Developing Nanozyme-based Optical Analytical Methods To Detect Phosphate In Aquatic Environment

Phosphate ion（Pi）is an important class of inorganic salts widely utilized in food,agricultural and chemical industries.However,excess phosphate in water can cause the eutrophication of rivers and lakes,which can cause serious ecological and environmental issues.As a result,phosphate content is an important indicator for assessing the safety of environmental water quality.To date,several methods for detecting phosphate have been developed,including colorimetric,fluorescence,ion chromatography,and electroanalytical methods.Although conventional phosphate detection technologies are adequate for laboratory measurements,they are either expensive,time-consuming to detect,or require pretreatment,and cannot meet the demand for on-site,quick,low-cost,and high-performance detection.To avoid water quality deterioration and maintain ecological balance,it is critical to design and construct novel methods and techniques for the in-field detection of phosphate with high sensitivity and specificity.Nanozymes are a type of nanomaterials with catalytic activity similar to enzymes.Nanozymes,in comparison with natural enzymes,are affordable and stable,allowing them to efficiently overcome the difficulties of bioenzymes’easy inactivation and high cost.Nanozymes also combine the intrinsic physicochemical features of nanomaterials,such as fluorescence,magnetic response,photothermal properties,and surface-enhanced Raman scattering,to create multifunctional and multimodal sensors.In order to meet the requirement for phosphate detection in water,we explored novel optical approaches for phosphate measurement based on nanozyme catalysis.The effects of phosphate on the catalytic activity of various nanozymes were extensively investigated,and the regulatory mechanisms of phosphate on the catalytic activity of nanozymes were discovered,resulting in new methodologies and techniques for phosphate detection.Main contents of this thesis include the following items:1.A highly sensitive and specific colorimetric sensor for Pi determination via utilizing Zr⁴⁺to synergistically inhibit the peroxidase-like activity of Fe₃O₄ nanocubes was developed.The decoration of hydrophilic 3,4-dihydroxyhydrocinnamic acid（DHCA）on Fe₃O₄ endows it with enhanced ability to catalyze the 3,3’,5,5’-tetramethylbenzidine（TMB）chromogenic reaction.Because of electrostatic interaction,Zr⁴⁺is easy to be adsorbed onto the negatively charged Fe₃O₄-DHCA nanozyme.When Pi is further added,it can interact with Zr⁴⁺rapidly and form a layer of coating on Fe₃O₄-DHCA surface,significantly suppressing its peroxidase-mimicking activity.As a result,the TMB chromogenic reaction is dramatically inhibited in the presence of Pi.Based on this attractive strategy,Pi in the wide scope of 0.066～33.3μM could be linearly determined,providing a detection limit down to 49.8 n M.Thanks to the strong and specific interaction between Zr⁴⁺and Pi,the colorimetric sensor could be employed to detect Pi with favorable selectivity.Reliable analysis of the target in tap and river water was also verified by our assay,indicating its great potential as a simple but efficient tool for environmental monitoring.2.A smartphone-assisted off-on photometric approach for Pi analysis based on the analyte-promoted peroxidase-mimicking catalytic activity of porous Ce_xZr_1-xO₂（x≥0.5）nanocomposites was created.The Ce⁴⁺/Ce³⁺redox pair in Ce_xZr_1-xO₂ endowed it with certain activity to catalyze the TMB color reaction with the participation of H₂O₂,and both the existing Zr⁴⁺and Ce⁴⁺species enabled the nanozyme to specifically recognize Pi.It was observed that the bonded Pi could greatly promote the peroxidase-like activity of the Ce_xZr_1-xO₂ nanocomposite towards positively charged TMB.According to the new finding,high-performance sensing of Pi with wide detection range,high sensitivity and good selectivity was realized,giving a detection limit down to 0.09μM.Further,a3D-printed smartphone-based signal reading system was designed and coupled with the sensing method,enabling the rapid,convenient,in-field and instrument-free analysis of Pi for environmental monitoring.3.A high-performance dual-channel ratiometric colorimetric sensing of Pi was proposed based on the analyte-triggered differential oxidase-mimicking activity changes of oxidized Ce-Zr bimetal-organic frameworks（oxidized UiO-66（Ce/Zr））.By integrating two types of metal nodes with different functions into one framework structure,the proposed oxidized UiO-66（Ce/Zr）not only exhibits excellent activity to catalyze the chromogenic oxidation of both TMB and 2,2’-azino-bis（3-ethylbenzothiazoline-6-sulfonic acid）（ABTS）due to oxidase-like active Ce⁴⁺/Ce³⁺sites but also provides main Zr⁴⁺sites to specifically recognize Pi.When Pi is in presence,it is rapidly adsorbed onto oxidized UiO-66（Ce/Zr）and changes the surface chemistry of the latter,leading to differential enzyme-like activity changes towards the two chromogenic substrates TMB and ABTS.Based on this finding,a dual-channel ratiometric colorimetric method was established for the sensitive determination of Pi.Compared to conventional single-signal colorimetric sensors,our method offered a widened linear detection scope from 3.3 to 666.7μM.Also,robust and accurate analysis of Pi in various water matrices was demonstrated,indicating our method’s great potential in practical applications.4.A Fe-Zr bi-metal organic frameworks（UiO-66（Fe/Zr）-NH₂）with three functions（self-fluorescence,peroxidase-mimicking activity,and specific recognition）was designed to establish a ratiometric fluorescent platform for high-performance Pi sensing.The use of a fluorescent organic ligand endows the MOF material with strong self-fluorescence at 435 nm.The presence of Fe³⁺/Fe²⁺nodes offer good enzyme-like capacity to catalyze the o-phenylenediamine（OPD）substrate to fluorescent OPDox（555 nm）,which then quenches the self-fluorescence of UiO-66（Fe/Zr）-NH₂ due to photoinduced electron transfer.The Zr⁴⁺nodes in the MOF material act as selective sites for Pi recognition.When Pi exists,it specifically adsorbs onto UiO-66（Fe/Zr）-NH₂and decreases the latter’s peroxidase-mimetic activity,resulting in the less production of fluorescent OPDox.As a consequence,the self-fluorescence of UiO-66（Fe/Zr）-NH₂at 435 nm is restored,and the fluorescence signal from OPDox at 555 nm is reduced inversely.With the ratiometric strategy,efficient determination of Pi with outstanding sensitivity and selectivity has been realized,giving a detection limit down to 85 n M in the concentration scope of 0.2-266.7μM.Accurate measurement of the target in practical water matrices has also been validated,indicating its promising application for Pi analysis in environmental and other fields.5.Single-atom iron doped carbon dots（SA Fe-CDs）with both photoluminescence and oxidase-mimicking catalytic activity was fabricated to enable the dual-mode colorimetric and fluorometric quantification of Pi.The SA Fe-CDs,obtained via a facile in-situ pyrolysis process of a Fe-coordinated small molecule precursor,not only exhibit adjustable fluorescence originating from the nature of small-sized carbon dots,but also provide excellent oxidase-mimetic activity with highly dispersed monoatomic Fe as active sites.By taking TMB as a common substrate,the SA Fe-CDs can catalyze the direct conversion of colorless TMB to a blue oxide TMBox with no addition of H₂O₂.Meanwhile,their photoluminescence is quenched due to the inner filter effect（IFE）caused by the TMBox species produced.However,Pi can inhibit the oxidase-mimicking activity of SA Fe-CDs by specifically coordinating with single-atomic Fe sites,leading to a significant suppression of the catalytic chromogenic reaction,and as such the photoluminescence of SA Fe-CDs is recovered again because of the relieved IFE.According to this principle,a dual-mode colorimetric and fluorescence assay of Pi with high sensitivity,selectivity and rapid response was established.Its reliability and practicability were verified by detecting environmental water samples.Our work paves a new path to design and develop multifunctional enzyme-like catalysts,and it also offers a simple but efficient dual-mode method for phosphate monitoring,which will inspire the exploration of multi-mode sensing strategies based on enzyme-like catalysis.