Stress-dependent macromolecular crowding in the mitochondrial matrix.

Macromolecules of various sizes induce crowding of the cellular environment. This crowding impacts on biochemical reactions by increasing solvent viscosity, decreasing the water-accessible volume and altering protein shape, function, and interactions. Although mitochondria represent highly protein-rich organelles, most of these proteins are somehow immobilized. Therefore, whether the mitochondrial matrix solvent exhibits macromolecular crowding is still unclear. Here, we demonstrate that fluorescent protein fusion peptides (AcGFP1 concatemers) in the mitochondrial matrix of HeLa cells display an elongated molecular structure and that their diffusion constant decreases with increasing molecular weight in a manner typical of macromolecular crowding. Chloramphenicol (CAP) treatment impaired mitochondrial function and reduced the number of cristae without triggering mitochondrial orthodox-to-condensed transition or a mitochondrial unfolded protein response. CAP-treated cells displayed progressive concatemer immobilization with increasing molecular weight and an eightfold matrix viscosity increase, compatible with increased macromolecular crowding. These results establish that the matrix solvent exhibits macromolecular crowding in functional and dysfunctional mitochondria. Therefore, changes in matrix crowding likely affect matrix biochemical reactions in a manner depending on the molecular weight of the involved crowders and reactants.

Experimental modification in thermal stability of oligomers by alanine substitution and site saturation mutagenesis of interfacial residues.

For certain industrial applications, the stability of protein oligomers is important. In this study, we demonstrated an efficient method to improve the thermal stability of oligomers using the trimeric protein chloramphenicol acetyltransferase (CAT) as the model. We substituted all interfacial residues of CAT with alanine to detect residues critical for oligomer stability. Mutation of six of the forty-nine interfacial residues enhanced oligomer thermal stability. Site saturation mutagenesis was performed on these six residues to optimize the side chains. About 15% of mutations enhanced thermal stability by more than 0.5 °C and most did not disrupt activity of CAT. Certain combinations of mutations further improved thermal stability and resistance against heat treatment. The quadruple mutant, H17V/N34S/F134A/D157C, retained the same activity as the wild-type after heat treatment at 9 °C higher temperature than the wild-type CAT. Furthermore, combinations with only alanine substitutions also improved thermal stability, suggesting the method we developed can be used for rapid modification of industrially important proteins.

Membranome-based identification of amino acid substitution in Haemophilus influenzae multidrug efflux pump HmrM for reduced chloramphenicol susceptibility.

A decreased chloramphenicol susceptibility in Haemophilus influenzae is commonly caused by the activity of chloramphenicol acetyltransferases (CATs). However, the involvement of membrane proteins in chloramphenicol susceptibility in H. influenzae remains unclear. In this study, chloramphenicol susceptibility testing, whole-genome sequencing, and analyses of membrane-related genes were performed in 51 H. influenzae isolates. Functional complementation assays and structure-based protein analyses were conducted to assess the effect of proteins with sequence substitutions on the minimum inhibitory concentration (MIC) of chloramphenicol in CAT-negative H. influenzae isolates. Six isolates were resistant to chloramphenicol and positive for type A-2 CATs. Of these isolates, A3256 had a similar level of CAT activity but a higher chloramphenicol MIC relative to the other resistant isolates; it also had 163 specific variations in 58 membrane genes. Regarding the CAT-negative isolates, logistic regression and receiver operator characteristic curve analyses revealed that 48T > G (Asn16Lys), 85 C > T (Leu29Phe), and 88 C > A (Leu30Ile) in HI_0898 (emrA), and 86T > G (Phe29Cys) and 141T > A (Ser47Arg) in HI_1177 (artM) were associated with enhanced chloramphenicol susceptibility, whereas 997G > A (Val333Ile) in HI_1612 (hmrM) was associated with reduced chloramphenicol susceptibility. Furthermore, the chloramphenicol MIC was lower in the CAT-negative isolates with EmrA-Leu29Phe/Leu30Ile or ArtM-Ser47Arg substitution and higher in those with HmrM-Val333Ile substitution, relative to their counterparts. The Val333Ile substitution was associated with enhanced HmrM protein stability and flexibility and increased chloramphenicol MICs in CAT-negative H. influenzae isolates. In conclusion, the substitution in H. influenzae multidrug efflux pump HmrM associated with reduced chloramphenicol susceptibility was characterised.

Highly enhanced chloramphenicol adsorption performance of MIL-53-NH2(Al)-derived porous carbons modified with tannic acid.

The worldwide demand for antibiotics has experienced a notable surge, propelled by the repercussions of the COVID-19 pandemic and advancements in the global healthcare sector. A prominent challenge confronting humanity is the unregulated release of antibiotic-laden wastewater into the environment, posing significant threats to public health. The adoption of affordable carbon-based adsorbents emerges as a promising strategy for mitigating the contamination of antibiotic wastewater. Here, we report the synthesis of novel porous carbons (MPC) through a direct pyrolysis of MIL-53-NH2(Al) and tannic acid (TANA) under N2 atmosphere at 800 °C for 4 h. The effect of TANA amount ratios (0%-20%, wt wt-1) on porous carbon structure and adsorption performance was investigated. Results showed that TANA modification resulted in decreased surface area (1,600 m2 g-1-949 m2 g-1) and pore volume (2.3 cm3 g-1-1.7 cm3 g-1), but supplied hydroxyl functional groups. Adsorption kinetic, intraparticle diffusion, and isotherm were examined, indicating the best fit of Elovich and Langmuir models. 10%-TANA-MPC obtained an ultrahigh adsorption capacity of 564.4 mg g-1, which was approximately 2.1 times higher than that of unmodified porous carbon. 10%-TANA-MPC could be easily recycled up to 5 times, and after reuse, this adsorbent still remained highly stable in morphology and surface area. The contribution of H bonding, pore-filling, electrostatic and π-π interactions to chloramphenicol adsorption was clarified. It is recommended that TANA-modified MIL-53-NH2(Al)-derived porous carbons act as a potential adsorbent for removal of pollutants effectively.

Research on promoting the asymmetric reaction of phenylacetylene with aldehydes by chloramphenicol base ligands.

A series of chiral ligands were synthesized using chloramphenicol base as starting materials. These ligands were applied to the asymmetric catalytic reactions of terminal alkynes with aldehydes to obtain a propargyl alcohol product in high yield (80-94%) with excellent enantioselectivities (82-96%).

Chloramphenicol alleviates 5-fluorouracil-induced cellular senescence through activation of autophagy.

5-Fluorouracil (5-FU) is a first-line treatment for colorectal cancer, but side effects such as severe diarrhea are common in clinical use and have been linked to its induction of normal cell senescence. Chloramphenicol (CAP) is an antibiotic commonly used to treat typhoid or anaerobic infections, but its senescence-related aspects have not been thoroughly investigated. Here, we used 5-FU to induce senescence in human umbilical vein endothelial cells (HUVECs) and investigated the relationship between CAP and cellular senescence at the cellular level. In a model of cellular senescence induced by 5-FU treatment, we discovered that CAP treatment reversed the rise in the percentage of senescence-associated galactosidase (SA-ß-gal)-positive cells and decreased the expression of senescence-associated proteins (p16), senescence-associated genes (p21), and senescence-associated secretory phenotypes (SASPs: IL-6, TNF-α). In addition, CAP subsequently restored the autophagic process inhibited by 5-FU and upregulated the levels of autophagy-related proteins. Mechanistically, we found that CAP restored autophagic flux by inhibiting the mTOR pathway, which in turn alleviated FU-induced cellular senescence. Our findings suggest that CAP may help prevent cellular senescence and restore autophagy, opening up new possibilities and approaches for the clinical management of colorectal cancer.

Effect of Chloramphenicol as Antibiotic on the Structure and Function of Pepsin and Its Mechanism of Action.

The interaction between chloramphenicol (CHL) and pepsin (PEP), as well as the impact of CHL on PEP conformation, were investigated using spectroscopic techniques and molecular docking simulations in this study. The experimental results demonstrate that CHL exhibits a static quenching effect on PEP. The thermodynamic parameters indicate that the reaction between CHL and PEP is spontaneous, primarily driven by hydrogen bonding and van der Waals forces. Moreover, the binding distance of r<7 nm suggests the occurrence of Förster's non-radiative energy transfer between these two molecules. In the synchronous fluorescence spectrum, the maximum fluorescence intensity of PEP produced a redshift phenomenon, indicating that CHL was bound to tryptophan residues of PEP. The addition of CHL induces changes in the secondary structure of PEP, as confirmed by the observed alterations in peak values in three-dimensional fluorescence spectra. The UV spectra reveal a redshift of 3 nm in the maximum absorption peak, indicating a conformational change in the secondary structure of PEP upon addition of CHL. Circular dichroism analysis demonstrates significant alterations in the α-helix, ß-sheet, ß-turn, and random coil contents of PEP before and after CHL incorporation, further confirming its ability to modulate the secondary structure of PEP.

Chloramphenicol-imprinted polychitosan bounded with carbon dots as fluorescent sensor, dispersive sorbent, and drug carrier.

Chitosan, an abundant natural polysaccharide, was conjugated with carbon dots (CDs) and self-polymerized with chloramphenicol (CAP) templates to synthesize CD-incorporated and molecularly CAP-imprinted polychitosan (CD-MIC). The CD-MIC was used for fluorescent sensing, dispersive sorption, and dosage release of CAP at different pH levels. The sphere of action mechanism, approved by emission and excitation fluorescence, UV-Vis absorption, and fluorescence lifetime measurements, regulated the fluorescence static quenching. By the Perrin model, the quenching extent was linearly correlated to CAP within 0.17 - 33.2 µM (LOD = 37 nM) at pH 7.0. With an imprinting factor of 3.1, the CD-MIC was more selective for CAP than CD, although it was less sensitive to CAP. The recoveries of 5.0 µM CAP from milk matrix were 95% (RSD = 2.3%) for CD-MIC probes and 62% (RSD = 4.5%) for CD. The Langmuir and pseudo-second-order models preferably described the isothermal and kinetic sorptions of CAP into the imprinted cavities in CD-MICs, respectively. The Weber - Morris kinetic model showed three stages involved in intraparticle diffusion, which was pH-dependent and gradually arduous at the later stage, and showed external diffusion partly engaged in the diffusion mechanism. The 20 - 70% of CAP formulated in CAP-embedded CD-MICs were released in 8 - 48 h. The release percentage was lower at pH 7.0 than at pH 5.0 and 9.0, but the equilibrium time was shorter. At pH 7.0, the release percentage reached 45% at 10 min and slowly increased to 51% at 24 h.

Design, optimization and pharmaceutical characterization of wound healing film dressings with chloramphenicol and ibuprofen.

OBJECTIVE: The aim of the present study was to develop and optimize a wound dressing film loaded with chloramphenicol (CAM) and ibuprofen (IBU) using a Quality by Design (QbD) approach. SIGNIFICANCE: The two drugs have been combined in the same dressing as they address two critical aspects of the wound healing process, namely prevention of bacterial infection and reduction of inflammation and pain related to injury. METHODS: Three critical formulation variables were identified, namely the ratios of Kollicoat SR 30D, polyethylene glycol 400 and polyvinyl alcohol. These variables were further considered as factors of an experimental design, and 17 formulations loaded with CAM and IBU were prepared via solvent casting. The films were characterized in terms of dimensions, mechanical properties and bioadhesion. Additionally, the optimal formulation was characterized regarding tensile properties, swelling behavior, water vapor transmission rate, surface morphology, thermal behavior, goniometry, in vitro drug release, cell viability, and antibacterial activity. RESULTS: The film was optimized by setting minimal values for the folding endurance, adhesive force and hardness. The optimally formulated film showed good fluid handling properties in terms of swelling behavior and water vapor transmission rate. IBU and CAM were released from the film up to 80.9% and 82.5% for 8 h. The film was nontoxic, and the antibacterial activity was prominent against Micrococcus spp. and Streptococcus pyogenes. CONCLUSIONS: The QbD approach was successfully implemented to develop and optimize a novel film dressing promising for the treatment of low-exuding acute wounds prone to infection and inflammation.

Obtaining New Biocompatible Composite Materials with Antibacterial Properties Based on Diatomite and Biologically Active Compounds.

This study aimed to create new composite materials based on diatomite-a non-organic porous compound-through its surface modification with bioactive organic compounds, both synthetic and natural. Chloramphenicol, tetrahydroxymethylglycoluril and betulin were used as modifying substances. Composite materials were obtained by covering the diatomite surface with bioactive substance compounds as a solution and material dispersion in it. The materials were characterized by IR spectroscopy, SEM and X-ray photoelectron spectroscopy. For the biocomposites, the hemolytic effect, plasma proteins' adsorption on the surface and the antibacterial activity of the obtained materials were studied. Results show that the obtained materials are promising for medicine and agriculture.

Synergistic Effect of Fe Single-Atom Catalyst for Highly Efficient Microwave-Stimulated Remediation of Chloramphenicol-Contaminated Soil.

Chloramphenicol (CAP) has long been used extensively in agriculture and is severely toxic to the biological environment. Microwave catalysis appears a promising method for soil remediation due to its fast and effective heat transfer, but it is challenging to prepare catalysts with good electromagnetic wave absorption and robust catalytic activity. In this study, atomically dispersed Fe on three-dimensional N-doped carbon supports (3D Fe-NC) is firstly used for microwave remediation of soil. Thanks to the synergistic effect of microwave "hot spots" and reactive oxygen species (•OH, •O2 - ), 3D Fe-NC can completely remove 99.9% of CAP in 5 min. The removal rate constant is nearly twice that of commercial activated carbon. Significantly, the germination rate of lettuce seeds in microwave-repaired soil contaminated by CAP reaches 70%. This work demonstrates the application of Fe single-atom catalyst in microwave remediation of contaminated soil, providing a novel insight for agricultural soil remediation.

Bacterial Dehydrogenases Facilitate Oxidative Inactivation and Bioremediation of Chloramphenicol.

Antimicrobial resistance represents a major threat to human health and knowledge of the underlying mechanisms is therefore vital. Here, we report the discovery and characterization of oxidoreductases that inactivate the broad-spectrum antibiotic chloramphenicol via dual oxidation of the C3-hydroxyl group. Accordingly, chloramphenicol oxidation either depends on standalone glucose-methanol-choline (GMC)-type flavoenzymes, or on additional aldehyde dehydrogenases that boost overall turnover. These enzymes also enable the inactivation of the chloramphenicol analogues thiamphenicol and azidamfenicol, but not of the C3-fluorinated florfenicol. Notably, distinct isofunctional enzymes can be found in Gram-positive (e. g., Streptomyces sp.) and Gram-negative (e. g., Sphingobium sp.) bacteria, which presumably evolved their selectivity for chloramphenicol independently based on phylogenetic analyses. Mechanistic and structural studies provide further insights into the catalytic mechanisms of these biotechnologically interesting enzymes, which, in sum, are both a curse and a blessing by contributing to the spread of antibiotic resistance as well as to the bioremediation of chloramphenicol.

Molecular Mechanism of Chloramphenicol and Thiamphenicol Resistance Mediated by a Novel Oxidase, CmO, in Sphingomonadaceae.

Antibiotic resistance mediated by bacterial enzyme inactivation plays a crucial role in the degradation of antibiotics in the environment. Chloramphenicol (CAP) resistance by enzymatic inactivation comprises nitro reduction, amide bond hydrolysis, and acetylation modification. However, the molecular mechanism of enzymatic oxidation of CAP remains unknown. Here, a novel oxidase gene, cmO, was identified and confirmed biochemically. The encoded CmO oxidase could catalyze the oxidation at the C-1' and C-3' positions of CAP and thiamphenicol (TAP) in Sphingobium sp. strain CAP-1. CmO is highly conserved in members of the family Sphingomonadaceae and shares the highest amino acid similarity of 41.05% with the biochemically identified glucose methanol choline (GMC) oxidoreductases. Molecular docking and site-directed mutagenesis analyses demonstrated that CAP was anchored inside the protein pocket of CmO with the hydrogen bonding of key residues glycine (G) 99, asparagine (N) 518, methionine (M) 474, and tyrosine (Y) 380. CAP sensitivity tests demonstrated that the acetyltransferase and CmO could enable a higher level of resistance to CAP than the amide bond-hydrolyzing esterase and nitroreductase. This study provides a better theoretical basis and a novel diagnostic gene for understanding and assessing the fate and resistance risk of CAP and TAP in the environment. IMPORTANCE Rising levels of antibiotic resistance are undermining ecological and human health as a result of the indiscriminate usage of antibiotics. Various resistance mechanisms have been characterized-for example, genes encoding proteins that degrade antibiotics-and yet, this requires further exploration. In this study, we report a novel gene encoding an oxidase involved in the inactivation of typical amphenicol antibiotics (chloramphenicol and thiamphenicol), and the molecular mechanism is elucidated. The findings provide novel data with which to understand the capabilities of bacteria to tackle antibiotic stress, as well as the complex function of enzymes in the contexts of antibiotic resistance development and antibiotic removal. The reported gene can be further employed as an indicator to monitor amphenicol's fate in the environment, thus benefiting risk assessment in this era of antibiotic resistance.

Context-Specific Action of Ribosomal Antibiotics.

The ribosome is a major antibiotic target. Many types of inhibitors can stop cells from growing by binding at functional centers of the ribosome and interfering with its ability to synthesize proteins. These antibiotics were usually viewed as general protein synthesis inhibitors, which indiscriminately stop translation at every codon of every mRNA, preventing the ribosome from making any protein. However, at each step of the translation cycle, the ribosome interacts with multiple ligands (mRNAs, tRNA substrates, translation factors, etc.), and as a result, the properties of the translation complex vary from codon to codon and from gene to gene. Therefore, rather than being indiscriminate inhibitors, many ribosomal antibiotics impact protein synthesis in a context-specific manner. This review presents a snapshot of the growing body of evidence that some, and possibly most, ribosome-targeting antibiotics manifest site specificity of action, which is modulated by the nature of the nascent protein, the mRNA, or the tRNAs.

A Novel Electrocatalyst Pd(II)@Ni3 (HITP)2 for Ultrasensitive Detection of Chloramphenicol: Experimental and Computational Investigation.

Ultrasensitive electrochemical detection on hazardous substances like antibiotics and pesticides is essential but still challenging in rapid test technology. Herein, the first electrode using highly conductive metal-organic frameworks (HCMOFs) for electrochemical detection of chloramphenicol is proposed. The design of electrocatalyst Pd(II)@Ni3 (HITP)2 with ultra-sensitivity in detection of chloramphenicol is demonstrated by loading Pd onto HCMOFs. An ultra-low limit of detection (LOD) of 0.2 nM (64.6 pg/mL) was observed for these materials, which is 1-2 orders of magnitude lower than the other reported materials for chromatographic detection. Moreover, the proposed HCMOFs showed long-time stability over 24 h. The superior detection sensitivity can be attributed to the high conductivity of Ni3 (HITP)2 , and the large Pd loading. The experimental characterizations and computational investigation determined the Pd loading mechanism in Pd(II)@Ni3 (HITP)2 , revealing PdCl2 adsorption on the abundant adsorption sites of Ni3 (HITP)2 . The proposed electrochemical sensor design using HCMOFs was demonstrated to be both effective and efficient, showing that the adoption of HCMOFs decorated with other effective electrocatalysts, which combine high conductivity and high catalytic activity, is of great advantage for ultrasensitive detection.

An Advanced Molecularly Imprinted Photochemical Sensor Based Carbon Quantum dots for Highly Sensitive Detection of Chloramphenicol in Food.

A facile method which combines the advantages of carbon quantum dots and molecular imprinting technology to design a fluorescence molecular imprinting sensor for the high sensitivity and selective detection of chloramphenicol. The fluorescent molecule imprinted polymers are synthesized by sol-gel polymerization using carbon quantum dots as functional monomers and fluorescent sources, TEOS as crosslinkers, breaking with the traditional understanding of an additional functional monomer. Under optimal experimental, as the concentration of chloramphenicol increases, the fluorescence intensity of the fluorescence molecule imprinting sensor gradually decreases. The concentration of chloramphenicol is linear in the range of 5-100 µg/L and the detection limit is 1 µg/L (N/S = 3). The sensor is able to detect chloramphenicol in milk, enabling the application of real samples. The results show that this work provides an easy method to preparing fluorescent molecular imprinting sensors for the detection of chloramphenicol in milk.

Gas-phase stability and thermodynamics of ligand-bound, binary complexes of chloramphenicol acetyltransferase reveal negative cooperativity.

The biological role of the bacterial chloramphenicol (Chl)-resistance enzyme, chloramphenicol acetyltransferase (CAT), has seen renewed interest due to the resurgent use of Chl against multi-drug-resistant microbes. This looming threat calls for more rationally designed antibiotic derivatives that have improved antimicrobial properties and reduced toxicity in humans. Herein, we utilize native ion mobility spectrometry-mass spectrometry (IMS-MS) to investigate the gas-phase structure and thermodynamic stability of the type I variant of CAT from Escherichia coli (EcCATI) and several EcCATI:ligand-bound complexes. EcCATI readily binds multiple Chl without incurring significant changes to its gas-phase structure or stability. A non-hydrolyzable acetyl-CoA derivative (S-ethyl-CoA, S-Et-CoA) was used to kinetically trap EcCATI and Chl in a ternary, ligand-bound state (EcCATI:S-Et-CoA:Chl). Using collision-induced unfolding (CIU)-IMS-MS, we find that Chl dissociates from EcCATI:S-Et-CoA:Chl complexes at low collision energies, while S-Et-CoA remains bound to EcCATI even as protein unfolding occurs. Gas-phase binding constants further suggest that EcCATI binds S-Et-CoA more tightly than Chl. Both ligands exhibit negative cooperativity of subsequent ligand binding in their respective binary complexes. While we observe no significant change in structure or stability to EcCATI when bound to either or both ligands, we have elucidated novel gas-phase unfolding and dissociation behavior and provided a foundation for further characterization of alternative substrates and/or inhibitors of EcCATI.

Catalytically enhanced direct degradation of nitro-based antibacterial agents using dielectric barrier discharge cold atmospheric pressure plasma and rhenium nanoparticles.

The common utilization of antimicrobial agents in medicine and veterinary creates serious problems with multidrug resistance spreading among pathogens. Bearing this in mind, wastewaters have to be completely purified from antimicrobial agents. In this context, a dielectric barrier discharge cold atmospheric pressure plasma (DBD-CAPP) system was used in the present study as a multifunctional tool for the deactivation of nitro-based pharmacuticals such as furazolidone (FRz) and chloramphenicol (ChRP) in solutions. A direct approach was applied to this by treating solutions of the studied drugs by DBD-CAPP in the presence of the ReO4- ions. It was found that Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS), generated in the DBD-CAPP-treated liquid, played a dual role in the process. On the one hand, ROS and RNS led to the direct degradation of FRz and ChRP, and on the other hand, they enabled the production of Re nanoparticles (ReNPs). The produced in this manner ReNPs consisted of catalytically active Re+4, Re+6, and Re+7 species which allowed the reduction of -NO2 groups contained in the FRz and ChRP. Unlike the DBD-CAPP, the catalytically enhanced DBD-CAPP led to almost FRz and ChRP removals from studied solutions. The catalytic boost was particularly highlighted when catalyst/DBD-CAPP was operated in the synthetic waste matrix. Re-active sites in this scenario led to the facilitated deactivation of antibiotics, achieving significantly higher FRz and ChRP removals than DBD-CAPP on its own.

Repurposing streptomycin and chloramphenicol against bacterial pathogens by combination with diminazene aceturate.

Bacterial resistance is a threat to health worldwide, mainly due to reduced effective treatment. In this context, the search for strategies to control such infections and suppress antimicrobial resistance is necessary. One of the strategies that has been used is combination therapy. In the present work, we investigated the in vitro efficacy of the antimicrobials diminazene aceturate (DA), chloramphenicol (CHL), and streptomycin (STP) alone and in combination against Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus clinical isolates. DA was capable of inhibiting all strains with MIC of 25-400 µg mL-1, while STP and CHL showed antibacterial activity with minimum inhibitory concentration (MICs) of ≤3.12-400 µg mL-1. The combination of aceturate with STP showed synergism toward almost all Gram-negative bacteria, with fractional inhibitory concentration index (FICIs) of 0.09-0.37. In addition, for CHL and aceturate, synergisms for Gram-negative and -positive strains were observed. A time-kill assay against E. coli revealed that the aceturate and STP combination can inhibit bacterial growth in a shorter time when compared with single antibiotics. In addition, antimicrobials did not show hemolytic activity even at the highest concentrations used. Therefore, the antimicrobial combinations presented in this work showed important results, demonstrating that combined therapy can be used as an alternative strategy for pathogen control.

A graphene-oxide-based aptasensor for fluorometric determination of chloramphenicol in milk and honey samples utilizing exonuclease III-assisted target recycling and Nb.BbvCI-powered DNA walker cascade amplification.

Herein, a graphene oxide (GO)-based fluorescence aptasensor was developed for the sensitive and selective detection of chloramphenicol (CAP), based on exonuclease III (Exo III)-assisted target recycling and Nb.BbvCI-driven DNA walker cascade amplification. Interactions between CAP, hairpin1(HP1), hairpin2 (HP2), and 3'-amino modified hairpin3 (HP3) labeled with carboxyfluorescein (FAM) and covalently coupled to GO enabled efficient CAP detection. CAP was quantitatively assayed by measuring fluorescence at excitation/emission wavelengths of 480/514 nm, resulting from the accumulation of released FAM. A good linear range of 1 fM to 1 nM and a limit of detection (LOD) of 0.875 fM (signal-to-noise (S/N)= 3) were achieved. This aptasensor can distinguish the CAP from interference antibiotics with good specificity and selectivity, even if the concentration of the interfering substance is ten-fold higher than the target concentration. Moreover, the developed fluorescence aptasensor was successfully applied for the detection of CAP in spiked milk and honey samples. Thus, this method is potentially applicable for assaying CAP in foods and provides a promising strategy for the development of fluorescence aptasensors for environmental sample analysis.