Team:CSMU Taiwan/Design
Introduction
Nowadays, various public health problems regarding to Influenza control exists, varying from prediction, prevention, to screening and treatment.
The problems are shown in the table below:
| Screening | High false negativity High price High time cost Not enough added value |
|---|---|
| Prediction | Insufficient spatial resolution Insufficient time resolution Low diversity of data |
| Prevention | Unwanted price Lack of people’ compliance |
| Treatment | Side effect |
There are many advantages of the aptamer. If we utilize the features of the aptamer well, we can solve these problems.
Thus, we searched papers and brainstormed for designing a series of products combined with the aptamer that include all the aspects.
Features and advantages of the aptamer are listed below, and we also list the corresponding ideas of products that are very likely to implement.
| Advantge | Correspondong products | |
|---|---|---|
 screening |
Low False-negative Roate low cost Fast Production and Development More sample to get data |
Influsensor |
 Prediction |
High spatial solution by low cost and high stability High temporal solution by low cost and high stability |
Improvement of AI efficiency according to higher qualitied data |
 Prevention |
Low cost High Stability Animal Free |
Air purifier |
 Treatment |
Low antigenicity Low environmental pollution |
Anti-HA drug |
With the combination of listed products, we consider it as a system that can solve the problems below, strengthen the function of the four aspects, and connect the aspects efficiently. The systemis called CSMU_Taiwan influsystem.
The aspect with problems at most is the screening. Therefore, we consider the screening as a beachhead, using the rapid diagnostic strip as a start to design into aptamer-type strip, prove thefeasibility of utilizing the features of the aptamer, invade the potential market for rapid influenza diagnostic tests (please see our Entrepreneurship page),and develop our CSMU_Taiwan influsystem.
The rapid diagnostic kit we design is called Influsensor. We divide the design of Influsensor into three parts: strip, casing, and swab.
After the description of the three parts of Influsensor respectively, we explain the characteristics of Influsensor in detailed.
Finally, we describe every aspect of the CSMU_Taiwan influsystem. We also take avian flu as a example of developmental potential to embed into the CSMU_Taiwan influsystem.
Overview
We developed this rapid screening tool kit called influsensor. This tool kit contains aptamer-type rapid screening test strip and a specially designed throat swab.
For the strip, influsensor aims to distingush influenza A and influanza B, so there are three line on the strip which we will depict detailedly in the "Strip design".
there are two versions of influsensor: Easy-to-use version and Professional version
| Easy-to-use version of influsensor | Professional version of influsensor | |
|---|---|---|
| Composition | strip and simple casing | strip and casing with camera and computer cable |
| User | Doctors in countryside or developing country | Clinics and hospitals |
| Function | Rapid screening | Data uploading and precise surveillance |
| Advantage | fast and easy diagnosis | accurance, widening database |
Easy-to-use version of influsensor
There are three check points on the strip:
- The aptamer for detecting influenza A as test group
- The aptamer for detecting influenza B as test group
- The avidin for binding with biotin as control group. Because of the design of control group, we can assure that the reagent flow through the whole strip.
Professional version
The function of the outer casing makes two difference between the easy-to-use version and the professional version of influsensor:
First, there is a camera inside the casing of the professional version of influsensor that enable us to acquire the quantitative data and help doctors to acquire more precise results.
Second, the function of submitting the inspection data to the Cloud database in the casing of the professional version of influsensor enables us to obtain key data through the quantitative information and coordinates, effectively lowering the situations that doctors forget to report. Thus, the inspection data can be upload to the Cloud data base simultaneously after acquiring the result.
Strip design
Structure
The selected aptamers are labeled with biotins first because biotins bind streptavidins with high specificity and high affinity. We can make good use of this feature to design the control group of the rapid diagnostic strip, assuring whether the strip is functional or not.
Next,we make the selected aptamers labeled with biotins bind nanogolds thatl abel the aptamers with strawberry red. We can make good use of the feature of the nanogold to design the test group of the rapid diagnostic strip, indicating the result of positive and negative.
There are four components on the strip: sample pad, released pad, nitrocellulose paper and absorbent pad.
- Sample pad: The function of sample pad is to absorb the sample.
- Released pad: The function of the released pad is to mix the complex of nanogold and aptamer labeled with biotin( hereinafter referred to as complex) when the sample flows through the pad.
- Nitrocellusose paper: Nitrocellulose paperis the main active site for the complex to react and the result of testing will be shownin this area.
- Absorbent pad: The function of the absorbent pad is to absorb the liquid, resulting in a continuous flow from the sample pad to the absorbent pad which means the sample flows through the nitrocellulose paper.
About the nitrocellulose paper, we explain more in details. There two kinds of line designed on the nitrocellulose paper for showing the result: the test line and the control line.
The test line is coated with complementary DNAs of the selected aptamer for binding the complex. The test line will show color reaction because of the binding, indicating the result of negativity.
The control line is coated with streptavidins that can bind with biotins with high specificity and high affinity, so complexes will bind streptavidins.The control line always shows color reaction, indicating the sample flow through the whole strip indeed.
The mechanism of strips:
Negative:
The aptamers in the solution will bind with the complementary DNA at the check point of test group, and the biotin which labeling aptamer will bind with the avidin at the control group.
The result of negative displays two lines because the aptamer is carried with nanogold.
Positive:
In the existence of influenza virus, the aptamers are bond with the virus.
This situation leads to the configuration change of aptamers and the unbinding of complementary DNA at the check point of test group.
However, the biotin will still bind with the avidin at the check point of control group.
Thus, the result of positive displays only one line.
Casing design
In the purpose to
- Keep the loaded samples at a restricted area.
- Prevent the loaded samples from flowing away or spread into the environment.
- Prevent the samples from being contaminated.
- Create a clear user interface to observe the screening result.
- Mix the reagent and sample evenly
A case to hold the test scrip was designed according to these concepts.
Our casing includes three parts: the upper cover, lower casing, and a small plastic sheet.
Upper cover: Includes two openings respectively the sample loading area and observing window. (Purpose 4)
Lower casing: A restricted space to place the strip, limiting the loaded sample inside the case and keep the samples from being contaminated.(Purpose1,2,3)
Small plastic sheet: To pre-mix the reagent buffer and the sample collected evenly on the sample area first. Then, pull off this small plastic sheet so that the mixed sample- reagentcontacts the sample pad, then flow through the test strip to obtain the result.(Purpose 5)
The casing of the rapid diagnostic strip is 3D printed with PLA( polylactic acid) which is green material for the protection of the Earth.
The pictures below is the exhaustive calculation of the details on the casing.
Because we need precise assembly of the upper cover and lower casing, we asked CCU iGEM team to help us accurately 3D printing the casing we want.
The video is the 3D model of the casing constructed by CCU iGEM team.
This is the 3D printing casing!
Swab design
A swab was designed in the purpose to obtain sample from tonsil, which should be easy and safe to operate for users.
Our swab contains four parts: rigidly non-hygroscopic cotton, hollow tube, squeezing cap, and the squeezing cap protecting shell.
- Rigidly non-hygroscopic cotton→
Samples will stick onto the cotton if it was hydroscopic. Hence, the ideal cotton can’t be absorptive, but should possess high permeability so that the reagent buffer could flow through rapidly and thoroughly, taking the samples onto the sample pad of test strip. - Hollow tube→
To hold the reagent buffer(about 700ul). - Squeezing cap→
To squeeze the reagent buffer from the hollow tube and flow through the sample cotton. - Cap protecting shell→
To avoid accidentally squeezing out the reagent buffer before the sample was collected by adding a protecting shell onto the squeezing cap. Moreover, we hope that users could take out self-test, hence the swab was designed long enough (20cm) for people to reach into the tonsil easily.
Please check Demonstrationfor the User guide.The picture below is our draft of swab.
The picture below is assembly of swab.
The picture below is final product of swab.
Characteristic of influsensor
Low Cost, Fast Production and Development, High Stability, Low False-negative Rate, and Animal Free.
Low Cost
Description:
Aptamers are composed of nucleic acid, and are able to mass product via chemical synthesis.
In comparison, antibodies are protein, which can only mass product via biological synthesis. Since in most cases protein don’t express as expected, it takes more rounds of trials to debug. Moreover, the product usually contains an amount of bio-by-products since cell and animals are involved during the production, resulting in more procedures to purify it in order to meet the quality demand. Complicated steps indicates more cost, making the production of antibody expensive.
The advantage for the public health:
With the advantage of low cost, it means we can use the aptamer rapid diagnostic kit as a routine examination.
When we doubt that a patient is infected by influenza, the kit can be used as a primary diagnostic test to increase the spatial and time resolution of the data and prevent the spread of the virus under unclear clinical symptoms which are because of the differences of Individual physique.
The huge data which include the position and time can be submitted to the database as the use of epidemic prevention.Fast Production
Description:
The component of the aptamer is nucleic acid so that we can synthesize the aptamer through PCR.
However, antibodies are proteins that need the processes of cell culture, protein extraction, and protein purification.
That is the reason why the aptamers can be produced fast.
The advantage for the public health: For emerging infectious disease such as Ebola, the time cost of one month may pay the price of thousands of people’s lives.Fast Development
Description:
Aptamers are selected by the method of SELEX, so we can find out the target nucleic acid with only two weeks.
However, if we want specific antibody, we need to inject the antigen to the mouse. After taking out the spleen, make the B cells and the tumor cells to fuse together, then we can finally start to produce the antibody. It takes about three months to get the target antibody.
That is the reason why one of the advantages of the aptamer is fast development.
The advantage for the public health:
The advantage of fast development is the same as the fast production. For emerging infectious disease such as Ebola, the time cost of one month may pay the price of thousands of people’s lives.High Thermal Stability
Description:
The aptamer is a short DNA fragment so that it can afford the denaturation pressure of high temperature and turn back to functional structure after the ambient pressure is removed.
However, the antibody cannot turn back to functional structures after denaturing because its structure is protein.
The advantage for the public health:
With high thermal stability, the storage life of products can be extended and the amount of damage can be reduced. We can provide highly efficient medical care to the people lived in remote districts or under extraordinary circumstance.Low False-Negative Rate
Description:
The biggest problem of the antibody- type diagnostic is the high false-negative rate. The high affinity of the aptamer can effectively lower the possibility of false negatives.
The advantage for the public health:
We can reduce the potential infectious source to suppress the prevalence of the disease and acquire the accurate information for epidemic prevention.Animal Free
Description From the paragraph of Fast Production and Development, we know that the production of antibodies is using the spleen of mouse as raw material, and it will consume a large amount of animal’s lives.
As a medical product of the new era, when we use animals as tools for production, we should adopt the idea of animal welfare theory.
We should use animals as experiment materials only under necessary circumstance and should ease their pain.
Our product indeed reduce the unnecessary sacrifice of animal’s lives.
We achieve the goal of living in harmony with the animals.User’s manual
The Public:
Using rapid diagnostic strip as household appliance for prevention of influenza.
Uploading the inspection data to the Cloud.Doctors:
Using rapid diagnostic strip for diagnosing and preventing influenza.
Uploading the inspection data to the Cloud.Government:
Using rapid diagnostic strip, and ecological monitor of wetlands as long-term surveillance system.
Uploading the inspection data to the Cloud.
CSMU_Taiwan Influsystem
Screening
Because we have described it above, this part will tell about the advantage of aptamer comparing with antibody.
| Characteristic of Product | Aptamer | Antibody |
|---|---|---|
| Cost | Low | High |
| Speed of Production and Development | Fast(1-2week) | Slow (12 weeks) |
| Stability | High | Low |
| Inspection time | 5 minutes | 10-15 minutes |
| False-negative rate | Low | High |
| Using animals as material | No | Yes |
Prediction
AI
Background
The region with traffic congestion can be the breeding sites of viruses. If we want to predict the outbreak of the disease, we need data with high resolution to reduce the amount of incorrect information.
Efficacy
By the location that virus rage through currently to judge which region the virus might emerge. For example, if we find the influenza virus emerging in certain area of a town, there is a chance that the disease breaks out in other towns or countries nearby. Thus, we can take preventive measures to prevent the outbreak.
Design
Upper Figure: Arrange all the collected data into three dimension graphics: The X-Y coordinates represent the spatial distribution, whereas the Z coordinate represents the timeline. Fill in the missing data, in other words, the data in the future by using Image Inpainting. The future is then predicted.
Lower Figure: The upper part of the picture can be stimulated when the lower part of the picture is given.
The core concept:
It originates from GAN (Generative Adversarial Network)— By allowing it to identify the features of disease transmission pathway from the given data, to further create and distinguish three-dimension graphics. It includes two parts: Generator and Discriminator.
Generator:
Creates data from latent variable while applying the concept of Convolutional Neural Networks to analyze the graphics. The purpose was to confuse the Discriminator from distinguishing the created data and the actual data.
Discriminator:
Determines whether the figure is an actual data or not in the purpose of excluding the data created by Generator.
During this process, Discriminator summarizes the features among these data, for example the point, line , plane, or margin. The association between these features were then analyized by using Fully Connected Layer. For example, when a flu outbreaks in one town, how high is the possibility that it would trigger another outbreak in another town.
Another application during the learning process of GAN is translator, which connects the latent variable of Generator and the features Discriminator concluded, establishing the association between features and latent variable.
Finally
the system was developed into:
The Discriminator analyzes the graphic→The features were sent to the Generator via the translator→Superpose the features of latent variable together. →Superpose another random vector →Create variable possible future graghics.
The advantage for the public health
Intensive data base enables the prediction of affect an area could cause to the next area when a specific characteristic appears.
In our project, the advantage is that it is able to predict the next outbreak area without virus latent, hence the government could implement containment action such as injecting vaccine.
This is especially useful for countries with high popularity density: when it encounters influenza, they can use the predict system and spend money in the critical areas, thus reducing medical expenditure.
Furthermore, medical deficiency areas could be detected by this method, and amend the loophole before an outbreak. In addition, the virus outbreak origin could be calculated when the database was giant.
User’s manual
Government:
Using AI to predict the subsequent flu trend of expandingby utilizing the Cloud database of influenza.
Prevention
Air purifier
Background
The outbreak of flu usually starts from a group of people with weak immune system, such as the people at school, hospital, and nursing home. Besides, the latent period of virus is 2 days. The viruses was spread before the symptom appear, and the flu outbreaks. If we can find out the virus in the latent period, we can surely prevent the outbreak of a flu epidemic. That is why we design the air purifier which can detect the influenza virus promptly.
Design
Inside the air purifier, we can place the chip of aptamer that can sense the influenza virus. When the droplets with virus are in the air, the virus will connect the aptamer on the chip, and then the connection will change the electrical characteristics such as changing the resistance of the resistor. The air purifier will tell the users that there are viruses in the environment and take preventive measures against the virus.
Advantage
We can combine the data collected from the air purifier with the Cloud database. In contrast to the inspection data, the locale data include the data of the range of clinics. The data of fixed surveillance system (spot monitoring) can also be used for another aspect of analyzing. With these two kinds of data, we can give abundant information to the Cloud database for acquiring more precise prediction.
User’s manual
The Public:
Using air purifieras
household appliance for prevention of influenza.
Doctors:
Using air purifierfor
preventing influenza and administer the prophylactic drug.
Treatment
Anti-HA drug
Background
If the influenza virus want to invade cells, they all need a common key to open the door of cells. The key is hemagglutinin (HA). Therefore, if we can block the hemagglutinin of the influenza virus, we can stop the invasion of virus and prevent the expansion of the disease.
Design
We use the method of SELEX (systematic evolution of ligands by exponential enrichment) to select out the aptamer for H1 and H3. The way that we test the efficacy is observing the level that the aptamer affect the TCID50 of the virus. The efficacy testing method is hemagglutination assay. Using different concentration of the aptamer to mix with quantified amount of the virus and observe the level of hemagglutination.
Advantage
Compared with the intracellular drugs, the anti-HA drug composed of aptamer act extracellularly so that we can reduce the side effects of biochemical metabolism that occur in most of the drugs. In addition, drugs are located in the body fluid, so we also take the allergy problem into consideration. It is the advantage of the aptamer! When it comes to general medicine and therapeutic antibody, they are all macromolecular complex compounds. By contrast, the molecular weight of the aptamer is small and DNA is a common substance in the body, so the antigenicity of the aptamer is low. Therefore, the possibility of having side effects is reduced significantly when compared with general medicine.
User’s manual
Doctors:
Using Anti-HA drugto
save the patient with lower cost.
Developmental potential
other application of aptamer
Anti-virus spray
Background
The influenza virus is usually spread by the way of disease cluster in the enclosed places such as hospitals, schools, and offices. If we can stop the invasion of the virus, we can reduce the possibility of the outbreak. That is the reason we have the idea of anti-virus spray.
Design
Select out the aptamer for the hemagglutinin of influenza then add them into the water. We can sprinkle the droplets containing the aptamer by sprayers or air coolers. When the droplets meet the virus in the air, the aptamer can bind the hemagglutinin of influenza, which means we destroy the key for virus to invade and inactivate the virus, achieving the efficacy for preventing the disease.
Advantage
General disinfectants cannot be sprayed into the air for preventing. Take alcohol as example, the nature of flammability may cause dangers. Take liquid bleach as another example, the high toxicity will stimulate the respiratory tract. Yet, the aptamer we selected is just a fragment of DNA. The aptamer will affect the virus only and there is no harm for human beings.
Obstacle
When releasing the free DNA fragment, we cannot assure the influence of the enviornment. However, we consider the product promising as long as the safety problem can be solved.
Broad application of aptamer
Taking influenza as a start, we can construct a medical system based on aptamers. We can expand our system horizontally and vertically. In horizontal aspect, we can expand the system to different species. In vertical aspect, we can develop aptamer-based systems of various kinds of diseases. Highly efficient suppression of species originally should be the natural enemy of creatures, mostly various kinds of disease. Diseases even attack the conservative target of different kinds of species to solve the problem of the competition relationship between hosts and parasites. The Red Queen hypothesis says “Now, here, you see, it takes all the running you can do, to keep in the same place. If you want to get somewhere else, you must run at least twice as fast as that!”
That is the concept!
We take avian flu as example below.
Background
We can classify the bird species into two group: wild fowl and poultry.
Wild fowl:
We can classify the wild fowl into the groups of migratory birds and resident birds.
For migratory birds, they are the source of introducing new virus gene, causing other species of birds transforming the low pathogenic avian influenza strain into highly pathogenic avian influenza strain.
For resident birds, they act as the bridge between the migratory birds and the poultry. They are also the source for viruses to recombine their genes.
Poultry:
We can classify the poultry into the groups of waterfowl and terrestrial bird.
For waterfowl, they are hosts for storing the virus and usually appear asymptomatically. Therefore, it is difficult for veterinarians to find out, and the disease spreads silently. What is worse is that the breeding equipment of waterfowl are displayed openly, so that the waterfowl contacts the wild birds more frequently and increase the possibility of spreading the disease. Moreover, waterfowls are like a culture room of viruses because of the low surveillance ratio.
For terrestrial bird, they are the primary victim that we care about. It always cause a large amount of deaths when breaking out the avian flu. Because of the enclosed environment for breeding the terrestrial bird such as chicken, they are not easy to be infected by the wild birds. The main route of infection is because of the individual causes, such as the cross contamination of feed carriers and transport vehicles. It is difficult to judge which one is the route of infection because the complicated interaction. Furthermore, the labor cost and time cost of screening are considerably high. It is hard to do the general survey, causing the leakage of epidemic prevention.
Screening
Poultry:
The government can regulate the host of hennery to set the air purifier on the fence. We can detect the droplets of the chicken and upload the inspection data to the Cloud database simultaneously. Not only we avoid the inconvenience of census and the asymptomatic infection spreading, but we acquire higher quality data for analyzing subsequently.
Migratory bird
:
The aim to inspect the group of migratory bird is collecting the strains of the virus. Usually, only 2 to 3 percent of the stool samples collected in traditional way are positive. Applying the model of rapid screening can significantly lower the transportation cost, time cost, and the time pressure for the referral laboratory.
Prediction
We can collect the data from henneries and construct a three-dimensional graphic with a number of cases and a timeline. Afterwards, we can get the characteristic through AI and predict every possible situation. In conclusion, when a hennery have cases of avian flu, we can know how many henneries are in the high risk to be infected. Cleaning, disinfecting, or taking the prevention measure below are the method we can use to minimize the loss of henneries.
Prevention
Anti-virus spray: In the high risk period, we can spray the droplets containing selected aptamer into the air to stop the invasion of the avian influenza and reduce the use of antibiotic prophylaxis. As a result, we can prevent the generation of the antibiotic resistant bacteria and make chickens become healthier.
Treatment
For the epidemic prevention in animals, treatment is not the main point because of the high cost.
References and sources:
1. Wu S, Liu L, Duan N, Li Q, Zhou Y, Wang Z. Aptamer-Based Lateral Flow Test Strip for Rapid Detection of Zearalenone in Corn Samples. Journal of Agricultural and Food Chemistry 2018 Feb 28;66(8):1949-1954. doi:0.1021/acs.jafc.7b05326. Epub 2018 Feb 20.
2. Li J, Jing L, Song Y, Zhang J, Chen Q, Wang B, Xia X, Han Q. Rapid Detection of Rongalite via a Sandwich Lateral Flow Strip Assay Using a Pair of Aptamers.Nanoscale Research Letters 2018 Sep24;13(1):296. Doi:110.1186/s11671-018-2709-9.
3. Yanhong Zeng, Jianlong Fu, Hongyang Chao, Baining Guo Learning Pyramid-Context Encoder Network for High-Quality Image Inpainting Cornell University Submitted on 16 Apr 2019 (v1), last revised 11 Jul 2019 (this version, v4)
4. Alec Radford, Luke Metz, Soumith Chintala Unsupervised Representation Learning with Deep Convolutional Generative Adversarial Networks Cornell University Submitted on 19 Nov 2015 (v1), last revised 7 Jan 2016 (this version, v2)
5. Song W, Li H, Liang H, Qiang W, Xu D. Disposable electrochemical aptasensor array by using in situ DNA hybridization inducing silver nanoparticles aggregate for signal amplification. Analytical Chemistry 2014 Mar 4;86(5):2775-83. doi: 10.1021/ac500011k. Epub 2014 Feb 14.
6. Giamberardino A1, Labib M, Hassan EM, Tetro JA, Springthorpe S, Sattar SA, Berezovski MV, DeRosa MC. Ultrasensitive norovirus detection using DNA aptasensor technology. PLOS ONE 2013 Nov 14;8(11):e79087. doi: 10.1371/journal.pone.0079087. eCollection 2013.
7. Kratschmer C1, Levy M1. Effect of Chemical Modifications on Aptamer Stability in Serum Nucleic Acid Therapeutics 2017 Dec;27(6):335-344. doi: 10.1089/nat.2017.0680. Epub 2017 Sep 25.
8. Li W, Feng X, Yan X, Liu K, Deng L. A DNA Aptamer Against Influenza A Virus: An Effective Inhibitor to the Hemagglutinin-Glycan Interactions. Nucleic Acid Therapeutics 2016 Jun;26(3):166-72. doi: 10.1089/nat.2015.0564. Epub 2016 Feb 23.
9.Marangoni K, Neves AF, Rocha RM, Faria PR, Alves PT, Souza AG, Fujimura PT, Santos FA, Araújo TG, Ward LS, Goulart LR. Prostate-specific RNA aptamer: promising nucleic acid antibody-like cancer detection.Scientific Reports 2015 Jul 15;5:12090. doi: 10.1038/srep12090.