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1.
A one-step electrochemical aptasensor using the thiol- and methylene blue- (MB-) dual-labeled aptamer modified gold electrode for determination of ochratoxin A (OTA) was presented in this research. The aptamer against OTA was covalently immobilized on the surface of the electrode by the self-assembly effect and used as recognition probes for OTA detection by the binding induced folding of the aptamer. Under the optimal conditions, the developed electrochemical aptasensor demonstrated a wide linear range from 0.1 pg mL−1 to 1000 pg mL−1 with the limit of detection (LOD) of 0.095 pg mL−1, which was an extraordinary sensitivity compared with other common methods for OTA detection. Moreover, as a practical application, this proposed electrochemical aptasensor was used to monitor the OTA level in red wine samples without any special pretreatment and with satisfactory results obtained. Study results showed that this electrochemical aptasensor could be a potential useful platform for on-site OTA measurement in real complex samples.  相似文献   

2.
An amplified electrochemical impedimetric aptasensor for ochratoxin A (OTA) was developed with picomolar sensitivity. A facile route to fabricate gold nanoparticles covalently bound reduced graphene oxide (AuNPs–rGO) resulted in a large number of well-dispersed AuNPs on graphene sheets with tremendous binding sites for DNA, since the single rGO sheet and each AuNP can be loaded with hundreds of DNA strands. An aptasensor with sandwich model was fabricated which involved thiolated capture DNA immobilized on a gold electrode to capture the aptamer, then the sensing interface was incubated with OTA at a desired concentration, followed by AuNPs–rGO functionalized reporter DNA hybridized with the residual aptamers. By exploiting the AuNPs–rGO as an excellent signal amplified platform, a single hybridization event between aptamer and reporter DNA was translated into more than 107 redox events, leading to a substantial increase in charge-transfer resistance (Rct) by 7∼ orders of magnitude compared with that of the free aptamer modified electrode. Such designed aptasensor showed a decreased response of Rct to the increase of OTA concentrations over a wide range of 1 pg mL−1–50 ng mL−1 and could detect extremely low OTA concentration, namely, 0.3 pg mL−1 or 0.74 pM, which was much lower than that of most other existed impedimetric aptasensors. The signal amplification platform presented here would provide a promising model for the aptamer-based detection with a direct impedimetric method.  相似文献   

3.
Here we demonstrate for the first time that by physically adsorbing aptamer onto conductive film assembled via alternate adsorption of graphene/polyelectrolyte and methylene blue/polyelectrolyte, a label-free electrochemical aptasensor with high sensitivity and selectivity for peptide detection is constructed. Graphene multilayer derived from layer-by-layer assembly has played significant roles in this sensing strategy: allowing accumulation of methylene blue, facilitating electron transfer and providing much more adsorption site. As compared to previous electrochemical aptasensors, the current sensor based on graphene multilayer alternated with electroactive molecule layer offers extremely high capability for sensitive detection of target without interference of environmental surrounding. This electroactive probe-confined graphene multilayer confers great flexibility to combine with differential pulse voltammetry (DPV) together. In the presence of target d entiomer of arginine vasopressin (d-VP), the binding of peptide to aptamer block the electron transfer process of MB, leading to decreased current peak of DPV. By this way, this electrochemical aptasensor based on electroactive molecule-intercalated graphene multilayer provide highly sensitive and specific detection of d-VP with the lowest detectable concentration of 1 ng mL−1 and a wide detection range from 1 to 265 ng mL−1.  相似文献   

4.
Xiluan Yan  Masaaki Kai 《Talanta》2009,79(2):383-4519
We have developed a novel sensitive chemiluminescence (CL) aptasensor for the target assay as exemplified by using adenosine as a model target. In this work, we have demonstrated the signaling mechanism to make detection based on magnetic separation and 3,4,5-trimethoxyl-phenylglyoxal (TMPG), a special CL reagent as the signaling molecule, which reacts instantaneously with guanine nucleobases (G) of adenosine-binding aptamer strands. Briefly, amino-functioned capture DNA sequences are immobilized on the surface of carboxyl-modified magnetic beads, and then hybridized with label-free G-rich (including 15 guanine nucleobases) adenosine-binding aptamer strands to form our CL aptasensor. Upon the introduction of adenosine, the aptamer on the surface of magnetic beads is triggered to make structure switching to the formation of the adenosine/aptamer complex. Consequently, G-rich aptamer strands are forced to dissociate from magnetic beads sensing interface, resulting in a decrease of CL signal. The decrement of peak signal is proportional to the amount of adenosine. The effects of the amounts of capture DNA, aptamer, magnetic beads are investigated and optimized. It was found that the CL intensity had a linear dependency on the concentration of adenosine in the range of 4 × 10−7 to 1 × 10−5 M. With a low detection limit of 8 × 10−8 M and simplicity in CL detection, this novel technique will offer a great promise for future target/aptamer analysis.  相似文献   

5.
In this study we propose a chromogenic platform for rapid analysis of organophosphate (OP) and carbamate (CM) insecticide residues, based on recombinant Drosophila melanogaster acetylcholinesterase (R-DmAChE) as enzyme and indoxyl acetate as substrate. The visible chromogenic strip had the advantages identical to those of commonly used lateral flow assays (LFAs) with utmost simplicity in sample loading and result observation. After optimization, depending on the color intensity (CI) values, the well-established assay has the capabilities of both qualitative measurement via naked eyes and quantitative analysis by colorimetric reader with the desirable IC50 values against the tested six insecticides (0.06 μg mL−1 of carbofuran, 0.28 μg mL−1 of methomyl, 0.03 μg mL−1 of dichlorvos, 31.6 μg mL−1 of methamidophos, 2.0 μg mL−1 of monocrotophos, 6.3 μg mL−1 of omethoate). Acceptable matrix effects and satisfactory detection performance were confirmed by in-parallel LC–MS/MS analysis in different vegetable varieties at various spiked levels of 10−3 to 101 μg g−1. Overall, the testified suitability and applicability of this novel platform meet the requirements for practical use in food safety management and environmental monitoring, especially in the developing world.  相似文献   

6.
In this work, a repeatable assembling and disassembling electrochemical aptamer cytosensor was proposed for the sensitive detection of human liver hepatocellular carcinoma cells (HepG2) based on a dual recognition and signal amplification strategy. A high-affinity thiolated TLS11a aptamer, covalently attached to a gold electrode through Au–thiol interactions, was adopted to recognize and capture the target HepG2 cells. Meanwhile, the G-quadruplex/hemin/aptamer and horseradish peroxidase (HRP) modified gold nanoparticles (G-quadruplex/hemin/aptamer–AuNPs–HRP) nanoprobe was designed. It could be used for electrochemical cytosensing with specific recognition and enzymatic signal amplification of HRP and G-quadruplex/hemin HRP-mimicking DNAzyme. With the nanoprobes as recognizing probes, the HepG2 cancer cells were captured to fabricate an aptamer-cell-nanoprobes sandwich-like superstructure on a gold electrode surface. The proposed electrochemical cytosensor delivered a wide detection range from 1 × 102 to 1 × 107 cells mL−1 and high sensitivity with a low detection limit of 30 cells mL−1. Furthermore, after the electrochemical detection, the activation potential of −0.9 to −1.7 V was performed to break Au–thiol bond and regenerate a bare gold electrode surface, while maintaining the good characteristic of being used repeatedly. The changes of gold electrode behavior after assembling and desorption processes were investigated by electrochemical impedance spectroscopy and cyclic voltammetry techniques. These results indicate that the cytosensor has great potential in disease diagnostic of cancers and opens new insight into the reusable gold electrode with repeatable assembling and disassembling in the electrochemical sensing.  相似文献   

7.
Four simple, rapid, accurate, precise, reliable and economical spectrophotometric methods have been proposed for simultaneous determination of salbutamol sulphate (SS), bromhexine hydrochloride (BH) and etofylline (ET) in pure and commercial formulations without any prior separation or purification. They were first derivative zero crossing spectrophotometry (method 1), simultaneous equation method (method 2), derivative ratio spectra zero crossing method (method 3) and double divisor ratio spectra derivative method (method 4). The ranges for SS, BH and ET were found to be 1-35 μg mL−1, 4-40 μg mL−1 and 5-80 μg mL−1. For methods 1 and 2, the values of limit of detection (LOD) were 0.2314 μg mL−1, 0.4865 μg mL−1 and 0.2766 μg mL−1 and the values of limit of quantitation (LOQ) were 0.7712 μg mL−1, 1.6217 μg mL−1 and 0.9221 μg mL−1 for SS, BH and ET, respectively. For method 3, LOD values were 0.3297 μg mL−1, 0.2784 μg mL−1 and 0.7906 μg mL−1 and LOQ values were 0.9325 μg mL−1, 0.9282 μg mL−1 and 2.6352 μg mL−1 for SS, BH and ET, respectively. For method 4, LOD values were 0.3161 μg mL−1, 0.2495 μg mL−1 and 0.2064 μg mL−1 and LOQ values were 0.9869 μg mL−1, 0.8317 μg mL−1 and 0.6879 μg mL−1 for SS, BH and ET. The precision values were less then 2% R.S.D. for all four methods. The common excipients and additives did not interfere in their determinations. The results obtained by the proposed methods have been statistically compared by means of Student t-test and by the variance ratio F-test.  相似文献   

8.
A novel aptamer biosensor for cancer cell assay has been reported on the basis of ultrasensitive electrochemical detection. The assay uses the aptamer as a capture probe to recognize and bind the tumor marker on the surface of the cancer cells, forming an aptamer-based sandwich structure for MCF-7 cells detection. Functionalized nanoporous materials, porous graphene oxide/Au composites (GO/Au composites) and porous PtFe alloy have been introduced into the biosensor. Owing to the large surface area and versatile porous structure, the use of nanoporous materials can significantly improve the analysis performance of the biosensors by loading of large amounts of molecules and accelerating diffusion rate. Under the optimized experimental conditions, the proposed aptamer biosensor exhibited excellent analytical performance for MCF-7 cells determination, ranging from 100 to 5.0 × 107 cells mL−1 with the detection limit of 38 cells mL−1. The biosensor showed good selectivity, acceptable stability and reproducibility, and developed a highly sensitive and selective method for cancer cells detection.  相似文献   

9.
This work reports the advantages of a label free electrochemical aptasensor for the detection of lysozyme. The biorecognition platform was obtained by the adsorption of the aptamer on the surface of a carbon paste electrode (CPE) previously blocked with mouse immunoglobulin under controlled-potential conditions. The recognition event was detected from the decrease in the guanine and adenine electro-oxidation signals produced as a consequence of the molecular interaction between the aptamer and lysozyme. The biosensing platform demonstrated to be highly selective even in the presence of large excess (9-fold) of bovine serum albumin, cytochrome C and myoglobin. The reproducibility for 10 repetitive determinations of 10.0 mg L−1 lysozyme solution was 5.1% and 6.8% for guanine and adenine electro-oxidation signals, respectively. The detection limits of the aptasensor were 36.0 nmol L−1 (if considering guanine signal) and 18.0 nmol L−1 (if taking adenine oxidation current). This new sensing approach represents an interesting and promising alternative for the electrochemical quantification of lysozyme.  相似文献   

10.
Cancer is one of the most serious and lethal diseases around the world. Its early detection has become a challenging goal. To address this challenge, we developed a novel sensing platform using aptamer and RNA polymerase-based amplification for the detection of cancer cells. The assay uses the aptamer as a capture probe to recognize and bind the tumor marker on the surface of the cancer cells, forming an aptamer-based sandwich structure for collection of the cells in the microplate wells, and uses SYBR Green II dye as a tracer to produce strong fluorescence signal. The tumor marker interacts first with the recognition probes which were composed of the aptamer and single-stranded T7 RNA polymerase promoter. Then, the recognition probe hybridized with template probes to form a double-stranded T7 RNA polymerase promoter. This dsDNA region is extensively transcribed by T7 RNA polymerase to produce large amounts of RNAs, which are easily monitored using the SYBR Green II dye and a standard fluorometer, resulting in the amplification of the fluorescence signal. Using MCF-7 breast cancer cell as the model cell, the present sensing platform showed a linear range from 5.0 × 102 to 5.0 × 106 cells mL−1 with a detection limit of 5.0 × 102 cells mL−1. This work suggested a strategy to use RNA signal amplification combining aptamer recognition to develop a highly sensitive and selective method for cancer cells detection.  相似文献   

11.
Enriching the surface density of immobilized capture antibodies enhances the detection signal of antibody sandwich microarrays. In this study, we improved the detection sensitivity of our previously developed P-Si (porous silicon) antibody microarray by optimizing concentrations of the capturing antibody. We investigated immunoassays using a P-Si microarray at three different capture antibody (PSA – prostate specific antigen) concentrations, analyzing the influence of the antibody density on the assay detection sensitivity. The LOD (limit of detection) for PSA was 2.5 ng mL−1, 80 pg mL−1, and 800 fg mL−1 when arraying the PSA antibody, H117 at the concentration 15 μg mL−1, 35 μg mL−1, and 154 μg mL−1, respectively. We further investigated PSA spiked into human female serum in the range of 800 fg mL−1 to 500 ng mL−1. The microarray showed a LOD of 800 fg mL−1 and a dynamic range of 800 fg mL−1 to 80 ng mL−1 in serum spiked samples.  相似文献   

12.
Despite the wide applicability and specificity of lectins to carbohydrate moieties, there are few lectin specific biosensors. This is attributed to the difficulty in defining the relevant experimental parameters to measure for sensing. We hereby describe the development of direct and indirect electrochemical sensors to determine the exact trace amounts of probarley lectin (ProBL) and its conversion product wheat germ agglutinin (WGA). In addition to WGA, the antigens (ProBL) employed in this study were over expressed in bacteria, isolated from protein bodies, and purified using immobilized N-acetylglusamine in order to obtain correctly folded active lectins. The amperometric immunosensor uses cell lines producing monoclonal antibody (mAB) to the pro-region of ProBL over expressed from Escherichia coli. The efficacy and sensing characteristics of the lectin were optimized using monoclonal antibody to WGA and the resulting sensor was found to detect only ProBL in the linear range 10−3-102 μg mL−1 and a detection limit of 10−3 μg mL−1.  相似文献   

13.
A novel fluorescent probe for Cu2+ determination based on the fluorescence quenching of glyphosate (Glyp)-functionalized quantum dots (QDs) was firstly reported. Glyp had been used to modify the surface of QDs to form Glyp-functionalized QDs following the capping of thioglycolic acid on the core–shell CdTe/CdS QDs. Under the optimal conditions, the response was linearly proportional to the concentration of Cu2+ between 2.4 × 10−2 μg mL−1 and 28 μg mL−1, with a detection limit of 1.3 × 10−3 μg mL−1 (3δ). The Glyp-functionalized QDs fluorescent probe offers good sensitivity and selectivity for detecting Cu2+. The fluorescent probe was successfully used for the determination of Cu2+ in environmental samples. The mechanism of reaction was also discussed.  相似文献   

14.
Liu W  Zhang Z  Liu Z 《Analytica chimica acta》2007,592(2):187-192
In this paper, a chemiluminescence (CL) micro-flow system combined with on-line solid phase extraction (SPE) is presented for determination of β-lactam antibiotics (penicillin, cefradine, cefadroxil, cefalexin) in milk. It is based on the enhancement effect of β-lactam antibiotics on the luminol-K3Fe(CN)6 CL system. The micro-flow system was fabricated from two polymethyl methacrylate (PMMA) plates (50 mm × 40 mm × 5 mm) with the microchannels of 200 μm wide and 150 μm deep. C18-modified silica gel was packed into the microchannel (length: 10 mm; width: 1 mm; depth: 500 μm) to serve as SPE device. Extraction and preconcentration of the analytes were carried out using on-line SPE micro-flow system and the selectivity of CL detection was improved. The detection limits were 0.5 μg mL−1 of penicillin, 0.04 μg mL−1 of cefradine, 0.08 μg mL−1 of cefadroxil and 0.1 μg mL−1 of cefalexin. The proposed method was also applied to analyze the β-lactam antibiotics in milk. Experimental results were in good agreement with those obtained by high performance liquid chromatography (HPLC) method with UV detection.  相似文献   

15.
A homogeneous aggregation immunoassay involving the use of gold nanoparticles (AuNPs) and light scattering detection is described for soy protein determination in food samples. AuNPs act as enhancers of the precipitate that appears when the antigen-antibody complex is formed. The AuNPs-antibody conjugate has been synthesized by physical adsorption of polyclonal anti-soy protein antibodies onto the surface of commercial AuNPs with a nominal diameter of 20 nm. The direct assay is based on the reaction of the conjugate with soy protein, which reaches the equilibrium in about 10 min, and the measurement of the light scattering intensity at 530 nm, which is proportional to the analyte concentration. The dynamic range of the calibration graph is 0.2-20 μg mL−1 and the detection limit value is 65 ng mL−1. The precision, expressed as relative standard deviation, has been assayed at two different concentrations, 0.2 and 1 μg mL−1, giving values ranging from 4.7 to 5.9%. The interference of other proteins has been assayed. The usefulness of this method has been shown by its application to the analysis of fruit juice and “nonmilk yoghourt” samples. The results obtained with the proposed method are similar to those obtained by using a commercial ELISA kit, but the assay time is significantly shorter and the detection limit was about 10 times lower. A recovery study has been also performed, giving values in the range of 84.0-119.3%.  相似文献   

16.
A simple, precise, accurate and validated, acetonitrile-free, reverse phase high performance liquid chromatography (HPLC) method is developed for the determination of melamine in dry and liquid infant formula. The separation is performed on a Kromasil C18 column (150 mm × 3.2 mm I.D., 5 μm particle size) at room temperature. The mobile phase (0.1% TFA/methanol 90:10) is pumped at a flow rate of 0.3 mL min−1 with detection at 240 nm. Melamine elutes at 3.7 min. A linear response (r > 0.999) is observed for samples ranging from 1.0 to 80 μg mL−1. The method provides recoveries of 97.2-101.2% in the concentration range of 5-40 μg mL−1, intra- and inter-day variation in <1.0% R.S.D. The limit of detection (LOD) and limit of quantification (LOQ) values are 0.1 μg mL−1 and 0.2 μg mL−1, respectively.  相似文献   

17.
Y?ld?z Uluda? 《Talanta》2010,82(1):277-383
A simple and sensitive sensor method for cancer biomarkers [prostate specific antigen (PSA) and PSA-alpha 1-antichymotrypsin (ACT) complex] analysis was developed, to be applied directly with human serum (75%) by using antibody modified quartz crystal microbalance sensor and nanoparticles amplification system. A QCM sensor chip consisting of two sensing array enabling the measurement of an active and control binding events simultaneously on the sensor surface was used in this work. The performance of the assay and the sensor was first optimised and characterised in pure buffer conditions before applying to serum samples. Extensive interference to the QCM signal was observed upon the analysis of serum. Different buffer systems were then formulated and tested for the reduction of the non-specific binding of sera proteins on the sensor surface. A PBS buffer containing 200 μg mL−1 BSA, 0.5 M NaCl, 500 μg mL−1 dextran and 0.5% Tween 20, was then selected which eliminated the interfering signal by 98% and enabled the biomarker detection assay to be performed in 75% human serum. By using Au nanoparticles to enhance the QCM sensor signal, a limit of detection of 0.29 ng mL−1 PSA and PSA-ACT complex (in 75% serum) with a linear dynamic detection range up to 150 ng mL−1 was obtained. With the achieved detection limit in serum samples, the developed QCM assay shows a promising technology for cancer biomarker analysis in patient samples.  相似文献   

18.
Hoogerheide JG  Scott RA 《Talanta》2005,65(2):453-460
A rapid and sensitive method for the determination of alkylating agents in complex reaction mixtures was developed and characterized. Analyses are based on the alkylation of 2-mercaptopyridine by the analyte; the derivative is separated by RP-HPLC and measured by fluorescence detection. When applied to the determination of dimethyl sulfate, the method is linear over four orders of magnitude: 0.01-10 μg mL−1. By using recrystallized 2-mercaptopyridine, quantitation limits of 10 ng mL−1 can be achieved. Precision of the assay is 2% R.S.D. in the 1-10 μg mL−1 range and about 15% R.S.D. at 10 ng mL−1. Studies on the pH dependence of the derivatization reaction were key to minimizing interference from the dimethyl sulfate degradation product, monomethyl sulfate, in quenched reaction samples.  相似文献   

19.
A novel method for determination of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) in an extract from mung bean sprouts using high performance liquid chromatography (HPLC) with chemiluminescence (CL) detection is described. The method is based on the CL reaction of auxin (indole-3-acetic acid and indole-3-butyric acid) with acidic potassium permanganate (KMnO4) and tris(2,2′-bipyridyl)ruthenium(II), which was immobilized on the cationic ion-exchange resin. The chromatographic separation was performed on a Nucleosil RP-C18 column (i.d.: 250 mm × 4.6 mm, particle size: 5 μm, pore size: 100) with an isocratic mobile phase consisting of methanol-water-acetic acid (45:55:1, v/v/v). At a flow rate of 1.0 mL min−1, the total run time was 20 min. Under the optimal conditions, the linear ranges were 5.0 × 10−8 to 5.0 × 10−6 g mL−1 and 5.0 × 10−7 to 1.0 × 10−5 g mL−1 for IAA and IBA, respectively. The detection limits were 2.0 × 10−8 g mL−1 and 2.0 × 10−7 g mL−1 for IAA and IBA, respectively. The relative standard deviation (RSD) of intra-day were 3.1% and 2.3% (n = 11) for 2 × 10−6 g mL−1 IAA and 2 × 10−6 g mL−1 IBA; The relative standard deviations of inter-day precision were 6.9% and 4.9% for 2 × 10−6 g mL−1 IAA and 2 × 10−6 g mL−1 IBA. The proposed method had been successfully applied to the determination of auxin in mung bean sprouts.  相似文献   

20.
Haiping Zhou  Jinghe Yang 《Talanta》2009,78(3):809-813
It is found that Al(III) can further enhance the intensity of resonance light scattering (RLS) of the silver nanoparticles (AgNPs) and nucleic acids system. Based on this, a novel method of determination of nucleic acids is proposed in this paper. Under optimum conditions, there are linear relationships between the enhancing extent of RLS and the concentration of nucleic acids in the range of 1.0 × 10−9-1.0 × 10−7 g mL−1, 1.0 × 10−7-2.0 × 10−6 g mL−1 for fish sperm DNA (fsDNA), 1.0 × 10−9-7.0 × 10−8 g mL−1 for calf thymus DNA (ctDNA) and 1.0 × 10−9-1.0 × 10−7 g mL−1 for yeast RNA (yRNA). The detection limits (S/N = 3) of fsDNA, ctDNA and yRNA are 4.1 × 10−10 g mL−1, 4.0 × 10−10 g mL−1 and 4.5 × 10−10 g mL−1, respectively. The studies indicate that the RLS enhancement effect should be ascribed to the formation of AgNPs-Al(III)-DNA aggregations through electrostatic attraction and adsorption bridging action of Al(III). And the sensitivity and stability of the AgNPs-fsDNA system could be enhanced by Al(III).  相似文献   

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