Purpose of hardware

Our team designed a test kit for biomarkers of breast cancer. The kit contains all the materials needed for a CRISPR-Cas system to work, and when there is the target circRNA biomarker in the tester’s sample, the kit can give a fluorescence signal.

 

The fluorescence signal can be viewed under UV light which can damage human tissues and eyes. So, we designed a device to make sure that the test results can be observed safely and conveniently. Recipients of our detection kit can also have this device. They can use the kit immediately by adding their own blood sample to the test tubes, placing the test tubes in the device and observe the results safely. The end users of this detection kit are mainly composed of general public, especially women population, given the fact that 99 percent breast cancer incidence in America happens among woman population. (Robinson, J., 2008)

 

Structure of Hardware

Our device contains an outer box, a UV lamp, a glass sheet, and a strip of 8 tubes.

 

As we try to bring our idea into practice, we made several attempts, as shown in Graph 1 to 2.

 

Graph 1: A concept model we built as our first trial to present our idea

 

 

 

Graph 2: 3D model of our device

 

The UV lamp is placed vertically in the back of the box with the switch locating outside the box. The 8 tubes/strip can be placed in a shelf that is 2-3cm in front of the UV lamp. The UV rays can reach the bottom of the tube. An opening is designed on the right side of the box, which is a drawer-like structure enabling the picking and placing of the 8 tubes. A window is design in the front side of the box, which allows the tester to observe the fluorescence through it. The window is made of a glass sheet to protect the user from UV light.

 

Application of the hardware

The usage of our kit with this hardware included following steps. The user collects their blood sample, adds all reagents into the 8 test tubes in the device as instructed by the manual, and adds blood samples in the tube 5 to 8 (Graph 3). The user then closes the box and waits for 15 minutes. Afterwards, the user turns on the UV lamp to check the fluorescence signal. When observing the tubes, the user should make sure that the negative control tubes do not give out fluorescence signals and that strong fluorescence can be seen in positive control tubes (Graph3).

The final conclusion should be made based on all four testing tubes. Positive results of the tube 5 and 7 mean a detection of hsa_circ_0001785 and positive results of the tube 6 and 8 mean a detection of hsa_circ_0001982. When the results are released, patients with positive results should go to hospitals to get further diagnosis because this detection kit cannot replace the role of standard diagnosis.

 

 

 

Graph 3: Illustration of reagents in a sample of 8 tubes/strip 

 

 

Theory

The device is mainly designed to keep a dark background for fluorescence observation and shielding UV light. The test tubes are placed in the dark box with only UV light shining on the tube bottom. Viewing glass sheet can protect the user from UV light while enable a clear vision of fluorescence signals.

 

Limitation

This equipment has some limitations when used in reality. Firstly, the environment has to be relatively dark in order to clearly observe the intensity of the fluorescence.

 

Also, the optimal temperature for the Cas12a protein to function is at 28℃(Graph 4), so our  test kit is affected by environmental temperature(Malzahn. et al., 2019). Therefore, in places that is too cold and do not have the condition to own heating devices, the test results might not be accurate.

 

In order to maintain the enzymatic activity of Cas12a protein, we need to create a relative low-temperature environment during the delivery of the test kit with our device. Therefore, ice bags are needed.

 

Safety

Dealing with the waste sample blood is a hard thing. For one thing, the blood may contain some unknown disease and might become a source of infection. “Blood stasis is both a pathological product and a pathogenic factor”(Peng, 2008). It is a very hard thing to make sure that the blood will not leak and cause biosafety issues for people who are not professional.

 

Moreover, the UV light can cause damage to the tester when the tester is exposed to it during a long time period. Therefore, the tester should prevent opening the UV lamp when the box is not fully closed.

 

The most important thing to mention is to safely deal with enzymes. When handling enzyme, it is worth to note that the tester should avoid inhalation of enzymes, which might cause immune response and result in respiratory allergy, and direct contact with skin and eyes, which can cause irritation. According to Enzyme Technical Association(ETA), “mild to severe symptoms may occur such as sneezing, nasal or sinus congestion, coughing, watery eyes, and runny nose; more serious symptoms may occur with the progression and onset of occupational asthma such as tightness of the chest, wheezing, and shortness of breath”(ETA, 2017). Therefore, when working with the reagents, masks and gloves are needed, and that children should be kept away from the reagents.

 

Future Development

This detection kit enables people to test the biomarkers quickly, with no requirement for professional personnel and laboratory equipment. It is especially suited to those in rural areas. Moreover, because of its simplicity, it is also financially friendly to anyone who is willing to assess their risk of getting breast cancer. The device is simple and cheap and can be used for other kits that required UV light for signal observation. 

 

There are still numerous ways to improve the design of the device. For example, it is a good direction to change the material of the box to increase its lightproofness. Pressing the reagents into dry powder can simplify the delivery of the detection kit since there is no need for ice bags.

 

Reference

[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] 2021, Cordis EU research results, non-invasive and accurate diagnosis and treatment of breast cancer. https://cordis.europa.eu/article/id/435358-accurate-but-non-invasive-test-for-breast-cancer-in-a-kit

[6] Liu, M. C., Oxnard, G. R., Klein, E. A., Swanton, C. S. M. C. C., Seiden, M. V., Liu, M. C., ... & Yip, A. S. (2020). Sensitive and specific multi-cancer detection and localization using methylation signatures in cell-free DNA. Annals of Oncology, 31(6), 745-759.

[7] Galleri official website, https://www.galleri.com/

[8] Zhang Yanna, et al."Effect of Knockdown circular RNA hsa_circ_1785 on the Proliferation, Migration and Invasion of Breast Cancer Cell" Journal of Modern Oncology 27.02(2019):201-206.

[9] Malzahn AA, Tang X, Lee K, Ren Q, Sretenovic S, Zhang Y, Chen H, Kang M, Bao Y, Zheng X, Deng K, Zhang T, Salcedo V, Wang K, Zhang Y, Qi Y. Application of CRISPR-Cas12a temperature sensitivity for improved genome editing in rice, maize, and Arabidopsis. BMC Biol. 2019 Jan 31;17(1):9. doi: 10.1186/s12915-019-0629-5. PMID: 30704461; PMCID: PMC6357469.

[10]Enzyme Technical Association(2017). Working safely with enzymes. https://www.enzymetechnicalassociation.org/wp-content/uploads/2017/11/Working-Safely-with-Enzymes-Nov2017-English.pdf 

[11]Yin WB, Yan MG, Fang X, Guo JJ, Xiong W, Zhang RP. Circulating circular RNA hsa_circ_0001785 acts as a diagnostic biomarker for breast cancer detection. Clin Chim Acta. 2018 Dec;487:363-368. doi: 10.1016/j.cca.2017.10.011. Epub 2017 Oct 16. PMID: 29045858.

[12]Tang YY, Zhao P, Zou TN, Duan JJ, Zhi R, Yang SY, Yang DC, Wang XL. Circular RNA hsa_circ_0001982 Promotes Breast Cancer Cell Carcinogenesis Through Decreasing miR-143. DNA Cell Biol. 2017 Nov;36(11):901-908. doi: 10.1089/dna.2017.3862. Epub 2017 Sep 21. PMID: 28933584.