A microfluidic chip based ratiometric aptasensor for antibiotic detection in foods using stir bar assisted sorptive extraction and rolling circle amplification.

A ratiometric and sensitive microfluidic chip based aptasensor was developed for antibiotic detection with kanamycin (Kana) as a model analyte. A novel stir bar assisted sorptive extraction and rolling circle amplification strategy was designed to largely amplify the signal and overcome complex matrix interference in food samples. The detection mechanism was as follows: firstly, many duplex DNA probes (a single-stranded DNA as a primer hybrid with an aptamer sequence) were modified on a stir bar. In the presence of Kana, the probes on the bar could specifically capture Kana and release the primer to trigger RCA in the presence of a circular DNA template (CDT). As the reaction proceeds, the amount of CDT decreased and the number of RCA products increased. It is worth mentioning that they can be efficiently separated and detected using a microfluidic chip. The signal ratio of RCA products and CDT (IR/IC) can be employed to qualify Kana in a wide linear range from 0.8 pg mL-1 to 10 ng mL-1 with a low detection limit of 0.3 pg mL-1. This method exhibited excellent sensitivity and selectivity and can obviously reduce the matrix interference through a ratiometric strategy combined with stir bar extraction. The aptasensor was successfully tested in milk and fish samples, confirming that it can be applied for on-site quantitation of antibiotic residues in foods.

Multiplex detection of quality indicator molecule targets in urine using programmable hairpin probes based on a simple double-T type microchip electrophoresis platform and isothermal polymerase-catalyzed target recycling.

Recently, it has been crucial to be able to detect and quantify small molecular targets simultaneously in biological samples. Herein, a simple and conventional double-T type microchip electrophoresis (MCE) based platform for the multiplex detection of quality indicator molecule targets in urine, using ampicillin (AMPI), adenosine triphosphate (ATP) and estradiol (E2) as models, was developed. Several programmable hairpin probes (PHPs) were designed for detecting different targets and triggering isothermal polymerase-catalyzed target recycling (IPCTR) for signal amplification. Based on the target-responsive aptamer structure of PHP (Domain I), target recognition can induce PHP conformational transition and produce extension duplex DNA (dsDNA), assisted by primers & Bst polymerase. Afterwards, the target can be displaced to react with another PHP and initiate the next cycle. After several rounds of reaction, the dsDNA can be produced in large amounts by IPCTR. Three targets can be simultaneously converted to dsDNA fragments with different lengths, which can be separated and detected using MCE. Thus, a simple double-T type MCE based platform was successfully built for the homogeneous detection of multiplex targets in one channel. Under optimal conditions, the assay exhibited high throughput (48 samples per hour at most, not including reaction time) and sensitivity to three targets in urine with a detection limit of 1 nM (ATP), 0.05 nM (AMPI) and 0.1 nM (E2) respectively. The multiplex assay was successfully employed for the above three targets in several urine samples and combined the advantages of the high specificity of programmable hairpin probes, the excellent signal amplification of IPCTR, and the high through-put of MCE which can be employed for screening in biochemical analysis.

Separation and enrichment of six indicator polychlorinated biphenyls from real waters using a novel magnetic multiwalled carbon nanotube composite absorbent.

A novel and effective magnetic multiwalled carbon nanotube composite for the separation and enrichment of polychlorinated biphenyls was developed. Fe3 O4 @SiO2 core-shell structured nanoparticles were first synthesized, then the poly(sodium 4-styrenesulfonate) was laid on its surface to prepare the polyanionic magnetic nanoparticles. The above materials were then grafted with polycationic multiwalled carbon nanotubes, which were modified by polydiallyl dimethyl ammonium chloride through the layer-by-layer self-assembly approach. Its performance was tested by magnetic solid-phase extraction and gas chromatography with mass spectrometry for the determination of six kinds of indicator polychlorinated biphenyls in water samples. Under optimal conditions, the spiked recoveries of several real samples for six kinds of polychlorinated biphenyls (PCB28, PCB52, PCB101, PCB138, PCB153, PCB180) were in the range of 73.4-99.5% with relative standard deviations varying from 1.5 to 8.4%. All target compounds showed good linearities in the tested range with correlation coefficients higher than 0.9993. The limits of quantification for six kinds of indicator polychlorinated biphenyls were between 0.018 and 0.039 ng/mL. The proposed method was successfully applied to analyze polychlorinated biphenyls in real water samples. Satisfactory results were obtained using the effective magnetic absorbent.

A universal assay strategy for sensitive and simultaneous quantitation of multiplex tumor markers based on the stirring rod-immobilized DNA-LaMnO3 perovskite-metal ions encoded probes.

It was extremely urgent to develop some simultaneous and sensitive biosensors for detecting multiplex serum tumor markers (TMs) for early screening of cancers. Herein, a multiplex assay was developed based on the DNA-LaMnO3 (DNA-LMO) perovskite encoded probes and targets mediated competitive replacement strategy. Alpha fetoprotein (AFP), carcinoembryonic antigen (CEA) and prostate specific antigen (PSA) markers were employed as representative target TMs. Aptasensor is prepared by a series of DNA-LMO-M encode probes which were prepared by three hyperbranched DNA firstly immobilized on LMO encapsulating Pb, Cd or Cu ions. Then, three TMs aptamers were labeled on the stirring-rod and hybridized with the probes. After the developed encoded probes was incubated the TMs, the encoded probes corresponding to different TMs can be released into the supernatant through the competitive replacement. The inner metal ion can be simultaneously detected by square wave voltammetry corresponding to various TMs. Since the stirring rod can enrich many encoded probes containing a lot of metal ions, multiplex signal amplification can be realized. Due to the enrichment and easy separation of the stirring rod, the signal-to-noise ratio was also obviously improved and thus to results in good sensitivity and accuracy. Moreover, it took only 20 min to detect three targets which much faster than many same types of aptasensor. Under the optimal conditions, the low detection limit for CEA (3.6 × 10-4 ng/mL), AFP (3.4 × 10-4 ng/mL) and PSA (2.8 × 10-4 ng/mL) were obtained. Therefore, this method is likely to be used for early and sensitive screening of tumors.

A universal signal-on electrochemical assay for rapid on-site quantitation of vibrio parahaemolyticus using aptamer modified magnetic metal-organic framework and phenylboronic acid-ferrocene co-immobilized nanolabel.

Sensitive and rapid detection of pathogenic bacteria remains important and challenging for food safety and preventing outbreaks of foodborne disease. The major limitations of standard analytical methods for detecting vibrio parahaemolyticus (V.P) lie in their bulky equipment and tedious and long-time operation. This study presents an electrochemical aptasensor for the rapid on-site quantification of V.P in seafood. Magnetic nanoscale metal-organic frameworks (Fe3O4@NMOF) labeled with an aptamer against V.P served as capture probes, while gold nanoparticles combined with phenylboronic acid and ferrocene acted as the nanolabels. When detecting V.P, the sandwich-type complex of capture probe-V.P-nanolabel was formed and magnetically attached to a screen-printed electrode (SPE) for signal measurement. Under optimal conditions, the increase in the ferrocene electrochemical signals could assess the V.P amount; the quantified concentration range was 10-109 cfu/mL. Then, the developed signal-on sensor successfully detected V.P in real seafood samples, exhibiting many advantages. It could not only specifically enrich and rapidly separate the V.P in complex samples but also largely amplify the signal. Moreover, using compact SPE with a detection time of maximum 20 min as the measurement platform allows rapid on-site assays. Thus, the proposed method is a feasible strategy for screening V.P in seafood.

A Multicolor Fluorescence Nanoprobe Platform Using Two-Dimensional Metal Organic Framework Nanosheets and Double Stirring Bar Assisted Target Replacement for Multiple Bioanalytical Applications.

Multicolor fluorescence probes can show fluorescence of different colors when detecting different targets, and the excellent feature can create a highly differentiated multicolor sensing platform. However, most of the previously reported multicolor luminescent materials usually suffer from high toxicity and photobleaching, complex preparation procedures, and poor water solubility, which may not be conducive to bioanalytical applications. Two-dimensional metal organic frameworks (2D MOFs), which have large specific surface areas with long-range fluorescence quenching coupled with biomolecular recognition events, have encouraged innovation in biomolecular probing. Here, we propose a 2D-MOF-based multicolor fluorescent aptamer nanoprobe using a double stirring bar assisted target replacement system for enzyme-free signal amplification. It utilizes the interaction between 2D MOFs and DNA molecules to detect multiple antibiotics quickly, sensitively, and selectively. Since 2D MOFs have excellent quenching efficiency for luminescence of fluorescent-dye-labeled single-strand DNA (ssDNA), the background fluorescence can be largely reduced and the signal-to-noise ratio can be improved. When the adsorbed ssDNA formed double helix double-stranded DNA with its complementary ssDNA, its fluorescence can be almost fully recovered. The assay was tested by detecting chloramphenicol (CAP), oxytocin (OTC), and kanamycin (KANA) in biological samples. The developed aptasensor was sufficiently sensitive to detect the antibiotic residues as low as 1.5 pM CAP, 2.4 pM OTC, and 1 pM KANA (S/N = 3). It has been preliminarily used for multicolor imaging of three different antibiotics in fish tissue slices with satisfactory results.

Multiplexed electrochemical aptasensor for antibiotics detection using metallic-encoded apoferritin probes and double stirring bars-assisted target recycling for signal amplification.

Simultaneous and sensitive detection of various antibiotic residues in one sample is essential to evaluation of food safety status. Herein, a multiplexed electrochemical aptasensor for multiplex antibiotics detection, with kanamycin (KANA) and ampicillin (AMP) as representative analytes, was designed by using metal ions encoded apoferrtin probes and double stirring bars-assisted target recycling for signal amplification. The encoded probes were prepared by apoferritin loading Cd2+ and Pb2+ ions and labeling with duplex DNAs (aptamers corresponding to KANA and AMP hybrid with its complementary DNA sequence), respectively. In the presence of KANA and AMP, the targets can recurrently react with the probes on the bars, and then replace a lot of Apo-Mencoded signal tags into supernatant. The peak currents of Cd2+and Pb2+from the tags corresponding with the concentrations of KANA and AMP were detected by square wave voltammetry in one run. As a result, KANA and AMP can be detected simultaneously within the range from 0.05 pM to 50 nM. And the detection limits were 18 fM KANA and 15 fM AMP (S/N = 3). The assay was testified to detect KANA and AMP residues with consistent results of ELISA in samples, e.g. milks and fishes. The assay was highly-sensitive, selective, cost-effective and easy-to-operate due to Apo-M encoded probes with high loading capacity of signal source substances. Moreover, double stirring bar-assisted target recycling, which was enzyme-free and could overcome matrix interference, was fabricated for signal amplification. Thus, the assay showed potential advantages for sensitively screening of antibiotic residues in foods.

Microchip electrophoresis based multiplexed assay for silver and mercury ions simultaneous detection in complex samples using a stirring bar modified with encoded hairpin probes for specific extraction.

It is crucially important to rapidly, simultaneously, and sensitively determine trace amounts of heavy metal ions in complex samples. Herein, a stirring bar modified with two kinds of encoded hairpin DNA probes (H0 and H0') was used in a multiplexed strategy allowing for specific extraction of Hg2+ and Ag+ coupled to microchip electrophoresis (MCE) separation and LED induced fluorescence (LIF) detection. The extraction step utilizes stir bars, which are functionalized with designed hairpin DNA probes (H0 with TT and H0' with CC mismatches in stems). This allows the specific capture of Hg2+ and Ag+ through CAg+C and THg2+T interactions. These complexes are then enzymatically degraded by the action of exonuclease III (Exo III). The ions released during this enzymatic reaction can initiate a new cycle of interactions with hairpin structures and enzymatic reactions and so on. This cyclic step is specific to the presence of Hg2+ and Ag+ and represents the first round of amplification of the presence of the selected ions. The resulting single strand DNAs on the stirring bars after enzymatic degradation were used in the second step as primers to trigger the catalytic hairpin assembly (CHA) in the presence of a couple of hairpin structures in solution. Such a reaction allows producing duplexes that can be monitored by MCE-LIF. The fluorescence intensity of CHA products (IP) increased and that of hairpin DNAs (IR) decreased with the increase of target concentrations. The signal ratios (IP/IR and IP'/IR') consisted of targets. The assay was employed for Hg2+ and Ag+ detection in several mediums including water, milk, and fish samples with complex matrices. The results showed that the assay could avoid matrix interference to increase the sensitivity. Therefore, the multiplexed assay was ideal to simultaneously and quickly detect metal ions in complex samples.

A microchip electrophoresis-based assay for ratiometric detection of kanamycin by R-shape probe and exonuclease-assisted signal amplification.

Excessive intake of kanamycin (KANA) can cause some serious drug-resistant diseases, so it is urgent to develop some accurate and rapid analytical methods for monitor KANA residues in foodstuffs with complex matrix. Recently, many ratiometric assays were reported to be capable of overcoming matrix interference. Herein, a ratiometric and homogeneous assay for KANA detection based on microchip electrophoresis (MCE) was developed. First, by one single strand DNA (S-DNA) and one hairpin DNA (H-DNA), a novel R shape DNA probe (R-DNA) was prepared. After the probe was incubated with KANA, the S-DNA-KANA complex was formed, and H-DNA was released. Moreover, in the presence of exonuclease I (Exo-I), S-DNA-KANA complex would be digested to release the captured KANA for triggering target recycling and signal amplification. With the reaction going on, the fluorescence intensity of H-DNA (IH) increased and that of R-DNA (IR) decreased. They can be separated at different voltage intensities and converted to fluorescent signals for signal readout by MCE. The signal ratio of IH/IR was found to be linear toward target from 0.5 pg mL-1 to 10 ng mL-1, and the limit of detection was 150 fg mL-1. Moreover, it was successfully employed for KANA detection in milk and fish samples with consistent results of enzyme linked immune sorbent assay (ELISA). The R-DNA probe can quantitatively convert the amount of target to the intensity of DNA without label by MCE, and achieved exonuclease assisted signal amplification in homogenous solution. It was valuable to detect antibiotics residues in foodstuff with complex matrix. This approach broadened the application field of MCE to detect antibiotics without derivatization, which provided a promising platform for rapid screening of antibiotic residues in food.

Enzyme- and label-free electrochemical aptasensor for kanamycin detection based on double stir bar-assisted toehold-mediated strand displacement reaction for dual-signal amplification.

It is critically important to detect antibiotic residues for monitoring food safety. In this study, an enzyme- and label-free electrochemical aptasensor for antibiotics, with kanamycin (Kana) as a typical analyte, was developed based on a double stir bar-assisted toehold-mediated strand displacement reaction (dSB-TMSDR) for dual-signal amplification. First, we modified two gold electrodes (E-1 and E-2) with different DNA probes (S1/S2 hybrid probe in E-1 and DNA fuel strand S3 in E-2). In the presence of Kana, an S1/S2 probe can be disassembled from E-1 to form an S2/Kana complex in supernatant. The S2/Kana could react with S3 on E-2 to form S2/S3 hybrid and release Kana through TMSDR. After then, the target recycling was triggered. Subsequently, the formed S2/S3 hybrid can also trigger a hybridization chain reaction (HCR). Consequently, the dual-signal amplification strategy was established, which resulted in many long dsDNA chains on E-2. The chains can associate with methylene blue (MB) as redox probes to produce a current response for the quantification of Kana. The assay exhibited high sensitivity and specificity with a detection limit at 16 fM Kana due to the dual-signal amplification. The double stir bars system can both increase phase separation and prevent leakage of DNA fuel to reduce background interference. Moreover, it allows flexible sequence design of the TMSDR probes. The assay was successfully employed to detect Kana residues in food and showed potential application value in food safety detection.

A multiple signal amplified colorimetric aptasensor for antibiotics measurement using DNAzyme labeled Fe-MIL-88-Pt as novel peroxidase mimic tags and CSDP target-triggered cycles.

An ultrasensitive colorimetric aptasensor was developed for antibiotics detection, with chloramphenicol (CAP) as model target, using DNAzyme labeled Fe-MIL-88-Pt as novel peroxidase mimic signal tags and target-triggered circular strand-displacement polymerization (CSDP) for signal amplification. The system consists of two components which can partially hybridize with each other: one is capture probe which was formed through immobilizing hairpin DNA containing aptamer sequence on magnetic beads (MB-cDNA), another is signal tag which was constructed through labeling single strand DNAzyme (G-quadruplex/Hemin) which can partially hybrid with cDNA on platinum nanoparticles functionalized Fe-MIL-88 (MIL-88-Pt-DNAzyme). All components of MIL-88, Pt and DNAzyme in the tag can act as peroxidase mimic to triply catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by H2O2 into a blue-colored oxidized TMB (oxTMB) for the colorimetric readout. Thus distinctive signal can be observed by naked eye even in presence of 0.02 nM tags. In the presence of target and primer, cDNA loop can open to form cDNA/CAP intermediates, enabling primer to hybridize with the exposed sequences of the cDNA, which initiated target assisted CSDP recycles. Then numerous signal tags were released into supernatant to catalyze TMB for color development. There was a liner relationship between the absorbance and the concentration of CAP in the range of 0.1 pM (0.0323 pg/mL) to 1000 pM (323 pg/mL) with the detection limit of 0.03 pM (0.0097 pg/mL). The ultra-high sensitivity was ascribed to the multiplex catalytic activities from the tags and CSDP based signal amplification. Furthermore, this method can produce signals being observed by naked eye to facilitate in-situ detection and be further extended to detect other antibiotics in food just by simply replacing cDNA on the sensing system.

A two dimensional metal-organic framework nanosheets-based fluorescence resonance energy transfer aptasensor with circular strand-replacement DNA polymerization target-triggered amplification strategy for homogenous detection of antibiotics.

In the study, a novel two dimensional metal-organic framework (Cu-TCPP nanosheets) based fluorescence resonance energy transfer (FRET) aptasensing platform was developed for detecting antibiotics. Cu-TCPP nanosheets were employed for quenching the background fluorescence and circular strand-replacement DNA polymerization (CSRP) for signal amplification. To fulfill the purpose, we designed an aptamer hairpin probe (HP) whose stem can be opened while specifically binding to target. Then the opened HP would bind with the primer. Under the action of polymerase, extension reaction was induced to generate double-stranded DNA (dsDNA), and then the target was released for the next cycle. Finally, SYBR Green I (SG) can bind with dsDNA to produce strong fluorescence response for quantification of target. It's worth mentioning that the fluorescence of HP/SG complex and free SG could be completely quenched by Cu-TCPP nanosheets while that of dsDNA/SG complex wouldn't be affected. Thus, the sensor produced negligible background signals. It can produce 7.5-fold improved S/N compared to a graphene oxide (GO)-based FRET aptasensor. Chloramphenicol (CAP) was chosen as the model analyte to demonstrate the feasibility of the sensor system. The detection range is broad from 0.001 to 10 ng mL-1 with a detection limit down to 0.3 pg mL-1. The proposed assay was label free and can be used in homogenous detection which greatly simplifies the complexity of operations. The strategy opens a new way to develop sensitive, in-situ and simple assay for antibiotics in foods.

ß-cyclodextrin functionalized meso-/macroporous magnetic titanium dioxide adsorbent as extraction material combined with gas chromatography-mass spectrometry for the detection of chlorobenzenes in soil samples.

A high-performance and selective adsorbent was developed for simultaneous extraction of 6 chlorobenzenes residues in soil samples by using magnetic solid phase extraction (MSPE) combined with automated SPE followed by gas chromatography-mass spectrometry (GC-MS). The adsorbent was synthesized by grafting carboxymethyl-ß-cyclodextrin (CM-ß-CD) on the surface of porous core-shell magnetic Fe3O4@flower like TiO2 microspheres (Fe3O4@fTiO2-CMCD), used as a carrier. The main factors (adsorbent amount, adsorption time, elution solvent, elution volume, and elution flow rate) affecting the extraction efficiency were investigated in detail. The adsorbent exhibited high loading capacity (25.6 mg g(-1) for 1,3-dichlorobenzene). This maybe due to meso-/macroporous TiO2 having high specific surface area; as a carrier of the ß-cyclodextrin film, it could obviously increase the number of recognition sites. The newly developed adsorbent also showed good selectivity towards chlorobenzenes based on host-guest interactions between ß-cyclodextrin (on adsorbent's surface) and targets, which can minimize complex matrix interference in soil samples. The proposed method was successfully applied for the analysis of environmental soil samples with recovery ranging from 87.3 to 104.3%. All target compounds showed good linearities with correlation coefficients (r) higher than 0.996. The limits of quantitation for the 6 CBs were 0.03-0.09 µg kg(-1). These findings confirmed meso-/macroporous structure Fe3O4@fTiO2-CMCD as a highly effective extraction material for use in trace CB analyses in complex soil samples.

Employment of a novel magnetically multifunctional purifying material for determination of toxic highly chlorinated polychlorinated biphenyls at trace levels in soil samples.

In this study, we developed a magnetically multifunctional purifying material for efficient removal of matrix interferences, especially certain organochlorine pesticide (DDT, DDE, and DDD), during the determination of toxic highly chlorinated polychlorinated biphenyls (PCBs) at trace levels in soil samples. The multifunctional adsorbent (CMCD-NH2-MNPs) was prepared by grafting carboxymethyl-ß-cyclodextrin on the surface of amino-functionalized magnetite (Fe3O4) nanoparticles. CMCD-NH2-MNPs has stronger host-guest complexation with DDT, DDE, and DDD, but the same adsorbent shows weaker adsorption ability toward highly chlorinated PCBs (from tetra- to octa-chlorinated PCBs) owing to their steric hindrance effect. Based on this principle, a simple and rapid gas chromatography-mass spectrometry (GC-MS) method was developed for six indicator PCBs (PCB28, PCB52, PCB101, PCB138, PCB153, and PCB180) in soil. Comparative studies were conducted to determine the clean-up efficiency of the following three techniques: (i) Oasis-HLB, (ii) multi-layer silica column, and (iii) dSPE employing CMCD-NH2-MNPs. The results indicate that CMCD-NH2-MNPs as the purification material can easily and effectively remove DDT, DDE, and DDD in soil samples within a short duration of time. The recoveries for highly chlorinated PCBs were in the range of 85.4-102.2%, with RSDs varying between 1.0 and 6.5%. The proposed method was verified as one of the most effective clean-up procedures for the analysis of highly chlorinated PCBs in real soil samples.

Synthesis and characterization of a molecularly imprinted polymer for the determination of trace tributyltin in seawater and seafood by liquid chromatography-tandem mass spectroscopy.

Analysis of tributyltin chloride (TBT) in environmental samples, such as seawater, is important in order to evaluate the TBT contamination and accumulation in the trophic chain. The environmental impact of organotin compounds has been a particular focus of analytical studies. The present study reports the use of molecular imprinting technology coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine trace amounts of TBT in seawater and seafood (mussel tissue samples). The imprinted polymer was synthesized by a non-covalent free-radical approach using acrylamide (AM) as a monomer and TBT as a template molecule in acetonitrile solvent (polymerization media). The imprinted polymer synthesized by this approach exhibited good adsorptive capacity and allowed specific retention of TBT. Recoveries of TBT in seawater samples spiked with different TBT concentrations ranged from 67.2% to 81.1% with peak area precision (RSD)<3.7%, and recoveries of TBT in mussel tissue samples ranged from 75.0% to 94.2% with RSD<4.8%.

Novel one-step route for synthesizing CdS/polystyrene nanocomposite hollow spheres.

CdS/polystyrene nanocomposite hollow spheres with diameters between 240 and 500 nm were synthesized under ambient conditions by a novel microemulsion method in which the polymerization of styrene and the formation of CdS nanoparticles were initiated by gamma-irradiation. The product was characterized by transmission electron microscopy (TEM), field-emission scanning electron microscopy (FESEM), X-ray powder diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, and thermogravimetric analysis (TGA), which show the walls of the hollow spheres are porous and composed of polystyrene containing homogeneously dispersed CdS nanoparticles. The quantum-confined effect of the CdS/polystyrene nanocomposite hollow spheres is confirmed by the ultraviolet-visible (UV-vis) and photoluminescent (PL) spectra. We propose that the walls of these nanocomposite hollow spheres originate from the simultaneous synthesis of polystyrene and CdS nanoparticles at the interface of microemulsion droplets. This novel method is expected to produce various inorganic/polymer nanocomposite hollow spheres with potential applications in the fields of materials science and biotechnology.