Difference between revisions of "Team:DLUT China B/Demonstrate"

Aldehyde and C18 connection verification

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

Fig.2. Analysis of dye-free electrophoresis

The molecular weight changes for (b) and (c) do not appear to be 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 Nanobodies is separated from the supernatant, so the total protein concentration decreased significantly and the relative abundance of Nanobodies increased significantly.

After processing, the molecular weight of the Nanobody can be calculated to be about 18 KDa.

Nanobody concentration determination

Fig.3. Protein concentration - absorbance standard curve

Based on the standard curve of protein concentration and then based on the molecular weight of the nanobody measured by electrophoresis, we can determine the concentration of the Nanobody for further experiments.

We measured the absorbance of three groups of protein concentrations:

$$ y_1 = 0.520,\quad y_2 = 0.536,\quad y_3 = 0.495 \quad \mathrm{(Abs)}$$

According to the standard curve, the corresponding concentration was obtained.

$$ c_1=392.7,\quad c_2=410.9,\quad c_3=363.2 \quad \mathrm{(μg/mL)}$$

We designed a liquid crystal cell as an observation supporter. First, the upper glass slide was modified with 0.2%DMOAP to induce the vertical alignment of the liquid crystals and make the optical imaging of liquid crystals appear black at the first time.Meanwhile, we modified the lower slide with a mixture solution of 3% (v) APTES and 1% (v) DMOAP and 1% (v) GA, so that it could connect to the target protein. Then We mixed the sample with the antibody and added it to the modified slide. Finally, the upper and lower slides were assembled, and then the liquid crystals were added. We could observe the optical imaging of the liquid crystals pool under the microsco-pe, so as to determine the concentration of β₂-MG of the sample. The following paragraphs are the results of our investigation on the fixed antigen and antibody concentration, and the brightne-ss contrast of β₂-MG nano-antibody modified by C18 and the normal β₂-MG nano-antibody combined with the liquid crystal.

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

Keeping other conditions unchanged,the concerntration of β₂-MG is in the range of 250ng/mL to 1000ng/mL, and the results are shown in Fig. 4.

(a)	(b)
(c)	(d)
(e)	(f)
(g)	(h)
(i)
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

From the experimental results, the results show 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 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, the results show that the optical imaging of the liquid crystal cell becomes gradually bright with the increase of the concentration of fixed β₂-MG under other conditions unchanged. Therefore, we can determine the change trend of the brightness of the liquid crystal. The concentration of β₂-MG increases in the same trend. The relationship between the luminance of liquid crystal and the concentration of β₂-MG can be established, 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 of the experiment, the maximum immobilized β₂-MG concentration of 500 ng/mL is selected to be fixed on the substrate surface, which can keep the optical imaging background dark.

Determination of the concentration of β₂-MG nano antibody

1. Screening criteria for the concentration of β₂-MG nano-antibody

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

Under the condition of 500ng/mL of β₂-MG, 250ng/mL, 250ng/mL, 500ng/mL, and 600ng/mL of β₂-MG nano-antibodies were fixed on the glass slides, and the color and brightness changes of the liquid crystal film were compared, as shown in Fig. 5.

(a)	(b)
(c)	(d)
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 (c) 500 ng/mL of β2-MG nano-antibodies (d) 600 ng/mL of β2-MG nano-antibodies

The results shows that when the concentration of β₂-MG nano-antibody was 250 ng/mL, the optical imaging spot of liquid crystal cell was star-shaped, whose quantity was more than that of the non-fixed β₂-MG antibody, but the change was not obvious. 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 the phenomena of the concentration of β₂-MG nano-antibody which is 400 ng/mL.

3. Determination of the concentration of β₂-MG nano antibody

Through the experimental results, we could find that as the concentration of antibody increased , the crystal molecular orientation is getting more messy, and the bright spots gradually enhance in the optical imaging. However, 400ng/mL is the threshold of the concentration of β₂-MG nano-antibodies. When the concentration of nano-antibodies increases again, the bright spots in the liquid crystal imaging did not enhance significantly. Therefore, the lowest antibody concentration at 400ng/mL was selected for the immobilization of β₂-MG on the substrate surface, which could maintain the maximum bright spots in the optical imaging background.

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 β₂-MG nano-antibody modified by the long chain of C18 is larger than that of β₂-MG nano-antibody. 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

Under the condition of 500ng/mL of β₂-MG, 250ng/mL C18 modified of β₂-MG nano-antibodies, 250ng/mL and 400ng/mL of β₂-MG nano-antibodies were fixed on the glass slides, and color and brightness changes of liquid crystal film were compared, as shown in Fig. 6.

(a)	(b)
(c)
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

From the experimental results, fixed with the same concentration of β₂-MG nano antibody (both with a concentration of 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.

Through experiments, we finally decided to immobilize 500ng/mL β₂-MG on the substrate , binding with 400ng/mL β₂-MG nano-antibody. In addition, we determined that the detection sensitivity of the antibody at β₂-MG modified by C18 was higher. Therefore, we can detect the concentration of β₂-MG in the range of 100ng/ ml-400ng /mL (a theoretical estimate) by this method, which meets our requirements for the early diagnosis of nephropathy patients.

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.