Concentration Of Streptavidin Research Articles

An ultrasensitive sensor based on quantitatively modified upconversion particles for trace bisphenol A detection.

Bisphenol A (BPA) is one of the endocrine-disrupting chemicals which might cause reproductive and endocrine system diseases, and poses a serious threat to the ecosystem and human health. This paper reports an ultrasensitive sensor for trace BPA detection employing fluorescence resonance energy transfer (FRET) between modified upconversion nanoparticles (UCNPs) and tetramethylrhodamine. To circumvent the problems of low luminous efficiency of FRET and low sensitivity of sensor, the upconversion nanoparticles with very strong fluorescence efficiency were prepared and quantitatively modified. Results showed that the concentrations of amino groups and streptavidin were 43nmol/mg and 6.12μg/mg on the surface of the UCNPs, respectively. Under the optimal detection conditions, the peak intensity of UCNPs at 547nm was linear with the logarithm of the BPA concentration with the detection limit of 0.05ng/mL. Without complicated pre-processing, the recoveries were in general between 91.0 and 115.0% in tap water, river water, and disposable paper cup water. Therefore, the proposed sensor is suitable for effective sensing of trace BPA in water samples. Graphical abstract ᅟ.

Large-Area Silver-Stibnite Nanoporous Plasmonic Films for Label-Free Biosensing.

The development of various plasmonic nanoporous materials has attracted much interest in different areas of research including bioengineering and biosensing because of their large surface area and versatile porous structure. Here, we introduce a novel technique for fabricating silver-stibnite nanoporous plasmonic films. Unlike conventional techniques that are usually used to fabricate nanoporous plasmonic films, we use a room-temperature growth method that is wet-chemistry free, which enables wafer-scale fabrication of nanoporous films on flexible substrates. We show the existence of propagating surface plasmon polaritons in nanoporous films and demonstrate the extreme bulk refractive index sensitivity of the films using the Goos-Hänchen shift interrogation scheme. In the proof-of-concept biosensing experiments, we functionalize the nanoporous films with biotin-thiol using a modified functionalization technique, to capture streptavidin. The fractal nature of the films increases the overlap between the local field and the immobilized biomolecules. The extreme sensitivity of the Goos-Hänchen shift allows femtomolar concentrations of streptavidin to be detected in real time, which is unprecedented using surface plasmons excited via the Kretschmann configuration.

Highly sensitive label-free antibody detection using a long period fibre grating sensor

An optical fibre long period grating (LPG) biosensor is appealing in the detection of biomolecules because of the high sensitivity, label-free and real-time measurement. The miniaturized size, ability of remote sensing and immunity to electromagnetic interference of the LPG biosensor provide various possibility of single-point sensing in situations such as point of care diagnostics and in vivo measurement. Two optical fibre LPG based biosensors are reported for detection of streptavidin (SV) and immunoglobulin M (IgM) respectively. The LPG is coated with a film containing three layers of Poly(allylamine hydrochloride)/gold coated silica nanoparticles via the layer-by-layer method. Biotin is covalently bonded to the surface of the gold shell by means of the formation of an amide bonds for detection of streptavidin. The concentration of SV in water for detection varied from 1.25 nM to 2.7 μM. The LPG sensor, operating close to the phase matching condition shows a high sensitivity of 3.88 (ng/mm2)−1 and a detection limit of 0.86 pg/mm2 for the detection of SV. The limit of detection is 22 times lower than previously demonstrated with this type of sensor. The developed IgM sensor has the same configuration of film but has anti-IgM embedded on the LPG instead of biotin, demonstrating versatility of the sensing platform. This was used for the detection of human IgM with concentrations from 15.6 μg/ml to 1 mg/ml. The LPG sensor exhibits a sensitivity of 11 nm (ng/mm2)−1 for the detection of IgM with a detection limit of 15 pg/mm2. The developed highly sensitive IgM sensor shows the potential application of clinical point of care for detection of lower concentration of IgM in vitro. The proposed biosensor exhibits high sensitivity and rapid detection of low concentrations biomolecules from the small size of SV to the large size of IgM.

Engineered Phage Matrix Stiffness-Modulating Osteogenic Differentiation.

Herein, we demonstrate an engineered phage mediated matrix for osteogenic differentiation with controlled stiffness by cross-linking the engineered phage displaying Arg-Gly-Asp (RGD) and His-Pro-Gln (HPQ) with various concentrations of streptavidin or polymer, poly(diallyldimethylammonium)chloride (PDDA). Osteogenic gene expressions showed that they were specifically increased when MC3T3 cells were cultured on the stiffer phage matrix than the softer one. Our phage matrixes can be easily functionalized using chemical/genetic engineering and used as a stem cell tissue matrix stiffness platform for modulating differential cell expansion and differentiation.

A cationic conjugated polymer coupled with exonuclease I: application to the fluorometric determination of protein and cell imaging.

A strategy is described for the detection of protein by using a cationic fluorescent conjugated polymer coupled with exonuclease I (Exo I). Taking streptavidin (SA) as model protein, it is observed that Exo I can digest single-stranded DNA conjugated with biotin and carboxyfluorescein (P1) if SA is absent. This leads to the formation of small nucleotide fragments and to weak fluorescence resonance energy transfer (FRET) from the polymer to P1. If, however, SA is present, the high affinity of SA and biotin prevents the digestion of P1 by Exo I. This results in the sorption of P1 on the surface of the polymer through strong electrostatic interaction. Hence, efficient FRET occurs from the fluorescent polymer to the fluorescent label of P1. Fluorescence is measured at an excitation wavelength of 370nm, and emission is measured at two wavelengths (530 and 425nm). The ratio of the two intensities (I530/I425) is directly related to the concentration of SA. Under the optimal conditions, the assay has a detection limit of 1.3ng·mL-1. The method was also applied to image the folate receptor in HeLa cells, thus demonstrating the versatility of this strategy. Graphical abstract A fluorometric strategy is described for protein detection and cell imaging based on a cationic conjugated polymer (PFP) coupled with exonuclease I (Exo I) trigged fluorescence resonance energy transfer (FRET).

Highly sensitive electrochemical biosensor for streptavidin detection based on CdSe quantum dots

An electrochemical biosensor was developed based on a steric hindrance hybridization assay to allow the highly sensitive detection of streptavidin. In the steric hindrance hybridization assay, the signaling strand DNA (sig-DNA) was labeled at the 3′ end with CdSe quantum dots (QDs) and at the 5′ end with biotin, and capturing strand DNA (the complementary strand of sig-DNA) was labeled at the 5′ end with thiol. The steric hindrance effect generated by streptavidin which was bound with the signaling DNA strand. The streptavidin limited the ability of the sig-DNA to hybridize with the cap-DNA, which were linked on the surface of a gold electrode. Therefore, the concentration of streptavidin was detected indirectly based on the concentration of CdSe QDs on the electrode surface. The concentration of CdSe QDs on the electrode surface was detected by differential pulse anodic stripping voltammetry. Under optimal conditions, the streptavidin detection range using the as-prepared biosensor was 1.96pg/mL to 1.96µg/mL and the detection limit was 0.65pg/mL. The experimental results showed that the electrochemical biosensor could detect streptavidin rapidly and accurately.

A Highly Sensitive Real-time Immuno-PCR Assay for Detecting Benzo[a]pyrene in Food Samples by Application of Biotin-Streptavidin System

Benzo[a]pyrene (BaP), a kind of polycyclic aromatic hydrocarbon, has great potential hazards to the ecosystem and human health. In this study, based on the preparation of BaP immunogen and coating antigen, a specific polyclonal antibody targeting BaP was generated, and a highly sensitive real-time immuno-PCR assay using the biotin-streptavidin system (BA-IPCR) was developed for detecting BaP in vegetables and barbecue food. Several detection conditions were optimized, such as blocking reagent, the concentration of streptavidin, and the concentration of biotinylated DNA (bio-DNA). Under the optimal conditions, a calibration curve was constructed in the concentration range of 5 pg L−1 to 10 ng L−1. The limit of detection was 2.85 pg L−1. Recovery of the spiked samples ranged from 92.1 to 109.6%. This immunoassay showed high specificity, with low cross-reactivity values with BaP analogues. The consistency between the results obtained from BA-IPCR and gas chromatography and mass spectrometer (GC-MS) was 98.96%. This proposed method is reliable and has great potential to detect trace BaP in environmental and food samples.

Homogenous assay for protein detection based on proximity DNA hybridization and isothermal circular strand displacement amplification reaction.

This work proposed a homogenous fluorescence assay for proteins, based on the target-triggered proximity DNA hybridization in combination with strand displacement amplification (SDA). It benefited from target-triggered proximity DNA hybridization to specifically recognizethe target and SDA making recycling signal amplification. The system included a molecular beacon (MB), an extended probe (EP), and an assistant probe (AP), which were not self-assembly in the absence of target proteins, due to the short length of the designed complementary sequence among MB, EP, and AP. Upon addition of the target proteins, EP and AP are bound to the target proteins, which induced the occurrence of proximity hybridization between MB, EP, and AP and followed by strand displacement amplification. Through the primer extension, a tripartite complex of probes and target was displaced and recycled to hybridize with another MB, and the more opened MB enabled the detection signal to amplify. Under optimum conditions, it was used for the detection of streptavidin and thrombin. Fluorescence intensity was proportional to the concentration of streptavidin and thrombin in the range of 0.2-30 and 0.2-35nmol/L, respectively. Furthermore, this fluorescent method has a good selectivity, in which the fluorescence intensity of thrombin was ~37-fold or even larger than that of the other proteins at the same concentration. It is a new and simple method for SDA-involved target protein detection and possesses a great potential for other protein detection in the future. Graphical abstract A homogenous assay for protein detection is based on proximity DNA hybridization and strand displacement amplification reaction.

Highly sensitive biosensors based on all-dielectric nanoresonators.

Biosensing based on nanophotonic structures has shown a great potential for cost-efficient, high-speed and compact personal medical diagnostics. While plasmonic nanosensors offer high sensitivity, their intrinsically restricted resonance quality factors and strong heating due to metal absorption impose severe limitations on real life applications. Here, we demonstrate an all-dielectric sensing platform based on silicon nanodisks with strong optically-induced magnetic resonances, which are able to detect a concentration of streptavidin of as low as 10-10 M (mol L-1) or 5 ng mL-1, thus pushing the current detection limit by at least two orders of magnitudes. Our study suggests a new direction in biosensing based on bio-compatible, non-toxic, robust and low-loss dielectric nanoresonators with potential applications in medicine, including disease diagnosis and drug detection.

Effects of particle addition to Streptomyces cultivations to optimize the production of actinorhodin and streptavidin

Streptomyces is a genus of Gram-positive bacteria that grow in various environments, and its shape resembles filamentous fungi. The most interesting property of Streptomyces is the ability to produce bioactive secondary metabolites, such as antifungals, antivirals, antitumorals, and especially antibiotics. In submersed culture Streptomyces and other filamentous microorganisms can grow as free mycelium, pellets or intermediate growth forms. All growth forms can be beneficial for product formation depending on the microbial strain and the product of interest. Here, the effects of addition of particles on the growth and product formation of two Streptomyces strains were investigated. Streptomyces coelicolor was used as a model organism for the production of the antibiotic actinorhodin and Streptomyces avidinii was used to produce streptavidin. In all experiments, the addition of broken and porous SiO2 particles (120–200µm) to the culture medium led to an increased productivity. In order to evaluate the particle effect on actinorhodin production three different media with and without the addition of particles were compared. The productivity was strongly influenced by the choice of media and was further improved by the addition of particles. The enhancement factor between un-supplemented and supplemented cultures varied between 85% by using a complex medium and 160% in a minimal medium. The highest product concentration of 855mg actinorhodin L−1 was measured after 12 days in a complex medium by addition of 5g glass particles L−1. In contrast, the final concentration of streptavidin was not affected by the supplementation of glass beads (diameter: 0.25–0.5mm) to cultures of Streptomyces avidinii. Nevertheless, a kinetic effect occurred upon supplementation by particles, allowing an acceleration of product formation. The impact of the particle addition to the Streptomyces cultures on the sustainability of the bioprocess was proven by using the E-factor. The E-factor denotes the amount of waste generated per product equivalent. This evaluation shows the beneficial effects of the particle addition in both production systems. Due to the fact that the particle addition leads to a decreased E-factor a life cycle assessment should be performed to further evaluate the particle effect on the sustainability of the complete process chain.

Time-resolved homo-FRET studies of biotin-streptavidin complexes

Förster resonance energy transfer is a mechanism of fluorescence quenching that is notably useful for characterizing properties of biomolecules and/or their interactions. Here we study water-solutions of Biotin-Streptavidin complexes, in which Biotin is labeled with a rigidly-bound fluorophore that can interact by Förster resonance energy transfer with the fluorophores labeling the other, up to three, Biotins of the same complex. The fluorophore, Atto550, is a Rhodamine analogue. We detect the time-resolved fluorescence decay of the fluorophores with an apparatus endowed with single-photon sensitivity and temporal resolution of ~30ps. The decay profiles we observe for samples containing constant Biotin-Atto550 conjugates and varying Streptavidin concentrations are multi-exponential. Each decay component can be associated with the rate of quenching exerted on each donor by each of the acceptors that label the other Biotin molecules, depending on the binding site they occupy. The main features that lead to this result are that (i) the transition dipole moments of the up-to-four Atto550 fluorophores that label the complexes are fixed as to both relative positions and mutual orientations; (ii) the fluorophores are identical and the role of donor in each Biotin-Streptavidin complex is randomly attributed to the one that has absorbed the excitation light (homo-FRET). Obviously the high-temporal resolution of the excitation-detection apparatus is necessary to discriminate among the fluorescence decay components.

Visual microarray detection for human IgE based on silver nanoparticles

A rapid, visual protein detection method of human immunoglobulin E (hIgE) was developed for 96-well microplate arrays by using aptamer modifying silver nanoparticles. An aptamer specifically recognizing IgE and biotinylated oligonucleotides were used to modify AgNPs and the resulting nanoprobes were employed to assay model analyte hIgE. When addition of the analyte as well as the probes to the microplate array on which Goat anti-human IgE was first immobilized, the signal of this “sandwich” format was further amplified by silver enhancement technique based on silver ions reductive reagents and achieved a visual dots in the microplates. To achieve quantitative analysis, a series of gradient concentration streptavidin (SA) was immobilized in the same well. As a result, an internal standard curve was plotted by the signal of different concentration of SA based on streptavidin-biotin coupling reaction. With the help of this curve, a quantitative detection of IgE was established and a detection limit of 20ngmL−1 could be obtained. The presented method was further carried out for detection of human serum samples and relative consistent results were found.

A simple and efficient design to improve the detection of biotin-streptavidin interaction with plasmonic nanobiosensors

In this manuscript we propose a simple and efficient strategy to improve the sensitivity of localized surface plasmon resonance (LSPR) shift-based biosensors using biotin-streptavidin recognition interaction as a proof-of-concept. Specifically, biotin molecules are immobilized on a low-cost plasmonic LSPR biosensor based on annealed self-assembled spherical gold nanoparticles (AuNSs) and successively incubated with increasing concentrations of streptavidin, achieving a limit of detection (LOD) of 5nM. Interestingly, when the detection is performed by the same biotin-functionalized plasmonic AuNSs substrate but against streptavidin previously conjugated to gold nanorods, the LSPR shift is 26-fold enhanced. Moreover, we confirm these results through numerical simulations and demonstrate that the proposed sensing architecture can operate as transducer not only to confirm the adsorption of bioanalyte but also to provide the chemical identity of the capture and targeted molecules from their vibrational Raman fingerprints. Therefore, we are confident that the development of such plasmonic biosensors that use metallic labels for improving the sensitivity of detection could become highly promising for future point-of-care diagnostic assays, pushing sensitivity towards single-molecule detection limit.

Fed-batch production and secretion of streptavidin by Hansenula polymorpha: Evaluation of genetic factors and bioprocess development

Streptavidin – a protein secreted by the filamentous bacterium Streptomyces avidinii – is applied in a variety of methods, leading to numerous studies on its heterologous production. Development and characterization of a novel expression system for streptavidin genes by Hansenula polymorpha is described utilizing different target gene variants along with the two methanol-inducible promoters PMOX and PFMD. Extracellular product concentrations were higher for cultivation at 30 instead of 37°C. The best performing strain carrying the full-length streptavidin gene under control of PFMD was characterized in the bioreactor applying a synthetic medium and oxygen-controlled feeding of glucose. Derepression resulted in an extracellular concentration of 1.31±0.07μM of tetrameric streptavidin after 48h (27.3nMh−1). Feeding of glycerol improved biomass formation, but lowered the product concentration. By combining derepression and methanol induction the final extracellular streptavidin concentration increased to 11.42±0.22μM (approx. 751mgL−1), yielding a productivity of 52.5nMh−1. Despite supplementing biotin the proportion of biotin-blocked binding sites in the supernatant dropped from 54.4±5.0 % after 18h to 17.2±6.5 % towards the end of glucose feeding to a final value of 1.1±3.8 %, indicating a highly bioactive product. Thus, H. polymorpha proved to be a suitable host for the production of streptavidin.

Constitutive production and efficient secretion of soluble full-length streptavidin by an Escherichia coli ‘leaky mutant’

Due to its various applications the protein streptavidin is a highly interesting target for heterologous production. This study focuses on different Escherichia coli-based constructs targeting a high-level expression and secretion of streptavidin to the medium. The effect of various promoters, variants of the target gene, leader sequences and host strains on expression and secretion into the culture broth was analyzed. Constitutive production of full-length streptavidin fused with the leader sequence of the bglA gene from Bacillus amyloliquefaciens by the periplasmic ‘leaky mutant’ E. coli JW1667-5 (Δlpp-752:kan) at 30°C generated the highest yield of the conditions tested, surpassing the extracellular concentration of a conventional T7-based expression system. Supplementation of the medium by the non-ionic surfactants Triton® X-100 and X-45 led to an improved secretion of the protein to the culture supernatant. Tetrameric concentrations of streptavidin of 2790±166nM were reached in shake flasks at a productivity of 49.6nMh−1. Optimization of conditions led to a successful transfer to the bioreactor, yielding a maximal concentration of 2608±169nM and a productivity of 65.2nMh−1 in fed-batch operation. The proportion of biotin-blocked binding sites of 8.3±4.3% indicated a highly bioactive product.

Artificial Formation and Tuning of Glycoprotein Networks on Live Cell Membranes: A Single‐Molecule Tracking Study

We present a method to artificially induce network formation of membrane glycoproteins and show the precise tuning of their interconnection on living cells. For this, membrane glycans are first metabolically labeled with azido sugars and then tagged with biotin by copper-free click chemistry. Finally, these biotin-tagged membrane proteins are interconnected with streptavidin (SA) to form an artificial protein network in analogy to a lectin-induced lattice. The degree of network formation can be controlled by the concentration of SA, its valency, and the concentration of biotin on membrane proteins. This was verified by investigation of the spatiotemporal dynamics of the SA-protein networks employing single-molecule tracking. It was also proven that this network formation strongly influences the biologically relevant process of endocytosis as it is known from natural lattices on the cell surface.

Tunable nanogap devices for ultra-sensitive electrochemical impedance biosensing

A wealth of research has been available discussing nanogap devices for detecting very small quantities of biomolecules by observing their electrical behavior generally performed in dry conditions. We report that a gold nanogapped electrode with tunable gap length for ultra-sensitive detection of streptavidin based on electrochemical impedance technique. The gold nanogap is fabricated using simple monolayer film deposition and in-situ growth of gold nanoparticles in a traditional interdigitated array (IDA) microelectrode. The electrochemical impedance biosensor with a 25-nm nanogap is found to be ultra-sensitive to the specific binding of streptavidin to biotin. The binding of the streptavidin hinder the electron transfer between two electrodes, resulting in a large increase in electron-transfer resistance (Ret) for operating the impedance. A linear relation between the relative Ret and the logarithmic value of streptavidin concentration is observed in the concentration range from 1 pM (picomolar) to 100 nM (nanomolar). The lowest detectable concentration actually measured reaches 1 pM. We believe that such an electrochemical impedance nanogap biosensor provides a useful approach towards biomolecular detection that could be extended to a number of other systems.

Liposomes with High Refractive Index Encapsulants as Tunable Signal Amplification Tools in Surface Plasmon Resonance Spectroscopy.

One major goal in the surface plasmon resonance (SPR) technique is the reliable detection of small molecules as well as low analyte concentrations. This can be achieved by a viable signal amplification strategy. We therefore investigated optimal liposome characteristics for use as a signal enhancement system for SPR sensors, as liposomes excel not only at versatility but also at colloidal stability and ease of functionalization. These characteristics include the encapsulation of high refractive index markers, lipid composition, liposome size, and surface modifications to best match the requirements of the SPR system. Our studies of the binding of biotinylated liposomes to surface-immobilized streptavidin show that the refractive index of the encapsulant has a major influence on the SPR signal and outweighs the influence of the thin lipid bilayer. Thus, the signal amplification properties of liposomes can be adjusted to the respective needs of any analytical task by simply exchanging the encapsulant solution. In this work, a maximum enhancement factor of 23 was achieved by encapsulating a 500 mM sucrose solution. Dose-response studies with and without liposome enhancement revealed an improvement of the limit of detection from 10 nmol L(-1) to 320 pmol L(-1) streptavidin concentration with a much higher sensitivity of 3 mRIU per logarithmic unit of the concentration between 500 pmol L(-1) and 10 nmol L(-1).

Fluorescence enhancement and multiple protein detection in ZnO nanostructure microfluidic devices.

In this study, different morphological ZnO nanostructures, those of sharp nanowires (NWs), rod NWs, and hexahedral-puncheon nanostructures, were grown in microfluidic channels on the same glass substrate. Characterizations of correspondent biomolecule binding properties were simulated and demonstrated. The surface was modified using 3-ammineopropyl-triethoxysilane (3-APTES) and biotin-N-hydroxysuccinimide ester (NHS-biotin). Different concentrations (4.17pM to 41.7nM) of dye-conjugated streptavidin were simultaneously infused through the second microfluidic channels, which lie 90° from the first microfluidic channels. The florescent intensity at the crossover areas showed good agreement with simulations, with sharp ZnO NWs exhibiting the largest dynamic range and the highest fluorescent intensity. We further characterize correspondent protein detection using sharp ZnO NWs. The surfaces of these ZnO NWs were modified with mouse immunoglobulin G (IgG), infused through the second microfluidic channels with dye-conjugated (Alexa 546) anti-mouse IgG in different concentrations. Concentrations ranging from 417fM to 41.7nM can be resolved using sharp ZnO NWs. Finally, multiple protein detection was demonstrated using a five-by-eight microfluidic channel array. Fluorescence images present clear multiple detections at the crossover areas when using the sharp ZnO NWs for simultaneous dye-conjugated anti-mouse IgG and dye-conjugated anti-rabbit IgG (Alexa 647) detection.

Highly sensitive optical fibre long period grating biosensor anchored with silica core gold shell nanoparticles.

An optical fibre long period grating (LPG), modified with a coating of silica core gold shell (SiO2:Au) nanoparticles (NPs) deposited using the layer-by-layer method, was employed for the development of a biosensor. The SiO2:Au NPs were electrostatically assembled onto the LPG with the aid of a poly(allylamine hydrochloride) (PAH) polycation layer. The LPG sensor operates at the phase matching turning point to provide the highest sensitivity. The SiO2:Au NPs were modified with biotin, which was used as a ligand for streptavidin (SV) detection. The sensing mechanism is based on the measurement of the refractive index change induced by the binding of the SV to the biotin. The effect on sensitivity of increasing the surface area by virtue of the SiO2:Au nanoparticles' diameter and film thickness was studied. The lowest measured concentration of SV was 2.5nM, achieved using an LPG modified with a 3 layer (PAH/SiO2:Au) thin film composed of SiO2 NPs of 300nm diameter with a binding constant of k=1.7(pM)(-1), sensitivity of 6.9nm/ng/mm(2) and limit of detection of 19pg/mm(2).