Simultaneously fluorescence detecting thrombin and lysozyme based on magnetic nanoparticle condensation

In this protocol, a fluorescent aptasensor based on magnetic separation for simultaneous detection thrombin and lysozyme was proposed. Firstly, one of the anti-thrombin aptamer and the anti-lysozyme aptamer were individually immobilized onto magnetic nanoparticles, acting as the protein captor. The other anti-thrombin aptamer was labeled with rhodamine B and the anti-lysozyme aptamer was labeled with fluorescein, employing as the protein report. By applying the sandwich detection strategy, the fluorescence response at 515 nm and 578 nm were respectively corresponding to lysozyme and thrombin with high selectivity and sensitivities. The fluorescence intensity was individually linear with the concentration of thrombin and lysozyme in the range of 0.13-4 nM and 0.56-12.3 nM, and the detection limits were 0.06 nM of thrombin and 0.2 nM of lysozyme, respectively. The preliminary study on simultaneous detection of thrombin and lysozyme in real plasma samples was also performed. It shows that the proposed approach has the good character for simultaneous multiple protein detection.     

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.     

Analytical methods for pesticide residues in rice

Rice consumption has increased worldwide over recent decades, as it has become one of the most common foods. Although the analysis of environmental samples coming from rice areas has been well documented, there is less information regarding the analysis of pesticide residues in rice-grain samples.Rice (paddy, brown and white) can be considered a complex matrix, leading to difficulties in the application of the different multiresidue methods described in the literature. This review addresses and compares the principal extraction and clean-up methodologies [e.g., liquid-liquid extraction, solid-phase extraction, pressurized-liquid extraction, QuEChERS (quick, easy, cheap, effective, rugged and safe), gel-permeation chromatography and supercritical-fluid extraction - with QuEChERS-based methods being the most frequently employed].Traditionally, the determination of pesticide residues in rice has been based on gas chromatography with mass spectrometry (MS). But the application of new classes of pesticides has driven laboratories to increase the use of liquid chromatography with tandem MS. The limits of detection and quantification are in the ranges 0.09-90 μg/kg and 1-297 μg/kg, respectively, for the methodologies reported. These values agree with the current internationally-accepted maximum residue limits (MRLs).Based on the European Union (EU) database, more than 3000 analyses of pesticide residues in rice have been performed by official EU laboratories over the past decade. Of these, 6% reported pesticide residues above the MRLs.Physico-chemical properties can explain the occurrence of pesticides in rice commodities: lipophilic pesticides are frequently found in brown rice, whereas fungicides are mainly found in milled rice. Carbendazim, malathion, iprodione, tebuconazole, quinclorac and tricyclazole are the pesticides most frequently found in white rice, while buprofezin, hexaconazole, chlorpyrifos and edifenphos are most commonly found in paddy rice.Pesticide-residue concentrations can be affected during rice processing - with concentrations generally lower in the final products. However, few studies focusing on primary processing have addressed the setting of precise values applicable for the processing factors.     

Single-walled carbon nanotubes based quenching of free FAM-aptamer for selective determination of ochratoxin A

Ochratoxin A, a toxin produced by Aspergillus ochraceus and Penicillium verrucosum, is one of the most abundant food-contaminating mycotoxins in the world. It has been classified by the International Agency for Research on Cancer (IARC) as a possible human carcinogen. In this paper, a sensitive and selective fluorescent aptasensor for ochratoxin A (OTA) detection was constructed, utilizing single-walled carbon nanotubes (SWNTs) as quencher which can quench the fluorescence of free unfolded toxin-specific aptamer attached with FAM (carboxyfluorescein). Without any coating materials as compared to graphene-oxide based sensor, we obtained the detection limit of our sensing platform based on SWNTs to be 24.1 nM with a linear detection range from 25 nM to 200 nM. This technique responded specifically to OTA without interference from other analogues (N-acetyl-l-phenylalanine, warfarin and OTB). It has also been verified for real sample application by testing 1% beer containing buffer solution spiked with a series of concentration of OTA.     

Aptamer-functionalized solid phase microextraction–liquid chromatography/tandem mass spectrometry for selective enrichment and determination of thrombin

In this publication, a novel solid phase microextraction (SPME) coating functionalized with a DNA aptamer for selective enrichment of a low abundance protein from diluted human plasma is described. This approach is based on the covalent immobilization of an aptamer ligand on electrospun microfibers made with the hydrophilic polymer poly(acrylonitrile-co-maleic acid) (PANCMA) on stainless steel rods. A plasma protein, human α-thrombin, was employed as a model protein for selective extraction by the developed Apt-SPME probe, and the detection was carried out with liquid chromatography/tandem mass spectrometry (LC–MS/MS). The SPME probe exhibited highly selective capture, good binding capacity, high stability and good repeatability for the extraction of thrombin. The protein selective probe was employed for direct extraction of thrombin from 20-fold diluted human plasma samples without any other purification. The Apt-SPME method coupled with LC–MS/MS provided a good linear dynamic range of 0.5–50 nM in diluted human plasma with a good correlation coefficient (R² = 0.9923), and the detection limit of the proposed method was found to be 0.30 nM. Finally, the Apt-SPME coupled with LC–MS/MS method was successfully utilized for the determination of thrombin in clinical human plasma samples. One shortcoming of the method is its reduced efficiency in undiluted human plasma compared to the standard solution. Nevertheless, this new aptamer affinity-based SPME probe opens up the possibility of selective enrichment of a given targeted protein from complex sample either in vivo or ex vivo.     

Optimization of aptamer microarray technology for multiple protein targets

Aptamer-based microarrays for the quantitation of multiple protein analytes have been developed. A multiplex aptamer microarray was generated by printing two RNA aptamers (anti-lysozyme and anti-ricin) and two DNA aptamers (anti-IgE and anti-thrombin) on to either streptavidin (SA) or neutravidin (NA)-coated glass slides. However, substantial optimization was required in order to ensure the simultaneous function of the aptamer:analyte pairs. The effects of protein labeling, assay buffer, surface coating, and immobilization chemistry and orientation were investigated. A single buffer (PBS buffer containing 5 mM MgCl₂ and 0.1% Tween 20) was found to work well with all the aptamers, even though this was not the buffer originally used in their selection, while neutravidin-coated slides yielded a lower detection limit, wider detection range, and more uniform background than streptavidin-coated slides. Incubation with Cy3-labeled proteins yielded sensitive, target-specific, and dose-dependent responses to each protein. Target protein concentrations as low as 72 pg/mL (5 pM, lysozyme), 15 ng/mL (0.5 nM, ricin), 1.9 ng/mL (0.01 nM, IgE), and 170 ng/mL (5 nM, thrombin) could be detected. These results show that aptamer arrays can potentially be used with numerous proteins in parallel, furthering the notion that aptamer arrays may be useful in proteomics.     

Extraction and determination of organophosphorus pesticides in water samples by a new liquid phase microextraction-gas chromatography-flame photometric detection

A rapid, sensitive and efficient liquid phase microextraction (LPME) method was developed to determine trace concentrations of some organophosphorus pesticides in water samples. This method combines liquid phase microextraction with gas chromatographic (GC) analysis in a simple and inexpensive apparatus involving very little organic solvent consumption. It involves exposing a floated drop of an organic solvent on the surface of aqueous solution in a sealed vial. Experimental parameters which control the performance of LPME such as type of organic solvent, organic solvent and sample volumes, sample stirring rate, sample solution temperature, salt addition and exposure time were investigated and optimized. Finally, the enrichment factor, dynamic linear range (DLR), limit of detection (LOD) and precision of the method were evaluated by the water samples spiked with organophosphorus pesticides. Using optimum extraction conditions, very low detection limits (0.01-0.04 μg L⁻¹) and good linearities (0.9983 < r² < 0.9999) were achieved. The LPME was performed for determination of organophosphorus pesticides in different types of natural water samples and acceptable recoveries (96-104%) and precisions (3.5 < R.S.D.% < 8.9) were obtained. The results suggested that the newly proposed LPME method is a rapid, accurate and effective sample preparation method and could be successfully applied for extraction and determination of organophosphorus pesticides in water samples.     

A highly sensitive enzyme immunoassay for evaluation of 2′-deoxycytidine plasma level as a prognostic marker for breast cancer chemotherapy

A highly sensitive competitive enzyme immunoassay (EIA) has been developed and validated for the determination of the plasma level of 2′-deoxycytidine (dCyd), the potential prognostic marker for breast cancer chemotherapy. This assay employed a monoclonal antibody that recognizes dCyd with a high specificity, and 5′-succinyl-dCyd (5′sdCyd) conjugate of bovine serum albumin (5′sdCyd-BSA) immobilized onto microplate wells as a solid phase. The assay involved a competitive binding reaction between dCyd, in plasma sample, and the immobilized 5′sdCyd-BSA for the binding sites of the anti-dCyd antibody. The bound antibody was quantified with horseradish peroxidase-labeled anti-immunoglobulin second antibody and 3,3′,5,5′-tetramethylbenzidine as a peroxidase substrate. The concentration of dCyd in the sample was quantified by its ability to inhibit the binding of the antibody to the immobilized 5′sdCyd-BSA and subsequently the color formation in the assay. The assay limit of detection was 8 nM and the effective working range at relative standard deviations (R.S.D.s) of ≤10% was 20-800 nM. No cross-reactivity from the structurally related nucleobases, nucleosides, and nucleotides was observed in the proposed assay. Mean analytical recovery of added dCyd was 98-100 ± 3.2-8.2%. The precision of the assay was satisfactory; R.S.D. was 3.4-4.2 and 4.3-8.9% for intra- and inter-assay precision, respectively. The proposed EIA was compared favorably with HPLC method in its ability to accurately measure dCyd spiked into plasma samples. The analytical procedure is convenient, and one can analyze 200 samples per working day, facilitating the processing of large-number batch of samples. The proposed EIA is expected to contribute in further evaluation of dCyd as a prognostic marker for breast cancer chemotherapy and elucidation of the role of dCyd in various biological and biochemical systems.     

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.     

Development of a new microextraction method based on a dynamic single drop in a narrow-bore tube: application in extraction and preconcentration of some organic pollutants in well water and grape juice samples

A novel sample preparation technique, the microextraction method based on a dynamic single drop in a narrow-bore tube, coupled with gas chromatography-flame ionization detection (GC-FID) is presented in this paper. The most important features of this method are simplicity and high enrichment factors. In this method, a microdrop of an extraction solvent assisted by an air bubble was repeatedly passed through a narrow-bore closed end tube containing aqueous sample. It has been successfully used for the analysis of some pesticides as model analytes in aqueous samples. Parameters affecting the method's performance such as selection of extraction solvent type and volume, number of extractions, volume of aqueous sample (tube length), and salt effect were studied and optimized. Under the optimal conditions, the enrichment factors (EFs) for triazole pesticides were in the range of 141-214 and the limits of detection (LODs) were between 2 and 112 μg L⁻¹. The relative standard deviations (C = 1000 μg L⁻¹, n = 6) were obtained in the range of 2.9-4.5%. The recoveries obtained for the spiked well water and grape juice samples were between 71 and 106%. Low cost, relatively short sample preparation time and less solvent consumption are other advantages of the proposed method.     

Highly sensitive determination of recombinant human erythropoietin-α in aptamer-based affinity probe capillary electrophoresis with laser-induced fluorescence detection

Recombinant human erythropoietin-α (rHuEPO-α) has been widely used in clinic for anemia treatment. The detection and quantification of rHuEPO-α is essential for monitoring this widespread recombinant glycoprotein pharmaceutical. In this paper, we developed a new affinity probe capillary electrophoresis/laser-induced fluorescence (APCE/LIF) method for the detection of rHuEPO-α by using a specific single-stranded DNA aptamer probe for the first time. In this method, the complex of aptamer-rHuEPO-α and the free aptamer can be well separated and identified by their migration and fluorescence intensity after systematic optimization. The existence of sodium cation in the sample buffer and running buffer played a critical role for stabilizing complex and enhancing the separation efficiency, additionally, suitable high voltage and sample buffer additives were also important for improving the peak height of the complex. Under the optimized conditions, the method was successfully applied for the quantification of rHuEPO-α in physiological buffer, artificial urine and human serum. The linear range for rHuEPO-α was from 0.2 to 100 nM and the limit of detection was 0.2 nM (i.e. 7.4 ng/mL). Further binding experiments using fluorescein isothiocyanate-labeled rHuEPO-α (F-rHuEPO-α) and N-deglycosylated F-rHuEPO-α demonstrated that the oligosaccharides moiety was of importance in the specific interaction between rHuEPO-α and its aptamer.     

Low-density extraction solvent-based solvent terminated dispersive liquid–liquid microextraction combined with gas chromatography-tandem mass spectrometry for the determination of carbamate pesticides in water samples

A simple and fast method of low-density extraction solvent-based solvent terminated dispersive liquid–liquid microextraction (ST-DLLME) was developed for the highly sensitive determination of carbamate pesticides in the water samples by gas chromatography-tandem mass spectrometry (GC-MSMS). After dispersing, the obtained emulsion cleared into two phases quickly when an aliquot of acetonitrile was introduced as a chemical demulsifier into the aqueous bulk. Therefore, the developed procedure does not need centrifugation to achieve phase separation. It was convenient for the usage of low-density extraction solvents in DLLME. Under the optimized conditions, the limits of detection for all target carbamate pesticides were in range of 0.001–0.50 ng mL⁻¹ and the precisions were in the range of 2.3–6.8% (RSDs, 2 ng mL⁻¹, n = 5). The proposed method has been successfully applied to the analysis of real water samples and good spiked recoveries over the range of 94.5–104% were obtained.     

Aptamer Molecular Beacon Sensor for Rapid and Sensitive Detection of Ochratoxin A

Ochratoxin A (OTA) is a carcinogenic fungal secondary metabolite which causes wide contamination in a variety of food stuffs and environments and has a high risk to human health. Developing a rapid and sensitive method for OTA detection is highly demanded in food safety, environment monitoring, and quality control. Here, we report a simple molecular aptamer beacon (MAB) sensor for rapid OTA detection. The anti-OTA aptamer has a fluorescein (FAM) labeled at the 5′ end and a black hole quencher (BHQ1) labeled at the 3′ end. The specific binding of OTA induced a conformational transition of the aptamer from a random coil to a duplex–quadruplex structure, which brought FAM and BHQ1 into spatial proximity causing fluorescence quenching. Under the optimized conditions, this aptamer sensor enabled OTA detection in a wide dynamic concentration range from 3.9 nM to 500 nM, and the detection limit was about 3.9 nM OTA. This method was selective for OTA detection and allowed to detect OTA spiked in diluted liquor and corn flour extraction samples, showing the capability for OTA analysis in practical applications.     

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 multi-residue method for the determination of 203 pesticides in rice paddies using gas chromatography/mass spectrometry

This work reports a rapid, specific and sensitive multi-residue method based on the Quick Easy Cheap Effective Rugged and Safe (QuEChERS) sample preparation method and gas chromatography with mass spectrometric detection in the selected ion monitoring mode (GC-MS-SIM) using one quantification ion and two identification ions for the routine analysis of 203 pesticides in rice paddies. Analyses of fortified rice paddy samples were performed at different levels (0.05, 0.20 and 0.50 mg kg⁻¹). Mean recoveries from five replicates ranged from 75% to 115%, with coefficients of variation lower than 17%. The limit of quantification was in the range of 0.002-0.05 mg kg⁻¹ for the pesticides. 1040 rice paddy samples were analyzed for method application.     

Streptavidin binding bifunctional aptamers and their interaction with low molecular weight ligands

This paper describes the measurement of the binding affinities of two bifunctional RNA aptamers to their respective ligands. The aptamers comprise either a theophylline or malachite green binding sequence fused to a streptavidin binding sequence. These bifunctional aptamers are shown to bind simultaneously to both the small ligand and to streptavidin whether in free solution or on gold surfaces. Binding isotherms for both interactions were measured by different physiochemical techniques: surface plasmon resonance, fluorescence spectroscopy and dynamic light scattering. Both qualitatively and quantitatively there is little difference in binding affinities between the bifunctional aptamers and their monofunctional components. The respective K_d values for streptavidin binding in the monofunctional aptamer and in the theophylline bifunctional aptamer were 12 nM and 65 nM, respectively whilst the K_d values for theophylline binding in the monofunctional aptamer and the streptavidin bifunctional aptamer were 300 nM and 120 nM. These results are consistent with treating each aptamer sequence as a module that can be combined with others without significant loss of function. This allows for the use of streptavidin based immobilization strategies without either the cost of biotinylated dNTPs or the variable yields associated with the chemical biotinylation of RNA.     

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.     

Label-free detection of adenosine based on fluorescence resonance energy transfer between fluorescent silica nanoparticles and unmodified gold nanoparticles

A sensitive and convenient strategy was developed for label-free assay of adenosine. The strategy adapted the fluorescence resonance energy transfer property between Rhodamine B doped fluorescent silica nanoparticles (SiNPs) and gold nanoparticles (AuNPs) to generate signal. The different affinities of AuNPs toward the unfolded and folded aptamers were employed for the signal transfer in the system. In the presence of adenosine, the split aptamer fragments react with adenosine to form a structured complex. The folded aptamer cannot be adsorbed on the surface of AuNPs, which induces the aggregation of AuNPs under high ionic concentration conditions, and the aggregation of AuNPs leads to the decrease of the quenching ability. Therefore, the fluorescence intensity of Rhodamine B doped fluorescent SiNPs increased along with the concentration of adenosine. Because of the highly specific recognition ability of the aptamer toward adenosine and the strong quenching ability of AuNPs, the proposed strategy demonstrated good selectivity and high sensitivity for the detection of adenosine. Under the optimum conditions in the experiments, a linear range from 98 nM to 100 μM was obtained with a detection limit of 45 nM. As this strategy is convenient, practical and sensitive, it will provide a promising potential for label-free aptamer-based protein detection.     

High-performance liquid chromatography with paired ion electrospray ionization (PIESI) tandem mass spectrometry for the highly sensitive determination of acidic pesticides in water

A novel method based on the paired ion electrospray ionization (PIESI) mass spectrometry has been developed for determination of acidic pesticides at ultratrace levels in surface and ground waters. The proposed approach provides greatly enhanced sensitivity for acidic pesticides and overcomes the drawbacks of the less sensitive negative ion mode ESI-MS. The limits of detection (LODs) of 19 acidic pesticides were evaluated with four types of dicationic ion-pairing reagent (IPR) in both single ion monitoring (SIM) and selected reaction monitoring (SRM) mode. The LOD of 19 pesticides obtained with the use the optimal dicationic ion-pairing reagent ranged from 0.6 pg to 19 pg, indicating the superior sensitivity provided by this method. The transition pathways for different pesticide-IPR complexes during the collision induced dissociation (CID) were identified. To evaluate and eliminate any matrix effects and further decrease the detection limits, off-line solid-phase extraction (SPE) was performed for DI water and a river water matrix spiked with 2000 ng L⁻¹ and 20 ng L⁻¹ pesticides standards respectively, which showed an average percent recovery of 93%. The chromatographic separation of the acidic pesticides was conducted by high-performance liquid chromatography (HPLC) using a C18 column (250 mm × 2.1 mm) in the reversed phase mode using linear gradient elution. The optimized HPLC–PIESI-MS/MS method was utilized for determination of acidic pesticide at ng L⁻¹ level in stream/pond water samples. This experimental approach is 1–3 orders of magnitude more sensitive for these analytes than other reported methods performed in the negative ion mode.