Antibody- biodot conjugation as detection platform: in fluorescence immunoassay
,1 Mohammad pourhassan moghaddam
,2 Abolfazl akbarzadeh
,3 soodabeh davaran
,4 Nosratollah zarghami
1. Department of Medical Biotechnology, Faculty of Advanced Medical Science, Tabriz University of Medical Sciences
2. Department of Medical Biotechnology, Faculty of Advanced Medical Science, Tabriz University of Medical Sciences
3. Department of Medical Nanotechnology, Faculty of Advanced Medical Science, Tabriz University of Medical Sciences
4. Department of Medical Nanotechnology, Faculty of Advanced Medical Science, Tabriz University of Medical Sciences
5. Department of Medical Biotechnology, Faculty of Advanced Medical Science, Tabriz University of Medical Sciences
Fluorescence immunoassays are generally used in medical diagnostics, life science investigations. detection of biological molecules is considered as the main task in medical diagnosis procedures. various tags of different chemical composition are used for tracking biological target molecules present in the specimen, and regarding the several biosafety issues associated with the application of chemical tags for biodiagnosis purposes, the application of biocompatible tags is needed to avoid such issues. moreover, biocompatible tags can be safely used for in vivo diagnosis, particularly in vivo imaging procedures. therefore, in this work, the aim of the study was to prepare and apply a novel biocompatible dot in bimolecular detection.
Biodots were prepared by heating dna as starting material at 80৹c overnight. after preparing dna- dot and antibody, conjugation occurs in pbs buffer. moreover, the absorbance and intensity of the as-prepared dots were measured using uv-vis spectrophotometer and in vivo imaging equipment. the size and surface chemical groups were determined using high resolution electron microscopy, xps, ftir, zata potential and dls. the biocompatibility of the dots were evaluated by mtt assay. biodots were conjugated to the anti-igg antibody using coupling the surface free phosphate group of biodot and –nh2 group of antibody by edc/nhs chemistry. dot blotting and gel electrophoresis was confirmed antibody bio-dot conjugation. the bioconjugates were used to detect igg using an immunoassay test. anti- egfr bio dot photoluminescence images of the lung cancer cell line were obtained on cytation5 system.
The synthesized dots showed a spherical morphology and have an average size of about 20 nm. xps showed the presence of po41- and co groups on the biodots. mtt assay did not demonstrate any cytotoxicity on the human cells. the dots were highly photolumincent and they had a maximum absorbance in 258nm. the observed lifetime of bio-dot was 10.44ns. for biodetection, the bio-dot conjugates could detect anti- goat igg on paper blotting and egfr on cancer cell line with 5 deferent concentrations. this probe was used for fluorescent labeling of the cells approximately 95%.
The developed biodot conjugates can be easily used for biodetection purposes and they are great promise in designing novel biocompatible in-vivo imaging tests in the future.