Tài liệu Sythesis of conjugated molecules based on dithienopyrrole derivatives and pyrene as chemosensor for nitroaromatic pesticides detection

VIETNAM NATIONAL UNIVERSITY - HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY BAO KIM DOAN SYNTHESIS OF CONJUGATED MOLECULES BASED ON DITHIENOPYRROLE DERIVATIVES AND PYRENE AS CHEMOSENSOR FOR NITROAROMATIC PESTICIDES DETECTION Major: Chemical Engineering Major Code: 8520301 M$67(5¶6 THESIS HO CHI MINH CITY, February 2022 THIS RESEARCH IS COMPLETED AT: HO CHI MINH UNIVERSITY OF TECHNOLOGY ± VNU-HCM Instructor: Assoc. Prof. Dr. Nguyen Tran Ha Examiner 1: Dr. Nguyen Quoc Thiet Examiner 2: Dr. Nguyen Thanh Tung The master¶s thesis is defended at the National Key Laboratory of Polymer and Composite Materials, Ho Chi Minh city University of Technology (HCMUT), VNUHCM on February 24th, 2022. The board of the Master¶V7KHVLV'HIHQVHCouncil includes: 1. Assoc. Prof. Dr. Nguyen Truong Son Chairman 2. Dr. Cu Thanh Son Member, Secretary 3. Dr. Nguyen Quoc Thiet Reviewer 1 4. Dr. Nguyen Thanh Tung Reviewer 2 5. Assoc. Prof. Dr. Nguyen Tran Ha Member Verification of the Chairman of the Master¶V7KHVLV'HIHQVH&ouncil and the Dean of faculty of Chemical Engineering after the thesis being corrected (If any). CHAIRMAN OF THE COUNCIL DEAN OF FACULTY OF CHEMICAL ENGINEERING Assoc. Prof. Dr. Nguyen Truong Son VIETNAM NATIONAL UNIVERSITY HCM SOCIALIST REPUBLIC OF VIETNAM HO CHI MINH UNIVERSITY OF Independence ± Freedom - Happiness TECHNOLOGY __________________ __________________ THE TASK SHEET OF 0$67(5¶6 THESIS Full name: BAO KIM DOAN Student code: 2070162 Date of birth: 10/16/1997 Place of birth: Ho Chi Minh city Major: Chemical engineering Major code: 8520301 I. THESIS TOPIC: Synthesis of conjugated molecules based on dithienopyrrole derivatives and pyrene as chemosensor for nitroaromatic pesticides detection II. TASK AND CONTENTS: III. a. Synthesis of two conjugated molecules 2PDTP and 2PDOF based on pyrene derivative combined with either 4-(2-ethylhexyl)-4h-dithieno[3,2-b:2',3'-d]pyrrole or 9,9-dioctyl-9H-fluorene. b. Characterize the properties of these conjugated molecules by 1HNMR spectroscopy, FTIR spectroscopy, UV-Vis absorption spectrum, Photoluminescence Spectroscopy« c. Evaluate the applicability of the molecules as a fluorescent sensor for detecting NACs pesticides. TASKS STARTING DATE: 02/2021 IV. TASK ENDING DATE: 12/2021 V. SUPERVISORS: Assoc. Prof. Dr. Ha Tran Nguyen HCM, February « DEPARTMENTAL APPROVAL Signature ADVISER APPROVAL $GYLVHU¶VVLJQDWXUH Assoc. Prof. Dr. Ha Tran Nguyen ACKNOWLEDGEMENT I would like to acknowledge Assoc. Prof. Dr. Ha Tran Nguyen for his guidance, encouragement, and for allowing me the opportunity to work in an advanced group during my research. I thank all the members of the research group in National Key Laboratory of Polymer and Composite Materials for their support, it has been a pleasure to work with them. I would like to acknowledge M.Sc. Dat Hung Tran and M.Sc. Anh Duc Song Nguyen for their assistance with the optical and FTIR measurements. Also, I would like to express my gratitude to all my friends who have provided assistance and support during my past years at Ho Chi Minh University of Technology (HCMUT). I could not have done this without each and every one of you. Finally, last but no means least, I would like to express my special thankfulness to my family for all of their love and support. Without the encouragement of my parents, this goal would not have been possible. Thank you all with love! Ho Chi Minh City, February 2022 i ABSTRACT Nitroaromatic (NACs) pesticides have been broadly used in agriculture because of their high insecticidal and herbicidal efficiency. However, the most NACs pesticides are highly toxic, since they can trigger a series of neurological diseases, and even death. Therefore, the rapid and sensitive detection of NACs pesticides is one of great significance to human health and environmental protection. In this work, novel conjugated molecules based on pyrene, dithienopyrrole and fluorene derivatives including 4-(2-ethylhexyl)-2,6di(pyren-1-yl)-4h-dithieno[3,2-b:2',3'-d]pyrrole (2PDTP DQG ¶-(9,9-dioctyl-9H- fluorene-2,7-diyl)dipyrene (2PDOF) have been successfully designed and synthesized. The chemical structures of these conjugated molecules were determined via Fourier-transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR). The optical properties of conjugated molecules were investigated via UV±vis and fluorescence spectroscopies. The conjugated molecules exhibited the efficient fluorescence quenching toward herbicide mesotrione as nitroaromatic pesticide that could be promising candidates as chemosensor for tracing of NACs pesticides. TÓM TҲT Thuӕc trӯ sâu hӑ QLWURDURPDWLF1$&Vÿmÿѭӧc sӱ dөng rӝng rãi trong nông nghiӋp vì hiӋu quҧ diӋt côn trùng và diӋt cӓ cao. Tuy nhiên, hҫu hӃt các loҥi thuӕc trӯ sâu NACs ÿӅXFyÿӝc tính cao, vì chúng có thӇ gây ra bӋnh OLrQTXDQÿӃn thҫn kinh, và thұm chí tӱ YRQJ'Rÿy viӋc phát hiӋn nhanh và nhҥy các loҥi thuӕc trӯ sâu NACs là mӝt trong nhӳng viӋFFyêQJKƭDWROӟQÿӕi vӟi sӭc khӓHFRQQJѭӡi và bҧo vӋ P{LWUѭӡng. Trong nghiên cӭu này, các phân tӱ liên hӧp mӟi dӵa trên các dүn xuҩt pyrene, dithienopyrrole và fluorene bao 4-(2-ethylhexyl)-2,6-di(pyren-1-yl)-4h-dithieno[3,2-b:2',3'-G@S\UUROH 3'73 Yj ¶(9,9-dioctyl-9H-fluorene-2,7-diyl)dipyrene (2PDOF) ÿmÿѭӧc thiӃt kӃ và tәng hӧp thành công. Cҩu trúc hóa hӑc cӫa các phân tӱ liên hӧSQj\ÿѭӧF[iFÿӏnh thông qua quang phә hӗng ngoҥi Fourier (FTIR) và cӝQJKѭӣng tӯ hҥt nhân (NMR). Tính chҩt quang hӑc cӫa các phân tӱ liên hӧSÿѭӧc khҧo sát thông qua phә UV ± vis và phә huǤnh quang (PL). Các phân tӱ liên hӧp thӇ hiӋn khҧ QăQJGұp tҳt huǤnh quang hiӋu quҧ ÿӕi vӟi thuӕc diӋt cӓ PHVRWULRQHGRÿyFiFSKkQWӱ liên hӧp này có thӇ là nhӳng ӭng cӱ YLrQÿҫy hӭa hҽn cho viӋc thiӃt kӃ cҧm biӃn hóa hӑc ӭng dөng phát hiӋn thuӕc trӯ sâu NACs. ii DECLARATION Full name of author: Bao Kim Doan Name of thesis: ³6\QWKHVLV RI FRQMXJDWHG PROHFXOHs based on pyrene, dithienopyrrole and fluorene derivatives as optical chemosensor for nitroaromatic SHVWLFLGHVGHWHFWLRQ´ This thesis is a summary of the work carried out in the National Key Laboratory of Polymer and Composite Materials under the supervision of Associate Professor Ha Tran Nguyen. All work is orginal except where states otherwise by reference or acknowledgment. Parts of this work have been published in Journal of the Brazilian Chemical Society ZLWK WLWOH ³Synthesis of Conjugated Molecules Based on Dithienopyrrole Derivatives and Pyrene as Chemosensor for Mesotrione Detection´ Ho Chi Minh City, February 2022 Author Bao Kim Doan iii TABLE OF CONTENTS ACKNOWLEDGEMENT ............................................................................................ i ABSTRACT .................................................................................................................. ii DECLARATION ......................................................................................................... iii TABLE OF CONTENTS ............................................................................................ iv LIST OF FIGURES ..................................................................................................... vi LIST OF TABLES ..................................................................................................... viii ABBREVIATIONS ...................................................................................................... ix CHAPTER 1 : INTRODUCTION ...............................................................................1 1.1 Background of pesticides detection. .................................................................. 1 1.2 Fluorescence sensor for the detection of nitroaromatic pesticides .............. 3 1.3 Thesis Objective ................................................................................................... 4 CHAPTER 2 : LITERATURE REVIEW ...................................................................6 2.1 Fluorescence quenching theory ......................................................................... 6 2.1.1. Static and Dynamic quenching mechanism ................................................6 2.1.2. Energy transfer mechanism .........................................................................9 2.1.3. Photoinduced electron transfer mechanism ..............................................10 2.2 Fluorescent materials for chemosensor .......................................................... 10 2.2.1 Small molecule fluorophores ....................................................................11 2.2.2 Conjugated fluorescent polymers ..............................................................13 2.2.3 Supramolecular systems ............................................................................14 2.2.4 Aggregation induced emission (AIE)- active materials and bio-inspired fluorescent materials ...............................................................................................15 2.3 Molecular design of fluorophores ................................................................... 16 2.4 Overview of thesis .............................................................................................. 19 CHAPTER 3 : EXPERIMENTAL.............................................................................21 iv 3.1 Materials and Reagents .................................................................................... 21 3.2 Analysis and measurement methods ............................................................... 21 3.3 Synthesis of 4-(2-ethylhexyl)-4h-dithieno[3,2-b:2',3'-d]pyrrole (DTP) [73] . ............................................................................................................................... 25 3.4 Synthesis of 1-Bromopyrene [74] .................................................................... 26 3.5 Synthesis of 4-(2-ethylhexyl)-2,6-di(pyren-1-yl)-4h-dithieno[3,2-b:2',3'- d]pyrrole (2PDTP). ...................................................................................................... 26 3.6 6\QWKHVLVRI¶-(9,9-dioctyl-9H-fluorene-2,7-diyl)dipyrene (2PDOF) .... 27 3.7 Nitroaromatic pesticide detection ................................................................... 28 CHAPTER 4 : RESULTS AND DISCUSSION ........................................................29 4.1 Synthesis and structure characterization....................................................... 29 4.1.1 Direct Arylation synthesis of 4-(2-ethylhexyl)-2,6-di(pyren-1-yl)-4h- dithieno[3,2-b:2',3'-d]pyrrole (2PDTP) ..................................................................29 4.1.2 Suzuki cross-FRXSOLQJV\QWKHVLVRI¶-(9,9-dioctyl-9H-fluorene-2,7- diyl)dipyrene (2PDOF) ...........................................................................................37 4.2 Optical properties of 2PDTP and 2PDOF. .................................................... 42 4.3 Fluorescence quenching studies with Mesotrione in solution ..................... 44 CHAPTER 5 : CONCLUSION ..................................................................................52 LIST OF PUBLICATIONS ........................................................................................53 REFERENCES ............................................................................................................54 APPENDICES .............................................................................................................. xi CURICULUM VITAE ............................................................................................. xvii v LIST OF FIGURES Fig. 1-1: Schematic illustration of the working principle of the fluorescence quenching based sensor for NACs pesticide detection .....................................................................3 Fig. 2-1: Quenching mechanisms of fluorescent sensor which is used in the process of detecting analytes. And the effect of temperature on the effectiveness of dynamic (a) and static (b) quenching. [17] .........................................................................................6 Fig. 2-2: The schematic illustration of molecular wire theory. ....................................11 Fig. 2-3: Fluorescence enhancement of sensor sensor benzimidazo[2,1-a]benz[de] isoquinoline-7-one-13-(N-butylthioamide) by reaction with Hg2+ ...............................12 Fig. 2-4: Two conjugated polymers P1 and P2 with three-dimensional iptycene units. .......................................................................................................................................13 Fig. 2-5: 2,1,3-benzooxadiazole-alt-fluorene (PFBO) structure. .................................14 Fig. 2-6: Structures of dendrimers for three generations defined as G1±G3. ..............15 Fig. 2-7: Small molecules for AIE materials such as tetraphenylethene (TPE), triphenylbenzene (TPB) and siloles ...............................................................................16 Fig. 2-8: (top) Structures of fluorophores; (bottom) HOMO-LUMO of TAP and TEP. .......................................................................................................................................18 Fig. 2-9: Structure of 2PDTP and 2PDOF ...................................................................20 Fig. 3-1: Jablonski diagram of electronic transitions, absorption spectrum and emission spectrum..........................................................................................................24 Fig. 3-2: Synthesis of DTP ............................................................................................25 Fig. 3-3: Synthesis of 1-Bromopyrene ...........................................................................26 Fig. 3-4: Synthesis of 2PDTP ........................................................................................26 Fig. 3-5: Synthesis of 2PDOF .......................................................................................27 Fig. 4-1: Synthetic routes for the conjugated molecular 2PDTP. Reagents and conditions: (i) Pd2(dba)3, BINAP, t-BuONa, toluene, 110 °C; (ii) DMF, r.t.; (iii) Pd(OAc)2, P(Cy)3.HBF4, Cs2CO3, PivOH, toluene, 110 °C. .........................................29 Fig. 4-2: Direct arylation mechanism of 2PDTP synthesis reaction. ...........................31 Fig. 4-3: The 1H NMR spectrum (500 MHz, CDCl3) of 1-Bromopyrene ......................32 Fig. 4-4: The 1H NMR spectrum (500 MHz, CDCl3) of DTP ........................................33 Fig. 4-5: The FTIR spectrum of 2PDTP ........................................................................34 vi Fig. 4-6: The 1H NMR spectrum (500 MHz, CDCl3) of 2PDTP ...................................36 Fig. 4-7: Synthetic routes for the conjugated molecular 2PDOF. Reagents and conditions: (i) DMF, r.t.; (ii) Pd(PPh3)4, K2CO3, toluene/ethanol/H2O (10:2:1.5, v/v), 80 °C. .............................................................................................................................37 Fig. 4-8: Suzuki cross-coupling mechanism of 2PDOF synthesis reaction. .................38 Fig. 4-9: The FTIR spectrum of 2PDOF .......................................................................39 Fig. 4-10: The 1H NMR spectrum of 2PDOF ................................................................41 Fig. 4-11: UV-Vis absorption spectra of 2PDTP (A) and 2PDOF (B) at different concentrations in CHCl3 with a path length of 1 cm. ....................................................42 Fig. 4-12: Estimate the molar absorption coefficients Hof triad 2PDTP and 2PDOFaccording to Lambert-Beer law at the maximum wavelength of absorption Omax .................................................................................................................................43 Fig. 4-13: Fluorescence quenching of 2PDTP (A) and 2PDOF (B) in CHCl3 ȝ0 upon addition of Mesotrione (0-75 PM). .......................................................................45 Fig. 4-14: Fluorescence intensities of 2PDTP (top) and 2PDOF (bottom) decrease when 1 PM Mesotrione was added. ...............................................................................45 Fig. 4-15: Fluorescence emission spectrum of 2PDTP (A) and 2PDOF (C) in CHCl3 (1 µM) with concentration increase of mesotrione; and the corresponding SternVolmer plot of 2PDTP (B) and 2PDOF (D). ................................................................47 Fig. 4-16: UV±vis absorption spectra of 2PDTP/2PDTP with mesotrione (A) and 2PDOF/2PDOF with mesotrione (B) in CHCl3 with a path length of 1 cm. ................49 Fig. 4-17: Proposed schematic diagram of 2PDTP interacted with mesotrione via the static quenching mechanism. .........................................................................................50 Fig. 4-18: Image of 2PDTP coated on the cellulose paper under UV irradiation (365nm). .........................................................................................................................51 vii LIST OF TABLES Table 1-1: Common NACs pesticides [8] .......................................................................2 Table 4-1: IR absorption wavenumbers (cm-1) and their functional group for 2PDTP .......................................................................................................................................34 Table 4-2: IR absorption wavenumbers (cm-1) and their functional group for 2PDOF. .......................................................................................................................................40 Table 4-3: Optical properties of triad 2PDTP and 2PDOF.........................................44 viii ABBREVIATIONS Abbreviation names Full explanation 2PDTP 4-(2-ethylhexyl)-2,6-di(pyren-1-yl)-4h-dithieno[3,2b:2',3'-d]pyrrole 2PDOF ¶-(9,9-dioctyl-9H-fluorene-2,7-diyl)dipyrene AChE Acetylcholinesterase AuNPs Gold nanoparticles AIE Aggregation induced emission BINAP ¶-bis(diphenylphosphino)-¶-binaphthyl C Celsius CQDs Carbon quantum dots CPs Conjugated polymers DET Dexter energy transfer DTP 4-(2-ethylhexyl)-4h-dithieno[3,2-b:2',3'-d]pyrrole DOF ¶-(9,9-dioctyl-9H-fluorene-2,7-dityl)bis(4,4,5,5tetramethyl-1,3,2- dioxaborolane) DMF Dimethylfomamide DCM Dicloromethane FTIR Fourier-transform infrared FRET Förster resonance energy transfer 1 Proton nuclear magnetic resonance H NMR HOMO Highest occupied molecular orbitals LUMO Lowest unoccupied molecular orbitals MIP Molecularly imprinted polymer MOFs Metal-organic framework NACs Nitroaromatic NMOF1 Nanocrystal metal-organic framework NBS N-bomosuccinimide OLEDs Organic Light Emitting Diodes PET Photoinduced electron transfer ix PFBO 2,1,3-benzooxadiazole-alt-fluorene PL Photoluminescence Pd Palladium SET Surface energy transfer TNT Trinitrotoluene TMS Tetramethylsilane UV-Vis UV-visible WHO ± FAO World Health Organization ± Food and Agriculture Organization x CHAPTER 1 : INTRODUCTION 1.1 Background of pesticides detection. Pesticides are widely used in agricultural production, and the pesticides demand is increasing due to population growth leading to a priority in promoting high agricultural productivity. The ingredients of pesticides are chemicals that can control pests or plant growth have become effective tools in boosting agricultural productivity. However, pesticides are also the world's leading concern in food safety, which are the main cause of more than 200,000 deaths each year [1]. Furthermore, the health concerns associated with the consumption of products containing pesticides are becoming more noticeable in developing countries due to the lack of knowledge and understanding of farmers using pesticides in cultivation. At present, many pesticides with nitro-aromatic compounds (NACs) such as parathion, nitrofen, fenitrothion and mesotrione have been commonly used to protect crops. NACs pesticides are one of the leading causes of environmental pollution due to their high biological activity and toxicity [2]. Most pesticides do not distinguish between plants and pests, they have the potential to harm humans, animals and other living organisms if used incorrectly. One of the most important aspects of minimizing potential risks to humans and the environment is the monitoring of these pesticide residues. Up to now, the methods of detecting pesticides have made remarkable progress. Typical are the traditional chromatographic detection methods, including gas chromatography (GC) [3], high-performance liquid chromatography (HPLC) [4] and gas chromatographymass spectrometry (GC ± MS) [5], besides other methods such as electrochemical analysis [6], biosensors based on AChE [7], etc. All of these methods have their own advantages and disadvantages, in which the chromatographic method shows accurate and high sensitivity but requires expensive equipment investment, time-consuming, complicated operation and poor portability. There is a reason why developing a new method to detect pesticide residues with advantages: fast, sensitive, reliability and lowcost for timely handling is one of the important issues that need to be focused on research. 1 Table 1-1: Common NACs pesticides [8] No Names Structure 1 Parathion methyl Ia 2 Fenitrothion II 3 Mesotrione III 4 2,6dinitroaniline _ 5 Nitralin O 6 Pendimethalin III 7 Nitrofen O 8 Oxyfluorfen U 2 WHO Acute Hazard 9 Bifenox U Ia = Extremely hazardous; II = Moderately hazardous; III = slightly hazardous; U = Unlikely to present acute hazard in normal use; O = Obsolete as pesticide, not classified. 1.2 Fluorescence sensor for the detection of nitroaromatic pesticides In recent years, a fluorescence-based method has been approached due to better efficiency, simplicity, fast, and reliability. This technique is based on colorimetric and fluorimetric responses which are mainly focused on by vast fluorescence sensors. Optical sensors based on photoluminescence (PL) quenching have attracted great interest due to their sensitivity, low cost and ease of operation [9]. Fig. 1-1: Schematic illustration of the working principle of the fluorescence quenching based sensor for NACs pesticide detection In 2009, Mallard-Favier et al [10] synthesized a new peracetylated cyclodextrin trimer bearing three 1,2,3-triazole linkers that the fluorescent tripod exhibits a very good variation of emission fluorescence by the addition of pendimethalin with extremely low detection limits (0.8-4 µM). In 2014, Kumar et al [11] synthesized a luminescent nanocrystal metal-organic framework (NMOF1) for chemosensing 3 of the nitroaromatic-containing organophosphate pesticides such as parathion, methyl parathion, paraoxon and fenitrothion. In 2018, Hergert et al [12] reported the molecular chain effect in phenylene ethynylene oligomers to detect insecticides. The results show a significant increase in Stern-Volmer quenching - beyond the molecular wire effect. In 2018, Zhao et al [13] developed a pyranine-EDVHGIOXRUHVFHQW³WXUQ-RII´PHWKRG for paraquat sensor. Under the optimized conditions, this method is sensitive, selective, and could be used for paraquat detection in the real-world sample. In 2019, a molecularly imprinted polymer (MIP)-based fluorescent probe was synthesized by Sun et al [14] using biomass-derived carbon quantum dots (CQDs) encapsulated into MIPs via a sol-gel method, for rapid and sensitive mesotrione detection, based on the capture capacity of CQDs@MIPs to target mesotrione to quench fluorescence. In 2019, Hu et al [15] also reported a colorimetric chemosensor for the simple and rapid detection of dimethoate pesticides in agricultural products based on the inhibition of the peroxidase-like activity of gold nanoparticles (AuNPs). Optical chemosensing techniques based on functional materials/nanomaterials such as conjugated molecules/oligomers/polymers, macrocycle and luminescent metal-organic frameworks have shown great potential for sensitive and selective detection of pesticide. In 2020, Zhang et al [16] reported that a carbazole-containing polymer with significant inherent porosity exhibited apparent fluorescence quenching upon raising the concentration of trifluralin, and the fluorescence quenching with the increase of concentration is up to 84%. 1.3 Thesis Objective Functionalization of conjugated fluorophore and understanding of molecular interaction of electron donors and electron acceptors are very important to produce efficient fluorescence sensors. The main objective of this thesis is to investigate the applicability of fluorescence conjugated molecular as a fluorescent chemosensor to detect nitroaromatic pesticides. The design of conjugated molecular using in this thesis 4 is based on pyrene derivative combined with either 4-(2-ethylhexyl)-4h-dithieno[3,2b:2',3'-d]pyrrole or 9,9-dioctyl-9H-fluorene. These fluorescent chemosensors are expected to provide critical insight of the detection mechanism, thus contributing to the future development of fluorescent chemosensors that will detect more efficiently. 5 CHAPTER 2 : LITERATURE REVIEW 2.1 Fluorescence quenching theory The applications of the fluorescence sensor were based on the principle that the molecular contact between analytes and fluorophore either decreases the fluorescence by quenching, or increases fluorescence by suppressing the quenching effect. This contact can be the result of the complex formation (static quenching), the diffusive encounter (dynamic quenching), energy transfer and photoinduced electron transfer (PET). Fig. 2-1: Quenching mechanisms of fluorescent sensor which is used in the process of detecting analytes. And the effect of temperature on the effectiveness of dynamic (a) and static (b) quenching. [17] 2.1.1. Static and Dynamic quenching mechanism Static quenching occurs when a non-emissive ground-state is formed through the interaction between the sensor and the quencher. The complex immediately returns to the ground state without emission of the photon when absorbing light leading to quenching of the initial fluorescence intensity. However, an excited-state electron 6 transfer takes place from the excited state of the sensor to the quencher through collision in the dynamic quenching with the mechanism of energy transfer or charge transfer. [18] The two quenching mechanisms mentioned above possess very different natures that can be distinguished by time-resolved measurements of the fluorescence decays of the sensing materials (߬଴ Τ߬). For static quenching, the fluorescence decay lifetime of material will remain unchanged as the concentration of the quencher is increased (߬଴ Τ߬ ൌ ͳ). The formation of a non-emissive fluorophore±quencher complex is the source of the quenching, and any molecules that did not bind to an analyte will decay with their natural lifetime. For dynamic quenching (diffusive encounter), a collision of the quencher molecules to the excited fluorescent sensor is necessary, and thereby dynamic quenching is a diffusion-controlled process (߬଴ Τ߬ ൌ ‫ܨ‬଴ Ȁ‫)ܨ‬. The sensor and the quencher are unbound and quenching occurs when a photoexcited material interacts briefly with a colliding analyte molecule. Therefore, it reduces the average fluorescence lifetime as the concentration of the quencher is increased. The measurement of fluorescence lifetime change in the absence and presence of quenchers represents the most common way to examine whether the quenching is a static or dynamic process. [19] Dynamic quenching of fluorescence is described by the Stern±Volmer equation and the correlation of lifetime with quencher concentration can be expressed as: ߬଴ ൌ ͳ ൅ ‫ܭ‬஽ ሾܳሿሺͳሻ ߬ ‫ܨ‬଴ ߬଴ ൌ  ሺʹሻ ‫ܨ‬ ߬ Where ‫ܨ‬଴ and ‫ ܨ‬are the fluorescence intensities in the absence and presence of a quencher, respectively; ߬଴ and ߬ are the fluorescence life times of the sensors before and after addition of quencher at a given concentration [Q], respectively; ‫ܭ‬஽ are the dynamic Stern±Volmer quenching constant. For static quenching, the dependence of the fluorescence intensity upon quencher concentration is easily derived by consideration of the association constant for complex formation, and the equation is shown as follows: 7