RESUMEN
In recent years, there has been growing concern among consumers about pesticide contamination in fruits. Therefore, rapid, reliable, and consistent detection methods for OPPs, especially dimethoate, are crucially needed. The existing quantitative methods for detecting dimethoate are not suitable for rapid measuring system such as the dimethoate samples from two channels. Hence this paper examines the utilization of a dual-channel system for utilize the absorption variations of the Localized Surface Plasmon Resonance (LSPR) bands of gold nanoparticles (AuNPs) were investigate for detection of dimethoate. Under optimized conditions, the relationship between concentrations of dimethoate and absorbance ratios (A(520)/A(640)) was linearly found in the concentration range of 10-100 nM. Result from the experiment shows that both channels exhibit a linear correlation coefficient as high as 0.97 and a limit of detection (LOD) as low as 5.5 nM. This LSPR detection system was characterized by testing the dimethoate in apple samples and the recovery rates were found to be in the range of 85.90% to 107.37%. The proposed dual-channel LSPR system for detecting dimethoate creating a new approach for detecting organophosphate insecticide in agricultural fields. It could lay the foundation for designing a high-throughput analysis of the insecticides using a wavelength division multiplexing switch (WDMS).
Asunto(s)
Productos Agrícolas/normas , Dimetoato/análisis , Análisis de los Alimentos/métodos , Contaminación de Alimentos/análisis , Frutas/normas , Insecticidas/análisis , Resonancia por Plasmón de Superficie/métodos , Productos Agrícolas/química , Análisis de los Alimentos/normas , Frutas/química , Sensibilidad y Especificidad , Resonancia por Plasmón de Superficie/normasRESUMEN
The ongoing outbreak of the novel coronavirus disease (COVID-19) has spread globally and poses a threat to public health in more than 200 countries. Reliable laboratory diagnosis of the disease has been one of the foremost priorities for promoting public health interventions. The routinely used reverse transcription polymerase chain reaction (RT-PCR) is currently the reference method for COVID-19 diagnosis. However, it also reported a number of false-positive or -negative cases, especially in the early stages of the novel virus outbreak. In this work, a dual-functional plasmonic biosensor combining the plasmonic photothermal (PPT) effect and localized surface plasmon resonance (LSPR) sensing transduction provides an alternative and promising solution for the clinical COVID-19 diagnosis. The two-dimensional gold nanoislands (AuNIs) functionalized with complementary DNA receptors can perform a sensitive detection of the selected sequences from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through nucleic acid hybridization. For better sensing performance, the thermoplasmonic heat is generated on the same AuNIs chip when illuminated at their plasmonic resonance frequency. The localized PPT heat is capable to elevate the in situ hybridization temperature and facilitate the accurate discrimination of two similar gene sequences. Our dual-functional LSPR biosensor exhibits a high sensitivity toward the selected SARS-CoV-2 sequences with a lower detection limit down to the concentration of 0.22 pM and allows precise detection of the specific target in a multigene mixture. This study gains insight into the thermoplasmonic enhancement and its applicability in the nucleic acid tests and viral disease diagnosis.
Asunto(s)
Betacoronavirus/química , Técnicas Biosensibles/métodos , Resonancia por Plasmón de Superficie/métodos , Betacoronavirus/genética , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/normas , ADN Complementario/química , ADN Complementario/genética , Oro/química , Calor , Nanopartículas del Metal/química , Hibridación de Ácido Nucleico/métodos , SARS-CoV-2 , Resonancia por Plasmón de Superficie/instrumentación , Resonancia por Plasmón de Superficie/normasRESUMEN
Transparent conducting oxides (TCOs) have appeared in the past few years as potential plasmonic materials for the development of optical devices in the near infrared regime (NIR). However, the performance of biosensors with TCOs has been limited in sensitivity and figure of merit (FOM). To improve the performance of the biosensors with TCOs, a biosensor based on long-range surface plasmon with Ga-doped zinc oxide (GZO) is proposed. It is shown that a larger FOM with a 2~7 times enhancement compared to the traditional surface plasmon polaritons (SPPs) sensor and higher detection accuracy (DA) can be realized in our proposed sensor compared with the surface plasmon resonance (SPR) sensor with GZO. Therefore, this sensor can be used to detect biological activity or chemical reactions in the near infrared region.
Asunto(s)
Galio , Resonancia por Plasmón de Superficie/métodos , Óxido de Zinc , Dispositivos Ópticos , Resonancia por Plasmón de Superficie/normasRESUMEN
Surface Plasmon Resonance Biosensors (SPR) are one of the most powerful tools to characterize protein binding, e.g. for drug discovery, like target identification, ligand fishing, assay development, lead selection and manufacturing quality control. However, there is increasing concern about its reproducibility in the light of the reproducibility crisis. Therefore an appropriate analytical instrument qualification (AIQ) is required for quality assurance of SPR instruments. AIQ is a prerequisite for analytical method validation and it is consisting of four parts, Design Qualification (DQ), Installation Qualification (IQ), Operational Qualification (OQ) and Performance Qualification (PQ). PQ regularly executed is supposed to continuously control the performance of the instrument under actual running conditions. In this work a performance qualification method was developed for the SPR instrument Biacore X100. This method is suitable for the routinely control of the instrument performance for antibody-antigen binding measurements. Control charts were designed to get a clearly representable and easy implementable tool to check the critical parameters. These control charts and a straightforward protocol now allow the design and application of an individual performance qualification procedure that can be used in the laboratory routine. They serve as reference for individual standard operation procedures (SOPs).
Asunto(s)
Resonancia por Plasmón de Superficie , Reacciones Antígeno-Anticuerpo , Reproducibilidad de los Resultados , Resonancia por Plasmón de Superficie/normasRESUMEN
The introduction of nanomaterials as detection reagents has enabled improved sensitivity and facilitated detection in a variety of bioanalytical assays. However, high nanoprobe densities are typically needed for colorimetric detection and to circumvent this limitation several enhancement protocols have been reported. Nevertheless, there is currently a lack of universal, enzyme-free and versatile methods that can be readily applied to existing as well as new biosensing strategies. The novel method presented here is shown to enhance the signal of gold nanoparticles enabling visual detection of a spot containing <10 nanoparticles. Detection of Protein G on paper arrays was improved by a 100-fold amplification factor in under five minutes of assay time, using IgG-labelled gold, silver, silica and iron oxide nanoprobes. Furthermore, we show that the presented protocol can be applied to a commercial allergen microarray assay, ImmunoCAP ISAC sIgE 112, attaining a good agreement with fluorescent detection when analysing human clinical samples.
Asunto(s)
Técnicas Biosensibles/métodos , Nanopartículas del Metal , Técnicas Biosensibles/normas , Oro/química , Humanos , Proteínas Inmovilizadas/análisis , Resonancia por Plasmón de Superficie/métodos , Resonancia por Plasmón de Superficie/normasRESUMEN
There is increasing clinical interest in extracellular vesicles (EV) for diagnostic and treatment purposes. This review provides an overview of bulk immunoassays to analyse EV. Western blot and enzyme-linked immunosorbent assay are still the two predominant bulk immunoassays. Recently, new assays have become available that can detect exposure to EV concentrations that are up to 10,000-fold lower. This is advantageous for applications that detect rare EV. Other important parameters are the detectable concentration range, the required sample volume, whether simultaneous presence of different antigens on a single EV can be detected, size selectivity of each assay and practical considerations. In this review, we will explain the working principles of the traditional and novel assays together with their performance parameters. The most sensitive assays are micro-nuclear magnetic resonance, surface plasmon resonance, and time-resolved fluorescent immunoassay.
Asunto(s)
Plaquetas/metabolismo , Western Blotting/normas , Ensayo de Inmunoadsorción Enzimática/normas , Vesículas Extracelulares/metabolismo , Fluoroinmunoensayo/normas , Resonancia por Plasmón de Superficie/normas , Biomarcadores/análisis , Plaquetas/citología , Western Blotting/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Vesículas Extracelulares/química , Fluoroinmunoensayo/instrumentación , Fluoroinmunoensayo/métodos , Humanos , Límite de Detección , Resonancia Magnética Nuclear Biomolecular/instrumentación , Resonancia Magnética Nuclear Biomolecular/métodos , Activación Plaquetaria , Reproducibilidad de los Resultados , Resonancia por Plasmón de Superficie/instrumentación , Resonancia por Plasmón de Superficie/métodosRESUMEN
Three advanced methods, high performance affinity chromatography (HPAC), surface plasmon resonance (SPR) and surface plasmon resonance imaging (SPRi) were compared and evaluated for determining the drug-cyclodextrin (CD) interactions herein. In total, 18 sparingly soluble drugs were selected for this comparative study. The three methods share a unique connection in the working principles and strategies. The same strategies of CD fixation onto solid phase were used in HPAC and SPR for the measurements, whereas, the SPR and SPRi share identical working principles. However, whilst these relationships are evident, no strong correlation was found between kinetic constants obtained from the three methods: Four drugs, namely, prednisolone, pseudolaric acid B, diazepam and gramisetron failed to show any response on SPR, whereas, the kinetics parameters from SPRi and HPAC were successfully measured. From a comparative review of all the kinetic data, random results without any trends were observed (ka, kd and KA) regardless of the relationships between the three methods: It is apparent that the measurement conditions (volume, flow rate, buffers), non-specific adsorption and experimental procedures had a strong impact on the generated data. The relative advantages and limitations of each method are critically presented on the basis of generated data. This comparative study provides a basis to further upgrade these techniques for confident measurement of drug-CDs interactions.
Asunto(s)
Ciclodextrinas/análisis , Ciclodextrinas/metabolismo , Resonancia por Plasmón de Superficie/métodos , Espectrometría de Masas en Tándem/métodos , Interacciones Farmacológicas , Resonancia por Plasmón de Superficie/normas , Espectrometría de Masas en Tándem/normasRESUMEN
Surface Plasmon Resonance biosensors measure the interaction between a molecule in solution and its interaction partner attached to a sensor surface. Under certain conditions, the observed binding rate can be used directly to obtain the concentration of the molecule in solution, without the use of any standard. This type of assay is referred to as Calibration Free Concentration Analysis, CFCA. By examining experimental conditions, including immobilization levels and temperature, for a range of analytes, and by using global analysis of several sample dilutions, conditions that gave the most robust results were identified. These conditions provided the concentration values that were on average â¼15% lower than those obtained using other methods. The accuracy of the concentration determined may be related to how the analyte is distributed in the dextran matrix and to its distance from the gold surface, and may thereby depend on the conversion of the SPR signal to mass. A good precision of CFCA, â¼8% (n = 21), was demonstrated when this method was used to efficiently guide purification procedures of Interferon α-2a. In this paper, the theory behind CFCA and the future developments, as well as the application of CFCA for absolute and relative concentration measurements (including the assessment of the potency of a biotherapeutic medicine) are discussed, and new evaluation tools that broaden the range of applications, are introduced.
Asunto(s)
Interferón-alfa/análisis , Modelos Químicos , Programas Informáticos , Resonancia por Plasmón de Superficie/métodos , Calibración , Humanos , Interferón-alfa/química , Interferón-alfa/aislamiento & purificación , Resonancia por Plasmón de Superficie/normasRESUMEN
BACKGROUND: Method evaluation of new assays for the detection of antiphospholipid antibodies (aPL) such as anti-cardiolipin (aCL) or anti-ß2-glycoprotein I (aß2-GPI) is challenging, as no internationally accepted reference material is available yet. Besides a lack of standardization, unacceptable inter-laboratory comparability of established tests is regularly observed. Owing to the absence of a commonly accepted reference standard, the evaluation of two research surface plasmon resonance (SPR) biosensor assays was performed using statistical methods from latent class analysis (LCA). METHODS: aCL and aß2-GPI IgG and IgM were measured in sera from 63 antiphospholipid syndrome patients, fulfilling the Sydney criteria, and in 34 healthy controls with four commercial assays. LCA was performed on the results and sera were assigned to the antibody-positive or antibody-negative group. Sera were subsequently evaluated in the SPR assays for aCL and aß2-GPI. Optimal cutoffs and diagnostic performances of the research systems were established employing the LCA-derived gold standard. RESULTS: With area under the curve results of 0.96 and 0.89 for the detection of aCL and aß2-GPI, the research SPR assays discriminated well between antibody-positive and antibody-negative sera. Their sensitivities and specificities were comparable to the investigated commercial immunoassays. CONCLUSIONS: SPR assays are a suitable tool for the detection of aCL and aß2-GPI with diagnostic performances not different from currently available commercial tests. LCA enabled the calculation of sensitivities and specificities for aPL assays in absence of a reference standard.
Asunto(s)
Anticuerpos Antifosfolípidos/sangre , Modelos Estadísticos , Resonancia por Plasmón de Superficie/métodos , Adulto , Femenino , Humanos , Masculino , Estándares de Referencia , Resonancia por Plasmón de Superficie/normasRESUMEN
Low accuracy is a big obstacle in the dark-field microscopy imaging (iDFM) technique in practical applications. In order to reduce the deviations and fluctuations in the observed or snapped scattered light in the iDFM technique caused by unavoidable measurement errors, bare gold nanoparticles (AuNPs) were introduced as an internal reference (IR). The feasibility of using AuNPs as the IR in iDFM in theory was verified. The function of the IR in improving the precision of the acquired data through post data analysis was identified by three kinds of experiments: monitoring the oxidation process of silver nanoparticles (AgNPs) at room temperature, quantifying the level of glucose with AgNPs used as probes and quantifying the change in the light intensity of AuNPs after the plasmon resonance energy transfer (PRET) between AuNPs and tetramethylrhodamine (TAMRA).
Asunto(s)
Oro , Nanopartículas del Metal , Microscopía/métodos , Calibración , Nanopartículas del Metal/normas , Nanopartículas del Metal/ultraestructura , Microscopía/normas , Microscopía/estadística & datos numéricos , Nanotecnología , Estándares de Referencia , Resonancia por Plasmón de Superficie/métodos , Resonancia por Plasmón de Superficie/normas , Resonancia por Plasmón de Superficie/estadística & datos numéricosRESUMEN
Surface plasmon resonance (SPR) is the gold standard for determining rate and equilibrium constants of bimolecular complexes. Accuracy of these parameters depends on the correct determination of the concentration of the injected analyte. Calibration free concentration analysis (CFCA) has been developed to overcome the limitation of measuring protein concentrations spectroscopically, which may overestimate the fraction of the protein that really binds to the immobilized ligand, i.e. the active concentration. In this work, we demonstrate that CFCA can also be implemented in a capture format for measuring active concentrations. Capture CFCA (CCFCA) was first validated by measuring the concentration of a HLA-B*44:02 antigen solution. The active concentration of this molecule determined by CCFCA was similar to that obtained by covalent CFCA. CCFCA was then used to determine the concentration of the W6/32 pan class I HLA monoclonal antibody over three different HLA molecules captured by another specific antibody. This could not have been performed by covalent CFCA because immobilized HLA molecules cannot withstand regeneration. By exploring different capture levels we also show that CCFCA gives consistent results even at low capture levels. Knowing the active concentration of W6/32, we then determined the rate and equilibrium constants of W6/32-HLA complexes on the same flow cell. CCFCA is of general use for measuring active concentrations and of great interest for analytes recognizing ligands that cannot be covalently immobilized on sensor chips. The capture mode also allows determining the kinetic constants of multiple analyte-ligand complexes on the same flow cell. This increases experiments throughput and reduces sensor chip consumption.
Asunto(s)
Anticuerpos/análisis , Técnicas Biosensibles/métodos , Antígenos HLA/análisis , Resonancia por Plasmón de Superficie/métodos , Técnicas Biosensibles/normas , Calibración , Humanos , Resonancia por Plasmón de Superficie/normasRESUMEN
Despite its importance and all the considerable efforts made, the progress in drug discovery is limited. One main reason for this is the partly questionable data quality. Models relating biological activity and structures and in silico predictions rely on precisely and accurately measured binding data. However, these data vary so strongly, such that only variations by orders of magnitude are considered as unreliable. This can certainly be improved considering the high analytical performance in pharmaceutical quality control. Thus the principles, properties and performances of biochemical and cell-based assays are revisited and evaluated. In the part of biochemical assays immunoassays, fluorescence assays, surface plasmon resonance, isothermal calorimetry, nuclear magnetic resonance and affinity capillary electrophoresis are discussed in details, in addition radiation-based ligand binding assays, mass spectrometry, atomic force microscopy and microscale thermophoresis are briefly evaluated. In addition, general sources of error, such as solvent, dilution, sample pretreatment and the quality of reagents and reference materials are discussed. Biochemical assays can be optimized to provide good accuracy and precision (e.g. percental relative standard deviation <10 %). Cell-based assays are often considered superior related to the biological significance, however, typically they cannot still be considered as really quantitative, in particular when results are compared over longer periods of time or between laboratories. A very careful choice of assays is therefore recommended. Strategies to further optimize assays are outlined, considering the evaluation and the decrease of the relevant error sources. Analytical performance and data quality are still advancing and will further advance the progress in drug development.
Asunto(s)
Bioensayo/normas , Exactitud de los Datos , Descubrimiento de Drogas , Calorimetría/normas , Bases de Datos Factuales , Electroforesis Capilar/normas , Fluorescencia , Inmunoensayo/normas , Ligandos , Espectroscopía de Resonancia Magnética/normas , Preparaciones Farmacéuticas/metabolismo , Sensibilidad y Especificidad , Resonancia por Plasmón de Superficie/normasRESUMEN
We study how the size of spherical gold nanoparticles (AuNPs) influences their ability to enhance the response of optical biosensors based on surface plasmon resonance (SPR). We present a theoretical model that relates the enhancement generated by the AuNPs to their composition, size, and concentration, thus allowing for accurate predictions regarding the SPR sensor response to various AuNPs. The effect of the AuNP size is also investigated experimentally using an SPR biosensor for the detection of carcinoembryonic antigen (CEA) in which AuNPs covered with neutravidin (N-AuNPs) are used in the last step of a sandwich assay to enhance the sensor response to biotinylated secondary antibody against CEA. The experimental data are in excellent agreement with the results of the theoretical analysis. We demonstrate that the sensor response enhancement generated by the N-AuNPs is determined by (i) the sensor sensitivity to N-AuNP surface density (Sσ) and (ii) the ability of the N-AuNPs to bind to the functionalized surface of the sensor. Our results indicate that, while Sσ increases with the size of the N-AuNP, the ability of the functionalized surface of the sensor to bind the N-AuNPs is affected by steric effects and decreases with the size of N-AuNP.
Asunto(s)
Técnicas Biosensibles/métodos , Oro/química , Nanopartículas del Metal/química , Resonancia por Plasmón de Superficie/normas , Límite de Detección , Tamaño de la PartículaRESUMEN
A highly sensitive broad specificity monoclonal antibody was produced and characterised for microcystin detection through the development of a rapid surface plasmon resonance (SPR) optical biosensor based immunoassay. The antibody displayed the following cross-reactivity: MC-LR 100%; MC-RR 108%; MC-YR 68%; MC-LA 69%; MC-LW 71%; MC-LF 68%; and Nodularin 94%. Microcystin-LR was covalently attached to a CM5 chip and with the monoclonal antibody was employed in a competitive 4 min injection assay to detect total microcystins in water samples below the WHO recommended limit (1 µg/L). A 'total microcystin' level was determined by measuring free and intracellular concentrations in cyanobacterial culture samples as this toxin is an endotoxin. Glass bead beating was used to lyse the cells as a rapid extraction procedure. This method was validated according to European Commission Decision 96/23/EC criteria. The method was proven to measure intracellular microcystin levels, the main source of the toxin, which often goes undetected by other analytical procedures and is advantageous in that it can be used for the monitoring of blooms to provide an early warning of toxicity. It was shown to be repeatable and reproducible, with recoveries from spiked samples ranging from 74 to 123%, and had % CVs below 10% for intra-assay analysis and 15% for inter-assay analysis. The detection capability of the assay was calculated as 0.5 ng/mL for extracellular toxins and 0.05 ng/mL for intracellular microcystins. A comparison of the SPR method with LC-MS/MS was achieved by testing six Microcystis aeruginosa cultures and this study yielded a correlation R(2) value of 0.9989.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Cianobacterias/metabolismo , Líquido Intracelular/metabolismo , Microcistinas/metabolismo , Péptidos Cíclicos/metabolismo , Resonancia por Plasmón de Superficie/métodos , Animales , Cianobacterias/química , Líquido Intracelular/química , Líquido Intracelular/microbiología , Ratones , Ratones Endogámicos BALB C , Microcistinas/análisis , Microcystis/química , Microcystis/metabolismo , Péptidos Cíclicos/análisis , Reproducibilidad de los Resultados , Resonancia por Plasmón de Superficie/normasRESUMEN
A simple, small size, and low cost sensor based on a Deferoxamine Self Assembled Monolayer (DFO-SAM) and Surface Plasmon Resonance (SPR) transduction, in connection with a Plastic Optical Fiber (POF), has been developed for the selective detection of Fe(III). DFO-SAM sensors based on appropriate electrochemical techniques can be frequently found in the scientific literature. In this work, we present the first example of a DFO-SAM sensor based on SPR in an optical fiber. The SPR sensing platform was realized by removing the cladding of a plastic optical fiber along half the circumference, spin coating a buffer of Microposit S1813 photoresist on the exposed core, and finally sputtering a thin gold film. The hydroxamate siderophore deferoxamine (DFO), having high binding affinity for Fe(III), is then used in its immobilized form, as self-assembled monolayer on the gold layer surface of the POF sensor. The results showed that the DFO-SAM-POF-sensor was able to sense the formation of the Fe(III)/DFO complex in the range of concentrations between 1 µm and 50 µm with a linearity range from 0 to 30 µm of Fe(III). The selectivity of the sensor was also proved by interference tests.
Asunto(s)
Costos y Análisis de Costo , Hierro/análisis , Resonancia por Plasmón de Superficie/economía , Resonancia por Plasmón de Superficie/instrumentación , Deferoxamina/análisis , Oro , Fibras Ópticas/economía , Estándares de Referencia , Análisis Espectral , Resonancia por Plasmón de Superficie/normasRESUMEN
One of the main challenges in the development of new analytical platforms for ultrasensitive bioaffinity detection is jointly achieving a wide dynamic range in target analyte concentration, especially for approaches that rely on multistep processes as a part of the signal amplification mechanism. In this paper, a new surface-based sandwich assay is introduced for the direct detection of B-type natriuretic peptide (BNP), an important biomarker for cardiac failure, at concentrations ranging from 1 aM to 500 nM. This was achieved using nanoparticle-enhanced surface plasmon resonance (SPR) where a DNA aptamer is immobilized on a chemically modified gold surface in conjunction with the specific adsorption of antiBNP coated gold nanocubes in the presence of the biomarker target. A concentration detection range greater than eleven orders of magnitude was achieved through dynamic control of only the secondary nanoparticle probe concentration. Furthermore, detection at low attomolar concentrations was also achieved in undiluted human serum.
Asunto(s)
Aptámeros de Nucleótidos/química , Insuficiencia Cardíaca/sangre , Nanopartículas del Metal/química , Péptido Natriurético Encefálico/sangre , Resonancia por Plasmón de Superficie/métodos , Resonancia por Plasmón de Superficie/normas , Biomarcadores/análisis , Biomarcadores/sangre , Oro/química , Insuficiencia Cardíaca/diagnóstico , Humanos , Masculino , Péptido Natriurético Encefálico/análisisRESUMEN
Modern photonics is being revolutionized through the use of nanostructured plasmonic materials, which confine light to sub-diffraction limit resolution providing universal, sensitive, and simple transducers for molecular sensors. Understanding the mechanisms by which light interacts with plasmonic crystals is essential for developing application-focussed devices. The strong influence of grating coupling on electromagnetic field distribution, frequency and degeneracy of plasmon bands has now been characterized using hexagonal nanohole arrays. An equation for nanohole arrays was derived to demonstrate the strong influence of incidence and rotation angle on optical properties of 2D plasmonic crystals such as nanohole arrays. Consequently, we report experimental data that are in strong agreement with finite difference time-domain (FDTD) simulations that clearly demonstrate the influence of the grating coupling conditions on the optical properties (such as plasmon degeneracy and bandwidth), and on the distribution of the plasmon field around nanohole arrays (including tuneable penetration depths and highly localized fields). The tuneable 3D plasmon field allowed for controlled sensing properties and by increasing the angle of incidence to 30 degrees, the resonance wavelength was tuned from 1000 to 600 nm, and the sensitivity was enhanced by nearly 300% for a protein assay using surface plasmon resonance (SPR) and by 40% with surface-enhanced Raman scattering (SERS) sensors.
Asunto(s)
Nanoestructuras/química , Nanotecnología , Resonancia por Plasmón de Superficie/normas , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/normas , Oro/química , Humanos , Nanotecnología/instrumentación , Nanotecnología/métodos , Compuestos de Sulfhidrilo/química , Resonancia por Plasmón de Superficie/métodos , Propiedades de SuperficieRESUMEN
BACKGROUND: Quantitating total levels of monoclonal antibody (mAb) biotherapeutics in serum using ELISA may be hindered by soluble targets. RESULTS: We developed two low-pH-sample-pretreatment techniques to minimize target interference. The first procedure involves sample pretreatment at pH <3.0 before neutralization and analysis in a target capture ELISA. Careful monitoring of acidification time is required to minimize potential impact on mAb detection. The second approach involves sample dilution into mild acid (pH â¼4.5) before transferring to an anti-human capture-antibody-coated plate without neutralization. Analysis of target-drug and drug-capture antibody interactions at pH 4.5 indicated that the capture antibody binds to the drug, while the drug and the target were dissociated. Using these procedures, total biotherapeutic levels were accurately measured when soluble target was >30-fold molar excess. CONCLUSION: These techniques provide alternatives for quantitating mAb biotherapeutics in the presence of a target when standard acid-dissociation procedures are ineffective.
Asunto(s)
Ácido Acético/química , Anticuerpos Monoclonales/sangre , Monitoreo de Drogas/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/normas , Ácido Acético/análisis , Angiopoyetina 2/inmunología , Animales , Humanos , Concentración de Iones de Hidrógeno , Límite de Detección , Macaca fascicularis , Control de Calidad , Estándares de Referencia , Solubilidad , Resonancia por Plasmón de Superficie/métodos , Resonancia por Plasmón de Superficie/normasRESUMEN
The combination of stable biorecognition elements and robust quantum dots (QDs) has the potential to yield highly effective reporters for bioanalyses. Llama-derived single domain antibodies (sdAb) provide small thermostable recognition elements that can be easily manipulated using standard DNA methods. The sdAb was self-assembled on dihydrolipoic acid (DHLA) ligand-capped CdSe-ZnS core-shell QDs made in our laboratory through the polyhistidine tail of the protein, which coordinated to zinc ions on the QD surface. The sdAb-QD bioconjugates were then applied in both fluorometric and surface plasmon resonance (SPR) immunoassays for the detection of ricin, a potential biothreat agent. The sdAb-QD conjugates functioned in fluoroimmunoassays for the detection of ricin, providing equivalent limits of detection when compared to the same anti-ricin sdAb labeled with a conventional fluorophore. In addition, the DHLA-QD-sdAb conjugates were very effective reporter elements in SPR sandwich assays, providing more sensitive detection with a signal enhancement of ~10-fold over sdAb reporters and 2-4 fold over full sized antibody reporters. Commercially prepared streptavidin-modified polymer-coated QDs also amplified the SPR signal for the detection of ricin when applied to locations where biotinylated anti-ricin sdAb was bound to target; however, we observed a 4-fold greater amplification when using the DHLA-QD-sdAb conjugates in this format.
Asunto(s)
Puntos Cuánticos , Ricina/análisis , Ricina/inmunología , Anticuerpos de Dominio Único/química , Resonancia por Plasmón de Superficie/métodos , Fluoroinmunoensayo/métodos , Fluoroinmunoensayo/normas , Unión Proteica/inmunología , Ricina/metabolismo , Resonancia por Plasmón de Superficie/normasRESUMEN
Protein-protein interactions identified through high-throughput proteomics efforts continue to advance our understanding of the protein interactome. In addition to highly specific protein-protein interactions, it is becoming increasingly more common for yeast two-hybrid, pull-down assays, and other proteomics techniques to identify multiple protein ligands that bind to the same target protein. A resulting challenge is to accurately characterize the assembly of these multiprotein complexes and the competition among multiple protein ligands for a given target. The Association of Biomolecular Resource Facilities-Molecular Interactions Research Group recently conducted a benchmark study to assess participants' ability to correctly describe the interactions between two protein ligands and their target protein using primarily biosensor technologies, such as surface plasmon resonance. Participants were provided with microgram quantities of three proteins (A, B, and C) and asked to determine if a ternary A-B-C complex can form or if protein-B and protein-C bind competitively to protein-A. This article will summarize the experimental approaches taken by participants to characterize the molecular interactions, the interpretation of the data, and the results obtained using different biosensor instruments.