New SMC single molecule detection technology

Although many existing biomarker detection technologies have enriched people's understanding of biomarkers, acquired a large amount of data and experience, and solved many biological or medical problems, many bottlenecks as mentioned above still limit biomarkers. Application of the object. Especially for the early diagnosis or prevention of diseases, especially tumors, it is necessary to find the presence of biomarkers in samples such as serum/plasma that are easily available and far from the lesion during the incubation period or early stage of the disease. Studies have shown that the levels of these markers are likely to be as low as sub-picograms per milliliter. Almost all of the above techniques are powerless for this detection interval. Second, according to reports in the literature, about 75% of the known 400,000 human proteins, or 300,000, are difficult to detect by the prior art. A large number of biomarkers are under the detection limits of the prior art. Third, the prior art is subject to lower sensitivity in many cases, and only a certain biomarker can be measured in a disease individual, and at this time the disease may have entered the actual stage of occurrence. Although these markers are extremely low in healthy individuals or individuals at risk for disease, they are likely to have predictive effects, and the prior art cannot effectively utilize this. Fourth, medical researchers have obtained various types of body fluid samples, such as cerebrospinal fluid, cervical fluid, alveolar lavage fluid, tear fluid, urine, etc. These body fluid samples often have very low biomarker content, but they have The unique research value of a particular field, and the valuable biological information contained therein is often ignored by existing technologies. Therefore, there is an urgent need to develop more sensitive and specific biomarker detection techniques to better meet the research needs of basic or translational medicine.

Figure 5. Principle of single molecule detection technology

The birth of new technologies provides an important opportunity for the realization of these research needs. Single Molecule Counting (SMC) produces qualitative changes in antibody-dependent protein biomarker detection. The detection technology for single-molecule detection is represented by a single-molecule detection platform such as Erenna (also known as Singulex). The basic principle is to use a capillary to perform loading of fluorescein-labeled molecules, and to perform single-molecule excitation detection by laser focusing. . When a fluorescein-labeled molecule passes through a high-energy laser focus, the photoflash signal emitted by a single protein molecule-conjugated fluorescein is measured by a detector. The number and intensity of the light scintillation signal are positively correlated with the molecular concentration, so that a standard curve can be established. The concentration of the solution can be quantitatively detected by counting the light scintillation signal within a certain period of time (Fig. 5). The origin of this technology is controversial. Many scholars used single-molecule detection technology to perform single-molecule detection on different molecules in the same period, and achieved success. Representative work includes: In 1996, Chen and other scholars successfully carried out single molecule detection of phycoerythrin; in the same year, Craig and other scholars used single molecule detection technology to detect alkaline phosphatase; in 1998, Fister and other scholars conducted Single molecule detection of rhodamine 6G molecule. ^[3-5]

For the detection of protein biomarkers, the single-molecule immunoassay kit still uses a typical double-antibody immunological sandwich method. Unlike the common ELISA method for uniformly irradiating the 96-well plate bottom, the SMC technology kit dissociates the molecules forming the double-antibody immunosand complex, and then uses the single-molecule detection method to measure the fluorescein-labeled secondary antibody. Figure 6). The biggest advantage of this technology is that the sensitivity is significantly improved. In practice, the sensitivity of a few picograms per milliliter is often achieved (Fig. 7), which is roughly equivalent to about 1000 times the normal ELISA.

Figure 6. Experimental flow of single molecule immunoassay

Figure 7. Sensitivity comparison of antibody-dependent biomarker detection techniques (cited from references)

Compared with the traditional detection techniques reviewed above, the application of SMC single molecule detection technology is at a high speed. Despite the general optimism in the industry, the changes that this technology brings to biomarker testing still take time to prove. This technology has shown very important value in the case of Troponin I (cTnI) detection. Unlike traditional techniques that only detect cTnI factors in individuals with heart disease, this technique measures trace amounts of cTnI in healthy individuals. The 12-year study showed surprising findings (Fig. 8 ^[6] ) that the background cTnI of healthy individuals is inversely proportional to the cumulative incidence of future heart disease. In another high-level study, trace amounts of mutant mHTT protein in the cerebrospinal fluid of Huntington's disease-free individuals (mutant gene carriers) are stably measured, providing a new dimension for the diagnosis and treatment of Huntington's disease. Scheme ^[8] .

Figure 8. Single-molecule detection technology for detection of cTnI in healthy individuals predicts cumulative incidence of heart disease

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