Nowadays, the thrombosis risk analysis is still not a convenient device for home-aid detection. We not only aim to build up a convenient detector, but also integrate our device with the Nattokinase release. D-dimer a type of fibrin degradation products. It’s a common and effective marker especially for the deep vein thrombosis (DVT) [1]. Fortunately, D-dimer is also occurring in human saliva [2], so we can measure its level by non-intrusive methods. We applied latex enhanced immunoturbidimetric assay (LEIA) [3], where antibody agglutination with the target protein to measure the absorbance reflecting the concentration. In addition, we designed a smartphone application that could control the measurement of d-dimer level and release the IR by a simple tap. The IR release induces our optogenetic system and produces Nattokinase immediately.
Aims
1. Quantification of D-Dimer concentration with absorbance. 2. Build up a convenient device with complete function. 3. Design a simple application to control our device.
Design
1. Prepare 112 L reaction buffer, 64 L Latex reagent in reaction well. 2. Collect and pipet 8 L saliva sample into reaction well. 3. Let the sample stand still for 10 mins to react completely. 4. Turn on the Arduino-controlled light source and detect the transmission of 600nm light. 5. The signal can be sent to smartphone app by the blue tooth module. 6. Trigger the Infrared by tapping the app instruction. 7. Nattokinase production and release.
Part 1. Quantification D-Dimer standard curve
LEIA assay illustration
The anti-D-Dimer antibody could capture the D-Dimer to form agglutination. Further, the Latex beads enhanced the turbidity that could be measured by a spectrophotometer. The 600 nm wavelength absorbance could transfer to the concentration of D-Dimer and show thrombosis risk level.
Artificial saliva recipe:
components
concentration
glycerol
2%
KCl
0.625 (g/L)
MgCl2 ∙6H2O
0.06 (g/L)
CaCl2 ∙2H2O
0.166 (g/L)
K2HPO4
0.804 (g/L)
KH2PO4
0.326 (g/L)
Adjust pH to 6.75 with KOH
We construct the standard curve with TechnoLEIA® D-Dimer kit and prepare artificial saliva for simulating the saliva environment. To determine whether volume of sample affects the measurement. We design different experimental sets. We finally choose the third one for further measurement, 8 L D-Dimer sample with 112 L reaction buffer and 64 L Latex reagent.(X-axis is the D-Dimer concentration, y-axis is the absorbance normalization intensity.)
We construct the standard curve by measuring different concentrations of D-Dimer absorbance.It shows a linear relation that conforms to beer’s law.
Part 2. Build up our hardware
Our device consists of some critical components. First of all, the Arduino UNO board is the core of the whole device and the bluetooth module (Arduino HC-06) could receive and release signals with a cell phone app. Second, the spectrophotometer (TCS3200 light sensor) with our modification could act as an orange light (around 600 nm) source and detect the transmission light intensity. Last, the red LED bulb act as a IR release simulation, we can easily control it by our application.
3D print model and our device
Movie demonstration of our device
We modeled our simplified final product design on Sketchup online. A plastic disposable reaction holder with preloaded anti-d-dimer beads dissolved in buffer can be plug into the device easily after the addition of saliva sample.
Part 3. Design Natto it out application
We designed a user-friendly interface application for controlling the device by MIT app inventor. The application is a key element for connecting the D-Dimer thrombosis risk detection with the Nattokinase release. The user could finish all steps from detection to decrease fibrin accumulation in 10~20 mins.
[1] Alejandro Lazo-Langner, Diagnosis of Pulmonary Embolism with D-Dimer Adjusted to Clinical Probability, The new England journal of medicine 2019
[2] Zhang, X., Wan, Y., Cooper-White, J., Dimeski, G., Atherton, J., & Punyadeera, C. (2013). Quantification of D-Dimer levels in human saliva. Bioanalysis, 5(18), 2249–2256.
[3] Diao D, Wang Z, Cheng Y, Zhang H, Guo Q, Song Y, Zhu K, Li K, Liu D, Dang C. D-Dimer: not just an indicator of venous thrombosis but a predictor of asymptomatic hematogenous metastasis in gastric cancer patients. PLoS One. 2014 Jul.
