Acute myocardial infarction is a common disease in Europe and the United States. In China, the number of people suffering from the disease has increased significantly in recent years, with at least 500,000 new cases and at least 2 million existing patients each year. Despite the high prevalence, commercially available tests are expensive and time-consuming. Worldwide, coronary heart disease causes about 170,000 deaths per year. China has shown a significant increase in mortality in recent years [1]. Acute myocardial infarction (AMI) has many negative effects on humans, along with complications, leading to arrhythmias, heart rupture, heart failure, etc., and death if the symptoms are severe; to date, there are many deaths due to acute myocardial infarction disease, with a lethality rate of about 20%. The early stages of the disease, if left untreated, pose a serious threat to the health and life of the patient.

Introduction to Acute Myocardial Infarction

define

Acute myocardial infarction is a disease caused by acute, persistent ischemia and hypoxia of the coronary arteries.

This condition persists for more than 30 minutes with a crushing or suffocating sensation in the precordial region, and the patient has a feeling of near death. The clinical presentation is usually severe and persistent retrosternal pain not relieved by rest and nitrates, accompanied by elevated serum cardiac enzyme activity and progressive electrocardiographic changes, which may be complicated by arrhythmia, shock, or heart failure and is often life-threatening. Precursors: Some patients may have symptoms such as fatigue, chest discomfort, palpitations, shortness of breath, irritability, and precordial pain for several days before the onset of the disease.

The cost of detecting and treating this disease is very high, even in areas with a high level of economic development in health care. At the diagnostic stage, the testing process is complex, the testing equipment is expensive, and most of the methods are also extremely demanding, technically challenging, and risky, while simple tests take more time and are less sensitive, making it impossible to accurately determine whether a patient has the disease or not. To address these shortcomings, a rapid, convenient, and reliable test for acute myocardial infarction is urgently needed. We have designed a method for early specific detection: a kit for miRNA detection, which is a colloidal gold-modified gene fragment with different hairpin structures targeting different miRNAs to trigger an amplification reaction, and adding the amplification product to the kit can determine whether there is a possibility of myocardial infarction disease. This kit can help more people to check their health condition in time, and also reduce their financial burden so that more people can enjoy better medical services.

Our Solutions

miRNAs as biomarkers

The treatment of acute myocardial infarction has also attracted increasing attention in recent years. After suffering an acute myocardial infarction, cardiomyocytes respond to extracellular stimuli from ischemia, which can then activate a variety of pathological gene programs and reprogramme gene expression. Some miRNAs, such as miRNA-133a and miRNA-499, play a key role in regulating overall cardiac function. Plasma levels of miRNA-133a have been reported to increase dramatically after the onset of AMI symptoms and return to normal levels within 15 hours. As these specific miRNAs have been shown to be critical in regulating important cardiomyocyte activities after an acute myocardial infarction outbreak, abnormally high levels of them in the circulating blood are often considered a marker of acute myocardial infarction, especially in its early stages. Therefore, the detection of miRNAs associated with AMI in human blood samples is of particular concern. Although strategies and techniques have been developed to detect miRNAs associated with various diseases [1, 6].

We have designed a kit that can detect miRNA expression to predict heart attack. By taking blood to extract total RNA using the kit it will be possible to detect whether the suspected patient is at risk of developing heart attack disease, the kit requires some technical manipulation before use, it is a gene fragment targeting different miRNAs to trigger an amplification reaction, and finally the amplification product will be dripped into the kit to determine this. This would provide a rapid, proactive, and immediate method of assessing risk, allowing early treatment and potential disease reversal [1]. This experiment targets different hairpin structures of gene fragments on colloidal gold modification to different miRNAs [1-2], which bind to the miRNAs and trigger the HCR isothermal amplification reaction, and simultaneously use the reverse cleavage function of cas12a to occur the chromogenic reaction to complete the detection of miRNAs [3]. Our project combines colloidal gold technology, HCR isothermal amplification technology, and the function of CRISPR reverse cleavage to establish a specific detection method for miRNA [4-5]. These antibodies can be integrated into easy-to-read test strips to detect the presence of miRNAs in blood. Our product will provide a much-needed low-cost early detection method for heart attacks, improving healthcare worldwide, especially in resource-poor rural communities.

A CRISPR-Cas12a system was developed, comprising of Cas12a and crRNA vectors [6]. To acquire the Cas12a protein, we created a Cas12a plasmid and transferred it into BL21 E. coli colonies. After cultivation, the Cas12a protein was purified and extracted. Our Cas12a protein acted as a reverse cleavage in the assay, triggering the HCR amplification reaction by colloidal gold targeting various gene fragments corresponding to different miRNAs [7]. Under the action of Cas12a, the corresponding fragments of the amplification products are reverse-cut, and the color reaction occurs, completing the detection of miRNAs [4, 8].

It is finally expressed in the form of a kit:

Negative: the T-line is not colored, indicating that the reporter molecule has not been cleaved by Casase and Casase has not been activated; Positive: the T-line is visible to the naked eye and is considered positive, indicating that the nucleic acid probe has been cleaved by Casase and Casase has been activated;