Molecular level design
Nanobody
Some cartilage fish such as sharks, rat sharks, etc. have antibodies called Ig-NAR in vivo, which consist of only two heavy chains but each has multiple constant domains (Fig. 1A). There is also a heavy chain antibody naturally deleted light chain, only two heavy chains, each heavy chain has three domains, including a variable domain, so this antibody (natural) has a heavy chain variable region Fragments (Fig. 1B), and found that such antibodies are abundant in some camelids, such as llamas, glamas, alpacas, llamas, and the like. Comparing hcIgG and Ig-NAR, it is not difficult to find that both antibodies use the variable region domain of the heavy chain N-terminus to specifically bind antigen, and the crystal structure of this part presents a flat shape resembling a football (Fig.2), approximately 2.5 nm in diameter and 4.2 nm in height. Antibodies of this size at the nanometer level are called Nanobodies (Nanobody, Nb), and now Nanobodies are commonly referred to as immunoglobulins of this hcIgG heavy chain variable region domain in camelids.[1]
|
|
Fig.1 The comparison between ordinary antibody and nanobody |
|
|
Fig.2 The crystal structure of nanobody |
In vivo catalysis
Because nanobodies can be expressed in prokaryotic expression and have strong stability, we used nanobodies instead of traditional antibodies for experiments. However, since the anisotropy of the nano-antibody molecules is not obvious, the ability to cause changes in the arrangement of liquid crystal molecules is limited. Therefore, we need to perform long-chain modification on Nanobodies to increase their ability to respond to liquid crystals.
|
|
Fig.3 Nanobody recognition beta 2 microglobulin |
Here we use a two-plasmid system: insert the Formylglycine-Generating Enzyme(FGE) gene into one plasmid; insert the Nanobody gene and the aldehyde-based precursor tag into the other plasmid.
|
|
Fig.4 FGE specifically recognizes amino acid sequences and catalyzes the process of specific sites |
At the same time, FGE can specifically recognize this tag, which in turn catalyzes the oxidation of cysteine to the aldehyde group. Thus co-expression of these two plasmids in E. coli yields FGE and Nb-Tag, while FGE catalyzes Nb-Tag, and finally we will obtain Nanobodies with aldehyde-based labels.[2] In this way, the Nanobodies we obtained have specific labels that are not found in all other proteins: aldehyde groups. This greatly simplifies our subsequent operations without the need for complicated purification steps; there is no need to worry about non-specific reactions; modularization of Nanobodies greatly simplifies the subsequent steps while increasing the choice of modification. We tried to bind the Nanobody to C18-NH-NH2 because the C18 molecule itself has a good ability to induce liquid crystal molecules. It can be said that our work combines the specific binding ability of Nanobodies with the ability to induce C18. Our subsequent liquid crystal experiment also proved this.
|
|
Fig.5 FGE specifically recognizes amino acid sequences and catalyzes the process of specific sites |
[1] [29] De Genst E, Saerens D, Muyldermans S, et al. Antibody repertoire development in camelids[J]. Dev Comp Immunol, 2006, 30(1-2): 187-198.
[2] Barfield R.M., Rabuka D. Leveraging Formylglycine-Generating Enzyme for Production of Site-Specifically Modified Bioconjugates. Methods in Molecular Biology, 2018, 1728(1): 3-16.
LCD sensor design
Overview
Recently, liquid crystal sensor, as a novel and unique optical sensor, has the advantages of simple structure, low cost, no need for marking and portability, and provides a simple and sensitive online real-time monitoring platform for biomolecule detection. The liquid crystal sensor uses the object to be tested to change the orientation of the vertically aligned liquid crystal molecules induced by the surface active molecules (Fig. 6),then making liquid crystal optical imaging change, therefore we can complete the detection of the target.
|
|
|
(a) Liquid crystal orientation without antibody |
(b) Liquid crystal orientation of antibody |
|
|
|
(c) Antigen, antibody |
(d) DMOAP, Liquid crystal molecule |
|
Fig.6 Schematic diagram of the change in orientation of the liquid crystal alignment of the analyte (a) Schematic diagram when the liquid crystal is not disturbed. (b) Schematic diagram when the liquid crystal is disturbed. (c)The introduction of Antigen and antibody. (d) The introduction of DMOAP andLiquid crystal molecule. |
|
We designed a liquid crystal cell as the observation carrier.First, we modify the slides so that they can induce vertical alignment of the liquid crystals, making the initial state of the optical imaging of the liquid crystal cell black; at the same time, we modify the slides so that they can be attached to the target protein; Then we add the sample to be tested and the antibody to the modified slide. Finally, the upper and lower slides are assembled, and then the liquid crystal is added. We can observe the optical imaging of the liquid crystal cell under the microscope to judge the β2 of the detected sample. - MG concentration.
The surface of a plain glass slide substrate was decorated with the mixed self-assembled monolayers of N, N-dimethyl-N-octadecyl(3-aminopropyl) trimethoxysilyl chloride (DMOAP) and (3-aminopropyl) trimethoxysilane (APTES) with both long and short alkane thiols (Fig. 7). DMOAP is used to induce vertical uniform orientation of liquid crystal molecules. The amino group in APTES reacts with an aldehyde group in GA to immobilize β2-MG as a functional molecule. When a certain concentration of β2-MG nanobody solution and different concentrations of the β2-MG antigen solution to be tested are sequentially added, the β-2-Microglobulin (β2-MG) immobilized on the slide is competitively combined with the β2-MG to be tested. The limited reaction site on the nanobody, after the substrate-bound β2-MG specifically binds to the β2-MG nanobody, the nanobody molecule has a certain spatial stereo structure and molecular size effect, which can disturb the orientation of the liquid crystal 5CB molecule. It is arranged in an oblique or nearly parallel manner. As the concentration of β2-MG to be measured in the sample changes, the amount of the nanobody bound to the substrate also changes, thereby changing the color and brightness of the liquid crystal film (Fig. 8) to achieve β2 - Specific detection of MG.
|
|
Fig.7 Process of liquid crystal molecules being disturbed |
|
|
Fig.8 Immobilized antigen 500ng/m, nanobody 1500ng/ml |
Design of self-assembled film
It has been investigated that the effective birefringence, which is relative to the color and brightness of LC film, varied with alignment layers. Accordingly, the surface modification of substrate is critical for the detection of biomolecules by LC biosensor, in the current process of LC biosensor construction. The substrate sensitive membrane was constructed by APTES and DMOAP hybrid self-assembly method. The long-chain alkyl chain in DMOAP can induce liquid crystal molecules to be oriented in the long-chain direction and arranged vertically. At this time, the polarized light cannot pass through the liquid crystal cell, and the optical image is uniform. Black pattern. The amino group in APTES reacts with an aldehyde group in GA to immobilize biomolecules as functional molecules. Since APTES is a short-chain alkyl group, liquid crystal molecules cannot be effectively aligned vertically, and background interference can be caused by polarized microscope imaging. Therefore, it is necessary to explore the influence of the ratio of APTES and DMOAP on liquid crystal orientation, determine the optimal ratio, and on this basis, further determine the optimal content of GA.
Using immunocompetitive detection to detect β2-MG
Based on the principle of competitive immunity, the lower the concentration of β2-MG, the more β2-MG nano-antibody molecules bound to the substrate surface, the brighter the image; otherwise, the higher the concentration of β2-MG, the fewer β2-MG nano-antibody molecules bound to the substrate surface, the more black the image tends to be. When the concentration of β2-MG mixed with nano-antibody was 400 ng/mL, the optical imaging of liquid crystal was almost uniform black pattern (Fig. 9 (a)); when the concentration of β2-MG mixed with nano-antibody was 100 ng/mL, the optical imaging of liquid crystal had large area color (Fig. 9 (b)). Therefore, we need to design experiments to explore the maximum antigen concentration fixed to the surface of the base slide and the most suitable antibody concentration.
|
|
|
(a) 400ng/mL |
(b) 100ng/mL |
|
Fig. 9 Schematic diagram of mixing beta 2-MG with beta 2-MG nano-antibody (a) Mixing of beta 2-MG with beta 2-MG nano-antibody at 400 ng/mL; (b) Mixing of beta 2-MG with beta 2-MG nano-antibody at 100 ng/mL. |
|
According to Jeffrey M. Brake et al, we found that the change of long chain phospholipid molecular arrangement induced the change of liquid crystal arrangement when the copper net with liquid crystal was covered by phospholipid molecules. According to the commonly used inducers of liquid crystal molecules, such as DMOAP, polyimide and other molecules, they are long chain structures. Therefore, we speculate that long chain molecules may affect the alignment of liquid crystal molecules. Therefore, we designed to increase the sensitivity of liquid crystal detection by modifying long chain molecules on ordinary nano-antibodies.
|
|
|
(a) |
(b) |
|
Fig.10 Comparison of disruption of liquid crystal molecules by ordinary nano-antibodies and C18-modified nano-antibodies (a) Nano-antibodies modified by ordinary nano-antibodies (b) and C18-modified nano-antibodies |
|
Based on the above principles and designs, we can construct a liquid crystal biosensor. Using a competitive immunoassay, we can induce the vertical homogeneous orientation of liquid crystal molecules by modifying self-assembled monolayers on the glass substrate to observe the changes of color and brightness of the liquid crystal film before and after the binding of β2-MG nano-antibodies to β2-MG, and design the liquid crystal through modeling. A non-labeled, highly sensitive and specific liquid crystal detector for β2-MG was constructed based on the relationship between the color and brightness of the membrane and the concentration of β2-MG.
[1]Jiao Zhang, Xiuxia Su, Dong Yang, Chonglin Luan. Label-free liquid crystal biosensor for cecropin B detection[J]. Talanta, 2018, 186.
[2]Li G, Li X, Yang M, et al. A gold nanoparticles enhanced surface plasmon resonance immunosensor for highly sensitive detection of ischemia-modified albumin[J]. Sensors, 2013, 13(10): 12794-12803.
[3]Piotr Popov, Lawrence W. Honaker, Edgar E. Kooijman, Elizabeth K. Mann, Antal I. Jákli, A liquid crystal biosensor for specific detection of antigens, Sensing and Bio-Sensing Research, Volume 8, 2016, Pages 31-35
