Concentration Of Streptavidin Research Articles

A homogeneous assay for protein analysis in droplets by fluorescence polarization

We present a novel homogeneous ("mix-incubate-read") droplet microfluidic assay for specific protein detection in picoliter volumes by fluorescence polarization (FP), for the first time demonstrating the use of FP in a droplet microfluidic assay. Using an FP-based assay we detect streptavidin concentrations as low as 500 nM and demonstrate that an FP assay allows us to distinguish droplets containing 5 μM rabbit IgG from droplets without IgG with an accuracy of 95%, levels relevant for hybridoma screening. This adds to the repertoire of droplet assay techniques a direct protein detection method which can be performed entirely inside droplets without the need for labeling of the analyte molecules.

Improved localized surface plasmon resonance biosensing sensitivity based on chemically-synthesized gold nanoprisms as plasmonic transducers

Supporting substrate attached gold nanostructures display localized surface plasmon resonance (LSPR) properties at the visible and near-infrared (NIR) wavelengths. The LSPR wavelength is very responsive to the refractive index of the surrounding medium, allowing simple label-free biosensing when biomolecules are irreversibly adsorbed onto nanostructures. Herein, we show that chemically synthesized gold nanoprisms with 22 nm average edge lengths are very efficient plasmonic transducers for label-free biosensing. The LSPR properties of gold nanoprisms on silanized glass substrate were investigated and readily influenced by bulk refractive index changes and local changes very close to the sensing surface due to the analyte adsorption. The nanoprisms had a 583 nm/RIU (1.62 eV/RIU) refractive index sensitivity and a 4.9 figure of merit (FOM). In addition, the nanoprism surface was modified with straight chain alkanethiols. The LSPR wavelength (λmax) of chemisorbed nanoprisms on glass was very sensitive to surface modification by straight chain alkanethiols and linearly red-shifted by 2.5 nm with every methylene unit of the alkanethiol chain. Importantly, the biotin functionalized nanoprisms displayed red-shifts in λmax upon streptavidin (SA) binding. The lowest SA concentration was measured to be 50 pM, much lower than gold nanospheres, nanorods, bipyramids, and nanostars.

High sensitivity AlGaN/GaN field effect transistor protein sensors operated in the subthreshold regime by a control gate electrode

We demonstrate high sensitivity AlGaN/GaN field effect transistor biosensors with a control gate electrode for streptavidin detection. The device active area is functionalized with 3-Aminopropyltriethoxysilane and N-hydroxysulfosuccinimide-biotin for streptavidin binding. Without any electrochemical side effects, a gate voltage is applied through a Pt control electrode to the solution so that the device operates sensitively in the subthreshold regime. Due to the logarithmic relationship between the channel current and gate voltage in the subthreshold regime, at a concentration of 4.73 pM streptavidin, the device exhibits 9.97% current change in the subthreshold regime compared with the current in phosphate buffered saline solution. In the linear regime, the current change is 0.49% at the same streptavidin concentration.

Integrated polymer-based Mach-Zehnder interferometer label-free streptavidin biosensor compatible with injection molding

We report the development of a Mach-Zehnder interferometer biosensor based on a high index contrast polymer material system and the demonstration of label-free online measurement of biotin–streptavidin binding on the sensor surface. The surface of the polyimide waveguide core layer was functionalized with 3-mercaptopropyl trimethoxy silane and malemide tagged biotin. Several concentrations of Chromeon 642-streptavidin dissolved in phosphate buffered saline solution were rinsed over the functionalized sensor surface by means of a fluidic system and the biotin-streptavidin binding process was observed in the output signal of the interferometer at a wavelength of 1310 nm. Despite the large wavelength and the comparatively low surface sensitivity of the sensor system due to the low index contrast in polymer material systems compared to inorganic material systems, we were able to resolve streptavidin concentrations of down to 0.1 μg/ml. The polymer-based optical sensor design is fully compatible with cost-efficient mass production technologies such as injection molding and spin coating, which makes it an attractive alternative to inorganic optical sensors.

Label-free optical biosensing using a horizontal air-slot SiN_x microdisk resonator

We demonstrate label-free optical biosensing using a horizontal air-slotted silicon-rich silicon nitride (SiNx) microdisk resonator. Due to the strong confinement of light in the air-slot, a large resonance shift of 6.2 nm is observed upon reaction of the biotin-functionalized disk with a streptavidin solution with concentration of 2.5 μg/ml. Assuming a linear relationship between resonance shift and streptavidin concentration, we estimate the sensitivity to be 2.5 ± 0.2 nm/(μg/ml). Comparing this value with surface sensitivity of 5.7 nm/nm calculated using FDTD simulations, a detection limit of 30 ± 2 ng/ml, is extrapolated.

Software-based quantitation of bioassays on CD

Compact discs (CDs) can be used as rapid, low-cost, high-capacity screening platforms for running bioassays with no modification of the detection hardware (conventional standard optical drive). We describe herein a new protocol to read and quantitate biotin–streptavidin binding assays with a standard optical drive by using a CD-data analysis software (IsoBuster), which identifies erroneous sectors by locating the exact error position bit-by-bit and allows various data formats to be used. The numbers of erroneous sectors increase as a function of the streptavidin concentration in the tested solutions. High spatial accuracy and detection sensitivity (0.3 μg/mL) were achieved.

Microtubule bundle formation driven by ATP: the effect of concentrations of kinesin,streptavidin and microtubules

Recently, a method was established for the formation of microtubule (MT) assemblies by anactive self-organization (AcSO) process, in which MTs were crosslinked duringsliding motion on a kinesin-coated surface, and this was coupled with adenosinetriphosphate (ATP) hydrolysis. Streptavidin (ST) was the glue used to crosslinkbiotin-labeled MTs. Although most of the MT assemblies were in the bundle form, theyvaried in size, shape and motility, depending on the initial conditions used. In this paper,we systematically examined the effects of the concentrations of kinesin, ST andMT on the formation of MT bundles under the initial conditions of the process.

Single-bead-based consecutive biochemical assays using a dielectrophoretic microfluidic platform

In this paper, we describe a method for performing single-bead-based, consecutive biochemical assays on a dielectrophoretic microfluidic chip using the interaction of FITC-labeled streptavidin with biotin-coated 2-μm polystyrene (PS) beads as a model system. A microfluidic device with four sets of electrodes on a glass slide was used to trap a single bead at the center of each set of electrodes through negative dielectrophoresis. Each set of electrodes was composed of four electrodes arranged in a cross-like manner. The electrodes were prepared using a simple process consisting of UV lithography and aluminum etching. A laser-induced fluorescence system detected the fluorescence emission from the FITC-labeled streptavidin conjugated with the biotin-coated PS beads. We investigated the effects of (i) the magnification/numerical aperture of the objective used for collecting fluorescence signals and (ii) the cut-off frequency of the low-pass digital filter, which filtered out high-frequency noise, on the signal-to-noise ratio. The average measured fluorescence maximum, determined from 10 consecutive fluorescence signals, increased with increasing concentrations of streptavidin (up to 0.3μg/mL); it leveled off when the concentration of streptavidin was greater than 0.3μg/mL. The limit of detection (signal-to-noise ratio=3.0) was 8.3ng/mL.

High-Throughput DNA Assays Using Picoliter Reactor Volumes

The online characterization and detection of individual droplets at high speeds, low analyte concentrations, and perfect detection efficiencies is a significant challenge underpinning the application of microfluidic droplet reactors to high-throughput chemistry and biology. Herein, we describe the integration of confocal fluorescence spectroscopy as a high-efficiency detection method for droplet-based microfluidics. Issues such as surface contamination, rapid mixing, and rapid detection, as well as low detections limits have been addressed with the approach described when compared to conventional laminar flow-based fluidics. Using such a system, droplet size, droplet shape, droplet formation frequencies, and droplet compositions can be measured accurately and precisely at kilohertz frequencies. Taking advantage of this approach, we demonstrate a high-throughput biological assay based on fluorescence resonance energy transfer (FRET). By attaching a FRET donor (Alexa Fluor 488) to streptavidin and labeling a FRET acceptor (Alexa Fluor 647) on one DNA strand and biotin on the complementary strand, donor and acceptor molecules are brought in proximity due to streptavidin-biotin binding, resulting in FRET. Fluorescence bursts of the donor and acceptor from each droplet can be monitored simultaneously using separate avalanche photodiode detectors operating in single photon counting mode. Binding assays were investigated and compared between fixed streptavidin and DNA concentrations. Binding curves fit perfectly to Hill-Waud models, and the binding ratio between streptavidin and biotin was evaluated and found to be in agreement with the biotin binding sites on streptavidin. FRET efficiency for this FRET pair was also investigated from the binding results. Efficiency results show that this detection system can precisely measure FRET even at low FRET efficiencies.

Localized surface plasmon resonance (LSPR) of polyelectrolyte-functionalized gold-nanoparticles for bio-sensing

Gold-nanoparticles can be used as a robust tool for bio-sensing based on localized surface plasmon resonance (LSPR). Both local refractive index change and self-assembly of the nanoparticles can lead to some spectral shifts of the LSPR extinction peak. Using different mechanisms of LSPR peak shifts, two types of transducers are set up to detect the streptavidin concentration through the specific recognition with biotin moleculars, which are conjugated to gold-nanoparticles. One is based on the self-assembly of gold-nanoparticles in solution, and the other is based on the increase of the local refractive index near the surface of the immobilized gold-nanoparticles after streptavidin–biotin binding. The sensitivity and efficiency of the two transducers are compared. Gold-nanoparticles are functionalized with polyelectrolyte, which can improve significantly the stability of the nanoparticles and reduce the unspecific binding of streptavidin. Polyelectrolyte functionalization also provides a simple way to conjugate gold-nanoparticles with charged molecules such as biotin and BSA–biotin.

A micromachined microcalorimeter with split-flow microchannel for biochemical sensing applications

This paper reports a micromachined microcalorimeter based on a split-flow microchannel structure for biochemical applications. It is characterized by measuring the reaction heat between biotin and streptavidin. For enhancing its sensitivity, the thermal components of the proposed microcalorimeter are fabricated on a high-thermal resistivity layer, SU-8 in this case, which leads to reduce the parasitic heat transfer to the silicon substrate. For removing additional heating elements, the split-flow microchannel is used. The split-flow microchannel scheme constantly keeps the output of the microcalorimeter near zero level when there is no biochemical reaction; in this state, a heater becomes unnecessary. The microchannel is based on the principle that target biomolecules applied to both the reaction and the reference chambers maintain the chambers at the same temperature, and then capture biochemicals are applied to the reaction chamber on hot junctions, only. Finally, the voltage generated by the biochemical reaction heat in the reaction chamber is measured. In order to verify characteristics of the fabricated sensor, reaction heat due to the biotin–streptavidin reaction was measured. For the experiment, 3.4 mg/ml biotin was used with various streptavidin concentrations of 1.0, 0.5, 0.25, 0.1, and 0.01 mg/ml. Three microliters of titrated biotin and streptavidin of various concentrations were injected into the sensor through a split-flow microchannel. The measurement results show that its sensitivity was 33.7 V/molarity (=0.21 V/cal) as the concentrations of streptavidin.

Universal liposomes: preparation and usage for the detection of mRNA

Dye-encapsulating liposomes can serve as signaling reagents in biosensors and biochemical assays in place of enzymes or fluorophores. Detailed here is the use and preparation of streptavidin-coupled liposomes which offer a universal approach to biotinylated target detection. The universal approach provides two advantages, i.e. only one type of liposome is necessary despite varying target and probe sequences and the hybridization event can take place in the absence of potential steric hindrance occurring from liposomes directly conjugated to probes. One objective of this work was to optimize the one-step conjugation of SRB-encapsulating liposomes to streptavidin using EDC. Liposome, EDC, streptavidin concentrations, and reaction times were varied. The optimal coupling conditions were found to be an EDC:carboxylated lipid:streptavidin molar ratio of 600:120:1 and a reaction time of 15 min. The second goal was to utilize these liposomes in sandwich hybridization microtiter plate-based assays using biotinylated reported probes as biorecognition elements. The assay was optimized in terms of probe spacer length, probe concentration, liposome concentration, and streptavidin coverage. Subsequently, the optimized protocol was applied to the detection of DNA and RNA sequences. A detection limit of 1.7 pmol L(-1) and an assay range spanning four orders of magnitude (5 pmol L(-1)-50 nmol L(-1)) with a coefficient of variation </=5.8% was found for synthetic DNA. For synthetic RNA the LOQ was half that of synthetic DNA. A comparison was made to alkaline phosphatase-conjugated streptavidin for detection which yielded a limit of quantitation approximately 80 times higher than that for liposomes in the same system. Thus, liposomes and the optimized sandwich hybridization method are well suited for detecting single-stranded nucleic acid sequences and compares favorably to other sandwich hybridization schemes recently described in the literature. The assay was then used successfully for the clear detection of mRNA amplified by nucleic acid sequence-based amplification (NASBA) isolated from as little as one Cryptosporidium parvum oocyst. The detection of mRNA from oocysts isolated from various water sample types using immunomagnetic separation was also assessed. Finally, to prove the wider applicability and sensitivity of this universal method, RNA amplified from the atxA gene of Bacillus anthracis was detected when the input to the preceding NASBA reaction was as low as 1.2 pg. This highly sensitive liposome-based microtiter plate assay is therefore a platform technology allowing for high throughput and wide availability for routine clinical and environmental laboratory applications.

Detection in near‐field domain of biomolecules adsorbed on a single metallic nanoparticle

In this paper, we study the performances of nanosensors based on Localized Surface Plasmon Resonance in the context of biological sensing. We demonstrate the sensitivity and the selectivity of our designed nanosensors by studying the influence of the concentration of Streptavidin on the shift of Localized Surface Plasmon Resonance wavelength. In addition, to study the detection of biomolecules on a single Au nanoparticle, we used a Scanning Near-field Optical Microscope. These results represent new steps for applications in biological research and medical diagnostics.

Label-Free Plasmonic Detection of Biomolecular Binding by a Single Gold Nanorod

We report the use of individual gold nanorods as plasmonic transducers to detect the binding of streptavidin to individual biotin-conjugated nanorods in real time on a surface. Label-free detection at the single-nanorod level was performed by tracking the wavelength shift of the nanorod-localized surface plasmon resonant scattering spectrum using a dark-field microspectroscopy system. The lowest streptavidin concentration that was experimentally measured was 1 nM, which is a factor of 1000-fold lower than the previously reported detection limit for streptavidin binding by biotinylated single plasmonic nanostructures. We believe that the current optical setup is able to reliably measure wavelength shifts as small as 0.3 nm. Binding of streptavidin at 1 nM concentration induces a mean resonant wavelength shift of 0.59 nm suggesting that we are currently operating at close to the limit of detection of the system.

Optimization of an amperometric biosensor for the detection of hepatitis C virus using fractional factorial designs

Fractional factorial design and factorial with center point design were applied to the development of an amperometric biosensor for the detection of the hepatitis C virus. Biomolecules were immobilized by adsorption on graphite electrodes modified with siloxane-poly(propyleneoxide) hybrid matrix prepared using the sol-gel method. Several parameters were optimized, such as the streptavidin concentration at 0.01 mg mL-1 and 1.0% bovine serum albumin, the incubation time of the electrodes in the complementary DNA solution for 30 minutes and a 1:1500 dilution of the avidin-peroxidase conjugate, among others. The application of chemometric studies has been efficient, since the best conditions have been established with a restricted number of experiments, indicating the influence of different factors on the system.

A label-free biosensor based on gold nanoshell monolayers for monitoring biomolecular interactions in diluted whole blood

Gold nanoshells (GNSs) were self-assembled on the surface of transparent glasses modified with 3-aminopropyltrimethoxysilane (APTES) to form GNS self-assembled monolayers (SAMs). Because the localized surface plasmon resonance (LSPR) of GNSs can be controlled in the near-infrared (NIR) region of the spectrum, where the optical transmission through tissue and whole blood is optimal, GNSs would be used as an effective signal transduction in whole blood. Accordingly, after modified with cystamine and biotin-NHS (N-hydroxy succinimide), GNS SAMs were used as a novel optical biosensor for real-time detection of streptavidin–biotin interactions in diluted human whole blood within short assay time, without any sample purification/separation. An UV–vis–NIR spectrophotometer was used to monitor the absorbance changes at 730 nm as a function of time for different concentrations of streptavidin in 20% whole blood, and the results showed that the biosensor displayed low detection limit of ∼3 μg/mL and wide dynamic range of ∼3–50 μg/mL. This approach provides an opportunity to construct LSPR biosensor for protein sensing and cellular analysis in diluted whole blood.

High-Throughput DNA Droplet Assays Using Picoliter Reactor Volumes

The online characterization and detection of individual droplets at high speeds, low analyte concentrations, and perfect detection efficiencies is a significant challenge underpinning the application of microfluidic droplet reactors to high-throughput chemistry and biology. Herein, we describe the integration of confocal fluorescence spectroscopy as a high-efficiency detection method for droplet-based microfluidics. Issues such as surface contamination, rapid mixing, and rapid detection, as well as low detections limits have been addressed with the approach described when compared to conventional laminar flow-based fluidics. Using such a system, droplet size, droplet shape, droplet formation frequencies, and droplet compositions can be measured accurately and precisely at kilohertz frequencies. Taking advantage of this approach, we demonstrate a high-throughput biological assay based on fluorescence resonance energy transfer (FRET). By attaching a FRET donor (Alexa Fluor 488) to streptavidin and labeling a FRET acceptor (Alexa Fluor 647) on one DNA strand and biotin on the complementary strand, donor and acceptor molecules are brought in proximity due to streptavidin-biotin binding, resulting in FRET. Fluorescence bursts of the donor and acceptor from each droplet can be monitored simultaneously using separate avalanche photodiode detectors operating in single photon counting mode. Binding assays were investigated and compared between fixed streptavidin and DNA concentrations. Binding curves fit perfectly to Hill-Waud models, and the binding ratio between streptavidin and biotin was evaluated and found to be in agreement with the biotin binding sites on streptavidin. FRET efficiency for this FRET pair was also investigated from the binding results. Efficiency results show that this detection system can precisely measure FRET even at low FRET efficiencies.

An optical biosensor based on localized surface plasmon resonance of silver nanostructured films

Ag nanostructured films fabricated by the reduction of sputtered AgOx thin films are shown to exhibit a strong localized surface plasmon resonance (LSPR) at wavelengths around 370 nm. The spectral position of the LSPR, defined as the wavelength of minimal reflectance, shifts linearly depending on the refractive index n of the surrounding medium in a range of n = 1.0–1.5. By the immobilization of biotin molecules at the Ag surface, events of binding between the immobilized biotin and solution-phase streptavidin were probed for streptavidin concentrations in a range of 16 nM–8 µM. From the sigmoidal fitting of the data, the surface-confined binding affinity constant was estimated as Ka,surf = 3.0 × 106 M−1. These results experimentally demonstrate the great potential of the Ag nanostructured films for application as cost-effective LSPR biosensors.

Sublithographic vertical gold nanogap for label-free electrical detection of protein-ligand binding

Label-free electrical detection of protein-ligand binding using a vertical gold nanogap is presented. A sublithographic nanogap was created using a sacrificial ultrathin film deposited by atomic layer deposition (ALD) in a process similar to the formation of a cantilever in microelectromechanical system processing. Due to the atomic precision of the sacrificial Al2O3 thickness by ALD, a 7nm nanogap was successfully fabricated. After binding streptavidin to biotin on the gold surface, an electrical current was measured for various voltages. A dramatic current increase was observed in the case of biotin-streptavidin binding in comparison with the other two cases: a control group filled with air and a biotin-only binding group. There was a minimal current change in the cases of the biotin-PBST group, the biotin-BSA group, and the biotin-saturated streptavidin group, as compared with the biotin-streptavidin group. At a 0.1μg∕ml concentration of streptavidin (1.5nM), the current difference before and after the protein binding was amplified by approximately 3000-fold with 17nm nanogap. Also, the detection sensitivity of the vertical nanogap as the gap size varied was investigated. As the size of biotin-streptavidin binding is the most comparable to 12nm nanogap, the highest sensitivity was shown in the 12nm gap device. 7nm nanogap can be used to detect smaller size of biomolecule than that of biotin streptavidin. This arrayable, two-terminal microdevice could be tested for use on a wide range of other biomolecules.

Kinetic and Equilibrium Binding Analysis of Protein−Ligand Interactions at Poly(amidoamine) Dendrimer Monolayers

The interaction of streptavidin (SA) with a biotinylated surface has been of great interest in the development of an interfacial layer for protein immobilization based on self-assembled monolayers (SAMs) and polymeric layers. Here, we demonstrate the unique characteristics of protein-ligand interactions on dendrimer monolayers based on kinetic and equilibrium binding analyses. With amine-ended poly(amidoamine) dendrimers from the first (G1) to fourth (G4) generation, the formation of even, compact dendrimer monolayers on gold was confirmed using FT-IR spectroscopy and ellipsometry. For the SA-biotin interaction, quantitative analysis of bound SA using surface plasmon resonance showed that the saturation binding level of SA was fairly higher in all dendrimer layers when compared to other tested systems of 11-mercaptoundecylamine SAMs and a poly(L-lysine) layer. Kinetic studies revealed that the initial binding rate of SA up to the saturation level was 2-fold higher in all dendrimer layers than in the SAMs regardless of the surface density of functionalized biotin. Concurrently, the dendrimer layers led to much higher values of sticking probability, which is defined as the probability that the SA molecule adsorbs upon collision with a biotinylated surface, at a fixed SA coverage, and prolonged the significant levels around the maximum probability with increasing SA coverage. Plots of the saturation coverage of SA versus the SA concentration in solution showed that SA binding onto the biotinylated G1 and G3 layers fit to a Langmuir isotherm model. Taken together, faster binding of SA and highly ordered packing of the molecules seems to be achieved through typical properties of the dendrimer monolayers such as surface distribution of functionalized biotin, surface corrugation, and flexibility of highly branched larger dendrimers, which provides a guideline for the construction and analysis of an interfacial layer in biosensing applications.