Biotin Group Research Articles

Structurally responsive oligonucleotide-based single-probe lateral-flow test for detection of miRNA-21 mimics.

A single-probe strip test for the rapid and sensitive detection of miRNA-21 mimics is reported herein. Highly specific structurally responsive bi-functional, thiol and biotin, DNA/LNA oligonucleotide probes (molecular beacons-MB) were designed and conjugated with gold nanoparticles (AuNPs) (i.e. biotin-MB-AuNPs). The proposed design had the ability to modulate the accessibility of the biotin group as a function of the presence of a miRNA target allowing the interaction of the boilable with the streptavidin test zone only in the presence of the miRNA-21 mimics. For quantitative evaluation, images of the strip tests were recorded using a flatbed scanner (Epson Perfection V370 Photo). The colour intensities of the test zones of the strip tests were analysed with the ImageJ software (Scion Corp., USA) and quantified as a function of pixel intensity. The response of the strip test was linear over the range 0.5 to 20 nM miRNA-21 (limit of detection of 115 pM) and showed good reproducibility (intra and inter CVs below 8%); furthermore, the assay was shown to be highly selective, discriminating other interference miRNAs mimics (e.g. miRNA-221 and miRNA-205). Finally, the proposed strip test was used for detection of miRNA-21 mimics in spiked serum samples, demonstrating its potential for point-of-care clinical applications. Main advantages of the single-probe strip test design are its versatility, simplicity and robustness, which can be easily extended to other miRNA targets by tuning the sequence of the single probe. Furthermore, the use of the structurally responsive single probe is particularly relevant in the case of short-length targets, such as miRNA, whereas a conventional sandwich approach might require a careful control of assay conditions such as hybridization temperature and salt concentration.

A 7-Deazaadenosylaziridine Cofactor for Sequence-Specific Labeling of DNA by the DNA Cytosine-C5 Methyltransferase M.HhaI.

DNA methyltransferases (MTases) catalyze the transfer of the activated methyl group of the cofactor S-adenosyl-l-methionine (AdoMet or SAM) to the exocyclic amino groups of adenine or cytosine or the C5 ring atom of cytosine within specific DNA sequences. The DNA adenine-N6 MTase from Thermus aquaticus (M.TaqI) is also capable of coupling synthetic N-adenosylaziridine cofactor analogues to its target adenine within the double-stranded 5′-TCGA-3′ sequence. This M.TaqI-mediated coupling reaction was exploited to sequence-specifically deliver fluorophores and biotin to DNA using N-adenosylaziridine derivatives carrying reporter groups at the 8-position of the adenine ring. However, these 8-modified aziridine cofactors were poor substrates for the DNA cytosine-C5 MTase from Haemophilus haemolyticus (M.HhaI). Based on the crystal structure of M.HhaI in complex with a duplex oligodeoxynucleotide and the cofactor product, we synthesized a stable 7-deazaadenosylaziridine derivative with a biotin group attached to the 7-position via a flexible linker. This 7-modified aziridine cofactor can be efficiently used by M.HhaI for the direct, quantitative and sequence-specific delivery of biotin to the second cytosine within 5′-GCGC-3′ sequences in short duplex oligodeoxynucleotides and plasmid DNA. In addition, we demonstrate that biotinylation by M.HhaI depends on the methylation status of the target cytosine and, thus, could provide a method for cytosine-C5 DNA methylation detection in mammalian DNA.

Site-Specific N-Terminal Labeling of Peptides and Proteins using Butelase 1 and Thiodepsipeptide.

An efficient ligase with exquisite site-specificity is highly desirable for protein modification. Recently, we discovered the fastest known ligase called butelase 1 from Clitoria ternatea for intramolecular cyclization. For intermolecular ligation, butelase 1 requires an excess amount of a substrate to suppress the reverse reaction, a feature similar to other ligases. Herein, we describe the use of thiodepsipeptide substrates with a thiol as a leaving group and an unacceptable nucleophile to render the butelase-mediated ligation reactions irreversible and in high yields. Butelase 1 also accepted depsipeptides as substrates, but unlike a thiodesipeptide, the desipeptide ligation was partially reversible as butelase 1 can tolerate an alcohol group as a poor nucleophile. The thiodesipeptide method was successfully applied in N-terminal labeling of ubiquitin and green fluorescent protein using substrates with or without a biotin group in high yields.

Site‐Specific N‐Terminal Labeling of Peptides and Proteins using Butelase 1 and Thiodepsipeptide

AbstractAn efficient ligase with exquisite site‐specificity is highly desirable for protein modification. Recently, we discovered the fastest known ligase called butelase 1 from Clitoria ternatea for intramolecular cyclization. For intermolecular ligation, butelase 1 requires an excess amount of a substrate to suppress the reverse reaction, a feature similar to other ligases. Herein, we describe the use of thiodepsipeptide substrates with a thiol as a leaving group and an unacceptable nucleophile to render the butelase‐mediated ligation reactions irreversible and in high yields. Butelase 1 also accepted depsipeptides as substrates, but unlike a thiodesipeptide, the desipeptide ligation was partially reversible as butelase 1 can tolerate an alcohol group as a poor nucleophile. The thiodesipeptide method was successfully applied in N‐terminal labeling of ubiquitin and green fluorescent protein using substrates with or without a biotin group in high yields.

The unusual and dynamic character of PX-DNA.

PX-DNA is a four-stranded DNA structure that has been implicated in the recognition of homology, either continuously, or in an every-other-half-turn fashion. Some of the structural features of the molecule have been noted previously, but the structure requires further characterization. Here, we report atomic force microscopic characterization of PX molecules that contain periodically placed biotin groups, enabling the molecule to be labeled by streptavidin molecules at these sites. In comparison with conventional double stranded DNA and with antiparallel DNA double crossover molecules, it is clear that PX-DNA is a more dynamic structure. Furthermore, the spacing between the nucleotide pairs along the helix axis is shorter, suggesting a mixed B/A structure. Circular dichroism spectroscopy indicates unusual features in the PX molecule that are absent in both the molecules to which it is compared.

Thermal-induced Immuno-nephelometry Using Gold Nanoparticles Conjugated with a Thermoresponsive Polymer for the Detection of Avidin.

Thermoresponsive immunonephelometry was achieved with biotinylated poly(acrylate) and thermoresponsive gold nanocomposites composed of 13-nm gold nanoparticles and thermoresponsive polymers containing triethylenetetramine and biotin groups. The avidin-biotin interaction was used to model an immunoreaction in order to demonstrate thermoresponsive immunonephelometry. In the absence of avidin, positively charged gold nanocomposites electrostatically interacted with biotinylated poly(acrylate) to form binary complexes, in which the charges canceled each other out. The charge cancelation resulted in the binary complexes precipitating when the solution was heated above the phase-transition temperature. However, adding avidin formed ternary sandwich complexes through the avidin-biotin interaction. The ternary complexes remained sufficiently soluble above the phase-transition temperature because of the spatial isolation of the positive and negative charges. The transmittance of the solution containing the thermoresponsive gold nanocomposites and biotinylated poly(acrylate) at 37°C increased as the avidin concentration increased. A sigmoidal profile was observed from 10(-6.5) to 10(-5.5) mol/L. The concentration of avidin spiked in bovine serum was determined by our method.

Detection of Peptide-based nanoparticles in blood plasma by ELISA.

AimsThe aim of the current study was to develop a method to detect peptide-linked nanoparticles in blood plasma.Materials & MethodsA convenient enzyme linked immunosorbent assay (ELISA) was developed for the detection of peptides functionalized with biotin and fluorescein groups. As a proof of principle, polymerized pentafluorophenyl methacrylate nanoparticles linked to biotin-carboxyfluorescein labeled peptides were intravenously injected in Wistar rats. Serial blood plasma samples were analyzed by ELISA and by liquid chromatography mass spectrometry (LC/MS) technology.ResultsThe ELISA based method for the detection of FITC labeled peptides had a detection limit of 1 ng/mL. We were able to accurately measure peptides bound to pentafluorophenyl methacrylate nanoparticles in blood plasma of rats, and similar results were obtained by LC/MS.ConclusionsWe detected FITC-labeled peptides on pentafluorophenyl methacrylate nanoparticles after injection in vivo. This method can be extended to detect nanoparticles with different chemical compositions.

Aptamer-based fluorescent solid-phase thrombin assay using a silver-coated glass substrate and signal amplification by glucose oxidase

We describe an aptamer-based solid-state biosensor for the fluorometric determination of thrombin. The surface of silver-coated glass was modified with the thrombin-binding aptamer 1 (TBA 1) of the sequence 5′-HS-TTT TTT TTT TTT TTT GGT TGG TGT GGT TGG-3′. A second (and biotinylated) thrombin -binding aptamer (TBA 2) with the sequence 5′-biotin-AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-3′ was applied as the signaling aptamer. Following binding of thrombin by TBA 1 on the surface, TBA 2 is added and then binds to the thrombin on the surface of the silver-coated glass to form the thrombin-aptamer complex. The biotin groups on TBA 2 are then coated with streptavidin, and biotin-labeled glucose oxidase (biotin-GOx) is added to bind to streptavidin. The quantity of GOx immobilized in this way is directly related to the quantity of thrombin bound on the surface. Following cleavage of the aptamer with DNase I, glucose is added and oxidized by GOx to yield H2O2. Horseradish peroxidase is added and causes the oxidation of 3-p-hydroxyphenylpropanoic acid to yield a fluorescent product. The intensity of the blue fluorescence is directly related to the thrombin concentration in the 300 pM to 6500 pM range, and the detection limit is as low as 82 pM. The assay has good selectivity and practicability.

An electrochemical microRNAs biosensor with the signal amplification of alkaline phosphatase and electrochemical–chemical–chemical redox cycling

MicroRNAs (MiRNAs) have been regarded as clinically important biomarkers and drug discovery targets. In this work, we reported a simple and ultrasensitive electrochemical method for miRNAs detection based on single enzyme amplification and electrochemical–chemical–chemical (ECC) redox cycling. Specifically, upon contact with the target miRNAs, the hairpin structure of biotinylated DNA immobilized on gold electrode was destroyed and the biotin group in DNA was forced away from the electrode surface, allowing for the coupling of streptavidin-conjugated alkaline phosphatase (SA-ALP). Then, ascorbic acid (AA, the enzymatic product of ALP) triggered the ECC redox cycling with ferrocene methanol (FcM) and tris(2-carboxyethyl)phosphine (TCEP) as the redox mediator and the chemical reducing reagent, respectively. The method was more sensitive than that with horseradish peroxidase (HRP) or glucose oxidase (GOx) triggered recycling since one ALP molecule captured by one target miRNA molecule promoted the production of thousands of AA. Analytical merits (e.g., detection limit, dynamic range, specificity, regeneration and reproducibility) were evaluated. The feasibility of the method for analysis of miRNA-21 in human serum has also been demonstrated.

Dietary intake of high-dose biotin inhibits spermatogenesis in young rats.

To characterize a new function of the water-soluble vitamin, biotin, in reproduction and early growth in mammals, the effects of high dietary doses of biotin on early spermatogenesis were biochemically and histologically investigated in male rats. Weaned rats were fed a CE-2 (control) diet containing 0.00004% biotin, or a control diet supplemented with 0.01%, 0.1%, or 1.0% biotin. Pair-fed rats were fed a control diet that was equal in calories to the amount ingested by the 1.0% biotin group, because food intake was decreased in the 1.0% biotin group. Food intake and body weight gain were lower in the 1.0% biotin group than in the control group. The kidney, brain and testis weights were significantly lower in the 1.0% biotin group than in the pair-fed group after 6 weeks of feeding. The accumulation of biotin in the liver and testis increased in a dose-dependent manner. In the 1.0% biotin group, the number of mature sperm was markedly lower, that of sperm with morphologically abnormal heads, mainly consisting of round heads, had increased. In addition, the development of seminiferous tubules was inhibited, and few spermatogonia and no spermatocytes were histologically observed. These results demonstrated that the long-term intake of high-dose biotin inhibited spermatogenesis in young male rats.

Enhancement of Colorimetric Response of Enzymatic Reactions by Thermally Evaporated Plasmonic Thin Films: Application to Glial Fibrillary Acidic Protein.

We report the enhancement of the colorimetric response of horseradish peroxidase (HRP) and alkaline phosphatase (AP) in bioassays by thermally evaporated silver, gold, copper and nickel thin films. In this regard, a model bioassay based on biotin-avidin interactions was employed. Biotin groups and enzymes were introduced to all surfaces using a biotinylated linker molecule and avidin, respectively. The colorimetric response of HRP in the model bioassay carried out on the plasmonic thin films were up to 4.4-fold larger as compared to control samples (i.e., no plasmonic thin films), where the largest enhancement of colorimetric response was observed on silver thin films. The colorimetric response of AP on plasmonic thin films was found to be similar to those observed on control samples, which was attributed to the loss of enzymes from the surface during the bioassay steps. The extent of enzymes immobilized on to plasmonic thin films was found to affect the colorimetric response of the model bioassay. These findings allowed us to demonstrate the use of silver thin films for the detection of glial fibrillary acidic protein (GFAP), where the colorimetric response of the standard bioassays for GFAP was enhanced up to 67% as compared to bioassays on glass slides.

Developing a Universal Steric Trapping Strategy for Studying Folding and Stability of Helical Membrane Proteins

“Steric trapping” is a method that links binding of monovalent streptavidin (mSA) to unfolding of a biotinylated protein (MP). It allowed the measurements of high affinity protein–protein interactions and thermodynamic stability of polytopic helical MPs in a native environment, which had been difficult to achieve using more conventional methods. In the current steric trapping framework, a conformation-sensitive chromophore or an enzymatic activity in a target MP is required for monitoring mSA-induced unfolding. This target-specific approach is a limiting factor that hinders its application to various MP systems, i.e. MPs in a misfolded conformation, MPs with an assembly role, or MPs without convenient unfolding readout. To further advance the steric trapping strategy for more general application, we have developed novel tripartite probes possessing a thiol-reactive group, a biotin group and a fluorescent or paramagnetic group for sensitization of unfolding or binding of mSA. We applied the new strategy to investigating the stability and unfolding mechanism of an intramembrane protease GlpG. By combining FRET between fluorescently labeled GlpG and quencher-labeled mSA as a measure of mSA binding and the proteolytic activity of GlpG as a measure of unfolding, we proved the thermodynamic coupling between binding and unfolding, and determined the thermodynamic stability and unfolding rate of GlpG in a native micellar environment. While the stabilities were similar independent of the location of biotin pairs, the unfolding was 20 times faster when the biotin pair was placed near the proteolytic active site. This result suggests a subdomain-organization of the helical bundle architecture of GlpG. Steric trapping may serve as a useful tool for elucidating the local versus global flexibility of helical MPs.

Histopathological Study Of The Effect Of Low Level Laser And Biatain Ag Non-Adhesive Dressing On The Avulsion Wound Healing In Rabbits

The aims of this study was to compare between the effect of Low level Laser and Biatain Ag non-adhesive dressing on healing of avulsion wound in rabbits. Eighteen adult healthy rabbits were used. It is divided in to two equal groups , Laser group , and Biatain Ag group, also each group divide into three equal sub group for histopathological study at 1 week , 2 week and 3 week post operation. The avulsion wound was done on the back(dorsal) after general anesthesia by blade 1 cm . post-operation , in laser group, the rabbits were exposed to low level laser for 5 minute/daily to period 1week, 2week ,3week continuously , but in Biatain Ag group, the avulsion wound was dressing by Biatain Ag non-adhesive dressing and it fixed on wound by blaster and continuous to 1week, 2week, 3week.The histopathological examination appear in first week, both groups showed ulceration but in biotin group showed more ulceration than laser group. In two week, laser group some animals showed absence of ulceration with dermal fibrosis. In two week, in biatain group showed reduce the size ulcer with dermal fibrosis. In three week, laser group showed complete healing with moderate thickening of epidermis and hyperkeratosis with dermal fibrosis. While biatain group showed complete healing with marked dermal fibrosis but presence of foci of hair folliculitis. Conclusion In two week, laser group all showed healing of ulcer except one animal while biatain group there was only reduce size of ulcer but not complete healing..

A novel adenosine-based molecular beacon probe for room temperature nucleic acid rapid detection in cotton thread device

We used cotton thread as substrate to develop a novel room temperature DNA detection device for low-cost, sensitive and rapid detection of a human genetic disease, hereditary tyrosinemia type I related DNA sequences. A novel adenosine based molecular beacon (ABMB) probe modified on gold nanoparticle was used as reporter probe. In the presence of coralyne, a small molecule which can react with adenosines, the ABMB would form a hairpin structure just like traditional molecular beacon used extensively. In the presence of target DNA sequences, the hairpin structure of ABMB modified on gold nanoparticles will be opened and the biotin group modified at one end of the DNA probes will be released and react with the streptavidin immobilized on the test zone of the cotton thread. The response of the thread based DNA test device is linear over the range of 2.5–100nM complementary DNA. The ability of our developed device for discriminating the single base mismatched DNA related to a human genetic disease, hereditary tyrosinemia type I, was improved comparing with previous report. It is worth mentioning that the whole assay procedure for DNA test is performed under room temperature which simplified the assay procedures greatly.

An ultrasensitive electrochemical miRNAs sensor based on miRNAs-initiated cleavage of DNA by duplex-specific nuclease and signal amplification of enzyme plus redox cycling reaction

In this work, we reported a simple and ultrasensitive electrochemical sensing platform for microRNAs (miRNAs) detection by combination of duplex-specific nuclease (DSN) with signal amplification of alkaline phosphatase (ALP) plus redox cycling reaction. Specifically, biotinylated single-stranded DNA capture probes (CPs) immobilized on gold electrodes allowed streptavidin-conjugated alkaline phosphatase (SA-ALP) to be attached, facilitating the production of electrochemically active p-aminophenol (p-AP) from p-aminophenyl phosphate (p-APP) substrate. The resulting p-AP could be cycled by tris(carboxyethyl)phosphine (TCEP) after its electro-oxidization, enabling an increase in the anodic current of p-AP (known as p-AP redox cycling). If the CPs formed duplexes with miRNAs by hybridization, DNA in the DNA-miRNAs duplexes would be cleaved by DSN, making the biotin groups stripping from the electrode surface and the SA-ALP molecules incapable of attachment onto the electrode. In this process, miRNAs strands remained intact and could be released back to the sample solution for recycling. The oxidation current of p-AP decreased significantly with the increase of miRNAs concentration. As a result, a detection limit of 0.2fM for miRNA-21 detection was achieved.

Creating highly amplified enzyme-linked immunosorbent assay signals from genetically engineered bacteriophage

For early detection of many diseases, it is critical to be able to diagnose small amounts of biomarkers in blood or serum. One of the most widely used sensing assays is the enzyme-linked immunosorbent assay (ELISA), which typically uses detection monoclonal antibodies conjugated to enzymes to produce colorimetric signals. To increase the overall sensitivities of these sensors, we demonstrate the use of a dually modified version of filamentous bacteriophage Fd that produces significantly higher colorimetric signals in ELISAs than what can be achieved using antibodies alone. Because only a few proteins at the tip of the micron-long bacteriophage are involved in antigen binding, the approximately 4000 other coat proteins can be augmented—by either chemical functionalization or genetic engineering—with hundreds to thousands of functional groups. In this article, we demonstrate the use of bacteriophage that bear a large genomic fusion that allows them to bind specific antibodies on coat protein 3 (p3) and multiple biotin groups on coat protein 8 (p8) to bind to avidin-conjugated enzymes. In direct ELISAs, the anti-rTNFα (recombinant human tumor necrosis factor alpha)-conjugated bacteriophage show approximately 3- to 4-fold gains in signal over that of anti-rTNFα, demonstrating their use as a platform for highly sensitive protein detection.

Quantification of plasma HIV RNA using chemically engineered peptide nucleic acids.

The remarkable stability of peptide nucleic acids (PNAs) toward enzymatic degradation makes this class of molecules ideal to develop as part of a diagnostic device. Here we report the development of chemically-engineered PNAs for the quantitative detection of HIV RNA at clinically relevant levels that are competitive with current PCR-based assays. Using a sandwich hybridization approach, chemical groups were systematically introduced into a surface PNA probe and a reporter PNA probe to achieve quantitative detection for HIV RNA as low as 20 copies per milliliter of plasma. For the surface PNA probe, four cyclopentane groups were incorporated to promote stronger binding to the target HIV RNA compared to PNA without the cyclopentanes. For the reporter PNA probe, 25 biotin groups were attached to promote strong signal amplification after binding to the target HIV RNA. These general approaches to engineer PNA probes may be used to detect other RNA target sequences.

A facile method for interfering with off-target silencing mediated by the sense strand.

Although siRNA duplexes are widely used for gene silencing, several unwanted effects such as activation of innate immunity and off-target gene silencing can limit their therapeutic use. Off-targeting can be identified for both the sense and antisense siRNA strands. Some avenues of obstructing the incorporation of the sense strand into the RNA-induced silencing complex (RISC) are currently being pursued. Herein, a biotin group at the 5'-end of the sense strand was used to inhibit its incorporation into the RISC complex. In contrast to chemical modifications, biotin is a naturally occurring compound and its presence in siRNA sequences will not induce side effects.

Specific visualization and identification of phosphoproteome in gels.

The applicability of gel-based proteomic strategies in phosphoproteomics has been largely limited by the lack of technologies for specific detection of phosphoproteins in gels. Here for the first time we report a strategy for simultaneous visualization and identification of phosphoproteome in gels (VIPing) through coupling specific detection of phosphoproteins with protein identification and phosphorylation site mapping by tandem mass spectrometry. The core of the strategy is a novel compound multifunctionalized with a titanium ion(IV) for outstanding selectivity toward phosphorylated residues, a fluorophore for visualization, and a biotin group for phosphopeptide enrichment. The sensitivity and specificity of the VIPing strategy was demonstrated using standard protein mixtures and complex cell extracts, and the method was applied to study the phosphorylation changes of an essential tyrosine kinase Syk and interacting proteins upon B-cell stimulation. The novel technique provides a powerful platform for gel-based phosphoproteomic studies.

Comparative study of the ToF‐SIMS, FT‐IR and XPS techniques for quantitative analyses of mixed self‐assembled monolayers

We present a comparative study of the time‐of‐flight‐secondary ion mass spectrometry (ToF‐SIMS), Fourier transform infrared (FT‐IR) spectroscopy and X‐ray photoelectron spectroscopy (XPS) techniques on a mixed self‐assembled monolayer (SAM) of biotinylated polyethylene glycol alkane thiol (BPT) and 11‐mercaptoundecanol (MUD) as a model system. This mixed SAM was employed to maximize specific binding between the biotin groups in the mixed SAM and the streptavidin, while minimizing nonspecific adsorption of the streptavidin. For a better quantitative analysis of our mixed SAM by using ToF‐SIMS, we performed a principal component analyses (PCA) with various peak sets and found that the negative ions of the MUD complementary peak set showed good correlation between the XPS and FT‐IR quantification results and the scores for principal component 1 from a PCA of the ToF‐SIMS data. These findings show that ToF‐SIMS analysis along with PCA would make a good surface analysis technique for identifying intact molecules of adsorbates and for quantifying at once each adsorbate in a mixed SAM. Copyright © 2014 John Wiley & Sons, Ltd.