Nanomaterials Combined With Nucleic Acid Amplification Technology For Highly Sensitive Analysis Of Antibiotics

Nowadays,antibiotics play an increasingly important role as bacteriostatic or antiseptic agent,and thus been widely utilized in various fields including agriculture and animal husbandry.In recent years,the ultra-sensitive detection of residual antibiotics(such as kanamycin and chloramphenicol)in genuine samples has attracted great interest in the realm of biological research.The accumulation of antibiotics in human body is mainly caused by long-term intake of food such as dairy products with residual antibiotics.Therefore,detecting the exact concentration of residual antibiotics in dairy products is crucial.However,the concentration of residual antibiotics in milk is generally quite low.Thus,it is difficult to use conventional methods to achieve accurate detection of antibiotics in milk.Hence,there is an urgent demand to establish analytical methods for highly sensitive and selective detection of residual antibiotics in milk and body fluids.Accordingly,this thesis introduces several novel bioanalytical techniques the highly sensitive and efficient detection of residual antibiotics in cow milk samples.The main contents are as follows.1.According to the challenge faced by the highly sensitive detection of ultra-trace amount of kanamycin(Kana)in biological samples,a highly sensitive colorimetric method,based on the formation of three-dimensional nickel-ferric layered double oxide(Ni-Fe LDO)/DNA networks triggered by antibiotic aptamers with excellent specificity,was established in Chapter 3.Taking advantage of the positively charged characteristics of the Ni-Fe LDO surface,the negatively charged G-quadruplex was adsorbed upon.XPS analysis proved that the Ni and Fe elements in Ni-Fe LDO coordinated with the G-quadruplex on its surface,and the electrons in the G-quadruplex transferred to Ni and Fe elements during catalytic oxidation,enduing Ni-Fe LDO with stronger peroxidase-like catalytic activity.Under the optimal experiment conditions,the limit of detection(LOD)of this method is as low as 0.31 fM(S/N=3).Finally,this method was utilized and detected the residual Kana in milk and urine accurately,exhibiting great potential application value for the detection of Kana residues in real samples.2.Relying on dual enzyme-free amplification strategies,i.e.,catalytic hairpin assembly(CHA)and hybridization chain reaction(HCR),three-dimensional Ni-Fe LDO/DNA networks(NWs)was formed and an ultrasensitive colorimetric assay for the detection of chloramphenicol(CAP)was developed.Since the G-quadruplex was absorbed on the surface of Ni-Fe LDO NSs,its peroxidase-like catalytic activity could be significantly enhanced.Moreover,the background signal was effectively decreased through magnetic separation.This method successfully produced an ultrasensitive signal for the detection of target CAP.Under optimal conditions,it showed a wide linear range between 0.1 fM to 0.1 n M with an LOD of 11.6 aM(S/N=3).In addition,this method also possesses the advantages of high specificity and stability,and was successfully applied in the quantitative detection of target CAP in cow milk sample.3.In Chapter 5,an ultrasensitive quad-amplification method for the colorimetric determination of Kana was established.Through the combination of two non-enzymatic signal amplification strategies(CHA and HCR)with Ni-Fe LDO,a novel Ni-Fe LDO/DNA frame network(FNW)structure with enhanced peroxidase-like activity was formed.The obtained DNA/LDO FNW was applied to catalyze the oxidation of3,3',5,5'-tetramethylbenzidine(TMB)and further release the oxidative TMB²⁺.The latter was subsequently used to etch silver@gold nanostars(Au@Ag NSR),thereby exposing the cusp(horn)of Au NSR and resulting in a significant change of localized surface plasmon resonance(LSPR)signal.The peak of UV-vis absorbance curve presents an obvious red shift,which agrees with a computational simulation result provided by COMSOL software.Since a minor silver-etching will also induce distinct red shift,this method showed outstanding sensitivity.Under the optimal conditions,it achieved a low LOD of 3 aM(S/N=3)for Kana,and a linear range between 0.01 fM and 0.1 n M.The practical applicability of this method was successfully demonstrated by the determination of Kana in genuine cow milk and human samples.4.In Chapter 6,an innovative method for sensitive detection of CAP was successfully developed by the application of a solvent-free magnetic single-drop(MSD)-DSPE with a pipette tip-based magnetic bar.Target CAP was utilized to trigger the formation of DNA walkers,which further opened the hairpin modified on the surface of magnetic Fe3O4 nanosheets.HCR was then triggered by the opened hairpin,constructing long double-stranded product with G-rich sequence.A single-drop microextraction mold was utilized to separate the Fe3O4 nanosheets from the sample solution,achieving high enrichment in quite small volume(4?L).Finally,the fluorescence signal of G-quadruplex/PPIX in the droplet formed at the bottom of the the magnetic microextraction rod was used to detection CAP.Based on a simple device,this method realized solvent-free DSPE for the first time.The formed stable droplet adsorbent-containing droplet achieved fast separation and enrichment,thus decreased the matrix interference and increased the signal.The LOD of this analytical method is as low as 26.5 aM(S/N=3),the linear dynamic range is from 0.1 fM to 1 n M with good linearity.This novel method exhibited its applicability for the detection of CAP in cow milk.5.In Chapter 7,a highly sensitive detection method for Kana was designed based on magnetic three-phase single droplet microextraction(MTP-SDME)combined with CHA-HCR cascade signal amplification method as well as of silver@gold nanostars(Au@Ag NSR).Au and Ag nanomaterials have specific LSPR characteristics.Moreover,Au NSR has have stronger LSPR characteristics due to the large number of horns.The deposition of a small amount of Ag on the surface of Au NSR caused a significant shift of its ultraviolet-visible absorption peak,which significantly enhanced the sensitivity of the analytical method.Meanwhile,magnetic separation can effectively decrease the matrix effects,minimize the background signal,and enhance the signal-to-noise ratio of the analyte.Under the optimal conditions,the LOD of this method is as low as 33 aM(S/N=3),and a wide linear range from 0.1 fM to 1 n M is obtained.Finally,this analytical method has been successfully applied to the detection of Kana in dairy products.