The Construction Of Nanofiber-Based Colorimetric Biosensor And Its Antibiotic Detection Application

Antibiotics,a class of inexpensive broad-spectrum medicine service in treating animal infectious disease and promoting growth.In recent years,driven by economic interest,antibiotic-resistant has been considered as a significant public health concern,due to the growing appearance of strains of bacteria resistant to antibiotics.The overuse of antibiotics can result in losing their effectiveness to kill bacteria which leads to the difficulty of treating diseases due to the new generation of bacteria have genes with resistance to some antibiotics.Therefore,the continuous application of antibiotic in animal husbandry can lead to an accumulation of resistant bacteria in the food of animal origin which causes illnesses that are difficult to treat.Besides antibiotic-resistant,the overuse of antibiotic can occur in several diseases including liver damage,ototoxicity,nephrotoxicity,allergic reactions,and yellowish discoloration of teeth.Subsequently,the World Health Organization?WHO?has considered antibiotic as one of the international concern due to the highest health risk to humanity.Henceforward,WHO has recognized the acceptable daily intake?ADI?and the maximum residue limit?MRL?to control antibiotic residues in the food of animal origin and environment.Up to now,the commonly used antibiotic detection methods mainly include high-performance liquid chromatography,electrophoresis,electrochemical analysis,and microbiological techniques.Although the above methods have high sensitivity,low detection limit,and high accuracy,however,the instruments used are expensive and require a particular operation.Besides,the sample preparation process is complicated and cumbersome,time-consuming,and it cannot be universally promoted,and it is unable to meet the detection requirements of a large number of samples on-site.On the other hand,colorimetric sensors are the most promising for antibiotic detection technology because of their advantages such as simple operation,detection can be achieved directly by naked eye observation,low cost,small size,good selectivity,and no need for expensive instruments.Therefore,researchers have conducted much research on the colorimetric detection of antibiotic from liquid and solid phases.However,existing liquid antibiotic detection systems generally have problems such as insufficient anti-interference ability,harsh storage conditions,and poor portability.Although the solid phase system improves portability and stability to a certain extent,the obtained colorimetric sensor still has low sensitivity,and the naked eye detection limit cannot meet the actual application requirements.In this thesis,systematic research and exploration are carried out on the above vital issues.Based on the high specific surface area,good pore structure and controllable bulk density of electrospun nanofiber membranes,the antibiotic colorimetric sensors based on electrospun nanofibers were proposed for the first time.The design and construction of the colorimetric sensor can fill the blank of the electrospinning nanofiber material in the research field of antibiotic sensor application.The main research results obtained are summarized as follows:?1?A fast-response and reusable oxytetracycline colorimetric sensing system was prepared by immobilization of nickel?II?ions on carboxymethylcellulose?CMC?coated polyacrylonitrile nanofibrous membranes?PAN NFMs?.Firstly,PAN NFMs were successfully prepared by using electrospinning technology with a specific surface area of 11.39 m²g^-1.Then CMC was coated on the PAN NFMs?CMC/PAN?.The coated PAN NFMs surface was a suitable platform to achieve effective immobilization of the nickel?II?ions.Due to the strong-complex behavior between nickel?II?ions and oxytetracycline,the constructed sensing platform has shown a colorimetric signal response from light green to yellow toward oxytetracycline,with a naked eye limit of5 nM at S/N=3,rapid response?2 min?,good reversibility?10 cycles?and selectivity.?2?Motivated by the nickel ions(Ni²⁺)colorimetric based system and according to the pharmacological theory,iron ions(Fe³⁺)and copper ions(Cu²⁺)can also strongly complex with tetracycline?TC?owing to numerous O-and N-containing groups.Therefore,the impact of such colorimetric sensing system was studied to improve the sensitivity and naked eye detection limit in this part.A strategy to prepare free-standing,nanofibrous structured test strips with tortuous porous structure and large sur face area by combining PAN NFMs,sodium alginate,and Fe³⁺was presented.Besides,Cu²⁺impregnated strips with the nanofibrous structure using ethylenediamine?EDA?grafted porous PAN?EPAN?was also presented.To construct tetracycline?TC?-sensing surface alginate was used to functionalize the PAN NFMs firstly,then Fe³⁺were assembled into alginate surface?FAPAN?.On the other hand,porous PAN NFMs were modified by ethylenediamine?EDA?firstly,then Cu²⁺were immobilized on NFMs to construct another TC-sensing surface?CEPAN?.The resultant FAPAN test strips exhibit the integrated properties of fast sensing process?10 min?,low naked eye detection limit?5?g/kg at S/N=3?,excellent anti-interference ability,and certain reusability.Furthermore,the TC concentration-dependent color change?yellow to maroon?were quantitatively visualized by an iPhone read-outed hue parameter.However,the CEPAN colorimetric system has not shown any color response toward TC,because the nanofibers and TC have the same chemical groups which are coordinated with copper ions.?3?In addition,compared with metals ions,AuNPs have recognized as an excellent agent for the developments of colorimetric sensors due to the ease of preparation,high molar absorptivity,high surface area,biocompatibility,and uniform shape and size;which make them have higher extinction coefficients than metals ions.Therefore,the non-enzymic melamine-AuNPs?MA@Au?conjugated probe system was also developed and applied for colorimetric detection of metronidazole?MTZ?.Firstly,AuNPs with an average diameter of 20.1±1.76 nm were successfully prepared by using sodium citrate reduction method with HAuCl₄.The non-enzymic MA@AuNPs probe was successfully developed by conjugation of the melamine with AuNPs.The polyamide?PA6?nanofibers membrane was prepared by electrospinning technology.Then the non-enzymic?MA@Au?was immobilized on the PA6 NFMs.The sensing performance showed a colorimetric response to detect metronidazole,with low necked eye limit of detection?2 nM at S/N=3?,a rapid detection time?2.5 min?and linear response in the 2 to 100 nM MTZ concentration range.?4?Furthermore,aptamers are synthetic biomolecules that can be suggested as a sensing agent to detect any type of antibiotics,due to high affinity and specificity bind to a specific target.Here,kanamycin?KMC?was selected as a representative target antibiotic agent,and a non-aggregated aptamer-AuNPs bio-conjugates probe system was proposed.The KMC-responsive aptamer was used as a response element to improve the selectivity of the system.The selected colorimetric agent was AuNPs with an average particle size of 17.5 nm synthesized using sodium citrate reduction method.The non-aggregated AuNPs probe was successfully prepared by bio-conjugation of kanamycin-responsive aptamer with AuNPs.The probe-supported platform was selected as glutamic acid grafted electrospun cellulose acetate?CA?nanofiber membrane?G-CA NFMs?.The carboxylic acid groups on the NFMs surface were acquired,which is an order of the effective immobilization of the Apt@Au probe is achieved,which was constructed a sandwich structured sensing platform.Subsequent sensing performance has shown a colorimetric signal response to kanamycin,due to Apt@Au conjugates disassembly.The biosensor strips displayed high sensitivity for KMC determination in a range 2.5⁸0 nM,with a naked eye limit of 2.5 nM at S/N=3.In addition to the excellent selectivity,reusability,and long-term storability,the strips also can be applied in real samples detection.