Development And Application Of A Ms-based Method For Specific Detection Of The SHV Family Of ?-lactamases By Mass Spectrometry Based On A Magnetic Solid-phase Extraction Procedure

Objective To prepare a magnetic nanocomposite material that can quickly and specifically recognize the?-lactamase SHV family.Establish a detection method for selective separation,enrichment and rapid identification of SHV combined with matrix-assisted laser desorption ionization time-of-flight mass spectrometry?MALDI-TOF MS?to achieve rapid detection of bacterial resistance.Methods?1?Fe₃O₄ nanoparticles?NPs?with a particle size of about 300 nm were synthesized by solvothermal method,and Fe₃O₄ was modified with polyacrylic acid?PAA?to prepare Fe₃O₄-COOH NPs.Then,the SiO₂ film was coated on the surface of Fe₃O₄-COOH NPs by sol-gel reaction to prepare Fe₃O₄@SiO₂.Fe₃O₄@SiO₂@MIP,the SHV family-specific molecularly imprinted material,was obtained under the crosslinking of TEOS through a mild sol-Gel polymerization with Fe₃O₄@SiO₂ as the base,SHV family-specific epitope VDAGDEQLER as the template molecule,three silane coupling agents?3-aminopropyl?triethoxysilane?APYES?,?-ureidopropyltriethoxysilane?UPTES?and bis?2-hydroxyethyl?amino propyltriethoxysilane?BHAPTES?as functional monomers.Then we characterized the materials and evaluated the adsorption performance.?2?The target genes bla_SHV-1,bla_SHV-18 and bla_TEM-1 had been amplified by PCR and combined with the expression vector pET-28a?+?,and then transformed the recombinant plasmid into the host strain E.coli BL21?DE3?.Transformed bacteria induced by Isopropyl-beta-D-thiogalactopyranoside?IPTG?highly expressed water-soluble drug-resistant proteins:SHV-1,SHV-18 and TEM-1.The purified protein were purified by Ni²⁺agarose affinity chromatography column and desalted by desalting column.Isothermal adsorption experiments and adsorption kinetic experiments were carried out on the purified protein using materials to observe the adsorption properties of the materials.?3?Using 0.05 mg/mL SHV-1 water/propylene glycol solution as the treatment object,Fe₃O₄@SiO₂@MIP as the extraction adsorbent,water as the washing liquid,and water/ammonia?25?28 wt%?solution as the eluting liquid,systematically optimize the amount of adsorbent and the volume fraction of ammonia in the eluent that affects the efficiency of MSPE.Taking the non-enzyme producing standard strain E.coli ATCC 25922 as the research object,the bacterial lysate was mixed with propylene glycol,and 0.05 mg/mL SHV-1 or SHV-18 was added.After MSPE treatment under optimal conditions,a little eluent was deposited on the target plate and detected SHV-1 and SHV-18 by MALDI-TOF MS.The established MSPE procedure was used for the purification and enrichment of SHV produced by the strain K.pneumonia HFK 417?production of SHV-1 verified by gene sequencing?and the standard strain K.pneumonia ATCC 700603?producing SHV-18?to evaluate the detection performance of the MSPE-MALDI-TOF MS method for SHV.Results?1?Fe₃O₄ NPs with a particle size of about 320 nm were successfully synthesized,and obtained Fe₃O₄-COOH NPs uniformly dispersed in water.A layer of SiO₂ film was further wrapped on the surface of Fe₃O₄-COOH NPs to obtain Fe₃O₄@SiO₂ with core-shell structure.By molecular imprinting technique,the molecular imprinting material Fe₃O₄@SiO₂@MIP specific for SHV family was obtained.Characterization results are as follows.Transmission electron microscopy showed that Fe₃O₄@SiO₂@MIP showed a clear core-shell structure with uniform size distribution and no agglomeration.The FT-IR and XPS spectra indicated that the material was successfully prepared.The XRD spectrum showed that the material had the same crystal phase as Fe₃O₄.It showed that the preparation process of MIP did not change the original crystal structure of Fe₃O₄.The hysteresis curve showed that the material has superparamagnetic characteristics and high magnetic response,can be quickly separated by an external magnetic field.The adsorption isotherm curve shows that Fe₃O₄@SiO₂@MIP adsorbs template molecules up to 50 mg/g and the imprinting factor as high as 7.33.Compared with the TEM family-specific epitope VDAGQEQLGR?VDR-10?,the material showed a good selectivity for template molecules.It indicated that the specific adsorption of the material to the template was based on shape matching and functional group recognition,which laid the foundation for its specific adsorption of SHV through epitope recognition basis.?2?The recombinant plasmid was successfully constructed and introduced into the host bacteria.The recombinant bacteria were induced by IPTG to obtain highly expressed water-soluble proteins and successfully purified to obtain three kinds of drug-resistant protein aqueous solutions isothermal adsorption experiments showed that Fe₃O₄@SiO₂@MIP exhibited higher adsorption capacity for SHV-1 and SHV-18 than non-imprinted materials,and lower adsorption capacity for TEM-1.It indicated that the imprinted material has good selectivity to SHV-1 and SHV-18.The evaluation of adsorption kinetics revealed that the material can reach adsorption equilibrium for SHV-1and SHV-18 within 90 minutes,but for TEM-1 within 210 minutes.And the later showed low adsorption capacity.The high adsorption capacity,favorable selectivity and fast adsorption rate of Fe₃O₄@SiO₂@MIP for SHV laid the foundation for the establishment of magnetic solid phase extraction technology based on this material and the specific detection of SHV by MALDI-TOF MS.?3?MSPE optimization results showed that the peak intensity of SHV-1 detected by mass spectrometry was the highest when the adsorbent dosage was 10 mg and the ammonia water volume fraction was 20%.In addition,the mass spectrometry results obtained that detected actual sample by the established MSPE-MALDI-TOF MS method showed that the mass spectrum peak of SHV is clear and the molecular weights correspond to the theoretical molecular weights of SHV-1 and SHV-18,respectively,which confirmed the accuracy and practicality of the resistance detection method established in this study.Conclusions In this study,we successfully prepared the magnetic material MIP that can specifically adsorb SHV,and optimized the MSPE technology using MIP as the adsorbent and then a new method for detecting the?-lactamase SHV family specific mass spectrometry was established based on MALDI-TOF MS.It had been verified that this method can realize the direct detection of drug resistance mechanism,which may provide a new method for the rapid detection of bacterial resistance,and extend the medical application value of magnetic nanomaterials.