Rapid detection of fluorescence: the application of biosensor is not a dream —— Interview with Professor Jin Zhenhua, technical director of Zhongke Maliang Bioengineering Co., Ltd.
ATP fluorescence method is a rapid detection technology based on the principle of firefly luminescence. Fireflies glow because they can synthesize biocatalysts that convert chemical energy of chemicals into light energy, such as firefly luciferase, D- fluorescein and ATP (adenosine triphosphate, an energy substance that all cell organisms can produce). Under aerobic conditions, luciferase catalyzes fluorescein to produce oxidized fluorescein and emit fluorescence. If the above conditions are met, even systems other than fireflies can emit fluorescence. As early as the late 1950s, NASA established a rapid ultramicro ATP detection technology using firefly luciferase. At the end of 1960s, ATP in soil samples of the earth was detected and applied to detect whether there is life on the moon and Mars in the future. The commercial application of ATP detection based on insect luciferase began in the late 1980s. Luciferase was originally obtained by cultivating firefly larvae. With the development and application of genetic engineering technology, scientists extracted genes encoding firefly enzymes from firefly cells and introduced them into bacterial cells. In this way, a large number of insect luciferases can be obtained in just over ten hours through biological fermentation.
In 2003, Zhong Ke Ma Liang introduced the patent project of "New strain containing luciferase gene and production of insect luciferase" invented by Professor Jin Zhenhua from Institute of Development of Chinese Academy of Sciences. After several years of industrialization exploration, we finally started mass production of insect luciferase and insect luciferase enzyme preparation with independent intellectual property rights after 2005, and completed the matching of related detection products in 2007, realizing the rapid detection of the total number of bacteria in food samples. Principle of ATP fluorescence detection based on insect luciferase Only when ATP exists, insect luciferase can catalyze the reaction of insect luciferase and produce fluorescence. Therefore, if there are too many luciferases and luciferases, the fluorescence intensity will only depend on the amount of ATP in the reaction system. Taking the bacteria in the sample as an example, the more cells, the more ATP and the stronger fluorescence. That is to say, under certain conditions, the fluorescence value is linearly related to the amount of ATP; In addition, the fluorescence value can be easily and accurately measured by a photometer. Therefore, under the condition of excluding the interference of non-bacterial ATP in the sample, as long as the relationship curve between the relative fluorescence value and the number of bacterial cells is obtained, the number of bacterial cells in the sample can be quickly determined according to the standard curve between the relative fluorescence value of pure ATP and the number of bacterial cells.
Composition and function of ATP rapid detection system At present, there are many application systems for detecting biomass, enzymes and metabolites based on the principle of firefly luminescence. Although the application purposes, operation methods and sample sources of various detection systems are different, they all include the following parts in composition.
1. reagent system The reagent system includes all reagents needed to promote the fluorescence reaction and effective ATP extraction reagents. Specifically, it includes somatic cell lysis reagent group, bacterial cell lysis reagent group and fluorescence reaction reagent group. The somatic cell lysis reagent group can specifically and effectively lyse the non-bacterial cells (somatic cells) in the sample and release ATP, and then remove it through the filter membrane at the bottom of the cuvette. The main function of bacterial cell lysis reagent group is to effectively lyse bacterial cells and release ATP for detection. At the same time, in order to avoid ATP degradation and affect the authenticity of the test results, bacterial cell lysis reagents should be able to quickly inactivate ATP degrading enzymes. The fluorescent reagent group includes fluorescein and fluorescein which can interact with bacterial ATP in the sample and emit fluorescence.
2. Sampler and sample pretreatment device This part includes sampler that can quantitatively obtain samples to be tested in different physical states, pressurizer that pressurizes ATP discharged from somatic cells, cuvette with membrane for reaction, cup holder for sample concentration and special concentrator.
3. Microphotometer Microphotometer, that is, miniature fluorescence detection instrument. According to the specific needs, the photometer not only has a screen for measuring and displaying fluorescence values, but also has an external interface combined with a personal computer or an automatic printer, so as to record and store relevant data for product quality or hygiene monitoring trend analysis.
Advantages of ATP rapid detection technology Generally speaking, the realization of rapid detection depends on the sound, light, magnetic and other signals that can be easily detected by the sample to be detected during the reaction. The rapid detection of ATP fluorescence is realized by generating fluorescence that is easy to detect. In the aspect of health monitoring, it has incomparable advantages over conventional detection. Both practice and literature data show that using the fluorescence value detected by the rapid detection system, that is, the total ATP value (ATP from bacteria and food residues) as a health monitoring index is much better than visual inspection or bacterial plate counting in terms of timeliness and actual effect. Therefore, some people think that the total ATP can be used as a criterion to judge the health status.
1. The total number of bacteria was detected by fast and accurate conventional plate counting method, even if the bacteria grew into colonies visible to the naked eye on LB medium. Due to the uneven dilution of samples, a colony is often composed of two or more bacterial cells, and LB medium is only suitable for the growth of aerobic or semi-aerobic bacteria. So we can only get the number of such bacteria. The ATP fluorescence rapid detection method can detect all bacteria, fungi and algae, and the real-time detection results can be obtained in 1-5 minutes, with simple operation and short time. The plate counting method takes 1-5 days, and the results are difficult to reflect the actual hygiene situation.
2. The sample size is small. Because luciferase is very sensitive to ATP, ATP below 0. 1 picogram molecule (10- 12 mole) can be detected by luminescence reaction, so it can be used to detect samples with low bacterial content. For example, the bacterial content in beer sterilized by 0.45um membrane filtration is very low, which can not be detected by conventional methods, but can be detected by ATP fluorescence concentration. Application of ATP Rapid Detection Technology "ATP Fluorescence Rapid Detection System" has been used for the whole process monitoring since 1990s. There are many products successfully developed and put on the market according to the principle of firefly luminescence. For example, the ATP fluorescence rapid detection system developed by Zhongke Maliang can be used to monitor the whole process of food, beverage and dairy products: before production and processing, it can be used to detect the cleaning and disinfection effect of production facilities and the total number of bacteria in raw materials; In the process of processing and production, it can be used for hygienic monitoring of key control points of production equipment (such as water supply and ventilation valves), which affects the detection of the total number of special microorganisms in dairy fermented drinks and wines; After processing and production, it can be used to detect the total number of contaminated bacteria in finished products. In addition, it can also be used for rapid detection of microorganisms (total bacteria) in various samples by law enforcement departments such as environmental protection, water administration, customs and quarantine. The application of ATP rapid detection in HACCP Through the study of food production line or surrounding sanitary environment, it is found that if the total number of bacteria at a certain point on the production line obtained by plate counting method is very small or sterile, but the ATP value obtained by ATP fluorescence detection method is very high, then a large number of microorganisms will easily breed at the later stage of the detection point. After analysis, it is considered that the total ATP value obtained by ATP fluorescence detection comes from residues and microorganisms. And in most cases, the proportion from microorganisms is very small. Therefore, the ATP value of ATP fluorescence detection can be used to judge whether the detection point will be polluted again, rather than the total number of bacterial colonies converted according to the ATP value of bacterial cells, but the key is to determine the ATP critical value of different detection points in different production lines. Based on this, developed countries have used ATP fluorescence detection in HACCP to evaluate whether the contact surfaces of food raw materials and cleaned and disinfected food raw materials meet the hygiene standards. Then focus on monitoring the key parts that are easy to cause microbial pollution. For example, through the rapid detection of ATP, it can be judged whether there is the possibility of pollution in the intermittent time from routine disinfection to new production; Then, the ATP value range which is not only in line with the situation of the unit, but also in line with the health requirements is worked out. Application of pocket ATP fast detector At present, the pocket ATP fast detector has been applied to all aspects of life, and its application in food is mainly to detect the sanitary condition of tableware in restaurants and trains. The Ministry of Railways of China decided to use the portable rapid detector of Shenyang Zhongke Maliang Bioengineering Co., Ltd. on the train before the 2008 Olympic Games to replace the indirect chlorine ion detection method for tableware hygiene inspection. In addition, the rapid detection of ATP based on luciferase can also be applied to other fields and uses, such as tap water detection, barreled water sterility detection, anthrax detection in military field diet, environmental monitoring, clinical detection and so on.