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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.

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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 Cd 2+ and Pb 2+ 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 Cd 2+ and Pb 2+ 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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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.

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 H 2 O 2 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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

An ultrasensitive electrochemical aptasensor for multiplex antibiotics detection based on endonuclease and exonuclease assisted dual recycling amplification strategy was proposed. Kanamycin and chloramphenicol were selected as candidates. Firstly, aptamers of the antibiotics were immobilized on bar A and then binding with their endonuclease labeled complementary DNA strands to construct enzyme-cleavage probes. Secondly, The nano zirconium-metal organic framework (NMOF) particles with 1,4-benzene-dicarboxylate (BDC) as linker was defined as UiO-66. And its updated version, hierarchically porous UiO-66 (HP-UIO-66) decorated with different electroactive materials as signal tags were synthesized. Then they were immobilized on bar B linked by two duplex DNA strands which can be specifically cleaved by corresponding enzyme-cleavage probes in bar A. Once targets were introduced into system, aptamers can capture them and then release enzyme-cleavage probes. In the presence of exonuclease-I, exonuclease assisted target recycling amplification was triggered and more enzyme-cleavage probes were released into solution. The probes can trigger endonuclease assisted recycles and repeatedly cleave their corresponding duplex DNA strands on bar B then released numerous signal tags into supernatant. Thus two recycling amplification was performed in the system. Finally, MB and Fc in the signal tags were detected by square wave voltammetry after removing bar A/B and the current intensities were correspondent with the concentration of KANA and CAP respectively. Under the optimum condition, the limits of detection for the KANA and CAP were 35fM and 21fM respectively with a wide linear range from 1 × 10 -4 to 50nM. This dual recycling amplification detection system exhibited high sensitivities and specificity. It can be easily extended to detect other targets if changing the corresponding aptamers and has potential application values for screening of multiplex antibiotics residues in food safety., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A mimicking-enzyme-based colorimetric aptasensor was developed for the detection of kanamycin (KANA) in milk using magnetic loop-DNA-NMOF-Pt (m-L-DNA) probes and catalytic hairpin assembly (CHA)-assisted target recycling for signal amplification. The m-L-DNA probes were constructed via hybridization of hairpin DNA H1 (containing aptamer sequence) immobilized magnetic beads (m-H1) and signal DNA (sDNA, partial hybridization with H1) labeled nano Fe-MIL-88NH 2 -Pt (NMOF-Pt-sDNA). In the presence of KANA and complementary hairpin DNA H2, the m-L-DNA probes decomposed and formed an m-H1/KANA intermediate, which triggered the CHA reaction to form a stable duplex strand (m-H1-H2) while releasing KANA again for recycling. Consequently, numerous NMOF-Pt-sDNA as mimicking enzymes can synergistically catalyze 3,3',5,5'-tetramethylbenzidine (TMB) for color development. The aptasensor exhibited high selectivity and sensitivity for KANA in milk with a detection limit of 0.2 pg mL -1 within 30 min. The assay can be conveniently extended for on-site screening of other antibiotics in foods by simply changing the base sequence of the probes.

A novel label-free, universal, and high throughput aptasensor was developed based on a microchip electrophoresis (MCE) platform for automatic detection of antibiotic residues in food. Firstly, chloramphenicol (CAP) was employed as a model to be captured by its aptamer probe (Apt). Then, the partial complementary oligonucleotide of CAP's aptamer (C-DNA) was introduced into the reaction system. Because the Apt-CAP complex can't further hybrid with free C-DNA, the amount of hybrid Apt-C-DNA double strand DNA (dsDNA) was less than that without adding the target. Finally, the above mixture was introduced into the microchip electrophoresis (MCE) platform for detection, both dsDNA and Apt-CAP can be separated and produce different fluorescence signals in the MCE. In a certain concentration range, the ratio of signal between dsDNA and Apt-CAP (I dsDNA /I Apt-CAP ) was proportional to the concentration of targets. Under the optimum conditions, the ratio showed a satisfactory linearity range from 0.008 to 1ng/mL of CAP with a detection limit of 0.003ng/mL. Thus, a universal MCE-based assay was developed for quantifying CAP automatically. The method was also successfully applied in the different food samples for CAP detection, which showed a good recovery (Milk: 91.1-108%, Fish: 86.1-114%) and the results were consistent with that of ELISA. This method owned many merits as follows: firstly, MCE was a high throughput screening platform and the detection time is limited to 3min for each sample. Secondly, the aptamer probes can be directly used for detection without labeling any signal tag which can facilitate the preparation procedures of probes. Thirdly, the operation was easy just by the following steps: firstly, the mixture of aptamer probes were incubated followed adding C-DNA; then measurement was performed. Moreover, the assay with MCE platform can be used to detect other targets just by changing the corresponding aptamer probe; it can even realize simultaneous detection when the targets have aptamers with different number of base pairs. Above all, it's a high- throughput and prospective method which can be applied in high throughput screening of antibiotics in food safety., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Herein, a smart single-stranded DNA binding protein (SSB)-assisted fluorescence aptamer switch based on fluorescence resonance energy transfer (FRET) was designed. The FRET switch was synthesized by connecting SSB labeled quantum dots (QDs@SSB) as donor with aptamer (apt) labeled gold nanoparticles (AuNPs@apt) as acceptor, and it was employed for detecting chloramphenicol (CAP) in a homogenous solution. In the assay, the interaction between core-shell QDs@SSB and AuNPs@apt leads to a dramatic quenching (turning off). After adding CAP in the detection system, AuNPs@apt can bind the target specifically then separate QDs@SSB with AuNPs@apt-target, resulting in restoring the fluorescence intensity of QDs (turning on). Consequently, the fluorescence intensity recovers and the recovery extent can be used for detection of CAP in homogenous phase via optical responses. Under optimal conditions, the fluorescence intensity increased linearly with increasing concentrations of CAP from 0.005 to 100ngmL -1 . The limit of this fluorescence aptamer switch was around 3pgmL -1 for CAP detection. When the analyte is changed, the assay can be applied to detect other targets only by changing relative aptamer in AuNPs@apt probe. Furthermore, it has potential to be served as a simple, sensitive and portable platform for antibiotic contaminants detection in biological and environmental samples., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The present study aimed to explore a novel triple-amplification electrochemiluminescence (ECL) assay for detecting of chloramphenicol (CAP). This strategy was based on single-stranded DNA-binding protein (SSB) and horseradish peroxidase (HRP) enzyme-linked polymer (EnVision reagent, EV) labeled on Au nanoparticles (EV-Au-SSB) as nanotracer and exonuclease-assisted target recycling. The composite probes were prepared via immunoreactions between the CdS nanocrystal (CdS NC)-functionalized partial complementary DNA and aptamer (CdSNCs/Apt-ssDNA1) as capture probes, and EV-Au-SSB as nanotracer. When the composite probe solution co-existed with CAP and Exo I, the aptamer on the capture probes preferentially combined with CAP, and then CAP-Apt and nanotracer complex were released into the solution. Subsequently, Exo I in the solution could further digest the CAP-Apt from the 3'-end of the aptamer and release CAP, which could participate in further reaction with the probes. It was worth mentioning that EV contained a large number of HRPs on its dendritic chain. In the EV-Au-SSB, Au could enhance ECL intensity of CdS NCs by surface plasmon resonance. What's more, HRPs on EV could catalyze the reaction of H2O2, which could obviously enhance ECL intensity of CdS NCs. This study demonstrated excellent performance of the triple-amplification ECL assay, which makes this aptasensor system suitable and promising for the practical application of CAP residues in fish samples. Moreover, the assay might provide a promising avenue to develop efficient aptasensors to determine small-molecule harmful substances in environmental monitoring and food safety., (Copyright © 2016 Elsevier B.V. All rights reserved.)

An ultrasensitive electrochemical aptasensor for simultaneous detection of oxytetracycline (OTC) and kanamycin (KAN) has been developed based on metal ions doped metal organic frame materials (MOFs) as signal tracers and RecJ f exonuclease-catalyzed targets recycling amplification. The aptasensor consists of capture beads (the anti-single-stranded DNA Antibody, as anti-ssDNA Ab, labeled on Dynabeads) and nanoscale MOF (NMOF) based signal tracers (simplified as Apts-MNM, the NMOF labeled with metal ions and the aptamers). Particularly, the MOF (UiO-66-NH 2 ), with large internal surface areas, ultrahigh porosity and abundant amine groups in the pores, was employed as substrates to carry plenty of metal ions (Pb 2+ or Cd 2+ ) and label aptamers of OTC or KAN. Thus, the aptasensor is formed by the specific recognition between anti-ssDNA Ab and aptamers. In the presence of targets (OTC and KAN), aptamers prefer to form targets-Apts-MNM complexes in lieu of anti-ssDNA Ab-aptamer complexes, which results in the dissociation of Apts-MNM from capture beads. With the employment of RecJ f exonuclease, targets-Apts-MNM in supernatant was digested into mononucleotides and liberated the target, which can further participate in the next reaction cycling to produce more signal tracers. After magnetic separation, the enhanced square wave voltammetry (SWV) signals were produced from signal tracers. The aptasensor exhibited a linear correlation in the range from 0.5pM to 50nM, with detection limits of 0.18pM and 0.15pM (S/N=3) toward OTC and KAN respectively. This strategy provides specificity and sensitive approach for multiplex antibiotics detection and has promising applications in food analysis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A novel fluorescence aptasensor was successfully developed to respond to chloramphenicol (CAP) in food based on magnetic aptamer-liposome vesicle probe. In order to fabricate it, aptamer labeled on functionalized magnetic beads (MB) was firstly employed as capture adsorbent (MB-Apt), then SSB (single-stranded DNA binding protein) and DIL (1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanineperchlorate) coimmobilized liposomes (SSB/DIL-Lip) was employed as vesicle signal tracer. The composite vesicle probe is formed between SSB/DIL-Lip and MB-Apt based on SSB's specific recognition towards aptamer on vesicle signal tracer. Upon the vesicle probe solution reacted with CAP, the aptamer on the magnetic beads preferentially bounded with CAP, and then released SSB/DIL-Lip vesicle signal tracer in the supernatant after magnetic separation. The released tracer can emit fluorescence which was correspondence with the concentration of the analyte. At the optimum conditions, the aptasensor exhibited a good linear response for CAP detection in the range of 0.003-10nM with a detection limit of 1pM. Importantly, the methodology was further validated for analyzing CAP in fish samples with consistent results obtained by ELISA kit, thus providing a promising approach for quantitative monitoring of CAP and significant anti-interference ability in food safety., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A selective and facile fluorescence "switch-on" scheme is developed to detect antibiotics residues in food, using chloramphenicol (CAP) as model, based on a novel magnetic aptamer probe (aptamer-Pt-luminol nanocomposite labeled with hemin/G-quadruplex). Firstly, the composite probe is prepared through the immuno-reactions between the capture beads (anti-dsDNA antibody labeled on magnetic Dynabeads) and the nanotracer (nano-Pt-luminol labeled with double-strand aptamer, as ds-Apt, and hemin/G-quadruplex). When the composite probe is mixed with CAP, the aptamer preferentially reacted with CAP to decompose the double-strand aptamer to ssDNA, which cannot be recognized by the anti-dsDNA antibody on the capture probes. Thus, after magnetic separation, the nanotracer can be released into the supernatant. Because the hemin/G-quadruplex and PtNPs in nanotracer can catalyze luminol-H2O2 system to emit fluorescence. Thus a dual-amplified "switch-on" signal appeared, of which intensity is proportional to the concentration of CAP between 0.001 and 100ng mL(-1) with detection limit of 0.0005ng mL(-1) (S/N=3). Besides, our method has good selectivity and was employed for CAP detection in real milk samples. The results agree well with those from conventional gas chromatograph-mass spectrometer (GC-MS). The switch-on signal is produced by one-step substitution reaction between aptamer in nanotracer and target. When the analyte is changed, the probe can be refabricated only by changing the corresponding aptamer. Thus, all features above prove our strategy to be a facile, feasible and selective method in antibiotics screening for food safety., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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.