Team:GCGS China/Implementation

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Implementation

Design concept

For testing the Pseudomonas aeruginosa on the fish, we decided to use a test paper strip to perform the test. The previous product based on the test paper strip can be mainly divided into three types, the color bar, test cardboard set, and test paper stick.

Among all the products, the color bar is used for the test paper strip based on the colorimetry method only, which means that the tested sample should be fluid and has a color (not transparent); and the using method of the test paper stick is to use the sample pad of the test paper strip to attach the sample directly, that require the sample has fluid property(permeable).

However, the signals molecules on the Pseudomonas aeruginosa on the fish are mainly concentrated on the flesh of fish, and it does not contain any fluid properties, thus it is uneasy to be collected, therefore the test cardboard set should be a better choice, and it has better compatibility to any conditions in comparisons.

At the same time, a convenient sampler is required to be designed, because only the fluid can diffuse to the result window, the sample should be soaked in liquid to mix the signals molecules and the liquid in the sampling bottle, we decided to use a sampling bottle with buffer and a sampling spoon. (shown in Figure 1)

Fig 1.The sampler

Design principle

There are several different kinds of testing methods, and we chose the test paper strip based on the competition law. The main principle of the competition law depends on the process of the competition between the C4-HSL molecules and the probe that is complementary to the aptamer.

After our early lab verification, we found the aptamer that has the function of specific binding to the C4-HSL. Then, we designed to add a base sequence of AAA AAA AAA AAA AAA AAA AA-3 at the end of the C4-HSL aptamer, and modified it with Thiol at the 5' end, making it better combine with the colloidal gold particles.

The test paper strip is made up of the absorbent bent, control line, test line, and sample pad. The sequence of the test line is the one that is complementary to the C4-HSL aptamer and modified with biotin. The sequence of the control lines is 5'-biotin-TTT TTT TTT TTT TTT TTT TT-3'. Treat the probes with streptavidin, stabilize them on the NC membrane (the sequence is listed below).

Drip the tested solution onto the sample pad, as the solution contains C4-HSL, the C4-HSL would combine with the aptamer followed by the change of the structure of the aptamer. Therefore as the solution diffuses to the test line, the solution would not bind to the probe on the test line, then the control line presents color; Conversely, as there are no C4-HSL molecules in the solution, the aptamer combines with the test line, and binds to the probe of the control line, two lines both show color. (the results shown in Fig.2)

Fig 2. The schematic diagram of the competition law(from Wang, Libing et al.)

The sequnces were:

• C4-HSL-apt: 5’-Thiol-CGCTGCCCCCTGTCCTGGGTTAGCTAGCGAGAGCG AAA AAA AAA AAA AAA AAA AA-3'
• Test probe: 5'-Biotin-（CH2)6-CGGTCACTAGTACACCGCACCAGAAGCGGGCCCCG-3'
• Control probe: 5'-Biotin-TTT TTT TTT TTT TTT TTT TT-3'

Target audience

The target audience for this product can be mainly divided into three categories. The first one is the seller, for example, the fish/ seafood seller, the seafood restaurant. They can use our product to ensure the flesh is disinfected and prevent any food safety issues. The second client can be the consumers, such as the family that buys and cooks seafood. Our product would be useful for them to use especially after being stored for a long time or after freezing. And the third is the professional food detection organization and individuals, such as the national and local food safety-related agencies, or the professors in food preservation. For them, our product can help them to accelerate their detection and obtain valid data.

Application scenarios

Our product is designed to achieve the purpose of rapid, convenient, and accurate detection of fish spoilage, thus the application scenario of our product would be some places that require it, for instance, the restaurants that serve raw fish, the process of food at home, nursing homes, etc. We recommend our clients to use our product before the selling and cooking of fish. (more information in Hardware)

Safety and risks

Although this product itself does not have a sharp design, the sample may contain Pseudomonas aeruginosa or other microorganisms, which may cause infection when contacted with the sample. Therefore, don't let children get in touch with the product while using it. Please seal each component of the product in a bag and throw it in the trash after using it to avoid other people or items coming in contact with bacteria. After that, please use alcohol to disinfect your hands. If the liquid in the sample bottle accidentally flows to the desktop or elsewhere, please use alcohol to disinfect.

Product package

Here is our package for the final product(Fig.3): the picture above is the packaging on the front of the product. The picture of fish in the upper left corner represents our test paper used for fish, and the blank part in the middle is a layer of transparent plastic, allowing consumers to see the test paper's appearance directly. The four letters GCGS at the bottom right represent our team. We also wrote what the product is and what it is used for at the bottom. These elements make it easy for consumers to understand what it is and highlight the passionate characteristics of our team with bright colors and artistic words. When we look down, the second one is the packaging on the back of our product. On the upper left, there is a large "caution" sign, which uses a large red font to warn people that the followings are pieces of must-know information; the following lines are the warning content, especially the use of capital words and add The bold font gave people a second warning. On the right is the appearance of the test paper when it is positive or negative. People can directly look at the package for comparison when using it. At the bottom left, the team and product use are once again written.

Fig 3.Package of our product

User's manual

Home-used Pseudomonas aeruginosa testing paper strip

Fish spoilage testing

User’s instruction

The product is mainly divided into two parts: sampling bottle and test board, used to detect whether the fish is infected the Pseudomonas aeruginosa, so as to determine whether the fish is edible.

Using steps

• 1.Pull out the cap of the sampling bottle, rotate the sampler out.
• 2.Slightly scrape the surface of the fish with the sampling spoon to obtain the sample.
• 3.Put the cap of the sampling bottle and shake the sampling bottle for several times to make the sample completely mixed with the buffer, left the liquid for five minutes.
• 4.Remove the cap, drop the solution into the sample port of the sample board.
• 5.Wait for the solution diffuses to the result window, then wait for 10 minutes, and observe the color change of the test paper. If there is only one red line presents in the observation port, the other red line (test line) gets lighter, it means that the fish has been inflected, and the fish is not recommended to eat.
• 6.If the control line is colorless, it means that the result is invalid, and need to be tested again.

Caution

The product is for one use only, please dispose it after use, and do not use it for a second time. The product may contain harmful chemicals. Avoid children to touch it.

Fig 4.The user's manual

References:

• Wang, L., et al., An aptamer-based chromatographic strip assay for sensitive toxin semi-quantitative detection. Biosens Bioelectron, 2011. 26(6): p. 3059-62.