Development of nanogold as an immunomarker
As one of the four major modern labeling techniques, nanogold labeling techique is essentially an encapsulation process in which macromolecules, such as proteins, are adsorbed onto the surface of nanogold particles. The adsorption mechanism may be the negative charge on the surface of the nanogold particles, and the positive charge group of the protein due to electrostatic adsorption and the formation of a solid combination, and adsorption will not make the biomolecule denaturation, due to the gold particles with high electron density characteristics, in the gold labeling of the protein binding, the black-brown particles can be seen under the microscope, and when these markers are in a large number of aggregation of the corresponding ligands, the naked eye can be seen as a red or pink spots, and thus used for qualitative or semi-quantitative rapid immunoassay methods. Since the spherical gold nanoparticles have a strong adsorption function for proteins, they can bind to staphylococcal A proteins, immunoglobulins, toxins, glycoproteins, enzymes, antibiotics, hormones, bovine serum albumin, and other non-***valent binding, and thus have become a very useful tool in basic research and experiments.
1.1 As a microscopic tracer
In 1978, Geobegan et al. used nanogold-labeled antibodies for the detection of B-lymphocyte surface membrane immunoglobulin under ordinary light microscopy, and established immunogold staining at the light microscopic level (immunogold staining, IGS). 1981, Danscher used silver development to enhance the visibility of gold particles and improve the visibility of gold particles. The visibility of gold particles and improved the sensitivity.Holgate et al. established immunogold-silver staining (IGSS) for the visibility of gold particles at the light microscopic level using silver developer in 1983.Using the enhancement effect of silver to increase the radius of the visible particles of individual gold particles at the light microscopic level, increased the small-grained gold particles' In 1986, Fritz et al. successfully carried out the color IGSS method based on the IGSS method, which made the results more vivid and eye-catching. Nevertheless, the application of nanogold in light microscopy is decreasing due to the fact that silver nitrite compounds are photosensitive and need to be labeled in a dark room, which is very inconvenient for experimental operation, and the price of switching to non-photosensitive silver acetate compounds is too expensive. And the use of nanogold's high electron density, can clearly distinguish particles under the electron microscope, as in the transmission electron microscope (TEM), scanning electron microscope (sEM) and fluorescence microscopy tracer in the electron microscope immunochemistry and histochemistry has been widely used.
1.2 Application to homogeneous sol particle immunoassay
Homogeneous sol particle immunoassay (SPIA) is the use of immunological reaction of gold particles cohesion leads to the principle of color fading, the nanometer gold and antibody binding, the establishment of micro-agglutination test to detect the corresponding antigens, such as indirect hemagglutination, the same. The agglutinated particles can be directly observed with the naked eye. It has been successfully applied to the detection of PCG, and directly applied to the quantitative analysis by spectrophotometer.
l.3 Application to flow cytometry
Applying fluorescein-labeled antibodies to count and analyze cell surface antigens by flow cytometry (FCM) is one of the most important techniques in immunological research. However, due to the overlapping spectra of different fluorescein, it is difficult to distinguish different markers.Boehmer et al. found that nanogold can significantly change the scattering angle of the red laser, using nanogold-labeled goat anti-mouse Ig antibody applied in flow cytometry to analyze the surface antigens of different types of cells, the results of the nanogold-labeled cells in the wavelength of 632 nm, the 90-degree scattering angle can be enlarged more than 10-fold and at the same time does not affect the cell activity. Moreover, it is labeled with fluorescein*** and does not interfere with each other. Therefore, nanogold can be used as an effective marker for multiparameter cell analysis and sorting, analyzing various cell surface markers and cell inclusions.
1.4 Application to spot immunogold and silver staining technique
Dot-IGS (IGSS) is a method that combines spot ELISA with immunogold. The protein antigen is directly spotted on the nitrocellulose membrane, reacted with the specific antibody, and then drop the nanogold-labeled secondary antibody, as a result, gold particles aggregation occurs at the antigen-antibody reaction, forming red spots visible to the naked eye, which is known as spot immunogold staining (Dot-IGS). This reaction can be enhanced by silver developing solution, i.e., spot gold-silver staining (Dot-IGS/IGSS).
1.5 Application to immunoblotting
Immunoblotting (IBT), also known as immunotransfer technique, is based on the principle that the molecular weight of various antigens of different sizes, walking in electrophoresis at different speeds, and therefore occupy different positions on the nitrocellulose membrane; serum containing specific antibodies and the reaction of this film. Then the specific antigen-antibody reaction will show color. Compared with the enzyme labeling immunoblotting technique, the nanogold immunoblotting technique is simple, fast, and has a fairly high sensitivity. Moreover, the application of nanogold stains the unreacted antibody on the nitrocellulose membrane, evaluates the transmembrane efficiency, corrects the optical density curve of the antigen-antibody reaction, and then performs a quantitative immunoblotting assay.
1.6 Application to spotted gold immunofiltration assay
Spotted gold immuno-gold filtration assay (DIGFA) is one of the spotted immunoboding assays (DIBA), which was developed by Hawkes et al. in 1982. DIGFA is one kind of dot immunoboding assay (DIBA), which is a new immunological technique improved and developed on the basis of immunoblotting technique by Hawkes et al. in 1982. The principle is exactly the same as the spot immunogold staining assay, except that there is a pad of highly absorbent matting under the nitrate fiber membrane, which is the filtration device. After adding the antigen (antibody), the antibody (antigen) is added quickly, and then the gold-labeled second antibody is added. Due to the osmosis device, the reaction is very fast, and the color reaction can be shown within a few minutes. Compared with the spot immunotietration assay (d o t immunotietration assay, DIFA), the difference is that it is free of adding substrate solution, and the color is developed directly by the red colloidal gold probe, the result is vivid, the background is clearer, and it can be stored at room temperature. This method has been successfully applied to the examination of human immunodeficiency virus (HI) and the detection of alpha-fetoprotein in human serum. Currently in use are HCG kits, AFP kits, and GI tumor screening kits.
1.7 Application of immunochromatography
Immunochromatography assay (gold immunochromatography assay, GICA) is a variety of reaction reagents in the form of strips fixed in the same test strip, to be examined specimen is added to the end of the test strip, a reagent will be dissolved, through capillary action in the filtration of chromatography strips, moving and with the membrane on another reagent contact, the sample will be dissolved, and then filtered, moved and contacted with the membrane. After a reagent is dissolved, it percolates through the chromatographic strip by capillary action, moves and comes into contact with another reagent on the membrane, and the substance to be tested in the sample reacts with the receptor (e.g., antigen or antibody) directed against the substance to be tested on the chromatographic material in a specific immune reaction. During the chromatographic process, the immune complexes are trapped and accumulate in a certain area of the chromatographic material (the detection zone) and are visualized by visually detectable nanogold markers. The free marker crosses the detection zone and is automatically separated from the bound marker.GICA is characterized by a single reagent, one-step operation, and all reagents can be stored for a long period of time at room temperature. This new method advances the nanogold immunoassay test to a new stage.
1.8 Biosensor
Biosensor (biosensor) refers to the device or apparatus that can sense (or respond to) biological and chemical quantities and convert them into usable signals (including electrical signals, optical signals, etc.) output in accordance with a certain law. In terms of biosensors, nanogold is mainly designed as an immunosensor, which is designed to take advantage of the electrochemical changes caused by the specific binding of antigens and antibodies in living organisms. In addition, due to the redox potential of nanogold is +1.68V, has a very strong ability to capture electrons, can greatly improve as the determination of blood glucose biosensor glucose oxidase membrane activity, the finer the gold particles, the greater the activity.
1.9 Biochip
Biochip is a membrane, glass, silicon and other solid-phase media as a carrier, and its biggest advantage lies in the high throughput, parallelization, miniaturization. One experiment can detect multiple or more biological samples at the same time. Biochips include gene chips, protein chips, cell chips, and tissue chips. Currently, the application of biochips for food safety testing mainly includes pesticide and veterinary drug residue detection, food microbial detection, animal disease monitoring, genetically modified animal and plant detection, etc. In 2002, Park et al. introduced a new type of gene chip based on charge detection with nanogold as probe in Science, which has very good sensitivity and specificity and can detect single samples at a rate of 1 in 100,000, and can detect single samples at a rate of 1 in 100,000, and can detect single samples at a rate of 1 in 100,000, and can detect single samples at a rate of 1 in 100,000, which is very high. one-in-100,000 ratio to detect gene fragments with single-base mutations.
Application of nanogold technology in rapid food safety testing
At present, chemical analysis (CA), thin-layer chromatography (TLC), gas chromatography (GC), and high-performance liquid chromatography (HPLC) are generally used in food testing and analysis, but they require cumbersome and time-consuming pre-treatments, and the loss of the samples is also large. Compared with CA and TLC methods with lower sensitivity, GC and HPLC have higher sensitivity, but the operation technology requirement is high, the instrument is expensive, and it is not suitable for on-site rapid determination and popularization, while the detection technology with nanogold as the immuno-marker is making up for the shortcomings of these technologies, and it is increasingly used in the modern food analysis and detection.
2.1 Veterinary drug residues
The so-called veterinary drug residues refer to the residues of parent compounds of veterinary drugs and, or their metabolites, and impurities related to veterinary drugs in any edible part of animal products. Veterinary drug residues include both the original drug and the metabolites of the drug in the animal body. The main residual veterinary drugs are antibiotics, sulfonamides, furans, anticoccidials, hormones and dewormers. Veterinary drugs usually bring about contamination of foodstuffs through their use in the prevention and treatment of animal diseases, their use in feed additives and their introduction into food preservation. Long-term intake of animal food containing veterinary drugs will not only produce toxic effects on the human body and allergic reactions, but also drug-resistant strains of bacteria in the animal body can be transmitted to the human body, and when the human body has a disease, it will bring certain difficulties to the treatment of infectious diseases in the clinic and delay the normal treatment. In addition some residues are teratogenic, carcinogenic and mutagenic.
Verheijen used colloidal gold to label the purified anti-streptomycin monoclonal antibody, the detection limit of streptomycin is 160ng/ml, the detection is convenient and rapid, no other reagents and instruments, the time required is only lOmintl41. The use of colloidal gold immunochromatographic test strips in the detection of shrimp meat and other tissues in the specimen of the residual chloramphenicol ( chloramphenicol, CAP) residues in tissue specimens such as shrimp meat, the sensitivity can reach lng/ml in only 5-10min, and there is no cross-reactivity with analogs.Yong Jin et al. also used the gold standard method for the detection of neomycin residues in animal plasma and milk, and the detection limit was 10ng/mltl6J.Clenbuterol hydrochloride, the β2 receptor stimulant, is commonly known as Clenbuterol hydrochloride is a β2 receptor stimulant, commonly known as "leptin", which can enhance lipolysis and slow down proteolytic metabolism, and if used in livestock production, it can significantly increase the feed conversion rate and the lean meat rate; however, the use of too large a dose will have serious toxic side effects on the liver, kidneys, and other organs of the animal and human (indirectly). Although the European Union in 1996 banned the use of the drug in livestock production (EC Direc. tive 96/22/EC), China's Ministry of Agriculture also banned in 1997, but the domestic "leptin" poisoning incidents occur from time to time. Liu see the use of gold standard test paper method for rapid detection of clenbuterol hydrochloride, the minimum detection amount of 40ng/ml. now commercialized test strip products are now more mature, Belgium UCB Bio-products company developed the Tlhe Beta STAR test method is a specific β-lactam receptor is fixed in the test strips, colloidal gold colored particles as a marker, can be detected within 5min to detect penicillin. Penicillin and cephalosporin residues can be detected within 5min. And Ping Liu in China, in the detection of penicillin residues in milk with bioelectrochemical sensors, believes that the use of nanogold will help to improve the detection limit of the sensor.
2.2 Animal Infectious Diseases
Animal infectious diseases not only affect the economy of animal breeding, but also pose a threat to human health, the Food and Agriculture Organization of the United Nations and the World Health Organization have taken the prevention and control of serious animal epidemics as one of their priorities. Shrimp white spot virus (white spot syndrome virus, WSSV) is a major obstacle to the development of shrimp aquaculture industry, so far there is no effective drug, so the early detection of the virus, it is particularly important. Wang Xiaojie et al. have successfully studied the spot immunogold filtration assay (DIGFA) t19~ and the gold-standard paper method for the detection of shrimp white spot virus, of which the gold-standard paper method for the detection of white spot virus. The detection limit of the gold standard test strip method is 1 μg/ml, while the use of silver enhancement, can reach 0.0l μg/ml. Lai Qingjin et al. used the gold standard test strips to detect swine fever virus, 10 ~ 15min can be detected, and can be based on the results of the test to guide the reasonable immunization of swine fever and the establishment of appropriate immunization procedures. Avian influenza virus (AIV) is a virulent, viral infectious disease that causes acute death in poultry and can infect humans. Many regions of China have reported the occurrence of highly pathogenic avian influenza, which has caused significant economic losses to the poultry industry and seriously threatened human health. Liu Yongde et al. purified rabbit antibodies against avian influenza H5 and H9 subtypes of viruses, respectively, with the preparation of colloidal gold developed into an immunogold probe, with a modified osmosis method for safe and rapid detection of avian influenza H5 and H9 subtypes of viruses in the material being examined, the results can be obtained in 3min, the sensitivity of the detection of 1.62ug / ml and 1.25μg / ml, respectively.
2.3 Pesticide Residues
Difficulties in the analysis of pesticide residues include: complex background of the sample matrix, cumbersome pre-treatment process, which requires more time, low concentration of the measured components, limited qualitative ability of analytical instruments, insufficient instrumental sensitivity and a series of other problems, but the use of gold-labeled rapid detection can be a good solution to the above problems. Domestic Wang Shuo used nanometer gold immunochromatography and nanometer gold filtration method to detect carbaryl residues, the whole process of detection only takes 5min, and the detection limit reached 100ug/L and 50μg/L. Domestic biotechnology companies have also developed mature commercialized products, such as Kepavir pesticide residue rapid test strips.
2.4 Pathogenic microorganisms detection
Currently, the rapid detection of gold-based labeling research in pathogenic microorganisms is more, the detection of more types. The earliest Hasan to immunomagnetic separation technology based on immunocolloid gold technology has been successfully applied to the detection of 01 group of Vibrio cholerae (Vibriocholerae). Hong Bangxing et al. studied the oligonucleotide microarray technology with nitrocellulose membrane as the carrier of nanometer gold coloration, which provides the possibility of rapid and simple identification of pathogenic bacteria at the molecular level, and can even detect the drug-resistant variants of pathogenic bacteria. The microarray technology is highly sensitive and specific for 10 species (genera) of Escherichia coli, Salmonella, Shigella, Vibrio cholerae, Vibrio parahaemolyticus, Proteus, Listeria monocytogenes, Bacillus cereus, Clostridium botulinum and Campylobacter jejuni, and the level of detection can be as high as 10CFU/mlt251. Yin Chungguang et al. have demonstrated a rapid detection of Escherichia coli by using the integrated handheld Spreeta TM SPR When using the integrated handheld Spreeta TM SPR sensor for the rapid detection of E. coli, the introduction of colloidal gold complex antibody as a secondary antibody increased the quality significantly, further expanding the detection signal, and at the same time, prolonging the binding process between the colloidal gold complex antibody and microorganisms, so that the detection signal is further stabilized and amplified, thus significantly improving the detection accuracy, and increasing the detection precision of the sensor for E. coli from 10 6 CFU/ml to 10 1CFU/ml. ml. Gold immunodiafiltration of one of the important foodborne pathogens Escherichia coli 0157: H7, the current test is usually first sorbitol MacConkey agar (sMAC) for initial screening, and then biochemical and serological tests to do the identification of the general need for 24 ~ 48h, while the use of colloidal gold immunodiafiltration detection is very simple, in a short period of time can be obtained results.
In the rapid detection of pathogenic bacteria in the gold standard test strips are becoming more and more widely studied. Xie Zhaocong et al. application of colloidal gold immunochromatography detection of Vibrio cholerae in aquatic products in the study, the bacterial enrichment of Vibrio cholerae content of 1CFU / ml, through the bacterial enrichment of 12h, can be applied to colloidal gold immunochromatography diagnostic reagents detected, and the general detection of Vibrio cholerae in aquatic products used in the traditional conventional method, the detection of time limit is long, enrichment of the bacteria culture takes 8 ~ 16h, separation culture takes 14 ~ 20h, preliminary report takes more than 30h, the actual operation of the test. The preliminary report needs more than 30h, and in practice, it needs more than 3d to produce the report. Salmonella enterobacteriaceae can cause human salmonella food poisoning, Wang Zhongmin et al. used the immunofiltration method can detect 85% of the food poisoning caused by Salmonella, the sensitivity of 2.4 × 107CFU / ml, for the most common murine typhoid fever, swine cholera and Salmonella enteritidis, the detection rate of 100%, and the use of colloidal gold immunochromatographic method of sensitivity 2.1 × 106CFU / mlt30j. /mlt30j. By the United States as one of the seven major foodborne lethal pathogens Listeria monocytogenes, if in accordance with the traditional isolation and culture and identification techniques require l ~ 2 weeks, and the use of immune colloidal gold chromatography can be obtained in just 10min to detect the results of the sensitivity of 87.5%.
2.5 Detection of mycotoxins
Mycotoxin (Mycotoxin) is a toxic secondary metabolite produced by fungi (Fungi), which is widely present in food and feed, and if human beings accidentally consume contaminated food, they will be poisoned or induced by a certain disease, or even cancer. Detection of mycotoxins in food is often done by physicochemical or biological methods. However, physicochemical methods require expensive instruments and equipment and are complicated to operate. The use of immunological techniques for the detection of mycotoxins is highly sensitive and specific, which is very suitable for the detection of food samples.D.J.Chiao et al. used gold standard immunochromatography to detect 50ng/ml of botulinum toxin B (BoNT/B) within 10min, and if silver enhancement is used, the detection limit can be up to 50pg/ml and there is no cross-reactivity for botulinum toxins of type A and E. The detection limit can be reached to 50pg/ml if silver enhancement is used, and the detection limit can be reached to 50 pg/ml. Cross-reactivity. Raccoon mycotoxin is a class of mycotoxins produced by Aspergillus and Penicillium, of which the most toxic, the closest relationship with human health, the heaviest contamination of crops, the most widely distributed is ochratoxin A (OTA), Lai Weihua, et al. developed ochratoxin A rapid detection of colloidal gold test strips, the limit of detection reaches 10 ng / ml t331, much lower than the current China's ochratoxin limit requirements 5 The rapid detection of aflatoxin B z has also been researched in China, and the gold standard immunoassay strips for aflatoxin B developed by Sun Xiulan have a minimum detection limit of 2.5 ng/ml, which can qualitatively or semi-quantitatively detect the aflatoxin B content in food.
Conclusion
With the continuous development of science and technology, food analysis and detection technology is also constantly updated, improved and rapidly developed, especially rapid detection technology is more adaptable to the modern high efficiency, fast pace and meet the requirements of society. Instrumental analysis method can ensure the precision and accuracy of data, but its process is still relatively cumbersome. Although the development process of immunoassay with nanometer gold as the marker and other rapid detection technologies need to invest more money and longer time, they have the advantages of simplicity, rapidity, high sensitivity, high specificity, inexpensive, and less samples required, etc. The sensitivity is consistent with the conventional instrumental analysis and suitable for on-site screening, and the gold immunochromatographic technology among them is developing towards the direction of quantitative, semiquantitative detection and multi-dimensional detection. More reflect the advantages of gold standard technology. In short, the rapid detection technology of rapid, sensitive, simple and other advantages, so that it has a wide range of food hygiene and quarantine and environmental testing in the application of value and prospects for development.
Applications
Colorant for food, glass and organisms.
Used in genetic identification technology.
Used in the refining of environmental purification products.
Used as a preservative for food and cosmetics.
Added to cosmetics to play the role of whitening, anti-aging, moisturizing.
Produces antibacterial, antimicrobial, anti-inflammatory drugs, medical devices, health care products, beauty care devices.
Production and people's lives are closely related to all kinds of daily necessities, food, drinks and so on. Such as nano gold soap, toothbrush, a variety of beauty masks.