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Mass-spectrometry based assays in structural biology studies measure either intact or digested proteins. Typically, different mass spectrometers are dedicated for such measurements: those optimized for rapid analysis of peptides or those designed for high molecular weight analysis. A commercial trapped ion mobility-quadrupole-time-of-flight (TIMS-Q-TOF) platform is widely utilized for proteomics and metabolomics, with ion mobility providing a separation dimension in addition to liquid chromatography. The ability to perform high-quality native mass spectrometry of protein complexes, however, remains largely uninvestigated. Here, we evaluate a commercial TIMS-Q-TOF platform for analyzing noncovalent protein complexes by utilizing the instrument's full range of ion mobility, MS, and MS/MS (both in-source activation and collision cell CID) capabilities. The TIMS analyzer is able to be tuned gently to yield collision cross sections of native-like complexes comparable to those previously reported on various instrument platforms. In-source activation and collision cell CID were robust for both small and large complexes. TIMS-CID was performed on protein complexes streptavidin (53 kDa), avidin (68 kDa), and cholera toxin B (CTB, 58 kDa). Complexes pyruvate kinase (237 kDa) and GroEL (801 kDa) were beyond the trapping capabilities of the commercial TIMS analyzer, but TOF mass spectra could be acquired. The presented results indicate that the commercial TIMS-Q-TOF platform can be used for both omics and native mass spectrometry applications; however, modifications to the commercial RF drivers for both the TIMS analyzer and quadrupole (currently limited to m / z 3000) are necessary to mobility analyze protein complexes greater than about 60 kDa.

Glycoproteins play a central role in many biological processes including disease mechanisms. Nevertheless, because glycoproteins are heterogeneous entities, it remains unclear how glycosylation modulates the protein structure and function. Here, we assess the ability of tandem-trapped ion mobility spectrometry-mass spectrometry (tandem-TIMS/MS) to characterize the structure and sequence of the homotetrameric glycoprotein avidin. We show that (1) tandem-TIMS/MS retains native-like avidin tetramers with deeply buried solvent particles; (2) applying high activation voltages in the interface of tandem-TIMS results in collision-induced dissociation (CID) of avidin tetramers into compact monomers, dimers, and trimers with cross sections consistent with X-ray structures and reports from surface-induced dissociation (SID); (3) avidin oligomers are best described as heterogeneous ensembles with (essentially) random combinations of monomer glycoforms; (4) native top-down sequence analysis of the avidin tetramer is possible by CID in tandem-TIMS. Overall, our results demonstrate that tandem-TIMS/MS has the potential to correlate individual proteoforms to variations in protein structure.

Concentration-polarization (CP)-based biomolecule preconcentration is highly effective in enhancing the detection sensitivity yet fails to precisely and dynamically control the location of the preconcentrated biomolecule plug to ensure overlap with the sensing region (e.g., immobilized molecular probes). Here, we used electrothermal (ET) stirring as a means of controlling the location of a preconcentrated biomolecule plug. The applied microfluidic device consisted of a Nafion membrane to induce the CP and an array of individually addressable microscale heaters for active local ET stirring. The experimental results demonstrated that such a novel platform enabled active control of the location of the preconcentrated plug of target biomolecules, ensuring its overlap with the functionalized microparticles and ultimately yielding enhanced detection sensitivity and binding kinetics. This was demonstrated using avidin-biotin particles as a simple bead-based bioassay model.

Sensitive and specific visualization of cell surface biotin receptors (BRs) a class of clinically important biomarkers, remains a challenge. In this work, a dual-emission ratiometric fluorescent nanoprobe is developed for specific imaging of cell surface avidin, a subtype of BRs. The nanoprobe comprises a dual-emission quantum dot nanohybrid, wherein a silica-encapsulated red-emitting QD (rQD@SiO 2 ) is used as the "core" and green-emitting QDs (gQDs) are used as "satellites", which are further decorated with a new "love-hate"-type BR ligand, a phenanthroline-biotin conjugate with an amino linker. The nanoprobe shows intense rQD emission but quenched gQD emission by the BR ligand. Upon imaging, the rQD emission stays constant and the gQD emission is restored as cell surface avidin accrues. Accordingly, the overlaid fluorescence color collected from red and green emission changes from red to yellow and then to green. We refer to such a color change as a traffic light pattern and the nanoprobe as a fluorescent traffic light nanoprobe. We demonstrate the application of our fluorescent traffic light nanoprobe to characterize cancer cells. By the traffic light pattern, cervical carcinoma and normal cells, as well as different-type cancer cells including BR-negative colon cancer cells, BR-positive hepatoma carcinoma cells, breast cancer cells, and their subtypes, have been visually differentiated. We further demonstrate a use of our nanoprobe to distinguish the G2 phase from other stages in a cell cycle. These applications provide new insights into visualizing cell surface biomarkers with remarkable imaging resolution and accuracy.

We have reported a versatile nanopore method based on the combination of analyte-controlled liposome signal amplification and the nanopore detection of a reporter molecule, which largely extends the nanopore application range, and easily elevates the nanopore sensitivity to the fM level from the μM level.

Hybrid diamond-graphite nanoplatelet (DGNP) thin films are produced and applied to label-free impedimetric biosensors for the first time, using avidin detection as a proof of concept. The DGNPs are synthesized by microwave plasma chemical vapor deposition through H 2 /CH 4 /N 2 gas mixtures in a reproducible and rapid single-step process. The material building unit consists of an inner two-dimensional-like nanodiamond with preferential vertical alignment covered by and covalently bound to nanocrystalline graphite grains, exhibiting {111} diamond ||{0002} graphite epitaxy. The DGNP films' morphostructural aspects are of interest for electrochemical transduction, in general, and for Faradaic impedimetric biosensors, in particular, combining enhanced surface area for biorecognition element loading and facile Faradaic charge transfer. Charge transfer rate constants in phosphate buffer saline/[Fe(CN) 6 ] 4- solution are shown to increase up to 5.6 × 10 -3 cm s -1 upon N 2 addition to DGNP synthesis. For the impedimetric detection of avidin, biotin molecules are covalently bound as avidin specific recognition elements on (3-aminopropyl)triethoxysilane-functionalized DGNP surfaces. Avidin quantification is attained within the 10-1000 μg mL -1 range following a logarithmic dependency. The limits of detection and of quantitation are 1.3 and 6.4 μg mL -1 (19 and 93 nM), respectively, and 2.3 and 13.8 μg mL -1 (33 and 200 nM) when considering the nonspecific response of the sensors.

Native mass spectrometry is applied for the investigation of proteins and protein complexes worldwide. The challenge in native mass spectrometry is maintaining the features of the proteins of interest, such as oligomeric state, bound ligands, or the conformation of the protein complex, during transfer from solution to gas phase. This is an essential prerequisite to allow conclusions about the solution state protein complex, based on the gas phase measurements. Therefore, soft ionization techniques are required. Widely used for the analysis of protein complexes are nanoelectro spray ionization (nESI) mass spectrometers. A newer ionization method is laser induced liquid bead ion desorption (LILBID), which is based on the release of protein complexes from solution phase via infrared (IR) laser desorption. We use both methods in our lab, depending on the requirements of the biological system we are interested in. Here we benchmark the performance of our LILBID mass spectrometer in comparison to a nESI instrument, regarding sample conditions, buffer and additive tolerances, dissociation mechanism and applicability towards soluble and membrane protein complexes. Graphical Abstract ᅟ.

Since the importance of the high affinity between avidin and biotin, K d  = 3 × 10 -16  M, gained universal recognition, numerous chemical, biological and medical avidin-biotin based applications have been developed. However, in some cases the high affinity may be a disadvantage, as this interaction is irreversible under physiological conditions. The dye, 4'-hydroxyazobenzene-2-carboxylic acid (HABA), binds avidin, at the biotin binding site, as determined by X-ray, at a much lower affinity constant, K d  = 6 × 10 -6  M. We prepared a HABA affinity column (amber colored). Avidin bound to the column at a pH between 4 and 8.5, causing a change of color to red, and it could be eluted at mild conditions with buffers containing biotin, HABA, 1.5 M potassium chloride or a pH lower than 4.0 or higher than 8.5. Avidin eluted with HABA, created a red avidin-HABA complex, which was visualized. HABA free avidin was obtained by dialysis, which was followed by the loss of red coloration. The novel and easy to use HABA-affinity column was employed in our lab to prepare pure, fully glycosylated avidin from egg white. Most importantly, it may serve as an ideal tool for educational purposes, illuminating concepts of molecular recognition, reversible molecular binding, structure-based molecular design and solid phase chemical synthesis, as it is a reliable and visible reagent., (Copyright © 2018. Published by Elsevier B.V.)

This paper reported a novel colorimetric assay strategy for avidin and biotin interactions based on terminal protection of the biotinylated single-stranded DNA and the surface plasmon resonance adsorption of gold nanoparticles (AuNPs). In this assay, it was firstly found that biotin-ssDNA specifically bound to the target protein avidin with strong affinity could be protected from hydrolysis by exonuclease I (Exo I). Furthermore, a colorimetric strategy was designed for the detection of avidin and biotin interactions. In the process, in the presence of avidin, the interaction of avidin and biotin protected the digestion of Exo I towards the biotin-ssDNA. The biotin-ssDNA with negatively charged would attach to the surface of AuNPs with positively charge in high salt solution through electrostatic interactions, which prevented AuNPs to aggregate. With the increased addition of avidin, the absorbance of AuNPs in 520 nm increased gradually and the color showed gradually wine red. By taking advantage of terminal protection, the developed strategy could offer high sensitivity for detecting small molecule-protein interactions. The results revealed that the developed strategy was highly sensitive for detecting avidin in the concentration ranging from 0.01 to 0.2 μg/mL with the detection limit of 4 × 10 -3 μg/mL.The developed assay also showed highly specific, cost-efficient and convenient. Moreover, this strategy only required labeling the small molecule on a single-stranded DNA, circumventing protein modifications that might be harmful for activity. In view of these advantages, this new colorimetric method could have potential to become a universal, sensitive, and selective platform for detection of small molecule-protein interactions., (Copyright © 2018. Published by Elsevier B.V.)

A simple and homogeneous technology, the steric effect-regulated isothermal exponential amplification reaction (SER-EXPAR), was developed to sense proteins. By using a small molecule linked DNA nanostructure, termed enzyme-binding hairpin (EBH), the protein-small molecule binding events could be readily sensed by utilizing the steric effect generated between the protein and enzyme. It set free the enzyme to be active again, thus regulating the amplification rate of EXPAR.

When proteins are attached to microstructures such as a metal mesh device, changes in their optical properties occur. These changes have been characterized based on actual measurements in the infrared region of the spectrum. We have previously theoretically and experimentally demonstrated the optical changes associated with streptavidin. Here, we investigate three types of proteins: avidin, BSA, and lysozyme. The three proteins were adsorbed onto three types of metal mesh devices having different resonant frequencies, and the corresponding spectra were measured in the infrared region. The change in the frequency of the dip point in the spectrum was extracted to quantitatively determine the quantity of protein; these results were correlated with the quantitative measurements obtained by electrophoresis. By examining three types of different proteins, it was verified that a variety of proteins can be measured based on the optical characteristics of metal mesh devices.

The effects of charge state on structures of native-like cations of serum albumin, streptavidin, avidin, and alcohol dehydrogenase were probed using cation-to-anion proton-transfer reactions (CAPTR), ion mobility, mass spectrometry, and complementary energy-dependent experiments. The CAPTR products all have collision cross-section (Ω) values that are within 5.5% of the original precursor cations. The first CAPTR event for each precursor yields products that have smaller Ω values and frequently exhibit the greatest magnitude of change in Ω resulting from a single CAPTR event. To investigate how the structures of the precursors affect the structures of the products, ions were activated as a function of energy prior to CAPTR. In each case, the Ω values of the activated precursors increase with increasing energy, but the Ω values of the CAPTR products are smaller than the activated precursors. To investigate the stabilities of the CAPTR products, the products were activated immediately prior to ion mobility. These results show that additional structures with smaller or larger Ω values can be populated and that the structures and stabilities of these ions depend most strongly on the identity of the protein and the charge state of the product, rather than the charge state of the precursor or the number of CAPTR events. Together, these results indicate that the excess charges initially present on native-like ions have a modest, but sometimes statistically significant, effect on their Ω values. Therefore, potential contributions from charge state should be considered when using experimental Ω values to elucidate structures in solution.

Background: The production of recombinant proteins containing disulfide bonds in Escherichia coli is challenging. In most cases the protein of interest needs to be either targeted to the oxidizing periplasm or expressed in the cytoplasm in the form of inclusion bodies, then solubilized and re-folded in vitro. Both of these approaches have limitations. Previously we showed that soluble expression of disulfide bonded proteins in the cytoplasm of E. coli is possible at shake flask scale with a system, known as CyDisCo, which is based on co-expression of a protein of interest along with a sulfhydryl oxidase and a disulfide bond isomerase. With CyDisCo it is possible to produce disulfide bonded proteins in the presence of intact reducing pathways in the cytoplasm., Results: Here we scaled up production of four disulfide bonded proteins to stirred tank bioreactors and achieved high cell densities and protein yields in glucose fed-batch fermentations, using an E. coli strain (BW25113) with the cytoplasmic reducing pathways intact. Even without process optimization production of purified human single chain IgA 1 antibody fragment reached 139 mg/L and hen avidin 71 mg/L, while purified yields of human growth hormone 1 and interleukin 6 were around 1 g/L. Preliminary results show that human growth hormone 1 was also efficiently produced in fermentations of W3110 strain and when glucose was replaced with glycerol as the carbon source., Conclusions: Our results show for the first time that efficient production of high yields of soluble disulfide bonded proteins in the cytoplasm of E. coli with the reducing pathways intact is feasible to scale-up to bioreactor cultivations on chemically defined minimal media.

For the past 2 decades, emerging single-nanopore technologies have opened the route to multiple sensing applications. Besides DNA sensing, the identification of proteins and amyloids is a promising field for early diagnosis. However, the influence of the interactions between the nanopore surface and proteins should be taken into account. In this work, we have selected three proteins (avidin, lysozyme, and IgG) that exhibit different affinities with the SiNx surface, and we have also examined lysozyme amyloid. Our results show that the piranha treatment of SiNx significantly decreases protein adsorption. Moreover, we have successfully detected all proteins (pore diameter 17 nm) and shown the possibility of discriminating between denatured lysozyme and its amyloid. For all proteins, the capture rates are lower than expected, and we evidence that they are correlated with the affinity of proteins to the surface. Our result confirms that proteins interacting only with the nanopore surface wall stay long enough to be detected. For lysozyme amyloid, we show that the use of the nanopore is suitable for determining the number of monomer units even if only the proteins interacting with the nanopore are detected.

As metal nano-arrays show great potential on metal enhanced fluorescence (MEF) than random nanostructures, MEF of Ag zigzag nanorod (ZNR) arrays made by oblique angle deposition has been studied for biomolecule-protein interaction and DNA hybridization. By changing the folding number and the deposition substrate temperature, a 14-fold enhancement factor (EF) is obtained for biotin-neutravidin detection. The optimal folding number is decided as Z=7, owing to the high scattering intensity of Ag ZNRs. The substrate temperature T=25°C and 0°C slightly alters the morphology of Ag ZNRs but has no big difference in EF. Further, Ag ZNRs deposited on a layer of Ag film have been introduced to the DNA hybridization and a significant signal enhancement has been observed through the fluorescence microscope. Through a detailed quantitative EF analysis, which excludes the enhancing effect from the increased surface area of ZNRs and only considers the contribution of MEF, an EF of 28 is achieved for the hybridization of two single-stranded oligonucleotides with 33 bases. Furthermore, a limit of detection is determined as 0.01pM. We believe that the Ag ZNR arrays can serve as a universal and sensitive bio-detection platform., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We present a simple and inexpensive method for label-free detection of biomolecules. The method monitors the changes in streaming current in a fused silica capillary as target biomolecules bind to immobilized receptors on the inner surface of the capillary. To validate the concept, we show detection and time response of different protein-ligand and protein-protein systems: biotin-avidin and biotin-streptavidin, barstar-dibarnase and Z domain-immunoglobulin G (IgG). We show that specific binding of these biomolecules can be reliably monitored using a very simple setup. Using sequential injections of various proteins at a diverse concentration range and as well as diluted human serum we further investigate the capacity of the proposed technique to perform specific target detection from a complex sample. We also investigate the time for the signal to reach equilibrium and its dependence on analyte concentration and demonstrate that the current setup can be used to detect biomolecules at a concentration as low as 100pM without requiring any advanced device fabrication procedures. Finally, an analytical model based on diffusion theory has been presented to explain the dependence of the saturation time on the analyte concentration and capillary dimensions and how reducing length and inner diameter of the capillary is predicted to give faster detection and in practice also lower limit of detection., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Avidin is a glycoprotein with antinutritional property, which should be limited in daily food. We developed an affinity biosensor system based on resonance Rayleigh scattering (RRS) and using affinity biotin labeling Au nanoparticles (AuNPs). This method was selective and sensitive for quick avidin detection due to the avidin-biotin affinitive interaction. Under optimal conditions, RRS intensity of biotin-AuNPs increase linearly with an increasing concentration of avidin from 5 to 160 ng/mL. The lower limit of detection was 0.59 ng/mL. This rapid and selective avidin detection method was used in synthetic samples and egg products with recoveries of between 102.97 and 107.92%, thereby demonstrating the feasible and practical application of this assay., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Biosensors based on the localized surface plasmon resonance (LSPR) of individual metallic nanoparticles promise to deliver modular, low-cost sensing with high-detection thresholds. However, they continue to suffer from relatively low sensitivity and figures of merit (FOMs). Herein we introduce the idea of sensitivity enhancement of LSPR sensors through engineering of the material dispersion function. Employing dispersion and shape engineering of chiral nanoparticles leads to remarkable refractive index sensitivities (1,091 nm RIU(-1) at λ=921 nm) and FOMs (>2,800 RIU(-1)). A key feature is that the polarization-dependent extinction of the nanoparticles is now characterized by rich spectral features, including bipolar peaks and nulls, suitable for tracking refractive index changes. This sensing modality offers strong optical contrast even in the presence of highly absorbing media, an important consideration for use in complex biological media with limited transmission. The technique is sensitive to surface-specific binding events which we demonstrate through biotin-avidin surface coupling.

It is broadly interesting but remains a big challenge to explore nanomaterials-based methods to enable naked-eye observation and determination of ultratrace biomarkers and drugs. In this study, we developed a straightforward and extendable plasmonic nanosensor to enable visually quantitative determination of ultratrace target molecules through combining the use of enzyme-mimetic gold nanoclusters (AuNCs). Starting from sandwiched antibody-antigen (i.e., an analyte)-antibody structure, we conjugated AuNCs on the outer layer antibody to catalyze the decomposition of hydrogen peroxide used to reduce HAuCl4 into gold nanopartilces (AuNPs) for naked eye readout. This strategy is in theory applicable to all immunoreactions available and the protocol proposed to attach AuNCs onto an antibody is suitable to all proteins. The applicability of this type of nanosensor was validated by the determination of various ultratrace analytes such as protein avidin, breast cancer antigen, thyroid hormone, and even methamphetamine (MA), giving a naked-eye-readout limit of detection (LOD), down to 1.0 × 10(-20) M protein avidin, 7.52 × 10(-14) U/mL breast cancer antigen 15-3, 2.0 × 10(-15) mg/mL 3,5,3'-L-triiodothyronine and 2.3 × 10(-18) mg/mL MA. This strategy is thus considered an ultrasensitive way to fabricate plasmonic nanosensors, having wide and invaluable application potential in clinical, biological, and environmental studies, and in food quality control.

In this study, a new hydrazide derivative (UGF202) was synthesized and introduced as a highly sensitive and selective fluorescent probe to pre-stain glycoproteins in 1D and 2D SDS-PAGE. As low as 0.5-1 ng glycoproteins (transferrin, α1-acid glycoprotein, avidin) could be selectively detected, which is comparable to that of Pro-Q Emerald 300 stain, one of the most sensitive and commonly used glycoprotein staining kit. In addition, the specificity of the newly developed method was confirmed by the study of de-glycosylation, glycoproteins affinity enrichment and LC-MS/MS, respectively. According to the results, it is concluded that UGF202 pre-stain can provide an alternative for the visualization of gel-separated glycoproteins., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Herein, we describe a micro-nuclear magnetic resonance (μNMR) relaxometer miniaturized to palm-size and electronically automated for multi-step and multi-sample chemical/biological diagnosis. The co-integration of microfluidic and microelectronic technologies enables an association between the droplet managements and μNMR assays inside a portable sub-Tesla magnet (1.2 kg, 0.46 Tesla). Targets in unprocessed biological samples, captured by specific probe-decorated magnetic nanoparticles (NPs), can be sequentially quantified by their spin-spin relaxation time (T2) via multiplexed μNMR screening. Distinct droplet samples are operated by a digital microfluidic device that electronically manages the electrowetting-on-dielectric effects over an electrode array. Each electrode (3.5 × 3.5 mm(2)) is scanned with capacitive sensing to locate the distinct droplet samples in real time. A cross-domain-optimized butterfly-coil-input semiconductor transceiver transduces between magnetic and electrical signals to/from a sub-10 μL droplet sample for high-sensitivity μNMR screening. A temperature logger senses the ambient temperature (0 to 40 °C) and a backend processor calibrates the working frequency for the transmitter to precisely excite the protons. In our experiments, the μNMR relaxometer quantifies avidin using biotinylated Iron NPs (Φ: 30 nm, [Fe]: 0.5 mM) with a sensitivity of 0.2 μM. Auto-handling and identification of two targets (avidin and water) are demonstrated and completed within 2.2 min. This μNMR relaxometer holds promise for combinatorial chemical/biological diagnostic protocols using closed-loop electronic automation.

Two biotinylated coumarin-based fluorescent probes SPS3 and RC3 were designed for differentiating between structurally similar proteins streptavidin (SA) and avidin (AV). A substituted phenyl group is introduced onto SPS3, which may quench the fluorescence through twist intramolecular charge transfer (TICT). The fluorescence of SPS3 is turned on, by restraining the TICT process, when the fluorophore is buried at the surface of SA. RC3 is constructed by incorporating a biotin molecule to a coumarin fluorophore through a 4-atom spacer. The fluorescence intensity of RC3 is enhanced significantly when its fluorophore enters into the less polar binding pocket of AV. SPS3 and RC3 could be applied in distinguishing between SA and AV as well as in fluorescence imaging of biotin receptor over-expressed Hela cells.

The catalytic hairpin DNA assembly-programmed active Mg(2+)-dependent DNAzyme was proposed for dual-signal amplified detection toward protein and DNA. The protein detection was implemented with the further combination of an additional terminal protection strategy. The detection limit toward avidin and target DNA could be achieved as 2 pM and 0.5 pM, respectively, with a high selectivity.

Enzyme-catalyzed signal amplification with an antibody-enzyme conjugate is commonly employed in many bioanalytical methods to increase assay sensitivity. However, covalent labeling of the enzyme to the antibody, laborious operating procedures, and extensive washing steps are necessary for protein recognition and signal amplification. Herein, we describe a novel label-free and washing-free enzyme-amplified protein detection method by using dual-functional synthetic molecules to impose steric effects upon protein binding. In our approach, protein recognition and signal amplification are modulated by a simple dual-functional synthetic probe which consists of a protein ligand and an inhibitor. In the absence of the target protein, the inhibitor from the dual-functional probe would inhibit the enzyme activity. In contrast, binding of the target protein to the ligand perturbs this enzyme-inhibitor affinity due to the generation of steric effects caused by the close proximity between the target protein and the enzyme, thereby activating the enzyme to initiate signal amplification. With this strategy, the fluorescence signal can be amplified to as high as 70-fold. The generality and versatility of this strategy are demonstrated by the rapid, selective, and sensitive detection of four different proteins, avidin, O6-methylguanine DNA methyltransferase (MGMT), SNAP-tag, and lactoferrin, with four different probes.

Efficient diagnosis is very important for the prevention and treatment of diseases. Rapid disease screening in ambulatory environment is one of the most pressing needs for disease control. Despite there are many methods to detect the results of immunoassays, quantitative measurement for rapid disease screening is still a great challenge for point-of-care applications. In this study, a fabrication method for depositing carbon nanotube bundles has been successfully developed for realization of functional paper-based microfluidic sensing device. Quantitative detection of label-free immunoassay, i.e., biotin-avidin binding interaction, was demonstrated by direct measurement of the current change of the biosensor after single application of the target analyte. Sensitivity of 0.33 μA/ng mL(-1) and minimal detectable analyte concentration of 25 ng/mL were achieved. The time necessary for the detection was 500 s which is a large reduction compared with the conventional immunoassay. Such paper-based biosensor has the benefits of portability, fast response, simple operation, and low cost and has the potential for the development of rapid disease screening devices., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Inflammatory bowel diseases are chronic gastrointestinal pathologies causing great discomfort in both children and adults. The pathogenesis of inflammatory bowel diseases is not yet fully understood and their diagnosis and treatment are often challenging. Nanoparticle-based strategies have been tested in local drug delivery to the inflamed colon. Here, we have investigated the use of the novel avidin-nucleic acid nanoassembly (ANANAS) platform as a potential diagnostic carrier in an experimental model of inflammatory bowel diseases. Fluorescent- labeled ANANAS nanoparticles were administered to mice with chemically induced chronic inflammation of the large intestine. Localization of mucosal nanoparticles was assessed in vivo by dual-band confocal laser endomicroscopy. This technique enables characterization of the mucosal microvasculature and crypt architecture at subcellular resolution. Intravascular nanoparticle distribution was observed in the inflamed mucosa but not in healthy controls, demonstrating the utility of the combination of ANANAS and confocal laser endomicroscopy for highlighting intestinal inflammatory conditions. The specific localization of ANANAS in inflamed tissues supports the potential of this platform as a targeted carrier for bioactive moieties in the treatment of inflammatory bowel disease.

A new electrochemical label has been developed, which is made up of silver nanoparticles (AgNPs) coated with a mixture of zwitterionic and biotinylated zwitterionic polymers. These polymers improve colloidal stability in physiological medium and ensure biorecognition while direct electrochemical oxidation of silver nanoparticles strongly enhances the detection signal. The resulting hybrid nanomaterials are used as labels in the electrochemical sensing of avidin using sandwich assays elaborated using the biotin-avidin biorecognition system.

Thermoresponsive immunonephelometry was achieved with biotinylated poly(acrylate) and thermoresponsive gold nanocomposites composed of 13-nm gold nanoparticles and thermoresponsive polymers containing triethylenetetramine and biotin groups. The avidin-biotin interaction was used to model an immunoreaction in order to demonstrate thermoresponsive immunonephelometry. In the absence of avidin, positively charged gold nanocomposites electrostatically interacted with biotinylated poly(acrylate) to form binary complexes, in which the charges canceled each other out. The charge cancelation resulted in the binary complexes precipitating when the solution was heated above the phase-transition temperature. However, adding avidin formed ternary sandwich complexes through the avidin-biotin interaction. The ternary complexes remained sufficiently soluble above the phase-transition temperature because of the spatial isolation of the positive and negative charges. The transmittance of the solution containing the thermoresponsive gold nanocomposites and biotinylated poly(acrylate) at 37°C increased as the avidin concentration increased. A sigmoidal profile was observed from 10(-6.5) to 10(-5.5) mol/L. The concentration of avidin spiked in bovine serum was determined by our method.

Upconversion luminescence (UCL) detection based on rare-earth doped upconversion nanocrystals (UCNCs) as probes has been proved to exhibit a large anti-Stokes shift, no autofluorescence from biological samples, and no photobleaching. However, it is still a challenge to achieve a stable, reproducible solid-based UCL biosensor because of ineffective UCL of the UCNCs. In this work, we fabricated TiO2 inverse opal photonic crystals (IOPCs)/NaYF4:Yb(3+),Tm(3+) (Er(3+)) UCNC composite films, which can tremendously improve the overall UCL of Tm(3+) as high as 43-fold. Based on the fluorescence resonance energy transfer (FRET) and the specific interaction between biotin and avidin, a novel solid-based UC biosensor is presented for sensing avidin. This solid-based detection system is convenient for detection, and also can offer two parameters for detecting trace amounts of avidin, namely, the emission intensity and the fluorescence decay time. The sensor has a high sensitivity of 34 pmol(-1), a good linear relationship of 0.996 and a low detection limit of 48 pmol. It also exhibits excellent long-time photostability, and the absence of autofluorescence, and thus may have great potential for versatile applications in biodetection.

The first example of an all-organic oligofluorene truxene based distributed feedback laser for the detection of a specific protein-small molecule interaction is reported. The protein avidin was detected down to 1 μg mL(-1) using our biotin-labelled biosensor platform. This interaction was both selective and reversible when biotin was replaced with desthiobiotin. Avidin detection was not perturbed by Bovine Serum Albumin up to 50,000 μg mL(-1). Our biosensor offers a new detection platform that is both highly sensitive, modular and potentially re-usable., (Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.)

Fluorescence quenching effect of water-soluble anionic conjugated polymer (CP) (poly[5-methoxy-2-(3-sulfopoxy)-1,4-phenylenevinylene] (MPS-PPV)) by [Re(N-N)(CO)3(py-CH2-NH-biotin)](PF6) [N-N=2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline; py-CH2-NH-biotin=N-[(4-pyridyl) methyl] biotinamide] (Re-Biotin) and fluorescence recovery in the presence of streptavidin (or avidin) were investigated using Re-Biotin as quencher tether ligand (QTL) probe. Meanwhile, the mechanisms of fluorescence quenching and recovery were discussed to provide new thoughts to design biosensor based on water-soluble CPs. The results indicate that the sensing mechanisms of streptavidin sensor or avidin sensor, using Re-Biotin as QTL probe, are the same and stable, whether in non-buffer system (aqueous solution) or different buffer systems [0.01 mol·L(-1) phosphate buffered solution (pH=7.4), 0.1 mol·L(-1) ammonium carbonate buffered solution (pH=8.9)]. There exists specific interactions between streptavidin (or avidin) and biotin of Re-Biotin. Fluorescence quenching and recovery processes of MPS-PPV are reversible. Mechanisms of Re-Biotin quenching MPS-PPV fluorescence can be interpreted as strong electrostatic interactions and charge transferences between Re-Biotin and MPS-PPV. Fluorescence recovery mechanisms of Re-Biotin-MPS-PPV system can be interpreted as specific interactions between streptavidin (or avidin) and biotin of Re-Biotin making Re-Biotin far away from MPS-PPV. Avidin or strptavidin as re-Biotin probe can not only be quantitatively determinated, but also be identified., (Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.)

We present an organic semiconductor distributed feedback laser biosensor and demonstrate its sensing capabilities. Optimization of the gain layer thickness to maximize the response to refractive index changes and bulk sensing results are shown. Desthiobiotin-avidin sensing assay results are also presented and the potential to perform multiple, repeated sensing measurements is demonstrated.

Graphene related materials have been widely employed as highly efficient transducers for biorecognition. Here we show a conceptually new approach of using graphene oxide nanoplatelets (50 × 50 nm) as voltammetric inherently active labels for specific protein-target molecule recognition. This proof-of-principle is demonstrated by biotin-avidin recognition, which displays that graphene oxide nanoplatelet labels show excellent selectivity. Therefore, it is expected that inherently electroactive graphene oxide nanoplatelet labels will play a similar role as electroactive gold nanoparticle labels which were developed more than a decade ago.

Platforms which can display cell membrane ligands and receptors as a microarray library of probes for screening against a target are essential tools in drug discovery, biomarker identification, and pathogen detection. Membrane receptors and ligands require their native bilayer environment to retain their selectivity and binding affinity, and this complicates displaying them in a microarray platform. In this study, a design is developed in which the probes are first incorporated in supported lipid bilayers formed around micron-sized particles (lipobeads), and the microbeads themselves are then arrayed on a surface by hydrodynamic capture in a microfluidic obstacle course of traps. The traps are "V" shaped open enclosures, which are arranged in a wide channel of a microfluidic device, and capture the lipobeads (slightly smaller than the channel height) as they are streamed through the course. Screening assays are undertaken directly in the device after assembly, by streaming a fluorescently labeled target through the device and detecting the bead fluorescence. Conditions are first established for which the supported bilayers on the bead surface remain intact during the capture and assay steps, using fluorescent tags in the bilayer to infer bilayer integrity. Numerical calculations of the hydrodynamic drag coefficient on the entrapped beads are presented in conjunction with the stability experiments to develop criteria for the bilayer stability as a function of the screening assay perfusion rate. Simulations of the flow streamlines are also presented to quantify the trapping efficiency of the obstacle course. Screening assays are illustrated, assaying fluorescently labeled NeutrAvidin with biotin, and labeled cholera toxin with its ganglioside binding ligand, GM1. Sequential capturing of sets of lipobeads (one at a time, and with each set bearing a different probe), followed by indexing the bead positions after each set is entrapped, allows for the construction of an indexed array of multiple probes without the need for particle encoding and is illustrated using the NeutrAvidin-biotin pair. Finally, the lipobead platform is used for quantitatively measuring the kinetic rate constants for the binding of a probe (biotin) to a target (NeutrAvidin).

Carbon nanotube (CNT) has been utilized for the biological detection due to its extremely sensitive to biological molecules. A paper-based CNT sensing microfluidic device has been developed for the detection of protein, i.e., biotin-avidin, binding. We have developed a fabrication method that allows controlled deposition of bundled CNTs with well-defined dimensions to form sensors on paper. Then, polydimethyl siloxane (PDMS) was used to pattern the hydrophobic boundary on paper to form the reaction sites. The proposed fabrication method is based on vacuum filtration process with a metal mask covering on a filter paper for the definition of the dimension of sensor. The length, width, and thickness of the CNT-based sensors are readily controlled by the metal mask and the weight of the CNT powder used during the filtration process, respectively. Homogeneous deposition of CNTs with well-defined dimensions can be achieved. The CNT-based sensor on paper has been demonstrated on the detection of the protein binding. Biotin was first immobilized on the CNT's sidewall and avidin suspended solution was applied to the site. The result of the biotin-avidin binding was measured by the resistance change of the sensor, which is a label-free detection method. It showed the CNT is sensitive to the biological molecules and the proposed paper-based CNT sensing device is a possible candidate for point-of-care biosensors. Thus, electrical bio-assays on paper-based microfluidics can be realized to develop low cost, sensitive, and specific diagnostic devices.

In this research, we mainly focused on the micro-emulsion synthesis of biotinylated ZnS (zinc sulfide) nanocrystals for avidin recognition. Various samples of Zn(1-x)Mn(x) S, with x = 0.0001, 0.007, 0.02, 0.03, 0.055, 0.09 and 0.13, prepared by quaternary W/O (water-in-oil) microemulsion system. Cyclohexane was used as oil, Triton X-100 as surfactant, n-hexanol as a co-surfactant and mercaptoethanol and thioglycolic acid as linking agents. The obtained products were evaluated by commonly techniques such as: scanning electron microscopy (SEM), transmission electron microscopy (TEM), zeta meter for measurement ZP (zeta potential) and fluorescence spectroscopy analyses. The above-experimental results indicated that the optimum doping concentration of Mn was ~ 5.5% . The fluorescence spectra of the doped crystals consist of orange-red emissions. Eventually, this research showed with increasing more than 18 μl biotin to nanocrystals, no changes were observed in the emission intensity spectra.

This paper describes thin films comprising acenes dispersed in a conjugated polymeric host that have a ratiometric photoluminescence response to singlet oxygen. These films also respond to irradiation of protein-bound sensitizers, which represents a solution to the problem of protein-conjugated polymer non-specific interactions.

Avidin from egg white was migrated toward a cathode of nondenaturing electrophoresis and then immobilized on a polyvinylidene difluoride membrane. Adrenocorticotropic hormone (ACTH) was specifically captured after the biotinylated anti-ACTH antibody was bound to the membrane-immobilized avidin, and the captured ACTH was digested by the biotinylated trypsin on the membrane after extraction. The digested polypeptides from the ACTH were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). These results indicate that target substances can be specifically trapped and digested on membrane-immobilized avidin., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A new quantitative analysis methodology for localized surface plasmon resonance (LSPR) biosensing which determines surface-receptor fractional occupancy, as well as an LSPR imaging technique for the spatiotemporal mapping of binding events, is presented. Electron beam nanolithography was used to fabricate 20 × 20 arrays of gold nanostructures atop glass coverslips. A single biotinylated array was used to measure the association kinetics of neutravidin to the surface by spectroscopically determining the fractional occupancy as a function of time. By regenerating the same array, a reliable comparison of the kinetics could be made between control samples and neutravidin concentrations ranging from 1 μM to 50 nM. CCD-based imagery of the array, taken simultaneously with the spectroscopic measurements, reveals the binding of neutravidin to the surface as manifested by enhanced scattering over the majority of the resonance peak. The temporal resolution of the LSPR imaging technique was 200 ms and the spatial resolution was 8 μm(2)., (© 2011 American Chemical Society)

This report presents biotin-functionalized semiconducting polymers that are based on fluorene and bithiophene co-polymers (F8T2). Also presented is the application of these polymers to an organic thin film transistor used as a biosensor. The side chains of fluorene were partially biotinylated after the esterification of the biotin with corresponding alcohol-groups at the side chain in F8T2. Their properties as an organic semiconductor were tested using an organic thin film transistor (OTFT) and were found to show typical p-type semiconductor curves. The functionality of this biosensor in the sensing of biologically active molecules such as avidin in comparison with bovine serum albumin (BSA) was established through a selective decrease in the conductivity of the transistor, as measured with a device that was developed by the authors. Changes to the optical properties of this polymer were also measured through the change in the color of the UV-fluorescence before and after a reaction with avidin or BSA.

The paramagnetic microbead-based electrochemical binding assay was demonstrated for detecting two kinds of protein simultaneously. The principle of this assay is based on the sequestration electrochemistry. The protein binding electroactive magnetic microbeads which are conjugated with an electroactive compound and a ligand to bind specifically with a target protein were prepared. The avidin-biotin and soybean agglutinin (SBA)-galactosamine were chosen as model protein-ligand systems. The avidin binding electroactive magnetic microbead (ABEMMb) and SBA binding electroactive magnetic microbead (SBEMMb) are constructed by biotin/thionine and galactosamine/ferrocene modified on paramagnetic microbeads. The voltammetric response for these functionalized microbeads was measured by the Nd-Fe-B magnet-incorporating carbon paste rotating disk electrode. The measurements were performed in a microliter droplet using a rotating disk electrode system. Avidin and SBA were simultaneously detected by the decrease in the current responses from the reduction of ABEMMb and SBEMMb that was caused by the binding with target proteins. The limits of detection for avidin and SBA were 4 × 10(-10) and 2 × 10(-10) M, respectively.

This paper presents highly sensitive fluorescence detections of avidin and streptavidin using an optical interference mirror (OIM) slide consisting of a plane mirror covered with an optical interference layer. Compared with a common glass slide, the OIM slide can enhance the fluorescence from a dye by more than 100-fold. We fabricated an OIM slide by depositing an optical interference layer of Al(2)O(3) on an Ag mirror. To enhance the fluorescence maximally, the optimal thickness of the Al(2)O(3) layer was estimated from optical interference theory. For detections of protein, avidin/streptavidin labeled with fluorescein, Cy3, and Cy5 were detected with biotin immobilized on an OIM slide with the optimal Al(2)O(3) thickness. We achieved a sensitivity improvement of more than 50-fold, comparing with a glass slide. Such a high degree of improvement would be a significant contribution to further progress in biomedical research and medical diagnostics.

We have developed switchable lanthanide luminescence-based binary probe technology for homogeneous detection of avidin, which is a tetrameric protein. Two different nonluminescent label moieties--a light-absorbing antenna ligand and a lanthanide ion carrier chelate--were conjugated to separate biotins, which is known as avidin's natural ligand. The assay was based on binding of the two differently labeled biotins on separate binding sites on the target protein and consequent self-assembly of a luminescent complex from the two label moieties. Specific luminescence signal was observed only at the presence of the target protein. The characteristics of the switchable lanthanide luminescence assay were compared to the reference assay, based on lanthanide resonance energy transfer. Both assays had a limit of detection in the low-picomolar concentration range; however, the lanthanide chelate complementation-based assay had wider dynamic range and its optimization was more straightforward. The switchable lanthanide luminescence technology could be further applied to generic protein detection, using reagents that are analogous to the proximity ligation assay principle.

Introduction: Familial hemiplegic migraine type 1 (FHM-1) is caused by mutations in the CACNA1A gene, with the R192Q mutation being the most common. Elevated calcitonin gene-related peptide (CGRP) levels in acute migraine and clinical trials using CGRP receptor antagonists suggest CGRP-related mechanisms are important in migraine., Methods: Wild-type and R192Q knock-in mice were anaesthetized and perfused. Using immunohistochemical staining, the expression of CGRP in the trigeminocervical complex (TCC) and in the trigeminal and dorsal root ganglia was characterized., Results: There was a 38% reduction in the percentage of CGRP-immunoreactive cells in the trigeminal ganglia (p < 0.001) of R192Q knock-in mice compared to wild-type animals. The size distribution profile of CGRP-immunoreactive cells within the trigeminal ganglia demonstrated no significant difference in cell diameter between the two groups (p ≥ 0.56). CGRP expression was also reduced in thoracic ganglia of R192Q knock-in mice (21% vs. 27% in wild-type group; p < 0.05), but not in other ganglia. In addition, decreased CGRP immunoreactivity was observed in the superficial laminae of the TCC in R192Q knock-in mice, when compared to the control group (p < 0.005)., Conclusion: The data demonstrates that the FHM-1 CACNA1A mutation alters CGRP expression in the trigeminal ganglion and TCC. This suggests further study of these animals is warranted to characterize better the role of these mutations in the neurobiology of migraine.

We demonstrate the detection of dissolved avidin concentrations as low as 15 nM or 1 μg/ml using functionalized slotted photonic crystal cavities with integrated microfluidics. With a cavity sensing surface area of approximately 2.2 μm(2), we are able to detect surface mass densities of order 60 pg/mm(2) corresponding to a bound mass of approximately 100 ag. The ultra-compact size of the sensors makes them attractive for lab-on-a-chip applications where high densities of independent sensing elements are desired within a small area. The high sensitivity over an extremely small area is due to the strong modal overlap with the analyte enabled by the slotted waveguide cavity geometry that we employ. This strong overlap results in larger shifts in the cavity peak wavelength when compared to competing approaches., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We have employed the proposed Silica-Silver Core-Shell (SSCS) SERS-active substrates to detect four model proteins: lysozyme (a protein without chromophore), cytochrome c (a protein with chromophore of heme), fluorescein isothiocyanate (FITC)-anti human IgG (labeled with FITC) and atto610-biotin/avidin (recognition with labeled small molecules). SERS spectra of these proteins and Raman labels on the SSCS substrates show both high sensitivity and reproducibility, which are due to electromagnetic SERS enhancement with additional localization field within closely packed Ag nanoparticles decorated on the SiO(2) nanoparticles and the aggregation of SiO(2)@Ag particles. We have found that the SERS intensities of atto610-biotin/avidin adsorbed on the SSCS substrates are about 20 times stronger than those from Ag plating on Au-decorated substrate. Moreover, the broad surface plasmon resonance (SPR) of the proposed substrates will extend SERS applications to more biological molecules with different laser excitations., (Copyright © 2011 Elsevier Inc. All rights reserved.)

This paper presents an overview of development of a novel disposable plastic biochip for multiplexed clinical diagnostic applications. The disposable biochip is manufactured using a low-cost, rapid turn- around injection moulding process and consists of nine parabolic elements on a planar substrate. The optical elements are based on supercritical angle fluorescence (SAF) which provides substantial enhancement of the fluorescence collection efficiency but also confines the fluorescence detection volume strictly to the immediate proximity of the biochip surface, thereby having the potential to discriminate against background fluorescence from the analyte solution. An optical reader is also described that enables interrogation and fluorescence collection from the nine optical elements on the chip. The sensitivity of the system was determined with a biotin-avidin assay while its clinical utility was demonstrated in an assay for C-reactive protein (CRP), an inflammation marker.

In this study, we demonstrate that the protein binding capacity of a surface modified matrix-assisted laser desorption/ionization (MALDI) target can be increased significantly by architecturing the surface of the MALDI probe using gold microparticles. In the present approach, a MALDI target, initially modified via pulsed rf plasma deposition of an allyl amine polymer thin film, is subsequently architectured via reaction with 2-iminothiolane and surface attachment of gold microparticles. The modified probe is then exposed to thiolated biotin to introduce an avidin binding element on the surface of the gold beads. The protein binding capacity of this architectured target is compared with a similarly plasma polymer modified MALDI target that is directly biotinylated. Application of various surface concentrations of avidin to the two probes and MALDI-MS analysis of avidin contained in the solution removed from the probe reveals that saturation of the gold-particle architectured target occurs at a factor of 15-30 higher applied surface concentration, as compared with the unarchitectured target. Furthermore, MALDI-MS analysis of the avidin retained on the two probes reveals that the limit of detection is lowered by a factor of 15-20 on the gold-particle architectured target as compared with the unarchitectured target.

There has been immense interest in both instruments and methods to enhance fluorescence signal and achieve highly sensitive fluoroimmunoassay (FIA). In this paper, we present a facile, low-cost and general method of biotinylated colloidal photonic crystal (PC) to improve the FIA of avidin (avidin FIA). The fluorescence signal intensity of the avidin FIA on the colloidal PC can be enhanced over two orders of magnitude relative to the control sample, attributed to the large surface area, resonance field and coherent scattering effect of the colloidal PC. The detection limit is shrunk to 1/69 of that of the control sample. Furthermore, the signal to interference ratio (S/I ratio) is increased because the band-edge induced fluorescence enhancement is wavelength-selective. The interference fluorescence does not go up proportionally while the signal is significantly enhanced by the colloidal PCs. It is believed that the colloidal PC modified with biotin can act as an effective material for a general and sensitive fluoroimmunoassay., (Copyright © 2010 Elsevier B.V. All rights reserved.)