A local problem

We are fully aware of the current status of health checks for the population. Regular physical health check-ups are an important way to prevent diseases. However, resources such as hospital medical check-up centers and qualified third-party organizations are limited, and the process of booking and waiting for scheduling is cumbersome and puts greater financial pressure on residents.

我们充分了解了居民健康检查的现状。常态化的身体健康检查是预防疾病的重要途径。然而，医院体检中心和有资质的第三方机构等资源有限，预约、等候排期等流程较繁琐，给居民的经济压力也较大。

At present, community health centers are unable to undertake the routine antibody testing programs in medical checkups, which are usually entrusted to qualified companies, with longer testing cycles and higher costs; and the main indicators of the routine health checkups for residents and home visits by family doctors implemented in the community are still limited to blood pressure and blood sugar.

目前，社区卫生服务中心无法承担体检中常规的抗体检测项目，这些项目通常被委托于有资质的公司，检测周期较长，费用较高；而在社区中推行的居民常规健康普查、家庭医生上门检查的主要指标都仍限于血压、血糖。

Antibody tests such as autoantibodies, antiphospholipid antibodies and immunoglobulin sets are commonly used in health examinations. For various reasons, assisted diagnostic techniques have not yet been widely used in health examinations. We hope that assisted diagnostic techniques will facilitate the advancement of health screening in the direction of being faster, more accessible, and less expensive. (See Human Practices)

在健康检查中，自身抗体、抗磷脂抗体、免疫球蛋白全套等抗体检测项目是常用的。由于各种原因，辅助诊断技术尚未在健康检查中广泛应用。我们希望辅助诊断技术能促进健康检查向更快捷、更易获得、更廉价的方向进步。（见Human Practices）

Why E-pili?

Geobacter is a class of rod-shaped Gram-negative anaerobic bacteria belonging to the δ-Proteobacteria class [4-6]. They are important dissimilatory iron-reducing bacteria [7]. In order to survive in harsh anaerobic environments, certain species of Geobacter can utilize extracellular metal ions as electron acceptors for respiration, maintaining their own metabolism. This extracellular electron transfer process relies on a unique type of conductive appendage known as "electrically conductive pili"[8]. Electrons are transmitted through fibrous conductive structures on the cell surface, often with nanoscale diameters but lengths extending to the micrometer range, capable of facilitating electron transfer[9,10].

地杆菌（Geobacter）是一类呈棒状的革兰氏阴性厌氧细菌，属于δ-变形菌纲[4-6],是一种重要的异化三价铁还原菌[7]。为了在严苛的缺氧环境中生活，一些种类的地杆菌可以利用细胞外的金属离子作为电子受体进行呼吸，维持自身的新陈代谢。而这种细胞外的电子传递过程需要一种特殊的导电菌毛来介导[5]。电子通过细胞表面具有导电能力的纤维状附着物进行传递.这些纤维的直径往往只有纳米级别，但是长度却可以达到微米级别，并拥有传递电子的能力，因而被命名为导电菌毛（E-pili）[9,10]。

For instance, the type IV pili of Geobacter metallireducens exhibit an exceptional electrical conductivity of up to 277 S/cm at pH 7 [11], making them the most conductive biological nanomaterials known to date. Within these conductive bacterial nanowires, there is a high density of aromatic groups, and the overlapping Π-Π orbitals of aromatic moieties imparts metallic conductivity to these organic materials [12].

例如金属还原地杆菌的Ⅳ型菌毛在pH=7时的电导率高达277S/CM[11]，是人类目前所发现的电导率最高的生物纳米材料。这种导电菌毛内部分布着高密度的芳香族基团，而这些芳香族基团的轨道重叠可以赋予这些有机材料类似金属的导电性[12]。

As an emerging biomaterial, it have been utilized in various projects within the iGEM competition. In 2017, Franconia harnessed the excellent biocompatibility of these nanowires, employing them as conductive medium for transmitting electrical signals in their biological synthetic muscles.In 2020, Greatbay_SZ and Links_China employed these nanowires to create an air humidity generator based on the spontaneous water gradient formation on their surface[13].

我们所选用的导电菌毛为来源于金属还原地杆菌的菌毛，后者作为一种近期被发现的导电菌毛所表现出的超高的电导率能够很好的适配我们开发高精度定量检测系统的需求。

This type of material is considered a revolutionary "green" nanomaterial. The energy required to produce these protein nanowires through microbial processes is over two orders of magnitude less than the energy needed to produce an equivalent mass of silicon or carbon nanowires. Moreover, the production process does not generate any toxic chemicals [10]. Additionally, this material is relatively robust while being biodegradable and exhibits excellent biocompatibility [11].

这种导电菌毛被认为是一种革命性的“绿色”纳米材料。用微生物生产这种蛋白质纳米线所需的能量比生产同质量硅纳米线或者碳纳米线要少两个数量级以上,而且生产的过程中不会产生任何有毒害作用的化学物质[10]。除此之外，这种材料在相对坚固耐用的同时可以被生物降解，并且具有非常不错的生物相容性[11]。

What truly inspires us among these features is its high sensitivity of electrical conductivity to surface charge distribution. Through literature research, we've found that protonation can significantly enhance the nanowires' conductivity. For instance, the conductivity of the e-pili of Geobacter sulfurreducens increased by 100 times when the pH was lowered from 10.5 to pH 2 [12]. Additionally, researchers have investigated the effects of protein tag modifications on the conductivity of nanowires at the monomer's C-terminal. The results showed that protein tag modifications (such as His tag and HA tag) did not affect the nanowires' conductive capability, but did induce changes in their conductivity [13].

而在这些特性中真正启发我们的是它的电导率对于其表面电荷的分布情况高度敏感。通过文献调研，我们发现质子化可以大幅度的提高菌毛的电导率，例如当pH值从10.5降低到pH值2时硫还原菌菌毛的电导率增加了100倍[12]。除此之外，有研究人员测试了在菌毛单体的C端进行蛋白标签修饰对其电导率的影响，最后发现蛋白标签修饰（例如His-tag和HA-tag）不会影响菌毛的导电能力但会使其的电导率发生变化[13]。

Inspired by the properties mentioned above and in conjunction with approaches taken by other research teams in developing detection systems using this material [14,15], we have modified the C-terminal of the pilus and this modification allows for specific binding to the target antibodies. While surface adsorption often induces changes in surface charge, what we need to do is to detect the changes in its electrical conductivity. Finally, by establishing a standard curve, we can achieve real-time quantitative detection of the target antibody.

通过以上两点性质的启发并结合一些其他团队利用这种材料开发检测系统的方案[14,15]，我们今年选择通过对菌毛的C端进行修饰的使其能够特异性结合待测的物质，而表面吸附物通常会引起表面电荷的变化，我们只用去检测因为这种表面电荷变化而产生的电导率的变化，结合实现测定的标准曲线就能实现对于待测物质的即时定量检测。

Plink！

Plink旨在将导电菌毛作为连接生物信息和电子信息的桥梁，开发出同时具有快速、简单、经济、灵敏、便携等特点的实时定量抗体检测系统。我们的系统具有以下优点：
首先，它将抗原-抗体的特异性结合事件转化为电信号，相较于显色和荧光法，我们得出的曲线包含更多信息，具有更好的质控能力和定量能力，对于微量待测物也更加敏感。
其次，菌毛是一种经济、绿色、安全的生物材料，其能被简单地大规模生产，在较为稳定的条件下可以被降解，且对人与环境的安全风险很低。我们的设计使得该菌毛具有良好的可拓展性， 其检测阈值、目标检测物质都可根据检测需求变化。
第三，电导率仪是一种原理简单而技术成熟的电子仪器，其体积可小至随身便携水平，造价相对低廉，操作十分简单，可兼容于社区卫生服务站的配置。

Plink aims to utilize e-pili as a Link between biological and electronic information, developing a real-time quantitative antibody detection system with features like rapid, user-friendly , cost-effective, sensitive and portable. Our system offers several advantages: Firstly, it translates the specific binding events of antigen-antibody interactions into electrical signals. Compared to colorimetric and fluorescent methods, the curves generated from the conductivity meter contain more information, providing better quality control and quantification capabilities. Secondly, e-pili are an economical, green and safe biological nanomaterials. They can be easily produced on a large scale and pose low safety risks to both humans and the environment. Our design allows these nanowires to be highly scalable with detection thresholds and target substances adaptable to various testing needs. Thirdly, the conductivity meter is a mature electronic instrument with a simple principle. It can be as small as a portable device, is relatively inexpensive, and has user-friendly operation. It can also be easily integrated into community health service centers. This year, our project aims to achieve the following three major objectives:
1.Establish an e-pili based antibody quantitative detection system.
2.Propose a realistic solution that can be safely implemented.
3.Further unlock the application potential of this material and promote interdisciplinary collaboration.

How will we achieve these goals？

For goal one

我们对金属还原地杆菌菌毛的C端进行了Snoop-tag修饰，使其能够被连接有抗原表位标签的Snoop-catcher捕获，从而使得其能够在正确组装的情况下获得特异性结合待测抗体的能力 （除此之外，这种设计赋予了我们的项目对于不同种类检测需求的 快速响应能力和极高的可拓展性）。这种结合会影响到其表面的电荷分布情况进而改变这种纳米材料的电导率。通过测量这种材料的电导率变化我们就能实现对于抗体的定量检测。 （To learn more：Design）

We have performed Snoop-tag modification on the C-terminal of the pillus, allowing them to be captured by Snoop-catcher which carrying antigen epitope tags. This modification enables specific binding to the target antibodies when properly assembled. Additionally, this design provides our project with the capability to rapidly respond to different types of detection needs and high scalability. This binding interaction affects the surface charge distribution, subsequently altering the electrical conductivity of the e-pili. By measuring changes in electrical conductivity, we can achieve quantitative antibody detection. （To learn more：Design）

For goal two

在经过了大量的社会调研工作后（To learn more：Human practices）我们决定将我们的系统作为一种为未来的大流行病“疾病X”提供快速诊断方案的可选方案。 为了实现这个目标我们设计了一套由一次性导电菌毛检测耗材和高精度电导率仪相结合的硬件XXX（这个地方可能还需要给硬件起个名字） （To learn more：Hardware）并计算了我们 的方案在各个方面相对于现存方案可能存在的优势（To learn more：Implementation）。除此之外，我们还着重关注了我们项目中存在的安全风险，并给出了对应的解决方案。 （To learn more：Safety）

After conducting extensive social research (To learn more: human practices), we have decided to position our system as an alternative solution for providing rapid diagnostic capabilities for future pandemics. To achieve this goal, we have designed a set of hardware consisting of e-pili testing consumables and a high-precision conductivity meter（To learn more:Hardware）.We have also calculated the potential advantages of our approach compared to existing solutions across various aspects(To learn more:Implementation).Additionally, we have emphasized our project's focus on addressing safety risks and provided corresponding solutions.(To learn more:Safety)

For goal Three

作为一种革命性的绿色纳米材料，目前限制其在其他领域大规模取得应用的主要原因有以下两个：
1）缺少大规模的生产和纯化菌毛方法。
2）现有的菌毛的电导率仍然满足不了广泛的需求。
针对前者，我们使用了需钠弧菌---目前已知生长速度最快的工程菌作为底盘生物， 通过使用Crisperi抑制其原有的菌毛表达以促成一种可以满足大规模应用的纯化方案的实现（To learn more：Design）。 针对后者，我们通过分子动力学建模建立菌毛四级结构模型并使用相关算法去发掘菌毛中电子传递的实际路线并予以优化，结合蛋白质半理性定向进化的方法， 我们有望可以在最小程度影响菌毛的正确组装的基础上得到具有更高电导率的菌毛。（To learn more：Model）

As a revolutionary green nanomaterial, there are two main obstacles currently limiting its widespread application:

1. Lack of large-scaleproduction and purification methods.
2. The electrical conductivity ofexisting e-pili still does not meet broad demands.

Addressing the first issue, we have employed a fast-growing engineered bacterium, Vibrio natriegens as the chassis organism. By utilizing CRISPRi , we inhibited its native type Ⅳ pili expression, enabling a purification approach suitable for large-scale applications.(To learn more:Design) For the second issue, we conducted molecular dynamics modeling to create a structural model of the e-pili. Using relevant algorithms, we explored the actual pathways of electron transfer within the e-pili and optimized them. With a combination of semi-rational protein evolution techniques, we aim to enhance the e-pili's electrical conductivity while minimally affecting their proper assembly.(To learn more:Model)

Steps of Plink

Future prospects

抗体检测是确定人体或环境样本内是否存在特定病原体或疾病标志物的重要工具。 一般地，相较于定性检测，定量检测往往更精确，具有更佳的敏感性和特异性。然而，定量检测往往需要更严格的环境， 更复杂精密的设备，以及专业的操作人员。对于许多相对欠发达地区，卫生条件对居民健康造成威胁，而资源的缺乏和多种社会文化因素使得抗体检测技术运用困难。
本项目致力于为抗体的定量检测提供一种低成本的替代方案，通过利用导电菌毛自身的电导率随其表面电荷分布情况的变化这一特点， 将菌毛自身的电导率和待测分子的浓度相挂钩从而使得我们可以使用更加容易操作且价格低廉的电导率仪来进行定量。 这一原理上的转化有助于实现疾病标志物的定量检测技术的“前置”，从而使得原先不具有精确定量能力的个体或者组织能够获得该能力，从而促进人民的卫生健康公平。
由于项目的周期和我们团队的能力的有限，距离真正实现该目标还有非常远的距离。 但我们相信，今年我们的项目的设计和对于导电菌毛这一绿色纳米材料的应用潜力的解放，将会为后面的团队带来启迪。

Antibody detection is an important tool for determining the presence of specific pathogens or disease markers in human or environmental samples. Generally, quantitative detection is often more accurate, sensitive, and specific compared to qualitative detection. However, quantitative detection often requires stricter environmental conditions, more complex and precise equipment, as well as skilled operators. In many relatively underdeveloped areas, where health conditions pose a threat to residents, the lack of resources and various social and cultural factors make it difficult to apply antibody detection technology.
This project aims to provide a low-cost alternative for quantitative antibody detection by harnessing the property of conductive pili, which changes its electrical conductivity based on the surface charge distribution. By linking the pili's electrical conductivity to the concentration of the target molecule, we can use more easily accessible and cost-effective conductivity meters for quantitative measurements. This fundamental transformation in principle contributes to achieving the "Preposition" of quantitative detection technology for disease biomarkers. It allows individuals or organizations that previously lacked precise quantitative capabilities to acquire this ability, thus promoting fairness in public health.
Due to the limited project timeline and our team's capabilities, we are still far from fully realizing this goal. However, we believe that our project's design and the exploration of the application potential of e-pili as a green nanomaterial will inspire future teams.