Hydrogen peroxide sensors based upon photo-induced electron transfer
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Publication Number
US-9018377-B2
Publication Date
2015-04-28
Expiration Date
Abstract
The invention provides compounds of formula I F-L-Q (I) where F comprises a fluorophore capable of absorbing energy at an excitation wavelength and, in the absence of a quencher, emitting energy at an emission wavelength, which is different than the excitation wavelength; Q comprises a quencher; L comprises a linker moiety having two ends, one end being covalently bound to F and the other end being covalently bound to Q. The compounds are capable of undergoing a reversible reaction (1), provided below: (1) where Q+ is an oxidized form of Q representing the absence of a quencher, Ox comprises an oxidizing agent, which is capable of oxidizing Q to its oxidized form Q, and Red comprises a reducing agent, which is capable of converting Q back to its reduced form Q. The compounds can undergo photo-induced electron transfer when irradiated with energy and when Q exists in its oxidized form, Q+. The invention also provides methods of detecting and determining the presence of analytes and/or hydrogen peroxide in a sample, as well as a substrate that comprises the compound of formula I.
Core Innovation
The invention provides a compound of formula IA or a salt thereof, and the compound is used as a fluorescent probe to determine or detect hydrogen peroxide in a sample. The formula IA defines selectable substituents R2, R3, R1, X, Y, m, and n, with R2 selected from C1-8-alkyl, C1-8-alkoxy, C1-8-alkyl-aryl, and C1-8-alkyl-C1-8-alkoxy, and with optional substitution of alkyl or aryl groups with from one to four —C(O)OR′ substituents.
R3 is independently selected from OR1, SR1, and N(R1)2, and X and Y are independently selected from —NR1—, —O—, and —S—, with m being 1, 2, 3, 4, or 5 and n being 1, 2, 3, 4, 5, or 6. When n is at least 2, 2 and 4 hydrogen atoms in adjacent —CH2— groups in —X—(CH2)nY— are optionally not present so as to represent the moieties —CH=CH— and —C≡C—, respectively.
The invention relates to hydrogen peroxide-responsive photo-induced electron transfer (PET) sensor compounds having a fluorophore-linker-quencher architecture of the form F-L-Q. The compounds reversibly switch between quenched and unquenched oxidized form upon oxidation/reduction, and the disclosure provides fluorescence-based detection and quantification by irradiation at a fluorophore excitation wavelength and measurement of emitted energy at an emission wavelength.
Claims Coverage
The document includes one independent compound claim and two independent method claims. Across these claims, the core coverage is the formula IA compound scaffold, including salts and defined substituent choices, together with fluorescence-based excitation/emission measurement for determining hydrogen peroxide concentration or detecting its presence.
Formula IA compound and salts
A compound of formula IA, or a salt thereof, wherein R2 is selected from C1-8-alkyl, C1-8-alkoxy, C1-8-alkyl-aryl, and C1-8-alkyl-C1-8-alkoxy; any alkyl or aryl is optionally substituted with from one to four —C(O)OR′; each occurrence of R′ is independently selected from H, C1-8-alkyl and C3-8-heterocycloalkyl; R3 is independently selected from OR1, SR1, and N(R1)2; R1 is hydrogen or unsubstituted C1-6-alkyl; X and Y are independently selected from —NR1—, —O—, and —S—; m is 1, 2, 3, 4, or 5; n is 1, 2, 3, 4, 5, or 6; and when n is at least 2, 2 and 4 hydrogen atoms in adjacent —CH2— groups in —X—(CH2)nY— are optionally not present so as to represent the moieties —CH=CH— and —C≡C—.
Fluorescence-based determination of hydrogen peroxide concentration
A method of determining a concentration of hydrogen peroxide in a sample by contacting the sample suspected of containing hydrogen peroxide with a sufficient amount of a compound of formula IA or a salt thereof, irradiating the sample with electromagnetic radiation at an excitation wavelength at which the fluorophore absorbs the energy, measuring an intensity of energy emitted from the irradiated sample at an emission wavelength, if any, and correlating the measured intensity of the emitted energy with a concentration of the hydrogen peroxide in the sample.
Fluorescence-based detection of hydrogen peroxide presence
A method of detecting the presence of hydrogen peroxide in a sample by contacting the sample suspected of containing hydrogen peroxide with a sufficient amount of a compound of formula IA or a salt thereof, irradiating the sample with electromagnetic radiation at an excitation wavelength at which the fluorophore absorbs the energy, and detecting energy emitted from the irradiated sample at an emission wavelength, if any, wherein the detection of emitted energy supports a conclusion that at least some hydrogen peroxide is present in the sample.
The claims center on a defined fluorescence-active probe scaffold of formula IA, including salts, and on fluorescence excitation/emission measurement to correlate emission intensity with hydrogen peroxide concentration and to support presence or absence detection of hydrogen peroxide in a sample.
Stated Advantages
Hydrogen peroxide-responsive photo-induced electron transfer sensor activity.
Reversible switching between quenched and unquenched oxidized form upon oxidation/reduction.
Fluorescence-based detection and quantification of hydrogen peroxide.
Provides fluorescent response for hydrogen peroxide sensing using HRP with H2O2 and fluorescence measurement/calibration.
Enables covalent immobilization of an amino-reactive probe (B-NHS) onto an amino-modified polymeric support (O-(2-aminoethyl)-cellulose) for probe-bound comparison.
Supports calibration and time-course measurement behavior for H2O2 sensing, including described blank and photobleaching behavior.
Documented Applications
Determine or detect hydrogen peroxide in a sample.
Determine a concentration of hydrogen peroxide in a sample.
Detect the presence of hydrogen peroxide in a sample.
Fluorescence detection and determination of hydrogen peroxide (H2O2) concentration in a sample by contacting the sample with a compound of formula IA (or a salt), irradiating with electromagnetic radiation at a fluorophore-absorbing excitation wavelength, measuring emitted energy at an emission wavelength, and correlating the emitted intensity with H2O2 concentration.
Fluorescence detection of the presence of hydrogen peroxide in a sample by detecting emitted energy at an emission wavelength after irradiation at an excitation wavelength absorbed by the fluorophore, where emitted energy detection supports a conclusion that at least some hydrogen peroxide is present.
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