Team:ZJU-China/Hardware
The portable detection device
Overview
Nowadays, the most common way used for cervical cancer wide-range screening is human papillomavirus (HPV) infection detection based on DNA probe or PCR. Different from the traditional invasive HPV detection method, our team hopes to make a portable detection device in vitro with the advantages of high sensitivity, low price, and extreme convenience.
In our practice, our original idea was to make a device that be able to complete the experiment in the sanitary napkin and get the final result. Of course, this is the most ideal state, but in the course of our practice we found that this was not as simple as we imaged. Actually, there were so many problems needed to be solved. In the next step of the process, we designed three versions of the detection device. Though the first two editions did not perform their functions accurately in sanitary napkins, the third version finally achieved the goal to take blood from the sanitary napkin and display the result in an external portable detection device.
Version 1. A Microfluidic Chip in sanitary napkin
After our team came up with the idea to detect HPV using menstrual blood, we initially considered the technology of the microfluidic chip. At the time of design, we found it difficult to place the microfluidic chip which can complete the task of collecting menstrual blood and testing menstrual blood in a sanitary napkin. Firstly, the driving problem of the menstrual flow into the microfluidic chip was difficult to be solved. Since the chip was placed in the sanitary napkin, the external air pump was not suitable for us, so we considered the capillary driving method. Later, according to the experiment of the wet lab, the amount of menstrual blood required was relatively large which meant the capillary drive could not achieve the effect we wanted, and the microfluidic chip was not suitable for such a large quantity. In summary, we abandoned the method of microfluidic chip.
Version 2. A silicone detection device in sanitary napkin
Next, we considered whether we could create a vacuum environment with soluble materials to collect menstrual blood. Therefore, we designed the silicone detection device followed.
Figure1. The silicone detection device
Figure2. The silicone detection device in a sanitary napkin
However，we could not add distilled water in this device to make cells lyse or make sure the quantity of the menstrual blood the device collect. Moreover, after communicating with the sanitary napkin manufacturer and the sanitary napkin production visit, we learned that this design could not be incorporated into the sanitary napkin production process. We need to take a new way.
Version 3. An external portable detection device
1. Design
This device mainly consists of three parts: a sanitary napkin with a compression sponge, a sealed-off tube with defined volume of distilled water and an external portable detection device. The sanitary napkin with a compression sponge is used for the collection of menstrual blood. The sealed-off tube with a defined volume of distilled water is used for the first step of the experiment.
The distilled water will mix with the menstrual blood to make cells lysed. The design of the sealed-off tube is referenced to the design of the disposable eye drop; The external portable detecting device comprises three main parts: a pressing device, reaction chambers and a test paper. By squeezing the sponge, the collected menstrual blood flows into the first reaction chamber and finally we can obtain an observable result on the test paper or by fluorescence phenomenon.
Figure 3. The sanitary napkin with a compression sponge
Figure 4. The sealed-off tube with defined volume of distilled water
Figure 5. The external portable detection device.
2. Experimental test
2.1 Test of blood collection material
According to the wet lab experiment, we initially need at least 300 microliters of menstrual blood, so we should ensure that the menstrual blood collection material we use can provide enough menstrual blood. We conducted a series of experiments to select the best collection of menstrual blood for us.
In the experiment, our initial subjects were compressed sponges, medical sponges, and medical cotton balls.
Simple water cannot simulate the blood of the human body, and the speed of absorption will be much faster than menstrual blood. After all, the blood has a certain viscosity, and the menstrual blood will also be mixed with some discharged impurities. To simulate the viscosity of menstrual blood, we used a mixture of pure water and glycerol in a volume ratio of 43:7 to make the simulated liquid.
During the experiment, we placed the three subjects in a sanitary napkin, and injected the same 3.5 ml of the simulated liquid onto the sanitary napkin, waiting for absorption until the surface was dry, then we took out the experimental object and squeezed it to obtain the quantity of the simulated liquid.
From the results of the experiences, we found that we could get the most quantity of the simulated liquid from the compressed sponge, so we finally chose compressed sponge as our menstrual blood collection material.
2.2 Test of time control material
In terms of reaction time control, we chose to use polyvinyl alcohol (PVA), a soluble material, to print a 3-dimensional model.
According to the experiments of the wet lab, it took more than 20 minutes for the first RPA experiment and about 80 minutes for the Cas12a to detect the target fragment or fluorophore quencher (FQ)-labelled reporter assay. Therefore, our device must ensure the menstrual blood leaking to the next reaction chamber and also has enough reaction time in each reaction chamber.
At this stage, we only need to print multiple devices with reaction chambers as experimental devices, without having to print the entire device for experimentation. We print two reaction chambers separately, printing four for each reaction chamber. Next, we used PVA, soluble materials, to print a variety of wafers of different thicknesses as time control sheets, printing four for each thickness.
After that, we divided the time control sheets of the same thickness into a group and placed them in a series of parallel experiments. Similarly, we used a mixture of pure water and glycerol in a volume ratio of 43:7 to make the simulated liquid. The simulated liquid was injected into the reaction chamber, and the time of water leakage of each experimental device was recorded. Finally, compared the leakage time of time control sheets of different thicknesses to get the suitable thickness. At last we found that the 0.8mm was the best for our device.
Figure 6,7. The test of the time control material
3. The structure of the external portable detection device
Our team used SolidWorks Premium 2016 to design a 3D model of the external portable detection device for the system and print it with the 3D printer (Ultimaker3) provided by the Robotics Association of Zhejiang University.
The external portable detection device has a simple structure, compact space, and each structure realizes the integration of the three modules of the system: ① a pressing device to get the menstrual blood from the compressed sponge②reaction chambers③ a test paper to get the result/a portable UV flashlight to observe fluorescence
3.1 the pressing device
It can be seen from previous experiments that we can get enough menstrual blood samples from the compressed sponge by physical extrusion. So, we made a convenient pressing device to get the menstrual blood we need for our tests. Initially, the pressing device was designed to be cylindrical, but in the following practice, considering the size and cost of the device, we designed a new pressing device for the volume of the sponge filled with water. As shown in the figure below, the new extrusion device has been reduced in size.
Figure 8. The model of pressing device
3.2 The reaction chambers
According to the experimental design of our wet lab, two reaction chambers are needed.
If we use the test paper to detect, RPA should be put in the first reaction chamber while the reactants for the second reaction should be in the second reaction chamber. In order not to make RPA lost or inactivate, we sealed a layer of glutinous rice paper which can dissolved into water immediately at the opening. Since the quantity of reactants for the second reaction is so small, they were fixed on a circular sheet which would be in the second reaction chamber. If we use the fluorescence detection way, Only the reactants for the second reaction and the structure of reaction chambers are different
(All reactants are frozen into dry powder)
Figure 9. The model of chamber structure (L: test paper R: fluorescence detection)
3.3 The test paper/ fluorescence detection
Figure 10. Test paper
Figure 11. How to place the test paper
Figure 12. Portable UV flashlight
Figure 13. How to use the portable UV flashlight
3.4 Assembly process
4. Instructions for use
① Pour distilled water from the sealed-off tube into the reaction chamber.
② Remove the sponge from the sanitary napkin and place it into the pressing device.
③ Place the extrusion device in the inspection device and squeeze the menstrual blood from the sponge.
④ Let the external portable detection device stand for 1.3-2 hours, and the result is observed on the test paper/ use a portable UV flashlight to observe fluorescence.
5. Summary
From the experimental results, the accuracy of the test paper we established was not ideal enough(click for more information). In comparison, the result of fluorescence qualitative detection was better.
Figure 14. Result of the detection. (L: negative R: positive)
However, taking the cost into consideration, the cost of the HCR biosensing system is cheaper than that of the Cas12a biosensing system. Moreover, the HCR biosensing system can realize the multi-channel reaction. But the test paper is a must for HCR biosensing system, so after improving the accuracy of the test paper, we will choose the HCR biosensing system.
Figure 15. The cost of the main components
The current device is mainly designed for the Cas12a biosensing system. If we want to make a new one for the HCR biosensing system, we only need to modify the original device slightly.
The real-time fluorescence detection device
Overview
Since the portable detection device can only qualitatively detect the HPV, we hoped to have a device that can quantitatively detect the HPV for the intention to expand the scope of application of PaDetector. However, because of the shortage of time, we did not have enough quantitative data to support a quantitative detection device. We changed our idea and made a real-time fluorescence detection system which could help the wet lab to compare FnCas12a with LbCas12a as well as Tg1 with Tg2 quantitatively and got more convincing results.
System design
1. Optical module
In order to improve the uniformity of the spot and shorten the distance between the chip and the excitation light source, a strip-shaped ordinary blue LED array is used as the excitation light source.
The criterion for excitation filter selection is that the center wavelength is consistent with the center wavelength of the excitation source with the bandwidth as small as possible and not overlapping with the wavelength range of the emission filter at the center wavelength plus or minus bandwidth. In addition, the excitation light filter needs to cover the light-emitting area of the excitation light source. We used a strip-shaped ordinary blue LED array to perform four parallel fluorescence experiments at a time. The excitation light filter was also fabricated using a 3D printer, and the excitation filter was fixed directly in front of the LED flashlight.
Figure 16. The excitation filter with LED flashlight
The emission filter was placed between the camera and the lens, effectively circumventing the shortcomings of the filter's ability to resist light interference in a wide range of angles. Therefore, the emission filter was selected to have a circular shape, a diameter of 2.54 cm, and a filter having a center wavelength of 470 nm.
Figure 17. Main electrical components: camera, lens, filter
Figure 18. Optical module
2.Detection chip
Since the collected fluorescent photographs needed to be processed later, we should make a regular shaped container. In addition，because the excitation light filter needed to cover the light-emitting area of the excitation light source，so we made our detection chip according to the shape of the LED board. Here we made a simple detection chip. We used the hole opener to punch holes in the prepared polydimethylsiloxane (PDMS) plate, and then combined it with the glass plate. The figure below shows our detection chip.
Figure 19. The detection chips
3. Fluorescence image acquisition and analysis system
There are three main functions: real-time fluorescence image acquisition, fluorescence image processing, and image data analysis.
The main functions of the real-time fluorescence image analysis system include real-time fluorescence image acquisition, reaction localization in fluorescence images, real-time fluorescence image processing. The method uses grayscale image to obtain fluorescence intensity and obtain a curve by fitting real-time fluorescence intensity. The system algorithm flow chart is shown in the figure below.
work process
1.Prepare the chip to be reacted that has completed the injection of the reaction solution and place the chip on the platform.
2.Turn on the power, the system automatically controls the opening and closing state of the excitation light source and collects and stores the real-time fluorescence image to the specified path.
3.When the reaction is over, turn off the power. Process and analyze the collected real-time fluorescence images.
Results
The wet lab used the Real-time fluorescence detection device to compare FnCas12a with LbCas12a as well as Tg1 with Tg2 quantitatively and got more convincing results. The following shows the comparison results of FnCas12a with LbCas12a activity measured by this device.
Figure 20. Some of the real-time images
Figure 21. The curves of the FnCas12a and LbCas12a
From the result, it showed that our device has high accuracy and can provide a lot of help to the wet lab’s experiments.
