Superior stability, sensitivity, and precision and reproducibility of manufacture make gold suitable for use in membrane-based tests.

The demand for rapid membrane-based tests encompasses applications in a wide variety of fields (see box, below). The number of such potential applications will likely increase as these low-cost alternatives to expensive instrumented methods of testing become more sensitive and more specific.

    对快速诊断的需求正渗透到很多领域的应用当中（见下图）。随着较那些昂贵的检测方法廉价的检测的灵敏度和特异性的提高，这些潜在的应用数目很可能会增加。

This article focuses on the importance of a critical component of such tests—the detection label—and how its manufacture can affect the performance and reliability of the whole test system. Specifically, the article will address the advantages of using gold conjugates in this capacity.

    这篇文章主要讲述这些检测中重要物质——探测标记物的重要性，和它的制备如何影响整个检测系统的性能和可靠性。文章还特别阐述了在生产中用金标记物的 优点。

What Is a Rapid Test?

A rapid test is an inexpensive, disposable, membrane-based assay that provides visual evidence of the presence of an analyte in a liquid sample. Such tests can be formatted either as freestanding dipsticks or as devices enclosed within plastic housings. Typically, as little as 200 µl of liquid sample is required to perform the test, which is usually complete within 2–5 minutes. In clinical assays, the sample may be urine, blood, serum, saliva, or other body fluids. In nonclinical tests, the sample may be a small volume of solution prepared from soil, dust, plants, or food, and similarly applied directly to the membrane test strip. No instrumentation is required to perform such tests, which can be used in clinics, laboratories, field locations, and the home—often by inexperienced personnel.

什么是快速检测？

    快速检测是一种廉价的、可任意使用的、基于膜的检测方法，它能提供分析物存在于液体样品的视觉证明。这种检测可以以独立的试纸条，也可以以装在塑料盒中的形式进行。一般地，进行检测仅需要200ul的液体样品，一般在2－5分钟内完成。在临床检测中，样品可能有尿、血液、血清、唾液或其他体液。在非临床检测中，样品可能有由土壤、灰尘、植物或食物等准备好的小量的溶液，类似的直接应用在膜试纸条上。这种检测不需要任何仪器，能被用于临床、实验室、室外和家庭——经常由无经验的人员检测。

A large flask of gold conjugate. Photo by Hugh Burden, courtesy BB International
　

Rapid tests come in two forms—lateral flow and flow-through. The lateral-flow format is by far the most common because it is easier to manufacture and use. Although the same principles apply to both formats, the lateral-flow test is discussed here.

    快速检测分为两类——侧向流动和渗透。因为侧向流动易于生产和使用，所以它很普遍的存在。虽然两种形式的原理一样，但这里讨论的是侧向流动检测。

The base substrate of a rapid test is typically a nitrocellulose strip onto which is immobilized a capture binding protein, usually an antibody or antigen (see Figure 1). A pad (often glass fiber) containing dried conjugate is attached to the membrane strip. For the majority of currently available tests, this conjugate pad contains gold particles adsorbed with antibodies or antigens specific to the analyte being detected. A sample pad, usually paper, is attached to the conjugate pad. When applied to the sample pad, the liquid sample migrates by capillary diffusion through the conjugate pad, rehydrating the gold conjugate and allowing the interaction of the sample analyte with the conjugate. The complex of gold conjugate and analyte then moves onto the membrane strip and migrates towards the capture binding protein, where it becomes immobilized and produces a distinct signal in the form of a sharp red line. A second line, a control, may also be formed on the membrane by excess gold conjugate, indicating the test is complete.

    一个快速检测的底层一般是硝酸纤维素膜，在它上面固定了检测蛋白，通常是抗体或抗原（图1）。连接着膜的是包含有干燥金标的金标垫（通常是玻璃纤维）。在现有的大部分试纸条中，金标垫的金颗粒被能特异性作用于检测物的抗体或抗原吸附。样品垫通常是 纸，粘贴着金标垫。当液体样品用在样品垫上，液体由毛细作用力扩散通过金标垫，使金标复水并使得样品中的分析物特异性作用于金标。金标和分析物的复合物扩散到膜上，并向检测蛋白移动，在那里复合物被固定住，产生清楚的信号——一条红线。第二条线是控制线，在膜上由多余的金标记显色形成，用于表示检测完成。
　

Figure 1. Construction of a lateral-flow rapid test.

By definition, rapid tests should provide results in a short time, preferably minutes. Such tests must be convenient, accurate, reliable, inexpensive, disposable, and foolproof. They must also be easily and unambiguously interpreted, even by users without experience. From the manufacturers' point of view they should carry a large added value and be easily marketed worldwide to users who may be either experienced or inexperienced in the use of such tests. (see sidebar, below).

Rapid tests are versatile. By switching the antibodies and making small adjustments to the chemistry of the strip format, the same test design can be used for many applications.

   由定义可以看出，快速检测能在很短的时间内给出结果，准确的说是在几分钟内。这些检测必须是方便的、准确的、可靠的、廉价的、可任意使用的以及十分简单的。它们也必须容易和清楚的被说明，甚至是没有经验的使用者。从生产商的角度看，它们应该拥有大量附加价值，并且容易在全球销售给使用者，使用者可以是在试剂使用方面有经验的或没有经验的人员。

    快速检测是通用的，更换试纸条上的抗体并稍微调节其化学性质，同样的试纸条设计就能用于很多应用领域。

Why Use Gold?

Early rapid tests used colored latex to form the visual signal, and some current versions continue to use this method. Latex was originally, and still is, the prime labeling method used in agglutination tests. This is because of its predisposition to agglutinate in the presence of binding components. For rapid tests, in which stability of the conjugate is critical for avoiding false positives, this predisposition to agglutinate can become a major problem.

Because of their greater potential stability, gold labels were introduced into membrane-based rapid tests in the late 1980s. Gold particles of any accurately defined size can be manufactured reproducibly under the appropriate manufacturing conditions. Different sizes may be used for different applications. Its superior stability, sensitivity, and precision and reproducibility of manufacture make gold suitable for use in rapid tests. Gold is essentially inert and forms almost perfectly spherical particles when properly manufactured. Proteins bind to the surfaces of these gold particles with enormous strength when correctly coupled, thus providing a high degree of long-term stability in both liquid and dried forms. Also, when accurately stabilized during manufacture, nonspecific interaction of gold conjugates can be reduced to zero.

使用金标记的原因

    在初期，快速诊断用乳胶作为标记物来产生信号，现在仍有一些产品使用这个方法。过去和现在，乳胶法一直是凝集反应主要的标记方法，这是因为它在结合成分的存在下易于凝集。对于快速诊断试剂来说，标记物的稳定性对避免假阳性是非常重要的，因此易凝集的特性会成为大难题。

    因为具有强潜在稳定性，金标记在80年代后期被引入到快速诊断试剂中。在合适的生产条件下，任何精确大小的金颗粒都可以生产。不同的大小有不同的应用。生产的高稳定性、高灵敏度、高精度和高可重复性使得金适合用于在快速诊断试剂中。如正确制造，金是惰性的球型颗粒。当正确连接时，蛋白质能牢固地结合在金颗粒表面，无论是液态或固态都能保持长久的稳定性。稳定生产能使金标的非特异性结合为零。

A comparison of the benefits of different labels used to mark antibodies and antigens is shown in Table I.

Table I. Comparison of the characteristics of labels commonly used in rapid tests.

Manufacture of Gold Colloids

When producing large volumes of gold conjugate, sophisticated processing methods are required to attain reproducible batch-to-batch manufacture and to avoid instability. The manufacture of 100 liters of high-quality gold conjugate requires the utmost care and attention in order to achieve a final stable and sensitive product. Electron microscopy must be used to provide quality control at each step of the manufacture.

During the past 20 years, manufacturers have introduced a variety of different methods for synthesizing gold colloids. The goal has been to obtain colloids of a monodisperse nature, which are of a controlled and uniform diameter. Although all of the production methods rely upon the reduction of tetrachloric acid (HAuCl4) to form gold atoms, they vary considerably in the physical conditions, order of reagent addition, reducing agent used, and quality of the final colloid produced (size, shape, and coefficient of variance).

In general, all of the production methods use a reducer to donate electrons to the positively charged gold ions in solution and produce atomic gold. This is shown as follows:

Gold tetrachloric acid + reducer = gold colloid

HAuCl₄ + e– = Au⁰

Commonly used reducers include sodium citrate, yellow phosphorus, sodium borohydride, and sodium thiocyanate. Figure 2 shows the process of reduction at the ionic level.

Figure 2. Reduction of gold ions to form gold particles.

Before the addition of reducer, there are 100% gold ions in solution. The ordinate of the graph indicates the progress from gold ions to gold atoms as the reducer is added. Immediately after the reducer is added, there is a sharp rise in gold atom content in the solution until this level reaches supersaturation. Aggregation then occurs, in a process called nucleation, to form central icosahedral gold cores of 11 atoms at nucleation sites. The formation of nucleation sites, in order to reduce the supersaturation of gold atoms in solution, occurs extremely quickly. Once this is achieved, the remaining gold atoms in solution continue to bind to the nucleation sites under an energy-reducing gradient until all atoms are removed from solution.

The number of nuclei formed initially determines how many particles finally grow in the solution. This number, in turn, depends on the amount of reducer added. A large amount of reducer produces a large number of nucleation sites and hence a large number of gold particles. Clearly, the larger the number of nucleation sites for a given amount of gold chloride in solution, the smaller will be the final size of each gold particle. Particle size is thus carefully controlled by the amount of reducer added. If manufacturing conditions are optimized, then all nucleation sites will be formed instantaneously and simultaneously, resulting in all gold particles growing to exactly the same size (monodispersal). This is very difficult to do. Most manufacturing methods do not achieve instantaneous reduction and formation of nucleation sites, resulting in uneven growth and a multidisperse colloid that is virtually unreproducible and results in a very unstable conjugate.

A gold colloid comprises a suspension of gold particles individually surrounded by a negative charge layer arising from the residual negative ions in solution (see Figure 3). This charge layer, called the zeta potential, provides the means for the gold particles to repel one another and to stay in suspension indefinitely. The zeta potential can be compressed or expanded depending on the total ionic concentration of the surrounding solution.

Figure 3. Colloidal gold particle surrounded by a double ionic layer.

High-quality gold colloids and custom-manufactured conjugates are readily available commercially. By purchasing these components from a reputable supplier—instead of attempting to process large volumes of gold themselves—manufacturers can save time and considerably reduce their manufacturing risks.

胶体金的制备

    当生产大批量金标记物时，为保证批间重复性及避免不稳定性，成熟的工艺方法是必须的。生产100升高质量的金标需要非常小心以获得最终稳定及灵敏的产品。电子显微镜用于控制生产中每一阶段的质量。

    在过去20年中，生产者提出了多种不同方法来合成胶体金，目的是为了得到单分散性胶体金—受控的均一的胶体金。虽然所有的生产方法都是还原HAuCl4形成金原子，但是它们还受以下条件影响：物理条件、试剂的添加顺序、使用的还原剂和最终胶体的质量（大小、形状和变异系数）。

    一般来说，所有的生产方法是使用还原剂提供电子给溶液中的正价金离子而生成金原子，反应公式如下：

                                             HAuCl4 + e- =Au0

    通常使用的还原剂包括柠檬酸钠、黄磷、硼氢化纳、硫氰酸钠。

    在加还原剂之前,溶液中金离子是100%，曲线图的纵坐标表示加入还原剂后从金离子变为金原子的过程。加入还原剂后短时间内，溶液中的金原子含量直线上升直到饱和。开始出现聚集，这个过程叫做成核现象，形成11个原子构成的中央二十面体金核。为减少溶液中过饱和的金原子，晶核构架形成得非常块。一旦晶核形成，溶液中剩余的金原子依据能量递减梯度继续结合到晶核上直到所有原子从溶液中消失。

    最初形成晶核的数量决定最终溶液中有多少金颗粒。颗粒数量是由加入还原剂的量决定的。还原剂越多产生的晶核越多从而产生的金颗粒也越多。很明显，大量晶核必然导致金颗粒变小。因此，金颗粒的大小由加入还原剂的量多少控制。如果优化生产条件，所有的晶核将在瞬间同时形成，导致金颗粒达到均一的大小（单分散性）。但这是很难做到的。大部分的生产方法不能瞬间还原形成晶核，从而导致不均匀聚集，多分散性的胶体没有可重复性，导致形成非常不稳定的金标。

    胶体金由金颗粒悬浮液构成，金颗粒被一个负电荷层包被着（见图）。这个电荷层称作zeta电势，它能使金颗粒互斥并在悬浮液中任意分布。zeta电势能随溶液中的离子浓度收缩或扩张。

    高质量的胶体金和为客户生产的金标在市场上随处可见。购买知名供应商的原料——代替自行生产大批量胶体金——能节约时间并降低生产风险。

Good Gold, Bad Gold

The apparent ease with which gold colloids and conjugates can be manufactured has led to the commercial availability of many poor-quality, poorly characterized, and nonreproducible products. When incorporated into rapid tests, such products can lead to poor stability, sensitivity, and specificity. To prevent this, gold colloids should be evaluated ultrastructurally using a transmission electron microscope (TEM). Such an evaluation should enable the manufacturer to compare the diameter of the colloids to that of a calibrated standard and to obtain information about particle sphericity, particle irregularity, and overall variance in particle diameters.

Unevenly shaped particles occur when the nucleation and growth rate during the reduction process have been uncontrolled (see Figure 4). Particles may be seen to be of different sizes and shapes (oval, triangular, oblong, rhomboid, etc.). These particles will not coat evenly with protein during conjugation and will not evenly repel one another in solution. This can affect the color, sensitivity, specificity, and stability of the final product. A mere 5% of odd-shaped particles can influence a test result, making it completely nonreproducible. The signal formed by such "bad gold" is usually seen as having a bluish or purple color compared with the normal cherry red that typifies a well made 40-nm monodisperse colloid. Although the darker color may be easier to see against the white membrane of a test system, it indicates an inherent instability that is much more likely to give false results. Even worse, uneven particle shape and size produce very uneven protein coating, which leads to long-term clustering and aggregation of the conjugate. Such changes may occur within days or even hours of storage of the conjugate in solution.

Figure 4. Good gold colloids and bad (unstable) gold colloids.

Even drying the conjugate immediately onto the solid-phase substrate does not entirely overcome this problem. During the drying process, surface proteins that are not securely attached because of particle shapes, can easily become detached, yielding false negatives and high background.

One of the most common problems encountered in rapid-test performance is failure of the gold conjugate to release at the correct speed and with integrity from the glass-fiber conjugate pad. This failing is often the result of poorly coated gold particles being directly exposed to the glass-fiber material and not being able to release. This inevitably means that surface proteins have either become permanently attached to the fiber matrix or have left the gold particles and are floating free. With well-prepared gold conjugates, starting with evenly coated monodisperse and spherical particles, such dangers are greatly reduced.

Although TEM examination is the only true way to determine the quality of colloid, a quick method for determining whether a colloid or conjugate contains fused, aggregated, or heterogeneous particles, or a mixed-size population, is to examine its color visually. A good 40-nm colloid (the most frequently used size for diagnostic applications) should be cherry red. If the colloid or conjugate appears purple, it is likely to be of poor quality and unstable.

好胶体金和不好的胶体金

    胶体金和金标能在明显的简单条件下制备出来，可是简单的条件已经导致市面上的低质量、差特性和无可重复性的产品。当装配到快诊试剂上时，这种产品的稳定性、灵敏度和特异性都很差。为了防止这个问题，可用电子显微镜来检测胶体金的超细微结构。这种检测能使生产者对照标准比较胶体的直径，得到颗粒球状、颗粒不规则性和颗粒直径的变化。

    当还原过程中结晶和聚集速度不能控制时，形状不规则的颗粒将会产生（见图4）。颗粒呈现不同的大小和形状（椭圆形、三角形、长方形、斜长方形等等）。这些颗粒在标记时表面不能均匀包被蛋白质，也不能在溶液中均匀分布，这会影响成品的颜色、灵敏度、特异性和稳定性。仅仅5%的异性颗粒就会影响检验结果，使之没有可重复性。使用不好的胶体金产生的颜色是带蓝色或紫色，而用40nm单分散性胶体产生的是正常的粉红色。虽然在试剂白背景膜上深的颜色更容易看到，但暗示着潜在的不稳定性，即更可能得到错误的结果。更糟糕的是，不均一的颗粒形状和大小会产生很不均匀的蛋白质包被，会导致金标长期结块和聚合。这种变化在金标存放期的几天甚至几小时内发生。

    甚至将金标喷到膜上后快速干燥也不能彻底解决这个问题。在干燥过程中，因为颗粒形状而不能紧密结合，使得表面蛋白质很容易与胶体金分离，产生假阴性和深背景。

    在快诊试剂检测中经常遇到的问题之一是金标不能以正常速度并且完全地从金标垫上释放。这个问题常常是因为未完全包被的金颗粒直接暴露在垫上而不能释放。这就意味着金表面的蛋白质或永远吸附在基材上，或脱离金颗粒被冲走了。使用精制的金标，即使用均一包被的单分散球形颗粒，这些问题都会明显减少。

    虽然电镜检查是检验胶体金质量的唯一正确的方法，但有还有一种快速检验方法是目测它的颜色，它能用来检测胶体金或金标是否含有聚集的、融合的或异类的或混合大小的颗粒。一个好的40nm的胶体金（诊断试剂最常用的尺寸）应该是粉红色。如果胶体或金标呈现紫色，可能是低质量的、不稳定的。

Choice of Gold Particle Size

The signal is generated on the test strip by the accumulation of gold particles at the test or control line. These particles must be large enough to be seen. The greater the particle size, the easier it is to see an accumulation of these particles. For example, particles of 1 nm diameter would be virtually impossible to see, no matter how many had accumulated, because 1-nm particles do not have the bright red color of the larger sizes. It is not until particles reach 20 nm that a worthwhile signal can be seen. Steric hindrance becomes a problem as the particles increase in size (see Figure 5). For example, if an IgG molecule (160,000 daltons) is just 8 nm in length, only approximately 4 nm of this will extend from the surface of a gold particle. Particles of 100 nm will tend to dwarf the small surface molecules and make it difficult for them to interact with specific proteins. In addition, the larger the particle size, the fewer of them can be contained in a given volume of solution.

Figure 5. Choice of gold particle size for optimized signal.

This trade-off between required visibility and steric hindrance dictates that, for most immunoassay applications, the optimum particle size is 40 nm. In some cases where steric hindrance is a greater problem (e.g., for smaller antigens), particles of 20 nm are preferred. Larger particles may be preferred when a darker red color is desired or when the lower curvature of the particle surface would improve the molecular interaction between the antibody and antigen. Experiments should be performed to determine which particle size gives the highest sensitivity with the lowest background and greatest stability.

金颗粒大小的选择

    试剂条上信号的产生是因为金颗粒在C、T线的积累。这些颗粒必须大到能够被看到。颗粒越大，颗粒的积累越容易看到。例如，直径1nm的颗粒不管积累多少也不能被看到，因为1nm颗粒不具备大颗粒的粉红色。直到颗粒增大到20nm标记才可见。当颗粒增大时，空间障碍又成为一个问题（见图5）。例如，一个IgG分子（160,000daltons）在长度上只有8nm，大约4nm从金颗粒表面伸展开。100nm的颗粒有使表面分子变小的趋势，使它们难和特异蛋白结合。另外，颗粒越大，一定体积溶液中所含的数量越少。

    可见性和空间障碍的平衡如图，对多数免疫试剂应用来说，最佳的颗粒大小是40nm . 在一些情况下，空间障碍是一个大问题（如小抗原），20nm的颗粒更好。当需要暗红色或颗粒表面较小的曲率能增加抗原抗体间的分子引力时，大颗粒更好。应该由实验决定确定多少颗粒大小能带来高灵敏度、浅背景色和高稳定性。

Manufacturing Gold Conjugates

Manufacturers should understand the chemical and physical processes that permit a permanent coupling of their protein to the gold colloid. Armed with this knowledge, it is then simple to proceed from small test batches to large-scale manufacturing without compromising sensitivity, stability, specificity, or reproducibility. Simply throwing a conjugate together, while cheap and easy, will often lead to the formation of clusters. Viewed through a TEM, each cluster is a group of four or more gold conjugate particles. The presence of such clusters generally indicates an unstable product whose performance will change over time. Although initially appearing more sensitive than an unclustered product, such tests will soon begin to show stability and specificity problems.

A good conjugate is one where the protein is adsorbed onto the surface of the gold. Unlike latex, proteins are only adsorbed passively onto the surface; covalent coupling is not performed. However, hormones, drugs, and other small molecules (<10 kD) must first be coupled to a larger carrier protein, typically BSA, before passive conjugation can be performed. This is because there are not enough points of contact between the molecule and the gold surface. Steric hindrance would in any case prohibit the small molecule from rising above the zeta potential. The carrier should be inert, in that it should have no effect on detector protein activity.

Figure 6. Binding forces between an antibody and a gold particle.

The antibody will be firmly attached to the gold by the Fc region, leaving the Fab region protruding through the double-ionic layer surrounding the gold particle and able to bind the analyte (see Figure 6). There must be no excess of labeling antibody present. Such excess will cause unlabeled antibody to compete with the labeled antibody, resulting in the presence of false negatives, and may affect the long-term stability of the conjugate. Optimum binding of proteins to gold occurs at, or close to, the isoelectric point.

Adsorption of protein to gold occurs within a matter of seconds through the follwing mechanisms:

·    Initial charge attraction of the negative gold particle to the positively charged amino acids within the protein (e.g., lysine).

·    Hydrophobic adsorption of the protein to the particle surface, through certain amino acid residues including tryptophan.

·     Dative binding between sulphur residues on the protein (from cysteine residues) and the gold particle.

金标的制备

    生产者应清楚蛋白质永久结合到胶体金上的化学和物理过程。具备了这些知识，很容易从小批量生产过渡到大批量，而不牺牲灵敏度、稳定性、特异性或可重复性。仅仅将金标简单的混合在一起，既便宜又容易的，然而常常会导致结块。通过电镜观察，每个结块是四个或更多个金颗粒的聚合体。这些结块的存在意味着不稳定的产品性能会随时间改变。虽然比较未结块的产品刚开始具有更高的灵敏度，但这些产品很快会出现稳定性和特异性的问题。

    一个好的金标就是蛋白质吸附在金的表面。和乳胶不同，蛋白质只是被动吸附在表面，并没有共价键。然而，激素、毒品和其它一些小分子（<10kD）在被动结合前必须先和大分子载体偶合（最典型的是BSA）。这是因为在分子和金表面没有足够的连接点。空间障碍会阻碍小分子在金的zeta电位上出现。载体应该是惰性的，这样就不会影响检测蛋白质的活性。