A small-molecule-linked DNA–graphene oxide-based fluorescence-sensing system for detection of biotin

In this paper, we establish a novel fluorescence-sensing system for the detection of biotin based on the interaction between DNA and graphene oxide and on protection of the terminal of the biotinylated single-stranded DNA fluorescent probe by streptavidin. In this system, streptavidin binds to the biotinylated DNA, which protects the DNA from hydrolysis by exonuclease I. The streptavidin–DNA conjugate is then adsorbed to the graphene oxide resulting in the fluorescence being quenched. Upon the addition of free biotin, it competes with the labeled biotin for the binding sites of streptavidin and then the exonuclease I digests the unbound DNA probe from the 3′ to the 5′ terminal, releasing the fluorophore from the DNA. Because of the weak affinity between the fluorophore and graphene oxide, the fluorescence is recovered. Under optimal conditions, the fluorescence intensity is proportional to the concentration of biotin in the concentration range of 0.5–20 nmol/L. The detection limit for biotin is 0.44 nmol/L. The proposed fluorescence-sensing system was applied to the determination of biotin in some real samples with satisfactory reproducibility and accuracy. This work could provide a common platform for detecting small biomolecules based on protein–small molecule ligand binding.     

Endogenous biotin‐binding proteins: an overlooked factor causing false positives in streptavidin‐based protein detection

Biotinylation is widely used in DNA, RNA and protein probing assays as this molecule has generally no impact on the biological activity of its substrate. During the streptavidin‐based detection of glycoproteins in Lactobacillus rhamnosus GG with biotinylated lectin probes, a strong positive band of approximately 125 kDa was observed, present in different cellular fractions. This potential glycoprotein reacted heavily with concanavalin A (ConA), a lectin that specifically binds glucose and mannose residues. Surprisingly, this protein of 125 kDa could not be purified using a ConA affinity column. Edman degradation of the protein, isolated via cation and anion exchange chromatography, lead to the identification of the band as pyruvate carboxylase, an enzyme of 125 kDa that binds biotin as a cofactor. Detection using only the streptavidin conjugate resulted in more false positive signals of proteins, also in extracellular fractions, indicating biotin‐associated proteins. Indeed, biotin is a known cofactor of numerous carboxylases. The potential occurence of false positive bands with biotinylated protein probes should thus be considered when using streptavidin‐based detection, e.g. by developing a blot using only the streptavidin conjugate. To circumvent these false positives, alternative approaches like detection based on digoxigenin labelling can also be used.     

G‐rich sequence‐functionalized polystyrene microsphere‐based instantaneous derivatization for the chemiluminescent amplified detection of DNA

Herein, we develop a novel chemiluminescence (CL) approach with high sensitivity and excellent selectivity, by taking advantage of magnetic beads as preconcentration carriers and polystyrene microspheres as an amplification platform. Briefly, a ‘sandwich‐type’ detection strategy is employed in our design, which involves capture probe DNA immobilized on the surface of carboxyl‐terminated magnetic beads and multiple biotinylated reporter DNA self‐assembled on the surface of streptavidin‐modified polystyrene microspheres. The reporter DNA includes a guanine nucleobase‐rich (G‐rich) sequence domain for the generation of light and an additional tethered nucleic acid domain complementary with the target DNA. The CL signal is obtained via a novel instantaneous derivatization reaction between a specific CL reagent and the guanine nucleo­bases rich in the target and reporter DNA. As a result, we demonstrate that this DNA assay is reproducible, stable, easy to use, and can sensitively detect femtomolar target DNA related to anthrax lethal factors with excellent differentiation ability for single‐base mismatched sequences. Overall, this new CL protocol couples the high sensitivity of CL analysis with effective magnetic separation for discriminating against unwanted constituents such as mismatched sequences, and hence, offers great promise for DNA hybridization analysis. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of DNA nonradioactively labeled with photobiotin using streptavidin conjugates with peroxidase or alkaline phosphatase

A sensitive non-radioactive method for detecting biotinylated DNA is described. DNA was biotinylated with photobiotin, then processed by peroxidase and alkaline phosphatase covalently bound to streptavidin in the presence of the substrates of these enzymes. The formation of colored products was observed. The sensitivity of ca. 1.10(-9) g of DNA was achieved by using the conjugate of streptavidin and peroxidase, and that of ca. 1.10(-12) g of DNA--by using the cojugate of streptavidin and alkaline phosphatase. Due to rapidity, reproducibility and sensitivity of the method it can be used as an alternative to radioactive labeling with 32P.     

Detection of sub-picogram quantities of specific DNA sequences on blot hybridization with biotinylated probes.

A sensitive method for detecting biotinylated DNA probes on dot and Southern blots is described which is based on the principle outlined by Leary et al (1). This system has two main components: detection of biotinylated DNA by a two-step procedure with streptavidin and poly(alkaline phosphatase); and blocking background with Tween 20. 32fg and 80fg of lambda phage DNA was detected on dot and Southern blot hybridizations respectively. 150fg of beta-globin was detected on Southern blots of genomic DNA. This method is fast, reproducible and can detect single copy genes in 0.25 micrograms genomic DNA on Southern blots.     

Novel biotinylated nucleotide--analogs for labeling and colorimetric detection of DNA.

A series of dATP and dCTP nucleotide analogs have been synthesized which are modified by attachment of aliphatic linkers containing a functional group to the amino-nitrogen at the hydrogen bonding positions of the bases, that is, at the 6-position of adenine and the 4-position of cytosine. These nucleotides are incorporated into DNA probes by standard nick-translation protocols. DNA probes labeled with biotin derivatives of these nucleotides are effectively hybridized to target DNA sequences and can be detected by a streptavidin and calf intestinal alkaline phosphatase conjugate with a sensitivity (0.25 pg DNA) sufficient for reproducible and rapid detection of single copy genes in a Southern blot of mammalian DNA. Also, a procedure has been developed to allow reprobing of nylon filters that have been hybridized with biotinylated probes and developed with the streptavidin/alkaline phosphatase conjugate and a standard dye system.     

Affinity chromatography of DNA labeled with chemically cleavable biotinylated nucleotide analogs

DNA labeled with the chemically cleavable biotinylated nucleotide Bio-12-SS-dUTP was chromatographed on biotin cellulose affinity columns using either avidin or streptavidin as the affinity reagent. Although both proteins were equally effective in binding the Bio-12-SS-DNA to the affinity resin, two important differences were found. First, nonbiotinylated DNA bound to avidin, but not to streptavidin, in buffers containing 50 mM NaCl. Second, Bio-12-SS-DNA was released much more slowly from the streptavidin affinity column than from the avidin column upon washing with buffer containing dithiothreitol. This difficulty in reducing the disulfide bond of Bio-12-SS-DNA bound to streptavidin is most likely due to steric protection of the disulfide bond by the protein. This conclusion is supported by our finding that DNA labeled with another biotinylated nucleotide analog, Bio-19-SS-dUTP, is rapidly and efficiently recovered from a streptavidin column. In Bio-19-SS-DNA, the distance between the disulfide bond and the biotin group is approximately 10 A greater than that in Bio-12-SS-DNA. Therefore, Bio-19-SS-dUTP and streptavidin form the basis of an efficient affinity system for the isolation and subsequent recovery of biotinylated DNA in the presence of low ionic strength buffers.     

A conformational switch‐based aptasensor for the chemiluminescence detection of microRNA

A simple microRNA (miRNA) aptasensor has been developed combining the conformational switch of a streptavidin aptamer and isothermal strand displacement amplification. In the presence of its target miRNA, the allosteric molecular beacon (aMB) probe immobilized on the plate can be ‘switched on' and release the streptavidin aptamer. At the same time, Klenow fragment (3′→5′ exo‐) is utilized to initiate DNA‐strand displacement, which starts the target recycling process. Based on the aptamer' high binding affinity and subsequent catalytic chemiluminescence (CL) detection, this CL strategy is highly specific in distinguishing mature miRNAs in same family. It exhibits a dynamic range of four orders of magnitude with a detection limit of 50 fM, and shows great potential for miRNA‐related clinical practices and biochemical research.     

Hybrid selection of mRNA with biotinylated DNA

A novel, convenient, and highly efficient hybrid selection procedure is described. The method utilizes the polymerase chain reaction (PCR) in which one of two primers is biotinylated at the 5'-terminus. The concentration of the biotinylated primer is 100 times that of the other to synthesize biotinylated single-stranded DNA (asymmetric PCR). After hybridization of the biotinylated DNA with mRNA in solution, streptavidin agarose is used to trap the hybrid duplex of mRNA.DNA-biotin onto the solid matrix. The selected mRNA is then eluted from the streptavidin agarose. The quantitative physical recovery of selected mRNA is about 70% with about 33% retention of biological activity.     

免疫聚合酶链反应技术的建立及其对甲胎蛋白的检测

免疫ＰＣＲ是一种新的具高敏感性的抗原检测技术,它类似传统的ＥＬＩＳＡ法,若与抗体连接的酶用ＤＮＡ片段代替,则该ＤＮＡ片段可用于ＰＣＲ扩增。以链酶亲合素搭桥将生物素标记的抗体与ＤＮＡ相连建立了免疫ＰＣＲ技术,并将其用于检测甲胎蛋白（ＡＦＰ）,其敏感性比ＥＬＩＳＡ法高１０４。因此,免疫ＰＣＲ有可能作为一种具高敏感性的检测手段用于临床早期诊断     

A New Approach to Revealing Point Mutations in DNA Analyzed by Colorimetric Detection

A new approach to detection of point mutations in an amplified DNA was developed. The approach is based on highly selective ligation (T4 DNA ligase) of a tandem of short oligonucleotides one of which contains the biotin group. The ligation product is formed only when the hybridization complex DNA/tandem is formed and the tandem is perfect. The hybridization complex DNA/(biotinylated ligation product) was separated from the biotinylated component of the tandem by UV‐immobilization of the reaction mixture on a nylon membrane. The immobilized hybridization complex was detected colorimetrically by a streptavidin‐alkaline phosphatase cojugate with a chromogenic substrate.     

Preparation of single-stranded DNA from PCR products with streptavidin magnetic beads

The preparation of single-stranded DNA from double-stranded PCR products is an essential step in the identification of aptamers by Systematic Evolution of Ligands by EXponential enrichment (SELEX). The most widely used method for producing single-stranded DNA is alkaline denaturation of biotinylated PCR products attached to streptavidin-coated magnetic beads. Recently, it has been suggested that this method may be unsuitable due to the release of interfering amounts of streptavidin and biotinylated DNA. In this article, the alkaline method is compared with a thermal method that is known to release significant amounts of streptavidin and biotinylated DNA. Results show that trace amounts of streptavidin and biotinylated DNA are released in the alkaline method, but this can be curtailed by preconditioning the beads in aqueous sodium hydroxide. The main product in the alkaline method is single-stranded DNA, which is produced in high yield.     

Label-free and high-sensitive detection of Salmonella using a surface plasmon resonance DNA-based biosensor

A method based on surface plasmon resonance (SPR) DNA biosensor has been developed for label-free and high-sensitive detection of Salmonella. A biotinylated single-stranded oligonucleotide probe was designed to target a specific sequence in the invA gene of Salmonella and then immobilized onto a streptavidin coated dextran sensor surface. The invA gene was isolated from bacterial cultures and amplified using a modified semi-nested asymmetric polymerase chain reaction (PCR) technique. In order to investigate the hybridization detection, experiments with different concentration of synthetic target DNA sequences have been performed. The calibration curve of synthetic target DNA had good linearity from 5 nM to 1000 nM with a detection limit of 0.5 nM. The proposed method was applied successfully to the detection of single-stranded invA amplicons from three serovars of Salmonella, i.e., Typhimurium, Enterica and Derby, and the responses to PCR products were related to different S. typhimurium concentrations in the range from 10(2) to 10(10) CFU mL(-1). While with this system to detect E. coli and S. aureus, no significant signal was observed, demonstrating good selectivity of the method. In addition, the hybridization can be completed within 15 min, and the excellent sensor surface regeneration allows at least 300 assay cycles without obvious loss of performance.     

Determination of diethylstilbestrol based on biotin–streptavidin‐amplified time‐resolved fluoro‐immunoassay

A rapid and sensitive time‐resolved fluoroimmunoassay (TR–FIA) based on the biotin–streptavidin amplification system was developed for the determination of diethylstilbestrol (DES). Europium‐labelled streptavidin derivatives combined with europium and anhydride of diethylene triamine penta‐acetic acid were used to label streptavidin; biotin was coupled with goat anti‐rabbit IgG to form a biotin–goat anti‐rabbit IgG bridge between streptavidin–europium and the anti‐DES antibody in the immunoassay. The DES assay was carried out by measuring the fluorescence of Eu³⁺–SA at 615 nm. The presented method produced a wide linear range, 0.001–1000.0 ng/mL, and a detection limit up to 0.81 pg/mL for DES. The method was applied to determine DES in serum samples, with recoveries of 97.4–107.8% and RSD 1.32–4.04%. The assay results by the present method showed that biotin–streptavidin amplified TR–FIA for DES detection; it may offer high sensitivity and promising alternative special methods in biological samples. Copyright © 2011 John Wiley & Sons, Ltd.     

Validated spectrofluorimetric method for the determination of carbamazepine in pharmaceutical dosage forms after reaction with 4‐chloro‐7‐‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD‐Cl)

A sensitive and simple spectrofluorimetric method has been developed and validated for the determination of the anti‐epileptic drug carbamazepine (CBZ) in its dosage forms. The method was based on a nucleophilic substitution reaction of CBZ with 4‐chloro‐7‐nitrobenzo‐2‐ oxa‐1,3‐diazole (NBD‐Cl) in borate buffer (pH 9) to form a highly fluorescent derivative that was measured at 530 nm after excitation at 460 nm. Factors affecting the formation of the reaction product were studied and optimized, and the reaction mechanism was postulated. The fluorescence–concentration plot is rectilinear over the range of 0.6–8 µg/mL with limit of detection of 0.06 µg/mL and limit of quantitation of 0.19 µg/mL. The method was applied to the analysis of commercial tablets and the results were in good agreement with those obtained using the reference method. Validation of the analytical procedures was evaluated according to ICH guidelines. Copyright © 2015 John Wiley & Sons, Ltd.     

Binding of Streptavidin to Bacteria or Fungi and Its Applications in Detecting These Microbes

We have investigated the characteristics and utilities of streptavidin-binding to gram-negative and gram-positive bacteria and Candida spp. The pre-treatment of these microbes with chemical reagents such as CHCl₃, NaOH, and Tween 20 have allowed colorimetric visualization under light microscopy or quantitation on nitrocellulose membranes, using streptavidin/biotinylated alkaline phosphatase conjugates. Analysis of this binding was confirmed by western blot. These binding reactions were due to the specific interaction of streptavidin with biotinylated proteins present in the microbes. Competition assays with free biotin or inhibition by an antibiotin antibody confirmed binding to these proteins. With knowledge of these strongly specific interactions, we attempted to reveal the biotinylated proteins within these microbes using clinical specimens. Using phagocyte-smears from blood, urine, and ascites, these intracellular microbes were easily detected by light microscopy. One of the septic blood samples stained by our technique revealed semi-digested microbial signals despite the absence of a signal with routine staining. This detection system, which combines streptavidin as a probe and biotinylated proteins as a microbial marker, is useful in staining for intracellular bacteria or fungi (e.g., microbial infections in phagocyte-smears).     

Streptavidin-Aequorin Fusion Protein for Bioluminescent Immunoassay

The fusion protein of streptavidin to aequorin (STA-AQ) was highly purified from inclusion bodies in Escherichia coli cells and applied to a bioluminescent sandwich immunoassay. α-Fetoprotein (AFP), which is a serological marker of liver cancer, was used as a model analyte to test STA-AQ in an immunoassay. The measurable range of AFP by the sandwich immunoassay, using the complex of STA-AQ and the biotinylated anti-AFP antibody, was 0.02–200 ng/mL with an average coefficient of variation of 4.9%. The detection sensitivity with the complex of STA-AQ and the biotinylated anti-AFP antibody was similar to that with the complex of biotinylated aequorin, streptavidin and the biotinylated anti-AFP antibody. STA-AQ would be a useful reporter protein for immunoassays.     

Affinity thermoprecipitation and recovery of biotinylated biomolecules via a mutant streptavidin-smart polymer conjugate

A system has been developed for reversibly binding and thermoprecipitating biotinylated macromolecules. A high off-rate Ser45Ala (S45A) streptavidin mutant has been covalently conjugated to poly(N-isopropylacrylamide) (PNIPAAm), a temperature-responsive polymer. The resulting conjugate is shown to coprecipitate biotinylated immunoglobulin G (IgG) and a biotinylated oligonucleotide in response to a thermal stimulus. Thermally precipitated biotinylated macromolecules can be released from the S45A-PNIPAAm conjugate by simple treatment with excess free biotin. This release step has been shown to be unique to the mutant streptavidin conjugate-a conjugate of wild type (WT) streptavidin and PNIPAAm does not release bound biotinylated molecules upon treatment with excess free biotin. The capture efficiency (fraction of target molecule precipitated from solution) of the S45A-PNIPAAm conjugate is similar to that of the WT-PNIPAAm conjugate for the biotinylated IgG target molecule (near 100%), but significantly smaller for the biotinylated oligonucleotide target (approximately 60% for the S45A-PNIPAAm conjugate compared to 80% for the WT-PNIPAAm conjugate). The release efficiency (fraction of originally precipitated target molecule released after treatment with free biotin) of the S45A-PNIPAAm conjugate is 70-80% for the biotinylated IgG target and nears 100% for the biotinylated oligonucleotide target. This system demonstrates the use of a high off-rate streptavidin mutant to add reversibility to a system based on smart-polymer-streptavidin conjugates.     

Purification of CD47‐streptavidin fusion protein from bacterial lysate using biotin–agarose affinity chromatography

CD47 is a widely expressed transmembrane glycoprotein that modulates the activity of a plethora of immune cells via its extracellular domain. Therefore, CD47 plays important roles in the regulation of immune responses and may serve as targets for the development of immunotherapeutic agents. To make sure CD47 functionality is intact under the process of protein conjugation, CD47‐streptavidin fusion protein was expressed and purified because it can easily bind to biotin‐tagged materials via the unique biotin–streptavidin affinity. In this study, gene sequences of CD47 extracellular domain (CD47ECD) and core streptavidin (coreSA) with a total 834 bp were inserted into pET20b plasmid to construct recombinant plasmid encoding CD47‐SA fusion gene. After bacteria transformation, the CD47‐SA fusion protein was expressed by isopropyl‐β‐d ‐thiogalactopyranoside (IPTG) induction. The collected bacteria lysate was loaded on biotinylated agarose to proceed the purification of CD47‐SA fusion protein. Due to the unexpected high affinity between biotin and coreSA, standard washing and elution approaches (e.g., varying pH, using biotin, and applying guanidine hydrochloride) reported for biotin–streptavidin affinity chromatography were not able to separate the target fusion protein. Instead, using low concentration of the non‐ionic detergent Triton X‐100 followed with alkaline buffer could efficiently weaken the binding between biotin and coreSA, thereby eluting out CD47‐SA fusion protein from the biotin agarose column. The purified CD47‐SA fusion protein was further characterized by molecular biology methods and its antiphagocytic functionality was confirmed by the phagocytosis assay. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:949–958, 2016