NL2030373B1 - Chemiluminescence technology for detecting hydroxyl radical - Google Patents

Abstract

The present disclosure discloses a new chemiluminescence technology for detecting a hydroxyl radical, which is a new chemiluminescence technology using a tobacco extract as a luminescent probe to detect hydroxyl radicals in acidic, neutral and alkaline media. The technology has the advantages of simple operation, high detection speed, and no susceptibility to interference from the pH of the media, and has a great application prospect in the technical field of detection of a hydroxyl radical.

Description

P903/NLpd

CHEMILUMINESCENCE TECHNOLOGY FOR DETECTING HYDROXYL RADICAL

TECHNICAL FIELD

The present disclosure relates to the technical field of chemiluminescence detection, and in particular relates to a chemi- luminescence technology for detecting a hydroxyl radical.

BACKGROUND ART

The hydroxyl radical (OH), as a very active reactive oxygen species, exists widely in nature and organisms. Because ‘OH has a very strong oxidizing ability, it plays an important role (such as

Fenton reaction, photocatalytic reaction, and photochemical reac- tion) in degradation of natural environmental pollutants. In or- ganisms, ‘OH can cause damage to biomacromolecules (such as DNA, proteins, and lipids), leading to many diseases. Therefore, there is great practical needs for :OH detection. However, the lifetime of ‘OH is very short (microsecond level), which makes it a tech- nical problem to accurately detect it. Currently, ‘OH detection methods mainly include electron spin resonance (ESR), fluorescence methods, etc. These methods have their own defects and shortcom- ings, such as cumbersome and time-consuming operation, high detec- tion cost, low sensitivity, and poor specificity, which cannot fully meet the detection needs of ‘OH.

Chemiluminescence technology has the advantages of simple op- eration, high detection speed, low cost, high sensitivity, etc., and has unique advantages in the detection of reactive oxygen spe- cies. However, the current chemiluminescence technology is rarely used in ‘OH detection, mainly because the existing probes have poor specificity for ‘OH detection and are susceptible to the in- terference from the pH of a system. Therefore, it is great scien- tific and practical significance to develop a new chemilumines- cence technology for ‘OH detection.

SUMMARY

The objective of the present disclosure is to provide a chem-

iluminescence technology for detecting a hydroxyl radical.

The chemiluminescence technology for detecting a hydroxyl radical provided by the present disclosure is to use a tobacco leaf extract as a luminescent probe to detect ‘OH in a system con- taining ‘OH.

The tobacco leaf extract may be prepared by a method includ- ing the following steps: adding tobacco leaves to a reagent, and conducting extraction to obtain the tobacco leaf extract.

The tobacco leaves may be tobacco leaves for Virginian-type cigarettes, such as Yunyan 87, Yunyan 85, K326, Honghua dajinyuan, and ZhongyanlOQ.

The method may further include steps of drying and grinding the tobacco leaves into a powder before the tobacco leaves are added to the reagent.

A moisture content after drying may be less than or equal to 3%; a drying temperature is 40-60°C, and a drying time is 2-4 h; and a particle size of the powder is 20-100 mesh.

The reagent may be at least one from the group consisting of methanol, water, ethyl acetate, ethanol, acetone and chloroform.

The mass of the tobacco leaves to a volume of the reagent may be 1.0 g: (10-100) mL.

The extraction may be ultrasonic extraction.

The ultrasonic extraction may be conducted at a temperature of less than 70°C and a power of 200 W-500 W for 10 min-30 min.

The method may further include steps of filtering the ob- tained extract and collecting the filtrate after the extraction.

The filtration may use a filter membrane.

A pore size of the filter membrane may be less than 420 um.

The system containing ‘OH may be an acidic, neutral or alka- line system, and may have a pH of 0-14.

The system containing ‘OH may be a Fenton system, a Fenton- like system, an optical Fenton system, an electric Fenton system, a peroxidase catalytic system, a nanomaterial/H.O, "peroxidase- like" catalytic system and a halecguinone/H,0. system.

Substances replacing Fe(II) ions in the Fenton-like system may be Fe(III), iron-bearing minerals, and other transition metal ions such as Co, Cd, Cu, Ag, Mn, and Ni.

In the peroxidase catalytic system, peroxidase may include catalase and horseradish peroxidase.

In the nanomaterial/H:0: "peroxidase-1like" catalytic system, the nanomaterial may include iron-based nanomaterials (such as

Fe30:, Fe. 0;, FeS, BiFeO:, and CoFe,04), carbon-based nanomaterials (such as graphene oxide, and carbon nanotubes), and precious met- als and alloy nanoparticles (such as Au, AgPt, AgAu, and AgPd) of the precious metals.

In the haloquinone/H.0; system, the haloquinone may be tetra- chlorobenzoquinone (TCBQ), tetrabromobenzoguinone (TBrBQ), tetra- chlorohydroquinone (TCHQ), tetrabromohydroguinone (TBrHQ), O-TCB9,

Z-chlorobenzoguinone (2-CBQ), 2,3-dichlorobenzoquinone (2,3-DCB9}, 2,5-DCBQ, and 2,6-DCBOQ.

The system containing ‘OH may specifically be: a Fenton sys- tem (FeS0,/H.0,), a horseradish peroxidase (HRP) /H.0, system and/or a gold nanoparticle/H.0, system.

Use of the tobacco leaf extract in the detection of a hydrox- yl radical also belongs to the protection scope of the present disclosure.

The present disclosure successfully develops a new chemilumi- nescence technology which uses the tobacco leaf extract as a lumi- nescent probe to detect ‘OH in acidic, neutral and alkaline solu- tions. The technology has a great application prospect in ‘OH de- tection in various media.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows chemiluminescence kinetic curves of a tobacco leaf extract (16.3 mg/mL) and a FeSO,s (0.1 mmol/L) /H:0: (1.0 mmol/L) system under acidic (a), neutral (b) and alkaline {(¢) con- ditions before and after thiourea (1.0 mol/L) is added, where (a):

H.SO, (0.1 mol/L), (b): HO, and (c): NaOH (1.0x10° mol/L).

FIG. 2 shows chemiluminescence kinetic curves of the tobacco leaf extract (16.3 mg/mL) and a HRP (1.0 mg/mL)/H.0, (0.1 mol/L) system under acidic (a), neutral (b) and alkaline (c) conditions before and after thiourea (1.0 mol/L) is added, where (a): H.SO. (1.0x107* mol/L), (b): H;0, and (c): NaOH (0.01 mol/L).

FIG. 3 shows chemiluminescence kinetic curves of the tobacco leaf extract (16.3 mg/mL) and a gold nanoparticle (5 pg/mL) /H:0: (0.1 mol/L) system under acidic (a), neutral (b) and alkaline (c) conditions before and after thiourea (1.0 mol/L} is added, where (a): H,SO, (1.0%x107° mol/L), (b): H.0, and (c): NaOH (1.0x107* mol/L).

FIG. 4 shows chemiluminescence kinetic curves of the tobacco leaf extract (16.3 mg/mL) and systems containing TCBQ of different concentrations (1 mM, 2 mM, 5 mM and 10 mM) and H;0: (0.1 mol/L) under acidic (a), neutral (b) and alkaline {c) conditions, wherein (a): H-SO4 (1.0x107° mol/L), (b): H.0, and (c): NaOH (1.0x107* mol/L).

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present disclosure will be described below through spe- cific examples, but the present disclosure is not limited thereto.

The experimental methods used in the following examples are conventional methods unless otherwise specified; the reagents, ma- terials, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

A luminescence kinetic curve in the following examples is ob- tained by a static injection method in an ultra-weak luminescence analyzer (BPCL-GP15-TGC), and the negative high voltage is -1000

V.

A method for preparing a tobacco leaf extract in the follow- ing examples, unless otherwise specified, is conducted in an open system under natural light conditions and at room temperature (25°C).

Tobacco leaves for Virginian-type cigarettes (cured tobacco leaves) in the following examples can be prepared by a "three- stage (yellowing, color fixation, and killing-out)" curing pro- cess. Specific steps are as follows: In a yellowing stage, fresh ripe tobacco leaves {such as Yunyan 87, Yunyan 85, K326, Honghua dajinyuan, and Zhongyan 100) collected in the field are tied and loaded in a curing barn. After ignition, a temperature of the cur- ing barn is increased to 38°C at a rate of 1°C per hour, and a wet bulb temperature is kept 1.5°C lower than a dry bulb temperature, until more than 80% of the tobacco leaves at the bottom turn to about 80% yellow. Then the temperature of the curing barn is in- creased to 42°C, the wet bulb temperature is kept at 37°C, and moisture is properly removed to ensure that the tobacco leaves turn yellow and soft. In a color fixation stage, an amount of 5 moisture removal is increased, the temperature of the curing barn is increased to 55°C at a rate of 1°C per 2 hours, and the wet bulb temperature is slowly increased to and kept at 38°C to dry the leaves and fix a yellow color. In a killing-out stage, the temperature of the curing barn is increased to 69°C at a rate of 1°C per hour, the wet bulb temperature is kept at about 42°C, and curing is stopped after ensuring that the leaves are fixed in a yellow color and dried.

A tobacco leaf extract is prepared following steps below:

Main veins of the tobacco leaves for Virginian-type ciga- rettes (cured tobacco leaves) are removed, and the tobacco leaves are cut into slices and dried in an oven at 60°C for 2 hours. At this time, a moisture content of the tobacco leaves is about 3%.

After being taken out, the tobacco leaves are quickly ground for a duration of 2 min. After being ground, a tobacco leaf powder is sieved (with a 40-mesh screen). After sieving, the powder is quickly put into a clean and dry jar and sealed for later use. 1.0 g of the tobacco leaf powder is weighed, 40 mL of methanol is add- ed, and ultrasonic treatment (with a power 400 W) is conducted for 20 min. A mixture is filtered through a 0.45 pm organic phase fil- ter membrane, and finally a filtrate is collected to obtain the tobacco leaf extract.

Example

Firstly, chemiluminescence phenomena of a tobacco leaf ex- tract and the following three kinds of ‘OH-producing systems in- volving H:0, were investigated. (1) Fenton system (FeSO./H;0:;): A Fenton system is a strong oxidizing system, which is currently widely used in advanced oxi- dation technology (AOP), where ‘OH is a main oxide species, so de- tection of the ‘OH under different pH conditions is helpful to better understand a mechanism of action of a Fenton reaction in

AOP. For this reason, we investigated chemiluminescence signals of a tobacco leaf extract and a FeS0;/H;0: system.

Under acidic (a), neutral (b) and alkaline (c¢) conditions, the tobacco leaf extract (16.3 mg/mL) was used as a luminescent probe to detect ‘OH in a FeS0, (0.1 mmol/L) /H;0: (1.0 mmol/L) sys- tem and obtain chemiluminescence kinetic curves before and after thiourea (1.0 mol/L} was added, where (a): H:S04 (0.1 mol/L}, (b):

H.0, and (c): NaOH (1.0x107° mol/L).

As shown in FIG. 1, the tobacco leaf extract and FeS0,/H.0; could produce chemiluminescence signals under acidic, neutral and alkaline conditions. And the luminescence signals disappeared af- ter a ‘OH quencher thiourea was added, indicating that the ‘OH in the Fenton system could induce the tobacco extract to produce chemiluminescence signals regardless of the acidic, neutral and alkaline conditions. Therefore, the present disclosure can detect the ‘OH in the Fenton system under acidic, neutral and alkaline conditions by using the tobacco extract as the luminescent probe. (2) Horseradish peroxidase (HRP) /H,0, system: H;0. is decom- posed into strong oxidizing species such as ‘OH under catalysis of some peroxidases (such as HRP). At present, chemiluminescence bio- chemical detection based on the HRP/#H:0: enzyme catalytic system (such as ELISA) is widely used. However, the pH of some detection systems is neutral or even acidic, and luminescence reactions of most existing luminescent probes are conducted in alkaline or even strong alkaline environments, which limits the use of the chemilu- minescence technology. For this reason, we investigated a chemilu- minescence phenomenon of the tobacco extract and HRP/H:C: system under acidic, neutral and alkaline conditions.

Under acidic (a), neutral (bk) and alkaline (¢) conditions, the tobacco leaf extract (16.3 mg/mL) was used as a luminescent probe to detect ‘OH in an HRP (1.0 mg/mL) /H:0: (0.1 mol/L) system and obtain chemiluminescence kinetic curves before and after thio- urea (1.0 mol/L) was added, where (a): H.SO, (1.0x10% mol/L), (b):

H.0, and {c): NaOH (0.0lmol/L).

As shown in FIG. 2, the tobacco extract and HRP/H:0: system could produce chemiluminescence signals under acidic, neutral and alkaline conditions. And when the ‘OH quencher thiourea was added, the luminescence signals disappeared, indicating that the tobacco extract had a chemiluminescence reaction with ‘OH. Based on this,

we can establish a chemiluminescence system of tobacco extract-

HRP/H:0;: for analysis and detection by using the chemiluminescence phenomenon of the tobacco extract with the ‘OH in the HRP/H:0: en- zymatic catalytic system under acidic, neutral and alkaline condi- tions. (3) Gold nanoparticle/H:0; system: Studies have shown that some nanomaterials (such as nano-Fe;0,, and nanogold) have "peroxi- dase-like" properties on a surface, which can catalyze decomposi- tion of H,0. to produce ‘OH and other highly active species. Here we investigated the chemiluminescence phenomenon of the tobacco extract and nanogold/H:0: system under acidic, neutral and alkaline conditions.

Under acidic (a), neutral (b) and alkaline (c¢) conditions, the tobacco leaf extract (16.3 mg/mL) was used as a luminescent probe to detect ‘OH in a gold nanoparticle (5 ug/mL)/H,0, (0.1 mol/L) system and obtain chemiluminescence kinetic curves before and after thiourea (1.0 mol/L) was added, where (a): H:S04 (1.0x107° mol/L), (b): HO, and (c¢): NaOH (1.0x107* mol/L).

As shown in FIG. 3, the tobacco extract and nancgold/H:0; sys- tem could produce chemiluminescence signals under acidic, neutral and alkaline conditions. And when thiourea was added, the lumines- cence signals disappeared completely, which proved that the ‘OH produced under catalysis of a nanogold surface participated in a chemiluminescence reaction of the tobacco extract. Therefore, this technology can be used to study a "peroxidase-like" effect of sur- faces of various nanomaterials, and has a great application pro- spect.

The above chemiluminescence experiments of the tobacco ex- tracts and three :CH-producing oxidation systems show that the to- bacco extracts and ‘OH can undergo chemiluminescence reactions un- der acidic, neutral and alkaline conditions, thereby laying a sol- id theoretical foundation for detection of ‘OH in media with dif- ferent pH values. (4) In addition, studies have shown that TCBQ can react with

H:0, to produce ‘OH. In order to further verify feasibility of the present disclosure for detecting ‘OH, we tested the :OH produced in a TCBQ system under acidic, neutral and alkaline conditions.

Since ‘OH has a short life span and cannot be quantified with a standard curve, we conducted semi-quantitative detection by chang- ing a concentration of TCBQ in the system to produce different amounts of ‘OH.

Under acidic (a), neutral (b) and alkaline (c¢) conditions, the tobacco leaf extract (16.3 mg/mL) was used as a luminescent probe to detect ‘OH in systems containing TCBQ of different con- centrations (1 mM, 2 mM, 5 mM, and 10 mM) and H,0, (0.1 mol/L) to obtain chemiluminescence kinetic curves, where (a): HSO, (1.0x107° mol/L), (b): HO, and (c): NaOH {1.0x107% mol/L).

As shown in FIG. 4, the tobacco extract and TCBQ/H.0. could produce chemiluminescence signals under acidic, neutral and alka- line conditions, and a luminescence intensity increased linearly with an increase in a TCBQ concentration (R,=0.99). This indicates that there is a linear relationship between the luminescence in- tensity of the tobacco extract and an amount of ‘OH regardless of the acidic, neutral or alkaline conditions, so that semi- quantitative detection of ‘OH can be conducted under different pH conditions.

Claims (7)

CONCLUSIONS 1. Chemiluminescence technology to detect a hydroxyl radical, using a tobacco leaf extract as a luminescence probe to detect 'OH in a system containing 'OH. The technology according to claim 1, wherein the tobacco leaf extract is prepared by a method comprising the steps of: adding tobacco leaves to a reagent and performing extraction to obtain the tobacco leaf extract. The technology of claim 2, wherein the tobacco leaves are tobacco leaves for Virginian-type cigarettes; the reagent is at least one of the group consisting of methanol, water, ethyl acetate, ethanol, acetone and chloroform; the mass of the tobacco leaves to a volume of the reagent is 1.0 g : (10 - 100) ml; the extraction is ultrasonic extraction; and the ultrasonic extraction is performed at a temperature of less than 70 °C and a power of 200 W - 500 W for 10 min - 30 min. The technology according to any one of claims 1 - 3, wherein the system containing 'OH is an acidic, neutral or alkaline system and has a pH of 0 - 14. The technology according to any one of claims 1 to 4, wherein the system containing 'OH is a Fenton system, a Fenton-like system, an optical Fenton system, an electrical Fenton system, a peroxidase catalytic system, is a nanomaterial/H 2 O 2 . "peroxidase-like" catalytic system or a haloquinone/H 2 O 2 system. The technology according to any one of claims 1 to 4, wherein when a system under test contains a hydroxyl radical, after the tobacco leaf extract has been added, a chemiluminescence phenomenon sel occurs; and when a system under test does not contain a hydroxyl radical, no chemiluminescent phenomenon occurs after the tobacco leaf extract is added. 7. Use of a tobacco leaf extract in the detection of a hydroxyl radical.