Biotin-streptavidin Interaction Research Articles

Catalase-linked immunosorbent pressure assay for portable quantitative analysis.

In this study, catalase-linked immunosorbent pressure assay with a gas-generation reaction was established for quantitative detection of disease biomarker C-reactive protein (CRP) by a portable pressuremeter. The pressure-based detection system recognizes, transduces, and amplifies the target signal to a convenient target-correlated pressure signal reading in a closed chamber. Biotin molecules were modified on the surface of catalase in order to incorporate catalase into the pressure immunoassay by the streptavidin-biotin interaction. To improve the assay performance, the modification ratios of biotin molecules to catalase, and the concentrations of capture and detection antibodies were further optimized. The catalase-linked immunosorbent pressure assay allows portable and quantitation analysis of CRP with a limit of detection of 1.8 nM, which can satisfy the clinical needs for determining the risk of cardiovascular disease. The catalase-linked immunosorbent pressure assay also shows superior specificity and good accuracy. Compared to the previously reported assay catalyzed by PtNP nanozyme, catalase is not easily deactivated during storage and operation. With the merits of enzymatic efficiency, biocompatibility, low non-specific adsorption and facile modification, catalase can be reasonably used for reproducible, stable, simple quantitative detection of disease markers using a portable pressure-based assay in resource-limited settings.

Force spectroscopic detection of peptide cleavage by thrombin exploiting biotin–streptavidin interactions in a bio-sensing context

Atomic-force-microscopy-based single-molecule force spectroscopy (AFM-SMFS) has become an important technique as the basis of novel, label-free biosensing strategies.

Surface-tailored anti-HER2/neu-solid lipid nanoparticles for site-specific targeting MCF-7 and BT-474 breast cancer cells

CAB51, a compact antibody against human epithelial growth receptor 2 (HER2, ErbB2), has been linked to cationic Solid Lipid Nanoparticles (SLN) via streptavidin-biotin interaction and their targeting potential evaluated against breast cancer cells. The amount of streptavidin and biotinylated antibody was optimised by monitoring the mean complex size (intensity weighed average diameter), polydispersity index and immediate stability in phosphate buffer saline (PBS). The effect on MCF-7 and BT-474 cells was evaluated at concentrations of 0.01 mg/mL and 0.1 mg/mL (counted as solid lipid). Streptavidin adsorption onto SLN surface had no influence on cell viability. Linking the antibody showed a synergistic effect on cell viability at lowest concentration tested (0.01 mg/mL) which was lower than that observed after exposure to SLN alone or antibody alone. At the higher tested concentration (0.1 mg/mL), the observed toxicity was entirely governed by the inherent toxicity of the SLN themselves. Streptavidin adsorption had no effect on accumulation in cells, while the antibody-containing complexes showed clearly increased internalisation in both cell lines. In HER2/neu positive BT-474 higher internalisation was observed than in HER2/neu negative MCF-7.

Peptide-/protein-mediated nano-bio interface and its applications

Surface modification can impart nanostructures new interface properties. In this review, we summarize the representative surface modification methods of nanoparticles with peptides and proteins. The biomolecules can be conjugated with nanoparticles by noncovalent and covalent coupling. Both of these approaches have strengths and limitations. Physical adsorption is the most direct and simplest noncovalent method but peptides and proteins adsorbed on solid surface always loss their native structures and biological activity and the behavior of peptides and proteins on surface is still difficult to regulate precisely. A popular noncovalent conjugation with stability and selectivity is via biotin-streptavidin interaction. Covalent attachment is more stable and selective than noncovalent methods. The stability of covalent biomolecule-nanostructure conjugates makes this strategy useful for applications in biological media with other interfering species. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)/ N -hydroxysuccinimide (NHS) strategy is widely used for covalent bioconjugation with well-established protocol, but it could lead to hydrolysis problem, crosslinking and uncontrolled heterogeneous orientation. The click chemistry is another covalent strategy with higher selectivity and yield, while it takes an extended reaction time. In addition to considering stability, selectivity, reaction time, and yield mentioned above, covalent bioconjugation should proceed in mild condition and the reagents required should have no interference on properties of biomolecules and nanostructures. In recent years substantial progress has been made in the formation of bionanoconjugates, but there is still no completely reliable approach for biomolecule-nanosturcture conjugation. Heretofore fine-tuning the structure and orientation of peptides and proteins on surface has been challenging. The efforts on improving biostability, biodistribution and targeting of nanoparticles with peptides and proteins functionalization are introduced. Nanostructures are often used within body in biomedical applications, so they must overcome an ensemble of biological obstacles to perform their function. First they may suffer from clearance from the bodys immune system. Even though they can escape from clearance, they are required to arrive their correct battlefield. Whats more, nanostructures need to cross cell membrane barriers and reach specific organelles such as nucleus and mitochondria to function in most cases. Peptide- and protein-nanostructure conjugation has been emerged as a useful tool to address these problems mentioned above. They could provide nanostrctures protection from phagocytic clearance and promote persistent circulation. Nanostructures can penetrate cell membrane easily with cell-penetrating peptide modification. The specific recognition of biological molecules imparts nanostructures targeting ability. In addition, the applications in fields such as diagnostics are introduced based on antigen-antibody specific recognition. The problems that bionanoconjugates for medical applications may encounter in use are presented briefly. Once contacting with biological matrices, nanostructures will be immediately coated by proteins, forming a protein corona. This protein layer could make significant changes in nanostructure properties such as size, surface charge and even the bioactivity of the peptides and proteins conjugated on its surface, which provides nanostructure with a new biological identity. Some previous results suggested that protein-functionalized nanoparticles lost their targeting capabilities when a protein corona adsorbed on the surface. In short, not only should bioconjugation methods be picked carefully depending on the goals, but also the effects of the complex use environment on bionanoconjugates should be taken into consideration. This review is intended to help researchers get an idea of the progress and dilemma of bionanoconjugate construction and its applications, and provide some inspiration for design and synthesis of peptide and protein-modified nanostructures.

Evaluation of protein-ligand interactions using the luminescent interaction assay FlimPIA with streptavidin-biotin linkage

Post-translational modifications, such as phosphorylation, are crucial in the regulation of protein-protein interactions and protein function in cell signaling. Here, we studied the interaction between the transactivation domain peptide of cancer suppressor protein p53 and its negative regulator Mdm2 using a novel protein-protein interaction assay, based on the modified FlimPIA using the streptavidin-biotin interaction to link the p53 peptide and the probe enzyme. We succeeded in detecting an attenuation in the affinity of p53 towards Mdm2 caused by the phosphorylation at Thr18. It showed that the targets, which are not easy to fuse with the FlimPIA probes, such as phosphorylated peptides can be used in this system. Also, the use of streptavidin nanobeads was found effective to get clearer signal, probably due to concentration of the detection system onto the bead surface. The system was further applied to the detection of FKBP-FRB interaction using biotinylated FKBP domain, which suggested another potential merit of this system that allows to avoid misfolding and steric hindrance often observed for the fusion protein approach.

I-motif-coated exosomes as a pH-sensitive carrier for anticancer drugs

Nature-derived exosomes have been noted as emerging carriers for anticancer drugs. In this study, as a proof-of-concept, the anticancer drug doxorubicin (Dox) was loaded onto i-motif-modified exosomes (Exo-i-motif) to deliver Dox to cancer cells efficiently. The double-stranded biotin-i-motif/flare (ds-i-motif-bio)s efficiently released Dox in an acidic pH-responsive manner within 1 h. Based on gel electrophoresis, it was clearly confirmed that ds-i-motif-bio successfully interacts with biotin-conjugated exosomes and streptavidin (strep) via the biotin–streptavidin interaction. The particle sizes were below 150 nm without aggregation after strep-mediated modification of ds-i-motif-bio on the surfaces of the exosomes. In addition, released Dox had intact bioactivity for anti-proliferation after immobilization onto the exosomes. This study could serve as a new concept of pH-responsive delivery systems of anticancer drug using nature-derived exosomes with i-motifs.

Ultrasensitive Detection of Bacterial Protein Toxins on Patterned Microarray via Surface Plasmon Resonance Imaging with Signal Amplification by Conjugate Nanoparticle Clusters.

Sensitive detection and monitoring of biological interactions in a high throughput, multiplexed array format has numerous advantages. We report here a method to enhance detection sensitivity in surface plasmon resonance (SPR) spectroscopy and SPR imaging via the effect of accumulation of conjugated nanoparticles of varying sizes. Bacterial cholera toxin (CT) was chosen for the demonstration of enhanced immunoassay by SPR. After immobilization of CT on a gold surface, specific recognition is achieved by biotinylated anti-CT. The signal is amplified by the attachment of biotinylated 20 nm AuNP via streptavidin bridge, followed by attachment of 5 nm streptavidin-functionalized Fe3O4NP to the AuNP-biotin surface. The continuous surface binding of two differently sized conjugated nanoparticles effectively increases their packing density on surface and significantly improves SPR detection sensitivity, allowing quantitative measurement of CT at very low concentration. The dense packing of conjugated nanoparticles on the surface was confirmed by atomic force microscopy characterization. SPR imaging of the immunoassay for high-throughput analysis utilized an Au-well microarray that attenuated the background resonance interference on the resulting images. A calibration curve of conjugated nanoparticle binding signal amplification for CT detection based on surface coverage has been obtained that shows a correlation in a range from 6.31 × 10-16 to 2.51 × 10-13 mol/cm2 with the limit of detection of 5.01 × 10-16 mol/cm2. The absolute quantity of detection limit using SPR imaging was 0.25 fmol. The versatile nanoparticles and biotin-streptavidin interaction used here should allow adaptation of this enhancement method to many other systems that include DNA, RNA, peptides, and carbohydrates, opening new avenues for ultrasensitive analysis of biomolecules.

Gold NanoBipyramids Performing as Highly Sensitive Dual-Modal Optical Immunosensors

In this work, we demonstrate the feasibility of gold bipyramidal-shaped nanoparticles (AuBPs) to be used as active plasmonic nanoplatforms for the detection of the biotin-streptavidin interaction in aqueous solution via both Localized Surface Plasmon Resonance and Surface Enhanced Raman Scattering (LSPR/SERS). Our proof of concept exploits the precise attachment of the recognition element at the tips of AuBPs, where the electromagnetic field is stronger, which is beneficial to the surface sensitivity of longitudinal LSPR on the local refractive index and to the electromagnetic enhancement of SERS activity, too. Indeed, successive red shifts of the longitudinal LSPR associated with increased local refractive index reveal the attachment of para-aminothiophenol (p-ATP) chemically labeled Biotin to the Au surface and the specific capture of the target protein by biotin-functionalized AuBPs. Finite-Difference Time-Domain simulations based on the reconstructed index of refraction confirm LSPR measurements. However, the molecular identification of the biotin-streptavidin interaction remains elusive by LSPR investigation alone. Remarkably, we succeeded to complement the LSPR detection with reliable SERS measurements which permitted to (a) certify the molecular identification of biotin-streptavidin interaction and (b) extend the limit of detection of streptavidin in solution toward 10-12 M. Finally, to further probe the possibility to implement the AuBPs as dual LSPR-SERS based immunoassays in solution for real clinical diagnostics, we additionally investigated the AuBP's performance to transduce the specific antihuman IgG- human IgG binding event, providing thus a reference design for building unique plasmonic immunoassays for dual-optical detection of target proteins in aqueous solution.

Affinity capture of aflatoxin B1 and B2 by aptamer-functionalized magnetic agarose microspheres prior to their determination by HPLC.

A novel adsorbent is described for magnetic solid-phase extraction (MSPE) of the aflatoxins AFB1 and AFB2 (AFBs). Magnetic agarose microspheres (MAMs) were functionalized with an aptamer to bind the AFBs which then were quantified by HPLC and on-line post-column photochemical derivatization with fluorescence detection. Streptavidin-conjugated MAMs were synthesized first by a highly reproducible strategy. They possess strong magnetism and high surface area. The MAMs were characterized by transmission electron microscopy, scanning electron microscopy, optical microscopy, laser diffraction particle size analyzer, Fourier transform infrared spectrometry, vibrating sample magnetometry and laser scanning confocal microscopy. Then, the AFB-aptamers were immobilized on MAMs through biotin-streptavidin interaction. Finally, the MSPE is performed by suspending the aptamer-modified MAMs in the sample. They are then collected by an external magnetic field and the AFBs are eluted with methanol/buffer (20:80). Several parameters affecting the coupling, capturing and eluting efficiency were optimized. Under the optimized conditions, the method is fast, has good linearity, high selectivity, and sensitivity. The LODs are 25pg·mL-1 for AFB1 and 10pg·mL-1 for AFB2. The binding capacity is 350 ± 8ng·g-1 for AFB1 and 384 ± 8ng·g-1 for AFB2, and the precision of the assay is <8%. The method was successfully applied to the analysis of AFBs in spiked maize samples. Graphical abstract Schematic of novel aptamer functionalized magnetic agarose microspheres (Apt-MAM) as magnetic adsorbents for simultaneous and specific affinity capture of aflatoxins B1 and B2 (AFBs).

High-Throughput Incubation and Quantification of Agglutination Assays in a Microfluidic System.

In this paper, we present a two-phase microfluidic system capable of incubating and quantifying microbead-based agglutination assays. The microfluidic system is based on a simple fabrication solution, which requires only laboratory tubing filled with carrier oil, driven by negative pressure using a syringe pump. We provide a user-friendly interface, in which a pipette is used to insert single droplets of a 1.25-µL volume into a system that is continuously running and therefore works entirely on demand without the need for stopping, resetting or washing the system. These assays are incubated by highly efficient passive mixing with a sample-to-answer time of 2.5 min, a 5–10-fold improvement over traditional agglutination assays. We study system parameters such as channel length, incubation time and flow speed to select optimal assay conditions, using the streptavidin-biotin interaction as a model analyte quantified using optical image processing. We then investigate the effect of changing the concentration of both analyte and microbead concentrations, with a minimum detection limit of 100 ng/mL. The system can be both low- and high-throughput, depending on the rate at which assays are inserted. In our experiments, we were able to easily produce throughputs of 360 assays per hour by simple manual pipetting, which could be increased even further by automation and parallelization. Agglutination assays are a versatile tool, capable of detecting an ever-growing catalog of infectious diseases, proteins and metabolites. A system such as this one is a step towards being able to produce high-throughput microfluidic diagnostic solutions with widespread adoption. The development of analytical techniques in the microfluidic format, such as the one presented in this work, is an important step in being able to continuously monitor the performance and microfluidic outputs of organ-on-chip devices.

A fluorometric lateral flow assay for visual detection of nucleic acids using a digital camera readout.

A fluorometric lateral flow assay has been developed for the detection of nucleic acids. The fluorophores phycoerythrin (PE) and fluorescein isothiocyanate (FITC) were used as labels, while a common digital camera and a colored vinyl-sheet, acting as a cut-off optical filter, are used for fluorescence imaging. After DNA amplification by polymerase chain reaction (PCR), the biotinylated PCR product is hybridized to its complementary probe that carries a poly(dA) tail at 3΄ edge and then applied to the lateral flow strip. The hybrids are captured to the test zone of the strip by immobilized poly(dT) sequences and detected by streptavidin-fluorescein and streptavidin-phycoerythrin conjugates, through streptavidin-biotin interaction. The assay is widely applicable, simple, cost-effective, and offers a large multiplexing potential. Its performance is comparable to assays based on the use of streptavidin-gold nanoparticles conjugates. As low as 7.8 fmol of a ssDNA and 12.5 fmol of an amplified dsDNA target were detectable. Graphical abstract Schematic presentation of a fluorometric lateral flow assay based on fluorescein and phycoerythrin fluorescent labels for the detection of single-stranded (ssDNA) and double-stranded DNA (dsDNA) sequences and using a digital camera readout. SA: streptavidin, BSA: Bovine Serum Albumin, B: biotin, FITC: fluorescein isothiocyanate, PE: phycoerythrin, TZ: test zone, CZ: control zone.

A Y-Shaped Three-Arm Structure for Probing Bivalent Interactions between Protein Receptor–Ligand Using AFM and SPR

The goal of this research was to develop linkage chemistry for the study of bivalent interactions between a receptor and its ligand using atomic force microscopy (AFM) and surface plasmon resonance (SPR). We conceived a three-arm structure composed of flexible chains connected to a large rigid core with orthogonal functional groups at their ends for formation and attachment (or immobilization) of bivalent ligands. To demonstrate the principle, we chose the well-known biotin-streptavidin interaction as a model system. On the basis of a crystal structure of the biotin-streptavidin complex, we designed and synthesized a bisbiotin ligand to have a Y shape with two biotin motifs on its arms for binding and a functional group on its stem for immobilization or attachment, referred to as y-bisbiotin. First, we found that the y-bisbiotin ligand stabilized the streptavidin more than its monobiotin counterpart did in solution, which indicates that the bivalent interaction was synergistic. The y-bisbiotin was attached to AFM tips through a click reaction for the force measurement experiments, which showed that unbinding the bisbiotin from streptavidin needed twice the force of unbinding a monobiotin. For the SPR study, we added a ω-thiolated alkyl chain to y-bisbiotin for its incorporation into a monolayer. The SPR data indicated that the streptavidin dissociated from a mixed monolayer bearing y-bisbiotin much slower than from the one bearing monobiotin. This work demonstrates unique chemistry for the study of bivalent interactions using AFM and SPR.

Plasmonic Heterodimers with Binding Site-Dependent Hot Spot for Surface-Enhanced Raman Scattering.

A novel plasmonic heterodimer nanostructure with a controllable self-assembled hot spot is fabricated by the conjugation of individual Au@Ag core-shell nanocubes (Au@Ag NCs) and varisized gold nanospheres (GNSs) via the biotin-streptavidin interaction from the ensemble to the single-assembly level. Due to their featured configurations, three types of heterogeneous nanostructures referred to as Vertice, Vicinity, and Middle are proposed and a single hot spot forms between the nanocube and nanosphere, which exhibits distinct diversity in surface plasmon resonance effect. Herein, the calculated surface-enhanced Raman scattering enhancement factors of the three types of heterodimers show a narrow distribution and can be tuned in orders of magnitude by controlling the size of GNSs onto individual Au@Ag NCs. Particularly, the Vertice heterodimer with unique configuration can provide extraordinary enhancement of the electric field for the single hot spot region due to the collaborative interaction of lightning rod effect and interparticle plasmon coupling effect. This established relationship between the architecture and the corresponding optical properties of the heterodimers provides the basis for creating controllable platforms which can be exploited in the applications of plasmonic devices, electronics, and biodetection.

Evaluation of two sterically directed attachments of biomolecules on a coaxial nanofibre membrane to improve the development of optical biosensors

In this study, we have optimised the sterically directed attachment of biomolecules on the surface of coaxial membranes prepared by co-electrospinning which have been proved to be a material with very high performance for the development of biosensors with optical oxygen transduction. Uricase has been used as model enzyme.Two sterically directed strategies: a) covalent attachment via maleimide, and b) affinity bonding via biotin-streptavidin interaction, have been tested in order to preserve the enzymatic activity of uricase and to improve the analytical figures of merits on the determination of uric acid. The best results were obtained with biotin-streptavidin affinity interaction and using a biotinylation reagent containing a polyethylene glycol chain. The developed biosensor showed high sensitivity towards uric acid with a detection limit of 0.5 µM, a quantification limit of 1.8 µM and linear range from 1.8 to 250 µM. The applicability of the membrane as biosensor with optical oxygen transduction was proved by determining uric acid in serum samples. The obtained results showed a good correlation (0.999) with those obtained by an external reference laboratory.

Magnetic multiwalled carbon nanotubes as nanocarrier tags for sensitive determination of fetuin in saliva

This paper reports the development and performance of an electrochemical immunosensor using magnetic multiwalled carbon nanotubes (m-MWCNTs) as nanocarrier tags for the determination of human fetuin A (HFA), a relevant biomarker of obesity, insulin resistance, and type-2 diabetes as well as for pancreatic and liver cancers and inflammatory processes. Screen-printed carbon electrodes were grafted with p-aminobezoic acid and streptavidin was covalently immobilized on the electrode surface. A biotinylated capture antibody was immobilized through streptavidin-biotin interaction and a sandwich assay configuration was implemented using m-MWCNTs conjugated with HRP and anti-HFA antibodies as the detection label. The determination of HFA was accomplished by measuring the current produced by the electrochemical reduction of benzoquinone at −200 mV upon addition of H2O2 as HRP substrate. The prepared m-MWCNTs were characterized by SEM, TEM, XRD and EDS. All the steps involved in the immunosensor preparation were monitored by electrochemical impedance spectroscopy and cyclic voltammetry. A linear calibration plot for HFA was found between 20 and 2000 pg/mL with a LOD value of 16 pg/mL. This performance is notably better than that reported for an ELISA kit and a chronoimpedimetric immunosensor. The favorable contribution of m-MWCNTs in comparison with MWCNTs without incorporated magnetic particles to this excellent analytical performance is also highlighted. The immunosensor selectivity against other proteins and potentially interfering compounds was excellent. In addition, the usefulness of the immunosensor was demonstrated by the analysis of HFA in saliva with minimal sample treatment.

Egg white component-resolved diagnosis: Testing of serum ovalbumin-specific IgE by luminescent oxygen channeling immunoassay

Egg allergy is the second most common food allergy in children. Precise measurement of allergen component-specific IgE (sIgE) in serum contributes to molecular diagnosis of egg allergy, i.e. component-resolved diagnosis (CRD). Existing CRD methods, including ImmunoCAP and Immulite, have unavoidable drawbacks such as errors from solid-liquid phase separation and enzyme instability. Therefore, we take egg allergen ovalbumin as an example to establish a homogenous method−indirect luminescent oxygen channeling immunoassay (LOCI) to determine ovalbumin-sIgE precisely. In the indirect LOCI reaction system, ovalbumin-conjugated acceptor nanoparticles were brought close to streptavidin-conjugated donor nanoparticles through antigen-antibody and biotin-streptavidin interactions. The optimal conditions for LOCI were 16.67 μg/mL acceptor-ovalbumin, 1.047 μg/mL biotinylated anti-human IgE antibodies and a 1/20 dilution of serum in a 25-μL reaction volume for a 45-min incubation. We assessed the performance of of this LOCI system, including sensitivity, precision, and anti-interference ability, and compared the diagnostic performance of the LOCI with enzyme-linked immunosorbent assay (ELISA) through a receiver operator characteristic (ROC) analysis. Results revealed that LOCI was superior to ELISA in performance. In summary, we demonstrated that indirect LOCI was an excellent method for precise determination of allergen component-sIgE, contributing to CRD of food allergy.

A direct determination of AFBs in vinegar by aptamer-based surface plasmon resonance biosensor

Aflatoxin (AFB) is one of the most toxic fungal metabolites produced by Aspergillus flavus, which may contaminate food and agricultural products. Herein, an aptamer-based surface plasmon resonance (SPR) biosensor was developed to detect AFBs. The chosen aptamer showed comparable interaction with the two AFBs, namely aflatoxin B1 (AFB1) and aflatoxin B2 (AFB2). Such phenomenon was rarely reported, and might lead to a simultaneous detection of both AFBs. In this study, AFB1 was used to systematically establish the detection method. In the SPR system, streptavidin proteins were immobilized on the surface of a CM5 sensor chip as a cross-linker and biotin-aptamers were captured through streptavidin-biotin interaction. After optimization, the assay showed a dynamic range between 0.09 and 200 ng mL−1 (linear range from 1.5 to 50 ng mL−1 and a LOD of 0.19 ng mL−1) of AFB1 in buffer. As expected, the aptasensor showed high specificity towards AFB1 and AFB2, but hardly bound to other toxins with similar structures, including ochratoxin A (OTA), ochratoxin B (OTB), Zeralenone (ZEA) and T-2 toxin (T-2). Determination of AFB1 in vinegar was further performed using the SPR biosensor. Recoveries of AFB1 from spiked samples ranged from 96.3 to 117.8%. The developed SPR assay is a simple, fast and sensitive approach for the detection of residual AFBs in agricultural products and foodstuffs like vinegar.

Detection of biotin-streptavidin interactions based on poly(thymine)-templated copper nanoparticles coupled with Exo III-aided DNA recycling amplification

The recognition and analysis of small molecule–protein interactions plays an important role in chemical genetics, clinical diagnostics, and drug development. However, various established highly sensitive fluorescence strategies still involve the use of expensive fluorescence dye-labelled nucleic acids. Herein, we constructed a highly sensitive and label-free fluorescent assay for small molecule–protein interactions on the basis of exonuclease III (Exo III)-assisted signal amplification and poly(thymine)-templated fluorescent copper nanoparticles (CuNPs) as signal probes. Biotin–streptavidin system was taken as a model. It was designed that biotinylated double-strand DNA served as the binding probe. Biotin was labelled at the 3′-end of the trigger strand. One kind of stem-loop DNA, which consisted of the thymine rich sequence and the trigger DNA recognition domain, was also designed. Once streptavidin bind to the binding probe, with the help of Exo III digestion, the trigger strand was able to reach the cycle for multiple poly(thymine) sequences generation. The produced poly(thymine) sequences can be used as a template for the formation of CuNPs. Comparing with no Exo III-aided DNA recycling amplification, significantly improved signals for the quantitative determination of streptavidin were obtained.

A voltammetric immunosensor for clenbuterol based on the use of a MoS2-AuPt nanocomposite.

An ultrasensitive immunosensor for the direct detection of the illegally used livestock feed clebuterol (CLB) is described. It is based on the use of a glassy carbon electrode modified with an MoS2-AuPt nanocomposite and on biotin-streptavidin interaction. The use of MoS2-AuPt accelerates electron transfer, and this leads to a sharp increase in the electrochemical signal for the electrochemical probe hydrogen peroxide. Differential pulse voltammetry was used to record the current signal at a peak potential of -0.18V (vs SCE). Under optimal conditions, the electrode has a linear response in the 10pg·mL-1 to 100ng·mL-1 CLB concentration range and a 6.9pg·mL-1 detection limit (based on the 3σ criterium). This immunosensor is sensitive, highly specific and acceptably reproducible, and thus represents a valuable tool for the determination of CLB in pork. Graphical abstract Schematic of a voltammetric immunosensor for the determination of clenbuterol (CLB) based on the use of a nanocomposite prepared from molybdenum disulfide and a gold-platinum alloy (MoS2-AuPt), and making use ofthe biotin-streptavidin system.

Effect of linkers on immobilization of scFvs with biotin-streptavidin interaction.

Single-chain variable fragment antibodies (scFvs) are attractive for use in applications that require high specificity and binding to a target, such as biosensors. Previously, we demonstrated that a variety of scFvs can be immobilized onto a streptavidin surface through in vivo biotinylation of the biotin carboxyl carrier protein (BCCP) or smaller AviTag fused to the scFvs. However, the BCCP constructs showed better immobilization than the AviTag constructs. In this work, we investigated whether the discrepancy between the biotinylation tags could be alleviated by incorporating a flexible (G4 S)n linker of varying lengths or a rigid (EA3 K)3 linker between the biotinylation tags and the scFvs scFv13R4 and scFv5. Fusion of the (G4 S)5 linker or the (G4 S)3 linker to the AviTag construct of scFv13R4 or scFv5, respectively, and fusion of the (EA3 K)3 linkers to the AviTag constructs of both scFvs enhanced immobilization. Meanwhile, the robust immobilization of the BCCP construct of the scFv constructs remained unaffected. The positive to neutral effects of the linkers, with no adverse effects, make them beneficial tools to incorporate into fusion proteins that show poor immobilization without a linker.