Screening of broad-spectrum aptamer and development of electrochemical aptasensor for simultaneous detection of penicillin antibiotics in milk.

Penicillin antibiotics (PENs) play an important role in killing pathogenic bacteria. However, the residues of various penicillin antibiotics in milk gradually accumulate in the human body with the increase of milk intake, which causes direct harm to the human body. Aptamers can be used as recognition element of sensors. It is great significance to use broad-spectrum aptamers for simultaneous detection of PENs. In this study, we reported the screening and identification of DNA aptamers for PENs. The aptamers were screened by graphene oxide-systematic evolution of ligands by exponential enrichment (GO-SELEX). The broad-spectrum aptamers with high affinity and specificity were successfully obtained after 13 rounds of screening. The affinity and specificity of candidate aptamers were analyzed by a GO fluorescence competition method. Further sequence analysis revealed that a truncated 47 nt aptamer (P-11-1) had a higher affinity than the original 79 nt aptamer. The truncated aptamer P-11-1 was used as a recognition element, and an electrochemical aptasensor was prepared using gold nanoparticles (AuNPs) combined with ferroferric oxide-multi walled carbon nanotube (Fe3O4-MWCNTs) complex. The results showed that the developed aptasensor achieved the simultaneous detection of PENs in milk samples across a concentration range of 2 nM-10,000 nM, achieving a limit of detection of 0.667 nM. This methodology provided a simple and sensitive new thinking for antibiotic multi-residue detection.

Time-resolved fluorescence microspheres-antibody-penicillin-binding protein assisted construction of immunochromatographic assay for sensitive detection of 22 ß-lactams in milk.

A sensitive immunochromatographic assay (ICA) using time-resolved fluorescence microspheres (TRFMs) coupled with an indirect-labeling mode was developed for simultaneously determining 22 kinds of ß-lactams in milk samples. The TRFMs labeled anti-receptor monoclonal antibodies (mAbs) conjugated to penicillin-binding proteins (PBPs) as ternary TRFMs-mAb-PBPs (TMP) nanoscaffolds provide excellent solubility, brightness, and stability. Thanks to the fact that they not only fully expose the binding sites of PBPs, thereby enhancing the biological affinity of PBPs towards the target, but also generated superb fluorescence signals, the versatile TMP manifested unique possibilities as efficient probes for ICA with remarkable enhancement in sensitivity in ß-lactams screening. The results showed that the standard curves of the 22 varying ß-lactams displayed linearity in their respective concentration ranges (R2 > 0.98), with the cutoff values of 1-100 ng/mL. The constructed TMP-ICA was successfully applied to the analysis of real milk, with consistent results compared with liquid chromatography-tandem mass spectrometry (LC-MS), providing an effective method for sensing ß-lactams in food matrices.

Validation of the AnticFast® Beta-Lactams Rapid Test Kit for Detection of Beta-Lactams (Penicillins and Cephalosporins) in Raw Cow's Milk: AOAC Performance Tested MethodSM 032303.

BACKGROUND: AnticFast® Beta-Lactams Rapid Test Kit is a qualitative two-step (2 min + 5 min) rapid lateral flow assay to detect ß-lactam (penicillins and cephalosporins) antibiotic residues in raw commingled cow's milk. OBJECTIVE: The method performance was evaluated according to Commission Decision 2002/657/EC, Commission Implementing Regulation 2021/808, and Community Reference Laboratories Residues Guidelines for the Validation of Screening Methods for Residues of Veterinary Medicines. METHODS: The AnticFast Beta-Lactams Rapid Test Kit was evaluated for detection capability, selectivity, false-positive results, repeatability, robustness, suitability for various milk types and milk compositions, milks from various species, and test kit consistency and stability. Samples included milks spiked at concentrations bracketing the EU maximum residue limits (MRLs) for ß-lactams as well as bulk farm and tanker milks. RESULTS: The AnticFast Beta-Lactams Rapid Test Kit is specific for the detection of ß-lactams in milk and does not detect compounds from other antibiotic families. Interference was seen with clavulanic acid, a ß-lactamase inhibitor, which was expected. The test can detect all residues of ß-lactams (penicillins and cephalosporins) present on the EU-MRL list for milk at their respective MRL except for desfuroylceftiofur and cephalexin, which were above the MRL. No false positives were detected in the 602 (300 blank farm and 302 tanker load) samples tested. Robustness testing indicated that the detection in heat-treated milk types may be slightly hampered. For substances with a detection capability well below the MRL, this interference does not cause problems since detection at MRL remains guaranteed, but care should be taken for substances with a CCß at or near their MRL. Diminished sample flow was seen with reconstituted milk powder and blank ewes' milk, so sample flow should always be verified for these milk types. CONCLUSIONS: Results of this validation show that the AnticFast Beta-Lactams Rapid Test Kit is a reliable test for rapid screening of raw cows' milk for residues of ß-lactam antibiotics. HIGHLIGHTS: AnticFast Beta-Lactams Rapid Test Kit is an easy, realiable, robust and highly specific test for screening of raw cows' milk for residues of penicillins and cephalosporins.

Combinational Treatment Involving Decellularized Extracellular Matrix Hydrogels With Mesenchymal Stem Cells Increased the Efficacy of Cell Therapy in Pancreatitis.

Cell transplantation using mesenchymal stem cells (MSCs) has emerged as a promising approach to repairing and regenerating injured or impaired organs. However, the survival and retention of MSCs following transplantation remain a challenge. Therefore, we investigated the efficacy of co-transplantation of MSCs and decellularized extracellular matrix (dECM) hydrogels, which have high cytocompatibility and biocompatibility. The dECM solution was prepared by enzymatic digestion of an acellular porcine liver scaffold. It could be gelled and formed into porous fibrillar microstructures at physiological temperatures. MSCs expanded three-dimensionally in the hydrogel without cell death. Compared to the 2-dimensional cell culture, MSCs cultured in the hydrogel showed increased secretion of hepatocyte growth factor (HGF) and tumor necrosis factor-inducible gene 6 protein (TSG-6), both of which are major anti-inflammatory and anti-fibrotic paracrine factors of MSCs, under TNFα stimulation. In vivo experiments showed that the co-transplantation of MSCs with dECM hydrogel improved the survival rate of engrafted cells compared to those administered without the hydrogel. MSCs also demonstrated therapeutic effects in improving inflammation and fibrosis of pancreatic tissue in a dibutyltin dichloride (DBTC)-induced rat pancreatitis model. Combinational use of dECM hydrogel with MSCs is a new strategy to overcome the challenges of cell therapy using MSCs and can be used for treating chronic inflammatory diseases in clinical settings.

(Multivariate)-Metal-Organic Framework for Highly Efficient Antibiotic Capture from Aquatic Environmental Matrices.

Contamination of aquatic environments by pharmaceuticals used by modern societies has become a serious threat to human beings. Among them, antibiotics are of particular concern due to the risk of creating drug-resistant bacteria and, thus, developing efficient protocols for the capture of this particular type of drug is mandatory. Herein, we report a family of three isoreticular MOFs, derived from natural amino acids, that exhibit high efficiency in the removal of a mixture of four distinct families of antibiotics, such as fluoroquinolones, penicillins, lincomycins, and cephalosporins, as solid-phase extraction (SPE) sorbents. In particular, a multivariate (MTV)-MOF, prepared using equal percentages of amino acids l-serine and l-methionine, also exhibits outstanding recyclability, surpassing the benchmark material activated carbon. The good removal performance of the MTV-MOF was rationalized by means of single-crystal X-ray diffraction. These results highlight the situation of MOFs as a real and promising alternative for the capture of antibiotics from environmental matrices, especially wastewater streams.

Green Multiplex Chromatographic Determination of Nine Penicillin Antibiotics Residues in Industrial Air Dust and Wastewater Environmental Samples.

Determination of penicillin residues in different industrial effluents including wastewater and air samples is important to prevent exposure to residual amounts of penicillin and the development of antibiotic resistance. A green high performance liquid chromatography (HPLC) method coupled with diode array detection has been developed and validated for multiplex determination of nine penicillin antibiotics in the industrial air dust and wastewater environmental samples of penicillin facility in addition to the monitoring of facility surface cleaning. Separation was performed on C18 column with gradient elution of methanol and phosphate buffer (pH 4) at a flow rate of 1.5 mL min-1 and ultra violet (UV) detection at 220 nm. Low limits of detection were achieved (0.1-0.3 µg mL-1) indicating good sensitivity of the proposed. The method was applied for ensuring the efficiency of cleaning validation after worst-case selection. Recovery studies of the studied penicillins from fortified stainless steel and polycarbonate surfaces and swabs were between 91.91 and 100.22% with relative standard deviation 0.11-1.79%. The presence of any of the studied penicillins in wastewater samples from penicillin plant drainage was checked. Also, total air dust concentration (mg m-3) and % of penicillin active material residues in air dust were calculated from the area of the exposed group in suspension, tablet and vial production lines. The proposed method can be recommended for routine analysis of air and wastewater environmental samples for the detection of penicillin antibiotics at low levels as well as monitoring of facility surface cleaning with high accuracy and precision.

[Determination of three penicillin residues in milk by solid-phase extraction-ultra performance liquid chromatography-tandem mass spectrometry using a covalent triazine framework sorbent].

A method based on solid-phase extraction-ultra performance liquid chromatography-tandem mass spectrometry (SPE-UPLC-MS/MS) was established for the determination of gpenicillin, cloxacillin, ampicillin residues in milk. Using self-made covalent triazine frameworks (CTFs) as the solid-phase extraction sorbents, the main factors influencing the efficiency of the solid-phase extraction columns, such as the sorbent amount, eluent type, eluent volume, and flow rate, were optimized. The extraction and purification conditions for the samples were also investigated. The optimal extraction effect was achieved at a flow rate of 3 mL/min with 60 mg CTFs and 6 mL eluent solution (acetonitrile). Separation was carried out on a Waters ACQUITY UPLC BEH C18 column, and 0.1% formic acid aqueous solution-acetonitrile was used as the mobile phases for gradient elution. The filtrate was detected by ultra performance liquid chromatography-tandem mass spectrometry, identified by electrospray ionization (ESI) in the positive mode using multiple reaction monitoring, and quantified using external standards. The calibration curves of the three penicillins showed good linearity and the correlation coefficients of the linear regression equations for the three target analytes were all greater than 0.999. The limits of detection (LODs) and limits of quantification (LOQs) were 0.05-0.10 µg/kg and 0.1-0.4 µg/kg, respectively. The average recoveries of the three analytes were 84.9%-94.1%, and the relative standard deviations (RSDs, n=5) were 1.66%-3.27%. Moreover, the mechanism of interaction between the CTFs and the target analytes was analyzed. The results revealed the existence of π-π and hydrogen-bond interactions between the CTFs and analytes. The results further indicated that the CTFs could be successfully used for the enrichment and purification of penicillins in milk. The proposed method has the advantages of high precision, good reproducibility, high resolution, and short analysis time, and it is suitable for the qualitative and quantitative determination of trace targets in complex matrices.

Towards Multi-Analyte Detection with Field-Effect Capacitors Modified with Tobacco Mosaic Virus Bioparticles as Enzyme Nanocarriers.

Utilizing an appropriate enzyme immobilization strategy is crucial for designing enzyme-based biosensors. Plant virus-like particles represent ideal nanoscaffolds for an extremely dense and precise immobilization of enzymes, due to their regular shape, high surface-to-volume ratio and high density of surface binding sites. In the present work, tobacco mosaic virus (TMV) particles were applied for the co-immobilization of penicillinase and urease onto the gate surface of a field-effect electrolyte-insulator-semiconductor capacitor (EISCAP) with a p-Si-SiO2-Ta2O5 layer structure for the sequential detection of penicillin and urea. The TMV-assisted bi-enzyme EISCAP biosensor exhibited a high urea and penicillin sensitivity of 54 and 85 mV/dec, respectively, in the concentration range of 0.1-3 mM. For comparison, the characteristics of single-enzyme EISCAP biosensors modified with TMV particles immobilized with either penicillinase or urease were also investigated. The surface morphology of the TMV-modified Ta2O5-gate was analyzed by scanning electron microscopy. Additionally, the bi-enzyme EISCAP was applied to mimic an XOR (Exclusive OR) enzyme logic gate.

Combining Electrochemiluminescence Detection with Aptamer-Gated Indicator Releasing Mesoporous Nanoparticles Enables ppt Sensitivity for Strip-Based Rapid Tests.

The combination of electrogenerated chemiluminescence (ECL) and aptamer-gated indicator delivering (gAID) magnetic mesoporous silica nanoparticles embedded into glass fibre paper functionalised with poly(ethyleneglycol) and N-(3-triethoxysilylpropyl)diethanolamine allowed the development of a rapid test that detects penicillin directly in diluted milk down to 50±9 ppt in <5 min. Covalent attachment of the aptamer "cap" to the silica scaffold enabled pore closure through non-covalent electrostatic interactions with surface amino groups, while binding of penicillin led to a folding-up of the aptamer thus releasing the ECL reporter Ru(bpy)32+ previously loaded into the material and letting it be detected after lateral flow by a smartphone camera upon electrochemical excitation with a screen printed electrode inserted into a 3D-printed holder. The approach is simple, generic and presents advantages with respect to sensitivity, measurement uncertainty and robustness compared with conventional fluorescence or electrochemical detection, especially for point-of-need analyses of challenging matrices and analytes at ultra-trace levels.

Rapid and sensitive analytical method for the determination of amoxicillin and related compounds in water meeting the requirements of the European union watch list.

The presence of antibiotics in the aquatic environment is becoming one of the main research focus of scientists and policy makers. Proof of that is the inclusion of four antibiotics, amongst which is amoxicillin, in the EU Watch List (WL) (Decision 2020/1161/EU)) of substances for water monitoring. The accurate quantification of amoxicillin in water at the sub-ppb levels required by the WL is troublesome due to its physicochemical properties. In this work, the analytical challenges related to the determination of amoxicillin, and six related penicillins (ampicillin, cloxacillin, dicloxacillin, penicillin G, penicillin V and oxacillin), have been carefully addressed, including sample treatment, sample stability, chromatographic analysis and mass spectrometric detection by triple quadrupole. Given the low recoveries obtained using different solid-phase extraction cartridges, we applied the direct injection of water samples using a reversed-phase chromatographic column that allowed working with 100% aqueous mobile phase. Matrix effects were evaluated and corrected using the isotopically labelled internal standard or correction factors based on signal suppression observed in the analysis of spiked samples. The methodology developed was satisfactorily validated at 50 and 500 ng L - 1 for the seven penicillins studied, and it was applied to different types of water matrices, revealing the presence of ampicillin in one surface water sample and cloxacillin in three effluent wastewater samples.

Reversible Structural Transformation of CuI-TbIII Heterometallic MOFs with Highly Efficient Detection Capability toward Penicillin.

A CuI-TbIII heterometallic MOF, namely 1·DMF, was obtained via a coordination assembly process of isonicotinic acid with CuI and TbIII. 1·DMF can be switched to 1·MeOH in methanol with a luminescent emission response. Meanwhile, 1·MeOH exhibits a reversible single-crystal transformation to 1·DMF after immersion in DMF. Both MOFs have superior physicochemical stability. The 1·DMF-based biosensor has a remarkable sensing performance toward penicillin.

Penicillin biosensor based on rhombus-shaped porous carbon/hematoxylin/penicillinase.

In this experiment, we designed an electrochemical sensor using penicillinase (Pen X)-rhombus porous carbon (RPC) as the detection element and hematoxylin as the indicator to detect low concentrations of penicillin sodium (Pen G). A differential pulse voltammetry (DPV) method was used to detect Pen G in the concentration range of 10-8 -10-5 mg·mL-1 under optimal experimental conditions. The results showed that the peak current value and the logarithm of Pen G concentration showed a good linear relationship (R2 = 0.9915), and the LOD was 2.68 × 10-7 mg·mL-1 (S/N = 3). The actual milk samples were detected by the addition method and compared with the high-performance liquid phase method; no significant difference was found in the detection results. The working electrode prepared by cross-linking method not only extends the service life of the sensor, but also improves the sensitivity and reproducibility of the sensor. It can also be used to detect the Pen G residue in the actual milk samples repeatedly. PRACTICAL APPLICATION: In this study, an electrochemical sensor for the rapid detection of penicillin sodium in milk was prepared, which has good sensitivity and fast detection speed.

Green bioanalytical sample preparation: fabric phase sorptive extraction.

Fabric phase sorptive extraction (FPSE) is a recently introduced sample preparation technique that has attracted substantial interest of the scientific community dealing with bioanalysis. This technique is based on a permeable and flexible substrate made of fabric, coated with a sol-gel organic-inorganic sorbent. Among the benefits of FPSE are its tunable selectivity, adjustable porosity, minimized sample preparation workflow, substantially reduced organic solvent consumption, rapid extraction kinetics and superior extraction efficiency, many of which are well-known criteria for Green Analytical Chemistry. As such, FPSE has established itself as a leading green sample preparation technology of 21st century. In this review, we discuss the principal steps for the development of an FPSE method, the main method optimization strategies, as well as the applications of FPSE in bioanalysis for the extraction of a wide range of analytes (e.g., estrogens, benzodiazepines, androgens and progestogens, penicillins, anti-inflammatory drugs, parabens etc.).

Duplex Surface Enhanced Raman Scattering-Based Lateral Flow Immunosensor for the Low-Level Detection of Antibiotic Residues in Milk.

A duplex surface enhanced Raman scattering (SERS)-based lateral flow immunosensor was established for the simultaneous detection of two common antibiotic residues including tetracycline and penicillin in milk. The newly synthesized Au@Ag nanoparticles were labeled with different Raman molecules including 5,5-dithiobis-2-nitrobenzoic acid (DTNB) or 4-mercaptobenzoic acid (MBA), followed by the conjugation of anti-tetracycline monoclonal antibody or anti-penicillin receptor, forming two kinds of SERS nanoprobes. The two nanoprobes can recognize tetracycline-BSA and ampicillin-BSA, respectively, which facilitates the simultaneous detection of the two types of antibiotics on a single test line. After optimization, detection limits of tetracycline and penicillin as low as 0.015 ng/mL and 0.010 ng/mL, respectively, were achieved. These values were far below those of most of other documented bio-analytical approaches. Moreover, the spiking test demonstrates an excellent assay accuracy with recoveries of 88.8% to 111.3%, and satisfactory assay precision with relative standard deviation below 16%. Consequently, the results demonstrate that the SERS-based lateral flow immunosensor developed in this study has the advantages of excellent assay sensitivity and remarkable multiplexing capability, thus it will have great application potential in food safety monitoring.

Biosensors for penicillin quantification: a comprehensive review.

Penicillins represent most commonly prescribed and salient part of antibacterial armamentarium. Due to lack of proper guidance and regulations, especially in developing countries they are indiscriminately applied in agricultural sectors (livestock farming, aquaculture and plants). Major concerns related to remaining residues of penicillins in food or environment are allergy, development of antibacterial resistance, environmental pollution, economical losses to food industries, consumers and food safety. Researchers of different disciplines are developing reliable detection methods for penicillins in various samples. Among them biosensors are attracting considerable attention primarily for their instant detection, convenience in application, on-site monitoring and portability. Nowadays, they are becoming more sophisticated with the help of modern approaches such as nanotechnology. This review article summarizes the research literature on advancements and recent trends in the field of biosensors for penicillins quantification till date. Different domains of biosensors (electrochemical, optical, mass-sensitive and thermal) are discussed for penicillins along with their applicability.

Probeless non-invasive near-infrared spectroscopic bioprocess monitoring using microspectrometer technology.

Real-time measurements and adjustments of critical process parameters are essential for the precise control of fermentation processes and thus for increasing both quality and yield of the desired product. However, the measurement of some crucial process parameters such as biomass, product, and product precursor concentrations usually requires time-consuming offline laboratory analysis. In this work, we demonstrate the in-line monitoring of biomass, penicillin (PEN), and phenoxyacetic acid (POX) in a Penicilliumchrysogenum fed-batch fermentation process using low-cost microspectrometer technology operating in the near-infrared (NIR). In particular, NIR reflection spectra were taken directly through the glass wall of the bioreactor, which eliminates the need for an expensive NIR immersion probe. Furthermore, the risk of contaminations in the reactor is significantly reduced, as no direct contact with the investigated medium is required. NIR spectra were acquired using two sensor modules covering the spectral ranges 1350-1650 nm and 1550-1950 nm. Based on offline reference analytics, partial least squares (PLS) regression models were established for biomass, PEN, and POX either using data from both sensors separately or jointly. The established PLS models were tested on an independent validation fed-batch experiment. Root mean squared errors of prediction (RMSEP) were 1.61 g/L, 1.66 g/L, and 0.67 g/L for biomass, PEN, and POX, respectively, which can be considered an acceptable accuracy comparable with previously published results using standard process spectrometers with immersion probes. Altogether, the presented results underpin the potential of low-cost microspectrometer technology in real-time bioprocess monitoring applications. Graphical abstract.

Modularized Field-Effect Transistor Biosensors.

Field-effect transistors (FETs), when functionalized with proper biorecognition elements (such as antibodies or enzymes), represent a unique platform for real-time, specific, label-free transduction of biochemical signals. However, direct immobilization of biorecognition molecules on FETs imposes limitations on reprogrammability, sensor regeneration, and robust device handling. Here we demonstrate a modularized design of FET biosensors with separate biorecognition and transducer modules, which are capable of reversible assembly and disassembly. In particular, hydrogel "stamps" immobilizing bioreceptors have been chosen to build biorecognition modules to reliably interface with FET transducers structurally and functionally. Successful detection of penicillin down to 0.25 mM has been achieved with a penicillinase-encoded hydrogel module, demonstrating effective signal transduction across the hybrid interface. Moreover, sequential integration of urease- and penicillinase-encoded modules on the same FET device allows us to reprogram the sensing modality without cross-contamination. In addition to independent bioreceptor encoding, the modular design also fosters sophisticated control of sensing kinetics by modulating the physiochemical microenvironment in the biorecognition modules. Specifically, the distinction in hydrogel porosity between polyethylene glycol and gelatin enables controlled access and detection of larger molecules, such as poly-l-lysine (MW 150-300 kDa), only through the gelatin module. Biorecognition modules with standardized interface designs have also been exploited to comply with additive mass fabrication by 3D printing, demonstrating potential for low cost, ease of storage, multiplexing, and great customizability for personalized biosensor production. This generic concept presents a unique integration strategy for modularized bioelectronics and could broadly impact hybrid device development.

Human metallo-ß-lactamase enzymes degrade penicillin.

Nonribosomal peptides are assemblages, including antibiotics, of canonical amino acids and other molecules. ß-lactam antibiotics act on bacterial cell walls and can be cleaved by ß-lactamases. ß-lactamase activity in humans has been neglected, even though eighteen enzymes have already been annotated such in human genome. Their hydrolysis activities on antibiotics have not been previously investigated. Here, we report that human cells were able to digest penicillin and this activity was inhibited by ß-lactamase inhibitor, i.e. sulbactam. Penicillin degradation in human cells was microbiologically demonstrated on Pneumococcus. We expressed a MBLAC2 human ß-lactamase, known as an exosome biogenesis enzyme. It cleaved penicillin and was inhibited by sulbactam. Finally, ß-lactamases are widely distributed, archaic, and have wide spectrum, including digesting anticancer and ß-lactams, that can be then used as nutriments. The evidence of the other MBLAC2 role as a bona fide ß-lactamase allows for reassessment of ß-lactams and ß-lactamases role in humans.

Comparison of international legislation and standards on veterinary drug residues in food of animal origin.

Current legislation governing monitoring of drug residues in foodstuff of animal origin is being revised at the European level. This study provides a qualitative comparison of the legislation, public and private standards in the European Union, the United States of America (USA) and the Eurasian Customs Union/Russia. We made a quantitative comparison of Maximum Residue Levels (MRLs) applied in each region for pork kidneys for tetracycline (with a focus on doxycycline), penicillin and chloramphenicol. The Customs Union generally applied lower levels than the other regions, with MRLs for tetracyclines in pig kidneys being 1200 times lower than those applied in the USA. Growing consumer interest and concern about chemicals in their food could be leveraged to support and enhance the implementation of new initiatives to improve veterinary public health. Farmers and veterinarians could help reduce findings of drug residues in meat through the judicious use of preventive actions when using veterinary medicine.