Biotin End Research Articles

Construction of a 3D Quantum Dot Nanoassembly with Two-Step FRET for One-Step Sensing of Human Telomerase RNA in Breast Cancer Cells and Tissues.

Telomerase is an important biomarker for early diagnosis of cancers, but current telomerase assays usually rely on measuring the extension products of telomerase substrates, which increases the assay complexity. More evidence indicates that human telomerase RNA (hTR), as a core component of telomerase, is positively correlated with the telomerase activity. Herein, we demonstrate the development of a duplex-specific nuclease (DSN)-propelled 3D quantum dot (QD) nanoassembly with two-step Föster resonance energy transfer (FRET) for the one-step sensing of hTR in breast cancer cells and tissues. This assay involves only one hairpin probe modified with a Cy5 at the sixth base from the 5'-biotin end and a BHQ2 at the 3'-terminus, which integrates three functions of target recognition, target recycling amplification, and signal readout. The anchoring of the hairpin probe on the 605QD surface results in the formation of a 3D 605QD-Cy5-probe-BHQ2 nanoassembly in which two-step FRET occurs among the 605QD, Cy5, and BHQ2 quencher. Notably, the formation of 605QD-Cy5-probe-BHQ2 nanoassembly facilitates the reduction of background signal and the increase of signal-to-background ratio due to its dense, highly oriented nucleic acid shell-induced steric hindrance effect. This assay can achieve one-step and rapid detection of hTR with a detection limit of 2.10 fM, which is the simplest and most rapid hTR assay reported so far. Moreover, this assay can efficiently distinguish single-base mismatched sequences, and it can discriminate the hTR level between breast cancer patients and healthy donors with a high accuracy of 100%, with great prospects for early diagnosis of cancers.

Acoustic detection of a mutation-specific Ligase Chain Reaction based on liposome amplification.

Single nucleotide variants (SNVs) play a crucial role in understanding genetic diseases, cancer development, and personalized medicine. However, existing ligase-based amplification and detection techniques, such as Rolling Circle Amplification and Ligase Detection Reaction, suffer from low efficiency and difficulties in product detection. To address these limitations, we propose a novel approach that combines Ligase Chain Reaction (LCR) with acoustic detection using highly dissipative liposomes. In our study, we are using LCR combined with biotin- and cholesterol-tagged primers to produce amplicons also modified at each end with a biotin and cholesterol molecule. We then apply the LCR mix without any purification directly on a neutravidin modified QCM device Au-surface, where the produced amplicons can bind specifically through the biotin end. To improve sensitivity, we finally introduce liposomes as signal enhancers. For demonstration, we used the detection of the BRAF V600E point mutation versus the wild-type allele, achieving an impressive detection limit of 220 aM of the mutant target in the presence of the same amount of the wild type. Finally, we combined the assay with a microfluidic fluidized bed DNA extraction technology, offering the potential for semi-automated detection of SNVs in patients' crude samples. Overall, our LCR/acoustic method outperforms other LCR-based approaches and surface ligation biosensing techniques in terms of detection efficiency and time. It effectively overcomes challenges related to DNA detection, making it applicable in diverse fields, including genetic disease and pathogen detection.

L-Carnitine reduces the negative effects of formalin on sperm parameters, chromatin condensation and apoptosis in mice: An experimental study.

BackgroundFormalin is commonly applied as an antiseptic and tissue fixative. It has reactive molecules that lead to its cytotoxic effects. According to recent studies, formalin causes a change in the testicular and sperm structure and L-carnitine (LC) acts as an antioxidant to counteract its effects.ObjectiveThis study aimed to investigate the protective effects of LC on the parameters, chromatin condensation and apoptosis of mice sperm exposed to formalin.Materials and Methods In this experimental study, 24 balb/c mice (25-40 gr,10-12 wk) were divided into three groups (n = 8/each): group I without any injections or gavage; group II, received 10 mg/ kg formalin intraperitoneally (I.P); and group III was exposed to formalin and LC, where a dose of 10 mg/kg formalin was injected I.P daily and LC the dose of 100 mg/kg was kept in a solvent solution. After 31 days, the sperm examination was performed as follows: to evaluate chromatin and DNA quality of the sperm, we applied aniline blue (AB), toluidine blue (TB), chromomycin A3 (CMA3), and terminal transferase-mediated deoxy uridine triphosphate biotin end labeling (TUNEL) tests.Results Sperm parameters such as count, motility, morphology, and viability displayed a significant decrease in the formalin group. While the data exhibited a considerable augment in sperm parameters in the formalin + LC than the formalin and control groups (p 0.001), significant differences were detected between groups with respect to TB staining, TUNEL test, CMA3 test and AB staining in the formalin and formalin + LC groups.ConclusionLC can reduce the negative effects of formalin on sperm parameters, chromatin stability, and percentage of apoptosis in an animal model.

Enabling Indium Channels for Mass Cytometry by Using Reinforced Cyclam-Based Chelating Polylysine.

The synthesis of a polylysine polymer functionalized with the previously reported astonishingly inert [In(cb-te2pa)]+ chelate was performed. A biotin end group allowed the conjugation to biotinylated beads by the intermediary of a fluorescein isothiocyanate/neutravidin receptor. High quality imaging mass cytometry trials, based on 115In detection were performed to highlight the behavior of the material. Anti-CD20 antibody was labeled by the so-obtained In(III)-modified polylysine using the biotin/neutravidin interaction. Ramos (CD20[+]) and HL-60 (CD20[-]) cell lines were costained with the In(III)-modified bioconjugate by finding the best staining conditions. Both immunofluorescence microscopy (IF-M) and mass cytometry analyses confirmed the specific binding of anti-CD20 onto Ramos cells. CyTOF histograms constructed on the 115In detection allowed us to define and to separate, with a good signal-to-noise ratio, two populations (Ramos and HL-60). The inertness of In(III)-MCP-NAv over a three-month storage period was proved by performing new functionality tests involving Jurkat cells (CD20[-]) and multiparametric trials involving the 115In channel. The results ensure a promising future use of the previously announced [In(cb-te2pa)]+ complex-based polymers for mass cytometry.

Vitamin C attenuates negative effects of vitrification on sperm parameters, chromatin quality, apoptosis and acrosome reaction in neat and prepared normozoospermic samples

ObjectiveAim of this study was to evaluate the effects of vitamin C on sperm parameters, sperm chromatin quality and apoptosis resulted of vitrification in neat semen and prepared spermatozoa of normozoospermic samples. Material and methodsForty semen samples from normozoospermic men were included in this prospective study. Each sample was divided into five groups. Group I: control or fresh semen, group II: semen prepared by swim-up method and then vitrified, group III: neat semen was vitrified, group IV: vitamin C (600 μM) was added to prepared spermatozoa and then vitrified and group V: vitamin C (600 μM) was added to neat semen and then vitrified. After warming, sperm analysis was done accordingly. For evaluating the sperm chromatin/DNA integrity status and acrosome reaction, we used toluidine blue (TB), acridine orange (AO), terminal transferase mediated deoxyuridine triphosphate biotin end labeling (TUNEL) and double staining tests. ResultsAll of the sperm parameters (count, motility, morphology and viability) had significant differences (P < 0.05) between different groups, especially in group IV. Data showed sperm chromatin damages and acrosome reaction abnormality increased resulted of vitrification, but, in the groups that added vitamin C (IV, V) rate of damages was decreased and this was notable in the group IV. ConclusionVitamin C can attenuate the detrimental effects of vitrification on sperm parameters, chromatin quality and rate of apoptosis in both neat semen and prepared spermatozoa of normozoospermic samples.

Is there any correlation between sperm parameters and chromatin quality with embryo morphokinetics in patients with male infertility?

The main goal was to evaluate the correlation between sperm parameters and chromatin quality with embryo kinetics via time-lapse monitoring system (TLM). A total of 40 couples involved in the ICSI program as a result of male infertility. For assessment of sperm chromatin and DNA quality, we used aniline blue, toluidine blue, chromomycin A3, acridine orange and terminal transferase-mediated deoxyuridine triphosphate biotin end labelling assays. All mature oocytes were injected, and the generated zygotes (2PNs) were cultured in TLM. In day 3 after injection, single embryo transfer (SET) was carried out according to the morphology and morphokinetics. The patients were followed up until delivery. There were positive significant correlations between sperm count with CC2 (r=.330, p=.049), T4 (r=.329, p=.038), T6 (r=.342, p=.035) and T7 (r=.374, p=.025). Also, there were positive significant correlations between nonprogressive motility and T2 (r=0.323, p=.042), T3 (r=.411, p=.013) and T4 (r=.418, p=.007). Regarding the sperm chromatin quality assays, there were negative significant correlations between CMA3 and CC2 (r=-.272, p=.049) and between acridine orange and T5 (r=-.221, p=.040). It seems that the abnormal sperm parameters and chromatin alteration affect the normal embryo kinetics in ICSI program.

Three different signal amplification strategies for the impedimetric sandwich detection of thrombin

In this work, we report a comparative study on three highly specific amplification strategies for the ultrasensitive detection of thrombin with the use of aptamer sandwich protocol. The protocol consisted on the use of a first thrombin aptamer immobilized on the electrode surface, the recognition of thrombin protein, and the reaction with a second biotinylated thrombin aptamer forming the sandwich. Through the exposed biotin end, three variants have been tested to amplify the electrochemical impedance signal. The strategies included (a) silver enhancement treatment, (b) gold enhancement treatment and (c) insoluble product produced by the combination of the enzyme horseradish peroxidase (HRP) and 3-amino-9-ethylcarbazole (AEC). The properties of the sensing surface were probed by electrochemical impedance measurements in the presence of the ferrocyanide/ferricyanide redox marker. Insoluble product strategy and silver enhancement treatment resulted in the lowest detection limit (0.3 pM), while gold enhancement method resulted in the highest reproducibility, 8.8% RSD at the pM thrombin concentration levels. Results of silver and gold enhancement treatment also permitted direct inspection by scanning electron microscopy (SEM).

Vitamin C attenuates detrimental effects of diabetes mellitus on sperm parameters, chromatin quality and rate of apoptosis in mice

ObjectivesThe main goal was to examine the protective effects of vitamin C on sperm parameters, sperm chromatin condensation and apoptosis in experimentally-induced diabetic mice. Study design28 adult Syrian mice were divided into 4 groups. In Group1, the mice were diabetic that received a single dose of Streptozocin (STZ) (200mg/kg) intra-peritoneally (ip). Group 2 was included diabetic mice that received vitamin C (10mg/kg/daily, ip). Mice in group 3 were received vitamin C and group 4 was considered as control. After 35 days, sperm analysis was done accordingly. To asses sperm chromatin and DNA quality, we used aniline blue (AB), toluidine blue (TB), chromomycin A3 (CMA3), acridine orange (AO) and terminal transferase mediated deoxyuridine triphosphate biotin end labeling (TUNEL) tests. ResultsAll of the sperm parameters (count, motility, morphology and viability) had significant reduction in diabetic mice but, the data showed a significant increase in all of the sperm parameters in diabetic+vitamin C when compared with diabetic and control animals (P<0.05). There were significant differences (P<0.001) between groups regarding to TB staining (48.8±5.92 vs 34.3±4.13), AO test (35.9±6.11 vs 20.8±2.89) and TUNEL test (39.42±7.18 vs 22.00±3.65) in diabetic and diabetic+vitamin C groups, respectively. Nevertheless, in CMA3 and AB staining assays, there were not any significant differences between different groups. ConclusionVitamin C, as a potent antioxidant, can attenuate detrimental effects of diabetes mellitus on the sperm parameters, chromatin quality and apoptosis in an experimental model.

Effect of Pendant Group Structure on the Hydrolytic Stability of Polyaspartamide Polymers under Physiological Conditions

We describe the synthesis of metal chelating polymers based on polyaspartamide and polyglutamide backbones as carriers for (111)In in radioimmunoconjugates. These polymers [PAsp(DTPA), PGlu(DTPA)] have a biotin end group and diethylenetriaminepentaacetic acid (DTPA) chelators attached to the primary amines of the diethylenetriamine (DET) pendant groups of biotin-poly{N'-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} [PAsp(DET)] and of biotin-poly{N'-[N-(2-aminoethyl)-2-aminoethyl]glutamide} [PGlu(DET)]. Like Asn-containing proteins and polypeptides, polyaspartamides undergo uncatalyzed degradation under model physiological conditions (10 mM phosphate buffer, pH 7.4, 150 mM NaCl). We studied the uncatalyzed degradation of the polyaspartamide polymers by size exclusion chromatography and found that the degradation rate was sensitive to the nature of the pendant groups. The metal-free polymer underwent somewhat slower degradation than the corresponding polymers in which the DTPA groups were saturated with Eu(3+) or In(3+), but even after 14 days, substantial fractions of the polymers survived. We conclude that these polymers undergo negligible degradation on the time scale (24-48 h) of radioimmunotherapy treatment of tumors with (111)In. From a mechanistic perspective, we note that these degradation rates are on the order of the deamidation rates reported [J. Peptide Res. 2004, 63, 426] for Asn-containing pentapeptides, with half-times on the order of 10 days, but much slower than the rapid decay (hours) reported recently [Biomaterials 2010, 31, 3707] for poly{N'-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} itself. This variation in degradation rate can be explained in terms of the influence of positive charges on the pendant group enhancing the acidity of the side-chain amide nitrogen of the aspartamide repeat unit. The DET pendant group is positively charged at pH 7, but in indium-loaded PAsp(DTPA) this charge is offset by the net negative charge of the DTPA-In complex.

Thermoresponsive biotinylated star amphiphilic block copolymer: Synthesis, self-assembly, and specific target recognition

A novel star amphiphilic block copolymer star poly(ε-caprolactone)-b-poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)-DDAT [SPCL-b-P(NIPAAm-co-DMAAm)-DDAT] (DDAT: S-1-dodecyl-S′-(α,α′-dimethyl-α″-acetic acid)trithiocarbonate) was synthesized by combination of ring-opening polymerization (ROP) and reversible addition-fragment chain transfer (RAFT) polymerization. DDAT-terminated groups were further transformed into hydroxyl groups by one-pot strategy for aminolysis of DDAT and Michael addition reaction of an α,β-unsaturated ester of 2-hydroxyethyl acrylate (HEA). Biotinylated star copolymer SPCL-b-P(NIPAAm-co-DMAAm)-Biotin was obtained by coupling of biotin to the hydroxyl-terminated star copolymer using carbodiimide coupling chemistry. These star copolymers with DDAT, hydroxyl, and biotin end groups were capable of self assembling into core–shell structural micelles in aqueous solution. The variation of end groups significantly affected the micellar characters, such as hydrodynamic diameter (Dh), critical micellar concentration (CMC), and lower critical solution temperature (LCST). Biotinylated micelle exhibited a phase transition at 41.4 °C. The amount of biotin on the micelle surface as well as the specific recognition between biotinylated micelle and avidin was determined by 4′-hydroxyazobenzene-2-carboxylic acid/avidin (HABA/avidin) binding assay and dynamic light scattering (DLS). In addition, the biotinylated star copolymer displayed good biocompatibility according to a preliminary cytotoxicity study. The novel polymeric micelle with biodegradability, thermoresponse, and specific target recognition was expected to be a promising polymeric carrier material for targeted drug delivery.

Design and Synthesis of Monofunctionalized, Water-Soluble Conjugated Polymers for Biosensing and Imaging Applications

Water-soluble conjugated polymers with controlled molecular weight characteristics, absence of ionic groups, high emission quantum yields, and end groups capable of selective reactions of wide scope are desirable for improving their performance in various applications and, in particular, fluorescent biosensor schemes. The synthesis of such a structure is described herein. 2-Bromo-7-iodofluorene with octakis(ethylene glycol) monomethyl ether chains at the 9,9'-positions, i.e., compound 4, was prepared as the reactive premonomer. A high-yielding synthesis of the organometallic initiator (dppe)Ni(Ph)Br (dppe = 1,2-bis(diphenylphosphino)ethane) was designed and implemented, and the resulting product was characterized by single-crystal X-ray diffraction techniques. Polymerization of 4 by (dppe)Ni(Ph)Br can be carried out in less than 30 s, affording excellent control over the average molecular weight and polydispersity of the product. Quenching of the polymerization with [2-(trimethylsilyl)ethynyl]magnesium bromide yields silylacetylene-terminated water-soluble poly(fluorene) with a photoluminescence quantum efficiency of 80%. Desilylation, followed by copper-catalyzed azide-alkyne cycloaddition reaction, yields a straightforward route to introduce a wide range of specific end group functionalities. Biotin was used as an example. The resulting biotinylated conjugated polymer binds to streptavidin and acts as a light-harvesting chromophore to optically amplify the emission of Alexa Fluor-488 chromophores bound onto the streptavidin. Furthermore, the biotin end group makes it possible to bind the polymer onto streptavidin-functionalized cross-linked agarose beads and thereby incorporate a large number of optically active segments.

Mapping the Sensing Zone of Alpha-Hemolysin Using Immobilized DNA Containing a Single Abasic Residue

As efforts to sequence individual strands of DNA using biological nanopores continue to evolve, it has become increasingly important to understand the nature of the DNA-nanopore interaction. More specifically, characterizing the “sensing” zone inside the nanopore is an important part of understanding how nucleotide differentiation occurs. In order to characterize the sensing zone of the biological nanopore alpha-Hemolysin (αHL), we have examined how an adenine homopolymer containing a single abasic site blocks current flow through an αHL pore as the position of the abasic site is moved sequentially through the pore. In these experiments, single stranded DNA (ssDNA) oligonucleotides with a biotin (BTN) group at the 3’ terminus were allowed to complex with streptavidin. Due to the inability of the large streptavidin to translocate through the αHL, the attached oligonucleotide could be electrophoretically immobilized inside the pore. Creating and capturing numerous oligonucleotides in which the single abasic site was positioned progressively farther from the BTN termination (e. g. 7 adenines from the BTN end, 8 adenines from the BTN end, etc.) allowed a blocking current map of the αHL lumen to be developed. This blocking current map of αHL yielded a single peak, which is approximately two nucleotides in width, centered midway through the αHL pore where the beta-barrel meets the vestibule. This single peak corresponds to the primary sensing zone of αHL. Further experiments have demonstrated that both the width and location of the primary sensing zone can be altered by creating mutant αHL proteins in which the residues inside the αHL pore have been changed. These studies should prove integral in creating a class of mutant αHL pores capable of discriminating between single nucleotides in ssDNA.

Biotin functionalized poly(sulfonic acid)s for bioconjugation: In situ binding monitoring by QCM‐D

AbstractWe describe the synthesis of biotin end functionalized poly(sulfonic acid)s via living radical polymerization (LRP) for conjugation to Avidin. Quartz crystal microbalance (QCM‐D) and competitive binding studies were used to confirm this conjugation. A biotin initiator for copper‐mediated LRP was used to provide acrylamide and methacrylate based polymers with the functional end group. This investigation revealed that 2‐acrylamido‐2‐methyl‐1‐propanesulfonic acid was not a suitable monomer in its acid form but was successfully used in its sodium salt form. A second monomer, 3‐sulfopropylmethacrylate as the potassium salt was also studied and both monomers produced polymers with polydispersities &lt;1.3 and 1.4, respectively. Evolution of molecular weight with respect to time indicated that the polymerization of the acrylamide polymer is controlled. Quartz crystal microbalance with dissipation monitoring was used to confirm that the biotinylated polymers were able to bind to Avidin in situ. The gold surface of a quartz crystal was chemically modified resulting in a stable monolayer of Avidin; the biotinylated polymers were passed over the functionalized surface and their grafting ability was examined. A competitive binding evaluation was undertaken with 2‐(4‐hydroxyphenylazo)benzoic acid (HABA) dye to provide visual verification of conjugation. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011

Double Thermoresponsive Block Copolymers Featuring a Biotin End Group

A poly(oligo(ethylene glycol) monomethyl ether methacrylate)-block-poly(N-isopropyl methacrylamide) (POEGMA-b-PNIPMAM) block copolymer with a biotin end group on the PNIPMAM block as a biotarget was synthesized as a model system for temperature-controlled polymer immobilization. The synthesis was based on RAFT polymerization followed by postpolymerization modification of an activated ester precursor block and an exchange of the dithioester end group within one step. NMR, differential scanning calorimetry (DSC), dynamic light scattering (DLS), and turbidimetry measurements were performed to investigate the stimulus-responsive properties. The double thermoresponsive POEGMA-b-PNIPMAM with biotin end group showed a temperature-dependent multistage assembly behavior as it was completely soluble in water at temperatures below the LCST of both blocks, formed micellar structures above the LCST of PNIPMAM but below the LCST of POEGMA, or precipitated from solution above the LCST of both blocks. At room temperature, the polymer could be immobilized onto a streptavidin surface via its biotin end group, as shown in surface plasmon resonance (SPR) experiments. At 50 °C, at which the block copolymer formed micelles trapping the biotin target within the PNIPMAM core, no immobilization was observed, showing that the biological binding ability of the model could be controlled via external stimuli.

Synthesis of Hetero-Telechelic α,ω Bio-Functionalized Polymers

Reversible addition-fragmentation chain transfer (RAFT) polymerization was used to synthesize poly[diethylene glycol monomethylether methacrylate] (PDEGMA) (M(n) = 6250 g/mol, PDI = 1.14) with a pentafluorophenyl (PFP) activated ester and a dithioester end group. The hormone thyroxin (T4) was quantitatively attached to the PFP activated ester alpha end group via its amino group. The omega-terminal dithioester was not harmed by this reaction and was subsequently aminolyzed in the presence of N-biotinylaminoethyl methanethiosulfonate, yielding a polymer with a thyroxin and a biotin end group with very high heterotelechelic functionality. The polymer was characterized by (1)H, (13)C, and (19)F NMR, UV-vis, and IR spectroscopy and gel permeation chromatography. The thyroxin transport protein prealbumin with two thyroxin binding sites and streptavidin, which has four biotin binding sites, was conjugated using the biotarget labeled polymer, resulting in the formation of a protein-polymer network, confirming the heterotelechelic nature of the polymer. Polymer-protein microgel formation was observed with dynamic light scattering. To realize a directed protein assembly, prealbumin was immobilized onto a surface, exposing one of its two thyroxin binding groups and thus allowing the conjugation with the thyroxin alpha end group of the heterotelechelic polymer. The biotin omega end group of the attached polymer layer enabled the subsequent immobilization of streptavidin, yielding a defined multilayer system of two proteins connected with the synthetic polymer (efficiency of streptavidin immobilization 81% based on prealbumin). Without the polymer, no streptavidin immobilization occurred. The layer depositions were monitored by surface plasmon resonance. The synthetic approach of combining PFP activated esters with functional MTS reagents presents a powerful method for obtaining well-defined heterotelechelic (bio-) functionalized polymers.

Experimental Neuronal Migration Disorders Following the Administration of Ibotenate in Hamsters: The Role of the Subventricular Zone in the Development of Cortical Dysplasia

To examine the role of subventricular zone in cortical dysplasia, an experimental model was established using a neuronal tracing study. Ibotenate is a glutamate receptor agonist, and its intracerebral injection produces excitotoxic lesions mimicking microgyria and heterotopia. A total of 72 newborn hamsters were subjected. Biotinylated dextran amine was injected into the ganglionic eminence for the neuronal tracer. The cortical lesions and apoptotic cells were analyzed. In the microgyric cortex, terminal deoxynucleotidyl transferase-mediated 2' -deoxyuridine 5'-triphosphate-biotin end labeling-positive cells were increased and accumulated in the cortical folding. Biotinylated dextran amine-positive cells and fibers were derived from the dorsolateral subventricular zone and directed toward the cortical folding. In the heterotopic cortex, terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate- biotin end labeling-positive cells were distributed around the heterotopia, but number of cells was not remarkable. Our findings suggest that the subventricular zone plays an important role in the formation of cortical dysplasia, especially in the microgyria, after excitatory neuronal injury.

Directed Self-Assembly of Gold-Tipped CdSe Nanorods

Gold-tipped CdSe rods (nanodumbbells) were solubilized in an aqueous phase and self-assembled in a head-to-tail manner using biotin disulfide and avidin. The disulfide end of the biotin molecule attaches to the gold tip of the nanodumbbell, and the biotin end of the molecule is able to conjugate to an avidin protein. The avidin can strongly conjugate up to four biotin molecules. Changing the ratios of biotin to nanodumbbells leads to the formation of dimers, trimers, and flowerlike structures. To further improve the distribution of chain lengths, a separation method based upon weight was applied using a concentration gradient. The gold tips provide effective anchor points for constructing complex nanorod structures by self-assembly.

Synthesis and characterisation of a degradable poly(lactic acid)-poly(ethylene glycol) copolymer with biotinylated end groups.

Poly(lactic acid)-poly(ethylene glycol)-biotin (PLA-PEG-biotin) is a degradable polymer with protein resistant properties that can undergo rapid surface engineering in aqueous media to create biomimetic surfaces. Surface engineering of this polymer is dependent on biomolecular interactions between the biotin end group and the protein avidin. Given the vigorous conditions of synthesis, it is essential that the manufacture of the polymer does not alter the biotin structure or its molecular recognition. Equally, it is important that the incorporation of biotin does not adversely affect the physicochemical properties of the polymer. (1)H NMR provides evidence of biotin attachment and structural integrity. (1)H NMR, gel permeation chromatography (GPC), and differential scanning calorimetry (DSC) analysis shows there is no significant effect on bulk properties induced by the biotin end group. Surface plasmon resonance (SPR) and fluorescent spectroscopy studies using the 2-(4'-hydroxyazobenzene) benzoic acid (HABA)/avidin complex show that the biotin moieties binding capabilities are not impaired by the synthesis.

Biotinylated indoles as probes for indole-binding proteins.

Biotinylated indoles were prepared for application as bifunctional probes for the detection of indole-binding proteins which participate in the life processes of humans, animals, plants, and bacteria. The indole nucleus was functionalized, at ring positions 3, 5, or 6, by attachment of a 2-aminoethyl group, which was then coupled to the carboxyl moiety of biotin, via a spacer composed of 3 or 4 concatenated beta-alanine residues. The constructs thus obtained were able to inhibit tryptophanase activity, similarly to indole in a concentration-dependent manner. They also bound strongly to lysozyme and weakly to bovine and human serum albumins, in accordance with the known affinities of these proteins for indole and 3-(2-aminoethyl)indole (tryptamine). The biotin end of the protein-bound bifunctional probes could then be detected by coupling to (strept)avidin conjugated to alkaline phosphatase or horseradish peroxidase, followed by incubation with substrates which are converted by these enzymes to intensely colored or chemiluminescent products.

Conformational Study of Biotinylated DNA Oligonucleotides Utilizing Two-Dimensional Nuclear Magnetic Resonance and Molecular Dynamics

The solution structure of DNA oligonucleotides containing a biotin group covalently attached through a linker arm to the 5′ end has been studied by two-dimensional NMR and molecular modeling, in an attempt to determine whether the biotin end group is accessible to avidin binding. Such experiments are useful in suggesting that purification of synthetic DNA oligonucleotides by avidin-biotin affinity chromatography is carried out with a minimum amount of intramolecular association of biotin with potential binding sites within the DNA. Two DNA oligonucleotides that form hairpin structures have been proposed as possible worst case scenarios where biotin would not be available for avidin binding. These structures are d(XGCGCGTTTTCGCGC) and d(XGCGCGTTTTCGCGCAAAAA), where X represents the biotin and linker structures. No NOEs were detected between the biotin/linker portion of the molecule and the DNA hairpin. In addition, the magnitude of the NOEs between neighboring groups in the biotin/linker was approximately an order of magnitude smaller than that of the neighbors at a comparable distance within the DNA hairpin, suggesting that the biotin/linker undergoes considerable additional motion compared to the DNA hairpin. Molecular dynamics calculations also show that the biotin/linker undergoes a considerable range of motion. Thus, all data indicate that the biotin is not immediately associated with the hairpin and should be available for binding to avidin chromatographically relevant solvent conditions.