Integrated Human Practice

We wanted to learn the current detection methods to detect or deal with heavy metals so that we can study from them. Professor Shan said there were mainly 2 methods.

Dry digestion method

Using high temperature to make organic matter ash, and then through solvent extraction, to eliminate other ion interference effects. When using the dry digestion method for sample processing, reagent consumption is less, organic matter decomposition is complete, and multiple samples can be processed at the same time. However, the dry digestion method takes longer than other methods, and due to the absorption effect of crucible heating on the measured components, the recovery of heavy metals is low, which affects the accuracy of the determination results.

Wet digestion method

The organic compounds in the samples were digested by concentrated nitric acid, potassium permanganate, hydrogen peroxide and other acid solvents and oxidants. Compared with the single acid digestion sample, the mixed acid digestion sample has less volatilization loss, a higher recovery rate and a more thorough decomposition. However, the reagent dosage is large and the use of strong acid and strong oxidant may lead to harmful gases.

Q: What aspects should be considered in advance from the initial product to the final product?
A: There are 3 major aspects.

Consider or understand whether the product is needed in the real world, such as the target group (farmers, government agencies, people in the home, different groups of people with different goals), the market, who will buy it, and the user group. Second, the market situation, the advantages and disadvantages of similar products and their own products focus on testing which products are sold, and how big the market is, how many annual sales, how many annual interest rates, the price. Third, assuming that the product has advantages, then how to replace our products, how to publicize the advantages of our products, promote to the market.

Fluorescent protein itself is not easy to distinguish its luminous intensity. If our live bacterial expression has the need to detect different concentrations, what methods can be used to quantify (or distinguish different levels of) concentration expression? Our idea is: is it possible to design a set of color cards to correspond to the fluorescence expression of different colors, so as to express the concentration through different colors?

It is better to quantify, which will be more difficult because there are many considerations. The very standardized fixed amount, reaction time and temperature, microbial state time, and the product form of freeze-dried powder should be fixed. The higher expression will be faster, and cheaper is the color card. The fluorescence detector will be more accurate, mainly depending on the demand. If it is a rural site, the fluorescence detector will be better, because it will always be used.

Q: The product we made this time needs to be expressed by living bacteria. In the case of high requirements for bacterial activity, what are the possible solutions to ensure the convenience of the product. at present, our idea is to freeze-dried bacteria. If low temperature is required, how to ensure the reaction expression efficiency of bacteria?
A: Live bacteria must be freeze-dried. Now engineering bacteria are used for testing, which is a rare thing. Except for testing, engineering bacteria are mainly medical, but they are also in the clinical stage. For safety and regulatory regard, we may have to deal with the government, because the use of engineering bacteria is still limited and regulated by policies. Freeze drying doesn’t mean completely stable, it varies by species, protective agents, temperatures, etc. It is quite complex. We can look at the acellular system, which is very similar to active dry yeast, seeming dead, but still working like a live bacterium. Without the shell of the cell, the contents are still functioning and safer.

Q: What factors determine the expression of signals and sensing sequences in the design of biosensors?
A: In general, the expression signal is mainly determined by the object you detect. For example, the object you detect is colored. For example, there is a period of green algae outbreak in Taihu Lake, and you need to check whether the water in Taihu Lake is polluted by this heavy metal. Well, you might want to consider using globin. Or use this so-called yellow fluorescent protein. In general, it's mainly because there are so many signals that you can choose from.What it does is it takes this so-called heavy metal ion to get into the active center of this protein, and then this protein it recognizes this particular sensing sequence that we have. In fact, the process of our improvement is a process of protein recognition of DV, or recognition of I, in general, you basically have what is called protein determination, which is determined by what you detect, so in other words these two factors are summed up.

Q: If the expression of biological inductors has cross-influence factors, let's say a lot of colors are mixed together, what method will we use to adjust?
A: This is actually one of the biggest problems that we often have when we're doing this so-called biosensor. I wanted to express this green fluorescent protein, but actually, your expression of this protein is not in a vacuum, it's in an aqueous solution of your test sample. The background of it. It's just a little bit yellowish, even if it's a little bit cloudy. Maybe sometimes it's not easy to get it out of the background.

My suggestion, you need something called a double base color. I used a red and I also used a blue, and you know red, yellow, and blue, and there's a lot of variations of the colors. Let's say you take that red and you take that yellow, and these are two colors cross over and it shows up as the new color, and you know that you can adjust the intensity of these two different fluorescent proteins, and it shows up as a new color. Until you have this new color that will make a significant difference from your base color. You have to tune it up some other way, remotely, to get it out.

Q: What we want to do is that the test box is a biological test paper, but we can only do qualitative not quantitative. And do you have any solutions or suggestions?
A: It should be said that most of test papers, are qualitative. This is a recognized problem in the industry, including our nucleic acid test strips. But if you're willing to take more time and pay more money on this development, and someone is willing to buy it, it might be achievable. Usually it has a linear growth up to a certain range. When the concentration of heavy metals is relatively low, let's say you make a co-factor, or you make the central structure of a protein an accessory muscle. The more proteins that are activated, the more it recognizes this sensibility, and then, the more intense the fluorescent protein will be expressed. It's a linear growth process. But when you overload the extra heavy metal elements, the fluorescent protein might show no difference or be gone since it reaches the inflection point, the flat. That's why all of our test strips have effective test range.

In addition, in the real world, you might need an additional part - the activated charcoal. It can help you adsorb those confusing colors of the water sample before we take the heavy metal detection. You can attach this part to your detector and make it small, simple and easy to handle.