Polyacrylamide gel beads for the recognition of staphylococcal enterotoxin B

Protein‐imprinted polyacrylamide gel beads (IPGB) were synthesized via inverse suspension polymerization, using staphylococcal enterotoxin B (SEB) as template. The adsorption capacity of SEB‐IPGB was almost three times as much as that of non‐imprinted gel beads. The Langmuir adsorption models were applied to describe the equilibrium isotherms. The results showed that an equal class of adsorption was formed in the SEB‐IPGB with the maximum adsorption capacity of 8.40 mg SEB/g imprinted beads. The selectivity test of imprinted beads shows that they exhibited good recognition for SEB as compared with the other proteins. The formation of multiple hydrogen bonds and complementary shape between the imprinting cavities and the template proteins would be the two factors that led to the imprinting effect. The obtained SEB‐IPGB would be used as a potential material for protein toxin separation, extraction, and purification. Copyright © 2014 John Wiley & Sons, Ltd.     

An ultrasensitive method for the determination of thiouracil and phenylthiouracil using capillary zone electrophoresis and laser-induced fluorescence detection

This paper reports the development of a method based on capillary electrophoresis with laser-induced fluorescence detection for the simultaneous determination of thiouracil (TU) and phenylthiouracil (PhTU) with high sensitivity (nanomolar range, i.e., attomoles detected). After derivatization with 5-iodoacetamidofluorescein, the analytes were separated by capillary zone electrophoresis using 20 mM phosphate buffer (pH 10.0) and quantified by fluorescence detection. The linearity range, precision, recovery, and detection limits were determined, and the method was shown to be applicable for the determination of TU and PhTU in spiked feed samples and urine.     

Development of a tetramethylrhodamine-labeled probe for a capillary electrophoresis-based competitive immunoassay of staphylococcal enterotoxin B

Staphylococcal enterotoxin B (SEB) was labeled with tetramethylrhodamine isothiocyanate (TRITC) and used as a probe for a competitive immunoassay. Labeling conditions such as solution pH and time were varied to observe the effect on the fluorescent product. It was found that solution pH of the labeling reaction had little effect on the fluorescence signal of the resulting products. However, labeling at pH 7.0 produced a probe that had a higher affinity for the antibody used in this study than the probes produced at pH 8.0 and 9.0. The fluorescent probes were used to perform a competitive assay for SEB in model skim milk samples. Detection limit was approximately 300 fg of SEB. Quantitation was achieved by curve fitting of fluorescent signals for bound/free probe versus log[SEB] with logarithmic functions. Accuracy in the model skim milk samples was acceptable for 3 and 5 nM SEB, but decreased considerably for a concentration of less than 1 nM SEB. The error was attributed to deviation in linearity in the standard curve at lower concentrations. Reproducibility for the analysis of both standard solutions used for the calibration curves and the model skim milk samples was excellent, with standard deviations of approximately 10% from data collected over a 3-week period. No cross-reactivity was found when the assay was tested with a 700 nM sample of staphylococcal enterotoxin A. Although competitive immunoassays are usually used for small molecules, such as therapeutic drugs, the results demonstrate that relatively large molecules (SEB, 27 kDa) can also be assayed with the technique.     

Miniaturized 96-well ELISA chips for staphylococcal enterotoxin B detection using portable colorimetric detector

A previously developed fluorescence sensing platform, combining spatial illumination using electroluminescence (EL) semiconductor strips with charge coupled device (CCD)-based detection (EL-CCD), was adapted to a new 96-well chip for colorimetric immunological assays, enhancing the capabilities of the EL-CCD platform. The modified system was demonstrated using a colorimetric-based enzyme linked immunosorbent assay (ELISA) for detection of staphylococcal enterotoxin B (SEB). Limits of detection (LODs) of 3.9 ng/mL (±2.4 ng/mL) SEB were determined with the ELISA chip measured using the EL-CCD platform, following a standard 4-h ELISA protocol. The LODs were comparable to those obtained using standard 96-well ELISA plates measured using a standard laboratory 96-well plate reader. The miniature 96-well ELISA chip however required as little as 5-μL samples, representing a tenfold reduction in sample volume compared to a standard 96-well ELISA plates. The ELISA chip also demonstrated detection of SEB spiked into various food matrices (milk, mushrooms, and mayonnaise) using limited-to-no sample preparation, with LODs ranging from 3.9 to 18.5 ng/mL depending on the matrix. The EL-CCD platform is versatile, capable of multi-mode detection (e.g., fluorescent and colorimetric along with solution and solid phase assays), and could readily be applied to other field portable or point-of-care applications. Figure Detection of SEB using miniature ELISA chips coupled with a portable electroluminiscent-charge couple device (EL-CCD) detection system. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Surface plasmon resonance-based immunosensor with oriented immobilized antibody fragments on a mixed self-assembled monolayer for the determination of staphylococcal enterotoxin B

An immunosensor based on surface plasmon resonance (SPR) with a mixed self-assembled monolayer (SAM) was developed to determine staphylococcal enterotoxin B (SEB). The SAM on a gold surface was fabricated by adsorbing a mixture of 16-mercapto-1-hexadecanoic acid (16-MHA) and hexanethiol at various molar ratios. Initially, full-length anti-SEB was randomly immobilized onto the SAM to form the immunosensing surface. Through optimization of surface functionalization and anti-SEB immobilization, the SPR sensors can be applied to the determination of SEB in a linear range of 0.01?~?1.0 μg.mL^?1. Furthermore, a smaller antibody fragment (F(ab)’) was generated and immobilized randomly (via amino groups) or in an oriented manner (via ?SH groups) to form the immunosensing surface. The oriented immobilization of F(ab)’ led to a 50% increase in the antigen binding efficiency compared to randomly immobilized covalent F(ab’) fragments. The resulting calibration curve showed higher sensitivity. In addition, the specificity and applicability of the proposed immunosensor to milk samples were also demonstrated. Furthermore, the sensor can be regenerated using 0.1 M HCl, and 70% of the initial response was maintained over 3 cycles.     

Laser induced fluorescence and photochemical derivatization for trace determination of camptothecin

Laser-excited fluorescence was used for the selective determination of camptothecin in samples containing anti-cancer camptothecin-analogs (irinotecan and topotecan). The selectivity of the method was based on the UV photochemical derivatization in basic solution which increased the analyte fluorescence (337/450 nm) and eliminated fluorescence from the two campthotecin-analogs. The influence of UV exposure time and sodium hydroxide concentration was studied using an experimental design. Limit of detection was 4 × 10⁻¹⁰ mol L⁻¹ with linear fluorescence response up to 1 × 10⁻⁶ mol L⁻¹. Average recoveries of camptothecin (added to the samples to simulate a contamination) were 92 ± 4 and 94 ± 6% (n = 3) respectively in irinotecan and topotecan based pharmaceuticals.     

Six orders of magnitude dynamic range in capillary electrophoresis with ultrasensitive laser-induced fluorescence detection

An ultrasensitive laser-induced fluorescence detector was used with capillary electrophoresis for the study of 5-carboxy-tetramethylrhodamine. The raw signal from the detector provided roughly three orders of magnitude dynamic range. The signal saturated at high analyte concentrations due to the dead time associated with the single-photon counting avalanche photodiode employed in the detector. The signal can be corrected for the detector dead time, providing an additional order of magnitude dynamic range. To further increase dynamic range, two fiber-optic beam-splitters were cascaded to generate a primary signal and two attenuated signals, each monitored by a single-photon counting avalanche photodiode. The combined signals from the three photodiodes are reasonably linear from the concentration detection limit of 3 pM to 10 μM, the maximum concentration investigated, a range of 3,000,000. Mass detection limits were 150 yoctomoles injected onto the capillary.     

Colorimetric determination of staphylococcal enterotoxin B via DNAzyme-guided growth of gold nanoparticles

The authors describe a colorimetric method for the determination of the staphylococcal enterotoxin B (SEB) that also allows for visual readout. The assay is based on the growth of gold nanoparticles (AuNPs) mediated by a hemin/G-quadruplex DNAzyme which generates a color change from red to blue in the presence of SEB. The method is enzyme-free and does not require a label. The kinetics of the formation of the AuNPs is controlled by the hemin/G-quadruplex DNAzyme and this is key to the signal generation mechanism. In the presence of SEB, the reactions between aptamer and target modulated the amount of single probe G strands that form DNAzyme capable of consuming hydrogen peroxide. The growth process of AuNPs is influenced by the resulting concentration of H₂O₂ and leads to the color change. Under optimal conditions, a linear relationship exists between absorbance and SEB concentration in the range from 0.1 to 500 pg·mL￣¹ which covers the clinically relevant range. In case of visual detection, the lower limit of detection is 1 pg·mL^?1. The assay described here is sensitive, comparably inexpensive and can detect SEB rapidly without the need for sophisticated equipment. In our perception, the method has a wide scope in that it may be adapted to various nucleic acids, proteins and other biomolecules if respective aptamers are available.

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Graphical abstract Colorimetric determination of Staphylococcal enterotoxin B via DNAzyme-guided growth of gold nanoparticles

     

A capacitive biosensor for detection of staphylococcal enterotoxin B

A sensitive method for the detection of staphylococcal enterotoxin B (SEB) using a flow-injection capacitive biosensor is presented. SEB was purified from a crude culture filtrate of Staphylococcus aureus through three chromatographic steps. The first two steps were based on ion-exchange chromatography, and the last step was carried out on a gel filtration column. The SEB recovery values after the purification stages were 88%, 74%, and 12%, respectively. A horseradish peroxidase labeled antistaphylococcal enterotoxin B was prepared by the periodate method and was further employed in a sandwich-enzyme-linked immunosorbent assay (ELISA) for the determination of SEB concentrations in different samples obtained during the processing of the crude filtrate. The capacitive biosensor could detect SEB concentrations as low as 0.3 pg ml⁻¹ with a linearity ranging from 2.8 pg ml⁻¹ to 2.8 ng ml⁻¹ under optimized conditions. The response time was about 10 min. A good agreement was achieved between the developed capacitive biosensor system and ELISA as a reference method for detection of SEB levels in different purification samples. The newly developed sensor has the benefits of simplicity, high sensitivity, and multiple use capability.     

An immunoreactor-based competitive fluoroimmunoassay for monitoring staphylococcal enterotoxin B using bioconjugated quantum dots

Extensive research on avian systems has proved hens as an alternate source for polyclonal antibody generation necessary for immunosensing applications. Herein, we present the immobilization of avian antibody raised against staphylococcal enterotoxin B (SEB) and its applicability for a competitive fluoroimmunoassay technique. White leghorn hens immunized with SEB generated high affinity antibodies with a highest yield of 3.2 mg ml(-1) having affinity constant of 0.976 × 10(10) M l(-1). A competitive fluoroimmunoassay format was developed comprising CdTe(557) as a fluorescence detector for monitoring SEB, a bacterial super-antigen. CdTe(557) was bioconjugated to SEB according to the carbodiimide protocol and confirmed by absorption spectral analysis. An immunoreactor column was designed by immobilizing anti-SEB antibodies and was successfully employed as an efficient bio-recognition tool. An immuno-affinity reaction involving competitive binding between free SEB and CdTe(557)-bioconjugated SEB for immobilized antibody was relied upon to attain assay specificity and sensitivity. It was possible to quantify SEB from 1000 to 10 ng based on the integrated fluorescence of the SEB-CdTe(557) bioconjugate eluted from the immunoreactor column with a limit of detection of 8.15 ng and a regression coefficient R(2) = 0.9925. Thus, integration of QDs with immuno-affinity reactions revealed the versatility of nanoparticles as a potential fluorescence label for bioanalytical applications.     

Simultaneous determination of fermented milk aroma compounds by a potentiometric sensor array

The paper reports on the application of an electronic tongue for simultaneous determination of ethanol, acetaldehyde, diacetyl, lactic acid, acetic acid and citric acid content in probiotic fermented milk. The αAstree electronic tongue by Alpha M.O.S. was employed. The sensor array comprised of seven non-specific, cross-sensitive sensors developed especially for food analysis coupled with a reference Ag/AgCl electrode. Samples of plain, strawberry, apple-pear and forest-fruit flavored probiotic fermented milk were analyzed both by standard methods and by the potentiometric sensor array. The results obtained by these methods were used for the development of neural network models for rapid estimation of aroma compounds content in probiotic fermented milk.The highest correlation (0.967) and lowest standard deviation of error for the training (0.585), selection (0.503) and testing (0.571) subset was obtained for the estimation of ethanol content. The lowest correlation (0.669) was obtained for the estimation of acetaldehyde content. The model exhibited poor performance in average error and standard deviations of errors in all subsets which could be explained by low sensitivity of the sensor array to the compound. The obtained results indicate that the potentiometric electronic tongue coupled with artificial neural networks can be applied as a rapid method for the determination of aroma compounds in probiotic fermented milk.     

Micellar electrokinetic chromatography with laser-induced fluorescence detection for sensitive determination of ephedrine and pseudoephedrine

A selective and sensitive micellar electrokinetic chromatography method with laser-induced fluorescence detection was developed for the quantification of ephedrine (E) and pseudoephedrine (PE) derivatized with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole. After conducting a series of optimizations, a running buffer of 10 mM sodium borate + 16 mM SDS was used for separation of the derivatives. A linear relationship for E and PE was obtained in the range of 0.044-6.6 microg mL(-1) (correlation coefficient: 0.9943 for E, 0.9946 for PE), and the detection limits for E and PE were 0.70 and 0.30 ng mL(-1), respectively. The sensitivity of E and PE was improved by several multiples of ten over those of CZE-LIF method. The method was applied to the analysis of the two alkaloids in ephedra herbal medicine and preparations with recoveries in the range of 98.3-107.1%.     

An ultrasensitive chemiluminescence immunosensor for PSA based on the enzyme encapsulated liposome

A highly sensitive chemiluminescence immunosensor for the detection of prostate-specific antigen (PSA) was developed based on a novel amplification procedure with the application of enzyme encapsulated liposome. Horseradish peroxidase (HRP) encapsulated and antibody-modified liposome acts as the carrier of a large number of markers and specific recognition label for the amplified detection of PSA. In the detection of PSA, the analyte was first bound to the specific capture antibody immobilized on the microwell plates, and then sandwiched by the antibody-modified liposomes encapsulating HRP. The encapsulated markers, HRP molecules were released by the lysis of the specifically bound liposomes in the microwell with Triton X-100 solution. Then, the analyte PSA could be determined via the chemiluminescence signal of HRP-catalyzed luminol/peroxide/enhancer system. The “sandwich-type” immunoassay provides the amplification route for the PSA detection in ultratrace levels. The CL emission intensity exhibits dynamic correlation to PSA concentration in the range from 0.74 pg/ml to 0.74 μg/ml with readily achievable detection limit of 0.7 pg/ml.     

Microemulsion electrokinetic chromatography with laser-induced fluorescence detection for sensitive determination of ephedrine and pseudoephedrine

A selective and sensitive microemulsion electrokinetic chromatography with laser-induced fluorescence detection method was developed for the quantification of ephedrine (E) and pseudoephedrine (PE) derivatized with 4-chloro-7-nitrobenzo-2-oxa-1, 3-diazol. By a series of optimization, a running buffer composed of 20 mM borate + microemulsion (23.3 mM Sodium dodecyl sulfate/180.85 mM 1-butanol/16.4 mM n-heptane) +8% acetonitrile was applied for the separation of the derivatives. A linear relationship for E and PE was obtained in the range of 0.058-11.58 microg.mL(-1) (correlation coefficient: 0.9993 for E, 0.9995 for PE), and the detection limits for E and PE were 5.3 and 3.9 ng.mL(-1). The method was applied to the analysis of the two alkaloids in Chinese traditional herbal preparations with recoveries in the range of 96.9-105.4%.     

流动注射激光诱导荧光光度法测定食品中的铁

将流动注射与CCD阵列检测激光诱导荧光装置联用 ,形成连续自动荧光光谱分析系统 ,建立了在 1× 1 0 -3 mol L的HCl溶液中 ,Fe3 催化H2 O2 氧化罗丹明B(RhB)的催化荧光光度测定铁的新方法 ,对反应条件及流动注射各实验参数进行了优化 ,用于食品样品中铁的测定 ,测定结果与原子吸收分光光度法有一致性 ,方法的相对标准差不大于 8.0 % ,加标回收率为 95 .9%～ 1 0 6%。     

CCD阵列检测-激光诱导荧光光度法测定食品中锌

利用自行组装的激光诱导荧光 CCD阵列检测器 计算机联用装置 ,建立了测定食品中锌的灵敏方法。在乙酸 乙酸铵缓冲溶液 (pH 4 )中 ,Zn(Ⅱ )与SCN- 、若丹明B(RhB)反应生成Zn SCN RhB配合物 ,经乙醚萃取后 ,在激发波长 5 32nm和发射波长 5 80nm进行荧光强度测定。研究了表面活性剂对Zn SCN RhB配合物荧光强度的影响 ,优化了荧光反应及食品样品的处理条件 ,并用于食品试样中锌的测定。方法的检出限为 4ng·ml- 1,加标回收率为 90 .0 %～ 110 .0 % ,相对标准偏差为 2 .0 %～ 9.7% ,能满足食品卫生检验的要求。     

多道检测—激光诱导荧光猝灭法测定食品中磷

利用激光诱导荧光 二极管阵列检测 计算机联用装置 ,对磷钼杂多酸 罗丹明 6G荧光猝灭体系进行了研究 ,建立了一种测定食品中磷的新荧光光度法。试验表明 ,该法具有较好的灵敏度、选择性 ,检出限为 0 .1ng·ml- 1,加标回收率为 90 .6%～ 10 9.0 % ,相对标准偏差为 1.3%～3 0 % ,用于食品样品中磷的测定 ,结果满意     

Graphene-based materials for the electrochemical determination of hazardous ions

The use of graphene in the field of electrochemical sensors is increasing due to two main properties that make graphene and derivatives appealing for this purpose: their conductivity and high surface area. In addition, graphene materials can be easily functionalized with nanoparticles (Au, Pt, etc.) or organic molecules (DNA, polymers, etc.) producing synergies that allow higher sensitivity, lower limit of detection as well as increased selectivity. The present review focuses on the most important works published related to graphene-based electrochemical sensors for the determination of hazardous ions (such as As(III), Cd²⁺, Pb²⁺, Hg²⁺, Cr(VI), Cu²⁺, Ag⁺, etc.). The review presents examples of the use of graphene-based electrodes for this purpose as well as important parameters of the sensors such as: limit of detection, linear range, sensitivity, main interferences, stability, and reproducibility. The application of these graphene-based electrodes in real samples (water or food matrices) is indicated, as well. There is room for improvement of these type of sensors and more effort should be devoted to the use of doped graphene (doped for instance with N, B, S, Se, etc.) since electrochemically active sites originated by doping facilitate charge transfer, adsorption and activation of analytes, and fixation of functional moieties/molecules. This will allow the sensitivity and the selectivity of the electrodes to be increased when combined with other materials (nanoparticles/organic molecules).     

A hydrogel based fluorescent micro array used for the characterization of liquid analytes

A fluorimetric micro spot array using non-specific recognition function is described for the analysis of liquid samples. The array was composed of binary mixtures of various fluorescence dyes which were embedded in a hydrogel matrix. The interactions between the fluorescent dyes and their molecular surrounding inside the hydrogel, influence their fluorescence wave length and intensities. The array was used for the characterization of solvent mixtures. Developed fluorescence patterns of the complete array as well as the fluorescence intensity changes of single spots were analysed. It was proven, that specific analytical information can be gained using this non-specific recognition approach. The identification of some alcoholic beverages is described as an example of the application of this method when used for quality control purposes. Analogous to the appellation “electronic nose” and “electronic tongue” the described micro spot array acts as an “optochemical tongue”.     

An ellipsoidal mirror for detection of laser-induced fluorescence in capillary electrophoresis system: applications for labelled antibody analysis

An LIF detector was integrated into a CE system which uses a ball lens to focus the laser beam on the CE capillary. The detector employs an ellipsoid that is glued on the capillary window, to permit the collection of the fluorescence in the capillary. This 'trapped' fluorescence stays in the capillary because the angle of the silica/air interface is greater than the critical angle. The performance of this new detector setup is found to be identical to the collinear setup using the same ball lens. An application to the analysis of FITC-labeled IgG was optimized using a 14 cm effective length capillary. The LOD of an FITC-labeled IgG2 at an excitation wavelength of 488 nm was 150 pg/mL, which was 10 times better than the LOD recorded with slab gel silver staining. Using a tetramethylrhodamine (TAMRA)-labeled IgG2 and a 532 nm excitation wavelength the LOD is 50 pg/mL. The electropherograms of four different commercial FITC conjugates of IgG were studied. The presence of aggregates was observed in two samples while close kinetics of reduction was observed between free aggregates and high aggregates concentration samples. The integrated LIF detector provides an extremely powerful and convenient tool for antibody analysis and should be useful for therapeutic MAb control in pharmaceutical facilities.