Professor Meng Liu - Principles and Advancements of Single-Molecule Detection

Introduction: Professor Meng Liu is a doctoral supervisor and the dean of the School of Environment at Dalian University of Technology.

Field: He mainly works on the rapid analysis and detection of new pollutants and pathogens. He is involved in the development of fast diagnostic reagents, instruments, or products that meet the analytical and detection needs of "fast, accurate, simple" for environmental agencies, businesses, and medical institutions.

Why: As an expert in the field of biomacromolecule detection and biosensors, He showed a keen interest in our project. He took a detailed look into our ideas and introduced us to the principles and research progress of single-molecule detection, further clarifying our direction.

Takeaway: We established the idea of a biomolecular detection sensor based on the protein cage. By using the 60 sites on the protein cage and introducing a high ratio of eGFP fluorescent reporter molecules to illuminate the protein cage, along with the introduction of 1 to several specific antibody molecules using immunological recognition to capture biomarkers, this will achieve efficient detection of specific biomolecules.

Associate Professor Cheng Yang - Application of Aptamers

Introduction: Associate Professor and master's supervisor at Dalian University of Technology, College of Chemistry, Department of Analytical Chemistry.

Field: Carbon-based flexible sensors, nucleic acid aptamer biosensors.

Why: Although we had determined the distribution of the 60 sites, we still had questions about how to design the sites that capture biomarkers. Through literature review, we found that aptamers might be a solution to this problem. Hence, we decided to discuss with the analytical chemistry expert, professor Yang, to confirm if aptamers could be applied.

Takeaway: Aptamers can be used for the specific recognition of nucleic acid and protein biomarkers. However, aptamers specifically recognize target molecules through their secondary or tertiary structures to achieve specific interactions. It's challenging to develop a universal module, as well as in vitro screening. Therefore, we decided to give up on this approach.

Professor Meng Liu - Second Feedback

Why: Having learned that Professor Liu has expertise in the creation of paper strip tests, we consulted him again to understand the feasibility of semi-quantitative detection on the test strip. After a detailed discussion, he affirmed our idea and provided suggestions on how to implement the experimental plan.

Takeaway: We settled on the idea of producing a test paper with multiple "T" lines to achieve semi-quantitative detection, which is sequentially reducing the number of protein cages loaded based on the direction of the sample, aiming to achieve gradient detection.

Dr. Wen Sun - Solutions to Fluorescence Intensity Detection Issues

Introduction: Dr. Wen Sun is a researcher and doctoral supervisor at the State Key Laboratory of Fine Chemical Engineering at Dalian University of Technology. He currently serves as the Assistant Dean of the College of Chemical Engineering, Assistant Director of the Education Ministry's Frontier Science Center for Intelligent Materials in Chemical Engineering, and Assistant Director of the State Key Laboratory of Fine Chemical Engineering.

Field: Focusing on key scientific issues related to environmental and life sciences for analytical testing and tumor therapy, conducting research centered on functional dyes used for disease detection and photo-driven therapy.

Why: Our decision to utilize our device in community hospitals and homes means that our fluorescence detection equipment may not have the same accuracy as the large-scale devices in major hospitals and laboratories. This led us to question whether the fluorescence would be visible to the naked eye.

Takeaway: Dr. Sun is an expert in fluorescence molecule design, and he believes that the fluorescence of GFP is very stable. However, only when particle size reaches 500nm can it be observed with the naked eye. Based on this discussion, we determined that our approach is feasible. Combined with appropriate hardware tools and microarray technology, we could potentially achieve our testing goals more effectively. Also, considering that using RFP requires a more complex optical path and there isn't a suitable detection condition currently available, we decided to revert to using eGFP.

Dr. Yiheng Yang - Understanding Cardiovascular Diseases

Introduction: Dr. Yang is a cardiovascular specialist at the First Affiliated Hospital of Dalian Medical University. He also oversees post-treatment follow-up statistical surveys.

Why: The Cardiovascular Disease Diagnosis and Treatment Center affiliated with Dalian Medical University is a regional medical center for cardiovascular diseases in Liaoning Province. To ensure the scientific validity and universality of our project, we sought assistance from Dr. Yang, who has hands-on experience and extensive knowledge of the post-treatment outcomes of cardiovascular disease patients.

Details: During our discussions, we learned the following:

1. Current clinical detection equipment can conduct parallel tests and deliver results within half an hour, but it requires large equipment.

2. Most post-treatment follow-up studies for cardiovascular diseases involve large sample sizes, often between 5,000 to 10,000 participants. The prognosis for most cardiovascular diseases is generally poor. Phone follow-ups require careful time management and communication skills, limiting the type of questions that can be asked. This method of collecting data doesn't provide a comprehensive understanding of an individual patient's condition.

3. The hospital specializes in treating severe and rare cases. Especially for heart failure patients, the prognosis is generally poor, and they need long-term medication. Such patients have high rates of readmission, are more cooperative during follow-up calls, and tend to monitor their health more regularly.

4. Treating cardiovascular diseases can be expensive. Heart failure treatment, in particular, is even more so because patients with heart failure have high readmission rates, with each admission costing around 10,000 yuan. This places a heavy financial burden on families and uses significant medical resources.

5. Heart failure, as the final fortress of cardiovascular diseases, requires lifelong medication, and the dosage needs constant adjustments, demanding comprehensive management for chronic diseases throughout the entire process – from pre-hospitalization to in-hospital treatment, and post-hospitalization. Currently, while patients are in the hospital, doctors can adequately monitor and manage their condition continuously. However, early warnings before hospitalization and self-monitoring after discharge remain largely unaddressed. Patients lack a simple and feasible auxiliary monitoring method to provide early warnings, allowing them to adjust their lifestyle and medication dosage timely, and seek medical attention as early as possible. This can lead to a reduction in re-hospitalization rates, significantly improving the quality of life and long-term prognosis, ultimately conserving medical resources and medical insurance funds.

6. The immediate need for both cardiovascular doctors and patients is to improve the patient's ability to self-monitor their condition, thereby enabling timely intervention. This can reduce rehospitalization rates significantly. After getting to know our project in detail, Dr. Yang expressed high expectations. He indicated that our project addresses a current pain point in the management of heart failure patients. It can provide a straightforward solution for daily home laboratory tests for heart failure patients, making it highly valuable and practical for frontline clinical use and community chronic disease management.

Takeaway: Through our discussions with Dr. Yang, we gained a profound understanding of the severity and implications of heart failure. Recognizing the immense detrimental impact of this disease and the critical need for early detection, we decided to focus our project on heart failure as a case study. By doing so, we aim to explore the practical value of our project in real-world diagnostic scenarios, identify potential challenges, and uncover avenues for further optimization. The firsthand insights from frontline healthcare professionals provide invaluable guidance, ensuring our efforts are tailored to address the most pressing needs of the patient community.

Dr. Xin Zhao : Understanding Heart Failure

Introduction: Dr. Xin Zhao is the Director of the Cardiology Department at the Second Affiliated Hospital of Dalian Medical University, Director of the Cardiovascular Hospital, Director of the Heart Research Institute, and Head of the Cardiac Care Unit (CCU) Ward.

Why: We wanted to gain insight into the needs of the medical community and the current state of testing in hospitals. So, we reached out to Dr. Zhao Xin. We believe that someone like Dr. Zhao, who possesses extensive theoretical knowledge and practical experience, can provide valuable insights into the actual conditions of heart failure and the feasibility of our project. Additionally, we hope that Dr. Zhao, from a professional medical perspective, can offer directions and recommendations for our educational outreach efforts.

Details: During our discussions, we learned the following:

1.In the cardiology department, the incidence rate of heart failure is relatively high, reaching around 30%. Not only are there heart failure patients within the cardiology department, but also throughout various departments in the hospital. Additionally, heart failure patients are distributed across departments throughout the entire hospital.

2.We confirmed that NT-proBNP can serve as our target substance. We also understood that its concentration varies from person to person, and relying solely on NT-proBNP can only provide insight into the individual's heart condition.

3.Heart failure is categorized into four levels based on severity. Presently, patients arriving at the hospital are generally at level two or above, often seeking medical attention due to symptoms such as angina.

4.If our project can collaborate with artificial intelligence, it could potentially enable personalized, scientifically-based guidance through simple testing in community hospitals or even at home. This has the potential to significantly enhance the overall health of the population and save a substantial amount of healthcare resources.

Takeaway: Both heart failure and other cardiovascular diseases are showing a trend towards affecting younger individuals. Early detection is of great help in treatment. This further validates the necessity and feasibility of applying our project to early detection of heart failure. We have also taken in Dr. Zhao's advice, and in the future, collaborating with hardware and AI is a goal we will strive towards.

Professor Jingnan Cui: Try the Existing Portable Detection Technology

Profile: Professor and doctoral supervisor of the State key Laboratory of Fine Chemical Industry of Dalian University of Technology; mainly engaged in fluorescence analysis technology in food safety and environmental pollution in the rapid detection of trace harmful substances and fluorescence probe design, synthesis and biological enzyme activity detection.

The first exchange:

We are informed that Professor Jingnan Cui's team has designed a fluorescence analysis technology that can quickly detect trace harmful substances in food safety and environmental pollution. They taught us how to design and analyze the fluorescence detection. According to Mr. Cui, the detection approach employed by Professor Jingnan Cui's team utilizes two different wavelengths of excitation light to activate a designed chemical fluorescence probe and subsequently measures the emitted light through a sensor. The newly discovered fluorescence value is then converted into concentration value in accordance with the measured standard fluorescence curve to produce the detecting effect. We initially believed that this concept would be very beneficial to our project.

Dong Yiming: The possibility of fluorescence detection

Why: Shanghai Xinsu Medical Technology is committed to leveraging semiconductor technology to empower biomedicine. By harnessing the inherent advantages of miniaturization and high integration in silicon chips and microfluidic technologies, we aim to provide ultra-high throughput and ultra-high sensitivity solutions to meet the rapidly evolving needs in the fields of medical health, synthetic biology, DNA storage, and more. In the current market for instant diagnostics, paper chromatography tests hold a significant share. However, microfluidic chips, as an emerging technology, are not yet fully developed, and we are uncertain about which direction to choose to enhance our detection projects. That's when we reached out to Ms. Dong.

Detail: Ms. Dong emphasized to us that microfluidic chip technology presents high levels of complexity both in design and practical implementation. It requires extensive fine-tuning over a prolonged period to yield satisfactory results and is not well-suited for routine testing in everyday contexts. In contrast, chromatography paper, as a more mature technology, has gained widespread recognition and use among the public. It possesses a commercial appeal. If we can make certain optimizations to chromatography paper within our project, it could also represent a highly effective improvement.

Takeaway: Ms. Dong suggested that our team start with optimizing chromatography paper and in the future, when time and knowledge conditions allow, proceed with the design of microfluidic chip-based detection. In the subsequent interactive discussions, we once again reached out to Ms. Dong for in-depth exploration. As a seasoned iGEM participant, Ms. Dong provided us with invaluable insights and recommendations. Her input shattered our preconceived notions and rigid thinking about hp and edu work. We are deeply grateful for Ms. Dong's assistance, and we are committed to carrying forward the spirit and values of iGEM. We approach every individual we encounter in our interactions with a global, inclusive, respectful, and equal attitude. Our hope is that our project can genuinely and effectively address societal issues and assist those in need.

Professor Wang Baojun: The method of hierarchical characterization in microfluidic chips

Filed: Synthetic biology, gene components and circuit design, biosensor and intelligent diagnosis and treatment

Why：Professor Wang Baojun presented at the CCIC conference, introducing the design of multiple channels within microfluidic chips for conducting tests with varying concentration gradients, enabling a semi-quantitative calibration of the substances under examination. This inspired us, as our team had also contemplated semi-quantitative detection in the initial project conceptualization. Therefore, we engaged in discussions with Professor Wang.

Takeaway: We inquired about the possibility of designing multiple channels for semi-quantitative detection on chromatography paper， and Professor Wang indicated that there's a significant possibility for this approach to work. However, he also pointed out that there might be substantial differences in the specific design due to variations in the liquid flow dynamics between chromatography paper and microfluidic chips. He suggested that we should consult with experts specializing in chromatography paper design to validate the feasibility of this idea.

Wang Baojun: The cascade amplification method for detecting signals

Why：Professor Wang Baojun also presented at the CCiC conference the arsenic ion sensing array technology encapsulated in agarose gel. Building upon previous discussions with Professor Sun Wen, we were contemplating whether we could achieve visible results to the naked eye through fluorescence array cascade amplification. Consequently, we engaged in discussions with Professor Wang.

Takeaway: Professor Wang stated that achieving signal amplification through a fluorescence array is technically entirely feasible. However, he also pointed out that the specific implementation would depend on the fluorescence intensity of the substances we use and the medium in which they are immobilized.

Professor Wang Baojun: The method of hierarchical characterization in microfluidic chips

Second Feedback:

However, in the process of trying to learn this method, we found that the matching degree between the detection line and the control line of the chromatography test paper was not very high when the two wavelengths were excited simultaneously, and there were significant differences between the chemical probe and the biomolecule probe. Professor Cui agrees that we should start with single-wavelength light source excitation and modify the chromatography test paper's detection procedure. We draw some ideas from Professor Cui's design and determine to work on single-wavelength light source excitation.

Cardiovascular Disease Patients - Social Media

In the digital information era, many patients choose to use the internet to alleviate the psychological stress brought on by their illnesses and to share their personal battles against the disease. After actively searching, our team successfully established contact with some patients. Unfortunately, only three patients with heart failure were willing to share their journey of combating the disease with us. During our interactions, we took great care to consider the patients' perspectives and feelings, ensuring that our words did not unintentionally inflict any emotional harm to them， upholding the iGEM values of respect, honesty, responsibility, and equality. After our conversations, some of their words deeply resonated with us and tugged at our hearts:

All three patients were aged between 16 and 30. Surprisingly, one of them was even the same age as a member of our team. For many, this age may seem not related to cardiovascular diseases. However, through our direct interactions with these patients, our team members deeply felt the imminent trend of cardiovascular diseases affecting younger populations. It highlighted the urgency of raising awareness among young people about heart health. Ensuring regular monitoring for those with cardiovascular diseases is of paramount importance. This further reinforced the significance and purpose of our project.

During our discussions, we also learned that patients are likely to be hospitalized multiple times within a year. The cost of each stay ranges from 10,000 to 50,000 yuan, placing a heavy financial burden on families and posing challenges to healthcare resources.

Takeaway: We have realized the importance of early detection. Early treatment and regular monitoring for heart diseases cannot be overstated. The sooner a condition is detected, the lower the treatment cost, the lesser the pain endured, and the higher the likelihood of reversal to health. The increasing prevalence of cardiovascular diseases among younger individuals has drawn our attention. Due to their youth, they lack awareness and mindfulness about heart health. Cardiovascular diseases, as lifelong conditions, entailing high monitoring and treatment costs, need regular check-ups. We aim to identify a cost-effective, portable, efficient, and accurate testing method to assist those with cardiovascular diseases in keeping track of their heart health at all times, thereby reducing the chances of mortality.

Reflection I

After the aptamer approach failed, we brainstormed again. Following this meeting, we decided to use the SpyTag/SpyCatcher system to assemble the protein cage, providing a universal interface, and fusing the expression of RFP and streptavidin for capture. Our initial idea was to link only Streptavidin. However, we immediately raised a concern: each assembly doesn't guarantee 100% correct assembly of the protein cage's 60 sites at a 59:1 ratio, so how could we solve this situation? After discussions with our PI and instructor, we decided to add a RFP in the presence of streptavidin. By simultaneously measuring green and red fluorescence, we can determine the actual assembly ratio.

Community Hospitals

Why: The construction of community hospitals in China is still in steady progress. Through our research, we learned that in July 2020, China's National Health Commission issued the "Notice on Fully Promoting the Construction of Community Hospitals." In the "14th Five-Year Plan," there is also new constructive planning for the development of community hospitals. By the end of 2020, 1,410 community hospitals were established nationwide, an increase of 794 compared to the 616 in 2019. Of these 1,410 community hospitals, 739 (52.41%) were established by community health service centers, and 671 (47.59%) were established by township health centers. While the number of community hospitals is increasing, there is still a need to improve the quality of their medical services. We hope to enrich the functional capabilities of community hospitals by offering our portable heart health assessment kits or test strips, thus contributing to the construction of community hospitals in China.

To thoroughly understand the actual conditions and needs of community hospitals, we conducted field interviews and research in locations such as the main and branch stations of the Youjia Village Health Service Center in Ganjingzi District, Dalian, and the Luogou Village Health House in Wating Town, Nanyang, Henan Province. It's essential to fully understand how our project should be presented to serve the target population best. Identifying key factors to consider when promoting our achievements to community hospitals is crucial. This ensures that our project upholds the right values when implemented in real-world applications.

Details: Given that the general public still needs to a certain foundation in biology, we initially aimed our hardware for use in community hospitals during the early stages of our project. We hoped that with the assistance of professionals from community hospitals, we could achieve more precise and effective heart health assessments. Through discussions with doctors and professionals from various community hospitals, we understood the following:

1. Current community hospitals primarily focus on treating common ailments like colds and fevers, prescribing routine follow-up medications for patients, and conducting subsidized health check-ups for the elderly. These check-ups often require specialized doctors to assist.

2. Most community hospitals have been established for a long time and lack updates to their hardware. They occupy small areas, making them unsuitable for large equipment placement.

3. The main diseases treated are always mild conditions like colds and fevers. For severe conditions, patients are still advised to seek treatment at larger hospitals. The number of patients they see on average days is low.

4. Most doctors in community hospitals are general practitioners, and specialists are lacking. On average, there are two to three general practitioners per community hospital daily to meet basic medical needs, but they are not equipped to handle severe diseases or emergency cases.

5. Regarding diagnostic testing, most community hospitals have the capability for basic tests like ECGs and blood tests. Still, some cannot meet the needs of even these. There's a lack of infrastructure for advanced diagnostic equipment, such as CT or X-ray rooms.

6. Many patients have established perceptions about community hospitals and somewhat lack trust in them. As a result, fewer patients are willing to seek treatment in these hospitals actively.

7. The majority of patients treated in these locations are ordinary farmers. For them, blood tests, such as finger pricking, are more trustworthy and acceptable. On the contrary, some might find urine or saliva tests more uncomfortable. For the medical staff in community hospitals, blood tests are also preferred due to their simplicity and ease of operation.

These insights are vital for understanding the landscape of community health care and tailoring solutions to meet real-world needs.

Takeaway:We learned that the general public tends to trust blood tests more. Since community hospitals are primarily staffed with general practitioners and lack specialists, a clear, unambiguous, and detailed instruction manual remains essential even if the testing method is simple. Additionally, we should fully consider the equipment conditions in community hospitals, address safety issues before, during, and after testing, and develop a safe, efficient, portable, and high-precision testing tool.

Reflection II

1. It's crucial to give full consideration to the accuracy of the test, error control, the user's acceptance level, and the ease of operation. Consequently, we chose blood over saliva for testing.

2. During the implementation of our results, it's essential to create a comprehensive and detailed instruction manual. This ensures that different users can achieve reliable and consistent results when using the test strips and kits. In this way, we can guarantee that used materials are safely and hygienically disposed of.

3. For the waste generated during our testing process, we designed a distinctively colored medical waste collection bag. This bag, included within the kit, can also be sold separately or distributed as a public welfare initiative in communities. It not only allows for the centralized collection of test kits but also collects potential cross-contamination medical waste from daily life, such as masks and medical cotton swabs. Ultimately, these waste collection bags can be part of waste segregation and centralized collection, fulfilling our responsibility to the users, society, and the environment.

Reagent Kit Survey

We selected the widely used biomarker, NT-proBNP, for this reagent kit, and conducted a survey on the sources and prices of reagent kits from ten companies.

Takeaway:We found that the average price of reagent kits is 1835 RMB, with 40% of the reagent kits being imported. This realization underscores the high cost of reagent kits and emphasizes the necessity for developing a low-cost, portable, efficient, and accurate testing tool. Furthermore, we learned that the safety of reagent kit usage relies solely on users adhering to usage guidelines conscientiously. This raises higher standards for product safety, and the clarity and precision of hardware manuals to ensure the safety of the user community. In the production process, the production of antibodies and standard materials constitutes a major part of the finished product. The product is a non-cellular system, which minimizes the risk of infection for users to the greatest extent.

Dr. Guokun Wang: Optimization of the Detection Approach

Cardiovascular surgeon at Shanghai Changhai Hospital

Why: In order to gain a more comprehensive understanding of stakeholders' opinions, assess the practical application value of our project, and explore avenues for improvement, we aim to broaden our perspectives based on the recommendations of cardiologists. Therefore, we reached out to cardiovascular surgeon Dr. Guokun Wang.

Details: After discussions with Dr. Wang, we gained the following insights:

1.Heart failure is more of a condition than an independent disease. Typically, heart failure does not occur in isolation but is accompanied by other medical conditions.

2.NT-proBNP alone cannot serve as a single diagnostic criterion. When patients visit the hospital, diagnosis generally involves NT-proBNP, electrocardiograms, and clinical symptoms.

3.If preliminary diagnosis can be conducted at home or in an ambulance on the way to the hospital during cardiac emergencies, it can save diagnostic time, enable faster surgeries, and improve survival rates. Dr. Yang believes that diagnosing heart failure through NT-proBNP testing is not critical because heart failure is more of a long-term condition. It would be more meaningful to use cardiac troponin for myocardial infarction detection. In the case of a heart attack, surgery must be performed within 4 hours to ensure patient survival.

4.NT-proBNP testing can also be applied to follow-up examinations for patients in remote areas. After surgery, patients need to return to the hospital for follow-up within three to six months. However, many patients from outside Shanghai who seek medical treatment cannot return for follow-up and can only undergo follow-up examinations at their local hospitals.

5.With the rapid pace of modern life and unhealthy dietary habits characterized by high salt, sugar, and fat consumption, coronary heart disease is becoming more common at a younger age, affecting people in their 50s and even 40s. Among these cases, myocardial infarction poses the most serious challenge, as surgery must be performed within a 4-hour window.

6.If our hardware can be made portable and come with a corresponding smartphone app for real-time user reminders, it would be a significant game-changer.

Takeaway: Heart failure is more of a physiological condition and cannot be diagnosed solely through NT-proBNP testing. Diseases like myocardial infarction, which require rapid diagnosis, are in greater need of our project.

Reflection III

After interviews and discussions with the stakeholders mentioned above, our team has further positioned our project's practical clinical needs and improvement opportunities:

1.Our project will enable parallel testing of NT-proBNP, MR-proANP, and cardiac troponin, allowing for a comprehensive assessment of heart health in a multipoint and surface-based manner.

2.Taking Dr. Wang's advice into serious consideration and following discussions, we have decided to broaden the scope of our intended applications. We plan to improve our hardware to facilitate parallel testing of multiple cardiac indicators in ambulances, saving time for indicator testing. This will enable us to gain immediate insights into a patient's heart condition and intervene promptly.

3.After learning about the challenges faced by patients from remote areas who find it difficult to return to major cities for follow-up examinations, our team will design detailed instructions and simple, portable, and user-friendly hardware devices to help these individuals conduct rapid indicator monitoring at home or in local community hospitals. This will prevent delays in treatment due to outdated healthcare infrastructure.

4.We have taken into account the doctors' suggestion to design a smartphone app to assist in visually assessing indicator levels. We will collaborate with the hardware team to develop the software.

Cui Yonghua: Understanding the real reagent kits

Profile: General Manager of Roche Diagnostics Reagent Kits for the Dalian region

Why: In the interview with Dr. Zhao, we learned that Roche Diagnostics kits are currently used in the cardiovascular medicine department of the Second Hospital of Dalian Medical University. In order to further understand the necessary conditions for clinical detection kits, the accuracy of clinical detection, the attention we should pay to if our project is carried out, and what aspects need to be improved, we contacted the agent of Roche Diagnostics kits in Dalian for further interview and investigation.

Detail: During the interview, we gained the following insights:

1. Roche Diagnostics kit uses ECLIA for detection, which has the advantages of high accuracy and fast speed, but the cost is higher than that of ELISA.

2. The price of Roche Diagnostics' large-scale testing equipment costs more than a million yuan which is relatively expensive. What's more, it also occupies a large amount of ground. Therefore, it is obvious that community hospitals do not have the economic and space conditions to set up large-scale testing equipment.

3. The cost of kit production by manufacturers is mainly in technology development, human resources, experimental materials and equipment. If we want to industrialize our kits and test strips, we have to fully consider these factors to reduce costs and lower the prices on the basis of ensuring the accuracy of detection quality.

4. At the beginning of our project, we thought that saliva sampling was a simple and quick method for sampling, but after communication, we learned that saliva sampling faced the problems of inaccurate sampling and low content. From the perspective of being fully responsible for users, we agreed that blood sampling would be the optimal choice in terms of universality and accuracy.

5. Considering that we finally need to use fluorescence detection technology to quantify the detection results, we consulted Ms. Cui about this and learned that the use of microfluidic technology and the choice of luminescence mode should be considered in the fluorescence reading aspect. We communicated with the hardware group and the wet lab group to consider improving our fluorescence detection scheme.

6. In the process of communication, Ms. Cui said that our position at the community hospital may face the problem of low volume, which leads to greater cost than income. But we got a positive answer that community hospitals could take venous blood as samples. At home, the consumption is slightly higher, so the design of the test paper can be considered.

7. Ms. Cui affirmed the feasibility of our semi-quantitative test paper, and suggested that our software design could refer to the real-time detection of diabetes on mobile phones.

Takeaway: Through the communication with Ms. Cui, we further understood the environment of the current kit market. We clarified the use of blood as testing samples, confirmed the wet experiment verification scheme, cleared the direction of future comprehensive consideration of production costs and the fluorescence detection technology.

In order to investigate the public's understanding and cognition of cardiovascular diseases and daily maintenance of heart health more widely, we distributed questionnaires online through social media and offline with iGEM-DUT community as the carrier. Finally, 303 questionnaires were collected. We hope that in this way, we can understand the general situation of cardiovascular disease in the social non-cardiovascular disease group. The specific questionnaire analysis are as followed.

About Community Hospital

1. Under what circumstances would you go to a community hospital?

2. What do you think of the treatment in community hospitals?

It can be seen that more than half of the people choose to trust the community hospital, and only 2.31% of the people completely distrust the community hospital. Most people (74.92%) will give priority to the community hospital under certain circumstances. This is enough to prove the universality and importance of community hospitals in People's Daily life.

3. What concerns do you have about community hospitals?

The main concerns about community hospitals are Insufficient diagnostic criteria and Lack of expertise among doctors. This also confirms the sore points of insufficient diagnostic conditions and unprofessional community doctors that we know from community hospitals. Therefore, it is necessary to have equipment that can detect a variety of diseases in community hospitals. In addition, it would be even more icing on the cake if the corresponding diagnostic recommendations can be given based on the results.

About Heart

1. Do you stay up late or have irregular sleep patterns?

Up to 95.05% of the population have the situation of staying up late or having irregular sleep patterns, which will undoubtedly increase the burden on the heart and the risk of cardiovascular disease.

2. What is your attitude towards heart care?

3. Do you regularly undergo cardiovascular disease screening?

Most people also don't have the time or awareness to care for their hearts. Very few (3.63%) of them received cardiovascular disease detection. This will undoubtedly increase the probability of cardiovascular disease.

4. Have you ever experienced discomfort in your heart?

5. What measures do you take after experiencing pain or discomfort?

From the above questions, we know that more than half of people have felt heart discomfort, but only a small number of people will go to the hospital for detection, more people just rest, will not change their living habits. There is no doubt that this will lead to a deterioration of heart health, but if our device detects it in advance, it can give a warning to prevent the deterioration of the heart.

About Detection Method

1. What do you think about early detection of cardiovascular disease at home or in a community hospital?

2. What source of sample would you prefer?

3. What type of testing method would you prefer?

From the above questions, we know that there is a great desire to be able to conduct cardiovascular disease testing in the community and even at home. And people prefer to be able to test through blood, and to obtain more detailed information through small kits. This gave direction to our hardware design, from which we designed a small kit hardware for detecting blood. Please visit our Hardware page for details.