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1.
Anal Chem ; 92(6): 4459-4467, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32083467

RESUMO

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.


Assuntos
Avidina/análise , Espectrometria de Mobilidade Iônica , Conformação Proteica , Espectrometria de Massas em Tandem
2.
Anal Chem ; 92(3): 2476-2482, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31880149

RESUMO

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.


Assuntos
Avidina/análise , Biotina/análise , Dispositivos Lab-On-A-Chip , Temperatura
3.
Anal Chem ; 91(14): 8958-8965, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31251580

RESUMO

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@SiO2) 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.


Assuntos
Avidina/análise , Corantes Fluorescentes/química , Pontos Quânticos/química , Receptores de Fatores de Crescimento/análise , Biotina/análogos & derivados , Biotina/toxicidade , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Corantes Fluorescentes/toxicidade , Humanos , Ligantes , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Simulação de Acoplamento Molecular , Fenantrolinas/química , Fenantrolinas/toxicidade , Pontos Quânticos/toxicidade , Dióxido de Silício/química , Dióxido de Silício/toxicidade
4.
Chem Commun (Camb) ; 55(35): 5159-5162, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-30984931

RESUMO

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.


Assuntos
Avidina/análise , Proteínas Hemolisinas/química , Nanoporos , Hormônio Liberador de Tireotropina/análise , Lipossomas Unilamelares/química , Biotina/química , Fosfatidilcolinas/química , Ácido Fítico/química
5.
ACS Appl Mater Interfaces ; 11(8): 8470-8482, 2019 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-30694644

RESUMO

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 H2/CH4/N2 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 N2 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.


Assuntos
Avidina/análise , Técnicas Biossensoriais/métodos , Diamante/química , Grafite/química , Nanoestruturas/química , Técnicas Eletroquímicas , Ferricianetos/química , Gases/química , Limite de Detecção , Propilaminas/química , Silanos/química
6.
J Am Soc Mass Spectrom ; 30(1): 181-191, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30225732

RESUMO

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.


Assuntos
Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Proteínas de Membrana/química , Antiporters/análise , Antiporters/química , Avidina/análise , Avidina/química , Proteínas de Bactérias/análise , Proteínas de Bactérias/química , Soluções Tampão , Detergentes/química , Proteínas de Escherichia coli/análise , Proteínas de Escherichia coli/química , Glicerol/química , Lasers , Proteínas de Membrana/análise , Canais de Potássio/análise , Canais de Potássio/química , Espectrometria de Massas por Ionização por Electrospray/instrumentação
7.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1093-1094: 113-118, 2018 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-30015308

RESUMO

Since the importance of the high affinity between avidin and biotin, Kd = 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, Kd = 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.


Assuntos
Avidina , Compostos Azo/química , Cromatografia de Afinidade/métodos , Avidina/análise , Avidina/química , Avidina/isolamento & purificação , Avidina/metabolismo , Compostos Azo/metabolismo , Sítios de Ligação , Clara de Ovo/química
8.
Anal Sci ; 34(7): 765-770, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29998956

RESUMO

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.


Assuntos
Avidina/análise , Metais/química , Muramidase/análise , Soroalbumina Bovina/análise , Animais , Bovinos , Muramidase/metabolismo
9.
Talanta ; 185: 106-112, 2018 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-29759175

RESUMO

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.


Assuntos
Avidina/análise , Biotina/análise , Colorimetria , Ouro/química , Nanopartículas Metálicas/química
10.
Analyst ; 143(4): 829-832, 2018 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-29376175

RESUMO

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.


Assuntos
Sondas de DNA , Técnicas de Amplificação de Ácido Nucleico , Proteínas/análise , Avidina/análise , Humanos , Nanoestruturas , Soro
11.
Microb Cell Fact ; 16(1): 108, 2017 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-28619018

RESUMO

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 IgA1 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.


Assuntos
Citoplasma/química , Dissulfetos/química , Escherichia coli/genética , Animais , Avidina/análise , Avidina/biossíntese , Avidina/genética , Reatores Biológicos , Galinhas , Meios de Cultura/química , Citoplasma/metabolismo , Escherichia coli/química , Escherichia coli/citologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Feminino , Fermentação , Glucose/metabolismo , Glicerol/metabolismo , Hormônio do Crescimento Humano/biossíntese , Hormônio do Crescimento Humano/genética , Humanos , Fragmentos de Imunoglobulinas/biossíntese , Fragmentos de Imunoglobulinas/genética , Corpos de Inclusão/química , Corpos de Inclusão/metabolismo , Interleucina-6/biossíntese , Interleucina-6/genética , Oxirredução , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química
12.
Anal Chem ; 89(14): 7607-7614, 2017 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-28636334

RESUMO

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.


Assuntos
Álcool Desidrogenase/análise , Avidina/análise , Prótons , Albumina Sérica/análise , Estreptavidina/análise , Álcool Desidrogenase/metabolismo , Ânions/química , Cátions/química , Humanos , Espectrometria de Massas
13.
Langmuir ; 32(35): 8916-25, 2016 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-27506271

RESUMO

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.


Assuntos
Amiloide/análise , Avidina/análise , Imunoglobulina G/análise , Muramidase/análise , Compostos de Silício/química , Adsorção , Amiloide/química , Avidina/química , Técnicas Eletroquímicas , Imunoglobulina G/química , Cinética , Muramidase/química , Nanoporos/ultraestrutura , Soluções
14.
Biosens Bioelectron ; 82: 55-63, 2016 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-27040942

RESUMO

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.


Assuntos
Técnicas Biossensoriais/instrumentação , Proteínas/análise , Avidina/análise , Bacillus amyloliquefaciens/enzimologia , Proteínas de Bactérias/análise , Biotina/análise , Desenho de Equipamento , Humanos , Imunoglobulina G/análise , Ligantes , Ribonucleases/análise , Proteína Estafilocócica A/análise , Staphylococcus aureus/química , Estreptavidina/análise , Streptomyces/química
15.
Biosens Bioelectron ; 82: 240-7, 2016 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-27088369

RESUMO

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.


Assuntos
Técnicas Biossensoriais/métodos , DNA/análise , Nanotubos/química , Proteínas/análise , Prata/química , Avidina/análise , Biotina/análise , Fluorescência , Nanotecnologia , Nanotubos/ultraestrutura , Hibridização de Ácido Nucleico/métodos , Espectrometria de Fluorescência/métodos , Propriedades de Superfície
16.
Nat Commun ; 7: 11331, 2016 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-27090866

RESUMO

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.


Assuntos
Técnicas Biossensoriais/métodos , Ouro/química , Nanopartículas Metálicas/química , Nanotubos/química , Avidina/análise , Avidina/química , Biotina/análise , Biotina/química , Desenho de Equipamento , Nanopartículas Metálicas/ultraestrutura , Refratometria , Ressonância de Plasmônio de Superfície
17.
Artigo em Inglês | MEDLINE | ID: mdl-26978788

RESUMO

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.


Assuntos
Avidina/análise , Técnicas Biossensoriais/métodos , Biotina/química , Ouro/química , Nanopartículas Metálicas/química
18.
Anal Chem ; 88(2): 1412-8, 2016 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-26651518

RESUMO

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.


Assuntos
Técnicas Biossensoriais , Neoplasias da Mama/química , Ouro/química , Nanopartículas Metálicas/química , Nanotecnologia , Complexo Antígeno-Anticorpo/química , Antígenos de Neoplasias/análise , Avidina/análise , Feminino , Humanos , Metanfetamina/análise , Hormônios Tireóideos/análise
19.
Proteomics ; 15(21): 3623-8, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26256282

RESUMO

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.


Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Corantes Fluorescentes/química , Glicoproteínas/análise , Hidrazinas/química , Avidina/análise , Cromatografia Líquida , Eletroforese em Gel Bidimensional/métodos , Corantes Fluorescentes/síntese química , Humanos , Hidrazinas/síntese química , Orosomucoide/análise , Coloração e Rotulagem/métodos , Espectrometria de Massas em Tandem , Transferrina/análise
20.
Analyst ; 140(15): 5129-37, 2015 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-26034784

RESUMO

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.


Assuntos
Avidina/análise , Ferro/química , Espectroscopia de Ressonância Magnética/instrumentação , Nanopartículas Metálicas/química , Técnicas Analíticas Microfluídicas/instrumentação , Semicondutores , Biotinilação , Eletroumectação , Dispositivos Lab-On-A-Chip
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