Team:NCKU Tainan/Hardware
Hardware
Background
In Taiwan, National Health Insurance does not cover blood p-Cresol inspection fee. Additionally, each inspection costs about 1500 NTD[1] (50 USD) which is much more expensive compared to other blood tests[2] (Table1.). Since the accumulation of p-Cresol in patient’s blood is highly associated with the deterioration of chronic kidney disease and cardiovascular diseases, the monitoring of blood p-Cresol is necessary and important. The current method of measuring blood p-Cresol is high-performance liquid chromatography (HPLC) followed by mass spectrometry (MS). The cost of HPLC instrumentation and the complexity of the sample pretreatment makes p-Cresol inspection even harder to gain its universality. Hence, we created CreSense (a blood p-Cresol reader) making each test much affordable (1.6 USD per test) and easier, letting blood p-Cresol inspection more accessible to CKD patients and the public.
Increased level of p-Cresol in CKD patients is often related to the worsening of their condition and is associated with cardiovascular diseases, therefore the monitoring of patient blood p-Cresol is necessary and important. The current method for measuring blood p-Cresol is high-performance liquid chromatography (HPLC) followed by mass spectrometry (MS). HPLC instrumentation’s cost and the complexity of sample pretreatment makes p-Cresol inspection even harder to gain its universality, requiring around 1500 NTD (50 USD) for each test. Therefore, we created CreSense, a blood p-Cresol reader for making each test more affordable at just 50 NTD per test (1.6 USD) and easier, letting blood p-Cresol inspection more accessible to CKD patients and the public.
| Blood Tests | Testing Fee |
|---|---|
| HBS Ag antibody test | 160NTD |
| HAV IgM antibody test | 240NTD |
| GOT/GPT test | 90NTD |
| Total Cholesterol Test | 70NTD |
| Triglycerides Test | 120NTD |
Device Overview
Introducing CreSense - a user-friendly, affordable and customizable blood p-Cresol biosensing system. We designed CreSense to quantify blood p-Cresol concentration with a centrifugal platform and p-Cresol sensing bacteria (hyperlink: to “Demonstration”). CreSense consists of two parts - a centrifugal platform and a fluorescence detector. The centrifugal platform separates blood plasma from whole blood and directs separated plasma into the reaction chamber. In the second part, the user injects p-Cresol sensing bacteria into the reaction chamber where the presence of p-Cresol in the plasma will induce the expression of green fluorescent protein (GFP). The fluorescence emission can then be detected by a light-to-frequency converter (TSL 235R) located above the reaction chamber. The p-Cresol sensing bacteria can be changed to any kind of biomarker sensing bacteria, which gives CreSense a wide range of applications.
Device Budget
| Component | Quantity | Cost per unit | Cost |
|---|---|---|---|
| Arduino Uno | 1 | 160 NTD (5.23 USD) | 160 NTD (5.23 USD) |
| NodeMCU ESP8266 | 1 | 337 NTD (11 USD) | 337 NTD (11 USD) |
| Electric speed controller | 1 | 550 NTD (17.96 USD) | 550 NTD (17.96 USD) |
| Brushless motor | 1 | 450 NTD (14.70 USD) | 450 NTD (14.70 USD) |
| TSL 235R | 1 | 135 NTD (4.41 USD) | 135 NTD (4.41 USD) |
| Filter | 1 | 520 NTD (16.98 USD) | 520 NTD (16.98 USD) |
| Casing | 1 | 500 NTD (16.33 USD) | 500 NTD (16.33 USD) |
| Screws | 8 | 7.5 NTD (0.24 USD) | 60NTD (1.96 USD) |
| Lithium-ion battery | 1 | 300 NTD (9.80 USD) | 300 NTD (9.80USD) |
| Sensor holder | 1 | 200 NTD(6.53 USD) | 200 NTD (6.53 USD) |
| Total | 3212NTD (104.90 USD) | ||
Demonstration
To demonstrate our engineered system works, we have made a video to explain each part of CreSense and the operating process.
Blood Plasma Separation
White blood cells in human blood which could be lethal to the p-Cresol sensing bacteria. In order to make our p-Cresol sensing bacteria able to survive in the blood sample and produce fluorescent protein, filtering out blood cells beforehand is necessary. We searched for a simple way to separate blood plasma while integrating all the required steps. Finally, we chose a centrifugal microfluidic platform to achieve this goal[3].
In the microfluidic centrifugal platform, fluidic processing steps such as the mixing of reagents or the transfer of samples can be automated simply by implementing different spinning profiles[4]. Also, it can be easily operated by healthcare professionals in diagnostic centers. With all the advantages mentioned above, we’re able to separate the blood plasma and mix it with the p-Cresol sensing bacteria efficiently.
CreSense is equipped with a brushless motor to reduce the operational and mechanical noises, and also to obtain higher torque to weight ratio.
The microfluidic centrifugal platform disc is made by three layers of laser-cut-machine-processed acrylic disc which is combined by two layers of adhesives.
For each slot, we designed two chambers. The chamber on the right (trapezoidal chamber) is the blood separation chamber, the one on the left (square chamber) is the reaction chamber. The blood separation chamber is where we inject whole blood for plasma separation and the reaction chamber is where the p-Cresol sensing bacteria resides and expresses GFP.
By implementing different spinning profiles, plasma can be extracted from whole blood and directed into the reaction chamber.(hyperlink: to “Device demonstration”) After injecting blood sample, the disc spins at 3000RPM (600RCF) for 10 minutes where the plasma and blood cells would be separated in the blood separation chamber. Then, we lower the speed to 900RPM (50RCF) and capillary forces will direct plasma into the reaction chamber through the lower channel between two chambers.
At the beginning of designing the centrifugal microfluidic platform, we used polydimethylsiloxane (PDMS) to make microfluidic chips as sample holders and tested several versions. However, PDMS chip‘s tests continues to give unsatisfactory results due to bonding issues. In the middle of August, we consulted professor Ruey-Jen Yang (hyperlink: to “Integrated HP”), he suggested us to replace PDMS with acrylic due to its strength and deformation resistance properties. In the end, we choose acrylic as the material used in the device. Both methods are listed below.
We made acrylic molds for casting PDMS into the desired shape, then bonded another flat layer of PDMS onto it with the help of oxygen plasma treatment to cover up the channels, forming a three-dimensional structure. The chamber on the right (trapezoidal chamber) is blood separation chamber, the one on the left(square chamber) is reaction chamber. The blood separation chamber is where we inject whole blood to separate plasma. The reaction chamber is where the GFP production take place. From June to July, we made three different versions of molds with minor changes solving the problems we faced.
In version 1, we found out that when we insert fluid into the chip, there would be bubbles stuck at the corner of the chamber. We solved it by filleting every corner in version 2. When testing version 1 & 2, the bottom of whole blood chamber wasn’t strong enough to endure the force sample creates when the spinner’s rotational speed reaches 2500 RPM, we tried to overcome this by reinforcing the bottom part of the blood separation chamber in version 3.
After we made the mold for version 3, we consulted Professor Ruey-Jen Yang. He suggested us to use glass slides instead of another layer of PDMS to cover the channels, which the bonding will be stronger compared to PDMS-PDMS bonding. We took his advice and the result turned out to be unsatisfying - several bonding failed. However, after a week of debugging we obtained successful ones by adjusting the parameters of the oxygen plasma machine.
Final test for PDMS chip
In the final test, we put three microfluidic chips (PDMS-glass bond) on the chip holder and raise the spinning profile to 3000RPM for 10 minutes and then lowered 900 RPM for 5 minute[3]. The PDMS-glass bond chip is able to withstand the centrifugal force. With the adjusted parameters of the oxygen plasma machine, the bond between PDMS-PDMS layer was also strengthened and it too worked fine for blood plasma separation. We choose acrylic disc instead of PDMS chip in the end because PDMS chip has a longer fabrication time.
The major problem we encountered when we inject blood into the PDMS chip, blood often flew all the way into the reaction chamber instead of being confined in the reaction chamber. So we designed a passive valve in the connecting channel between the blood separation chamber and the reaction chamber.
Final test for acrylic disc
In the acrylic final test, we did the same experiment in PDMS chip final test. Acrylic disc’s ability to separate plasma turned out to be successful. We can see that there’s significant separation of plasma. Also, the plasma flew into the reaction chamber as we desired.
Blood p-Cresol Reader
For determining p-Cresol levels in patient’s blood, CreSense’s integrated fluorescent detector quantifies light intensity emitted by the p-Cresol sensing bacteria, which expresses GFP when in contact with blood plasma p-Cresol.
In our biobrick construct, we are using GFP as our reporter. To excite the emission of fluorescence by the GFP, we used a blue laser. The optimal fluorescence emission is at around 515 nm. A 510 nm filter is used to block the excitation light and allow only the emission light to pass through.
With the aid of a light-frequency converter - TSL 235R as our light sensor, we will be able to read different emission light intensity. Sensor readings will be converted to p-Cresol concentration and displayed on the LCD screen and uploaded onto our realtime database for further monitoring.
Our device is able to measure fluorescence intensity equivalent to 50nM of fluorescein. We used the Fluorescein Sodium Salt provided in the measurement kit for our calibration curve for the detector.
We also tested our fluorescence detector by adding autofluorescence E. coli (pSB1C3-j23100-sfGFP in DH5a) into blood plasma and we are now positive that our device can successfully senses the fluorescence emission from the bacteria in plasma.
At the beginning of designing our fluorescence detector, we used LDR as our light sensor. We soon realized that without a filter, our detector reading could not reflect the actual fluorescence intensity. From FigC2, we can see that the light sensor works fine for light transmittance measurement. The darker the surroundings is, the lower the LDR value.
This does not fit the purpose of our device which is fluorescence measurement. To measure only the fluorescence emission but not the total visible light transmission, we decided to add a narrow bandpass filter to block the excitation light (510nm) from the blue laser reaching the light sensor. We also changed the position of the light sensor and avoid a collinear arrangement. Besides, we replaced the light dependent resistor with our TSL 235R light-frequency converter thus improving the accuracy of our device.
Online Database
CreSense will be placed at the healthcare service provider. We connect Arduino Uno to a NodeMCU to upload detector readings onto an online realtime database with Firebase. These data can then be accessed by the healthcare professionals. In the future, we are hoping that we can integrate with Taiwan’s Ministry of Health and Welfare database. By doing so, our device can make a greater impact by helping to monitor the blood p-Cresol level of our CKD patients.
Arduino code and Circuits
We use Arduino IDE to code for our Arduino UNO and a NodeMCU board for our whole device. The complete code can be found on Github.
Below is our entire wiring sketch:
References
- 大安聯合醫事檢驗所. (n.d.). Retrieved from http://www.ucl.com.tw/webshop/shop/ServiceQueryInfo.asp?GoodsID=D0114066&GoodstypeID=D0&MiddleID=D011
- 新北市政府衛生局 . (n.d.). 門診資訊-自費抽血檢驗項目表. Retrieved from https://zhonghe.health.ntpc.gov.tw/content/index.php?parent_id=10054&type_id=10054
- Gorkin, R., Park, J., Siegrist, J., Amasia, M., Lee, B. S., Park, J.-M., … Cho, Y.-K. (2010). Centrifugal microfluidics for biomedical applications. Lab on a Chip, 10(14), 1758. doi: 10.1039/b924109d
- Amasia, M., & Madou, M. (2010). Large-volume centrifugal microfluidic device for blood plasma separation. Bioanalysis, 2(10), 1701–1710. doi: 10.4155/bio.10.140