| Antibiotics are widely used in animal husbandry and are excessive used,which had result in the antibiotic residues??-lactam antibiotics are the most common?in foods?such as dairy products?.Antibiotic residues can cause great harm to the human and the environment.At present,the common methods for detecting antibiotic residues exsit some disadvantages,therefore it is necessary to find a rapid and accurate detection method.In recent years,biosensors have become an important detection tool due to their high sensitivity and rapid response.Metal-doped mesoporous carbon materials have caused widely concerned in many fields due to their high specific surface area and excellent electrocatalytic properties.Methylene blue?MB?has highly conjugated ring structure and excellent electrical activity,and it can form a polymer semiconductor film with conductivity,which has been widely used for electrode modification.Penicillinase?Pen X?and New Delhi metal-?-lactamase-1?NDM-1?belong to?-lactamase and can hydrolyze?-lactam antibiotics by hydrolyzing the?-lactam ring.In particular,NDM-1 can hydrolyze all?-lactam antibiotics except for aztreonam,and it has less application in electrochemical detection aspect.Therefore,metal-doped mesoporous carbon materials were synthesized and three biosensors were designed based on Pen X and NDM-1 to detection of antibiotic model penicillin sodium?Pen G?and Ampicillin sodium?AMP?.1.Preparation and properties of two mesoporous carbon materialsCobalt-doped mesoporous carbon materials?Co@C?and cobalt sulfide doped mesoporous carbon materials?CoS2@C?were prepared by self-assembly method and impregnation method,and were functionalized by carboxyl groups to obtain COOH-Co@C and COOH-CoS2@C.The materials were characterized by Scanning electron microscopy?SEM?,transmission electron microscopy?TEM?,X-ray diffraction?XRD?,Nitrogen adsorption desorption,Fourier transform infrared spectroscopy?FTIR?,X-ray photoelectron spectroscopy?XPS?.The results demonstrated that Co@C and CoS2@C have good stability,pore structure,and were carboxyl modification successfully.2.Preparation and application of Immobilized penicillinase biosensorThe biosensor of Pen X-COOH-Co@C and Pen X-COOH-CoS2@C were obtained by immobilizing Pen X on a mesoporous carbon support by covalent bonding.MB was electropolymerized onto a glassy carbon electrode?GCE?to obtain PMB/GCE,and then Pen X-COOH-Co@C and Pen X-COOH-CoS2@C materials were respectively modified onto PMB/GCE to prepare two biosensors of Pen X-COOH-Co@C/PMB/GCE and Pen X-COOH-CoS2@C/PMB/GCE.After optimization of experimental conditions,the differential pulse voltammetry?DPV?was used to detect Pen G as an antibiotic model.The results showed that Pen X-COOH-Co@C/PMB/GCE responded to peak current and Pen G concentration at a Pen G concentration of 0.1-10 ng·mL-1.A good linear relationship was obtained,and the linear equation is y=0.3811x+17.20?r2=0.9974?;it also showed good linearity in the concentration range of 10-100 ng·mL-1 and the formula was y=0.1170x+19.73?r2=0.9959?,and the detection limit was 0.64 ng·mL-1?ppb?.In the Pen X-COOH-CoS2@C/PMB/GCE sensor,peak current and Pen G concentration showed good linearity in Pen G concentration range 0.1 to 10 ng·mL-1 and the linear equation was y=0.5642x+21.57?r2=0.9907?;it also showed good linearity in the concentration range of 20-100 ng·mL-1 and the formula was y=0.0904x+25.91?r2=0.9941?,and the detection limit is 0.61 ng·mL-1?ppb?.In addition,the two biosensors were successfully used to detect the actual sample milk by using the spike method.3.Preparation and application of NDM-1 enzyme biosensorPB was electropolymerized onto GCE to obtain PMB/GCE;NDM-1 and glutaraldehyde?GA?were mixed and dropped onto PMB/GCE to prepare NDM-1/PMB/GCE working electrode.The experimental conditions were optimizated and using Pen G and ampicillin?AMP?as antibiotic models to detect antibiotic.The results showed that NDM-1/PMB/GCE showed good linearity in the Pen G concentration range 1.0×10-55 to 1.0×10-2?g·mL-1 and the formula was y=326.98x+5.840?r2=0.9934?;it also showed good linearity in the concentration range of 1.0×10-1 to 1.0×101?g·mL-1 and the formula was y=0.3304x+9.9346?r2=0.9927?and the detection limit was 0.174 ppb.In addition,the response current of the NDM-1/PMB/GCE sensor was also linear with the AMP concentration range1.0×10-55 to 1.0×10-2?g·mL-1 and the linear equation is y=322.07x+22.82?r2=0.9920?;it also showed good linearity in the concentration range of 1.0×10-2 to 1.0×101?g·mL-1 and the formula was y=0.3420x+26.28?r2=0.9916?,and the detection limit was 0.35 ppb.The NDM-1/PMB/GCE sensor was successfully used to detect the actual sample milk by using the spike method.The research showed that the carboxyl functionalized metal-based mesoporous carbon material can be used as a good carrier for immobilizing Pen X.The prepared Pen X sensor and NDM-1 sensor have the advantages of high sensitivity,wide detection range and low detection limit,and can realize detection of actual samples. |
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