Cardiovascular diseases represent a leading global cause of disability and mortality, making early detection and intervention crucial for reducing the associated disease burden. Nitric oxide (NO), a key regulator of vascular function, is recognized as an early biomarker for cardiovascular risk.

However, existing NO detection technologies face significant limitations in accuracy, convenience, and cost-effectiveness, which restrict their widespread application in both clinical and home-based settings.

Through literature review, questionnaire surveys, and stakeholder interviews, this project systematically analyzes the current technological bottlenecks and user needs in NO detection. It proposes a novel concept for a fluorescent biosensor based on synthetic biology principles, with the goal of providing a more feasible and accessible solution for cardiovascular health monitoring.

The "double delay" in cardiovascular disease monitoring: From technical bottlenecks to public welfare predicaments

The sudden passing of Zhao Han, founder of Yun Hai Yao, from a myocardial infarction at just 40 has cast a sobering light on cardiovascular disease's most alarming feature: its tendency to remain undetected until advanced stages. As the leading cause of death globally year after year [1], this disease continues to show rising prevalence with disturbing trends toward younger onset ages [1], revealing critical gaps in our early prevention systems. What makes cardiovascular disease particularly devastating is how its silent progression plunges families into reactive crisis management when it's often too late for effective intervention.

The core challenge lies in the disease's pathological development - it progresses stealthily without specific early symptoms [2]. By the time patients seek help for traditional risk factors like blood pressure or lipid abnormalities, organic vascular changes have typically been developing for years, missing the optimal intervention window [2]. This creates a perfect storm for "dual delays" in diagnosis [5]: advanced detection technologies like coronary CTA remain inaccessible due to high costs [3], while widely available routine check-ups rely on lagging indicators that fail to provide timely warnings [4].

Our research at Shanghai Tongji Hospital has documented countless heartbreaking cases where patients hesitated due to prohibitive costs or received ambiguous reports that failed to convey urgency. This "precision tests are unaffordable, affordable tests lack precision" dilemma creates a systematic barrier to early detection, where manageable conditions inevitably progress to irreversible stages. The stories of patients who postponed 800 RMB vascular checks only to suffer heart attacks months later, or elderly couples who couldn't sustain specialized testing on their pensions, highlight how this dual delay mechanism perpetuates needless tragedies.

Breaking this cycle requires establishing a tiered screening and precise interpretation system [5]. We must make precision testing more accessible while ensuring routine examinations provide clear, actionable warnings. Rather than vague suggestions to "monitor" findings like "decreased vascular elasticity," we need concrete management plans. Zhao Han's legacy should inspire systemic reform that addresses both technological barriers and healthcare delivery models, ensuring early intervention becomes universally accessible rather than a privileged exception.

Therefore, the challenge of cardiovascular disease prevention and control is systemic in nature. It involves not only the accessibility and accuracy of detection technologies, but also the rational allocation of medical resources and the effectiveness of health management. We must break the vicious cycle of "late detection" by establishing a tiered screening system and precise interpretation mechanisms, thereby moving the focus earlier in the disease progression. This requires coordinated efforts in policy support, technological innovation, and public education - making early screening an inclusive public good and enabling precise prevention as a viable health choice. Only by building a new prevention-oriented healthcare system and promoting a fundamental shift from treatment to health management can we truly transform the landscape of cardiovascular disease prevention and treatment, avoiding more preventable life tragedies.

User demand research: Analysis of Unmet Needs for New Cardiovascular Monitoring Technologies

To accurately identify the core shortcomings and user expectations in the current cardiovascular health monitoring field, we conducted a specialized questionnaire survey aimed at providing direct and reliable decision-making basis for the development of new-generation detection technologies. The analysis results clearly reveal significant unmet needs in terms of accessibility, cost, and user experience of existing technologies. A total of 317 questionnaires were collected, and the analysis is as follows:

The questionnaire analysis clarified market expectations for new-generation cardiovascular monitoring technologies and identified unmet needs. Respondents were primarily aged 46-60 (42.59%) and 31-45 (28.39%), with over 60% having a family history of cardiovascular diseases. Most already suffer from related chronic conditions, yet this high-risk group currently lacks effective early-stage monitoring tools.

The research found that people generally face three main problems: the impact of diseases on daily life (46.37%), the economic pressure caused by medical expenses (43.53%), and the psychological anxiety of worrying about becoming a burden to family members (43.22%). These real issues reveal that the market urgently needs a new type of health monitoring solution that can not only help maintain quality of life but also alleviate economic and care pressures.

Regarding the new detection technology, what people most hope for is: the price should be affordable, and it should also be accurate and easy to use. Specifically, "affordable price" (51.10%) ranks first, while "accurate results" (46.69%) and "easy operation" (44.16%) are also highly valued by everyone. This points out the direction for our research - while ensuring accuracy, we should focus on reducing costs and making the detection process more convenient.

For a new detection technology, people hope that its price can be within an acceptable range. The research data clearly shows that 72.29% of the users expect the single test cost to be no more than 300 yuan. This price expectation directly reflects the urgent demand of the market for low-cost detection solutions. Therefore, during the technology development process, we must take cost control as the core consideration. By optimizing the detection process and material selection, we can truly reduce the economic burden on users and make the innovative technology truly benefit the large number of people in need.

In summary, this survey clearly demonstrates that the new generation of cardiovascular monitoring technology expected by the market must achieve the integration of cost controllability, user-friendly operation, and diagnostic accuracy - this represents the key to breaking the current "dual-delay" dilemma in cardiovascular disease management.

Based on solid research foundations, we plan to develop a novel dynamic monitoring solution for nitric oxide (NO). This project aims to establish NO—a key signaling molecule that directly reflects the functional state of vascular endothelium—as the core monitoring indicator. By creating a precise tracking system for its dynamic concentration changes, our solution will effectively overcome the limitations of traditional lagging indicators such as blood lipids and blood pressure.

Our Integrated Human Practice is the core driving force of project iteration. Through clinical interviews, academic exchanges and market research, we convert various feedbacks into specific technical optimizations and strategic adjustments, driving the project from the initial concept (Idea 1.0) to a systematic solution that integrates high-performance sensors (Idea 2.0) and practical hardware (Idea 3.0).

During the technical optimization phase (Phase 2), expert opinions from the clinical and academic communities directly guided five key improvements:

• Optimization 1: Establish a quantitative mathematical model between nitric oxide (NO) concentration and fluorescence intensity to achieve objective quantification of the detection results;

• Optimization 2: Introduce artificial plasma experiments to verify the sensor's anti-interference performance in complex biological matrices;

• Optimization 3: Conduct subcellular localization experiments to deeply explore the cellular origin and functional mechanism of NO;

• Optimization 4: Incorporate green fluorescent protein (GFP) into the fluorescence reporting system to enhance the diversity and reliability of signal detection;

• Optimization 5: Expand the application scenarios of the sensor in drug screening and disease mechanism research, and enhance the external value of the technology.

These optimizations have significantly enhanced the scientific rigor and application potential of Idea 2.0. Subsequently, based on Dr. Liu Feng's suggestions regarding the supporting equipment and Mr. Li's market insights, we formulated the Idea 3.0 hardware strategy for families and grassroots levels, ultimately forming a complete closed loop from molecular testing to terminal services, dedicated to resolving the "double delay" predicament in the early screening of cardiovascular diseases.

At the beginning of the project, the team systematically conducted brainstorming sessions and conducted literature research to clearly realize that the core difficulty in early screening of cardiovascular diseases lies in the lack of sensitive markers that can reflect vascular dysfunction before pathological changes. Nitric oxide (NO), as a key signaling molecule for endothelial function and vascular homeostasis, has a concentration change that is closely related to the early occurrence of various cardiovascular diseases. Therefore, it was established as the priority early risk indicator for this project.

To precisely understand clinical needs, we conducted interviews with laboratory technicians from Peking Union Medical College Hospital. Based on their over ten years of frontline experience, they indicated that current routine tests (such as blood lipids and glucose) primarily detect late-stage structural damage, while existing NO detection technologies face significant limitations: electrochemical methods are susceptible to blood matrix interference with poor reproducibility, and fluorescent probe methods struggle with photobleaching and stability issues, making it difficult to meet clinical requirements for long-term stable monitoring. They particularly emphasized that an ideal new-generation detection technology must balance specificity, interference resistance, operational convenience, and cost control.

Based on the above clinical pain points, we turned to the molecular recognition mechanisms that have been optimized through long-term evolution in nature. It was found that the bacterial transcription factor NorR can achieve highly specific binding to NO through its non-heme iron center, and has no cross-reaction with common molecules such as oxygen. The related structural mechanism has been resolved [6]. This natural recognition module provides a reliable molecular basis for constructing high-performance biosensors.

Based on our in-depth research, we have proposed an innovative solution (Idea 1.0): the development of a dual-platform system utilizing genetically encoded NorR monochromatic fluorescent sensors based on YFP and mCherry. In this design, the YFP sensor achieves highly sensitive detection through its high quantum yield and excellent signal-to-noise ratio, while the mCherry sensor demonstrates superior tissue penetration and reduced autofluorescence interference due to its long-wavelength emission characteristics.

This innovative technological platform offers three core advantages: First, the monochromatic sensor design significantly streamlines the detection process while reducing equipment requirements. Second, the genetic encoding strategy ensures excellent heritability and long-term stability of the sensors. Third, the natural NorR-based recognition mechanism provides exceptional selectivity, enabling effective discrimination between nitric oxide and other reactive nitrogen species. This technology successfully overcomes key technical limitations of existing detection methods in terms of specificity, stability, and cost control, offering a reliable and cost-effective innovative solution for clinical early diagnosis.

As the experiment progressed, we continued to communicate with experts to optimize the molecular design and expression strategies.

Interview

Liu Feng, Director of the Cardiovascular Department at Suzhou Kowloon Hospital, affiliated with Shanghai Jiao Tong University School of Medicine

During the interview, Dr. Liu Feng pointed out that there are significant differences in the diagnostic indicators for various cardiovascular diseases at present. Accurate diagnosis often requires the combination of systematic medical knowledge, in-depth understanding of professional guidelines, and rich clinical experience. Based on this insight, our product needs to establish a clear quantitative correspondence between nitric oxide (NO) concentration and fluorescence signals, that is, by detecting the fluorescence intensity to quantitatively reflect the NO concentration, thereby providing objective and quantifiable test results. To this end, we constructed a mathematical model, successfully achieving a linear correlation between NO concentration and fluorescence intensity, laying the theoretical foundation for subsequent precise detection (Optimization1).

Summary：Based on clinical insights from Dr. Liu Feng, we have established a precise quantitative relationship between NO concentration and fluorescence intensity through a mathematical model, enabling objective diagnostic results that effectively address variability in cardiovascular disease diagnosis (Optimization 1).

Dr. Zhiye Wu, a Physician at the Department of Cardiology, Zhujiang Hospital of Southern Medical University.

After talking with Dr. Wu, we learned that although the NorR-YFP probe developed in the laboratory performed well and had a strong binding ability to nitric oxide, this result was obtained under a relatively simple experimental environment. Only the nitric oxide donor fructose was added. We realized that if the product is to be used for blood testing in the future, the blood itself is extremely complex, and various substances in it may interfere with the detection. The actual effect may not be as ideal as in the laboratory. Therefore, we decided to add an "artificial plasma experiment" (Optimization2) at this step of functional verification. We wanted to simulate the real blood environment as closely as possible to see how the probe performs in complex situations. In this way, our evaluation of it will be more in line with reality, the results will be more persuasive, and we will feel more at ease.

Summary：Following discussions with Dr. Wu, we recognized that the probe's excellent performance was achieved in simplified lab conditions. To ensure reliability in real blood detection, we introduced artificial plasma experiments (Optimization 2) to evaluate its performance in complex environments, thereby enhancing clinical relevance.

Wu Dan, Deputy Director of the Pharmacy Department of Tongji Hospital Affiliated to Tongji University

During the communication with Professor Wu Dan, she affirmed the product of this project from three aspects: clinical value, promotion feasibility, and future research direction. At the clinical level, this tool can promote personalized medication. It can optimize the treatment plan by quickly assessing the patient's drug response and can also achieve real-time monitoring of therapeutic effects in a minimally invasive way, reducing the burden on patients. At the promotion level, although the product requires low-temperature storage, the grassroots level already has refrigeration conditions. Additionally, its portability and moderate cost make it in line with the guiding principle of hierarchical medical care and suitable for chronic disease management and screening at the grassroots level. At the same time, Professor Wu Dan pointed out that using nitric oxide as an early indicator for drug screening is only the first step. What we really need to do is to use more precise probes to delve into the cells and clearly understand where this nitric oxide signal originates from - only in this way can we truly understand the disease at its root. Therefore, we further conducted the subcellular localization experiment (Optimization3) to verify the functional localization of the constructed fluorescent protein in specific subcellular structures (including the nucleus, mitochondria, and cytoplasm).

Summary：Following discussions with Professor Wu Dan, we gained key insights across three dimensions: clinical value for personalized medication guidance, feasibility for primary healthcare deployment, and research advancement through subcellular localization experiments (Optimization 3) to trace NO signaling origins.

Academical Meet Up

At the Hangzhou iGEM academic exchange conference, we engaged in extensive presentations and discussions with multiple university teams, receiving highly valuable feedback. Professor Meng Qiu, an associate professor at the College of Biological Engineering, Zhejiang University of Technology, specifically suggested that alongside YFP and mCherry, we should incorporate the classic GFP for systematic comparison to achieve more comprehensive fluorescent protein screening. We promptly implemented this practical recommendation as Optimization 4 in our experimental design. Separately, Professor Qiao, also an associate professor at the same institution, highlighted the probe's potential applications beyond nitric oxide detection, noting its utility in drug development—particularly for evaluating drug efficacy and investigating pulmonary hypertension pathogenesis. These insights significantly expanded our research perspectives and were subsequently incorporated as Optimization 5 in our study.

Summary：At the Hangzhou iGEM conference, we received valuable suggestions from Professor Meng Qiu to incorporate GFP for systematic comparison (Optimization 4), and Professor Qiao highlighted the probe's potential in drug development and pulmonary hypertension research (Optimization 5), significantly expanding our project's scope.

Based on expert feedback, we have systematically upgraded our initial concept (Idea 1.0) to an enhanced version (Idea 2.0) through the following key improvements:

• Introduced plasma environment validation to enhance physiological relevance;

• Incorporated GFP into the probe screening system for expanded signal options;

• Established a quantitative NO-fluorescence intensity model for improved accuracy;

• Added subcellular localization experiments for functional verification;

• Explored application potential in drug development and pulmonary hypertension research.

These refinements significantly strengthened the project's scientific rigor and application potential.

Guided by expert insights and market research, we have formulated the IDea 3.0 hardware strategy based on the core principles of "intelligent detection, universal accessibility, and user-friendly design", with the aim of building a "device - consumables - service" closed-loop ecosystem for home and grassroots medical care.

Interview

Liu Feng, Director of the Cardiovascular Department at Suzhou Kowloon Hospital, affiliated with Shanghai Jiao Tong University School of Medicine

Doctor Liu Feng pointed out during the discussion on the application prospects of the product that although the current protein technology has good detection performance, for it to be truly easy to promote and use, dedicated equipment still needs to be developed. This equipment should be able to achieve automated operation, making the detection process more convenient and efficient, while maintaining portability to adapt to various scenarios such as grassroots medical institutions, communities, and even households. Through the "protein + equipment" overall solution, not only can the difficulty of use and operational errors be reduced, but the practicality and popularity of the product can also be greatly improved.

Summary：Based on Dr. Liu Feng's recommendation, we will develop a companion automated portable detection device to form an integrated "protein + equipment" solution, enhancing practicality and accessibility.

Mr. Li, Vazyme Distributor

Mr. Li has been engaged in the sales of various test kits for a long time. During the communication, he pointed out that the compact and portable test kits are more favored by consumers. At the same time, he also mentioned that some test kits that are considered to have good performance by researchers, actually have poor sales results. The fundamental reason lies in the lack of real market demand. In addition, some test kits can provide test results, but users still need to go to the hospital for further confirmation, resulting in limited practical application value and seeming to be rather useless. Therefore, Mr. Li strongly suggests that before our product officially enters the market, we must conduct thorough market research to accurately grasp the user needs.

Summary：Summary：Based on Mr. Li's market insights, we recognize that thorough market research is essential to ensure our product aligns with genuine user needs and delivers tangible practical value.

Survey

In the later stage of the project, to meet the demand for home NO (nitric oxide) detection, we initiated the development of supporting hardware. Based on Mr. Li's market-oriented suggestions, in order to further improve the product design from multiple dimensions such as packaging, functions and user needs, we conducted a second round of questionnaire surveys. A total of 178 valid responses were collected, providing a basis for subsequent improvements.

Based on the results of the questionnaire survey, this project has formed a systematic understanding of the market demand and user characteristics for home cardiovascular detection devices. Approximately 99% of the respondents have a positive attitude towards biotechnological products, indicating that the product has a broad social recognition base and is expected to have relatively low market promotion resistance.

Attitudes towards biological technology services and humans

In terms of usage scenarios, over 60% of users prefer to conduct tests at home, highlighting the core demands for convenience, privacy protection, and non-medical operations. The target users are mainly the elderly population aged 45 and above, accounting for over 90%. This group has clear health monitoring needs, and the product design must fully consider the elderly-friendly features and the purchasing logic driven by the "filial piety economy".

Testing location

The age of the user

In terms of behavior monitoring, approximately 75% of the users expect to have regular monitoring conducted every three months or half a year, indicating a high level of user loyalty and providing a continuous revenue stream for consumables and value-added services.

Frequency of use

In terms of functionality, users generally prefer qualitative results that are intuitive and easy to understand. Nearly 90% of the respondents value the intelligent analysis capabilities of the accompanying software and expect to receive comprehensive health management support through an integrated solution of "device + software + service".

User requirements

In terms of product form, over 60% of users expect the device to be small in size and easy to use at home. Nearly 90% of them have experience using fingertip blood collection devices, suggesting that the design concept of portability and integration similar to that of blood glucose meters can be adopted.

The size of the user's requirements

Has the user ever used any instrument similar to the fingertip blood collector?

Based on the survey results, we have drawn the following conclusions:

• Market Attitude: Over 90% of users hold a positive attitude towards biotech products, indicating broad market acceptance;

• Usage Scenario: Over 60% of users prefer home testing, with core demands being convenience and privacy.;

• Target Users: The user base is highly concentrated in the 45+ age group, necessitating a focus on elderly-friendly design and the "filial piety economy";

• User Loyalty: Approximately 75% of users are committed to regular monitoring, predicting a stable, long-term revenue from consumables and services;

• Functional Needs: Users prefer intuitive results and intelligent analysis, favoring an integrated "device + software + service" solution;

• Product Form Factor: Over 60% of users prefer compact devices, suggesting a design philosophy similar to widely adopted blood glucose meters.

Based on expert interviews and market research findings, we have developed the application-oriented Idea 3.0 solution. Centered on three core principles - "Intelligent Detection, Universal Accessibility, and User-Centric Design" - this solution establishes a comprehensive "device-consumables-service" closed-loop system:

1）Intelligent Detection: We have developed portable devices enabling automated operation and qualitative result output, complemented by intelligent software for health trend analysis.

2）Universal Accessibility: Through modular design, we achieve portability and cost-effectiveness while adapting to diverse scenarios from primary care to home use.

3）User-Centric Design: Inspired by glucose meters, we implement lightweight one-step operation with elderly-optimized interfaces for enhanced usability.

This integrated hardware-software ecosystem builds upon our molecular sensing technology to deliver practical and market-ready solutions.