Amplified impedimetric aptasensor based on gold nanoparticles covalently bound graphene sheet for the picomolar detection of ochratoxin A

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 (R_ct) by 7∼ orders of magnitude compared with that of the free aptamer modified electrode. Such designed aptasensor showed a decreased response of R_ct 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.     

Three different signal amplification strategies for the impedimetric sandwich detection of thrombin

In this work, we report a comparative study on three highly specific amplification strategies for the ultrasensitive detection of thrombin with the use of aptamer sandwich protocol. The protocol consisted on the use of a first thrombin aptamer immobilized on the electrode surface, the recognition of thrombin protein, and the reaction with a second biotinylated thrombin aptamer forming the sandwich. Through the exposed biotin end, three variants have been tested to amplify the electrochemical impedance signal. The strategies included (a) silver enhancement treatment, (b) gold enhancement treatment and (c) insoluble product produced by the combination of the enzyme horseradish peroxidase (HRP) and 3-amino-9-ethylcarbazole (AEC). The properties of the sensing surface were probed by electrochemical impedance measurements in the presence of the ferrocyanide/ferricyanide redox marker. Insoluble product strategy and silver enhancement treatment resulted in the lowest detection limit (0.3 pM), while gold enhancement method resulted in the highest reproducibility, 8.8% RSD at the pM thrombin concentration levels. Results of silver and gold enhancement treatment also permitted direct inspection by scanning electron microscopy (SEM).     

Fluorescence aptasensor based on competitive-binding for human neutrophil elastase detection

To our knowledge, we report the first fluorescence aptasensor for detecting human neutrophil elastase (HNE) in homogeneous solution. The biosensor contains a short DNA scrambled sequence strand (SS) complementary to part of the aptamer sequence or the loop of molecular beacon (MB). The aptamer-HNE recognition event involves competition between the molecular beacon and loose HNE aptamer for the binding the short DNA strand. The new biosensor can detect as little as 0.34 nM of HNE, and the response is linear in the tested concentration range of 0.34-68 nM with the detection limit of 47 pM.     

A label free aptasensor for Ochratoxin A detection in cocoa beans: An application to chocolate industries

Contamination of food by mycotoxin occurs in minute/trace quantities. Nearly 92.5% of the cocoa samples present Ochratoxin A (OTA) levels at trace quantity. Hence, there is a necessity for a highly sensitive and selective device that can detect and quantify these organic toxins in various matrices such as cocoa beans. This work reports for the first time, a facile and label-free electrochemical impedimetric aptasensor for rapid detection and quantitation of OTA in cocoa beans. The developed aptasensor was constructed based on the diazonium-coupling reaction mechanism for the immobilization of anti-OTA-aptamer on screen printed carbon electrodes (SPCEs). The aptasensor exhibited a very good limit of detection (LOD) as low as 0.15 ng/mL, with added advantages of good selectivity and reproducibility. The increase in electron transfer resistance was linearly proportional to the OTA concentration in the range 0.15–2.5 ng/mL, with an acceptable recovery percentage (91–95%, RSD = 4.8%) obtained in cocoa samples. This work can facilitate a general model for the detection of OTA in cocoa beans based on the impedimetric aptasensor. The analysis can be performed onsite with pre-constructed and aptamer modified electrodes employing a portable EIS set up.     

Ultrasensitive one-step rapid detection of ochratoxin A by the folding-based electrochemical aptasensor

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.     

Sensitive chemiluminescence aptasensor based on exonuclease-assisted recycling amplification

We report herein an exonuclease-assisted aptamer-based target recycling amplification strategy for sensitive and selective chemiluminescence (CL) determination of adenosine. This aptasensor is based on target-induced release of aptamers from capture probes immobilized on the 96-well plate surface, and thus leading to a decreased hybridization with gold nanoparticle-functionalized reporter sequences followed by a CL signal. The introduction of exonuclease III catalyzes the stepwise removal of mononucleotides from 3′-hydroxyl termini of duplex DNAs of aptamers, liberating the adenosine. Therefore, a single copy of target adenosine can lead to the release and digestion of numerous aptamer strands from the 96-well plates and ultimately an enhanced sensitivity is achieved. Experimental results revealed that the exonuclease-assisted recycling strategy enabled the monitoring of adenosine with wide working ranges and low detection limits (LOD: 0.5 nM). This new CL strategy might create a novel technology for the detection of various targets and could find wide applications in the environmental and biomedical fields.     

Non-instrumental immunochemical tests for rapid ochratoxin A detection in red wine

Gel-based and membrane-based flow-through immunoassay formats were investigated for rapid ochratoxin A (OTA) detection in red wine. The flow-through set-up consisted of an antibody containing gel or membrane placed at the bottom of a standard solid-phase extraction column (i.e. the flow-through column), combined with a clean-up column. Different clean-up methods were studied for red wine clarification and purification. The optimal method consisted of passing wine, diluted with an aqueous solution containing 1% polyethylene glycol (PEG 6000) and 5% sodium hydrogencarbonate, through strong anion exchange (SAX) silica. An immunoassay for OTA detection in red wine was optimized and a cut-off level at 2 μg L⁻¹ according to EU legislation was achieved with both formats. A more significant colour difference between blank and spiked samples was observed for the gel-based assay making this superior to the membrane-based assay. The proposed rapid gel-based test was compared with a standard immunoaffinity column - high-performance liquid chromatography - fluorescent detection (IAC-HPLC-FLD) method and a good correlation of the results was obtained for naturally contaminated wine samples.     

Label-free colorimetric aptasensor for sensitive detection of ochratoxin A utilizing hybridization chain reaction

The combination of high selectivity of aptamer with the peroxidase-mimicking property of DNAzyme has presented considerable opportunities for designing colorimetric aptasensor for detection of ochratoxin A (OTA). The activities of both aptamer (as biorecognition element) and DNAzyme (as signal amplification element) are blocked via base pairing in the hairpin structure. Hybridization chain reaction (HCR) between two hairpin DNAs was employed to further improve the sensitivity of this method. The presence of OTA triggers the opening of the hairpin structure and the beginning of HCR, which results in the release of many DNAzyme, and generates enhanced colorimetric signals, which is correlated to the amounts of OTA with linear range between 0.01 to 0.32 nM, and the limit of detection is 0.01 nM under optimal conditions. OTA in yellow rice wine and wheat flour samples was also detected using this method. We demonstrate that a new colorimetric method for the detection of OTA has been established, which is simple, easy to conduct, label-free, sensitive, high throughput, and cost-saving.     

Determination of ochratoxin A in wine using liquid-phase microextraction combined with liquid chromatography with fluorescence detection

A liquid-liquid microextraction technique (LPME) has been applied to the extraction of ochratoxin A (OTA) from wine prior to its quantification by HPLC-fluorescence detection. OTA was extracted from wine, through 1-octanol immobilized in the pores of a porous hollow fiber, and introduced into 1-octanol inside the fiber. Recovery was 77%. The method was adequate for quantification of OTA in wine at levels within the range 0.25-10 ng/ml with a LOD of 0.2 ng/ml, and can be a simple and inexpensive alternative to the use of inmunoaffinity columns in order to quantify OTA levels in wine.     

A novel aptasensor strategy for protein detection based on G-quadruplex and exonuclease III-aided recycling amplification

The detection of biomarkers is of great significance in the diagnosis of numerous diseases,especially cancer.Herein,we developed a sensitive and universal fluorescent aptasensor strategy based on magnetic beads,DNA G-quadruplex,and exonuclease Ⅲ(Exo Ⅲ).In the presence of a target protein,a label-free single strand DNA(ssDNA)hybridized with the aptamer was released as a trigger DNA due to specific recognition between the aptamer and target.Subsequently,ssDNA initiates the ExoⅢ-aided recycling to amplify the fluorescence signal,which was caused by N-methylmesoporphyrin IX(NMM)insertion into the G-quadruplex structure.This proposed strategy combines the excellent specificity between the aptamer and target,high sensitivity of the fluorescence signal by G-quadruplex and ExoⅢ-aided recycling amplification.We selected(50-1200 nmol/L)MUC1,a common tumor biomarker,as the proof-of-concept target to test the specificity of our aptasenso r.Results reveal that the sensor sensitively and selectively detected the target protein with limits of detection(LODs)of 3.68 and 12.83 nmol/L in buffer solution and 10%serum system,respectively.The strategy can be easily applied to other targets by simply substituting corresponding aptamers and has great potential in the diagnosis and monitoring of several diseases.     

A dual-amplification aptasensor for highly sensitive detection of thrombin based on the functionalized graphene-Pd nanoparticles composites and the hemin/G-quadruplex

In this work, an advanced sandwich-type electrochemical aptasensor for thrombin was proposed by integrating hemin/G-quadruplex with functionalized graphene-Pd nanoparticles composites (PdNPs-RGs). The hemin/G-quadruplex formed by intercalating hemin into thrombin binding aptamer (TBA), firstly acted as a NADH oxidase, assisting the oxidation of NADH to NAD⁺ accompanying with the generation of H₂O₂ in the presence of dissolved O₂. Subsequently, the hemin/G-quadruplex acted as HRP-mimicking DNAzyme that rapidly bioelectrocatalyze the reduction of the produced H₂O₂. At the same time, the Pd nanoparticles supported on p-iodoaniline functionalized graphene were also adopted to catalyze the reduction of H₂O₂. Thus, with the dual catalysis, a dramatically amplified electrochemical signal could be obtained. Besides, the avidin–biotin system for binding aptamer sequences on electrodes not only improved the sensitivity of thrombin analysis but also obtained an acceptable repeatability of the aptasensor. With several factors mentioned above, a wide linear ranged from 0.1 pM to 50 nM was acquired with a relatively low detection limit of 0.03 pM (defined as S/N = 3). These excellent performances provided our approach a promising way for ultrasensitive assay in electrochemical aptasensors.     

Validation of a liquid chromatography method for the simultaneous quantification of ochratoxin A and its analogues in red wines

A validated high-performance liquid chromatography (HPLC) method with fluorescence detection for the simultaneous quantification of ochratoxin A (OTA) and its analogues (ochratoxin B (OTB), ochratoxin C (OTC) and methyl ochratoxin A (MeOTA)) in red wine at trace levels is described. Before their analysis by HPLC-FLD, ochratoxins were extracted and purified with immunoaffinity columns from 50 mL of red wine at pH 7.2. Validation of the analytical method was based on the following parameters: selectivity, linearity, robustness, limits of detection and quantification, precision (within-day and between-day variability), recovery and stability. The limits of detection (LOD) in red wine were established at 0.16, 0.32, 0.27 and 0.17 ng L(-1) for OTA, OTB, MeOTA and OTC, respectively. The limit of quantification (LOQ) was established as 0.50 ng L(-1) for all of the ochratoxins. The LOD and LOQ obtained are the lowest found for OTA in the reference literature up to now. Recovery values were 93.5, 81.7, 76.0 and 73.4% for OTA, OTB, MeOTA and OTC, respectively. For the first time, this validated method permits the investigation of the co-occurrence of ochratoxins A, B, C and methyl ochratoxin A in 20 red wine samples from Spain.     

A highly sensitive aptasensor for on‐site detection of lipopolysaccharides in food

A rapid, low‐cost, highly sensitive, and specific capacitive aptasensor is presented for detection of lipopolysaccharides (LPS). Exposure to LPS could cause fever, gram‐negative sepsis, septic shock, and eventual death. Hence, rapid, low cost, and sensitive detection of LPS is pivotal for the safety of food, pharmaceutical, and medical devices and products. In this work, a capacitive sensing method based on alternating current electrokinetics is developed to achieve rapid and specific detection of LPS. This method uses an alternating current signal for two purposes. One is to induce positive dielectrophoresis, which attracts LPS toward the sensor electrodes’ surface and accelerates its binding with the immobilized aptamer probe. The other purpose is to simultaneously sense the binding reaction by measuring the interfacial capacitance change on the electrodes’ surface. The testing procedures and instrumentation setup of this sensing platform are significantly simplified while finding quantitative concentrations of both analytical and complex samples within 30 s. When testing analytical samples of LPS from Escherichia coli O55:B5, a LOD of 4.93 fg/mL is achieved. The recovery analysis is also performed with LPS spiked in a complex matrix and good recovery rates are demonstrated. This work provides an affordable and field‐deployable platform for highly sensitive and real‐time LPS detection.     

A electrochemiluminescence aptasensor for detection of thrombin incorporating the capture aptamer labeled with gold nanoparticles immobilized onto the thio-silanized ITO electrode

A novel electrochemiluminescence (ECL) aptasensor was proposed for sensitive and cost-effective detection of the target thrombin adopted an aptamer-based sandwich format. To detect thrombin, capture aptamers labeled with gold nanoparticles (AuNPs) were first immobilized onto the thio-silanized ITO electrode surface through strong Au-S bonds. After catching the target thrombin, signal aptamers tagged with ECL labels were attached to the assembled electrode surface. As a result, an AuNPs-capture-aptamer/thrombin/ECL-tagged-signal-aptamer sandwich type was formed. Treating the resulting electrode surface with tri-n-propylamine (TPA) and applying a swept potential to the electrode, ECL response was generated which realized the detection of target protein. Spectroscopy and electrochemical impedance techniques were used to characterize and confirm the fabrication of the ECL aptasensor. AuNPs amplification and smart sensor fabrication art were implemented for the sensitive and cost-effective detection purpose. Signal-to-dose curve excellently followed a sandwich format equation and could be used to quantify the protein, and the detection limit was estimated to be 10 nM. Other forms of thrombin such as β- and γ-thrombins had negligible response, which indicated a high specificity of α-thrombin detection. The aptasensor opened up new fields of aptamer applications in ECL domain, a highly sensitive technique, and had a promising perspective to be applied in microarray analysis.     

Optofluidics-based DNA structure-competitive aptasensor for rapid on-site detection of lead(II) in an aquatic environment

Lead ions (Pb²⁺), ubiquitous and one of the most toxic metallic pollutants, have attracted increasing attentions because of their various neurotoxic effects. Pb²⁺ has been proven to induce a conformational change in G-quadruplex (G4) aptamers to form a stabilizing G4/Pb²⁺ complex. Based on this principle, an innovative optofluidics-based DNA structure-competitive aptasensor was developed for Pb²⁺ detection in an actual aquatic environment. The proposed sensing system has good characteristics, such as high sensitivity and selectivity, reusability, easy operation, rapidity, robustness, portability, use of a small sample volume, and cost effectiveness. A fluorescence-labeled G4 aptamer was utilized as a molecular probe. A DNA probe, a complementary strand of G4 aptamer, was immobilized onto the sensor surface. When the mixture of Pb²⁺ solution and G4 aptamer was introduced into the optofluidic cell, Pb²⁺ and the DNA probe bound competitively with the G4 aptamer. A high Pb²⁺ concentration reduced the binding of the aptamer and the DNA probe; thus, a low-fluorescence signal was detected. A sensitive sensing response to Pb²⁺ in the range of 1.0–300.0 nM with a low detection limit of 0.22 nM was exhibited under optimal conditions. The potential interference of the environmental sample matrix was assessed with spiked samples, and the recovery of Pb²⁺ ranged from 80 to 105% with a relative standard deviation value of <8.5%. These observations clearly illustrate that with the use of different DNA or aptamer probes, the sensing strategy presented can be easily extended to the rapid on-site monitoring of other trace analytes.     

Determination of ochratoxin A in wine grapes: comparison of extraction procedures and method validation

A method for determination of ochratoxin A (OTA) in wine grapes is described, using extraction with a hydrogen carbonate and polyethylene glycol (PEG) solution (5% NaHCO₃ and 1% PEG 8000), followed by immunoaffinity clean-up and liquid chromatography with fluorescence detection. Validation was made with spiked samples, in levels of 0.05 and 1 μg kg⁻¹, with average recovery rates of 76% and relative standard deviations in repeatability and intermediate precision conditions of 8 and 12%, respectively. The limit of detection and limit of quantification in grapes were established at 0.004 and 0.007 μg kg⁻¹, respectively. To evaluate further the accuracy and efficiency of this method, naturally contaminated grapes were also analysed by another method that involves extraction with acidified methanol, at levels ranging from 0.05 to 37 μg kg⁻¹, and the results compared. A good correlation (r=0.9996) was found, with better performances in terms of precision for the new method. A survey was conducted on wine grapes from 11 Portuguese vineyards, during the harvest of 2002, using the proposed method. OTA was detected in three out of the 11 samples, at levels ranging from 0.035 to 0.061 μg kg⁻¹.The new method meets all the criteria of the European Commission directive 2002/26/CE, that lays down the sampling and the analysis methods for the official control of OTA levels in foodstuffs. It is reliable for low levels of contamination (ng kg⁻¹), and avoids the use of organic solvents in the extraction step.     

Determination of ochratoxin A in maize bread samples by LC with fluorescence detection

Ochratoxin A (OTA) is a secondary fungal metabolite produced by several moulds, mainly by Aspergillus ochraceus, A. carbonarius, A. niger and by Penicillium verrucosum. The present work shows the results of comparative studies using different procedures for the analysis of OTA in maize bread samples. The studied analytical methods involved extraction with different volumes of PBS/methanol, different extraction apparatus, and clean-up through immunoaffinity columns. The separation and identification were carried out by high-performance liquid chromatography with fluorescence detection. The optimized method for analysis of OTA in maize bread involved extraction with PBS:methanol (50:50), and clean-up with IAC column. The limit of quantification was 0.033 ng g⁻¹. Recoveries ranged from 87% to 102% for fortifications at 2.000 and 0.500 ng g⁻¹, respectively, within-day R.S.D. of 1.4% and 4.7%. The proposed method was applied to 15 samples and the presence of OTA was found in nine samples at concentrations ranging from nd to 2.650 ng g⁻¹.     

A novel aptasensor based on 3D-inorganic hybrid composite as immobilized substrate for sensitive detection of platelet-derived growth factor

A novel electrochemical detection approach for platelet-derived growth factor(PDGF) via "sandwich"structure is reported in this paper. 3D-4MgCO_3 Mg(OH)_2 4H_2O-Au NPs inorganic hybrid composite was utilized as immobilized substrate for sensitive PDGF detection and Pt-Au bimetallic nanoparticles were labelled on PDGF aptamer to indirectly detect PDGF for the first time. The proposed aptasensor exhibited a high catalytic efficiency towards reduction of H_2O_2, hence the sensitive detection of PDGF was achieved.Results showed that the aptasensor exhibited excellent linear response to PDGF, in the range of 0.1 pg/m L–10 ng/m L(4 fmol/L–400 pmol/L), with detection limit of 0.03 pg/m L(1.2 fmol/L).     

Occurrence of ochratoxin A in Turkish wines

A total of 95 wine samples including 34 white, 10 rosé and 51 red wines originating from four different Turkish areas were analysed for ochratoxin A (OTA). An analytical method based on immunoaffinity column (IAC) for clean-up and high performance liquid chromatography with fluorescence detection (HPLC-FD) was used to determine OTA in wines. The limit of detection (LOD) was estimated as 0.006 ng ml^− 1 for white wine and 0.010 ng ml^− 1 for rosé and red wines. The limit of quantification (LOQ) was estimated as 0.020 ng ml^− 1 in white wine and 0.030 ng ml^− 1 in rosé and red wines. Recovery experiments were carried out with spiked samples in the range 0.1–1 ng ml^− 1 of OTA. The average OTA recoveries from spiked white wine samples varied from 79.43% to 85.07%; while the mean recoveries for rosé and red wine samples were in the range of 77.48–83.96% and 76.61–83.55%, respectively. OTA was detected in 82 (86%) wine samples at levels of < 0.006–0.815 ng ml^− 1, which were below the maximum allowable limit established by the European Community. The mean OTA concentration in red wines was slightly higher than in white and rosé wines. Furthermore, our data indicate that the geographic region of origin has strong influence on OTA level for white, rosé and red wines: wines originating from Thrace (n = 44, mean = 0.158 ng ml^− 1) and Aegean (n = 28, mean = 0.060 ng ml^− 1) regions of Turkey were more contaminated with OTA compared with wines originating from central (n = 15, mean = 0.027 ng ml⁻¹) and east Anatolia (n = 8, mean = 0.027 ng ml^− 1) areas. This study showed that the occurrence of OTA in Turkish wines is high, but at levels that probably leads to a non-significant human exposure to OTA by consumption of wines.     

Ochratoxin A survey in Portuguese wine by LC-FD with direct injection

Wine and grape juices were identified as one of the most important sources of ochratoxin A (OTA), a mycotoxin with diverse toxic effects that naturally appears in food and foodstuffs all over the world.The aim of this study was to assess the OTA levels in Portuguese wines through the application of a simple and accurate method based on liquid chromatography (LC) with direct injection, followed by fluorescence detection (FD).Randomly selected wine samples were used to evaluate the performance of direct injection as efficient, fast, inexpensive and safe sample preparation method. The proposed method was successfully validated. The limit of quantification (LOQ) was 1.0 μg/L and OTA recoveries from wine samples, spiked at the three fortification levels, were higher than 85.4%, with RSDs lower than 9.6% for both red and white wines. The presence of OTA was confirmed by methyl ester derivatization followed by LC analysis.Data on OTA levels were obtained for 60 Portuguese red and white wine samples. OTA was found in 12 samples, nine (26%) red wine samples and three (12%) white wine samples. Only one red wine sample and one white wine sample presented a contamination level above the LOQ, with 1.23 and 2.4 μg/L, respectively. It should be pointed out that this white wine sample exceeded the EC maximum permitted level of 2.0 μg/L. The safe dose established as 120 ng/kg body weight/week was not exceeded by the weekly intake estimated for the samples contaminated above the LOQ.