Overview
Our hardware aims to develop a fast, convenient and affordable POCT device. Based on synthetic biology-based methods of HCR and CRISPR, it can assist doctors in pain classification, so as to facilitate accurate diagnosis and medication.
Up to now, the developed POCT platforms mainly include centrifugal microfluidic devices, MEMS, and paper-based analytical devices. Paper has become an attractive material for bioassay devices due to its simplicity, portability, and low cost, and the functions of paper materials are gradually developing in the functionalized films of various materials. Paper-based diagnostic devices have been accepted as an excellent alternative for future POC testing.
Therefore, our project constructed a POCT device based on a paper-based chip. HCR and CRISPR reactions can go on on paper-based chips. Heating, fluorescence detection, and software modules are provided to improve the portability and real-time performance of the device. Our device is characterized by high throughput, miniaturization, user-friendliness, and instant analysis. The entire detection process takes about an hour, which is convenient and quick.
System workflow:
For detection, we first add the patient’s blood sample to the paper-based chip and then put the chip on the heating instrument for HCR and CRISPR reactions. After about 60 minutes, we place the paper-based chip into the optical detection module for fluorescence detection. Then, the results can be displayed on the smartphone, and they can be transmitted to the doctor's computer through Bluetooth to assist the doctor in pain classification so that doctors can accurately diagnose and prescribe drugs.
Paper-based chip
To enable POC testing of HCR and CRISPR, we developed a fast, low-cost, and user-friendly paper-based chip. The capillary force and patterned hydrophobic channel design of the paper-based chip can control the directional flow of liquid and can be combined with the heating module and fluorescence detection module to complete the detection task. The paper-based chip consists of three functional layers, including the transfer pad, fluid channel pad, and reaction pad. When the sample solution is loaded into the sample injection hole, the sample solution wets the transfer pad and then flows through the fluid channel pad to the reaction pad. The asymmetric structure of the transfer pad and the presence of the fluid channel pad ensure the horizontal and vertical flow of the sample solution and the uniformity of the four channels. The fluid channel pad contains four patterned flow channels, and each reaction pad works independently and displays the test results simultaneously. The paper-based chip allows HCR reagents to be pre-embedded in the reaction pad by drying and can detect multiple miRNA targets simultaneously by embedding different HCR probes, which can be performed by adding CRISPR reagents and samples at the same time. In addition, the compatibility of the paper-based chip with biological reactions, the pre-embedding of HCR reagents, and the reactions of HCR and CRISPR were verified. The paper-based chip is compact and user-friendly, which has great potential for POC testing.
(A) Schematic diagram of the paper chip structure (B) Schematic diagram of the reaction flow on the paper chip (C) Schematic diagram of the biological reaction flow (D) Diagram of the test results (E) Physical diagram of the paper chip
For more details, please click>>Paper-Based Chip<<
Devices
Heating instrument
In order to meet the requirements of a constant temperature of 37°C and a miniaturized bioreaction system on the paper chip, we have made the following heating instrument. The heating instrument is mainly composed of four parts: a PT1000 thermal sensor; a silicone rubber heating film; a TCM1040 temperature control module, and structural housing. The user presets the parameters of the temperature control module through the host computer. After entering the working state, the temperature controller controls the heating of the silicone rubber heating film. The PT1000 thermal sensor collects the temperature data of the heating film and feeds the data back to the temperature controller. Through feedback regulation, the system can maintain the temperature around 37℃.
(A) Schematic diagram of temperature control structure; (B) Heating curve
For more details, please click>>Temperature Control Module<<
Optical detection hardware
As a good operation and detection platform, smartphones have been widely used in POCT. In order to realize the visualization and quantitative detection of four channels at the same time, we designed optical detection hardware combined with a smartphone. The fluorescence detection module adopts an oblique optical path, with the smartphone flash as the light source and the camera as the optical sensor. The device can work with the paper-based chip to achieve simpler and faster detection.
(a)The Optional Structure Of Our Equipment (b)Equipment Mode Diagram (c)Picture Of Linear Results Obtained By the Equipment (d)Physical Drawing Of Equipment
For more details, please click>>Optical detection hardware<<
Optical detection software
We developed an App for fluorescence result analysis. The core functions of the App are to analyze fluorescence information and display fluorescence intensity. We constructed the user interaction interface, photo interface, and diagnostic result presentation interface through Android Studio, IDEA, and PyCharm. The App can realize image acquisition, fluorescence region selection, fluorescence intensity measurement, and detection result analysis. We get the fluorescence intensity by analyzing the G value of the picture.
For more details, please click>>Optical detection software<<
FAQ
Does our hardware address a need or problem in synthetic biology?
Yes! CRISPR is an outstanding diagnostic technology in the field of synthetic biology. However, CRISPR in the laboratory still has some shortcomings, such as high cost and complex equipment. If CRISPR can be used in POCT, it will give full play to its advantages. The POCT device based on paper-based chips developed by us is user-friendly and portable, and it can be used without professionals, which helps to apply CRISPR in POCT.
Our project has constructed the HCR and CRISPR/Cas system, which aims to achieve the POCT of multiple miRNAs with few operation steps. First, fewer operation steps make demands on reagent pre-embedding. By selecting appropriate paper materials, HCR reagents can be drily stored without freeze-drying or other technologies, so that HCR reagents can be easily pre-embedded. Second, we solved the problem of simultaneous detection of multiple miRNAs through the patterned design of paper-based chips and the high throughput analysis capability of fluorescence devices. Third, we also designed a simple paper-based chip through the sequential stacking of functional layers and built a compact and user-friendly POCT device based on the paper-based chip, which realized the POCT of the HCR and CRISPR/Cas system and improved the ability of synthetic biology methods for POCT.
Did we conduct user testing and learn from user feedback?
Of course, our project did user testing and learned from user feedback during design, building, and final testing. We interviewed the staff of a third-party institution, who suggested that we distinguish different patient samples to better meet social needs. In response to this feedback, we came up with a way to label each patient sample with individual information. For example, the label is presented by a QR code. A smartphone using our optical detection device can scan the QR code to enter patient information. Second, we also learned from user feedback on operational issues. We randomly selected people as users to simulate the use of our hardware devices. In order to improve the user experience and reduce the difficulty of use, we added user guidance in the app. In addition, we constructed a split structure which was made by 3D printing to integrate each module in our project, which greatly reduced the operational difficulty of users.
For more details, please click>>implementation<<
Were we able to demonstrate utility and functionality in our hardware proof of concept?
Yes! We have verified the functions of each component module of the hardware to ensure that they can achieve their own functions and cooperate with other modules. We also demonstrated the complete use process of the system, which shows that our hardware can be used easily and realize the detection requirements. For the paper-based chip module, we have successfully verified that it can well control the liquid flow through the leakage test, uniformity test, and injection volume test experiments. We have conducted a step-by-step and complete biological reaction on the paper-based chip and compared it with the laboratory results, proving that the HCR and CRISPR reactions can go on in paper-based chips. For the optical detection module, we processed the image data, and successfully verified that the data have a good linear relationship with the fluorescence intensity, which provides a good basis for the accuracy of our detection results. For the temperature control module, we verified that the equipment can continuously and uniformly provide heat for the reaction through real-time temperature monitoring and uniformity, accuracy, and precision testing of the heating process. Finally, the operation demonstration of the whole system verifies that our hardware has high practicability.
For more details, please click>>proof of concept<<
Is there sufficient documentation of the hardware system for other teams to reproduce the device?
Yes! We are very happy to share the documentation, including the design and process of paper-based chips, as well as all the 3D models and software code, for future teams in need. You can view and download them in our contribution.
For more details, please click>>contribution<<
Reference
Seok, Youngung, et al. "A paper-based device for performing loop-mediated isothermal amplification with real-time simultaneous detection of multiple DNA targets." Theranostics 7.8 (2017): 2220.