Studies On The Carbon Nanomaterials Based Ratiometric Fluorescent Probe For Hydrolases Activity And Organophosphorus Pesticides Detection

With the continuous development of nanomaterials,various carbon nanomaterials have emerge as the times require.Carbon dots?CDs?and graphene quantum dots?GQDs?as fluorescent carbon nanomaterials have superior optical properties and biotoxicity.A series of advantages such as small size,simple synthesis method,and low cost make it widely used in biomedicine,photothermal therapy,catalysis and sensing.In addition,hydrolases is a functional protein with a hydrolysis effect on substrates,which is an indispensable part of living body with high efficiency and specificity.Alkaline phosphatase?ALP?can catalyze the hydrolysis of phosphates in alkaline media.The abnormal increase in activity of human serum ALP are related to diseases such as breast cancer,intestinal diseases and liver dysfunction.Trypsin?TRY?are a proteolytic enzyme produced in the pancreas.The abnormally high TRY concentration in human urine indicates the potential for pancreatitis.Acetylcholinesterase?Ach E?can quickly catalyze the hydrolysis of the acetyl group of the neurotransmitter acetylcholine,leading to the termination of nerve impulse transmission,which guarantees the normal physiological function of the human body.Organophosphorus pesticides?OPs?as inhibitors of Ach E,can cause metabolic disorders in the body's conduction media,causing headaches,dizziness and nausea.Therefore,the development of their detection methods is essential for the physical health of the human body.In the preface,the paper mainly introduces the preparation methods and properties of CDs and GQDs,and discusses the application of CDs and GQDs in detection and the significance of hydrolases and OPs on human physiology.The characteristics and advantages of the two types of materials were used to construct ratiometric fluorescent probe to detection of several hydrolases activities and OPs,rom the single-signal detection to dual-signal ratiometric detection,from evaluating enzyme activity to detecting enzyme inhibitors,from short-wavelength ratiometric detection to long-wavelength ratiometric detection,from laboratory fluorescence instrument detection to test kit detection,a total of 5 jobs have been performed.The main research contents of the paper are as follows:?1?Based on the characteristics of benzoquinones that can quench the fluorescence of GQDs by static quenching,a GQDs-based fluorescent probe was established to evaluate the activity of ALP.Hydrogen peroxide?H₂O₂?generates hydroxyl radicals?·OH?under the action of horseradish peroxidase?HRP?,and·OH are accompanied by strong oxidative properties to o-dihydroxybenzene?o-DHB?oxidates to o-benzoquinone and quenches GQDs fluorescence.The ascorbic acid?AA?produced by ascorbic acid 2-phosphat?AAP?under the action of ALP has strong reducing ability and removes H₂O₂and·OH from the solution,leading to the inhibition of the production of o-benzoquinone and the fluorescence recovery of GQDs.Therefore,a highly sensitive detection method for ALP was established.Under the optimal experimental conditions,the detection limit of ALP was 0.084 U/L.This method was successfully used to evaluate ALP activity in human serum.?2?Ionic liquid?IL?and water were used as electrolytes to electrochemically synthesize surface-functionalized CDs?IL-CDs?and design a ratiometric fluorescent probe based on IL-CDs to evaluation of ALP activity.H₂O₂are produced under the action of HRP,which can oxidize o-phenylenediamine?OPD?to 2,3-diaminophenazine?DAP?.The fluorescence of CDs are quenched through the RET process.Adding ALP can make AAP produce strong reducing AA and inhibit DAP formation by removing H₂O₂and·OH.Based on the fluorescence intensity ratio of DAP to IL-CDs,the detection limit of ALP was 0.012 U/L under the optimal experimental conditions.This method was successfully used to evaluate the activity of ALP in human serum.?3?Nitrogen-doped CDs?N-CDs?were synthesized by one-step hydrothermal method using TRY and ethylenediamine as carbon sources.A ratiometric fluorescent probe based on N-CDs was used to evaluate the activity of Ach E and detect OPs.Acetylcholine?Ach?produces H₂O₂through the combined enzymatic reaction of Ach E and choline oxidase?Ch O?.H₂O₂can oxidize OPD to DAP under the action of HRP.DAP quenches the fluorescence of N-CDs through the inner filter effect?I E?.OPs reduce the production of DAP by inhibiting the activity of Ach E,and the fluorescence of CDs are restored.The ratio of fluorescence intensity of DAP and N-CDs can be used to evaluate Ach E and detect OPs.Under the best experimental conditions,the limit of Ach E is 0.0271 U/L.The limits of dichlorvos and methyl parathion are0.229 ppb and 0.926 ppb,respectively.This method was successfully applied to the detection of OPs in food and water samples.?4?A ratiometric fluorescent probe for ultrasensitive detection of TRY concentration was established using green fluorescent CDs and red fluorescent Tetraphenylporphyrin tetrasulfonic acid?TPPS?.TPPS can effectively quench CDs fluorescence through I E.However,larger Hg²⁺can deform TPPS,which is beneficial to attack by smaller Mn²⁺and form metalloporphyrin compounds.The characteristic absorption and fluorescence intensity of TPPS decrease continuously to inhibit the I E effect of TPPS on CDs.When TRY is present,bovine serum albumin is cleaved into amino acid or peptide fragments and combines with Hg²⁺to form a more stable complex,which promotes the I E effect of TPPS on CDs.According to the ratio of the fluorescence intensity of TPPS and CDs,the concentration of TRY can be detected.Under the optimal experimental conditions,the detection limit of TRY was 0.0391 ng/m L.This method was successfully used for the detection of TRY in urine samples.?5?A test kit based on hydrogel-immobilized CDs and Cd Te QDs was established as a ratiometric fluorescent probe for on-site detection of OPs in food and water samples.The blue-green emission fluorescent CDs and red-emission fluorescent Cd Te QDs were embedded in agarose to form a mixed hydrogel,and they were fixed on the centrifuge tube cap.As an inhibitor of Ach E,OPs can prevent sulfur acetylthiocholine?ATCh?from generating thiocholine,?TCh?to prevent the binding of Cu²⁺,so that free Cu²⁺exists in the solution.After the centrifuge tube was inverted,Cu²⁺diffused into the mixed hydrogel to quench the fluorescence of Cd Te QDs,while the fluorescence intensity of CDs remained stable.In addition,the fluorescence color change of the centrifuge tube cover under the UV dark box is recorded by the built-in camera of the smartphone.The intensity values of the green channel?G?and the red channel?R?were obtained through the analysis of Image J software.According to the ratio of R and G,quantitative determination of OPs can be achieved without relying on professional detection equipment.Under the optimal conditions,the detection limit for dichlorvos is 0.672 ppb.This test kit was successfully applied to the detection of OPs in food and water samples.