Background
Now, the quality of our lives is increasing, and people with diabetes and high blood pressure are gradually increasing. Patients with diabetes for about 5 years or those with Hypertension for 5 to 10 years are at high risk of chronic kidney disease and are at risk of renal impairment at any time. Since diabetics and hypertensives are usually treated at home in early stages , routine blood and urine tests are rarely performed and the U-β2M indicator is unknown. This has led many patients to miss the opportunity for early intervention and control.
We hope to produce a test kit that can detect changes in β2-MG in the early stages of CKD and go to the hospital early to prevent further deterioration.
The main purpose of our experiment is to prove whether the change in β2-MG concentration in the home test box (Dr. Familict) can be reflected in the liquid crystal through a noticeable gray scale change. Therefore, we have constructed a molecule that is more suitable for the liquid crystal detection system based on the nano-antibody, a competitive combination which can disturb the liquid crystal to change the gray level of the image.
Experiment
Our experiments are mainly divided into two parts: molecular biology experiments and liquid crystal experiments.
We obtained nano-antibodies with aldehyde groups by adding aldehyde-based labels to nano-antibodies and by FGE treatment. After that, we added C18 to the Nanobody to enhance its interference. Finally, we verified that the interference of the antigen concentration on the liquid crystal can be detected.
In the molecular biology sector, we planned to connect a long-chain molecule (C18) on the nanobody in order to increase the interference of nano-antibodies on liquid crystals.
Firstly, We constructed two plasmids, pET28a and pBAD. The first plasmid pET28a contains a nanobody and a gene with an aldehyde-based catalytic substrate tag. The second plasmid, pBAD, contains the FGE (Formylglycine-generating enzyme) gene. Then we changed the conversion plan several times due to poor conversion efficiency, and finally got a satisfactory conversion rate.
We wanted to see that the substrate tag can be finally expressed at the inactive end of the nanobody in E. coli BL21, and has little effect on the conformation of the nanobody. In the cell, the substrate tag was converted to an aldehyde group by FGE treatment. Subsequently, after the transformation of aldehyde-based nanobody, we disrupted the cells by sonication and purified the disrupted cell suspension to obtain the nanobody we needed. Next step, we combined the Nanobody with C18. We have obtained a better experimental protocol by changing the conditions
We designed a liquid crystal cell as the observation carrier. Firstly, we modified the slides with DMOAP which can induce vertical alignment of the liquid crystals, making the initial state of the optical imaging of the liquid crystal cell black. Meanwhile, we modified the slides with APTES so that they can be attached to the β2-MG; Then we added β2-MG nanobody to the modified slide. Finally, the upper and lower slides were assembled, and the liquid crystals were added. We can observe the optical imaging of the liquid crystal cell under the microscope to judge the β2–MG of the detected sample concentration.
Verification part of molecular biology
Aldehyde and C18 connection verification
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Fig.1. Immunoassay for immunoglobulin after aldehyde aldolization (a) Fluorescent group (b) 25 ° C C18 (c) 37 ° C C18 (d) acidified supernatant (e) dye-free Marker1 (f) Defective Marker2(g) BL21 cell disrupted supernatant (h) BL21 cell disrupted pellet |
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Fig.2. Analysis of dye-free electrophoresis |
The molecular weight changes for (b) and (c) are not obvious, however, the C18 linkage at 37 ° C of (c) produces a certain amount of precipitation, which is not efficient. The acidification of the supernatant (g) in (d) serves as a purification, that is, the nano-antibody has separated from the supernatant, so the molecular weight remarkably lowered remarkably.
After processing, the molecular weight of the Nanobody can be calculated to be about 250 KDa.
Nanobody concentration determination
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Fig.3. Protein concentration - absorbance standard curve |
In conclusion, we can determine the concentration of the nanobody through further experiments based on the standard curve of protein concentration and the molecular weight of the nanobody measured by electrophoresis.
Liquid crystal verification section
Determination of immobilized β2-MG concentration
1. Standard for immobilized β2-MG concentration
We have known that the orientation of liquid crystal molecules is very sensitive to the topographic changes on the substrate surface. When the β2-MG and GA are crosslinked and fixed on the substrate surface, the topographic structure of the surface will be changed to a certain extent affecting the orientation of the liquid crystal molecules. Therefore, it is necessary to investigate the effect of the concentration of immobilized β2-MG on the optical imaging of the liquid crystal cell. With the decrease of the concentration of immobilized β2-MG, the perturbation to the orientation of liquid crystal molecule decreases, and the optical imaging of liquid crystal cell gradually darkens. Only a few speckles appear in the optical imaging of liquid crystal cell, which tend to be all-black background. The highest immobilized β2-MG concentration, which can keep the optical imaging background dark, is fixed on the substrate surface when sufficient antigen and antibody reactions are required.
2. Result
All the other conditions are unchanged. The concentration of β2-MG added from 250ng/mL to 700ng/mL is as follows:
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Fig. 4.Optical imaging of liquid crystal cell with different concentrations of immobilized β2-MG（a）1000ng/mL β2-MG （b）900ng/mL β2-MG（c）800 ng/mL β2-MG（d）700ng/mL β2-MG（e）600ng/mL β2-MG（f）500ng/mL β2-MG（g）400ng/mL β2-MG（h）300ng/mL β2-MG（i）250ng/mL β2-MG |
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From the experimental results, we found that when the concentration of β2-MG fixed to the substrate surface is under 500 ng/mL, only a few bright spots appear in the optical imaging of the liquid crystal cells gradually darkening, approaching the full black background. When the concentration of β2-MG is 500 ng/mL, the optical imaging of the liquid crystal cells shows obvious bright spots. When the concentration of β2-MG is above 500 ng/mL, the optical imaging appears obvious bright spot which is becoming brighter gradually.
3. Determination of immobilized β2-MG concentration
As a result, we can find that the optical imaging of the liquid crystal cell becomes brighter and brighter with the increase of the concentration of fixed β2-MG under other conditions. Therefore, we can determine the change trend of the brightness of the liquid crystal. The concentration of β2-MG increases in the same trend. We can establish the relationship between the luminance of liquid crystal and the concentration of β2-MG, and then determine the corresponding concentration of β2-MG according to the luminance of liquid crystal, so as to achieve the purpose of quantitative detection. Due to the need for sufficient β2-MG to react with antibodies, a maximum immobilized β2-MG concentration of 500 ng/mL, which can keep the optical imaging background dark is selected to be fixed on the substrate surface.
Determination of the concentration of β2-MG nano antibody
1. Screening criteria for the concentration of β2-MG nanoparticles
The influence of the concentration of the nano-antibody on the optical imaging of the liquid crystal cell was further investigated. If we don’t add β2-MG in the sample, there will has color spots in the polarizing microscope imaging. When the concentration of the nano-antibody in the sample was low, only a few bright spots approached the full black background. In the subsequent detection, the substrate immobilized β2-MG will compete with the tested sample β2-MG to bind the limited reaction sites on the β2-MG nano-antibody. When the concentration of the tested β2-MG approaches zero (detection limit is infinitely low), the β2-MG nano-antibody will combine with the substrate immobilized β2-MG to disturb the orientation of the liquid crystal molecule to the maximum extent, resulting in the optical imaging of the liquid crystal cell. With the increase of the concentration of β2-MG, the binding of β2-MG nano-antibody to the substrate immobilized β2-MG decreases, and the optical imaging becomes dark from, thus completing the detection of β2-MG.
2. Results
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Fig. 5. Change the concentration of fixed nano-antibodies to β2-MG under the condition of immobilizing 500 ng/mL of β2-MG on slide. (a) 250ng/mL of β2-MG nano-antibodies (b) 400 ng/mL of β2-MG nano-antibodies (d) 500 ng/mL of β2-MG nano-antibodies (c) 600 ng/mL of β2-MG nano-antibodies |
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From the experimental results, the fixed concentration of β2-MG was 500 ng/mL and the fixed antibody concentration was changed. When the concentration of β2-MG nano-antibody was 250 ng/mL, the optical imaging spot of liquid crystal cell was star-shaped, which was more than that of the non-fixed β2-MG antibody, but the change was not very large. When the concentration of β2-MG nano-antibody is above 400 ng/mL, the distribution of the optical imaging spot of the liquid crystal cell is similar to that of the β2-MG nano-antibody when the concentration of β2-MG nano-antibody is 400 ng/mL.
3. Determination of the concentration of β2-MG nano antibody
Through the experimental results, we can find that with the increase of antibody concentration, the orientation of liquid crystal molecules is disturbed, and the bright spots in optical imaging are gradually increased, but 400 ng/mL is the threshold of β2-MG nanobody concentration, when the concentration of Nano-antibody When it is increased, the bright spots of liquid crystal imaging are not significantly increased. Therefore, the minimum immobilized β2-MG nanobody concentration of 400 ng/mL, which can maintain the most bright spots in the optical imaging background, was fixed on the surface of the substrate.
Different effects of common β2-MG nano-antibodies and C18-modified β2-MG nano-antibodies on optical imaging of liquid crystal cells at the same concentration
1. Standard for the concentration of β2-MG antibody
The size of the antibody modified by C18 long chain is larger than that of the normal antibody, and the effect of the antibody on the orientation of the liquid crystal molecules is also greater. Therefore, it is necessary to further investigate the effects of ordinary β2-MG nano-antibodies and C18 long-chain modified β2-MG nano-antibodies on the optical imaging of liquid crystal cells.
2. Result
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Fig. 6. Change the concentration and types of fixed β2-MG nano-antibodies when 500 ng/mL β2-MG is immobilized on slide. (a) 250ng/mL modified C18 β2-MG nano-antibodies. (b) 250ng/mL β2-MG nano-antibodies. (c) 400ng/mL β2-MG nano-antibodies |
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From the experimental results, when the concentration of fixed β2-MG nano-antibody is same (250ng/mL), the brightness of the liquid crystal cell with C18 modified β2-MG nano-antibody is higher than the threshold of normal nano-antibody.
3. Comparison of sensitivity between normal β2-MG nano antibody and C18 modified β2-MG antibody
From the experimental results, we can find that the C18 modified β2-MG nano-antibody has greater disturbance to the liquid crystal molecules, so we can judge that the sensitivity of the C18 modified β2-MG nano-antibody is higher.
Conclusion
Through a series of Confirmatory experiment, we have proved that in the early stage of chronic kidney disease, our detection system can indeed reflect the changes in β2-MG concentration, which can remind users to go to the hospital for further testing.
In the future, we plan to establish a more complete back-office system, which can directly obtain more accurate test results based on the user's past physical indicators.
