Development And Application Of Biosensing Systems For The Metabolic Activity Indicators Of Microbial Biofilms

Microbial corrosion can occur on the surface of almost all marine engineering materials during resource exploitation in the marine environment,which seriously threatens the safe service of marine equipment and engineering facilities.Therefore,the study of microbially influenced corrosion in the marine environment is crucial.Rapid determination of the activity of corrosive microbial biofilms can not only reveal the development stage of biofilm,but also predict the development trend of microbial corrosion.In this study,the rapid detection methodsof characteristic metabolic active molecules of corrosive microbial biofilms were constructed for different application backgrounds,including a dual-signal ATP detection system based on artificial nanozymes inhibited by ZIF-90 nanoparticles,an instrument-free paper-based system for ATP detection,a intracellular and extracellular ATP discriminative detection system based on magnetically modified antimicrobial peptide,a simultaneous ultrasensitive ADP and ATP quantification system based on CRISPR/Cas12a integrated ZIF-90nanocomposites,a precise localization and simultaneous bacterial eradication system based on successive responsive towardspH and ATP,an instrument-free dual-readout viscosity flow system for cyt c detection,and a target-modulated competitive binding and exonuclease I-powered system for the simultaneous detection of ATP and cyt c.The construction and implementation process,signal feedback and optimization principle,and performance testing and evaluation system of each detection system were systematically investigated and validated in biofilms system.This study provides reliable tools to reveal the metabolic state of corrosive microorganisms in biofilms,and provides a technical guarantee to reveal the correlation between the metabolic activity of corrosive microorganisms and corrosion behavior,which is of great significance to reveal the microbial corrosion mechanism and develop protection technology.The main research results are as follows:（1）A dual-signal detection system for biofilm ATP based on ZIF-90 inhibition of MoS₂ nanoenzyme activity was developed The controlled release of MoS₂ mimic enzyme activity was achieved by using the response property of ZIF-90 nanoparticles to ATP,and the correlation between catalytic activity of H₂O₂-TMB reaction system and ATP concentration was established by optical and electrochemical methods.The proposed detection method holdsgood sensitivity and selectivity towardsATP,and the optical and electrochemical dual modes can meet the demand of ATP detection in different application environments.The prepared system enabled dual-signal detection of ATP in SRB biofilms.（2）A paper-based dual-signal ATP detection system based on ATP responsive cleavage of ChoA@ZIF-90 nanoparticles was designed,using the property of ATP-specific cleavage of ZIF-90 to release the encapsulated chitosanase,which cleaves chitosan hydrogels to produce solution viscosity changes.In addition,the cleavage of ZIF-90 releases the catalytic activity of the nanoenzyme,which is used to produce color changes.The proposed method is portable,rapid,and instrument-free for detection of ATP,and the detection are displayed directly on the filter paper results with distance and colorimetric readings,enabling qualitative on-site detection and quantitative visual measurement of ATP within microorganisms and biofilms.（3）A novel impedimetric aptasensor was designed for the discriminative detection of intracellular and extracellular ATP using antimicrobial peptide-functionalized magnetic particles（M-AMPs）.For discriminative intracellular and extracellular ATP detection,magnetically controlled M-AMPs were introduced into the detection system for mild and rapid bacterial disintegration.The intracellular and intracellular ATP levels were obtained via the difference of charge transfer resistance before and after M-AMPs release.The proposed aptasensor exhibited distinguished sensitivity and selectivity,which enabled the discriminative intracellular and extracellular ATP detection in various corrosive microorganism.（4）A nanoplatform based on dsDNA-ZIF-90@Ag₃AuS₂@Fe₃O₄ nanocomposites was established for the simultaneous detection of ADP and ATP,which was integrated with an ADP sensitive module based on aptamer recognition and DNA-activated CRISPR/Cas12a amplification,and an ATP responsive module based on ZIF-90nanocarrier fragmentation and quencher loading release.The absolute concentrations of nucleotides rather than the ADP/ATP ratio were successfully determined using the developed biosensor,which was helpful to exclude the interference of ADP during ATP detection.The proposed biosensor enabled accuracy detection of ADP and ATP,thus the nanoplatform provides a promising method for ultrasensitive dual-mode quantification of ADP and ATP in SRB biofilms.（5）A novel biofilm-targeted nanocontainer with successive responsive property towardspH and ATP for precise localization and simultaneous bacterial eradication.The biofilm-targeted nanocontainer was composed of a ATP-responsive zeolitic imidazolate framework-90（ZIF-90）core encapsulated in the pH-responsive amorphous calcium carbonate/poly（acrylic acid）（ACC/PAA）shell.Moreover,to meet the application requirements of different biofilm scenarios,the pH response ability of the nanocontainers could be adjusted by changing the metallic ions doped into the structure of the ACC/PAA shell.The precise localization and simultaneous bacterial eradication were successfully realized via a simple spray process and embedding in self-defensive coatings.The nanocontainer endowed the nanocontainers high precision for localization and simultaneous eradication of SRB biofilms.（6）A dual-readout viscosity flow sensor based on cyt c-triggered hyaluronidase（HAase）release was designed for on-site detection.The probe was fabricated using Ui O-67-NH₂ loaded with HAase as a nanocarrier,and the double-stranded DNA（dsDNA）as a gatekeeper.Release of HAase to lyse hyaluronic acid hydrogels was triggered by cyt c recognition,,and the changes in the diffusion distance and the color signal were used to quantify the biotarget concentration.The proposed sensor avoidsexpensive instruments and sophisticated operation procedures,making it practical for on-site cyt c detection.This sensor exhibited excellent specificity and reproducibility for detection of cyt c,enabling accurate detection of cyt c concentration in SRB biofilm.（7）A novel strategy based on target-modulated competitive binding and exonuclease I（Exo I）-powered signal molecule release was established for simultaneous multiple-target detection.Ui O-67 nanocarriers with high signal molecule loading characteristics were designed to release signal molecules using the specific recognition process of target molecules,and Exo I was introduced to accelerate the release of signal molecules.With the advantages of rapid response and high selectivity and sensitivity,this strategy provides guidance for simultaneous detection of ATP and cyt c in a variety of bacteria,including SRB.