Bioconjugations Dotaz Zobrazit nápovědu
Modification of DNA with reactive groups and their post-synthetic transformations are useful for labelling, imaging, bioconjugations and cross-linking with other (bio)molecules. This review summarizes the recent progress in this field and covers transformations of oxo groups, cycloadditions, conjugate additions, alkylations, cross-couplings and other reactions. Examples of applications are given and the practicability and scope of the reactions are discussed.
Upconversion nanoparticles (UCNPs) are an emerging class of optical materials with high potential in bioimaging due to practically no background signal and high penetration depth. Their excellent optical properties and easy surface functionalization make them perfect for conjugation with targeting ligands. In this work, capillary electrophoretic (CE) method with laser-induced fluorescence detection was used to investigate the behavior of carboxyl-silica-coated UCNPs. Folic acid, targeting folate receptor overexpressed by wide variety of cancer cells, was used for illustrative purposes and assessed by CE under optimized conditions. Peptide-mediated bioconjugation of antibodies to UCNPs was also investigated. Despite the numerous advantages of CE, this is the first time that CE was employed for characterization of UCNPs and their bioconjugates. The separation conditions were optimized including the background electrolyte concentration and pH. The optimized electrolyte was 20 mM borate buffer with pH 8.
- MeSH
- elektroforéza kapilární metody MeSH
- fluorescenční barviva chemie MeSH
- fluorescenční spektrometrie metody MeSH
- kyselina listová chemie MeSH
- limita detekce MeSH
- lineární modely MeSH
- nanokonjugáty chemie MeSH
- protilátky chemie MeSH
- reprodukovatelnost výsledků MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Publikační typ
- abstrakt z konference MeSH
A new simple electrochemical immunosensor approach for the determination of antibodies to tick-borne encephalitis virus (TBEV) in immunological products was developed and tested. The assay is performed by detecting the silver reduction signal in the bioconjugates with antibodies (Ab@AgNP). Here, signal is read by cathodic linear sweep voltammetry (CLSV) through the detection of silver chloride reduction on a gold-carbon composite electrode (GCCE). Covalent immobilization of the antigen on the electrode surface was performed after thiolation and glutarization of the GCCE. Specific attention has been paid to the selection of conditions for stabilizing both the silver nanoparticles and their Ab@AgNP. A simple flocculation test with NaCl was used to select the concentration of antibodies, and the additional stabilizer bovine serum albumin (BSA) was used for Ab@AgNP preparation. The antibodies to TBEV were quantified in the range from 50 IU·mL-1 to 1600 IU·mL-1, with a detection limit of 50 IU·mL-1. The coefficient of determination (r2) is 0.989. The electrochemical immunosensor was successfully applied to check the quality of immunological products containing IgG antibodies to TBEV. The present work paves the path for a novel method for monitoring TBEV in biological fluids.
- MeSH
- elektrochemické techniky metody MeSH
- elektrody MeSH
- imunoanalýza metody MeSH
- klíšťová encefalitida diagnóza imunologie MeSH
- kovové nanočástice chemie ultrastruktura MeSH
- protilátky virové imunologie MeSH
- sérový albumin hovězí MeSH
- skot MeSH
- spektrofotometrie ultrafialová MeSH
- stříbro chemie MeSH
- velikost částic MeSH
- viry klíšťové encefalitidy imunologie MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Electrophoretic mobility shift assay (EMSA) is a well-established technique to monitor interactions between biomolecules particularly DNA and proteins. Even though numerous variations of this method have been presented, challenges in the form of detection sensitivity and/or variations in the stability of the formed complex still remain. With advances in the area of nanomaterials improvements in EMSA have been also suggested. Recently, Zhang and Wang (Electrophoresis 2015, 36, 1011-1015) presented electrophoretic mobility shift method for determination of number of DNA molecules conjugated to quantum dots (QDs), which was further utilized for calculation of enzymatic activity, sequence specific DNA detection, and neutral molecule quantification.
- MeSH
- DNA analýza chemie MeSH
- kvantové tečky analýza chemie MeSH
- nanokonjugáty analýza chemie MeSH
- retardační test metody MeSH
- Publikační typ
- časopisecké články MeSH
This work reports for the first time a significantly improved and simplified electrochemical immunoassay to detect antibodies to tick-borne encephalitis virus (TBEV) using a 96-well microtiter plate as a platform for immobilization and silver nanoparticles (AgNPs) as electrochemical labels. The electrochemical assay is performed by detecting the elemental silver oxidation signal where the electroactive signalling silver species are released from the bioconjugates (Ab@AgNP, AbS@AgNP, and ProteinA@AgNP). For this purpose, AgNPs were synthesized and further tagged with biomolecules (antibodies to TBEV, cleaved antibodies to TBEV, and protein A). Signal is read by linear sweep anodic stripping voltammetry (LSASV) of silver ions (through the electrochemical stripping of accumulated elemental silver) on a graphite electrode (GE). AbS@AgNP was chosen as the best option for the new electrochemical immunoassay. The results of electrochemical measurements demonstrated that voltammetric signal increased with the increasing concentration of target antibodies to TBEV within the range from 100 to 1600 IU mL-1, with a detection limit of 90 IU mL-1. To verify the practical application of the novel electrochemical immunosensor, the quantity of immunoglobulins against TBEV in human serum was checked. The results may contribute to the development of alternative methods for monitoring TBEV in biological fluids.
- MeSH
- elektrochemické techniky metody MeSH
- imunoanalýza metody MeSH
- klíšťová encefalitida diagnóza virologie MeSH
- kovové nanočástice chemie MeSH
- lidé MeSH
- protilátky virové analýza MeSH
- stříbro chemie MeSH
- viry klíšťové encefalitidy imunologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The detection of cancer biomarkers in histological samples and blood is of paramount importance for clinical diagnosis. Current methods are limited in terms of sensitivity, hindering early detection of disease. We have overcome the shortcomings of currently available staining and fluorescence labeling methods by taking an integrative approach to establish photon-upconversion nanoparticles (UCNP) as a powerful platform for cancer detection. These nanoparticles are readily synthesized in different sizes to yield efficient and tunable short-wavelength light emission under near-infrared excitation, which eliminates optical background interference of the specimen. Here we present a protocol for the synthesis of UCNPs by high-temperature co-precipitation or seed-mediated growth by thermal decomposition, surface modification by silica or poly(ethylene glycol) that renders the particles resistant to nonspecific binding, and the conjugation of streptavidin or antibodies for biological detection. To detect blood-based biomarkers, we present an upconversion-linked immunosorbent assay for the analog and digital detection of the cancer marker prostate-specific antigen. When applied to immunocytochemistry analysis, UCNPs enable the detection of the breast cancer marker human epidermal growth factor receptor 2 with a signal-to-background ratio 50-fold higher than conventional fluorescent labels. UCNP synthesis takes 4.5 d, the preparation of the antibody-silica-UCNP conjugate takes 3 d, the streptavidin-poly(ethylene glycol)-UCNP conjugate takes 2-3 weeks, upconversion-linked immunosorbent assay takes 2-4 d and immunocytochemistry takes 8-10 h. The procedures can be performed after standard laboratory training in nanomaterials research.
- MeSH
- imunosorbenty MeSH
- lidé MeSH
- nádorové biomarkery MeSH
- nádory * diagnóza MeSH
- nanočástice * chemie MeSH
- oxid křemičitý chemie MeSH
- polyethylenglykoly chemie MeSH
- streptavidin MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Squaramate-linked 2'-deoxycytidine 5'-O-triphosphate was synthesized and found to be good substrate for KOD XL DNA polymerase in primer extension or PCR synthesis of modified DNA. The resulting squaramate-linked DNA reacts with primary amines to form a stable diamide linkage. This reaction was used for bioconjugations of DNA with Cy5 and Lys-containing peptides. Squaramate-linked DNA formed covalent cross-links with histone proteins. This reactive nucleotide has potential for other bioconjugations of nucleic acids with amines, peptides or proteins without need of any external reagent.
- MeSH
- DNA metabolismus MeSH
- lidé MeSH
- lysin metabolismus MeSH
- nukleotidy metabolismus MeSH
- peptidy chemie MeSH
- proteiny chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In this study, biotin-conjugated glutathione was synthesized using peptide bonding of the biotin carboxy group and amino group of the γ-glutamic acid to prepare an alternative coating for CdTe quantum dots (QDs). This type of coating combines the functionality of the biotin with the fluorescent properties of the QDs to create a specific, high-affinity fluorescent probe able to react with avidin, streptavidin and/or neutravidin. Biotin-functionalized glutathione-coated CdTe QDs were prepared by a simple one-step method using Na₂ TeO₃ and CdCl₂. Obtained QDs were separated from the excess of the biotin-conjugated glutathione by CE employing 300 mM borate buffer with pH 7.8 as a background electrolyte. The detection of sample components was performed by the photometric detection at 214 nm and LIF employing Ar⁺ ion laser (488 nm).
- MeSH
- biotin chemie metabolismus MeSH
- elektroforéza kapilární MeSH
- fluorescence MeSH
- glutathion chemie metabolismus MeSH
- kvantové tečky MeSH
- kyselina glutamová chemie MeSH
- sloučeniny kadmia chemie MeSH
- streptavidin chemie MeSH
- telur chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
