The Development Of Ratiometric Fluorescence Sensing Platforms Based On Manganese Dioxide Nanosheets And Their Application In Detection Of Alkaline Phosphatase Activity

Alkaline phosphatase（ALP）activity is a crucial indicator for disease diagnosis and nutrition regulation in livestock as well as for the successful pasteurization of milk,which is significant in food detection and food science.Fluorescence assays are ease to operate,highly sensitive,and capable to display visible detection results.And manganese dioxide（MnO₂）nanosheets have outstanding fluorescence signal modulation capabilities and can specifically recognize the catalytic products of ALP,such as ascorbic acid（AA）.Hence,some fluorescence sensing platforms based on MnO₂ nanosheets were developed to detect the activity of ALP.Two major limitations,however,exist with the current platforms of this kind.The first is that the modulation of fluorescence signals mostly relies on MnO₂ nanosheets’physical property of“light absorption ability”,less on their other properties.And the second is that the determination of ALP activity depends highly on the fluorescence intensity change of single emission channel,and the as-resulted brightness change of fluorescence image,which is susceptible to interference from non-ALP-related factors like instrument fluctuations.This paper additionally makes use of MnO₂ nanosheets’chemical properties of“oxidation ability”and“oxidase-like activity”to modulate fluorescence signals,overcoming the first limitation.And the ratiometric fluorescence’s self-correction function is adopted to get around the second limitation,which can reduce interference and improve the accuracy and sensitivity of the quantitative detection of ALP activity.In addition,the construction of multiple channels in ratiometric fluorescence induces the sensing platforms to show obvious fluorescence color changes,optimizing the visualization effect of the detection result.This paper investigated the modulation ability of MnO₂ nanosheets on fluorescence signals of various substances systematically,and developed 4 kinds of ratiometric fluorescence sensing platforms with two or/and three channels for ALP activity detection.All platforms were developed based on the properties of MnO₂nanosheets and adopted the ratiometric fluorescence strategy,with systematic coherence.The detection principle is that ALP in the sample could cause the degradation of MnO₂ nanosheets,i.e.,the specific recognition unit of the sensing platforms,and thus weakened their physicochemical properties and fluorescence modulation ability.It led to the changes in multiple-channel fluorescence intensities as well as fluorescence color,and therefore realized an accurate and sensitive quantitative detection of ALP activity with results visualization,which were practically applied to multiple categories of samples in the food field.The results of the study are summarized below.（1）Fluorescence signal modulation study of MnO₂ nanosheets on fluorescent and non-fluorescent substances.Firstly,functional nanomaterials including MnO₂nanosheets,Cd Te/Zn S quantum dots（Cd Te/Zn S QDs,or QDs）,carbon quantum dots（CDs）,and gold nanoclusters（Au NCs）were synthesized and characterized.Following that,the effects of MnO₂ nanosheets on a variety of fluorescent and non-fluorescent substances were investigated:first,using the physical property of light absorption ability to quench the aforementioned three fluorescent nanomaterials and organic fluorescent dye Alexa Fluor 488(AF₄₈₈)through inner filter effect（IFE）together with other quenching mechanisms,where the contribution of IFE was 23%–52%;second,using the chemical property of oxidation ability to oxidize non-fluorescent dopamine into green fluorescent polydopamine（PDA）nanoparticles(λ_em=485 nm);third,using the chemical property of oxidase-like activity to catalyze the oxidization of non-fluorescent o-phenylenediamine（OPD）and thiamine,to generate yellow fluorescent 2,3-diaminophenolazine(OPDyellow,λ_em=569 nm)and blue fluorescent thiochrome(λ_em=440 nm).The fluorescence emission intensities of a variety of substances could be controlled by MnO₂ nanosheets through these three interactions,which were dependent on the amount of MnO₂ nanosheets and thus could be used as fluorescence signal conversion units in MnO₂ nanosheet-based fluorescence sensing platforms to indicate ALP activity.（2）A two-channel ratiometric fluorescence sensing platform,MnO₂-CDs-Au NCs,was developed based on the light absorption ability of MnO₂ nanosheets to detect ALP activity.ALP could catalyze the dephosphorylation of 2-phospho-L-ascorbic acid（AA2P）to generate AA,and the generated AA reacts with MnO₂nanosheets in a redox reaction and degrades them to Mn²⁺ions,decreasing their light absorption ability and the fluorescence quenching capacity.Based on this principle,when the ALP activity was 0,the blue fluorescent CDs and red fluorescent Au NCs in the sensing platform were quenched at different degrees by the light absorption ability of MnO₂ nanosheets,with a small initial intensity ratio of red channel to blue channel and showing a blue fluorescence image.As the ALP activity increased,the quenching efficiency of MnO₂ nanosheets for CDs and Au NCs decreased.The fluorescence of CDs and Au NCs was therefore restored,and the intensity ratio of red channel to blue channel increased,changing the fluorescence color from blue to violet to red-violet.This platform could quantify ALP in the range of 10–150 m U/m L by the change of two-channel intensity ratio,with a limit of detection（LOD）of 4.2 m U/m L,and achieve the visualization of detection results by the fluorescence color change.And this platform was of high specificity and could be applied to the ALP activity detection in livestock sera,with a relative error of-4.66%to 9.17%compared to the commercial kit.The detection accuracy and visibility of the two-channel platform were considerably improved as compared to the one-channel platforms.However,the width of the quantitative detection range was insufficient and was lacking in the ALP activity detection in pasteurized milk.（3）In order to broaden the application,a ratiometric fluorescence-colorimetry dual-mode sensing platform was developed for ALP activity detection based on the（catalytic）oxidation ability of MnO₂ nanosheets.In the ratiometric fluorescence mode of MnO₂-dopamine-QDs,the oxidation ability of MnO₂ nanosheets was exploited to oxidize dopamine to green fluorescent PDA nanoparticles,which quenched the red fluorescent QDs and resulted in a green fluorescence image;ALP also caused the degradation of MnO₂ nanosheets via AA and the reduction of their oxidation ability,and thus decreased the generation of PDA nanoparticles and recovered the fluorescence of QDs.The fluorescence intensities of two channels changed,and the fluorescence color gradually shifted from green to yellow to red.Then the colorimetric mode was further introduced to extend the applicability.The oxidase-like activity of MnO₂ nanosheets was used to oxidize colorless 3,3’,5,5’-tetramethylbenzidine（TMB）to yellow TMBox;and after ALP detection,the oxidase-like activity of MnO₂ nanosheets decreased,the generation of TMBox reduced,the absorbance decreased,and the yellow color became lighter.This platform expanded the quantitative detection range of ALP activity from 0.04 to 80 m U/m L with an LOD as low as 0.015 m U/m L based on the changes in two-channel fluorescence intensities and absorbance value,and enabled the detection visualization by the（fluorescence）color changes,with high specificity.The ratiometric fluorescence mode and the colorimetric mode in this platform were applicable to the detection of livestock sera and pasteurized milk,respectively,and the detection results were consistent with those of commercial kits or test strips,enabling the compatible detection of multiple samples with large differences in ALP activity.（4）A three-channel ratiometric fluorescence sensing platform,MnO₂-OPD-QDs,was developed to detect ALP activity based on MnO₂ nanosheets’oxidase-like activity,aiming to improve the evolution range and richness of fluorescence color and to enhance the visualization accuracy.In the initial state,the oxidase-like activity of MnO₂ nanosheets was utilized to catalyze the oxidation of OPD and generate yellow fluorescent OPDyellow（channel 1）,which quenched the red fluorescent QDs and produced a yellow fluorescence image.Then,ALP degraded the MnO₂ nanosheets via AA and lowered their oxidase-like activity.The formation of OPDyellow was decreased as a result,and the red fluorescence of QDs was restored（channel 2）,eventually changing the fluorescence color to orange and red.Meanwhile,the excess enzyme-catalyzed product AA in the sensing platform reacted with OPD to produce blue fluorescent OPDblue（channel 3）,which further altered the fluorescent color to purple.The platform achieved the quantitative detection of 0.1–100 m U/m L ALP with an LOD of 0.06 m U/m L by the three-channel fluorescence signal change,and realized the detection visualization by the fluorescence color change in a wide range from yellow to orange to red to violet.And the platform was of high specificity and could be applied to ALP activity detection in livestock sera,with comparable detection results to commercial kits.In comparison to two-channel platforms,the variation range and richness of fluorescence color were improved by increasing the number of emission channels,thus improving the visualization effect.（5）A multiple-channel ratiometric fluorescence sensing platform,MnO₂-thiamine(-AF₄₈₈)-Au NCs,was developed based on MnO₂ nanosheets for the double detection of ALP activity,to improve the accuracy of quantitative detection and result visualization.The introduction of AF₄₈₈ or not corresponded to two detection modes,i.e.,a two-channel mode of MnO₂-thiamine-Au NCs without the introduction of AF₄₈₈,and a three-channel mode of MnO₂-thiamine-AF₄₈₈-Au NCs with the introduction of AF₄₈₈.For one sample,it offered two ratiometric fluorescence values（2-channel ratio vs.3-channel ratio）for the quantitative detection,and two fluorescence color images（“blue-violet–violet–red”evolution path vs.“blue–cyan–green–yellow–green”evolution path）for the visual detection.Therefore,a double detection of ALP activity was realized.The changes in multiple-channel fluorescence signal and fluorescence color were also originated from the decrease of light absorption capacity and oxidase-like activity of MnO₂ nanosheets caused by by AA,the catalytic product of ALP.The two quantitative/visual detection results could be verified against each other.When the ALP activities detected from two quantitative tests,or from two visual tests,have a big error,it means that the detection results may be disturbed;conversely,it means that the results are accurate.The sensing platform achieved quantitative detection of0.1–100 m U/m L ALP with LODs of 0.05 m U/m L and 0.06 m U/m L,respectively,and the results were visible,with high specificity and effective application in ALP detection in livestock sera and pasteurized milk.The above four MnO₂ nanosheets-based multiple-channel（two-or/and three-channel）ratiometric fluorescence sensing systems were successful in ALP activity detection in an accuracy,visualization,and wide application manner.