Streptavidin Conjugates Research Articles

Controlling the biochemical environment of extracellular matrix mimics over time to study and influence cell-matrix interactions for applications in regenerative medicine

Event Abstract Back to Event Controlling the biochemical environment of extracellular matrix mimics over time to study and influence cell-matrix interactions for applications in regenerative medicine Catherine Lambert1, Vincent Huynh1, Devang Nijsure1 and Ryan Wylie1 1 McMaster University, Department of Chemistry and Chemical Biology, Canada Introduction: Understanding the influence of the chemical environment within the extracellular matrix (ECM) on cellular activities is crucial to the fields of cancer and stem cell biology. ECM mimics are necessary to elucidate the role of cell-ECM interactions, which will lead to new biomaterials for regenerative therapies. The chemical composition of the ECM is spatially defined and dynamic, which are important regulators of cell fate[1]-[3]. In an effort to understand the dynamic features of the ECM that control cellular processes, we have developed ECM mimics from hydrogels where the biochemical environment can be temporally controlled. We are now expanding the system for the simultaneous spatial and temporal patterning of biomolecules within hydrogels using photochemistry. To achieve temporal control of the biochemical environment, peptides were immobilized in hydrogels through high affinity reversible physical interactions. Peptide-streptavidin conjugates were immobilized in agarose-desthiobiotin (AgD) hydrogels through the desthiobiotin-streptavidin interaction (KD 10-11M), and displaced by adding biotin, which binds streptavidin with greater affinity than desthiobiotin (KD 10-15M)[4]. This cycle was repeated to vary the chemical environment over time (Fig. 1). Photocaged peptides, which become bioactive upon irradiation, are now being immobilized using the desthiobiotin-streptavidin system to yield spatial control. Material and Methods: Synthesis of materials. AgD was synthesized by reacting amino-agarose with NHS-desthiobiotin. Peptide-streptavidin conjugates were synthesized by reacting maleimide-streptavidin with cysteine containing peptides such as CGRGDS. Peptide-streptavidin conjugates were fluorescently labelled with NHS-Alexa Fluor dyes. Reversible biomolecule immobilization. Immobilization experiments were performed by adding solutions of fluorescent streptavidin (with or without conjugated RGD) to 0.7 wt.% AgD gels. Biotin was added to unbind streptavidin. After a washing step to remove free biotin, streptavidin conjugates were re-immobilized within the same gels. Experiment were performed in PBS or media with 0.5 % BSA or 1 % calf bovine serum. Results and Discussion: Sequential immobilization of the adhesive RGD peptide in AgD hydrogels was achieved by varying media components. To demonstrate temporal control, we bound and unbound 2 different fluorescent molecules: 1) streptavidin modified with CGRGDS and Alexa Fluor 488 (AF488-SAv-RGD); and, 2) streptavidin modified with Alexa Fluor 647 (AF647-SAv) as a surrogate for a second biomolecule (Fig. 2). AF488-SAv-RGD was added at t=0h, which resulted in biochemically stable hydrogel (stable for over 20days). Biotin was introduced at t=47h (dashed line in Fig. 2) to remove AF488-SAv-RGD, as seen from the loss of hydrogel fluorescence. Simultaneous immobilization of 2 different molecules was achieved by adding a solution of AF488-SAv-RGD and AF647-SAv at t=170h. Unbinding was again achieved at 213h through the addition of biotin. Control experiments were conducted with agarose (Ag) hydrogels to quantify non-specific binding. This data demonstrates that it is possible to temporally control the biochemical environment of hydrogels using strong reversible physical interactions. It was also demonstrated that streptavidin-RGD is bioactive by performing fibroblast adhesions assays. Conclusion: We have developed a system to easily tune the biochemical environment of ECM mimics over time. We are currently expanding the system to control the biochemical environment in space and time through the use of photocaged peptides. This system will yield tailored biochemical environment to study and control cell-matrix interactions for applications in regenerative medicine. Furthermore, the method will be useful for many studies involving cell-matrix interactions due to its simplicity. This work was supported by NSERC USRA (C.L.), NSERC Discovery Grant and McMaster University.

Lectin-based protein microarray analysis of differences in serum alpha-2-macroglobulin glycosylation between patients with colorectal cancer and persons without cancer.

Glycosylation is co- and posttranslational modifications affecting proteins. The glycopattern changes are associated with changes in biological function and are involved in many diseases including cancer. We present the lectin-based protein microarray method enabling determination of differences in protein glycosylation. The method involves isolation of targeted protein from samples by immunoprecipitation, spotting of protein from multiple samples into arrays on a microarray slide, incubation with set of biotinylated lectins, the reaction with fluorescent conjugate of streptavidin, and detection of fluorescent intensities by microarray scanner. Lectin-based protein microarray was applied in investigation of differences in alpha-2-macroglobulin (α2M) glycosylation isolated from sera samples of healthy persons and patients with colorectal cancer (CC). From 14 lectins used in analysis, statistically significant differences (Student's t-test, P < 0.05) between two groups of samples (persons without cancer and CC patients) were found for 5 of them. α2M molecules isolated from sera of CC patients have higher content of α2,6 sialic acid, N-acetylglucosamine and mannose residues, and tri-/tetraantennary complex type high-mannose N-glycans. A novel lectin-based protein microarray developed and described can serve as a suitable analytical technique for sensitive, simple, fast, and high-throughput determination of differences in protein glycosylation isolated from serum or other samples.

Detection of carcinoembryonic antigen using single-domain or full-size antibodies stained with quantum dot conjugates

Compact single-domain antibodies (sdAbs) are nearly 13 times smaller than full-size monoclonal antibodies (mAbs) and have a number of advantages for biotechnological applications, such as small size, high specificity, solubility, stability, and great refolding capacity. Carcinoembryonic antigen (CEA) is a tumor-associated glycoprotein expressed in a variety of cancers. Detection of CEA on the tumor cell surface may be carried out using anti-CEA antibodies and conventional fluorescent dyes. Semiconductor quantum dots (QDs) are brighter and more photostable than organic dyes; they provide the possibility for labeling of different recognition molecules with QDs of different colors but excitable with the same wavelength of excitation. In this study, the abilities for specific detection of CEA expressed by tumor cells with anti-CEA sdAbs biotinylated in vitro and in vivo, as well as with anti-CEA mAbs biotinylated in vitro, were compared using flow cytometry and the conjugates of streptavidin with QDs (SA-QDs). The results demonstrated that either in vitro or in vivo biotinylated anti-CEA sdAbs are more sensitive for cell staining compared to biotinylated anti-CEA mAbs. The data also show that simultaneous use of biotinylated sdAbs with highly fluorescent SA-QDs can considerably improve the sensitivity of detection of CEA on tumor cell surfaces.

Markers for Immunofluorescence Analysis Based on Europium Complex with Fluorinated β-Diketones Carbazole Series

In order to develop new markers for immunofluorescence time-resolved luminescence analysis tetraketodiester carbazole series was obtained. Compound contains methoxycarbonyl reactive groups, separated from the 1,3-dicarbonyl chelating fragment by difluoromethylene spacers (CF2)4. Complexes of this compound with the ions Eu3+ are stable in aqueous solution and possess a long-living intensive luminescence with the main peaks: emission--in the area of 615 nm and excitation--in the 380-390 nm, that distinguishes them from most used analogues. Conjugate of streptavidin to compound was obtained and its fluorescent spectral characteristics allow to use it as a universal reagent for various schemes of biological microanalysis.

Functional gold nanoparticle coated surfaces for CA 125 cancer biomarker detection

Here we describe the detailed characterization of gold nanoparticle (Au Np) functionalized surfaces as a biosensing platform by studying a model streptavidin (SA)-biotin interaction. Conjugation of SA on Au Np immobilized on silicon (Si) and quartz surfaces and its interaction with biotin were characterized by X-ray photoelectron spectroscopy (XPS), UV-Vis spectroscopy, circular dichromism (CD) spectroscopy, and contact angle measurements. The immobilization method and atomic concentrations of Si 2p, Au 4f, S 2p, C 1s, N 1s, and O 1s of the resulting SA-biotin modified Si surface were determined by XPS. The CD spectrum and confocal microscopy imaging confirmed that step-by-step modification and bioconjugation can be monitored successfully. Such detailed and well-defined step-by-step characterization provides good information about the surface properties of biosensor platforms. In addition, the LSPR sensing ability of the Au Np based platforms was studied by using a model SA-biotin system. A 20 nm spectral red shift was detected when 150 nM SA was immobilized on to the Au Nps surface using the direct incubation/binding method on to the dry surface instead of the flow-injection method. The same platforms were also used to detect the CA 125 antibody-antigen system. Large spectral red shifts are very promising in terms of using these surfaces as LSPR biosensors.

A magnetic relaxation switching immunosensor for one-step detection of salbutamol based on gold nanoparticle–streptavidin conjugate

A magnetic relaxation switching immunosensor based on gold nanoparticles–streptavidin conjugate for the detection of salbutamol.

Quantitative PCR analysis of DNA aptamer pharmacokinetics in mice.

DNA aptamer oligonucleotides and their protein conjugates show promise as therapeutics in animal models of diseases such as multiple sclerosis. These molecules are large and highly charged, raising questions about their biodistribution and pharmacokinetics in mammals. Here we exploit the power of quantitative polymerase chain reaction to accurately quantitate the tissue distribution of 40-nucleotide DNA aptamers and their streptavidin conjugates after intraperitoneal injection in mice. We show remarkably rapid distribution to peripheral tissues including the central nervous system. Modeling of tissue distribution data reveals the importance of DNA aptamer sequence, 3′ modification, and protein conjugation in enhancing tissue exposure. These data help to interpret the previously observed effectiveness of aptamer conjugates, as opposed to free aptamers, in stimulating central nervous system remyelination in a mouse model of multiple sclerosis.

A novel paper-based microfluidic enhanced chemiluminescence biosensor for facile, reliable and highly-sensitive gene detection of Listeria monocytogenes

This paper describes the development of a paper-based microfluidic device that detects long DNA amplicons on the basis of hybridization reactions with a covalently immobilized DNA probe and biotin-labeled signal DNA strands, and chemiluminescent (CL) reactions catalyzed by a horseradish peroxidase (HRP)-streptavidin conjugate. The paper-based microfluidic CL biosensor is developed for detection of 198-bp DNA fragments obtained by PCR amplification of the Listeria monocytogenes hlyA gene, where the CL intensity is detected by a simple, inexpensive CCD. To our knowledge, the combined use of the CCD sensing and paper-based microfluidic CL detection is first proposed here. The paper-based device includes wax-screen printed channels, and further explores origami-type folds to create an easy-to-operate valve that makes it possible to successively wash the detection zone in-between hybridization steps. Compared with other paper-based wax-screen-printing methods, the proposed method is faster and needs lower heating temperature. In our DNA biosensor, CL signals generated using a HRP-luminol-H2O2 system are heightened with p-iodine phenol (PIP) and detected with a CCD system. Under optical conditions, a linear range of 1.94×10−1pmol/L to 1.94×104pmol/L is achieved and the limit of detection is found to be 6.3×10−2pmol/L. And, this detection limit is 3–5 orders of magnitude lower than those obtained from other CCD-based microfluidic CL DNA assays. The results show that the proposed integration of paper-based microfluidics, CL method and CCD sensing offers great promises in providing a highly-sensitive, reliable and cost-effective solution for gene biosensing applications.

Multiplexed modular genetic targeting of quantum dots.

While DNA-directed nanotechnology is now a well-established platform for bioinspired nanoscale assembly in vitro, the direct targeting of various nanomaterials in living biological systems remains a significant challenge. Hybrid biological systems with integrated and targeted nanomaterials may have interesting and exploitable properties, so methods for targeting various nanomaterials to precise biological locations are required. Fluorescence imaging has benefited from the use of nanoparticles with superior optical properties compared to fluorescent organic dyes or fluorescent proteins. While single-particle tracking (SPT) in living cells with genetically encoded proteins is limited to very short trajectories, the high photon output of genetically targeted and multiplexed quantum dots (QDs) would enable long-trajectory analysis of multiple proteins. However, challenges with genetic targeting of QDs limit their application in these experiments. In this report, we establish a modular method for targeting QD nanoparticles selectively to multiple genetically encoded tags by precomplexing QD–streptavidin conjugates with cognate biotinylated hapten molecules. This approach enables labeling and SPT of multiple genetically encoded proteins on living cells at high speed and can label expressed proteins in the cytosol upon microinjection into living cells. While we demonstrate labeling with three distinct QD conjugates, the approach can be extended to other specific hapten–affinity molecule interactions and alternative nanoparticles, enabling precise directed targeting of nanoparticles in living biological systems.

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.

Identification of protein phosphatase interacting proteins from normal and UVA-irradiated HaCaT cell lysates by surface plasmon resonance based binding technique using biotin–microcystin-LR as phosphatase capturing molecule

Identification of the interacting proteins of protein phosphatases is crucial to understand the cellular roles of these enzymes. Microcystin-LR (MC-LR), a potent inhibitor of protein phosphatase-1 (PP1), -2A (PP2A), PP4, PP5 and PP6, was biotinylated, immobilized to streptavidin-coupled sensorchip surface and used in surface plasmon resonance (SPR) based binding experiments to isolate phosphatase binding proteins. Biotin–MC-LR captured PP1 catalytic subunit (PP1c) stably and the biotin–MC-LR-PP1c complex was able to further interact with the regulatory subunit (MYPT1) of myosin phosphatase. Increased biotin–MC-LR coated sensorchip surface in the Surface Prep unit of Biacore 3000 captured PP1c, PP2Ac and their regulatory proteins including MYPT1, MYPT family TIMAP, inhibitor-2 as well as PP2A-A and -Bα-subunits from normal and UVA-irradiated HaCaT cell lysates as revealed by dot blot analysis of the recovered proteins. Biotin–MC-LR was used for the subcellular localization of protein phosphatases in HaCaT cells by identification of phosphatase-bound biotin–MC-LR with fluorescent streptavidin conjugates. Partial colocalization of the biotin–MC-LR signals with those obtained using anti-PP1c and anti-PP2Ac antibodies was apparent as judged by confocal microscopy. Our results imply that biotin–MC-LR is a suitable capture molecule in SPR for isolation of protein phosphatase interacting proteins from cell lysates in sufficient amounts for immunological detection.

Streptavidin conjugates with gold nanoparticles for visualization of single DNA interactions on the silicon surface

Applicability of scanning electron microscopy (SEM) for visualization of individual acts of DNA hybridization with oligonucleotide probes has been investigated using gold nanoparticles as a label. DNA or oligonucleotides were labeled with biotin molecules, which were then detected in DNA duplexes using a streptavidin conjugate with gold nanoparticles. Effective imaging of DNA duplexes was possible using the conjugate prepared by covalent binding. The detection limit of the model oligonucleotide of 19 bases was 20 pg.

Pre-hybridisation: An efficient way of suppressing endogenous biotin-binding activity inherent to biotin–streptavidin detection system

Endogenous biotin or biotinylated protein binding activity is a major drawback to biotin–avidin/streptavidin detection system. The avidin/streptavidin conjugate used to detect the complex of the biotinylated secondary antibody and the primary antibody binds to endogenous biotin or biotinylated proteins leading to non-specific signals. In Western blot, the endogenous biotin or biotinylated protein binding activity is usually manifested in the form of ~72kDa, ~75kDa and ~150kDa protein bands, which often mask the signals of interest. To overcome this problem, a method based on prior hybridisation of the biotinylated secondary antibody and the streptavidin conjugate was developed. The method was tested alongside the conventional biotin–streptavidin method on proteins extracted from zebrafish (Danio rerio) embryos. Results showed that the newly developed method efficiently suppresses the endogenous biotin or biotinylated protein binding activity inherent to the biotin–streptavidin detection system.

Conjugates of streptavidin with gold nanoparticles for the visualization of dna single interactions on the silicon surface

The potential of the method of scanning electron microscopy (SEM) to visualize the results of individual acts of DNA and oligonucleotides hybridization using gold nanoparticles as label was investigated. Molecule of biotin was introduced into DNA or oligonucleotide, and then it was detected in DNA duplex using a conjugate of streptavidin with gold nanoparticles. Effective imaging of DNA duplexes was possible using a conjugate prepared by covalent binding.. The detection limit of the model oligonucleotide of 19 bases was 20 pg.

Immunohistochemical staining of influenza virus in tissues.

Immunohistochemical methods are commonly used for studying the pathogenesis of influenza A virus by allowing the identification of sites of replication of the virus in infected tissues and the correlation with the histopathological changes observed. In this chapter, the materials and methods for performing immunohistochemical detection of influenza virus antigens in tissues are provided. The technique involves the following steps: heat-induced antigen retrieval; binding of a primary antibody to the virus antigen; antibody-antigen complex binding by a biotinylated secondary antibody; and binding of an enzyme-streptavidin conjugate. The enzyme is then visualized by application of the substrate chromogen solution to produce a colorimetric end product. Visualization of influenza virus antigen in tissues is based on chromogen deposition in the nucleus and cytoplasm of infected cells.

Signal Amplification via Biological Self-Assembly of Surface-Engineered Quantum Dots for Multiplexed Subattomolar Immunoassays and Apoptosis Imaging

The parallel and highly sensitive detection of biomolecules is of paramount importance to understand biological functions at the single cell level and for various medical diagnoses. Surface-engineered semiconductor quantum dots (QDs) have been demonstrated to act as a signal amplifiable reporter in immunoassays. This takes advantage of the QDs' robustness against self-quenching in proximity and the tunability of their surface properties. A streptavidin (SA) and biotin QD conjugate pair containing a zwitterionic surface modification was designed for QD self-assembly with minimal nonspecific adsorption. Typical sandwich-type immunoassay procedures were adopted, and the targeted protein binding events were effectively transduced and amplified by the fluorescence of the SA-biotin QD conjugates. The detection limit of myoglobin in 100% serum was determined to be at the subattomolar (tens of copies per milliliter) level, which was achieved by using 100 cycles of the layer-by-layer QD assembly. Adsorption kinetics studies and Monte Carlo simulations revealed that this highly sensitive signal amplification was accomplished by the zwitterionic surface, which gave equilibrium constants 5 orders of magnitude larger for specific binding than for nonspecific binding. The QD conjugates showed an effective multivalency of two, which resulted in a broad linear dynamic range spanning 9 orders of magnitude of target protein concentrations. The assay can be highly miniaturized and multiplexed, and as a proof-of-concept, parallel and rapid detection of four different cancer markers has been successfully demonstrated. To demonstrate that this QD signal amplification can be a universal platform, sensitive imaging and early detection of apoptotic cells were also showcased.

Ultrasensitive Detection and Quantification of Acidic Disaccharides Using Capillary Electrophoresis and Quantum Dot-Based Fluorescence Resonance Energy Transfer

Rapid and highly sensitive detection of the carbohydrate components of glycoconjugates is critical for advancing glycobiology. Fluorescence (or Förster) resonance energy transfer (FRET) is commonly used in detection of DNA, in protein structural biology, and in protease assays but is less frequently applied to glycan analysis due to difficulties in inserting two fluorescent tags into small glycan structures. We report an ultrasensitive method for the detection and quantification of a chondroitin sulfate disaccharide based on FRET, involving a CdSe-ZnS core-shell nanocrystal quantum dot (QD) streptavidin conjugate donor and a Cy5 acceptor. The disaccharide was doubly labeled with biotin and Cy5. QDs then served to concentrate the target disaccharide, enhancing the overall energy transfer efficiency, with unlinked QDs and Cy5 hydrazide producing nearly zero background signal in capillary electrophoresis using laser-induced fluorescence detection with two different band-pass filters. This method is generally applicable to the ultrasensitive analysis of acidic glycans and offers promise for the high-throughput disaccharide analysis of glycosaminoglycans.

Functionalization of high-moment magnetic nanodisks for cell manipulation and separation

Synthetic antiferromagnetic (SAF) nanoparticles are layer-structured particles with high single-particle magnetic moments. In order to covalently bind these nanoparticles to cells, they were coated with a silica shell followed by conjugation with streptavidin. The silica coating generates both SAF@SiO2 core-shell nanoparticles and silica core-free nanoparticles. Using a simple magnetic separation, silica nanoparticles were removed and SAF@SiO2 nanoparticles were purified. After streptavidin conjugation, these particles were used to stain lung cancer cells, making them highly magnetically responsive. The stained cells can rotate in response to an external magnetic field and can be captured when a blood sample containing these cells flows through the sifter. Open image in new window

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The use of Streptavidin Conjugates as Immunoblot Loading Controls and Mitochondrial Markers for use with Dictyostelium Discoideum

The loading controls used for quantitative immunoblotting of mammalian proteins are not appropriate for use with Dictyostelium discoideum. Actin levels, for example, change greatly during Dictyostelium development. In addition, Dictyostelium-specific antibodies for other potential control proteins are not commercially available. Here we demonstrate the use of labeled streptavidin to detect biotinylated mitochondrial 3-methylcrotonyl-CoA carboxylase α (MCCC1), providing a robust and convenient tool for quantitative normalization of Dictyostelium Western blots, as well as fluorescently labeling mitochondria for microscopy of fixed cells.