Enzymatic Protein Immobilization for Nanobody Array
Antibody arrays play a pivotal role in the detection and quantification of biomolecules, with their effectiveness largely dependent on efficient protein immobilization. Traditional methods often use heterobifunctional cross-linking reagents for attaching functional residues in proteins to correspond...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2024-01-01
|
| Series: | Molecules |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1420-3049/29/2/366 |
| _version_ | 1797339521966145536 |
|---|---|
| author | Zhuojian Lu Rui Ge Bin Zheng Peng Zheng |
| author_facet | Zhuojian Lu Rui Ge Bin Zheng Peng Zheng |
| author_sort | Zhuojian Lu |
| collection | DOAJ |
| description | Antibody arrays play a pivotal role in the detection and quantification of biomolecules, with their effectiveness largely dependent on efficient protein immobilization. Traditional methods often use heterobifunctional cross-linking reagents for attaching functional residues in proteins to corresponding chemical groups on the substrate surface. However, this method does not control the antibody’s anchoring point and orientation, potentially leading to reduced binding efficiency and overall performance. Another method using anti-antibodies as intermediate molecules to control the orientation can be used but it demonstrates lower efficiency. Here, we demonstrate a site-specific protein immobilization strategy utilizing <i>Oa</i>AEP1 (asparaginyl endopeptidase) for building a nanobody array. Moreover, we used a nanobody-targeting enhanced green fluorescent protein (eGFP) as the model system to validate the protein immobilization method for building a nanobody array. Finally, by rapidly enriching eGFP, this method further highlights its potential for rapid diagnostic applications. This approach, characterized by its simplicity, high efficiency, and specificity, offers an advancement in the development of surface-modified protein arrays. It promises to enhance the sensitivity and accuracy of biomolecule detection, paving the way for broader applications in various research and diagnostic fields. |
| first_indexed | 2024-03-08T09:49:23Z |
| format | Article |
| id | doaj.art-18f57cfa736a4d5c94102e2e259b3856 |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-08T09:49:23Z |
| publishDate | 2024-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-18f57cfa736a4d5c94102e2e259b38562024-01-29T14:07:54ZengMDPI AGMolecules1420-30492024-01-0129236610.3390/molecules29020366Enzymatic Protein Immobilization for Nanobody ArrayZhuojian Lu0Rui Ge1Bin Zheng2Peng Zheng3State Key Laboratory of Coordination Chemistry, Chemistry and Biomedicine Innovation Center (ChemBIC), School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, ChinaState Key Laboratory of Coordination Chemistry, Chemistry and Biomedicine Innovation Center (ChemBIC), School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, ChinaState Key Laboratory of Coordination Chemistry, Chemistry and Biomedicine Innovation Center (ChemBIC), School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, ChinaState Key Laboratory of Coordination Chemistry, Chemistry and Biomedicine Innovation Center (ChemBIC), School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, ChinaAntibody arrays play a pivotal role in the detection and quantification of biomolecules, with their effectiveness largely dependent on efficient protein immobilization. Traditional methods often use heterobifunctional cross-linking reagents for attaching functional residues in proteins to corresponding chemical groups on the substrate surface. However, this method does not control the antibody’s anchoring point and orientation, potentially leading to reduced binding efficiency and overall performance. Another method using anti-antibodies as intermediate molecules to control the orientation can be used but it demonstrates lower efficiency. Here, we demonstrate a site-specific protein immobilization strategy utilizing <i>Oa</i>AEP1 (asparaginyl endopeptidase) for building a nanobody array. Moreover, we used a nanobody-targeting enhanced green fluorescent protein (eGFP) as the model system to validate the protein immobilization method for building a nanobody array. Finally, by rapidly enriching eGFP, this method further highlights its potential for rapid diagnostic applications. This approach, characterized by its simplicity, high efficiency, and specificity, offers an advancement in the development of surface-modified protein arrays. It promises to enhance the sensitivity and accuracy of biomolecule detection, paving the way for broader applications in various research and diagnostic fields.https://www.mdpi.com/1420-3049/29/2/366protein immobilizationenzymatic ligation<i>Oa</i>AEP1nanobody |
| spellingShingle | Zhuojian Lu Rui Ge Bin Zheng Peng Zheng Enzymatic Protein Immobilization for Nanobody Array Molecules protein immobilization enzymatic ligation <i>Oa</i>AEP1 nanobody |
| title | Enzymatic Protein Immobilization for Nanobody Array |
| title_full | Enzymatic Protein Immobilization for Nanobody Array |
| title_fullStr | Enzymatic Protein Immobilization for Nanobody Array |
| title_full_unstemmed | Enzymatic Protein Immobilization for Nanobody Array |
| title_short | Enzymatic Protein Immobilization for Nanobody Array |
| title_sort | enzymatic protein immobilization for nanobody array |
| topic | protein immobilization enzymatic ligation <i>Oa</i>AEP1 nanobody |
| url | https://www.mdpi.com/1420-3049/29/2/366 |
| work_keys_str_mv | AT zhuojianlu enzymaticproteinimmobilizationfornanobodyarray AT ruige enzymaticproteinimmobilizationfornanobodyarray AT binzheng enzymaticproteinimmobilizationfornanobodyarray AT pengzheng enzymaticproteinimmobilizationfornanobodyarray |