Part 1: Overview of the product

With increasing number of patients being diagnosed with breast cancer worldwide, we aimed to design a biomarker detection kit to facilitate their early diagnosis and prognosis in order to reverse or, at least, mitigate the negative impact of breast cancer. In our project, we chose two circRNA biomarkers, hsa_circ_0001785 and hsa_circ_0001982, for the early diagnosis of breast cancer. In addition, hsa_circ_0001982 is also a biomarker for the prognosis of TNBC (triple negative breast cancer) patients for conventional chemotherapy. Our product utilizes a cell-free system containing all the necessary molecular machineries for a CRISPR-Cas12 system to detect the biomarkers. When a target circRNA is present in a patient’s sample such as blood or tissue, the CRISPR-Cas12 system will be activated and produce a fluorescent signal. Figure 1 shows how this system works. 

 

 

Figure 1: The graphical illustration of the CRISPR-Cas12a system used in our system

 

 

Part 2: End users

Our detection kit can detect hsa_circ_0001785 and hsa_circ_0001982.

 

Detection for hsa_circ_0001785 and hsa_circ_0001982 is useful for the general population to screen breast cancer, especially for the high-risk population such as those with a family history of breast cancer. A positive result of the kit indicates a huge possibility of early stage breast cancer and the user should immediately seek medical attention for further diagnosis and early treatment. Early diagnosis and early treatment are the keys to improving survival and ensuring quality of life for breast cancer patients.  

 

Detection for hsa_circ_0001982 is also valuable for TNBC patients. A positive result for TNBC patients means a poor prognosis with conventional chemotherapy and he/she is unlikely to benefit from this treatment. This can prevent TNBC patients from enduring unnecessary miserable side effects from a useless treatment and more importantly can lead physicians to choose other more efficient tailored treatments early for the patients before the cancer develops.

 

Therefore, the end users of this detection kit are mainly composed of general public and TNBC patients. Women population can especially benefit from our product, given the fact that 99% percent breast cancer incidence in America happens in women (Robinson, J., 2008). We hope that in the near future, this kit can save more breast cancer patients.

 

Part 3: Product Operation 

This kit includes a mixture of solution to initiate the reaction, a protection cover to avoid contamination from aerosol, and a device with a UV lamp and a shelf of 8 Eppendorf tubes. Hemostix and PVP-I solutions for taking blood samples should be prepared by users. The droppers (or Eppendorf pipette to achieve more accurate effect) should be used to transfer the liquid.

 

In general, this kit is a relatively safe and accurate product. And our device is a further proof for safety. The procedures for using this kit and specific warning points are addressed below.

 

First, users are supposed to use sterile hemostix to extract blood. This process could be operated by users themselves, while for elders, children, or coagulation disorder patients, it is better to be operated or supervised by professional doctors.

 

There are 8 tubes in the device for each test kit as shown in Graph 2. Negative and positive control samples are provided by the kit and should be added into Tube 1 to 4 (Graph 2). Blood samples should be added to Tube 5 to 8. The constituents of each reaction tube are listed in Figure 2. 

 

 

Figure 2: Ingrediants in the 8 reaction tubes provided by our kit

 

The user should homogenize the content inside the tubes, place the tubes in the shelf of the device (Figure 3), close the device and wait for about 15 minutes. Afterwards, the user can turn on the UV lamp to observe the results through the observation window of the device. The user can take a picture of the fluorescence and use softwares such as ImageJ to assist with the analysis.

 

 

Figure 3: 3D model of our device

 

It should be noticed that exposure to UV light can cause premature aging of the skin, eye problems, and signs of sun damage. Therefore, the device is offered to users to safely observe test results under UV light. Users must make sure the following things while using the hardware. First, users should check if the device is intact before use, especially the UV lamp and the observation window. Broken parts may leak UV light and cause damage. The observation glass and UV lamp need to be replaced if broken. Normal glass with thickness around 2-3cm can shield UV light and can be used for replacement.

 

In order to observe the test results immediately and accurately, the outline of the equipment should function well to ensure a dark environment for observation. Fluorescence signals of our kit under UV light are dim, so dark background can facilitate quick and precise detection of the signals.

 

Part 3: Real-World Implementation

In actual use, the main things to pay attention to are standardization and stability, because if you do not have a unified standard of operation, the results will be inconsistent, and the clinicians and the general public will be at a loss. The stability of the reagent kit is very important. Therefore, we set up positive and negative controls in our 8 tubes/strip to ensure the stability and standardization of the product. Furthermore, we installed four different tubes for sample testing using four different igRNA and dsDNA combinations to further confirm the reliability of the results.

 

The device can be made smaller and lighter for more convenience. And it can be made narrower for better fluorescence signal observation.

 

In practice, the most important thing to pay attention to is the processing steps before the sample is added to the system. Blood has many complex components, some of which may prevent the system from responding. Our project needs more experiments for real blood samples, such as experimenting with a gradient of circRNA concentrations in the actual blood samples. These experiments require animal models and human samples and should be conducted in compliance with ethical standards. However, such experiments are beyond the scope of our project

 

Part 4: Safety

The most accurate way people now use to diagnose breast cancer requires an invasive process called breast biopsy. Our product only needs a small amount of blood to complete the test, so it can be done in a non-invasive way and is quite safe.

However, we need to point out that after using the sample, the blood sample should go to a formal biochemical waste gather point to avoid harmful consequences. If the testers are unprofessional, they should be informed about these issues.

Furthermore, another thing to clarify is that the tester should avoid turning on the UV lamp when the device is not fully closed. Directly staring at the UV light without shield can cause damage to the skin and eyes of the tester.

Moreover, the tester should avoid direct contact with the reagents throughout the process. The contact can harm the tester and also may influence the testing results.  

 

Part 5: Challenges and prospect

This detection kit still needs optimization and further amelioration. In order to make it more convenient, time-conserving, and suitable for untrained people, we hope to make the kit in a test tube containing all gradients needed and can be preserved at room temperature demanding no special equipment for storage and deliver. This faces numerous challenges, for example, Cas12a protein needs a relatively strict environment to keep its activity, and it can be unstable and has a high probability to degrade at room temperature.

 

Therefore, we want to present a hypothesis to leave it for future teams who might be willing to work on this: to add all of the components in the system in an appropriate proportion and press them into dry powder. We were unable to bring it into life due to the budget and lack of technology. However, technically, when dripping treated blood into the dry powder, the system can get the moisture it needs and activate the whole process. This will make the whole process easier and therefore beneficial for rural areas where there are no labs and the residents are less educated.

 

Our second prospect for this detection kit is the addition of a fluorescence gradient reference to indicate the concentration of our biomarkers in the blood sample based on the intensity of the fluorescence. Due to the inability to get abundant real blood sample, we were unable to test and adjust. However, we believe it is a meaningful and beneficial work to be done by future iGEM teams. 

 

To sum up, we hope our project can further develop in two directions: simple usage and accuracy, due to the accuracy and rigor needed for disease diagnostics field.

 

 

Reference Page

[1] Robinson, J. D., Metoyer, K. P., & Bhayani, N. (2008). Breast cancer in men: a need for psychological intervention. Journal of Clinical Psychology in Medical Settings, 15(2), 134-139.

[2] Dialani, V., Chadashvili, T., & Slanetz, P. J. (2015). Role of imaging in neoadjuvant therapy for breast cancer. Annals of surgical oncology, 22(5), 1416-1424.

[3] Huber, S., Medl, M., Vesely, M., Czembirek, H., Zuna, I., & Delorme, S. (2000). Ultrasonographic tissue characterization in monitoring tumor response to neoadjuvant chemotherapy in locally advanced breast cancer (work in progress). Journal of ultrasound in medicine, 19(10), 677-686.

[4] Burkett, B. J., & Hanemann, C. W. (2016). A review of supplemental screening ultrasound for breast cancer: certain populations of women with dense breast tissue may benefit. Academic radiology, 23(12), 1604-1609.

[5] The American Cancer Society medical and editorial content team (2022). Breast Biopsy. https://cancer.org/cancer/breast-cancer

[6] The American Cancer Society medical and editorial content team (2019). Ultraviolet Radiation. https://cancer.org/healthy/cancer-causes/radiation-exposure/uv-radiation