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

m (修改文案。)
(修改文案。)
Line 23: Line 23:
 
                 <h2 class="title">Background</h2>
 
                 <h2 class="title">Background</h2>
 
                 <div class="mainText">
 
                 <div class="mainText">
−
                     <p>Now, the quality of our lives is increasing, and people with diabetes and high blood pressure are
+
                     <p>In order to fulfill the requirements of testing diseases at home for patients instead of going to
−
                         gradually increasing. Patients with diabetes for about 5 years or those with Hypertension for 5
+
                         hospital tested by high-tech testing equipment. We designed our products which can upload
−
                         to 10 years are at high risk of chronic kidney disease and are at risk of renal impairment at
+
                         grayscale data to the cloud and analyze it through mobile devices obviously. What we required is
−
                         any time. Since diabetics and hypertensives are usually treated at home in early stages ,
+
                         just a liquid crystal detection scheme, which is sufficient to reflect the change in
−
                        routine blood and urine tests are rarely performed and the U-β<sub>2</sub>M indicator is
+
                        β<sub>2</sub>-MG
−
                        unknown. This has led many patients to miss the opportunity for early intervention and control.
+
                         concentration.</p>
−
                    </p>
+
                     <p>The main purpose of our experiment is to prove whether the change in β<sub>2</sub>-MG
−
                    <p>We hope to produce a test kit that can detect changes in β<sub>2</sub>-MG in the early stages of
+
                        concentration in the
−
                         CKD and go to the hospital early to prevent further deterioration.</p>
+
−
                     <p>The main purpose of our experiment is to prove whether the change in β<sub>2</sub>-MG concentration in the
+
 
                         home test box (Dr. Familict) can be reflected in the liquid crystal through a noticeable gray
 
                         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
 
                         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
 
                         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.</p>
 
                         the liquid crystal to change the gray level of the image.</p>
−
                     <p>We designed a competitive binding method: First, immobilizing a certain amount of β<sub>2</sub>-MG in a
+
                     <p>We designed a competitive binding method : First, immobilizing a certain amount of β<sub>2</sub>-MG
 +
                        in a
 
                         liquid crystal, and then combining the test solution with an excess of Nanobody. The mixture is
 
                         liquid crystal, and then combining the test solution with an excess of Nanobody. The mixture is
 
                         then added to the liquid crystal, and due to the excess of the Nanobody, the excess is bound to
 
                         then added to the liquid crystal, and due to the excess of the Nanobody, the excess is bound to
−
                         the immobilized β<sub>2</sub>-MG, causing perturbation of the liquid crystal. Since the fixed β<sub>2</sub>-MG is a
+
                         the immobilized β<sub>2</sub>-MG, causing perturbation of the liquid crystal. Since the fixed
−
                         certain amount, the amount of β<sub>2</sub>-MG in the liquid to be tested can be calculated by the
+
                        β<sub>2</sub>-MG is a
 +
                         certain amount, the amount of β<sub>2</sub>-MG in the liquid to be tested can be calculated by
 +
                        the
 
                         difference method. However, during the experiment, we found that the nano-antibody itself has
 
                         difference method. However, during the experiment, we found that the nano-antibody itself has
 
                         little disturbance to the liquid crystal, so we hope to construct a more interfering molecule
 
                         little disturbance to the liquid crystal, so we hope to construct a more interfering molecule
−
                         based on the nano-antibody. At the same time, we tested the effects of different β<sub>2</sub>-MG and
+
                         based on the nano-antibody. At the same time, we tested the effects of different β<sub>2</sub>-MG
 +
                        and
 
                         nanobody concentrations on liquid crystal perturbations, and hope to find a set of solutions
 
                         nanobody concentrations on liquid crystal perturbations, and hope to find a set of solutions
 
                         with the most obvious gray scale changes.</p>
 
                         with the most obvious gray scale changes.</p>
Line 73: Line 75:
 
                         the disrupted cell suspension to obtain the nanobody we needed. Next step, we combined the
 
                         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
 
                         Nanobody with C18. We have obtained a better experimental protocol by changing the
−
                         conditions</p>
+
                         conditions.</p>
 
                     <p>We designed a liquid crystal cell as the observation carrier. Firstly, we modified the slides
 
                     <p>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
 
                         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
 
                         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 β<sub>2</sub>-MG; Then we added β<sub>2</sub>-MG nanobody to the modified
+
                         APTES so that they can be attached to the β<sub>2</sub>-MG; Then we added β<sub>2</sub>-MG
−
                        slide. Finally, the upper and lower slides were assembled, and the liquid crystals were added.
+
                        nanobody to the modified slide. Finally, the upper and lower slides were assembled, and the
−
                        We can observe the optical imaging of the liquid crystal cell under the microscope to judge the
+
                        liquid crystals were added. We can observe the optical imaging of the liquid crystal cell under
−
                        β<sub>2</sub>–MG of the detected sample concentration.</p>
+
                        the microscope to judge the β<sub>2</sub>–MG of the detected sample concentration.</p>
 
                 </div>
 
                 </div>
−
            </div>
+
                <div>
−
            <div>
+
                    <h2 class="title">Verification part of molecular biology</h2>
−
                <h2 class="title">Verification part of molecular biology</h2>
+
                    <div class="mainText">
−
                <div class="mainText">
+
                        <h3 class="title">Aldehyde and C18 connection verification</h3>
−
                    <h3 class="title">Aldehyde and C18 connection verification</h3>
+
                        <table class="img">
−
                    <table class="img">
+
                            <tr>
−
                        <tr>
+
                                <td><img src="https://static.igem.org/mediawiki/2018/b/bf/T--DLUT_China_B--Demonstrate3.png"
−
                            <td><img src="https://static.igem.org/mediawiki/2018/b/bf/T--DLUT_China_B--Demonstrate3.png"
+
                                        alt=""></td>
−
                                    alt=""></td>
+
                            </tr>
−
                        </tr>
+
                            <tr>
−
                        <tr>
+
                                <td>
−
                            <td>
+
                                    <p class="footnote">Fig.1. Immunoassay for immunoglobulin after aldehyde
−
                                <p class="footnote">Fig.1. Immunoassay for immunoglobulin after aldehyde aldolization
+
                                        aldolization
−
                                    (a) Fluorescent group (b) 25 ° C C18 (c) 37 ° C C18 (d) acidified supernatant (e)
+
                                        (a) Fluorescent group (b) 25 ° C C18 (c) 37 ° C C18 (d) acidified supernatant
−
                                    dye-free Marker1 (f) Defective Marker2(g) BL21 cell disrupted supernatant (h) BL21
+
                                        (e)
−
                                    cell disrupted pellet</p>
+
                                        dye-free Marker1 (f) Defective Marker2(g) BL21 cell disrupted supernatant (h)
−
                            </td>
+
                                        BL21
−
                        </tr>
+
                                        cell disrupted pellet</p>
−
                        <tr>
+
                                </td>
−
                            <td><img src="https://static.igem.org/mediawiki/2018/3/30/T--DLUT_China_B--Demonstrate2.png"
+
                            </tr>
−
                                    alt=""></td>
+
                            <tr>
−
                        </tr>
+
                                <td><img src="https://static.igem.org/mediawiki/2018/3/30/T--DLUT_China_B--Demonstrate2.png"
−
                        <tr>
+
                                        alt=""></td>
−
                            <td>
+
                            </tr>
−
                                <p class="footnote">Fig.2. Analysis of dye-free electrophoresis</p>
+
                            <tr>
−
                            </td>
+
                                <td>
−
                        </tr>
+
                                    <p class="footnote">Fig.2. Analysis of dye-free electrophoresis</p>
−
                    </table>
+
                                </td>
−
                    <p>The molecular weight changes for (b) and (c) are not obvious, however, the C18 linkage at 37 ° C
+
                            </tr>
−
                        of (c) produces a certain amount of precipitation, which is not efficient. The acidification of
+
                        </table>
−
                        the supernatant (g) in (d) serves as a purification, that is, the nano-antibody has separated
+
                        <p>The molecular weight changes for (b) and (c) are not obvious, however, the C18 linkage at 37
−
                        from the supernatant, so the molecular weight remarkably lowered remarkably.</p>
+
                            °C
−
                    <p>After processing, the molecular weight of the Nanobody can be calculated to be about 250 KDa.</p>
+
                            of (c) produces a certain amount of precipitation, which is not efficient. The acidification
−
                    <h3 class="title">Nanobody concentration determination</h3>
+
                            of
−
                    <table class="img">
+
                            the supernatant (g) in (d) serves as a purification, that is, the nano-antibody has
−
                        <tr>
+
                            separated
−
                            <td><img src="https://static.igem.org/mediawiki/2018/f/f0/T--DLUT_China_B--Demonstrate4.png"
+
                            from the supernatant, so the molecular weight remarkably lowered remarkably.</p>
−
                                    alt=""></td>
+
                        <p>After processing, the molecular weight of the Nanobody can be calculated to be about 250
−
                        </tr>
+
                            KDa.</p>
−
                        <tr>
+
                        <h3 class="title">Nanobody concentration determination</h3>
−
                            <td>
+
                        <table class="img">
−
                                <p class="footnote">Fig.3. Protein concentration - absorbance standard curve</p>
+
                            <tr>
−
                            </td>
+
                                <td><img src="https://static.igem.org/mediawiki/2018/f/f0/T--DLUT_China_B--Demonstrate4.png"
−
                        </tr>
+
                                        alt=""></td>
−
                    </table>
+
                            </tr>
−
                    <p>In conclusion, we can determine the concentration of the nanobody through further experiments
+
                            <tr>
−
                        based on the standard curve of protein concentration and the molecular weight of the nanobody
+
                                <td>
−
                        measured by electrophoresis.</p>
+
                                    <p class="footnote">Fig.3. Protein concentration - absorbance standard curve</p>
 +
                                </td>
 +
                            </tr>
 +
                        </table>
 +
                        <p>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.</p>
 +
                    </div>
 
                 </div>
 
                 </div>
−
            </div>
+
                <div>
−
            <div>
+
                    <h2 class="title">Liquid crystal verification section</h2>
−
                <h2 class="title">Liquid crystal verification section</h2>
+
                    <div class="mainText">
−
                <div class="mainText">
+
                        <p>We designed a liquid crystal cell as an observation supporter. First, the upper glass slide
−
                    <h3 class="title">Determination of immobilized β<sub>2</sub>-MG concentration</h3>
+
                            was modified with 0.2%DMOAP to induce the vertical alignment of the liquid crystals and make
−
                    <p>1. Standard for immobilized β<sub>2</sub>-MG concentration</p>
+
                            the optical imaging of liquid crystals appear black at the first time.Meanwhile, we modified
−
                    <p>We have known that the orientation of liquid crystal molecules is very sensitive to the
+
                            the lower slide with a mixture solution of 3% (v) APTES and 1% (v) DMOAP and 1% (v) GA, so
−
                        topographic changes on the substrate surface. When the β<sub>2</sub>-MG and GA are crosslinked and fixed on
+
                            that it could connect to the target protein. Then We mixed the sample with the antibody and
−
                        the substrate surface, the topographic structure of the surface will be changed to a certain
+
                            added it to the modified slide. Finally, the upper and lower slides were assembled, and then
−
                        extent affecting the orientation of the liquid crystal molecules. Therefore, it is necessary to
+
                            the liquid crystals were added. We could observe the optical imaging of the liquid crystals
−
                        investigate the effect of the concentration of immobilized β<sub>2</sub>-MG on the optical imaging of the
+
                            pool under the microsco-pe, so as to determine the concentration of β<sub>2</sub>-MG of the
−
                        liquid crystal cell. With the decrease of the concentration of immobilized β<sub>2</sub>-MG, the
+
                            sample. The following paragraphs are the results of our investigation on the fixed antigen
−
                        perturbation to the orientation of liquid crystal molecule decreases, and the optical imaging of
+
                            and antibody concentration, and the brightne-ss contrast of β<sub>2</sub>-MG nano-antibody
−
                        liquid crystal cell gradually darkens. Only a few speckles appear in the optical imaging of
+
                            modified by C18 and the normal β<sub>2</sub>-MG nano-antibody combined with the liquid
−
                        liquid crystal cell, which tend to be all-black background. The highest immobilized β<sub>2</sub>-MG
+
                            crystal.</p>
−
                        concentration, which can keep the optical imaging background dark, is fixed on the substrate
+
                        <h3 class="title">Determination of immobilized β<sub>2</sub>-MG concentration</h3>
−
                        surface when sufficient antigen and antibody reactions are required. </p>
+
                        <p>1. Standard for immobilized β<sub>2</sub>-MG concentration</p>
−
                    <p>2. Result</p>
+
                        <p>We have known that the orientation of liquid crystal molecules is very sensitive to the
−
                    <p>All the other conditions are unchanged. The concentration of β<sub>2</sub>-MG added from 250ng/mL to
+
                            topographic changes on the substrate surface. When the β<sub>2</sub>-MG and GA are
−
                        700ng/mL is as follows:</p>
+
                            crosslinked and fixed on the substrate surface, the topographic structure of the surface
−
                    <table class="img">
+
                            will be changed to a certain extent affecting the orientation of the liquid crystal
−
                        <tr>
+
                            molecules. Therefore, it is necessary to investigate the effect of the concentration of
−
                            <td><img src="https://static.igem.org/mediawiki/2018/f/f2/T--DLUT_China_B--Demonstrate5.png"
+
                            immobilized β<sub>2</sub>-MG on the optical imaging of the liquid crystal cell. With the
−
                                    alt=""></td>
+
                            decrease of the concentration of immobilized β<sub>2</sub>-MG, the perturbation to the
−
                            <td><img src="https://static.igem.org/mediawiki/2018/2/22/T--DLUT_China_B--Demonstrate6.png"
+
                            orientation of liquid crystal molecule decreases, and the optical imaging of liquid crystal
−
                                    alt=""></td>
+
                            cell gradually darkens. Only a few speckles appear in the optical imaging of liquid crystal
−
                        </tr>
+
                            cell, which tend to be all-black background. The highest immobilized β<sub>2</sub>-MG
−
                        <tr>
+
                            concentration, which can keep the optical imaging background dark, is fixed on the substrate
−
                            <td>
+
                            surface when sufficient antigen and antibody reactions are required. </p>
−
                                <p class="footnote">(a)</p>
+
                        <p>2. Result</p>
−
                            </td>
+
                        <p>Keeping other conditions unchanged,the concerntration of β<sub>2</sub>-MG is in the range of 250ng/mL to 1000ng/mL,
−
                            <td>
+
                            and the results are shown in Fig. 4.</p>
−
                                <p class="footnote">(b)</p>
+
                        <table class="img">
−
                            </td>
+
                            <tr>
−
                        </tr>
+
                                <td><img src="https://static.igem.org/mediawiki/2018/f/f2/T--DLUT_China_B--Demonstrate5.png"
−
                        <tr>
+
                                        alt=""></td>
−
                            <td><img src="https://static.igem.org/mediawiki/2018/c/c5/T--DLUT_China_B--Demonstrate7.png"
+
                                <td><img src="https://static.igem.org/mediawiki/2018/2/22/T--DLUT_China_B--Demonstrate6.png"
−
                                    alt=""></td>
+
                                        alt=""></td>
−
                            <td><img src="https://static.igem.org/mediawiki/2018/c/cd/T--DLUT_China_B--Demonstrate8.png"
+
                            </tr>
−
                                    alt=""></td>
+
                            <tr>
−
                        </tr>
+
                                <td>
−
                        <tr>
+
                                    <p class="footnote">(a)</p>
−
                            <td>
+
                                </td>
−
                                <p class="footnote">(c)</p>
+
                                <td>
−
                            </td>
+
                                    <p class="footnote">(b)</p>
−
                            <td>
+
                                </td>
−
                                <p class="footnote">(d)</p>
+
                            </tr>
−
                            </td>
+
                            <tr>
−
                        </tr>
+
                                <td><img src="https://static.igem.org/mediawiki/2018/c/c5/T--DLUT_China_B--Demonstrate7.png"
−
                        <tr>
+
                                        alt=""></td>
−
                            <td><img src="https://static.igem.org/mediawiki/2018/2/2b/T--DLUT_China_B--Demonstrate9.png"
+
                                <td><img src="https://static.igem.org/mediawiki/2018/c/cd/T--DLUT_China_B--Demonstrate8.png"
−
                                    alt=""></td>
+
                                        alt=""></td>
−
                            <td><img src="https://static.igem.org/mediawiki/2018/7/7d/T--DLUT_China_B--Demonstrate10.png"
+
                            </tr>
−
                                    alt=""></td>
+
                            <tr>
−
                        </tr>
+
                                <td>
−
                        <tr>
+
                                    <p class="footnote">(c)</p>
−
                            <td>
+
                                </td>
−
                                <p class="footnote">(e)</p>
+
                                <td>
−
                            </td>
+
                                    <p class="footnote">(d)</p>
−
                            <td>
+
                                </td>
−
                                <p class="footnote">(f)</p>
+
                            </tr>
−
                            </td>
+
                            <tr>
−
                        </tr>
+
                                <td><img src="https://static.igem.org/mediawiki/2018/2/2b/T--DLUT_China_B--Demonstrate9.png"
−
                        <tr>
+
                                        alt=""></td>
−
                            <td><img src="https://static.igem.org/mediawiki/2018/9/98/T--DLUT_China_B--Demonstrate11.png"
+
                                <td><img src="https://static.igem.org/mediawiki/2018/7/7d/T--DLUT_China_B--Demonstrate10.png"
−
                                    alt=""></td>
+
                                        alt=""></td>
−
                            <td><img src="https://static.igem.org/mediawiki/2018/7/75/T--DLUT_China_B--Demonstrate12.png"
+
                            </tr>
−
                                    alt=""></td>
+
                            <tr>
−
                        </tr>
+
                                <td>
−
                        <tr>
+
                                    <p class="footnote">(e)</p>
−
                            <td>
+
                                </td>
−
                                <p class="footnote">(g)</p>
+
                                <td>
−
                            </td>
+
                                    <p class="footnote">(f)</p>
−
                            <td>
+
                                </td>
−
                                <p class="footnote">(h)</p>
+
                            </tr>
−
                            </td>
+
                            <tr>
−
                        </tr>
+
                                <td><img src="https://static.igem.org/mediawiki/2018/9/98/T--DLUT_China_B--Demonstrate11.png"
−
                        <tr>
+
                                        alt=""></td>
−
                            <td colspan="2"><img
+
                                <td><img src="https://static.igem.org/mediawiki/2018/7/75/T--DLUT_China_B--Demonstrate12.png"
−
                                    src="https://static.igem.org/mediawiki/2018/5/5c/T--DLUT_China_B--Demonstrate13.png"
+
                                        alt=""></td>
−
                                    alt=""></td>
+
                            </tr>
−
                        </tr>
+
                            <tr>
−
                        <tr>
+
                                <td>
−
                            <td colspan="2">
+
                                    <p class="footnote">(g)</p>
−
                                <p class="footnote">(i)</p>
+
                                </td>
−
                            </td>
+
                                <td>
−
                        </tr>
+
                                    <p class="footnote">(h)</p>
−
                        <tr>
+
                                </td>
−
                            <td colspan="2">
+
                            </tr>
−
                                <p class="footnote">Fig. 4.Optical imaging of liquid crystal cell with different
+
                            <tr>
−
                                    concentrations of immobilized β<sub>2</sub>-MG（a）1000ng/mL β<sub>2</sub>-MG （b）900ng/mL β<sub>2</sub>-MG（c）800 ng/mL
+
                                <td colspan="2"><img
−
                                    β<sub>2</sub>-MG（d）700ng/mL β<sub>2</sub>-MG（e）600ng/mL β<sub>2</sub>-MG（f）500ng/mL β<sub>2</sub>-MG（g）400ng/mL β<sub>2</sub>-MG（h）300ng/mL
+
                                        src="https://static.igem.org/mediawiki/2018/5/5c/T--DLUT_China_B--Demonstrate13.png"
−
                                    β<sub>2</sub>-MG（i）250ng/mL β<sub>2</sub>-MG </p>
+
                                        alt=""></td>
−
                            </td>
+
                            </tr>
−
                        </tr>
+
                            <tr>
−
                    </table>
+
                                <td colspan="2">
−
                    <p>From the experimental results, we found that when the concentration of β<sub>2</sub>-MG fixed to the
+
                                    <p class="footnote">(i)</p>
−
                        substrate surface is under 500 ng/mL, only a few bright spots appear in the optical imaging of
+
                                </td>
−
                        the liquid crystal cells gradually darkening, approaching the full black background. When the
+
                            </tr>
−
                        concentration of β<sub>2</sub>-MG is 500 ng/mL, the optical imaging of the liquid crystal cells shows
+
                            <tr>
−
                        obvious bright spots. When the concentration of β<sub>2</sub>-MG is above 500 ng/mL, the optical imaging
+
                                <td colspan="2">
−
                        appears obvious bright spot which is becoming brighter gradually. </p>
+
                                    <p class="footnote">Fig. 4.Optical imaging of liquid crystal cell with different
−
                    <p>3. Determination of immobilized β<sub>2</sub>-MG concentration</p>
+
                                        concentrations of immobilized β<sub>2</sub>-MG（a）1000ng/mL β<sub>2</sub>-MG
−
                    <p>As a result, we can find that the optical imaging of the liquid crystal cell becomes brighter and
+
                                        （b）900ng/mL β<sub>2</sub>-MG（c）800 ng/mL
−
                        brighter with the increase of the concentration of fixed β<sub>2</sub>-MG under other conditions.
+
                                        β<sub>2</sub>-MG（d）700ng/mL β<sub>2</sub>-MG（e）600ng/mL β<sub>2</sub>-MG（f）500ng/mL
−
                        Therefore, we can determine the change trend of the brightness of the liquid crystal. The
+
                                        β<sub>2</sub>-MG（g）400ng/mL β<sub>2</sub>-MG（h）300ng/mL
−
                        concentration of β<sub>2</sub>-MG increases in the same trend. We can establish the relationship between
+
                                        β<sub>2</sub>-MG（i）250ng/mL β<sub>2</sub>-MG </p>
−
                        the luminance of liquid crystal and the concentration of β<sub>2</sub>-MG, and then determine the
+
                                </td>
−
                        corresponding concentration of β<sub>2</sub>-MG according to the luminance of liquid crystal, so as to
+
                            </tr>
−
                        achieve the purpose of quantitative detection. Due to the need for sufficient β<sub>2</sub>-MG to react
+
                        </table>
−
                        with antibodies, a maximum immobilized β<sub>2</sub>-MG concentration of 500 ng/mL, which can keep the
+
                        <p>From the experimental results, the results show when the concentration of β<sub>2</sub>-MG
−
                        optical imaging background dark is selected to be fixed on the substrate surface. </p>
+
                            fixed to the substrate surface is under 500 ng/mL, only a few bright spots appear in the
−
                    <h3 class="title">Determination of the concentration of β<sub>2</sub>-MG nano antibody</h3>
+
                            optical imaging of the liquid crystal cells gradually darkening, approaching the full black
−
                    <p>1. Screening criteria for the concentration of β<sub>2</sub>-MG nanoparticles</p>
+
                            background. When the concentration of β<sub>2</sub>-MG is above 500 ng/mL, the optical
−
                    <p> The influence of the concentration of the nano-antibody on the optical imaging of the liquid
+
                            imaging appears obvious bright spot which is becoming brighter gradually.</p>
−
                        crystal cell was further investigated. If we don’t add β<sub>2</sub>-MG in the sample, there will has color
+
 
−
                        spots in the polarizing microscope imaging. When the concentration of the nano-antibody in the
+
                        <p>3. Determination of immobilized β<sub>2</sub>-MG concentration</p>
−
                        sample was low, only a few bright spots approached the full black background. In the subsequent
+
                        <p>As a result, the results show that the optical imaging of the liquid crystal cell becomes
−
                        detection, the substrate immobilized β<sub>2</sub>-MG will compete with the tested sample β<sub>2</sub>-MG to bind the
+
                            gradually bright with the increase of the concentration of fixed β<sub>2</sub>-MG under
−
                        limited reaction sites on the β<sub>2</sub>-MG nano-antibody. When the concentration of the tested β<sub>2</sub>-MG
+
                            other conditions unchanged. Therefore, we can determine the change trend of the brightness
−
                        approaches zero (detection limit is infinitely low), the β<sub>2</sub>-MG nano-antibody will combine with
+
                            of the liquid crystal. The concentration of β<sub>2</sub>-MG increases in the same trend.
−
                        the substrate immobilized β<sub>2</sub>-MG to disturb the orientation of the liquid crystal molecule to the
+
                            The relationship between the luminance of liquid crystal and the concentration of
−
                        maximum extent, resulting in the optical imaging of the liquid crystal cell. With the increase
+
                            β<sub>2</sub>-MG can be established, and then determine the corresponding concentration of β<sub>2</sub>-MG
−
                        of the concentration of β<sub>2</sub>-MG, the binding of β<sub>2</sub>-MG nano-antibody to the substrate immobilized
+
                            according to the luminance of liquid crystal, so as to achieve the purpose of quantitative
−
                        β<sub>2</sub>-MG decreases, and the optical imaging becomes dark from, thus completing the detection of
+
                            detection. Due to the need of the experiment, the maximum immobilized β<sub>2</sub>-MG
−
                        β<sub>2</sub>-MG. </p>
+
                            concentration of 500 ng/mL is selected to be fixed on the substrate surface, which can keep
−
                    <p>2. Results</p>
+
                            the optical imaging background dark.</p>
−
                    <table class="img">
+
 
−
                        <tr>
+
                        <h3 class="title">Determination of the concentration of β<sub>2</sub>-MG nano antibody</h3>
−
                            <td><img src="https://static.igem.org/mediawiki/2018/4/4a/T--DLUT_China_B--Demonstrate14.png"
+
                        <p>1. Screening criteria for the concentration of β<sub>2</sub>-MG nano-antibody</p>
−
                                    alt="">
+
                        <p> The influence of the concentration of the nano-antibody on the optical imaging of the liquid
−
                            </td>
+
                            crystal cell was further investigated. If we don’t add β<sub>2</sub>-MG in the sample, there
−
                            <td><img src="https://static.igem.org/mediawiki/2018/7/7b/T--DLUT_China_B--Demonstrate15.png"
+
                            will has color spots in the polarizing microscope imaging. When the concentration of the
−
                                    alt="">
+
                            nano-antibody in the sample was low, only a few bright spots approached the full black
−
                            </td>
+
                            background. In the subsequent detection, the substrate immobilized β<sub>2</sub>-MG will
−
                        </tr>
+
                            compete with the tested sample β<sub>2</sub>-MG to bind the limited reaction sites on the
−
                        <tr>
+
                            β<sub>2</sub>-MG nano-antibody. When the concentration of the tested β<sub>2</sub>-MG
−
                            <td>
+
                            approaches zero (detection limit is infinitely low), the β<sub>2</sub>-MG nano-antibody will
−
                                <p class="footnote">(a)</p>
+
                            combine with the substrate immobilized β<sub>2</sub>-MG to disturb the orientation of the
−
                            </td>
+
                            liquid crystal molecule to the maximum extent, resulting in the optical imaging of the
−
                            <td>
+
                            liquid crystal cell. With the increase of the concentration of β<sub>2</sub>-MG, the binding
−
                                <p class="footnote">(b)</p>
+
                            of β<sub>2</sub>-MG nano-antibody to the substrate immobilized β<sub>2</sub>-MG decreases,
−
                            </td>
+
                            and the optical imaging becomes dark from, thus completing the detection of β<sub>2</sub>-MG.
−
                        </tr>
+
                        </p>
−
                        <tr>
+
                        <p>2. Results</p>
−
                            <td><img src="https://static.igem.org/mediawiki/2018/e/e8/T--DLUT_China_B--Demonstrate16.png"
+
                        <p>Under the condition of 500ng/mL of β<sub>2</sub>-MG, 250ng/mL, 250ng/mL, 500ng/mL, and
−
                                    alt="">
+
                            600ng/mL of β<sub>2</sub>-MG nano-antibodies were fixed on the glass slides, and the color
−
                            </td>
+
                            and brightness changes of the liquid crystal film were compared, as shown in Fig. 5.</p>
−
                            <td><img src="https://static.igem.org/mediawiki/2018/e/ef/T--DLUT_China_B--Demonstrate17.png"
+
                        <table class="img">
−
                                    alt="">
+
                            <tr>
−
                            </td>
+
                                <td><img src="https://static.igem.org/mediawiki/2018/4/4a/T--DLUT_China_B--Demonstrate14.png"
−
                        </tr>
+
                                        alt="">
−
                        <tr>
+
                                </td>
−
                            <td>
+
                                <td><img src="https://static.igem.org/mediawiki/2018/7/7b/T--DLUT_China_B--Demonstrate15.png"
−
                                <p class="footnote">(c)</p>
+
                                        alt="">
−
                            </td>
+
                                </td>
−
                            <td>
+
                            </tr>
−
                                <p class="footnote">(d)</p>
+
                            <tr>
−
                            </td>
+
                                <td>
−
                        </tr>
+
                                    <p class="footnote">(a)</p>
−
                        <tr>
+
                                </td>
−
                            <td colspan="2">
+
                                <td>
−
                                <p class="footnote">Fig. 5. Change the concentration of fixed nano-antibodies to β<sub>2</sub>-MG
+
                                    <p class="footnote">(b)</p>
−
                                    under the condition of immobilizing 500 ng/mL of β<sub>2</sub>-MG on slide. (a) 250ng/mL of
+
                                </td>
−
                                    β<sub>2</sub>-MG nano-antibodies (b) 400 ng/mL of β<sub>2</sub>-MG nano-antibodies (d) 500 ng/mL of β<sub>2</sub>-MG
+
                            </tr>
−
                                    nano-antibodies (c) 600 ng/mL of β<sub>2</sub>-MG nano-antibodies</p>
+
                            <tr>
−
                            </td>
+
                                <td><img src="https://static.igem.org/mediawiki/2018/e/e8/T--DLUT_China_B--Demonstrate16.png"
−
                        </tr>
+
                                        alt="">
−
                    </table>
+
                                </td>
−
                    <p>From the experimental results, the fixed concentration of β<sub>2</sub>-MG was 500 ng/mL and the fixed
+
                                <td><img src="https://static.igem.org/mediawiki/2018/e/ef/T--DLUT_China_B--Demonstrate17.png"
−
                        antibody concentration was changed. When the concentration of β<sub>2</sub>-MG nano-antibody was 250 ng/mL,
+
                                        alt="">
−
                        the optical imaging spot of liquid crystal cell was star-shaped, which was more than that of the
+
                                </td>
−
                        non-fixed β<sub>2</sub>-MG antibody, but the change was not very large. When the concentration of β<sub>2</sub>-MG
+
                            </tr>
−
                        nano-antibody is above 400 ng/mL, the distribution of the optical imaging spot of the liquid
+
                            <tr>
−
                        crystal cell is similar to that of the β<sub>2</sub>-MG nano-antibody when the concentration of β<sub>2</sub>-MG
+
                                <td>
−
                        nano-antibody is 400 ng/mL.</p>
+
                                    <p class="footnote">(c)</p>
−
                    <p>3. Determination of the concentration of β<sub>2</sub>-MG nano antibody</p>
+
                                </td>
−
                    <p>Through the experimental results, we can find that with the increase of antibody concentration,
+
                                <td>
−
                        the orientation of liquid crystal molecules is disturbed, and the bright spots in optical
+
                                    <p class="footnote">(d)</p>
−
                        imaging are gradually increased, but 400 ng/mL is the threshold of β<sub>2</sub>-MG nanobody concentration,
+
                                </td>
−
                        when the concentration of Nano-antibody When it is increased, the bright spots of liquid crystal
+
                            </tr>
−
                        imaging are not significantly increased. Therefore, the minimum immobilized β<sub>2</sub>-MG nanobody
+
                            <tr>
−
                        concentration of 400 ng/mL, which can maintain the most bright spots in the optical imaging
+
                                <td colspan="2">
−
                        background, was fixed on the surface of the substrate.
+
                                    <p class="footnote">Fig. 5. Change the concentration of fixed nano-antibodies to
−
                    </p>
+
                                        β<sub>2</sub>-MG
−
                    <h3 class="title">Different effects of common β<sub>2</sub>-MG nano-antibodies and C18-modified β<sub>2</sub>-MG
+
                                        under the condition of immobilizing 500 ng/mL of β<sub>2</sub>-MG on slide. (a)
−
                        nano-antibodies on optical imaging of liquid crystal cells at the same concentration</h3>
+
                                        250ng/mL of
−
                    <p>1. Standard for the concentration of β<sub>2</sub>-MG antibody</p>
+
                                        β<sub>2</sub>-MG nano-antibodies (b) 400 ng/mL of β<sub>2</sub>-MG
−
                    <p>The size of the antibody modified by C18 long chain is larger than that of the normal antibody,
+
                                        nano-antibodies
−
                        and the effect of the antibody on the orientation of the liquid crystal molecules is also
+
                                        (c) 500 ng/mL of β<sub>2</sub>-MG
−
                        greater. Therefore, it is necessary to further investigate the effects of ordinary β<sub>2</sub>-MG
+
                                        nano-antibodies (d) 600 ng/mL of β<sub>2</sub>-MG nano-antibodies</p>
−
                        nano-antibodies and C18 long-chain modified β<sub>2</sub>-MG nano-antibodies on the optical imaging of
+
                                </td>
−
                        liquid crystal cells. </p>
+
                            </tr>
−
                    <p>2. Result</p>
+
                        </table>
−
                    <table class="img">
+
                        <p>The results shows that when the concentration of β<sub>2</sub>-MG nano-antibody was 250
−
                        <tr>
+
                            ng/mL, the optical imaging spot of liquid crystal cell was star-shaped, whose quantity was
−
                            <td><img src="https://static.igem.org/mediawiki/2018/3/36/T--DLUT_China_B--Demonstrate18.png"
+
                            more than that of the non-fixed β<sub>2</sub>-MG antibody, but the change was not obvious.
−
                                    alt=""></td>
+
                            When the concentration of β<sub>2</sub>-MG nano-antibody is above 400 ng/mL, the
−
                            <td><img src="https://static.igem.org/mediawiki/2018/1/1b/T--DLUT_China_B--Demonstrate19.png"
+
                            distribution of the optical imaging spot of the liquid crystal cell is similar to the
−
                                    alt=""></td>
+
                            phenomena of the concentration of β<sub>2</sub>-MG nano-antibody which is 400 ng/mL.</p>
−
                        </tr>
+
 
−
                        <tr>
+
                        <p>3. Determination of the concentration of β<sub>2</sub>-MG nano antibody</p>
−
                            <td>
+
                        <p>Through the experimental results, we could find that as the concentration of antibody
−
                                <p class="footnote">(a)</p>
+
                            increased , the crystal molecular orientation is getting more messy, and the bright spots
−
                            </td>
+
                            gradually enhance in the optical imaging. However, 400ng/mL is the threshold of the
−
                            <td>
+
                            concentration of β<sub>2</sub>-MG nano-antibodies. When the concentration of nano-antibodies
−
                                <p class="footnote">(b)</p>
+
                            increases again, the bright spots in the liquid crystal imaging did not enhance
−
                            </td>
+
                            significantly. Therefore, the lowest antibody concentration at 400ng/mL was selected for the
−
                        </tr>
+
                            immobilization of β<sub>2</sub>-MG on the substrate surface, which could maintain the
−
                        <tr>
+
                            maximum bright spots in the optical imaging background.</p>
−
                            <td colspan="2">
+
 
−
                                <img src="https://static.igem.org/mediawiki/2018/3/3c/T--DLUT_China_B--Demonstrate20.png">
+
                        <h3 class="title">Different effects of common β<sub>2</sub>-MG nano-antibodies and C18-modified
−
                            </td>
+
                            β<sub>2</sub>-MG
−
                        </tr>
+
                            nano-antibodies on optical imaging of liquid crystal cells at the same concentration</h3>
−
                        <tr>
+
                        <p>1. Standard for the concentration of β<sub>2</sub>-MG antibody</p>
−
                            <td colspan="2">
+
                        <p>The β<sub>2</sub>-MG nano-antibody modified by the long chain of C18 is larger than that of β<sub>2</sub>-MG
−
                                <p class="footnote">(c)</p>
+
                            nano-antibody. Therefore, it is necessary to further investigate the effects of ordinary
−
                            </td>
+
                            β<sub>2</sub>-MG nano-antibodies and C18 long-chain modified β<sub>2</sub>-MG
−
                        </tr>
+
                            nano-antibodies on the optical imaging of liquid crystal cells. </p>
−
                        <tr>
+
                        <p>2. Result</p>
−
                            <td colspan="2">
+
                        <p>Under the condition of 500ng/mL of β<sub>2</sub>-MG, 250ng/mL C18 modified of β<sub>2</sub>-MG
−
                                <p class="footnote">Fig. 6. Change the concentration and types of fixed β<sub>2</sub>-MG
+
                            nano-antibodies, 250ng/mL and 400ng/mL of β<sub>2</sub>-MG nano-antibodies were fixed on the
−
                                    nano-antibodies when 500 ng/mL β<sub>2</sub>-MG is immobilized on slide. (a) 250ng/mL modified
+
                            glass slides, and color and brightness changes of liquid crystal film were compared, as
−
                                    C18 β<sub>2</sub>-MG nano-antibodies. (b) 250ng/mL β<sub>2</sub>-MG nano-antibodies. (c) 400ng/mL β<sub>2</sub>-MG
+
                            shown in Fig. 6.</p>
−
                                    nano-antibodies </p>
+
                        <table class="img">
−
                            </td>
+
                            <tr>
−
                        </tr>
+
                                <td><img src="https://static.igem.org/mediawiki/2018/3/36/T--DLUT_China_B--Demonstrate18.png"
−
                    </table>
+
                                        alt=""></td>
−
                    <p>From the experimental results, when the concentration of fixed β<sub>2</sub>-MG nano-antibody is same
+
                                <td><img src="https://static.igem.org/mediawiki/2018/1/1b/T--DLUT_China_B--Demonstrate19.png"
−
                        (250ng/mL), the brightness of the liquid crystal cell with C18 modified β<sub>2</sub>-MG nano-antibody is
+
                                        alt=""></td>
−
                        higher than the threshold of normal nano-antibody.</p>
+
                            </tr>
−
                    <p>3. Comparison of sensitivity between normal β<sub>2</sub>-MG nano antibody and C18 modified β<sub>2</sub>-MG
+
                            <tr>
−
                        antibody</p>
+
                                <td>
−
                    <p>From the experimental results, we can find that the C18 modified β<sub>2</sub>-MG nano-antibody has greater
+
                                    <p class="footnote">(a)</p>
−
                        disturbance to the liquid crystal molecules, so we can judge that the sensitivity of the C18
+
                                </td>
−
                        modified β<sub>2</sub>-MG nano-antibody is higher.</p>
+
                                <td>
 +
                                    <p class="footnote">(b)</p>
 +
                                </td>
 +
                            </tr>
 +
                            <tr>
 +
                                <td colspan="2">
 +
                                    <img src="https://static.igem.org/mediawiki/2018/3/3c/T--DLUT_China_B--Demonstrate20.png">
 +
                                </td>
 +
                            </tr>
 +
                            <tr>
 +
                                <td colspan="2">
 +
                                    <p class="footnote">(c)</p>
 +
                                </td>
 +
                            </tr>
 +
                            <tr>
 +
                                <td colspan="2">
 +
                                    <p class="footnote">Fig. 6. Change the concentration and types of fixed
 +
                                        β<sub>2</sub>-MG
 +
                                        nano-antibodies when 500 ng/mL β<sub>2</sub>-MG is immobilized on slide. (a)
 +
                                        250ng/mL modified
 +
                                        C18 β<sub>2</sub>-MG nano-antibodies. (b) 250ng/mL β<sub>2</sub>-MG
 +
                                        nano-antibodies.
 +
                                        (c) 400ng/mL β<sub>2</sub>-MG
 +
                                        nano-antibodies </p>
 +
                                </td>
 +
                            </tr>
 +
                        </table>
 +
                        <p>From the experimental results, fixed with the same concentration of β<sub>2</sub>-MG nano
 +
                            antibody (both with a concentration of 250ng/mL), the brightness of the liquid crystal cell
 +
                            with C18 modified β<sub>2</sub>-MG nano-antibody is higher than the threshold of normal
 +
                            nano-antibody.</p>
 +
                        <p>3 Comparison of sensitivity between normal β<sub>2</sub>-MG nano antibody and C18 modified
 +
                            β<sub>2</sub>-MG antibody</p>
 +
                        <p>From the experimental results, we can find that the C18 modified β<sub>2</sub>-MG
 +
                            nano-antibody has greater disturbance to the liquid crystal molecules, so we can judge that
 +
                            the sensitivity of the C18 modified β<sub>2</sub>-MG nano-antibody is higher.</p>
 +
                    </div>
 
                 </div>
 
                 </div>
−
            </div>
+
                <div>
−
            <div>
+
                    <h2 class="title">Conclusion</h2>
−
                <h2 class="title">Conclusion</h2>
+
                    <div class="mainText">
−
                <div class="mainText">
+
                        <p>Through experiments, we finally decided to immobilize 500ng/mL β<sub>2</sub>-MG on the
−
                    <p>Through a series of Confirmatory experiment, we have proved that in the early stage of chronic
+
                            substrate , binding with 400ng/mL β<sub>2</sub>-MG nano-antibody. In addition, we determined
−
                        kidney disease, our detection system can indeed reflect the changes in β<sub>2</sub>-MG concentration,
+
                            that the detection sensitivity of the antibody at β<sub>2</sub>-MG modified by C18 was
−
                        which can remind users to go to the hospital for further testing.</p>
+
                            higher. Therefore, we can detect the concentration of β<sub>2</sub>-MG in the range of
−
                    <p>In the future, we plan to establish a more complete back-office system, which can directly obtain
+
                            100ng/ ml-400ng /mL (a theoretical estimate) by this method, which meets our requirements
−
                        more accurate test results based on the user's past physical indicators.</p>
+
                            for the early diagnosis of nephropathy patients.</p>
 +
                        <p>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 β<sub>2</sub>-MG
 +
                            concentration,
 +
                            which can remind users to go to the hospital for further testing.</p>
 +
                        <p>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.</p>
 +
                    </div>
 
                 </div>
 
                 </div>
 
             </div>
 
             </div>

Revision as of 01:23, 18 October 2018

Demonstrate

Demonstrate

Background

In order to fulfill the requirements of testing diseases at home for patients instead of going to hospital tested by high-tech testing equipment. We designed our products which can upload grayscale data to the cloud and analyze it through mobile devices obviously. What we required is just a liquid crystal detection scheme, which is sufficient to reflect the change in β2-MG concentration.

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.

We designed a competitive binding method : First, immobilizing a certain amount of β2-MG in a liquid crystal, and then combining the test solution with an excess of Nanobody. The mixture is then added to the liquid crystal, and due to the excess of the Nanobody, the excess is bound to the immobilized β2-MG, causing perturbation of the liquid crystal. Since the fixed β2-MG is a certain amount, the amount of β2-MG in the liquid to be tested can be calculated by the difference method. However, during the experiment, we found that the nano-antibody itself has little disturbance to the liquid crystal, so we hope to construct a more interfering molecule based on the nano-antibody. At the same time, we tested the effects of different β2-MG and nanobody concentrations on liquid crystal perturbations, and hope to find a set of solutions with the most obvious gray scale changes.

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

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) 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

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

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.

Conclusion

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.