The point-of-care (POC) detection refers to medical testing that takes place at or near the location where patient care is provided, rather than in a centralized laboratory. It aims to provide rapid and convenient diagnostic results that can be immediately used for clinical decision-making. In recent years, with the threat of COVID-19, it is obvious that point-of-care detection has gained public attention. However, most of these POC detection devices do not have the ability to alleviate the patient's situation. Therefore, our project aims to construct a bio-device with the ability to elucidate the disease risk and to release the health benefits compound for the human body.
Inflammation, either the acute or slow term, is a health threat issue in today’s society. According to the World Health Organization (WHO), one such inflammatory disease, cardiovascular disease (CVD), accounted for approximately 17.9 million deaths in 2019, representing 32% of all global deaths. Needless to say, inflammatory diseases also include cancer, asthma, dermatitis, etc., affecting tens of thousands of people around the world. Our team aims to apply synthetic biology approaches to develop a bifunctional bio-device that will be a bio-detector capable of executing point-of-care detection of the inflammatory response. Upon the detection of calprotectin, a substance that can represent various inflammatory responses, our bio-device will release α-rhamnosidase, which is critical in converting hesperidin into the anti-oxidative compound hesperetin. The anti-inflammatory properties of hesperetin have been proven in several aspects including the inhibition of the inflammatory mediators production and the activity decreasing of pro-inflammatory enzymes in previous studies. Moreover, the results of our detection will be conveyed through both the fluorescent protein and electric pili. The results would be processed and then sent to the user's mobile devices and the database of medical facilities. In conclusion, with the help of our bio-device, inflammation-related conditions and diseases might be alleviated, even more, prevention.
The NYCU-Taipei team has long been focused on medical issues and therapeutic topics. This year, our team was assembled in December 2022, and we started our twice-a-week meetings and training from then on. During that time, we continued brainstorming and thinking about how to make breakthroughs that would also satisfy the third Sustainable Development Goal (SDG), ensuring healthy lives and well-being for people of all ages, as defined by the United Nations. This topic is of great concern to all members of our team. Moreover, the resource imbalance among people living in urban or rural areas, as well as between the rich and the poor, has become increasingly serious in recent years. This problem has garnered public attention and has been listed as the tenth goal of the SDGs, which aims to reduce inequalities within and among countries. We also hope that our bio-device can help address this issue.
After extensive discussion and numerous nights, we finalized our topic as a bi-functional biosensor with detection ability and therapeutic effect. In our plan, the biosensor will send the results to the user's electronic device and the cloud database of medical facilities. We believe that this bio-device can help to address the issue of lacking household disease detection and treatment devices that do not require cutting-edge technology. Furthermore, we hope that our device can be used in rural areas or distant mountains. In other words, our bio-device may facilitate the balance of medicine resources availability and reduce the disparity between urban and rural areas.
After years of development, medicine has made great progress in the diagnosis and treatment of diseases. However, there are still some shortcomings that need to be addressed. Here are a few examples:
The diagnosis of many diseases is limited to medical institutions due to the technological requirements, which raises the threshold for people to immediately know their physical condition.
Currently, only a limited number of diseases or conditions (e.g., COVID-19, diabetes) can be monitored at home. Many diseases cannot be detected in real-time in the early stages, making subsequent treatment difficult.
Due to the cost of technology and instruments, resources are unevenly distributed. Economic or geographical reasons can prevent residents in some areas from accessing quality medical care.
In response to the mentioned problems, we aim to achieve the following impact through the
development of a lightweight disease detector:
People can quickly assess their risk of diseases. With guidance from healthcare professionals, individuals can take early preventive measures. Additionally, our device also activates the production of hesperetin when it detects calprotectin, which is related to inflammation. Hesperetin is produced based on individual needs, enhancing the efficiency and effectiveness of healthcare.
As our device functions as a point-of-care bio-device, real-time detection of disease biomarkers enables early intervention and treatment. This promotes the efficiency of diagnosis and treatment, potentially improving cure rates and prognosis.
The costs of disease diagnosis may decrease, reducing the dependency on diagnostic technology exclusive to medical institutions. People can use the diagnostic device at home.
Our project aims to increase the accessibility and convenience of disease diagnosis, even in rural areas. It is expected to reduce the gap in medical care between urban and rural regions.
The application and development of EET technology provide novel electrical transmission pathways in biotechnology. This emerging technology, combining synthetic biology and electricity, holds great potential in both academia and business. Our project focuses on the application of EET and aims to lay the foundation for future development in this field.
Whether applied locally in Taiwan or globally, the aforementioned impacts are expected to bring positive benefits to human welfare.
Below are three major parts of our project, one is the detection system, one is the reporting module, and finally, the generation of health benefit hesperetin.
We aim to construct a bio-device that can detect the inflammatory state, release the signal for alarm health conditions, furthermore, supply the hesperetin for the anti-inflammatory purpose.
Early diagnosis and prediction of a disease can help improve therapeutic efficacy and prevent deterioration of the condition, and in turn, enhance the quality of medical care. Current diagnosis tools rely heavily on biomarker sampling from blood samples. The biochemical values in the blood are the standard evaluation approach today for biomarker tests. Biomarkers in the blood can indicate many diseases, not just cardiovascular disease. While it is proven to be useful in diagnosing many diseases, taking blood remains an intrusive method of retrieving biomarker samples. Furthermore, it has to be done by medics at healthcare facilities and is neither time nor cost-efficient. Recent studies are focusing on the development of non-intrusive approaches to replace traditional sampling methods. These include collecting body fluid samples from the urine, saliva, and sweat. Out of these studies, biomarkers from the saliva show promising preliminary data for predicting disease.
Calprotectin, a heterodimer of S100A8 and S100A9 EF-hand calcium-binding proteins, is an inflammation biomarker primarily derived from neutrophils and monocytes. Recent studies have investigated calprotectin in five contexts: serum, feces, urine, sweat, and saliva. However, due to the invasive procedure of blood tests, and the inconvenience of feces and urine sample collection, our research team primarily focuses on the detection of calprotectin from saliva. According to three recent studies, salivary calprotectin has been shown as a significant biomarker for periodontitis, systemic lupus erythematosus (SLE), and peritonsillar abscesses. In conclusion, calprotectin is a reliable and promising inflammation biomarker for several diseases and that’s why we chose calprotectin as our target biomarker.
To detect calprotectin, we plan to use the SaeRS system from Staphylococcus aureus. It is a two-component system that responds to human neutrophil peptides(HNP), calprotectin, and other components of the human immune system. Our project will use this TCS system to sense biomarkers and trigger further downstream gene circuits.
Hesperetin has been shown to have various benefits against inflammation, cardiovascular disease, and cancer. However, the ability of the human body to produce hesperetin is relatively low. Therefore, we wanted to figure out how to increase the rate of hesperetin production as a response to inflammatory signals.
To increase hesperetin production and help people avoid chronic inflammation, we propose three approaches.
The human gut microbiome is capable of producing hesperetin itself from hesperidin. Important microbial species including Bifidobacterium spp., Lactobacillus spp., etc. can perform this reaction. Furthermore, this information is mainly derived by the α-rhamnosidase. Therefore, we plan to clone the α-rhamnosidase gene into E. coli and try to increase its catalytic performance by adjusting the transcription level or modifying the protein structure.
In addition to rhamnoside, since the glucoside residues of hesperidin form a rutin structure, glucosidase is also required for converting hesperidin to hesperetin. Therefore, as a combination of rhamnosidase and glucosidase, rutinosidase is one of our candidate enzymes.
Hesperetin can be produced not only from hesperidin but also from other flavonoids such as naringin. The reaction from naringenin to hesperetin includes two steps, 3'-hydroxylation and 4'-O-methylation. The flavonoid 3'-hydroxylase, cytochrome P450 reductase, flavonoid 4'-O - Four enzymes, methyltransferase and methionine kinase are involved in the reactions We plan to clone these enzymes into E. coli to see whether this approach can improve the production rate of hesperetin or not.
Fluorescent Proteins (FPs) are a class of proteins that can absorb and emit light of specific wavelengths, resulting in vibrant fluorescence. They are powerful molecular tools that have been widely used to visualize and track the dynamics, localization, and expression of specific proteins in the molecular biology field. Some commonly used FPs include mCherry, AmilCP and mCerulean, which emit red, blue and cyan light respectively. We plan to use FPs as a reporter to represent the detection of calprotectin. Furthermore, we will design the experiments to elucidate the number of biomarkers.
Extracellular electron transfer (EET) is an anaerobic respiration process that couples carbon oxidation to the reduction of metal species, such as Fe3+、Mn5+. Shewanella oneidensis, Geobacter sulfurreducens and some of the bacteria utilize EET as their metabolism mechanism.
Geobacter sulfurreducens has electrically conductive pili(E-pili) on its cell surface, which are involved in long-range biological electron transfer and metal oxide reduction. Moreover, it is more efficient for electron transfer than the ones without E-pili. Therefore, we originally intended to transform a part of c-type cytochromes gene regarding E-pili expression into E.coli. However, due to the harsh requirement for cultivating Geobacter sulfurreducens, we decided to utilize the EET mechanism from Shewanella oneidensis instead.
In our project, We co-transform MtrCAB and ccmA-H plasmids into E.coli to construct our electrical signal reporting system. MtrA、MtrB and MtrC are the c-type cytochromes from Shewanella oneidensis for electron transfer. CcmA-H plasmid facilitate cytochrome maturation. Cytochrome maturation is a post-translational process involves covalent attachment of heme to the apocytochrome polypeptide by two thioether bonds. Therefore, MtrCAB will be workable after cytochrome maturation, and electrons will be transported from MtrA, MtrB, MtrC in order.
When the engineered E. coli detects the disease biomarker, then activates the downstream pathway and transports the electrons through EET to the extracellular surroundings. The electrons are accepted by our electrodes linked to cyclic voltammetry to test the current intensity. The current signal from EET is collected by the device we design and send to a mobile device, allowing us to monitor the patient's health status.
In this part, our ultimate goal is to detect disease biomarkers and utilize an EET system constructed in E. coli to release electrical signals outside the cell, allowing the analysis results to be delivered to our designed application. The testing results would be sent to the application installed on the user’s electronic device and uploaded to cloud databases of medical facilities. This can help users to track their health conditions, thus beneficial for doctors to observe patients' situations in an early time. With the advantage of faster detection and more accuracy of the results, it turns over a new leaf in diagnostics.
Our saliva health detection device has the potential to revolutionize the field of diagnosis. Meticulously conceived, it takes into account crucial factors such as portability and user-friendliness, making it accessible to a diverse user base. An outstanding feature of this device is its replaceable cartridge, which exhibits adaptability to a range of cartridges and detection methods, allowing the identification of various health indicators in saliva with remarkable versatility.
By incorporating cutting-edge hardware components, including a precision ADC, amplifier circuit, and visual-based fluorescence protein detection system, the device could ensure accurate and precise measurements of health parameters. Leveraging sophisticated hardware and software denoising algorithms, it enables robust signal processing, resulting in reliable data. The inclusion of multiple short-spectrum light sources and light intensity/color detectors enhances the device's analytical capacity for fluorescent protein, facilitating comprehensive health parameter analysis.
The accompanying software features an online database that enables real-time monitoring and tracking of health status. Paired with a sophisticated data analysis platform, users can gain valuable insights into their health trends. The interactive web app provides a user-friendly interface for convenient access to data and supports personalized interpretations. Furthermore, the device incorporates a warning system that promptly alerts users to any concerning trends or abnormalities, empowering them to take proactive healthcare measures.
This saliva health detection device represents a significant leap forward from current solutions. Its inherent portability, intuitive design, and comprehensive capabilities make it an invaluable tool for both individuals and healthcare professionals. By facilitating convenient and reliable health status tracking, this device empowers users to adopt proactive measures in managing their well-being. The potential for early detection of various health conditions would be greatly enhanced, enabling timely interventions, improved treatment outcomes, and ultimately, a notable advancement in public health.
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