Enzyme-free fluorometric assay for chloramphenicol based on double stirring bar-assisted dual signal amplification.

An enzyme-free fluorometric assay is described that accomplishes dual signal amplification by making use of a two stirring bars. Two Y-shaped DNA probes were designed and placed on the bars. When the target (with chloramphenicol as model analyte) is added, it triggers target recycling and simultaneously catalyzes hairpin assembly (CHA). A large fraction of DNA primers is released by the analyte from the bar to the supernatant and open hairpins with G-quadruplex DNA sequence. The G-quadruplex can specifically bind thioflavin T (ThT) to emit fluorescence (with excitation/emission maxima at 445 and 485 nm) for quantification of chloramphenicol. An enzyme is not needed. ThT is added to the system as a fluorescent DNA probe. All this strongly reduces the cost for sensor construction and usage. The dual signal amplification steps occur simultaneously which reduces the detection time. The assay was successfully employed to the determination of CAP in spiked milk and fish samples within 60 min and with a 16 pM limit of detection (at S/N = 3). Graphical abstract Schematic representation of a new method for the detection of chloramphenicol by using  two stirring bars. It is based on target recycling and catalyzed hairpin assembly amplification. CAP: chloramphenicol, ThT: thioflavin T, CHA: catalyzed hairpin assembly.

A fluorometric aptamer method for kanamycin by applying a dual amplification strategy and using double Y-shaped DNA probes on a gold bar and on magnetite nanoparticles.

A simple and highly sensitive fluorometric method is described for the determination of the antibiotic kanamycin (Kana) in food. Dual signal amplification is accomplished by making use of double Y-shaped aptamer DNA probes acting as a capture probes and signal amplification probes. The DNA probes were immobilized on a gold bar and on a magnetic bar, respectively. On addition of Kana, the Y-shaped aptamer probe captures Kana and then is disassembled to release two single-stranded DNAs. These trigger target recycling and HCR between the two bars simultaneously. As a result, many long duplex DNA chains are formed in the supernatant. After pulling out the bars and adding the fluorescent intercalating probe SYBR Green I, strong fluorescence (with excitation/emission peaks at 497/525 nm) is induced. The use of such double Y-shaped DNA probes obviously overcomes the unspecific signal amplification by HCR which increases selectivity and sensitivity. This is due to the fact that the hairpin of HCR is separated in being present in different arms of the Y-shaped probe. Under the optimal conditions, the assay has a limit of 0.45 pg·mL-1 for Kana. It was applied to analyze spiked milk, fish and pork samples. Graphical abstract The scheme represents a sensitive fluorometric aptamer-based method to detect kanamycin (Kana). It is making use of a double stirring bar-assisted dual amplification strategy with zero background. Abbreviations: apt: aptamer, AuNPs: gold nanoparticles, HCR: hybridization chain reaction.

Microfluidic chip electrophoresis for simultaneous fluorometric aptasensing of alpha-fetoprotein, carbohydrate antigen 125 and carcinoembryonic antigen by applying a catalytic hairpin assembly.

An aptamer based assay is presented that is making use of a catalytic hybrid assembly and a microfluidic chip electrophoresis format. It enables simultaneous determination of the biomarkers (BMs) α-fetoprotein (AFP), carbohydrate antigen 125 (CA125), and carcinoembryonic antigen (CEA). The respective aptamers were covalently bound to Fe3O4@AuNPs (AuMPs) magnetic beads and then used to capture the biomarkers on their surface. Different single-stranded DNA primers were then labeled with various antibodies as encoding and signaling tags. The signal tags reacted with AuMPs-BMs to form different antibody-BM-aptamer complexes. After magnetic separation, three pairs of hairpins as substrates were introduced to trigger catalytic hybrid assembly by the primers in the complex. This will form many duplex DNA products of different length in the supernatant. The products can be magnetically separated by microfluidic chip electrophoresis and determined by fluorometry at excitation/emission wavelengths of 495/525 nm. Several experimental conditions including the hairpin concentration, reaction time and temperature were systemically optimized. The method can simultaneously quantify AFP, CEA and CA125, respectively, with detection limits of 0.1, 0.2, 0.15 pg mL-1 (at S/N = 3). The aptamer functionalized magnetic beads can be reused for at least 20 times with a recovery of up to 80% after heat treatment. The method was employed to simultaneously detect the three BMs in serum samples. Graphical abstract Schematic presentation of the microfluidic chip electrophoresis and antibody-aptamer based multianalysis method for simultaneous detection of alpha-fetoprotein (AFP), carbohydrate antigen 125 (CA125) and carcinoembryonic antigen (CEA).

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.

Microfluidic electrophoretic non-enzymatic kanamycin assay making use of a stirring bar functionalized with gold-labeled aptamer, of a fluorescent DNA probe, and of signal amplification via hybridization chain reaction.

The authors describe an enzyme-free aptamer-based assay for the determination of the model antibiotic kanamycin (Kana). The method is making use of (a) microfluidic chip electrophoresis; (b) a stirring bar carrying a gold-labeled aptamer probe, and (c) the hybridization chain reaction (HCR) for signal amplification. Firstly, a stirring bar (length: 1 cm; diameter: 0.2 mm) was modified with a large amount of duplex DNA and then hybridized with aptamer and its partially complementary chains (cDNA). In the presence of Kana, the binding between the Kana and aptamer unwinds the duplex structures and releases a corresponding amount of cDNA into the supernatant. The released cDNA triggers the HCR in the presence of H1 and H2 DNA hairpin to produce a large amount of duplex DNA chains with different lengths. At the same time, the amounts of H1 and H2 are reduced. The decreased signal of H1/H2 after several HCR cycles can be used to quantify kana in the 1 pg·mL-1 to 10 ng·mL-1, with a detection limit of 0.29 pg·mL-1. The signal is generated by reading the fluorescence, best at excitation/emission maxima of 470/525 nm. The whole detection process takes 3 min only. The assay was employed to the detection of Kana in spiked milk and fish samples. Results are consistent with those of an enzyme linked immunosorbent assay. The assay has high throughput, high selectivity, and high amplification capability. Graphical abstract Schematic of a stirring bar functionalized with gold-labeled aptamer acting as the capture probe. It can capture the target and release primer simultaneously. The primer triggers the hybridization chain reaction inducing the consumption of H1 and H2. After a certain reaction time, the mixture is injected into the MCE platform for microfluidic electrophoretic separation and fluorometric detection.

A triple-amplification colorimetric assay for antibiotics based on magnetic aptamer-enzyme co-immobilized platinum nanoprobes and exonuclease-assisted target recycling.

Herein, an ultrasensitive and selective colorimetric assay for antibiotics, using chloramphenicol (CAP) as the model analyte, was developed based on magnetic aptamer-HRP-platinum composite probes and exonuclease-assisted target recycling. The composite probes were prepared through immunoreactions between the double stranded DNA antibody (anti-DNA) labeled on core-shell Fe3O4@Au nanoparticles (AuMNP-anti-DNA) as the capture probe, and the double stranded aptamer (aptamer hybrid with its complementary oligonucleotides) labeled on Pt@HRP nanoparticles as the nanotracer (ds-Apt-HRP-PtNPs). When the CAP samples were incubated with the probes for 30 min at room temperature, they could be captured by the aptamer to form a nanotracer-CAP complex, which was then released into the supernatant after magnetic separation. This is because the anti-DNA on the capture probes cannot recognize the single strand aptamer-CAP complex. The exonuclease I (Exo I) added into the supernatant can further digest the aptamer-CAP from the 3'-end of the aptamer and the CAP in the aptamer-CAP complex can be released again, which can further participate in a new cycling process to react with the probes. Pt and HRP in the nanotracer could both catalyze and dual amplify the absorbance at 650 nm ascribed to the 3,3',5,5'-tetramethylbenzidine (TMB)-H2O2 system. Moreover, Exo I can assist the target recycling, which can further amplify the signal. Thus, the triple amplified signal can be quantified by ultraviolet-visible spectroscopy. The experimental results showed that the CAP detection possessed a linear range of 0.001-10 ng mL(-1) and a detection limit of 0.0003 ng mL(-1) (S/N = 3). The assay was successfully employed to detect CAP in milk, which is much more facile, time saving, and sensitive than the commercial ELISA kits.

A portable and antibody-free sandwich assay for determination of chloramphenicol in food based on a personal glucose meter.

A portable and antibody-free sandwich assay was fabricated for determination of chloramphenicol (CAP) in animal-derived food by a personal glucose meter (PGM). The sandwich-type strategy was developed on the basis of magnetic molecularly imprinted probe (m-MIP) nanoparticles and a ß-cyclodextrin/invertase-functionalized signal tag. Firstly, the m-MIPs were fabricated using polydopamine molecularly imprinted film modified Fe3O4 nanoparticles with 2,2-dichloroacetamide as a template that could capture the 2,2-dichloroacetamide segment of CAP. Secondly, ß-cyclodextrin/invertase polymer bioconjungate was synthesized as a signal tag that could recognize the nitrobenzene segment of CAP through host-guest interaction. The dual-specificity recognition model relies on the formation of a sandwich between m-MIPs, different segments of CAP, and the ß-cyclodextrin-functionalized signal tag. The sandwich-type complex formed was then subjected to detection with a PGM. The complexes can hydrolyze sucrose to glucose, which can be used for detection with a PGM through invertase. According to our experiment, the concentration of CAP was proportional to the amount of glucose formed, which could quantitatively assess the CAP with a dynamic range of 0.5-50 ng mL(-1) and a detection limit of 0.16 ng mL(-1) (signal-to-noise ratio of 3). The detection time of the assay was about 1 h, which was obviously shorter than that of competitive ELISA. More importantly, we have successfully applied this on-site assay for CAP screening in animal-derived food.

A renewable and ultrasensitive electrochemiluminescence immunosenor based on magnetic RuL@SiO2-Au~RuL-Ab2 sandwich-type nano-immunocomplexes.

An ultrasensitive and renewable electrochemiluminescence (ECL) immunosensor was developed for the detection of tumor markers by combining a newly designed trace tag and streptavidin-coated magnetic particles (SCMPs). The trace tag (RuL@SiO(2)-Au~RuL-Ab2) was prepared by loading Ru(bpy)(3)(2+)(RuL)-conjuged secondary antibodies (RuL-Ab2) on RuL@SiO(2) (RuL-doped SiO(2)) doped Au (RuL@SiO(2)-Au). To fabricate the immunosensor, SCMPs were mixed with biotinylated AFP primary antibody (Biotin-Ab1), AFP, and RuL@SiO2-Au~RuL-Ab2 complexes, then the resulting SCMP/Biotin-Ab1/AFP/RuL@SiO2-Au~RuL-Ab2 (SBAR) sandwich-type immunocomplexes were absorbed on screen printed carbon electrode (SPCE) for detection. The immunocomplexes can be easily washed away from the surface of the SPCE when the magnetic field was removed, which made the immunosensor reusable. The present immunosensor showed a wide linear range of 0.05-100 ng mL(-1) for detecting AFP, with a low detection limit of 0.02 ng mL(-1) (defined as S/N = 3). The method takes advantage of three properties of the immunosensor: firstly, the RuL@SiO(2)-Au~RuL-Ab2 composite exhibited dual amplification since SiO(2) could load large amount of reporter molecules (RuL) for signal amplification. Gold particles could provide a large active surface to load more reporter molecules (RuL-Ab2). Accordingly, through the ECL response of RuL and tripropylamine (TPA), a strong ECL signal was obtained and an amplification analysis of protein interaction was achieved. Secondly, the sensor is renewable because the sandwich-type immunocomplexes can be readily absorbed or removed on the SPCE's surface in a magnetic field. Thirdly, the SCMP modified probes can perform the rapid separation and purification of signal antibodies in a magnetic field. Thus, the present immunosensor can simultaneously realize separation, enrichment and determination. It showed potential application for the detection of AFP in human sera.

A three-dimensional, magnetic and electroactive nanoprobe for amperometric determination of tumor biomarkers.

A novel electrochemical immunosensor for tumor biomarker detection based on three-dimensional, magnetic and electroactive nanoprobes was developed in this study. To fabricate the nanoprobes, negatively charged Fe(3)O(4) nanoparticles (Fe(3)O(4) NPs) and gold nanoparticles (Au NPs) were first loaded on the surface of multiple wall carbon nanotubes (MCNTs) which were functioned with redox-active hemin and cationic polyelectrolyte poly(dimethyldiallylammonium chloride) (PDDA). Using alpha fetoprotein (AFP) as a model analyte, AFP antibody (anti-AFP) was absorbed on the surface of Au NPs, bovine serum albumin (BSA) was then used to block sites against non-specific binding, and finally formed anti-AFP/Au NPs/Fe(3)O(4)/hemin/MCNTs named anti-AFP nanoprobes. When the target antigen AFP was present, it interacted with anti-AFP and formed an antigen-antibody complex on the nanoprobe interface. This resulted in a decreased electrochemical signal of hemin for quantitative determination of AFP when immobilized onto the screen-printed working electrode (SPCE). The results showed that the nanoprobe-based electrochemical immunosensor was sensitive to AFP detection at a concentration of 0.1 to 200 ng·mL(-1) with a detection limit of 0.04 ng·mL(-1), it also demonstrated good selectivity against other interferential substances. The electroactive nanoprobes can be massively prepared, easily immobilized on the SPCE for target detection and rapidly renewed with a magnet. The proposed immunosensor is fast, simple, sensitive, stable, magnet-controlled, nontoxic, label-free and reproducible.

An amperometric immunosensor based on a polyelectrolyte/gold magnetic nanoparticle supramolecular assembly--modified electrode for the determination of HIV p24 in serum.

A novel supramolecular amperometric immunosensor for the determination of Human Immunodeficiency Virus antigen p24 (HIV p24) was built up using the electrostatic layer-by-layer self-assembly technique upon a gold electrode with HIV p24 antibody (anti-p24) being immobilized on polyelectrolyte/gold nanoparticle multilayer films. The multilayer films were composed of poly(L-lysine) (pLys) and mercaptosuccinic acid (MSA) stabilized Fe(3)O(4)(core)/gold(shell) nano particles (GMPs).The immunosensor preparation steps were monitored by X-ray fluorescence spectrometry (XRFS), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In pH 6.5 PBS, after the immunosensor was incubated with HIV p24 solution at 25 degrees C for 5 min, the electron transfer access of FeCN is partially inhibited, which leads to a linear decrease of peak current. In addition, the performance of the immunosensor was studied in detail. It offers high-sensitivity for the detection of p24 and has good correlation for the detection of p24 in the range of 0.1 to 100.0 ng/mL with a detection limit of 0.05 ng/mL estimated at a signal-to-noise ratio of 3. The proposed immunosensor was used to analyze p24 in human serum specimens and the results showed the developed immunosensor provides a promising alternative approach for detecting p24 in the early diagnosis of AIDS patients.

Microchip electrophoresis based aptasensor for multiplexed detection of antibiotics in foods via a stir-bar assisted multi-arm junctions recycling for signal amplification.

Microchip electrophoresis (MCE) was a good available method for high-throughput and rapid detecting chemical pollutants in food samples. However, many of the reported MCE assays involve complex design of microchip, laborious operation and poor universality which limited its promotion in multiple antibiotics' detection. Herein, a multiplexed aptasensor was developed based on a universal double-T type microchip to one-step and simultaneously detect several antibiotics within 3 min using chloramphenicol (CAP) and kanamycin (Kana) as representatives. Besides, a novel stir-bar assisted DNA multi-arm junctions recycling (MAJR) strategy was designed for transducing and amplifying the signal. The brief detection mechanism was as following: the added CAP and Kana can specifically react with their aptamer probes on the stir-bar and produce different single-stranded DNA primer, respectively. Afterwards, the primers can trigger MAJR to form a lot of three- and four-arm DNA junctions corresponding to different targets. The DNA multi-arm junctions can be easily separated and detected by MCE for quantification. Moreover, the stir-bar can facilitate phase separation and obviously eliminate matrix interference in food. The assay was successfully applied in milk and fish samples, showing excellent selectivity and sensitivity with a detection limits of 0.52 pg mL-1 CAP and 0.41 pg mL-1 Kana (S/N = 3). Thus, the assay holds a great potential application for screening of antibiotics in food.

Zero background and triple-signal amplified fluorescence aptasensor for antibiotics detection in foods.

It's important to eliminate matrix interference for accurate detecting antibiotic residues in complex food samples. In this study, we designed a zero-backgrounded fluorescence aptasensor to achieve on-site detection of antibiotic residues, with chloramphenicol (CAP) as representative analyte. Moreover, a three stir-bars assisted target recycling system (TSBTR) was designed to achieve triple signal amplification and increase the sensitivity. The bars included one magnetic stir-bar modified with two kinds of long DNA chains, and two gold stir-bars modified with Y shape-duplex DNA probes respectively. In the presence of CAP, the target could recurrently react with the probes on the bars and replace a large amount of long DNA chains into supernatant. After then, the bars were taken out and SYBR green dye was added to the solution. The dye can specifically intercalate into the duplex structures of DNA chains to emit fluorescence while not emitting a signal in its free state. Under the optimized experimental conditions, a wide linear response range of 5 orders of magnitude from 0.001 ng mL-1 to 10 ng mL-1 was achieved with a detection limit of 0.033 pg mL-1 CAP. The assay was successfully employed to detect CAP in food samples (milk & fish) with consistent results with ELISA's. High selectivity and sensitivity were attributed to the zero background signal and triple signal-amplification strategy. Moreover, the detection time can be shortened to 40 min due to that three signal amplified process can occur simultaneously. The fluorescent aptasensor was also label- and enzyme-free. All these ensure the platform to be rapid, cost-effective, easily-used, and is especially appropriate for detection antibiotics in food safety.

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 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.

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 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.

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.

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.

Novel label-free and high-throughput microchip electrophoresis platform for multiplex antibiotic residues detection based on aptamer probes and target catalyzed hairpin assembly for signal amplification.

Novel label-free and multiplex aptasensors have been developed for simultaneous detection of several antibiotics based on a microchip electrophoresis (MCE) platform and target catalyzed hairpin assembly (CHA) for signal amplification. Kanamycin (Kana) and oxytetracycline (OTC) were employed as models for testing the system. These aptasensors contained six DNA strands termed as Kana aptamer-catalysis strand (Kana apt-C), Kana inhibit strand (Kana inh), OTC aptamer-catalysis strand (OTC apt-C), OTC inhibit strand (OTC inh), hairpin structures H1 and H2 which were partially complementary. Upon the addition of Kana or OTC, the binding event of aptamer and target triggered the self-assembly between H1 and H2, resulting in the formation of many H1-H2 complexes. They could show strong signals which represented the concentration of Kana or OTC respectively in the MCE system. With the help of the well-designed and high-quality CHA amplification, the assay could yield 300-fold amplified signal comparing that from non-amplified system. Under optimal conditions, this assay exhibited a linear correlation in the ranges from 0.001ngmL-1 to 10ngmL-1, with the detection limits of 0.7pgmL-1 and 0.9pgmL-1 (S/N=3) toward Kana and OTC, respectively. The platform has the following advantages: firstly, the aptamer probes can be fabricated easily without labeling signal tags for MCE detection; Secondly, the targets can just react with probes and produce the amplified signal in one-pot. Finally, the targets can be simultaneously detected within 10min in different channels, thus high-throughput measurement can be achieved. Based on this work, it is estimated that this detection platform will be universally served as a simple, sensitive and portable platform for antibiotic contaminants detection in biological and environmental samples.