Backgrounds
Acute myocardial infarction is very common in Europe and the United States, with approximately 1.5 million people in the United States suffering a myocardial infarction each year. In China, the prevalence of the disease has increased significantly in recent years, with at least 500,000 new patients and 2 million existing patients each year. The number of people suffering from the disease in Europe and the United States is also increasing every year [1]. Contemporary data from a large national database show that the rates of mechanical complications are low in patients presenting with STEMI and NSTEMI. Post–myocardial infarction mechanical complications continue to be associated with high mortality rates, which did not improve during the study period. The overall mortality rate of the disease can be as high as 20%, and 50% of patients die suddenly after an acute heart attack. There are many complications associated with this disease, but if the disease is detected early and prevented in time, the prevalence and mortality rates can be greatly reduced [2,4]. Our team initiated a project to predict and determine whether a suspected patient is likely to have an acute myocardial infarction by detecting miRNA. We have developed a quick, simple, and inexpensive kit that can be used by more people to contribute to mankind. According to a survey, every year in our country, people over the age of 50 regularly go for initial screening for acute myocardial infarction, and we know there is a huge market for the kit we have developed.
Products and Usage Procedure
（一）
1. Melt the reagents in the kit.
2. Prepare 20 μl of the reaction system according to the instructions (note: on ice).
3. Mix the reaction system by flicking several times, centrifuge slightly (avoid vortexing), repeat 3 times, and incubate at 37°C for 30 minutes.
（二）
1. Prepare the second step of the reaction system according to the instructions and add the reaction products from the above steps.
2. mix by flicking several times, centrifuge slightly (avoid vortexing), and repeat 3 times.
3. Place the reaction tube at 37 °C for 45 min.
4. Take 10µL of nucleic acid amplification product and dilute 5 times with 40µL ddH2O in a centrifuge tube and mix well.
5. Put all the diluted reaction products on the nucleic acid test strip (or insert the test strip into the diluted product) and record the result in the reading window within 5~10 minutes.
6. After recording the result, seal the test strip and dispose of it in a safe place.
7. Judgement of results
schematic analysis
The colloidal gold will be modified with gene fragments of different hairpin structures, and different gene fragments will target different miRNAs, which will be combined with miRNAs to trigger the HCR isothermal amplification reaction, and at the same time, the reverse cutting function of CRISPR technology will be used to release the signals and the color reaction to complete the miRNA detection, which will unite the colloidal gold technology, HCR isothermal amplification technology and CRISPR reverse cutting function to establish a specific detection method for miRNA and predict whether a patient suffers from heart attack based on the detection results. [3, 5]
As shown in the figure by comparing with blank and other categories of miRNAs, after the HCR amplification reaction, different miRNA amplification products produce different degrees of signals after binding to Cas12a protein. Therefore, our experiment successfully verified the good specificity of the assay. cas12a protein and CrRNA can recognize and cleave miRNA amplification products to generate signals.
Safety
In our experimental work, we use only one type of bacterial cell in our experiments: the E. coli BL21 receptor cell, but E. coli is a pathogenic strain. Considering the environment, all materials contaminated with E. coli are sterilized and discarded in a biohazardous disposal bin. As for our final product, since it is a test kit, the finished product will not pose any danger to the human body, the kit mainly contains our constructed and purified Cas12a protein and supporting experimental reagents. The production process is in strict compliance with laboratory safety regulations. Regarding the marketing of our products, if we want to work with hospitals and clinics and promote our products, we have to consider the price of our products and their practicality and safety, theoretically, our kits are not harmful to the human body.
Target user
1. Hospitals and clinics: These kits can be provided to hospitals and clinics for rapid screening of patients suspected of having an acute myocardial infarction. In the emergency department and in the laboratory, doctors can use this kit to make an initial assessment of whether the suspected patient is having an acute myocardial infarction, in order to provide an effective treatment plan for the patient.
2. Pharmacies: Pharmacies can sell these kits to people at risk of acute myocardial infarction, such as middle-aged and elderly people and people with high blood pressure, diabetes, and high cholesterol. These people can use the self-test to find out if they are at risk of acute myocardial infarction so that they can take preventive measures in good time.
3. Community: Community service organizations, such as community health centers and local health authorities, can make these kits available to community residents. For people in the community with a history or high risk of cardiovascular disease, the Acute Heart Attack Test can be carried out on a regular basis to allow early detection and intervention of underlying conditions.
It should be noted that the Acute Heart Attack Test Kit can only be used as an initial screening tool and the results are for reference only. Suspected cases require further investigation and confirmation of the diagnosis in the hospital. If the kit is used for self-testing, the product instructions should be followed to ensure accurate results.
reach for the marketplace
After successful product development, the next step was marketing. For the go-to-market strategy, we created a detailed business plan and carried out product promotion activities in human practices, distributed brochures with information about our team and the experimental project, as well as keychains and fans with logos to attract the public, and promoted the product at community sites through the production of easy-to-open posters and other promotional activities. We educate doctors, patients, and the public about the function and use of the kits. After the kits have been launched, after-sales service is also required, including answering users' questions, handling complaints, and updating product information.
future plans
For our products, there are operating conditions as well as time control, and the operation is complicated. In the future, our kits will be updated so that complex operations such as the amplification reaction process can be performed independently in the kit, simplifying the operation process so that more people, such as the elderly, can operate it smoothly. For the existing products, we will increase the publicity and promotion of the kit through advertising, and promotional activities update the product iteration, continue research and development, and improve the kit, improve its accuracy and reliability to meet market demand. We will expand sales channels to cooperate with hospitals, provide after-sales service, answer users' questions, and finally formulate and implement reasonable marketing strategies. We have created a business plan and would like to share our business plan and marketing strategy. You can find our business plan on the startup page.
Reference
[1] Wang, X.; Mu, X.; Li, J.; Liu, G.; Zhao, S.; Tian, J., A novel nanoparticle surface-constrained CRISPR-Cas12a 3D DNA walker-like nanomachines for sensitive and stable miRNAs detection. Anal Chim Acta 2023, 1251, 340950.
[2] A. Elbadawi, I.Y. Elgendy, K. Mahmoud, A.F. Barakat, A. Mentias, A.H. Mohamed, et al., Temporal Trends and Outcomes of Mechanical Complications in Patients With Acute Myocardial Infarction, JACC Cardiovasc Interv, 12(2019) 1825-36.
[3] R. Bruch, J. Baaske, C. Chatelle, M. Meirich, S. Madlener, W. Weber, et al., CRISPR/Cas13a-Powered Electrochemical Microfluidic Biosensor for Nucleic Acid Amplification-Free miRNA Diagnostics, Adv Mater, 31(2019) e1905311.
[4] S.S. Dani, A.N. Lone, Z. Javed, M.S. Khan, M. Zia Khan, E. Kaluski, et al., Trends in Premature Mortality From Acute Myocardial Infarction in the United States, 1999 to 2019, J Am Heart Assoc, 11(2022) e021682.
[5] K. Wright, K. de Silva, A.C. Purdie, K.M. Plain, Comparison of methods for miRNA isolation and quantification from ovine plasma, Sci Rep, 10(2020) 825.