Team:GreatBay United/Implementation

• Home
• Project
  1. Background
  2. Description
  3. Design
  4. Contribution
• Wetlab
  1. Proof of Concepts
  2. Engineering
  3. Result
  4. Measurements
  5. Parts
  6. Safety
  7. Lab Notebook
• Drylab
  1. Hardware
  2. Modeling
  3. Proposed Implementation
  4. Supporting Entrepreneurship
  5. Sustainability
• Human practices
  1. Integrated Human Practice
  2. Education
  3. Education and Communication
  4. Excellence in Other Area
• Team
  1. Team
  2. Attibution
  3. Collaboration
  4. Partnership

• Bacterial Infection
• Policies & Effect
• Our Original Purpose
• Safety
• Testing
• Further Extension

Implementation

Bacterial Infection:

At present, bacterial endotoxin infection has become a major problem in medical industry. The harmfulness of gram-negative bacterial infection is mainly due to the release of endotoxin. After entering the body, bacterial endotoxin induce the production of inflammatory mediators such as cytokines and adhesion molecules, resulting in endothelial cell injury and barrier function change, leading to systemic inflammatory response syndrome and sepsis. In severe cases, it can lead to hypotension, toxic shock, multiple organ failure (MOF) and even death.

Policies & Effect

Now there is an efficient and convenient method to detect the presence of bacterial endotoxin - LAL test. From the moment it was discovered, limulus agent has been widely used in a variety of possible detection of bacteria in the field. The demand for limulus reagent has been increasing over time. LAL testing is required for every drug licensed by the US Food and Drug Administration (FDA).

Limulus reagent is extracted from limulus blood, so the blood of horseshoe crab plays a pivotal role in the modern biological industry and its value seems to be irreplaceable in a period of time. The increase of limulus reagent demand undoubtedly leads to the demand increase of limulus blood, which threatens the lives of horseshoe crabs. But if the horseshoe crab disappears, it will have a huge impact on the modern biological industry.

Problems of attention and the huge testing requirements are in stark contrast is the lack of correlated detection technology. Although there are relatively well-functioning substitute of limulus reagent, but the bacterial endotoxin testing method such as recombinant C factor requires hours for incubation. ] It also depends on specific temperature for preservation, and needs to be operated in the laboratory, which means the limitation of the site of use.

There's a letter written by Zhanjiang A & C Biological LTD., the main idea is to inform the customers that their access to limulus has been largely reduced, and therefore the production of LAL would decrease steadily.

If we can develop an efficient artificial limulus reagent, not only can we reduce the probability of death due to infection of bacterial endotoxin, but also can save an endangered species in the future, which is of far-reaching significance.

Our Original Purpose

The whole system consists of three parts - detection system, cascade system and agglutination system. Agglutination system plays a role in showing the detection result. Considering the complexity of result presentation form of the existing endotoxin testing methods (recombinant C factor), we decided to use agglutination reaction to visualize the detection result, inspired by blood group test paper and limulus amebocyte lysate. In order to achieve this, we decided to use yeasts to build our own agglutination system. The yeast surface display technology is mature and helped us achieve cell-level agglutination, which results in results being seen by bare eyes easily.


Our tests verified that our agglutination system design is functional and feasible. These tests had confirmed that the agglutination system can work successfully. When HCV enzymes appear in the reaction system, agglutination will occur. .

Our hardware consists of two cubic receptacles binding together. Every plane is stuck together with waterproof glue to protect the electric circuits. The small, pink container is the water bath with a temperature sensor that detects the water temperature. The bottom of the water bath is a heater that heats the water and maintains water at optimum temperature. The lid has four holes in which four test tubes are placed. The four test tubes form the experiment group, repetition group, positive contrast group, and negative contrast group to reach the highest accuracy available. The external container mainly stores the processing unit and the monitor which shows the current water temperature to the user. This design helps the user to control the water temperature, which helps increase testing efficiency.

Safety:

Caring about the safety, we conducted the experiment based on the LPS mimic domains instead of real bacteria. This provided our team members and other teams that shared a same laboratory with us with a safe and regulated environment. Nobody needed to worry about bacteria leakage or any other circumstances that might disobey the rule of safety. All of the reagents that are used in the whole experiment do no harm to both the humans and the environment. Placed in the hardware before being used, the reagents are stored in tubes which are highly sealed. During the use of the hardware, following the instructions strictly, patients will not have chances to expose themselves to the reagents directly. This means that as long as they do not put their fingers in the tubes, they will not touch the reagents inside. The major components of the reagents consist of proteins, which can undergo denaturation automatically. This means that the reagents inside the tubes will not pollute the environment and harm humans even if they are accidentally leaked. No bacteria or any components that might cause infections are included in the tubes. However, we do recommend users to follow the rule of recycling strictly and dispose the used hardware according to local rule of rubbish categorization, because this helps save energy and resources, as what we expect in the sustainability part of our work.

Treating potential risks when utilizing electricity: <.br> Being concerned about the safety of using the hardware, which requires electricity to heat the water to the expected temperature, we looked into adapters and metal thermometers with different sizes and voltages. After careful calculations, shown in the modelling page, we decided to use the adapter with 12V and 2A. The maximum value of current that can be used in the hardware is 3A, so using a 2A adapter can ensure the safety of using electricity while ensuring the heating process does not last for too long. However, we do recommend users to use the hardware properly by following the safety rule of using electricity, as the same as how they use daily electronic appliances such as televisions and freezers.

Testing:

Currently, production of limulus reagent for endotoxin testing has been discontinued due to environmental concerns, and an alternative method is needed. GBU team wants to create an endotoxin testing system that people can test for even at home. Current alternatives require both reagents and expensive testing equipment. For example, recombinant factor C requires fluorescence to monitor product concentrations.

Scenarios - At Home

Because of the prevalence of bacterial infection, tests for bacterial infection are needed urgently. However, evidently people cannot detect endotoxins by these methods easily (for example, the recombinant C factor requires a fluorescence instrument to monitor the product concentration). So team GBU aimed at finding a way suitable for users at home, and we developed a system that used yeast surfaces to display agglutination that mimicking the natural limulus lysate.

Introduction-Intention:

Currently, limulus lysates used for endotoxin testing are discontinued due to environmental concerns, and an alternative method is needed. The GBU hopes to create an endotoxin testing system that will allow people to test for endotoxins at home, to see if food has excessive endotoxins, or if they have a bacterial infection themselves. However, current alternatives require both reagents and expensive testing equipment. So GBU developed a system that used yeast to display agglutination that mimicking the natural limulus lysate, but this reaction needs to be performed at a suitable temperature, usually in a water bath. In order to realize home testing

Introduction-Components of Our Hardware:

The container lid has four holes to hold four centrifuge tubes. No. 1 is negative control, No. 2 is positive control, and No. 3 and No. 4 are used to detect endotocxin. The protruding part on the side of the lid makes it convenient to open the lid. The heating plate is put on the bottom. There are three holes in the wall, one for plugging the temperature probe, and the poles of the heating plate protrude from the other two holes.

Specification of Details

Video

Further Extension:

Concerning about the environment, governments of many countries have adapted policies to control the production of LAL

We can see from the letter from a biotechnology company Zhanjiang A&C Biological LTD,which is show on the right). And now, our project, the alternation for LAL, fits well into this trend, and fills in the blank of LAL caused by the prohibition. What’s more, owed much to the suggestions given by CCiC judges and Dr.Guan(the doctor we interviewed with), we have solved the problem about achieving constant temperature and distractions caused by protein impurity. In the future, we can adopt our product into a simpler structure, and then put into a commercial use.