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Time：2020-08-17Click：
The status of colloidal gold in rapid detection
John Chandler, Tracey Gurmin, and Nicola Robinson
Rainylily comrades translation
Diagnostic Consulting Network (Irvine, CA), and Is currently the principal Diagnostic provider for Diagnostic Consulting. Dr. Jeff Bauer is the Principal scientist at BioDot Inc. bofarrell@dcndx.com and jbauer@dcndx.com
Rapid membrane testing has a wide range of requirements and can be applied in many fields (see table below). With the improvement of sensitivity and specificity, a large number of potential applications may be low cost alternatives to expensive instrument detection methods,
Application of rapid detection
Clinical medical
L allergy
L Markers of myocardial infarction
L Degenerative disease
L Substance abuse
L reproductive
L forensic
L Immunotyping
L Infectious diseases
L Serological tests
L Sexually transmitted diseases
L Stress response
L toxicology,
L Tumor marker
Agricultural application
L Food safety
L Plant and crop diseases
Environmental applications
L Biological contamination
L Environmental pollution
Veterinary application
(Similar to most areas of human clinical application)
This paper focuses on the very important part of this detection method -- the influence of the production of the detection marker on the performance and reliability of the whole detection system. In particular, the advantages of colloidal gold markers in this detection function are emphasized.
What is rapid detection?
Gold standard fluid in a flask
Hugh Burden Courtesy BB International
Rapid detection is an inexpensive, disposable, membrane-based method that provides visual evidence of the presence of an analyte in a liquid sample. The test can be done in a separate strip or in a plastic box. In general, this test requires at least 200ul of a liquid specimen and can be done in 2 to 5 minutes. In a clinical test, the sample may contain urine, blood, serum, saliva or other body fluids. In non-clinical tests, the sample may be a trace solution prepared from soil, dust, plants or food, which can also be tested directly with a film strip. The test requires no instrumentation and can be used in clinical, laboratory, outdoor, or home applications - usually for people with no operational experience.
The rapid detection method comes in two forms - chromatography and percolation. Chromatography is the most common form because it is relatively easy to make and use. The principle involved in both forms is the same, but we will discuss the method of chromatography here.
A substrate for rapid detection is usually a nitrocellulosic membrane on which the detection protein, usually an antibody or antigen, is fixed (Figure 1).
Attached to the membrane is a gold label pad (usually fiberglass) containing a dry gold label. For most tests currently available, the binding pad contains gold particles that adsorb specific antibodies or antigens against the analyte to be tested. The sample pad is usually paper with a binding pad attached. When the sample pad is used, the liquid sample passes through the binding pad by capillary diffusion and rehydrates the gold crosslinker, making the sample to be analyzed interact with the crosslinker. Then the gold crosslinker and the sample complex to be analyzed are transferred to the membrane strip and then to the protein binding compound, where the complex is fixed and produces a distinct signal in the form of a thin red line. The second line is the control line, formed by the remaining gold crosslinker on the film, indicating that the test is complete.
As the name suggests, rapid testing should provide results in a short period of time, minutes to be exact. The test must be convenient, accurate, reliable, inexpensive, disposable and easy to operate. They must also have easy and clear instructions that even inexperienced users can operate. From the manufacturer's point of view, this rapid testing will be of great value and easy to market to users around the world, regardless of their experience (see postscript below).
Rapid detection has a wide range of functions. The same design should be able to be used for multiple tests, either by changing the antibody or by making minor changes to the chemical formulation of the strip.
Why the gold mark?
Early rapid detection USES colored latex to form visual signals, and some current detection still USES this method. Latex has been and still is the main marker for agglutination because of its tendency to agglutinate in the presence of binding components. For rapid detection, the stability of cross-links is the key to avoid false positives, and the easy agglutination may be the main problem.
Because of their better potential stability, gold markers were introduced in the late 1980s for rapid membrane detection. Any particle of gold of precise size can be reproduced under the right conditions of production. Different sizes can be used for different purposes. Its excellent stability, sensitivity, accuracy and repeatability make it suitable for rapid detection. Gold is an inert element and can be properly processed to form a complete spherical particle. When properly connected, the protein is firmly attached to the surface of the gold particles, providing long-term stability in both liquid and solid form. Moreover, if the protein is precisely fixed during the manufacturing process, its non-specific binding to the gold crosslinker can be reduced to zero.
The comparison of the advantages and disadvantages of different markers of labeled antibodies and antigens is shown in Table 1:
Table 1. Comparison of features of commonly used markers in rapid detection
Characteristic of gold-silver carbon latex fuel enzyme
Visibility *** *** ***
Sensitivity is high
Stability *** ***
Color * -- *** *** *** *
Repeatability *** *** *** **
Magnification *** **
Step *** *** *** *** ** --
Multiple analysis test *** *** *** *** *** *** *
The results were clear
Easy to prepare *** *** *** *** *
Simple to use *** *** *** **
Adaptability *** *** ***
Low cost
* Limited application
** Suitable for partial testing
*** results were significant for most tests
Colloidal gold manufacturing
When colloidal gold is produced in large quantities, mature processes are required to ensure inter-batch repeatability and avoid instability. In order to obtain the final stable and sensitive product, the production of colloidal gold of 100 high quality requires extreme care and care to obtain the final stable and highly sensitive product. Every step in the production process requires the use of electron microscopy for quality control.
Over the past 20 years, manufacturers have introduced various methods to synthesize colloidal gold in order to obtain monodispersive colloids of a certain diameter and uniform uniformity. Although all production methods rely on reducing HAuCl4 to form gold atoms, the physical conditions vary considerably, such as the order in which the reactants are added, how much reductant is used, and the quality (size, shape, coefficient of variation, etc.) of the resulting colloids.
FIG. 2 Reduction of gold ions to form gold particles
In general, all production methods use reductants to provide electrons to positively charged gold ions in solution to produce gold atoms, as shown below:
Chloroauric acid + reducing agent = colloidal gold
HAuCl4 + e - = Au0
Reductants commonly used include sodium citrate, yellow phosphorus, sodium borohydride, and sodium thiocyanate. Figure 2 shows the horizontal reduction process of different ions.
Before adding the reductant, the solution contained 100% gold ions. The ordinate of the curve represents the process of converting gold ions into gold atoms when reductants are added. After adding the reductant, the amount of gold atoms in the solution immediately increases sharply until it reaches supersaturation. This is followed by agglutination in a process called nucleation, where the nucleation site forms a central icosahedral gold core consisting of 11 gold atoms. Nucleation sites are formed to reduce the over-saturation of gold atoms in solution, and they form very rapidly. Once this process is complete, the remaining gold atoms in the solution continue to bind to the nucleated site at a decreasing energy gradient until all atoms are removed from the solution.
The number of nuclei formed initially determines how many particles will eventually form in the solution, which in turn depends on the amount of reductant added. The more reductants, the more crystal nuclei are produced and the more gold particles are produced. Obviously, in a solution containing a given amount of gold chloride, the greater the number of nucleating sites formed, the smaller the final size of each gold particle formed. Thus the size of the particle can be finely regulated by the amount of reductant added. If the production conditions are optimized, all nucleation sites can occur simultaneously in an instant, so that the size of the formed gold particles is exactly the same (i.e. monodisperse type). This is a very difficult thing to do. Most production methods are unable to reduce all gold ions at the same time and form nucleation sites at the same time, resulting in uneven nucleation process and the generation of polydispersal colloid, which eventually leads to low repeatability of products and instability of cross-linking materials.
Figure 3 Colloidal gold particles surrounded by double electron layers
Each colloidal gold particle is surrounded by a suspension of gold particles derived from a negatively charged layer of residual anions in the solution (see Figure 3). This electron layer, known as zeta potential, causes the gold particles to repel each other and to be randomly distributed in the suspension. Zeta potential can be compressed or expanded by changing the concentration of ions in the surrounding solution.
High-quality colloidal gold and custom-made well-processed cross-links are readily available for commercial use. By purchasing these ingredients from reputable suppliers rather than trying to process large quantities of gold themselves, manufacturers can save time and reduce their manufacturing risk.
Good colloidal gold and bad colloidal gold
FIG. 4 Colloidal gold of high quality and low quality (unstable) colloidal gold
Colloidal gold and crosslinkers may appear to be easy to manufacture on the surface, but poor quality, poor performance and poor reproducibility can result in poor commercial applications. The application of such products to rapid detection may result in low stability, low sensitivity and low specificity of the results. To prevent such results, transmission electron microscopy (TEM) should be used to evaluate the ultrafine structure of colloidal gold. This test enables the producer to compare the diameter of the colloid against the standard, and to obtain the changes of particle size, particle size irregularity and particle size.
If nucleation and formation rate are not well controlled in the reduction process, the formation of particles will be uneven. (see Figure 4), the observed particles may have different sizes and shapes (such as ellipses, triangles, rectangles, rhomboids, and so on). Such particles will not be uniformly coated during binding to the protein, nor will they be uniformly distributed in the solution. Finally, the color, sensitivity, specificity and stability of the product will be affected. Only 5% of the particles with uneven shape will affect the test results, making them completely non-reproducible. In contrast to cherry red, which represents a well-made monodisperse colloidal particle of 40nm, the signal formed by "poor quality gold" is often seen as pale blue or purple. Although darker colors on the white background film are easier to detect in the system
Identify, but hint at, potential instability, which is more likely to lead to false results. More seriously, uneven particle shape and size will lead to uneven protein coating, which will lead to the accumulation and agglutination of cross-linked substances for a long time. Cross-linked substances stored in solution may show such changes within a few days or even hours.
This problem cannot be completely overcome even if the crosslinker is immediately dried on the solid matrix. During the drying process, surface proteins that do not bind stably due to particle shape are easily separated, resulting in false positives and high backgrounds.
One of the most common problems in rapid detection operations is that the gold crosslinker cannot be released from the fiberglass bonding pad at the appropriate speed and in a complete form. This problem is often caused by incomplete coated gold particles being directly exposed to the fiberglass material. This must mean that the surface proteins are either permanently attached to the fibrous matrix or float free from the gold particles. The existence of homogeneous monodispersed spherical particles at the beginning, coupled with the preparation of well-formed gold cross-links, can greatly reduce the occurrence of this dangerous problem.
Although TEM testing is the only correct method for determining the mass of a colloidal substance, a quick way to determine whether a colloidal substance or cross-linked substance contains fused, agglutinated, or heterogeneous particles, or groups of particles of different sizes, is to visually examine its color. Good grade colloids at 40nm (particle size most commonly used in diagnostic applications) should show cherry red. If the colloid or cross-linker appears purple, it is likely to indicate poor quality and instability.
Gold particle size selection
Figure 5: Size of gold particles selected due to optimized signal
Detection signal is the signal produced by the accumulation of gold particles on the test line or control line. The particles must be large enough to be seen, and the larger the particle size, the easier it will be to see them when they gather. Particles of 1nm diameter, for example, are almost impossible to see, no matter how many they aggregate, because 1nm particles don't
There's the bright red that larger particles have. Only particles up to 20nm in diameter show a visible signal. As particle size increases, steric hindrance becomes a problem again (see Figure 5). For example, if IgG (160,000 Dalton) molecules are only 8nm long and about 4nm extend from the surface of gold particles, then particles of 100nm will make these small surface molecules appear smaller and less likely to interact with specific proteins. Moreover, the larger the size of these particles, the smaller the number of them present in a given volume of solution.
This contradiction between the need for visibility and steric hindrance suggests that the optimal particle size is 40nm for most immunoassay applications. In some cases, when steric hindrance becomes a major problem (for example, for smaller antigens), 20nm particles are more appropriate. Larger particles are preferred when darker colors are desired or when the lower curvature of the molecular surface enhances the interaction between antigens and antibody molecules. It should be up to the experiment to determine how much particle size brings high sensitivity, light background color, and high stability.