Team:Rochester/Hardware
UteRus
Index
Overview
Menstrual blood collection
Collapsable Sterilizer
Lateral Flow Assay Imaging Station
DIY lab equipment
Therapeutics
Hardware
The process of creating a non-invasive method for the detection of endometriosis extends to providing the patients and testing clinics with an affordable and uncomplicated process. For the patient, this process starts with the collection of the menstrual blood sample, includes considering transportation of the sample, the sanitization of the collection tool, and possibly considers the use of a therapeutic tool through a vaginal ring. For the clinic, this process means receiving the menstrual blood sample, being able to centrifuge it, and then run it a comprehensive lateral flow assay.
The COVID-19 pandemic produced a lot of challenges for our hardware team. We had to follow limiting at home safety regulations set by the University of Rochester’s Environmental Health and Safety office as well as ones set by our departments. Although these restrictions hindered our ability to build some of our models, the unique circumstances of this year increased our creativity and enabled us to branch out our efforts since we had to come up with clever ways to design and test our models. This included using Lego building blocks to substitute for the lack of access to 3D printers and power tools, using alternative connections instead of soldering, and prototyping using LEDs instead of UV-C lights.
Our final results were divided into four main subcategories:
Menstrual blood collection and Menstrual cup sterilization
Lateral Flow Assay Imaging Station
DIY lab equipment
Therapeutics
Endo-cup
Our diagnostic product for endometriosis is a non-invasive test panel that diagnoses endometriosis by measuring the concentrations of biomarkers in menstrual blood. In order to allow for easy collection, our team proposed to use a menstrual cup, which is a small silicone cup positioned in the vaginal canal during menstruation, for collecting samples. Based on an interview with DivaCup, the current menstrual cup design has not significantly changed since its invention in the 1980s and very little research has been done to prove the functionality of the current design.
Endometriosis causes thickening of the vaginal wall and pelvic muscle fatigue (Natalie Yang, et al),(Ana Paula Santos Dos, et al, 2016). After talking with Dr. Gubbles, we realized that this causes conventional menstrual cups to be painful to endometriosis patients. Softer cups tend to be more comfortable for women with weaker vaginal muscles because they apply less pressure (Put A Cup In It). Therefore, we created a model of the pressure exerted on the vagina by the menstrual cup to study the effect of various cup designs.
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Endo Menstrual cup
Our diagnostic product for endometriosis is a non-invasive test panel that assesses the endometriosis risk of an individual by measuring the concentrations of biomarkers in menstrual effluent. In order to allow for easy collection, our team proposed to use a menstrual cup for collecting samples. A menstrual cup is a small silicone cup that is positioned in the vaginal canal during menstruation. Based on an interview with DivaCup, the current menstrual cup design has not significantly changed since its invention in the 1980s and very little research has been done to prove the functionality of the current design. Endometriosis alters the size and rigidity of the vagina (Bispo et al, 2016), and we took this into account with the softness, comfort, and ergonomic shape of our cup design. That fact makes it one of the first menstrual cups that tries to take anatomical change and the altered vaginal pressure caused by endometriosis into consideration.
After starting our project with the intention of providing a simple and noninvasive menstrual effluent collection method, we contacted Dr. Ashley Gubbels, a minimally invasive gynecologic surgeon, who has contributed a lot towards our project in other aspects than hardware. She informed us of how uncomfortable menstrual cups could be for endometriosis patients. This is in part due to the altered anatomy of the vaginal canal for individuals with endometriosis. Specifically, these patients have much stiffer vaginal muscles compared to individuals without endometriosis. As such, we decided to target the endometriosis demographic by designing a menstrual cup with thin walls, inverted rim, and bent shape to reduce pressure on the vaginal muscles. After talking to professor Gubbels, we decided to contact the menstrual cup company DivaCup. We were able to get in contact with Diva Cares, a branch of the DivaCup company, who provided us with further insight and advice for designing a lid for our menstrual cup in order to facilitate the transportation of the menstrual effluent samples to clinics. Transportation being an essential part to facilitate the process of collecting the sample by the patient on the second day of their period and then sending the sample to the clinic or doctor’s office. Their advice informed our choice to use a vial for the transportation of the menstrual effluent to minimize the risk of sample contamination and maintain the safety of patients and healthcare providers by reducing the chance of leakage. Our menstrual cup design was a critical aspect of our project as it allows clinics to obtain the samples and run the diagnostic test. DivaCup also advised us on our suggested cup models and provided a further explanation of how muscle spasms and contractions are very common among young endometriosis patients, which are signs of weakened vaginal muscles (Bispo et al, 2016).
We designed the menstrual cup using the CAD software Solidworks. Considering the weakened vaginal muscles of endometriosis patients (Bispo et al, 2016), we designed the cup to be 1.5 mm thick, which is the smallest recommended thickness (Bauer, 2019) because thinner cups are softer and more comfortable for women with weak vaginal muscles (Put A Cup In It). The rim, which is thicker than the cup body to make the cup unfold in the vagina, is the firmest part of the cup (Put A Cup In It). Therefore, we made the rim face inward to reduce the pressure it puts on the vagina. The cup body is bent at a 30-degree angle following the shape of the vagina, allowing easy insertion and minimizing misplacement (Barnhart et al, 2006). We chose a rounded toggle stem that both gives the user a grip point and does not irritate the labia upon removal (MCA Online, 2019). The rings around the base of the cup mark where users should pinch upon removal to break the vacuum keeping the cup to the vagina. The cup is 35mm in diameter, which is on the smaller side of commercial menstrual cups to reduce pressure on the vagina (Put A Cup In It). And the body is long enough for the cup volume to be 40 mL, which was recommended to us by Dr. Gubbels.
It is important to use medical grade silicone, which is FDA-approved and clinically tested for safe long-term interaction with the human body (Biodermis, 2018). There are a few risks that are associated with inserting hygiene products in the vaginal canal, one being Toxic Shock Syndrome (TSS) which is commonly caused by pathogenic bacteria such as Staphylococcus aureus. To address this issue, we proposed the use of a UV sterilizer to remove as much bacteria as possible from the surface of the cup. Read more about our sterilizer here.
Our team chose the menstrual cup as our method of collecting menstrual effluent for our endometriosis diagnostics. Taking into account the fact that endometriosis weakens the vaginal muscles, we designed a thin menstrual cup with inverted rim, rounded stem, and a volume of 40 mL.
Menstrual Cup Stems and their purpose: MCA Online Australia (2019, November 18). Retrieved from https://www.menstrualcupsaustraliaonline.com.au/purpose-of-menstrual-cup-stems/
Barnhart, K. T., Izquierdo, A., Pretorius, E. S., Shera, D. M., Shabbout, M., & Shaunik, A. (2006, February 14). Baseline dimensions of the human vagina. Retrieved from https://academic.oup.com/humrep/article/21/6/1618/724374
Bispo, A. P., Ploger, C., Loureiro, A. F., Sato, H., Kolpeman, A., Girão, M. J., & Schor, E. (2016). Assessment of pelvic floor muscles in women with deep endometriosis. Archives of Gynecology and Obstetrics, 294(3), 519-523. doi:10.1007/s00404-016-4025-x
Put A Cup In It. (2019, October 07). Menstrual Cup Firmness Guide. Retrieved from https://putacupinit.com/firmness/
US20190358077A1 - Hygiene product, in particular menstrual cup with an ergonomic shape. (n.d.). Retrieved from https://patents.google.com/patent/US20190358077A1/en
What is Medical Grade Silicone Used For? (n.d.). Retrieved from https://www.biodermis.com/what-is-medical-grade-silicone-used-for-s/225.htm
Sterilizer
As our team approached stakeholders including gynecologists, menstrual cup manufactures, and patients, we saw the need for cleaning menstrual cups when there is limited access to water, and when boiling water is not an option. Using the cup without proper cleaning would have a strong odor, with the potential of the individual developing infections. Furthermore, using a UV sterilization method could reduce the amount of water usage needed during cleaning. Therefore, our team decided to pursue the design of a UV sterilization chamber to improve sustainability and provide an alternative to the existing cleaning method.
Using a UV sterilization method could increase access to menstrual cups for patients and consumers in areas without proper access to clean water whether permanent or temporary (i.e: camping). We also learned that it might be a good alternative for consumers with access to water because the usual boiling method requires a much longer period of time to make sure the cup is clean and using a UV sterilizer that can remove the bacteria more efficiently from the cup between uses compared to washing it using soap (Mitchell, M. A., et al., 2015). Additionally, using a UV sterilizer can reduce the risk of having TSS which is normally rare, but we work to further decrease this risk (Mitchell, M. A., et al., 2015). While the team has designed a draft model for the sterilizer we are taking into consideration the comments we received from the menstrual cup company Diva Cares concerning alternatively using UVC light (less than 280nm wavelength) instead of using the full UV range. The main reason to use UVC instead of regular UVA or UVB is its weaker ability to penetrate the skin and can Another point we are currently on is making our UV sterilizer collapsable to make it more portable as the current sterilizer on the market are still relatively too large to be carried away.
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UV-C Portable Sterilizer
As our team approached stakeholders, including gynecologists, menstrual cup manufacturers, and patients, we saw the need to clean menstrual cups when there is limited access to water and when boiling water is not an option. Using a menstrual cup without proper cleaning could result in it having a strong odor (Diva Cup, Interview with Sophie Zivku) and could increase the risk of potential infections (Mitchell, M. A., Bisch, S., Arntfield, S., & Hosseini-Moghaddam, S. M., 2015). Furthermore, using a UV sterilization method could reduce the amount of water used during the cleaning process. Therefore, our team decided to design a UV sterilization method to improve sustainability and provide an alternative to the existing cleaning method (steamers and boiling). Using a UV sterilization method could increase patient and consumer access to menstrual cups in areas without proper access to clean water, whether permanent or temporary (i.e., camping). It is also a good alternative for consumers with access to water because the usual method requires boiling for an extended period to ensure the cup is clean. Additionally, some soaps could alter the cup’s pH, which might affect the vaginal canal’s pH. We have designed a model for the sterilizer. It takes comments from Diva Cup, a menstrual cup company, into account: adding a shaker device that includes liquid cleaning along with UVC light (wavelength between 200 to 280 nm) instead of using the full UV range, which can thoroughly clean the tiny pressure holes. Another point we are currently on is making our UV sterilizer collapsable to make it more portable. Additionally, using a UV sterilizer can reduce the risk of having TSS, which is usually rare (Mitchell, M. A., et al., 2015).
We made an initial design based on stakeholder interviews, including portability, safety, easy-to-use, and cost-effective. We used SolidWorks to make our designs. The initial design includes a UVC Light bulb on both the cap and bottom, as indicated by one blue circle on each side. To make it more portable than the design, we chose to design the cup to be collapsible to fit into any bag. The material of choice would be medical-grade silicone, which is safe, and with our design, it is foldable. It is powered by 2 AA batteries so that we do not have to have a power outlet to use it.
We consulted graduate students from the Department of Electrical and Computer Engineering and Department of Chemical Engineering for advice on feasibility, usability, and safety. Specifically, we initially only had one layer as the outer shell and had no transparent plastic cover over the bulbs to prevent liquid leakage. The graduate students gave us the advice of over layers to prevent short circuit due to liquid. The final design is shown below. Now the design has two layers of shell. The outer layer (see fig 2A) is made of hard materials such as hard plastic, and the inner layer of the shell (see fig 2B) is made of medical silicone as for the initial design. The wires that connect the UV light in the cap and the bottom are hidden between the two layers of shells for safety reasons to prevent water leakage and human touch. UV Lights are still in the lid and the base but with a transparent plastic cover to avoid circuit shortening due to potential water dripping from the menstrual cup.
We kept the setup minimal during prototyping and used all products accessible on the market, including a menstrual cup, LED bulb, wires, UV bulb, charging pin, batteries, battery holders micro Arduino for reproducibility purposes. UVC Light bulb was not used due to being unable to be in touch with the manufacturer that produces the small UV Light bulb. All safety training was conducted, and hardware team members took workshops on proper soldering techniques. The first step was to insert and solder the LEDs into both the top and bottom of the inner silicone shell. Wires were then connected to both LEDs and attached to the shell spirally. The inner shell is then put into the outer metal shell. Ideally, the outer shell would choose some non-conductive material. The gaps between the two shells were then glued with glass glue to ensure safety. The last step is to attach the Arduino and the battery holder at the bottom of the outer shell. The Sterilization process is controlled by the Arduino to be 2 mins preciously. Ideally, we would have chosen a charging pin to ensure maximum safety so that only the device turns itself on only when the top and bottom of the shell are closed. It was not able to be achieved due to our inability to find the manufacturer
There are safety aspects that should be considered for the UV light sterilizer itself. We had to consider the first safety aspect was the direct exposure of flesh with the UV light from the sterilizer, causing artificial sunburns. As such, we decided to use instead UVC light (207–222 nm), which the World Health Organization states are likely not harmful in the care of exposure to human tissue. However, due to a limited time frame, we could not build the preliminary prototype with a UVC bulb and instead built the model with regular LED Light. To further ensure safety, we will be providing clear guidelines and instructions on the use and warnings on not putting fingers inside the sterilizer or to look directly at the light with your eyes. Appropriate safety designs of the hardware are considered during the developing stage, including using a contact switch to ensure the device is only turned on once the cap is closed and a water leakage test to ensure that the sterilizer is fully sealed and no liquid could touch the electronic components.
As we talked with more stakeholders, some have expressed ethical concern about whether there could be some potential UV radiation residue in the cup after the sterilization. Traditional UV light does have the potential to penetrate human skin, which would cause all sorts of skin problems, even skin cancer; however, UVC, on the other hand, theoretically cannot penetrate the skin, even the eyes, so that it does not create a safety issue for humans. A UV radiation test was still conducted with a commercially available UV sterilizer to test the amount of UV radiation in the cup before, during, and after exposure to UV sterilization. A UV sensor (VME6070) was inserted into the menstrual cup, and the menstrual cup was put inside the menstrual cup sterilizer during and after sterilization. UV Index (Figure 3), an international standard measurement of the strength of UV, was used to measure the UV radiation. Indexes of 0 - 2 are considered very low risk of harm, while an index above ten is considered extremely dangerous if directly exposed to the skin. The result is shown below.
As seen above, the UV Index stays stable at one before and after the UV exposure, which is considered safe (Hendry, 2018). In comparison, the level goes up to 146 during exposure. Therefore, we concluded that the device is safe to use, and as long as we use the same UV light model, it would be safe as well.
A sanitization comparison experiment was also conducted in order to compare the strength in sanitizing between the traditional method, boiling, and the UVC sterilization. Each menstrual cup was inoculated with exponential growth-phase E. coli and allowed to dry in a 37 degrees Celsius incubator. The menstrual cups were then treated with a sanitization method. Efficiency of the sanitization method was determined by analyzing the colony forming units per millileter (CFU/mL) that remained on the menstrual cup after treatment compared to the untreated condition. We used two different sanitizing methods for this experiment: submerging the cup in boiling water for 5 minutes (the standard cleaning method) and using a commercially purchased UV sterilizer.
These methods demonstrated that placing the cup in boiling water for 5 minutes yielded approximately 0 CFU/mL, while we saw a 0.19-log decrease in CFUs with the UV sterilizer compared to the untreated condition when used as instructed by the manufacturer (cup was enclosed in the device for 2 minutes) (Figure 5).
As a result of this experiment, we wanted to figure out why such a low log-fold decrease of CFUs was observed when the menstrual cup was treated with UV light. Our hardware team had performed an in-depth literature review which suggested that UV light should be an efficient method for the sanitization of menstrual cups. Specifically, we wanted to test if increasing the time that the cup was exposed to the UV light would yield better results.
This experiment demonstrated that the commercially purchased UV sterilizer was ineffective for sanitizing the surface of the menstrual cup. Initially we thought that this might be due to low voltage, however, our hardware team determined that there was only a 7.1% decrease in voltage since we had purchased the sterilizer. As such, we hypothesize that the light might not be reaching all surfaces of the cup or the intensity of the light is not sufficient to kill bacteria on the surface of the cup. To address this issue, our hardware team decided to increase the number of UVC lights inside our sterilizer design and increase the voltage to the cup to ensure that the light reaches the proper intensity. We would also like to test the commercial sterilizer to check the wavelength of the light, as wavelengths outside of the range of 260 nm to 280 nm likely would not result in killing of the bacteria on the surface of the cup.
Throughout this project, we have prototyped a sterilizer that is portable, water-friendly, and safe by the UV Index experiment. Our next step would be to find smaller UV light bulbs, an ideal non-conductive outer shell material, and, more importantly, to test UV sterilization’s effectiveness with better control at the more physiological environment for more test rounds.
Reference
Buonanno, Manuela, et al. “Far-UVC Light (222 Nm) Efficiently and Safely Inactivates Airborne Human Coronaviruses.” Nature News, Nature Publishing Group, 24 June 2020, www.nature.com/articles/s41598-020-67211-2.
Hendry, Iain. “VEML6070 Ultraviolet Light Sensor and Arduino Example.” Arduino Learning, 18 July 2018, arduinolearning.com/code/veml6070-ultraviolet-light-sensor-and-arduino-example.php.
Mitchell, M. A., Bisch, S., Arntfield, S., & Hosseini-Moghaddam, S. M. (2015). A confirmed case of toxic shock syndrome associated with the use of a menstrual cup. Canadian Journal of Infectious Diseases and Medical Microbiology, 26.
As a result of this experiment, we wanted to figure out why such a low log-fold decrease of CFUs was observed when the menstrual cup was treated with UV light. Our hardware team had performed an in-depth literature review which suggested that UV light should be an efficient method for the sanitization of menstrual cups. Specifically, we wanted to test if increasing the time that the cup was exposed to the UV light would yield better results.
This experiment demonstrated that the commercially purchased UV sterilizer was ineffective for sanitizing the surface of the menstrual cup. Initially we thought that this might be due to low voltage, however, our hardware team determined that there was only a 7.1% decrease in voltage since we had purchased the sterilizer. As such, we hypothesize that the light might not be reaching all surfaces of the cup or the intensity of the light is not sufficient to kill bacteria on the surface of the cup. To address this issue, our hardware team decided to increase the number of UVC lights inside our sterilizer design and increase the voltage to the cup to ensure that the light reaches the proper intensity. We would also like to test the commercial sterilizer to check the wavelength of the light, as wavelengths outside of the range of 260 nm to 280 nm likely would not result in killing of the bacteria on the surface of the cup.
Throughout this project, we have prototyped a sterilizer that is portable, water-friendly, and safe by the UV Index experiment. Our next step would be to find smaller UV light bulbs, an ideal non-conductive outer shell material, and, more importantly, to test UV sterilization’s effectiveness with better control at the more physiological environment for more test rounds.
Buonanno, Manuela, et al. “Far-UVC Light (222 Nm) Efficiently and Safely Inactivates Airborne Human Coronaviruses.” Nature News, Nature Publishing Group, 24 June 2020, www.nature.com/articles/s41598-020-67211-2.
Hendry, Iain. “VEML6070 Ultraviolet Light Sensor and Arduino Example.” Arduino Learning, 18 July 2018, arduinolearning.com/code/veml6070-ultraviolet-light-sensor-and-arduino-example.php.
Mitchell, M. A., Bisch, S., Arntfield, S., & Hosseini-Moghaddam, S. M. (2015). A confirmed case of toxic shock syndrome associated with the use of a menstrual cup. Canadian Journal of Infectious Diseases and Medical Microbiology, 26.
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Gallery
Figure 1: Arduino with a UV sensor attached.
Figure 2: Position of the UV sensor inside the menstrual cup
Figure 3: UV test with commercial available sterilizer. The menstrual.cup is inside the sterilizer with the UV sensor inside the menstrual cup. Red LED lights up to indicates
Figure 4: Team member, Nello, connecting wires around the inner shell of the sterilizer.
Figure 5: Hardware manager, Helen, coding for the Arduino.
Figure 6: LED on the base
Figure 7: LED on the lid
Figure 8: Attachment of the battery holder at the bottom of the outer shell
Figure 9： Sterilizer folded
Figure 10: Sterilizer fully opened
Figure 1: Arduino with a UV sensor attached.
Figure 2: Position of the UV sensor inside the menstrual cup
Figure 3: UV test with commercial available sterilizer. The menstrual.cup is inside the sterilizer with the UV sensor inside the menstrual cup. Red LED lights up to indicates
Figure 4: Team member, Nello, connecting wires around the inner shell of the sterilizer.
Figure 5: Hardware manager, Helen, coding for the Arduino.
Figure 6: LED on the base
Figure 7: LED on the lid
Figure 8: Attachment of the battery holder at the bottom of the outer shell
Figure 9： Sterilizer folded
Figure 10: Sterilizer fully opened
Our wet lab team developed a lateral flow assay (LFA) using gold nanoparticles (GNPs) to create a sensitive point of care diagnostic. This assay produces a colorimetric signal that our hardware team was then tasked to quantify.
Our LFA is a paper-based assay for the detection and quantification of the biomarkers of endometriosis found in menstrual blood. The detecting device has a lateral flow membrane as the base that allows the sample to be soaked and flow from one end to the other. As the sample flows through the membrane, the biomarker contained in the sample will be specially bound to antibodies that are conjugated to the GNP. These biomarkers are then bound to a second immobilized antibody on the test line, whereas free gold-conjugated antibodies can travel through the test line and be bound to the control line. Binding of the biomarkers on the test line together with the cold-conjugated antibodies will cause a color change of the line. Therefore, positive results will have a color change in both the test line and the control line, whereas negative results will only have a color change in the control line but not the test line. This color change can then be detected and semi-quantified using our smartphone-based platform.
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Lateral Flow Assay Imaging Station
Our wet lab team developed a lateral flow assay (LFA) using gold nanoparticles (GNPs) to create a sensitive point of care diagnostic. This assay produces a colorimetric signal that our hardware team was then tasked to quantify.
Our LFA is a paper-based assay for the detection and quantification of the biomarkers of endometriosis found in menstrual blood. The detecting device has a lateral flow membrane as the base that allows the sample to be soaked and flow from one end to the other. As the sample flows through the membrane, the biomarker contained in the sample will be specially bound to antibodies that are conjugated to the GNP. These biomarkers are then bound to a second immobilized antibody on the test line, whereas free gold-conjugated antibodies can travel through the test line and be bound to the control line. Binding of the biomarkers on the test line together with the cold-conjugated antibodies will cause a color change of the line. Therefore, positive results will have a color change in both the test line and the control line, whereas negative results will only have a color change in the control line but not the test line. This color change can then be detected and semi-quantified using our smartphone-based platform.
Our wet lab team developed a lateral flow assay (LFA) to detect biomarkers present in the menstrual blood as a non-invasive diagnostic method for endometriosis. The direct readout of our lateral flow assay is the appearance of a colored band caused by the binding of biomarkers to gold-conjugated antibodies at the test line, similar to the output of a pregnancy test. Although this change can be observed by the naked eye, its reading is likely to be affected by the ambient light. In addition, since the intensity of the color change indicates the concentration of the biomarker, quantification of the color change will tell us how much biomarker is presented in the sample; however, this quantification cannot be done with the naked eye. Therefore, in order to image the LFA result in a standardized condition, quantify the results, and save the results as images, we decided to build a LFA imaging station.
We were inspired by a smartphone-based detector developed by Paterson et al., 2017. This detector is a smartphone attachment that would take advantage of the smartphone’s camera and flash light to image the LFA results. This device will allow the LFA strip to be inserted and thereby blocks the ambient light from the outside. This device takes a two-step protocol to take images of a fluorescence based LFA. First, we turn on the rear camera flash for photoexcitation of phosphors. Then, we turn off the camera flash for luminescence imaging. Since our LFA is based on the visible colorimetric changes caused by aggregation of gold nanoparticles, we decided to use the same strategy described in Paterson et al. 2017 with slight changes in imaging procedures to image our test results. In short, we will take the images of the LFA strip when the flashlight is on.
To increase the accessibility of our device, we decided to build our imaging station using Legos instead of 3D-printing used in Paterson et al 2017. To obtain images of the best quality, we decided to attach an extra 15X macro lens to the phone that increases the magnification along with the phone’s lens.
In summary, we wanted to use Legos to build the base of our imaging station. This imaging station will have a LFA channel that holds the LFA strip and blocks the ambient light at the same time. The station will also have a phone holder and a lens holder to further stabilize the phone while imaging.
Based on the original design, we built several prototypes and tested them (Picture 1). We tested them by imaging the LFA strip and assessed the image quality. Prototype 1 had a phone holder, a lens holder, and a LFA channel. A successful test should generate images that clearly show the color change of the LFA . However, we found, after blocking most of the ambient light, the image was too dark. Thus, we decided to add a light source that luminates the LFA channel with stable light.
Figure 1:Prototype 1, Prototype2, and Final Version of the imaging station
Figure 2:Different views of the final product.
In Prototype 2, We added a small light bulb controlled by a microcontroller onto the ceiling of the LFA channel. This light bulb is powered by three 1.5 V external batteries and can be controlled by a light switch. This external light source solved our problem faced in Prototype 1; however, we realized that the attaching microcontroller and battery set made the imaging station less portable. To improve portability, we integrated the microcontroller and the battery set at the bottom of our imaging station. Together with other small modifications, we made our final product. Different views of the final product are shown in Figure 2 and Figure 3.
Figure 3: Top, bottom, and back views of the station with parts being illustrated.
Once the overall design was finalized, next we tested several lenses to select the one that gives the best image. We tested a 180° fisheye lens, a 0.6X wide angle lens, a 15X macro lens, and a phone-based microscope. We found that the 180° fisheye lens and the 0.6X wide angle lens did not give enough magnification, and the phone-based microscope magnified the image too much. Therefore, we decided to use the 15X macro lens (Figure 4). This lens is compatible with multiple types of phones and fits the lens holder of our imaging station. A complete setup of the imaging station with phone, lens, and LFA strip is illustrated in Figure 5.
Figure 4: The 15X macro extra lens on a phone clamp.
Figure 5: A complete setup of the imaging station.
Unfortunately, we were unable to collaborate with wet lab team members to use a real LFA strip because of the social distancing rules enforced by the University of Rochester in response to the COVID-19 pandemic. Instead, we drew a red dot using a marker on the LFA strip and tested the performance of our final product by imaging this LFA strip proxy. We use the red dot as a proxy signal simply because it has a similar color as a LFA signal, and because we can draw the dot in a similar pattern as a LFA result. To test the consistency of our imaging station, we used three different types of phones, an iPhone 6P, iPhone 7, and iPhone XR to take images of the strip. We took three pictures for each phone under the same conditions, and in total nine images were obtained. We analyzed these images using the Analyze Particle function in ImageJ to measure the intensity of the red dot in pixels. We created box-plots to compare the consistency between phones and within phones (Figure 6). In the box-plots, x-axis illustrates the type of phones, and y-axis is the measurement of dot intensity in Pixels. We found that data taken with the same phone were consistent, whereas the consistency decreases between phones, likely due to differences in the camera resolution of different types of phones. Our results suggest that users should use the same phone to image their test results.
Figure 6: Quantification of LFA Proxy Result Imaged by Different Types of Phones
After experimenting with the design we decided to use an LED. We also made the decision of using a potentiometer to control the intensity of the light. See below in figure 7 the schematics and sketch for the circuit inside the imaging station.Also click below to find our code.
Figure 7: Circuit inside the imaging station
Figure 8: Budget and components for the LFA imaging station
In figure 7, it should be noted that some of the pieces and parts in column 2 are kits and sets and that does not portray the actual budget for the imaging station. Column 4 represents the budget required for building the imaging station much better. It’s also worth mentioning that not the whole Lego set was used, but there isn't a way to quantify the actual price of only the pieces that were used
We have created a smartphone-based LFA imaging station that allows users to image the LFA strip in standardized conditions and to store the test result as images. This imaging station is built by Lego and is low-cost and highly accessible. To further improve the consistency of the imaging station, one could possibly spray-color the Lego pieces in black to prevent light reflection. In addition, one could adjust the light intensity to make the imaging consistent for different types of phones. Moreover, to improve the data quantification process, more types of phone, i.e, non-iPhone phones, should be tested. Also, although ImageJ is widely used, it will be better to develop image analysis software specifically for our imaging station.
Paterson, Andrew S, Balakrishnan Raja, Vinay Mandadi, Blane Townsend, Miles Lee, Alex Buell, Binh Vu, Jakoah Brgoch, and Richard C Willson. 2017. "A low-cost smartphone-based platform for highly sensitive point-of-care testing with persistent luminescent phosphors." Lab on a chip 1051 - 1059.
Centrifuge
A centrifuge is a basic equipment for most biological experiments, however, a benchtop centrifuge tends to be expensive for a clinic that does not have a regular need for it, ranging from $1000 to $5000. Our project does not require all the features or precisions that come with the expensive centrifuge models. As such, our 'Do it yourself" (DIY) centrifuge will decrease the cost, eliminate non-relevant features, and will make such equipment easily available for clinics that do not normally have access to centrifuges. This encapsulates laboratories and clinics in more impoverished areas and teams with limited budgets. Our centrifuge design also contains a number of recycled items that are easily found around a house, further increasing its sustainability.
Creative methods of building centrifuges have emerged in the past few years. However, these DIY centrifuges lacked proper control of the speed. Previous models used potentiometer knobs instead of input number pads which decreased the accuracy and or alternatively increased time needed to reach the desired value. Our centrifuge is also meant to be more accessible, as all its features will take color blindness into consideration and utilize more visual cues for the hearing impaired. Finally, our centrifuge has two different screens, one being the LCD screen which would provide instructions and the information about the rpm, and another 4 digits, 7 segment display screen to provide a timer (which many DIY centrifuges lack). We are also trying to improve the 3D printing design for the sample holder for the centrifuge.
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DIY Centrifuge
A centrifuge is a basic equipment for most biological experiments, however, a bench-top centrifuge tends to be expensive for a clinic that does not have a regular need for it, ranging from $1000 to $5000. Our project does not require all the features or precisions that come with the expensive centrifuge models. As such, our 'Do it yourself" (DIY) centrifuge will decrease the cost, eliminate non-relevant features, and will make such equipment easily available for clinics that do not normally have access to centrifuges. This encapsulates laboratories and clinics in more impoverished areas and teams with limited budgets. Our centrifuge design also contains a number of recycled items that are easily found around a house, further increasing its sustainability
- Place your sample in the sample holder
- Plug in the battery to the ESC controller
- Input your desired rpm using the keypad.Press in the value followed by a hashtag. For example: if you want an rpm of 2000, the. you should input 2000#
- Once centrifugation time is completed, press 0#
Through testing we have learnt that the average time required to reach maximum speed is 6 seconds. The average time to slow down once the 0# combination is inputted would be 7 seconds.
Through experimentation we also learnt that the minimum centrifugation speed is 200 revolutions per minute, while the maximum safe speed is 4000 revolutions per minute.
In figure 1, it should be noted that some of the pieces and parts in column 2 are kits and sets and that is not the budget to make a centrifuge. Column 4 represents the budget required for building a centrifuge much better. It’s also noted that the hard drive and the brushless motor are marked, this is because they are alternatives and not part of the same centrifuge.
Upon completion of the centrifuge design and circuits, and once we finished preparing the detailed instructions for building the design. We collaborated with the Engineers World Health University of Rochester chapter to test out our instructions and how replicable they are for a member who has minimal previous experience in circuits and soldering.
Based on this testing process, the feedback we received was mostly positive about the instructions. The tester mentioned how they were clear, even if the soldering part is not very simply due to the narrow section on the recycled piece. They also mentioned that the circuit instructions and schematics were clear and easy to replicate.
Furthermore, once we were able to 3D our sample holder, we ran the centrifuge on our lowest rpm for about 30 minutes.The result is that we did see separation of the synthetic serum and the gold-conjugated antibodies -purple pellet that appears at the bottom. This shows that the product does its function of centrifugation. We also left a sample to settle for 12 hours and we observed that seperation did not happen further proving that our centrifuge works
During the design of our DIY centrifuge, the problem that shows with the highest frequency is debugging the code. Even though there are some examples online, combining these different examples together to build our DIY centrifuge, we still faced a lot of problems, such as the conflict of two examples, and not using the exact same equipment. To deal with these problems, the approach was to continue searching for different kinds of examples and learning during the mistakes.
For example, when we want to display the input from the keypad on LCD and then set this value as the speed of hard drive, we need to combine three parts of our project together. This is the hardest part for coding. I tried out many different kinds of combinations within the code to see in which way the input from the key can show appropriately on LCD and also be sent to the hard drive. I searched many codes online and finally figured out the solution.
The next challenge we faced was after printing our sample holder, the size was slightly different from the perfect fit with our hard drive. This resulted in us using the University's machine shop and use a lathe to fix the diameter of the sample holder.
The final challenge was creating a protective case as well as circuit covering using plexiglass.To achieve more accurate results we used a band saw, however a normal pair os scissors could have been used
In concern of the pandemic conditions, and due to all team members being unable to access lab space we had to follow all the online learning safety regulations that the university set. We submitted a safety proposal to follow such.
Suggested Experiments
- An acceleration sensor placed in one of the sample holders (Adafruit,2019). This is to check:
- If the rpm on the screen is correct
- If we can control acceleration asking what is time needed to reach a specific rpm
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Attaching a small piece of reflective tape -white- onto the top surface of the centrifuge chamber in between two consecutive tube slots.Then aiming a laser tachometer to where the tape is, this should measure the real RPM (Lab Manager, 2020).
- Check volume range that we can centrifuge at accurate speed by placing different volumes and performing the speed/ acceleration test at the same time
- Check how different voltages affect it by replacing the 5V battery with a 9V and a 12V
- Test if it affects heat of sample: Take a sample of ethyl glycerol solution, take temperature before and after centrifugation
- If the rpm on the screen is correct
- If we can control acceleration asking what is time needed to reach a specific rpm
Future Improvements
References
“2019 Adafruit: Mouser.” Mouser Electronics, www.mouser.com/ProductDetail/Adafruit/2019?qs=GURawfaeGuCOD7vRMbNtig==&gclid=EAIaIQobChMIvbXBsNTw6wIVFYTICh2qIgBhEAQYASABEgKA-fD_BwE.
“The Basics of Centrifuge Operation and Maintenance.” Lab Manager, www.labmanager.com/product-focus/the-basics-of-centrifuge-operation-and-maintenance-1433.
Inspired by the clinical trials that Ferring Pharmaceuticals are performing. The RAQUEL study is ‘Randomized Trial Assessing Quinagolide Vaginal Ring for Endometriosis-related Pain’. The study is a randomized, double-blind, Phase II clinical trial that is meant to evaluate the efficacy of quinagolide on the reduction of moderate to severe endometriosis-related pain compared to placebo. This project was introduced to us by Dr. Idhaliz Flores, who mentioned that targeted methods of drug delivery for endometriosis could be beneficial over current, generalized hormone treatment options.
The vaginal ring is a small, flexible ring constructed from plastic. The ring can be inserted into the vagina to work as a drug delivery method to provide a constant supply of therapeutic to the vaginal canal. This is typically used as a contraceptive method as it has an advantage over pills since it only needs to be used once a month. As such, we thought this provided a good drug delivery method for endometriosis as hormone treatments could be localized to the reproductive organs.
While we believe that this would be a very therapeutic method, we chose not to continue with the procedure of designing our own vaginal ring.
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Vaginal Ring for Endo-Aid
The vaginal ring is a small, flexible ring constructed from plastic. The ring can be inserted into the vagina to work as a drug delivery method to provide a constant supply of therapeutic to the vaginal canal. This is typically used as a contraceptive method as it has an advantage over pills since it only needs to be used once a month. As such, we thought this provided a good drug delivery method for endometriosis as hormone treatments could be localized to the reproductive organs
We focused on the design for this component of the project due to limited access to laboratory space. To develop a strong design, we worked on studying the different characteristics of vaginal rings. We were particularly interested in the possibility of using the pain reducers such as acetaminophen, ibuprofen, or NSAID instead of hormone suppressants since hormone suppressants have been associated with negative outcomes in endometriosis patients (Agarwal et al., 2015). In the end we chose not to continue with making the vaginal ring and instead chose to proceed with outlining what we would need for the design
Quinagolide is a non-ergot-derived selective dopamine receptor agonist specifically 4-[2-(dideuterioamino)ethyl]benzene-1,2-diol (PubChem, 2020) . This receptor agonist is commonly used for the treatment of hyperprolactinemia or generally high levels of prolactin. (DrugBank,2020) Quinagolide and the other dopamine receptor 2 agonist, cabergoline have both shown signs of reducing angiogenesis by dephosphorylation of VEGF2. (Neidler, 2019) Previous experiments on mice have shown that Quinagolide was effective in inhibiting angiogenesis and has reduced size of endometriotic lesions (Rafique,2018). This encouraged us to chose this specific drug for the vaginal ring.
How would we design the trials?
- Demographics
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Ideally, we are looking for a randomized diverse pool of menstruating individuals. Eligibility for the screening would require the patient to be (McLellan-Lemal, E. et al, 2014):
- 18-45 age range
- currently not undergoing any type of hormonal treatment especially not for endometreosis
- Patients should also not be using any other pain suppressants
- Patients can not be pregnant
- Dosages
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Current trials using Quinagolide in vaginal rings have suggested the tests of four different amounts and daily release rates: (Ferring Pharmaceuticals,2018)
- Experimental: A release rate of 4.5 μg/ day using a vaginal ring containing 360μg
- Experimental: A release rate of 9 μg/ day using a vaginal ring containing 720μg
- Experimental: A release rate of 13.5μg/ day using a vaginal ring containing 1080μg
- Placebo
- Tests:
- Daily tests for patients to rate their pain. Start a month before the insertion of the vaginal ring with all the patients rating their pain from 0 to 10. Continue tests daily till the end of the trials. So two comparison tests
- Any changes with the beginning of administration of the drug
- Any changes during the full period, did pain decrease more with the longer period of time
- Observe for possible side effects including vomiting, nausea, dizziness and fatigue. Less possible side effects (1-10%) could include abdominal pain, anorexia, constipation, diarrhea, insomnia, oedema, nasal congestion, flushing and hypotension (DrugBank,2020)
- Daily tests for patients to rate their pain. Start a month before the insertion of the vaginal ring with all the patients rating their pain from 0 to 10. Continue tests daily till the end of the trials. So two comparison tests
- Demographics