Team:ASIJ Tokyo/Implementation
When we first proposed to construct a detection kit for breast cancer, we wanted to make sure that our detection kit would be implementable in the real-world market. Therefore, we used a combination of our human practice interviews and consultation with entrepreneurs to help shape and influence our project design.
Using Aptamers and AuNPs as Detection Component
To develop a convenient test kit, we first performed research on different ways to help detect our biomarkers. Through our literature review, we discovered the prominence of aptamers, which are short oligonucleotides that can bind to a specific target, in this case, our biomarker. Aptamers have been compared to antibodies in their ability to specifically bind to target molecules, and are significantly cheaper and convenient compared to conventional antibodies (McKeague, 2017).
With our discovery of aptamers, we then thought of methods of utilizing aptamers to help detect our biomarkers in a cell-free environment. We came across methods like using ELISA and measuring fluorescence intensity, however, many of these methods are not portable and requires the use of expensive experiment. For example, not only does ELISA require the use of a 96-well plate reader to measure for absorbance, but it also requires the use of antibodies which defeats the purpose of utilizing aptamers. These limitations led us to use gold nanoparticles, which are versatile devices for detection based on their aggregate and non-aggregate modes. When gold nanoparticles undergo a change in mode, this results in a change in the color of the nanoparticle solution. Therefore, we hypothesized that by monitoring the color change of our AuNP assay, we will be able to detect our biomarkers using aptamers.
Using colorimetric detection has many benefits that can aid with the convenience and implementation of our detection device. Our AuNP assay can be constructed by just incubating the aptamers with the AuNP overnight, before adding the biomarker of interest. Furthermore, we can eliminate the use of specified equipment from laboratories by combining our AuNPs with lateral flow immunoassay strips, which allows for a portable and low-cost detection system (Pan et al., 2018). We plan on developing software next year that can help quantify the concentration of biomarkers based on the color of the AuNP solution, which will help test for and indicate signs of breast cancer.
Figure 1: Four-Step Approach to Detection Kit Implementation(Aptamers, Gold Nanoparticles, Lateral Flow Test, Analysis Software)
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Sensitivity and Specificity
One of the largest concerns for our project was the issue of false positives and inaccurate diagnostic results. With breast cancer stigma being a huge issue in Japan, we wanted to make sure our detection kit followed local and global guidelines to create a convenient device for women around the world.
While we have not been able to test the sensitivity of our AuNP assay, for next year, we will be making sure that our system can detect up to 15.2 ng/mL of Her-2, and similar levels for Mucin-1 and Mammaglobin B(Zhang et al., 2020). Unfortunately, a set benchmark for the amount of Mucin-1 or Mammaglobin B that indicates breast cancer still does not exist, but we will adapt our assay to detect for both minuscule or large concentrations of the biomarkers to allow for its versatility.
Our planned tests for specificity and selection of aptamers allow for our test to only detect biomarkers specific to breast cancer. This minimizes the chances of false or inaccurate results as our aptamers bind specifically to our biomarkers and no other proteins. By selecting aptamers, we have not only developed a convenient way for detection but a method that is highly sensitive and specific.
Biomarker Collection Methods
When designing our testing device and our experiments, the first area of concern was our method of collection.
Clinical tests like mammograms or biopsies have proven to be extremely accurate and reliable for the detection of breast cancer. However, they are invasive and based on our extensive interviewing for human practices, many women are uncomfortable getting these tests and would prefer a more convenient method for detection. This is why we designed a testing kit that is convenient and portable to target our proposed audience, which was the main focus of our project.
Therefore, when we researched our biomarkers, we selected Mammaglobin B and Mucin 1. This is because Mammaglobin B is found in high levels in the lacrimal and salivary glands, while Mucin 1 is found in high levels in sweat gland cells. We hypothesized that since both biomarkers are present in sweat and tears, they are effective for a noninvasive method of breast cancer detection. Our final biomarker, HER-2, while found in blood, is an established biomarker with high efficiency, which is why we selected this as a comparison with Mucin-1 and Mammaglobin B.
Our idea for convenient detection was praised by Professor Kazunori Ikebukuro of Tokyo University of Agriculture and Science when we met with him to discuss our project idea, as he mentioned that some people are unwilling to undergo procedures such as the drawing of blood(even if it is painless) and that using alternatives such as detection through sweat and tears would be effective and life-changing. However, he cautioned us on methods of collection for sweat and tears, as biomarker concentrations can vary greatly. Based on his advice, we performed the following research for the collection of our biomarker in order to show how our project would be implemented in the market.
Collection of Mucin-1 Biomarker
Mucin-1 is found in high levels in sweat gland cells, and we hypothesize that through collecting sweat and analyzing for biomarker concentration, we will be able to detect levels of Mucin-1. Our research has shown that sweat can be collected through using pads or capillary tubing, and many commercial products exist on the market. Therefore, our solution is to collect sweat through a pad, filter the content, and using our Au-NP system to detect our biomarker.
Figure 2:Sweat Collection
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Collection of Mammaglobin-B Biomarker
Mammoglobin-B is found in high levels in salivary and lacrimal gland cells. We hypothesize two methods to help collect samples for Mammaglobin-B.
First, we can collect salivary samples. With the COVID-19 pandemic, methods for salivary sample collection are very convenient and accessible. Additionally, saliva collection can be done at home without the use of specialized equipment or personnel, allowing for convenient at-home detection. However, when designing our collection system, we may need to consider methods to purify or monitor the quality of samples. Among the different methods for the collection of our biomarker, we hypothesize that this has the most potential due to the existing methods on the market and the prominence of this testing method. Therefore, we propose using a saliva collection technique for our physical testing device.
Figure 3:Saliva Collection
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Another method would be to collect tears. However, this method has various limitations and considerations. First, depending on the type of tears, the content of biomarker may vary greatly and research on the content of Mammoglobin-B in lacrimal gland cells is still limited. In addition, while tear collection kits do exist on the market, they are not firmly established and there is a significant barrier with how to get individuals to cry to collect the tears. However, despite the limitations, we want to present this as a potential method for collecting our biomarker.
Figure 4:Tear Collection
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Collection of Her-2 Biomarker
The concentration of HER-2 can be measured through a biopsy, but recent research suggests that there is enough concentration of HER-2 to be detected through blood.
Therefore, in order to collect HER-2, we recommend using an at-home blood testing kit, which widely exists on the market. However, at-home blood sample collection is prone to danger and can be potentially a safety hazard if users do not follow the procedures correctly. Nevertheless, blood sample collection is a prominent method for breast cancer detection due to the abundance of painless methods that exist. Combined with our Au-NP system, the sample collected from blood can be used to detect the amount of HER-2, and the presence of breast cancer can be determined based on the benchmark for HER-2. Therefore, we also propose using a blood collection technique for our physical testing device.
Figure 5:Blood Collection
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At-Home Testing Kit
While our at-home detection device has not been designed, we envision that it will be used and implemented in the following manner.
First, with our collection process(blood or saliva), we will collect and purify our samples to be tested. These samples will be then added to our detection kit, which will consist of a tube that contains our aptamer-AuNP solutions that were synthesized prior to distribution.
Next, the user will add the sample to the Au-NP solution and add NaCl to induce the color change of the gold nanoparticles. By preparing the aptamer-AuNP solution beforehand, users will only have to do three steps—collect the sample through blood or saliva, add the sample to the prepared aptamer Au-NP solution, and add NaCl to induce the color change. These steps will take place in less than 10 minutes, allowing for quick and easy detection for users.
