering simplified and accelerated testing regimes has impacted on many industries. Diagnostic tests are routinely run during clinical consultations, where a drop of blood, urine or saliva is used to give an accurate result while the patient is still present, and therapy commences immediately. Similarly, food manufacturing companies can run quality control tests at different stages of processing without a requirement for trained laboratory staff or interruption of the manufacturing process. Numerous other LFT applications, in veterinary practice, food storage and environmental monitoring, for example, require neither equipment nor training in performance or interpretation of result. These factors, combined with the speed of reaction, have made LFTs ideal for self-testing.

What Makes Gold so Suitable for LFTs?

There are two alternative types of particle commonly used in LFTs, namely dyed latex and gold. Many recently developed LFTs, including Phadia’s (formerly Pharmacia Diagnostics’) Immunocap Rapid, and Merck’s Singlepath and Duopath tests for food borne pathogens, have employed gold nanoparticles as the label of choice. Its strengths include the following:

Particle Size

Gold nanoparticles used in LFTs are generally smaller than latex. The size of ‘pores’ in the nitrocellulose membrane of the LFT is in the range 8 – 10 microns, enabling each to accommodate many particles at any one time. The sensitivity of this format of test is largely due to the intimate and vigorous mixing of particles and analyte which occurs as both pass along the length of the membrane en route to the capture line [Figure 1]. Yet another important factor is that the small size of the particles produces extremely dense packing at the capture line, and thus greater visibility.

It is essential that gold is obtained from a reputable supplier to guarantee that stringent QC procedures have been adhered to. This will result in particles which are uniform in size and shape, a major factor in the product remaining aggregate free and stable in storage. Gold colloid is a homogenous solution of particles of consistent surface area and charge, immediately meeting two of the key criteria for successful protein attachment.

Figure 1. View of lateral flow rapid test strip.

Range of Particle Sizes

The range of particle sizes which can be made to work in LFTs extends from 5nm to 250nm, making them extremely versatile for a number of different uses [Table 1], whilst even smaller particles have excellent properties within the field of lifesciences.

Table 1. Typical uses of different sized gold nanoparticles.

In order to fully exploit the strong visual properties of larger particles in LFT applications, uniformity of particles is essential. Good quality gold particles should be mono-disperse, spherical, and with less than 5% uneven shapes. Experience over many years has demonstrated that anything other than this specification can result in poor performance and wasted time and resource, as well-produced gold colloid will yield more tests per litre of gold.

Visibility

Gold in the range 20-120nm appears bright red because it is extremely efficient in scattering light. The scatter occurs despite the fact that the particles are smaller than the wavelength of incident light, and is due to the property surface plasmon resonance (SPR). The wavelength of visible light which is scattered varies with the size of the particles (see later).

Binding to Proteins

Unlike covalent conjugations which are generally used for latex, proteins such as antibodies are passively absorbed to gold. It is a relatively simple process and does not require the use of other reagents. Binding to gold particles occurs by three accepted modes:

·         Ionic binding. Gold particles as normally manufactured are surrounded by a layer of negatively charged ions, thus positively charged entities in any protein will bind firmly to the surface. Where citrate is used to produce the particles, the citrate will bind to amino acids such as lysine.

·         Hydrophobic binding. Amino acids with high hydrophobicity, such as tyrosine and tryptophan, bind to surfaces such as gold by this method. For proteins with a high content of these amino acids such as immunoglobulins, this forms an important component of total binding and is a reason why prolonged exposure of conjugated particles to detergent containing buffers will result in loss of reactivity.

·         Gold sulphur binding. The sharing of a lone pair of electrons by the gold and sulphur is termed dative binding, and the resulting linkage is extremely strong. The bonding of the gold to cysteine residues within a protein is possibly the most important component in coating the antibody/antigen to the particles.  It is for this reason that sulphur containing buffers or preservatives such as thiomersal need to be avoided.

Controllability

The manufacture of consistently round gold colloids over the whole useable size range is highly controlled via stringent quality control [Figure 2], and many years of development experience have enabled the elimination of problems of batch to batch variation and difficulties of bulk supply. The method of linkage of gold is simple and involves no reagents other than the protein, the diluent buffers and the particles. With quality raw materials – colloid and antibody – it is possible to calculate the exact amount of antibody required per 1ml of gold to give optimal performance in the assay, saving on cost and wasted raw materials.

Figure 2. Good round gold nanoparticles with low CV and uniform shape for optimal antibody loading and sensitivity.

Continuing Improvements in Lateral Flow Materials

The quality of materials used in lateral flow tests has improved significantly, which has had a positive impact on test performance.

Particles

Techniques have been developed that enable the production of large volumes of consistently round gold in large particle sizes up to 250nm, providing a versatile label for a number of markets. 40nm particles are often considered to be optimal in LFTs because this size is large enough to be highly visible, yet small enough not to create steric hindrance for proteins binding to the surface, thus optimizing the performance of the labelled material. As particle sizes increase in size, visibility improves, and initial evidence shows that the use of colloid of 60nm diameter can increase the end signal to optimal visibility in some assays, thus potentially increasing test sensitivity.

Nitrocellulose Membranes

An important feature of the membrane is the pore size which controls both the surface area available for protein binding, and the capillary flow rate at which a sample will migrate through the test strip. It is essential that the particles are consistent in size, as poor quality gold particles will cluster and will not flow freely through the membrane. It is also advisable to check that the gold colloid is the specified size. Size distribution comparisons have shown that, when manufactured incorrectly, reported 40nm gold can often contain a wide range of particle sizes with mean diameters of up to 143nm recorded [Figure 3 & 4], which can lead to false results.   Modern nitrocellulose membranes are made water-wettable without losing protein absorption and functionality. Post production treatments with surfactant and specific blockers can be used to enhance flow characteristics and remove non-specific interactions of assay components.

Figure 3. Size distribution comparison data, demonstrating mean diameter of 143nm in competitor gold labelled 40nm

Figure 4. Poor quality 40nm gold demonstrating non-round uneven shapes, clustering, and high CV 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, avoiding the use of complex developer/chase buffer solutions, enabling a truly ‘one-step’ assay.

Membranes

Blood separation membranes have advanced greatly enabling efficient separation of red blood cells and plasma from a fingerstick or venus whole blood sample, with little to no hemolysis. Both vertical and lateral flow membranes are now available, which are able to separate between 10-110ìl of whole blood sample enabling serum or plasma to be directly assayed without the need for sample manipulation and centrifugation.

Source: BBInternational

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