| Other Abstract | Immunity is the most important defense function of human beings and an
important mechanism for the body to maintain the stability of physiological environment. The process of specific immunity playing defense function in the body is called immune response, which is inseparable from the participation of immune molecules, especially the regulation of immune cells or immune molecules to itself.
Antibody is one of the most important immune molecules in immune response,and also a kind of universal glycoprotein in the body. The glycosylation in the antibodycan not only maintain the stability of the antibody structure, but also protect the antibody from enzymatic degradation. When the glucose level in the antibody is abnormal, the antibody will show immunogenicity, induce the immune response and be
attacked by other antibodies, and eventually cause damage to the body tissue, leading to the occurrence of autogenic immune diseases. Detection of glycosylated levels in antibodies is helpful to understand the status of antibodies in the body, evaluate the degree of immune response, and control the development of autoimmune diseases.
Exosomes, one of the regulators of immune responses, are vesicles (30-200nm) secreted by cells and contain biological macromolecules such as DNA, mRNA and protein. Exosomes affect biological functions through intercellular communication, and exosomes surface proteins are the structural basis for information communication.
Studies have shown that exosome surface proteins are regarded as new markers for early diagnosis of tumors due to their high specificity and sensitivity, and they have good fusion with cell membranes, and are also regarded as new carriers for drug therapy.
Detection of exosome surface proteins is a necessary way to study its application value. Currently, gel electrophoresis, mass spectrometry and lectin chip method are the main methods to detect glycosyl levels in antibodies. However, gel electrophoresis is prone to false positive results, mass spectrometry will destroy protein structure and lead to protein inactivation, and the results of lectin chip method cannot be absolutely
quantified. The detection methods for exotic surface proteins are mainly based on labeling rather than labeling. However, labeling method also has the problem of false positive results, and the sensitivity of non-labeling method is limited. Imaging ellipsometry biosensor technology is a highly sensitive, high-throughput, labeling free biosensor technology. It has relatively mature experience in biomolecular detection, the potential to detect glycol-group levels in antibodies and exosome surface proteins. The application steps of imaging ellipsometry biosensor are usually divided into three steps: surface design and assembly, surface signal detection and sensor signal transformation.
Surface modification is the basis of surface design and assembly. Self-assembled films represented by amino-silane are commonly used as surface modification coatings based on silicon substrates. It has been found that AUTES self-assembled membranes with
long carbon chains have stronger stability and more ordered orientation angles, but their performance in immobilization of biomolecules remains unknown.
The detection of sugar in the antibody level and secrete of body surface protein based on imaging ellipsometry biosensor technology and exploring AUTES in biomolecular fixed performance are cores of this article. In this paper, three steps of imaging ellipsometry biosensor technology were used to construct a technical route for detecting external surface proteins and a technical route for detecting glycosylated levels in antibodies. Therefore, the following scientific and technical problems had to be faced:
(1) How to evaluate the performance of AUTES in biomolecular fixation
efficiency?
(2) How to design and arrange an exosome surface protein detection surface?
How to convert sensing signals into surface protein signals?
(3) How to design and arrange the surface of an antibody for glycosyl-based detection? How to convert a sensing signal into a glycosyl-based signal?
In order to answer the first question, IgG (Immunoglobulin G) was selected as the target, glutaraldehyde was used as the signal amplification factor for IgG fixation with self-assembled membrane. The surface morphology characteristics, the number and activity of the fixed IgG in two kinds of self-assembled film (APTES/AUTES) were compared by using atomic force microscope, spectroscopic ellipsometer and imaging
ellipsometry biosensor. It was found that the efficiency of AUTES self-assembled membrane was less effective than the efficiency of APTES in immobilization of biomolecules. This work explored the relationship between the stability of aminosilane and the stability of biomolecules, and provided a new reference for the selection of surface modification methods using aminosilane as self-assembly membrane.
To answer the second question, we selected CD9 and CD63 on the surface of exosomes as target proteins, and designed and assembled ligands to capture exosomes with the help of Protein G and corresponding antibodies against target proteins. The signal of interaction between exosome and antibody of target protein was characterized in real time by total internal reverse imaging ellipsometry biosensor. By means of the quasi-first-order kinetic reaction equation, the affinity between two proteins on exosomes and corresponding antibodies was distinguished, and a system of imaging ellipsometry biosensor was established to detect exosomes surface proteins. This work not only provides a new way to detect exosome surface proteins, but also provides a basis for studying the interaction between exosomes and cell surface receptors.
In order to answer the third question, we premised on the difference in mannose glycosyl levels in the Fab and Fc ends of IgG, Con A lectin as mannose sugar-based signal amplification factor, the design of the two respectively to characterize IgG Fab side and Fc and mannose sugar base level surface, verify the effectiveness of the two ligands are arranged on the surface and specificity. With the help of external inverse imaging ellipsometry biosensor technology, the number of amplification factors on the two surfaces was detected, and the mannose levels at the Fab and Fc ends of IgG were estimated. The estimated qualitative results were consistent with the theoretical results.
A system based on imaging ellipsometry biosensor was developed for the detection of IgG mannose glycosyl level. This work provides a new possibility to study the relationship between abnormal glycosylation levels and protein physiological functions.
On the basis of answering the above questions, we optimized the setting of imaging ellipsometry biosensor technology by taking Anti-IgG as detection target from three aspects: azimuth angle of polarization device in optical path system, flow rate and flow time in microchannel chip reaction system. The sensitivity of imaging ellipsometry
biosensor was increased by 3.4 times. |
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