Chemiluminescent composition and method of regulating chemiluminescence

Abstract

The present application provides a chemiluminescent composition comprising (i) a luminophore compound; (ii) a metal ion; (iii) H2O2; and (iv) a carbonate-based buffer; the composition can achieve a significant increase in luminescent intensity and a significant increase in luminescent duration, and can further use a luminescent time adjusting agent to flexibly, efficiently and accurately adjust the starting time of chemiluminescence. The present application also provides a method for adjusting the starting time of chemiluminescence using the chemiluminescent composition and a method for generating a chemiluminescent pattern or multicolor chemiluminescence.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of chemiluminescence detection, and in particular, the present application provides chemiluminescence compositions and methods for adjusting the luminescence time, luminescence color and forming chemiluminescence patterns using the compositions. BACKGROUND

[0002] Chemiluminescence (CL) is a phenomenon of light emission accompanied by chemical reactions, which has a wide range of applications in cold light sources, biological analysis, biosensing, reporter genes, bioimaging, biological mapping and other fields. However, the CL technology reported so far still has some significant defects to be solved, for example, chemiluminescence usually needs to be carried out under alkaline conditions, which will greatly limit its application range; the light intensity generated is low, which cannot be effectively observed or identified in some applications, and cannot meet the related technical requirements; most known CL reactions are flash-type emission, after mixing the components, the CL reaction will immediately produce light emission, and the light emission process is too fast, the light emission time is extremely short, often only in seconds, minutes. Precise control of reaction time is a very difficult work, which limits the application of CL in biological analysis, biosensing and bioimaging. For example, if the CL reagent is mixed with the co-reactant manually to initiate the CL reaction, the reproducibility is poor, which seriously affects the precision and accuracy of chemiluminescence analysis. Therefore, automatic injectors and fluid dynamic injection techniques are often used in chemiluminescence analysis to control the occurrence of CL reaction, so as to improve the reproducibility of the analysis. Recently, microfluidic-assisted CL systems have also developed rapidly, which include pre-designed channels that can integrate multiple analysis steps and control the flow of small amounts of sample and co-reactants into the detection area in an automated manner. Although these technologies try to use some automated means to control the location and timing of CL light emission, these automated means need to use complex devices without exception, and the backflow and diffusion in the liquid mixing process also cannot be avoided. In addition, most detection techniques need to analyze samples one by one, and it is difficult to record multiple CL signals at the same time to realize one-time detection of multiple samples.

[0003] Therefore, researchers in the field are eager to develop a new CL technology that can achieve extremely high light intensity and non-instantaneous light emission under neutral conditions, and further hope to effectively adjust the light emission starting time and light emission color of CL, so as to realize controllable light emission curve, light emission pattern or multiple light emission colors. So far, the prior art has not reported any new progress in technology that can achieve the above effects. SUMMARY

[0004] In view of the above, the inventors of the present application have conducted intensive research and successfully developed a novel chemiluminescent composition, which achieves extremely high luminescent intensity and non-transient luminescence under neutral conditions through the combination of a carbonate-based buffer and metal ions, and further developed a novel technique for accurately and simply regulating the starting time of CL, and in addition, can realize complex CL patterns through the starting time regulation and the multiple color luminescence brought by fluorescence resonance energy transfer using a luminescence starting time regulator. The technique of the present application can at least achieve the following advantages: simple operation, neutral CL conditions, low biological toxicity, high luminescent intensity, prolonged luminescent time, controllable luminescent starting time, and can be very well applied to various purposes such as biological sample analysis and imaging, used to design novel CL systems and detection devices, and can be used for signal communication and encryption technology.

[0005] According to a first aspect of the present application, there is provided a chemiluminescent composition comprising:

[0006] (i) a luminophore compound;

[0007] (ii) metal ions, the metal ions comprising: Mn 2+ , Co 2+ , Cr 3+ , Fe 2+ , AuCl4 - , Fe 3+ , Ce 3+ , Cu 2+ , Ni 2 + , Pb 2+ , and any combination thereof;

[0008] (iii) H2O2; and

[0009] (iv) a carbonate-based buffer;

[0010] The chemiluminescent composition is in an aqueous solution or loaded on the surface of a solid carrier.

[0011] According to an embodiment of the first aspect of the present application, the composition further comprises: (v) a luminescence starting time regulator selected from the group consisting of: thiourea compounds, C4-C 16 sugar alcohols, C3-C 12 alkyl mono-, di- or triols, C6-C 16 aromatic carboxylic acids, carboxylic anhydrides, carboxylic esters or lactones, sulfone compounds, sulfoxide compounds, piperidine compounds, C6-C 16 aromatic amine compounds, benzoquinone compounds, superoxide dismutase (SOD), C6-C 16Aromatic hydrocarbons, phenylnitrone compounds, C6-C6 groups containing one to four hydroxyl groups and at least one alkenyl group. 16 Carboxylic acids, carboxylic anhydrides, carboxylic esters or lactones, C6-C containing one to four hydroxyl groups and at least one alkenyl group. 16 Cycloalkyl carboxylic acids, carboxylic anhydrides, carboxylic esters or lactones, C6-C containing one to four hydroxyl groups 16 Aromatic carboxylic acids, carboxylic anhydrides, carboxylic esters or lactones, C6-C containing one to six hydroxyl groups. 16 Phenolic compounds, C6-C6 compounds containing one to six hydroxyl groups. 16 Aromatic amines, amino acids, peptides, and any combination thereof.

[0012] According to another embodiment of the first aspect of the present invention, the luminescence onset time modifier is selected from: thiourea, ethylene thiourea, aminothiourea, 2-methyl-3-aminothiourea, mannitol, tert-butanol, isopropanol, benzoic acid, dimethyl sulfoxide (DMSO), tetramethylpiperidine (TEMPO), diphenylamine, p-benzoquinone, superoxide dismutase (SOD), 1,1-stilbene, 2,6-di-tert-butyl-4-methylphenol (BHT), tert-butylbenzyl nitrate. Ketones (PBN), 5,5-dimethyl-1-pyrrolino-N-oxide (DMPO), ascorbic acid, isoascorbic acid, ascorbic acid phosphate, ascorbate palmitate, ascorbate glucoside, gallic acid, pyrogallic acid, catechol, resorcinol, hydroquinone, pyrogallol, pyrogallol, pyrogallol, pyrogallol, tetraphenylethylene, o-phenylenediamine, p-phenylenediamine, m-phenylenediamine, catechin, dopamine, cysteine, glutathione, and any combination thereof. Preferred ingredients include thiourea, ethylene thiourea, aminothiourea, 2-methyl-3-aminothiourea, ascorbic acid, gallic acid, pyrogallol, catechol, hydroquinone, o-phenylenediamine, p-phenylenediamine, catechin, dopamine, cysteine, glutathione, and any combination thereof.

[0013] According to one embodiment of the first aspect of the invention, when the chemiluminescent composition is in an aqueous solution, the chemiluminescent composition further comprises (v) a carbonate-based buffer.

[0014] According to another embodiment of the first aspect of the present application, the light emitting compound includes an organic light emitter compound and an inorganic light emitter compound, the organic light emitter compound includes fluorescein-based (which can include fluorescein, erythrosin, eosin, phloxine) light emitters, oxadiazole-based light emitters, triazole-based light emitters, rhodamine-based light emitters, coumarin-based light emitters, 1,8-naphthalimide-based light emitters, pyrazoline-based light emitters, triphenylamine-based light emitters, polytriphenylamine-based light emitters, porphyrin-based light emitters, poly-porphyrin-based light emitters, carbazole-based light emitters, poly-carbazole-based light emitters, pyrazine-based light emitters, thiazole-based light emitters, perylene-based light emitters, polyphenyl-based light emitters, polythiophene-based light emitters, polyfluorene-based light emitters, polytriphenylamine-based light emitters, polypyrrole-based light emitters, poly-porphyrin[8]-based light emitters, boron fluoride complexed dipyrromethene (BODIPY)-based light emitters, quantum dot-based light emitters, cypridina luciferin (CLA)-based light emitters, coelenterazine fluorescein (CTZ)-based light emitters, aequorin-based light emitters, peroxyoxalate-based light emitters, adamantane-based light emitters, dioxetane-based light emitters, 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-A]pyrazin-3-one hydrochloride (MCLA)-based light emitters, lucigen, acridinyl ester-based light emitters, lophine-based light emitters, o-phenanthroline-based light emitters, gallic acid-based light emitters, N-bromosuccinimide light emitters, pyridine nucleotide flavin-based light emitters, a compound represented by formula (1) or a salt thereof, and a compound represented by formula (2) or a salt thereof,

[0015]

[0016] In formula (1), the A ring represents a C6-C 14 aromatic ring,

[0017] R1and R2independently represent hydrogen, straight chain or branched (C1-C 30 )alkyl substituted with an amino group at a terminal or unsubstituted, provided that the NR1R2has at least one NH2terminal;

[0018]

[0019] In formula (2), R3, R4, and R5independently represent C1-C6alkyl, C1-C6alkoxy, C6-C 16 aryl, C6-C 16 aryloxy, F, Cl, Br, I, -NH2, -OH, -NO2, -COOH, -C(=O)-C1-C6alkyl, and any combination thereof;

[0020] The inorganic luminescent compound includes a ruthenium ion complex luminescent, an iridium ion complex luminescent, an inorganic salt luminescent (which includes potassium ferricyanide, permanganate, dichromate, bromate, iodate, periodate, cerium (IV) salt, sulfite, nitrogen carbide), an inorganic gas luminescent (which includes sulfur dioxide, carbon dioxide), or any combination thereof.

[0021] According to another embodiment of the first aspect of the present application, the metal ion includes: Mn 2+ , Co 2+ , and combinations thereof. More preferably, it includes Mn 2+ .

[0022] According to another embodiment of the first aspect of the present application, the carbonate-based buffer is an aqueous solution containing carbon dioxide, sodium carbonate, and sodium bicarbonate, or an aqueous solution containing carbon dioxide and sodium bicarbonate, or an aqueous solution containing sodium bicarbonate.

[0023] The second aspect of the present application provides a method for adjusting the initiation time of chemiluminescence, which includes adding a luminescence initiation time adjusting agent to the chemiluminescent composition of the present application, the luminescence initiation time adjusting agent being selected from the group consisting of: thiourea compounds, C4-C 16 sugar alcohols, C3-C 12 alkyl mono-, di-, or triols, C6-C 16 aromatic carboxylic acids, carboxylic anhydrides, carboxylic esters, or lactones, sulfone compounds, sulfoxide compounds, piperidine compounds, C6-C 16 aromatic amine compounds, benzoquinone compounds, superoxide dismutase (SOD), C6-C 16 aromatic hydrocarbons, phenylnitrone compounds, C6-C 16 carboxylic acids, carboxylic anhydrides, carboxylic esters, or lactones, C6-C 16 cycloalkyl carboxylic acids, carboxylic anhydrides, carboxylic esters, or lactones, C6-C 16 aromatic carboxylic acids, carboxylic anhydrides, carboxylic esters, or lactones, C6-C 16 phenolic compounds, C6-C 16 aromatic amine compounds, amino acids, peptides, and any combination thereof.

[0024] According to another embodiment of the second aspect of the present invention, the luminescence onset time modifier is selected from: thiourea, ethylene thiourea, aminothiourea, 2-methyl-3-aminothiourea, mannitol, tert-butanol, isopropanol, benzoic acid, dimethyl sulfoxide (DMSO), tetramethylpiperidine (TEMPO), diphenylamine, p-benzoquinone, superoxide dismutase (SOD), 1,1-stilbene, 2,6-di-tert-butyl-4-methylphenol (BHT), tert-butylnitrone (PBN), 5,5-dimethyl-1-pyrrolidone-N-oxide (DMPO), and any combination thereof. Preferred ingredients include thiourea, ethylene thiourea, 2-methyl-3-aminothiourea, ascorbic acid, isoascorbic acid, ascorbic acid phosphate, ascorbate palmitate, ascorbate glucoside, gallic acid, pyrogallol, catechol, resorcinol, hydroquinone, pyrogallol, pyrogallol, pyrogallol, pyrogallol, tetraphenylethylene, o-phenylenediamine, p-phenylenediamine, m-phenylenediamine, catechin, dopamine, cysteine, glutathione, and any combination thereof.

[0025] According to another embodiment of the second aspect of the present invention, by adding different amounts or different types of the luminescence onset time modifier to multiple portions of the chemiluminescent composition, patterns composed of different chemiluminescences are generated.

[0026] According to another embodiment of the second aspect of the present invention, by adding the luminescence onset time modifier to multiple portions of the chemiluminescent composition containing different luminescent compounds, a pattern composed of different chemiluminescences is generated.

[0027] A third aspect of the invention provides a method for generating chemiluminescence of multiple colors, the method comprising using a chemiluminescent composition of the invention, the composition comprising multiple luminescent compounds, and fluorescence resonance energy transfer occurring between the multiple luminescent compounds, thereby generating chemiluminescence of multiple colors. Attached Figure Description

[0028] The following paragraphs discuss various embodiments of the invention in conjunction with the accompanying drawings. However, it should be noted that the embodiments shown in the drawings and described in detail below are merely some preferred embodiments of the invention, and the scope of protection of the invention is defined by the claims, and not limited to these preferred embodiments.

[0029] Figure 1A and Figure 1B The CL curves under different metal ion concentrations are shown according to some embodiments of the present invention.

[0030] Figure 2 The CL curves are shown based on a comparative example, under conditions where metal ions are not used.

[0031] Figure 3 CL curves under conditions using other buffers instead of carbonate buffer are shown according to one comparative example.

[0032] Figure 4A and Figure 4B CL photos obtained under conditions using different metal ions are shown according to one embodiment, respectively.

[0033] Figure 5A A graph showing the relationship between thiourea concentration and CL luminescence initiation time according to one embodiment of the present application is shown, Figure 5B A graph showing the relationship between the logarithm of thiourea concentration and the logarithm of CL luminescence delay time is shown.

[0034] Figure 6A and Figure 6B CL photos obtained under conditions using different thiourea concentrations are shown according to one embodiment, respectively.

[0035] Figure 7 CL photos obtained under conditions using different thiourea concentrations and the corresponding codes are shown according to one embodiment.

[0036] Figures 8A to 8C Adjustment of CL luminescence initiation time achieved by using ethylene thiourea, aminothiourea and 2-methyl-3-aminothiourea, respectively, according to one embodiment of the present application is shown.

[0037] Figures 9A to 9B Adjustment of CL luminescence initiation time achieved by using thiourea for different chemiluminescent organic molecules according to one embodiment of the present application is shown.

[0038] Figures 10A to 10C Multicolor luminescence effects achieved by FRET using multiple organic luminescent compounds according to some embodiments of the present application are shown. Figure 10D CL photos showing color luminescence gradient based on FRET are shown.

[0039] Figures 11A to 11C Patterns of multicolor CL luminescence continuous variation achieved by FRET using multiple organic luminescent compounds according to some embodiments of the present application are shown.

[0040] Figure 12 Adjustment of chemiluminescence initiation time of CL system loaded on solid support under the action of thiourea according to one embodiment of the present application is shown.

[0041] Figures 13A to 13FThe modulation of the chemiluminescence onset time of the CL system supported on a solid support by catechin, hydroquinone and p-phenylenediamine is shown according to further embodiments of the present application.

[0042] Figures 14A to 14C The modulation of the chemiluminescence onset time of the CL system supported on a solid support by catechin, hydroquinone and p-phenylenediamine is shown according to further embodiments of the present application.

[0043] Figure 15A and Figure 15B The modulation of the chemiluminescence onset time of the CL system supported on a solid support by catechin, hydroquinone and p-phenylenediamine is shown according to further embodiments of the present application. DETAILED DESCRIPTION

[0044] The ranges disclosed herein are presented in terms of specific lower and upper limits. There can be one or more lower limits and one or more upper limits. A given range is defined by selecting a lower limit and an upper limit. The selected lower and upper limits define the boundaries of a particular range. All ranges defined in this manner are inclusive and combinable, i.e., any lower limit can be combined with any upper limit to form a range. For example, where ranges of 60-120 and 80-110 are listed for a particular parameter, it is understood that ranges of 60-110 and 80-120 are also contemplated. Furthermore, if a minimum range value of 1 and 2 are listed, and if a maximum range value of 3, 4 and 5 are listed, then the following ranges are all contemplated: 1-3, 1-4, 1-5, 2-3, 2-4 and 2-5.

[0045] In the present application, unless otherwise stated, a numerical range "a-b" indicates a shorthand way of describing each and every individual number that is within the range, wherein a and b are both real numbers. For example, the numerical range "0-5" indicates that all real numbers between "0-5" have been listed herein, and "0-5" is merely a shorthand way of describing those numerical combinations.

[0046] In the present application, all embodiments and preferred embodiments mentioned herein can be combined with each other to form new technical solutions, if not specifically stated otherwise.

[0047] In the present application, all technical features and preferred features mentioned herein can be combined with each other to form new technical solutions, if not specifically stated otherwise.

[0048] In the present application, if not otherwise specified, all the steps mentioned herein can be performed in sequence or randomly, but preferably in sequence. For example, the method comprises steps (a) and (b) means that the method can comprise steps (a) and (b) performed in sequence, or steps (b) and (a) performed in sequence. For example, the method mentioned herein can further comprise step (c) means that step (c) can be added to the method in any order, for example, the method can comprise steps (a), (b) and (c), or steps (a), (c) and (b), or steps (c), (a) and (b), etc.

[0049] In the present application, if not otherwise specified, "comprising" mentioned herein means open-ended, and can also be closed-ended. For example, "comprising" can mean that it can further comprise other components not listed, or can only comprise the listed components.

[0050] According to one embodiment of the present application, the chemiluminescent composition described in the present application comprises: (i) a luminophore compound; (ii) a metal ion, the metal ion comprising: Mn 2+ , Co 2+ , Cr 3+ , Fe 2+ , AuCl 4- , Fe 3+ , Ce 3+ , Cu 2+ , Ni 2+ , Pb 2 + , and any combination thereof; (iii) H2O2; and (iv) a carbonate-based buffer; the chemiluminescent composition is in an aqueous solution or at least partially loaded on the surface of a solid carrier. Through this specially designed composition, the increase of luminescence intensity and the prolongation of luminescence duration can be effectively achieved.

[0051] According to one embodiment of the present application, the chemiluminescent composition is in an aqueous solution, in which case all components of the chemiluminescent composition, including the (iv) carbonate-based buffer, are in an aqueous solution. According to another embodiment of the present application, when the chemiluminescent composition is at least partially loaded on the surface of a solid support, for this embodiment, by "at least partially loaded on the surface of a solid support" is meant that at least a portion of components (i) to (iv) of the chemiluminescent composition are loaded on the surface of a solid support. Preferably, the organic or inorganic luminophore compound and the metal ion in the chemiluminescent composition are loaded on the surface of a solid support, while the (iii) H2O2 and the (iv) carbonate-based buffer are added to the organic or inorganic luminophore compound and metal ion components loaded on the surface of a solid support in the form of an aqueous solution.

[0052] According to another embodiment of the present application, whether the chemiluminescent composition is in an aqueous solution or at least partially loaded on the surface of a solid support, the chemiluminescent composition can further comprise (v) a luminescence initiation time adjusting agent, by selecting a suitable luminescence initiation time adjusting agent and its concentration, the CL luminescence initiation time can be precisely controlled. According to one embodiment of the present application, the luminescence initiation time adjusting agent can be loaded on the surface of a solid support. According to another embodiment of the present application, the luminescence initiation time adjusting agent can be in an aqueous solution.

[0053] According to one embodiment of the present application, the organic luminophore compound includes an organic luminophore compound commonly used in the CL field, examples of which include, but are not limited to, fluorescein-based luminophores (which include fluorescein, erythrosin, eosin, phloxine), oxadiazole-based luminophores, triazole-based luminophores, rhodamine-based luminophores, coumarin-based luminophores, 1,8-naphthalimide-based luminophores, pyrazoline-based luminophores, triphenylamine-based luminophores, polytriphenylamine-based luminophores, porphyrin-based luminophores, poly-porphyrin-based luminophores, carbazole-based luminophores, polycarbazole-based luminophores, pyrazine-based luminophores, thiazole-based luminophores, perylene-based luminophores, polyphenyl-based luminophores, polythiophene-based luminophores, polyfluorene-based luminophores, polytriphenylamine-based luminophores, polypyrrole-based luminophores, poly-porphyrin[8]-based luminophores, boron fluoride complexed dipyrromethene (BODIPY)-based luminophores, quantum dot-based luminophores, cypridina luciferin (CLA)-based luminophores, coelenterazine fluorescein (CTZ)-based luminophores, aequorin-based luminophores, peroxyoxalate-based luminophores, adamantane-based luminophores, dioxetane-based luminophores, 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-A]pyrazin-3-one hydrochloride (MCLA)-based luminophores, lucigen, acridinyl ester-based luminophores, lophine-based luminophores, o-phenanthroline-based luminophores, gallic acid-based luminophores, N-bromosuccinimide luminophores, pyridine nucleotide flavin-based luminophores, mixtures of the above materials, and the like. According to one embodiment of the present application, the inorganic luminophore compound includes a ruthenium ion complex luminophore, a yttrium ion complex luminophore, an inorganic salt-based luminophore (which includes potassium ferricyanide, permanganate, dichromate, bromate, iodate, periodate, cerium (IV) salt, sulfite, nitrous carbon), an inorganic gas-based luminophore (which includes sulfur dioxide, carbon dioxide), or any combination thereof. According to one embodiment of the present application, two or more of the above luminophores can be used to form a complexed fluorescence resonance energy transfer (FRET) system, which comprises a donor luminophore and an acceptor luminophore, the emission spectrum of the energy donor luminophore at least partially overlapping the absorption spectrum of the energy acceptor luminophore. In the case of a luminophore chemiluminescent reaction, a portion of the energy of the donor luminophore is transferred to the acceptor luminophore, causing a change in the wavelength and intensity of the light emitted by the entire system. According to one embodiment of the present application, the complexed FRET system comprises one donor luminophore and one acceptor luminophore, whereby one step of FRET occurs within the system. According to one embodiment of the present application, the complexed FRET system comprises one donor luminophore and two or more acceptor luminophores, whereby two steps of FRET, three steps of FRET, or more steps of FRET occur within the system.According to one embodiment of the present application, the donor luminophore can include one or more of the following: a CLA-based luminophore, a peroxodisulfate-based luminophore, an adamantane-dioxetane-based luminophore, lucigen, an acridinium ester-based luminophore, a lophine-based luminophore, a phenanthroline-based luminophore, a gallic acid-based luminophore, a compound represented by Formula (1) or a salt thereof, and a compound represented by Formula (2) or a salt thereof. The acceptor luminophore can include one or more of the following: a fluorescein-based luminophore (which can include fluorescein, erythrosin, eosin, and phloxine), an oxadiazole-based luminophore, a triazole-based luminophore, a rhodamine-based luminophore, a coumarin-based luminophore, a 1,8-naphthalimide-based luminophore, a pyrazoline-based luminophore, a triphenylamine-based luminophore, a polytriphenylamine-based luminophore, a porphyrin-based luminophore, a poly-porphyrin-based luminophore, a carbazole-based luminophore, a polycarbazole-based luminophore, a pyrazine-based luminophore, a thiazole-based luminophore, a perylene-based luminophore, a polyphenyl-based luminophore, a polythiophene-based luminophore, a polyfluorene-based luminophore, a polytriphenylamine-based luminophore, a polypyrrole-based luminophore, a poly-porphyrin[8]-based luminophore, a boron fluoride complex dipyrrylmethane (BODIPY)-based luminophore, and a quantum dot-based luminophore.

[0054] According to one embodiment of the present application, the organic luminophore compound includes a compound represented by Formula (1) or Formula (2). According to another embodiment of the present application, a compound represented by Formula (1) or Formula (2) is combined with one or more of the above-mentioned acceptor luminophores to be used as a complex FRET system. According to another embodiment of the present application, in the compound of the general Formula (1), the A ring is a benzene ring or a naphthalene ring, and the -NR1R2 moiety is an amino group or an N-(amino C1-C6 alkylene) N-(C1-C6 alkyl) amino group; more preferably, the A ring is a benzene ring, and the -NR1R2 moiety is an amino group, an N-(4-amino butyl) N-(ethyl) amino group, or an (N-4-amino hexyl) (N-ethyl) amino group. According to another embodiment of the present application, it is particularly preferred that the compound of Formula (I) has a structure represented by the following formula:

[0055]

[0056] According to another embodiment of the present application, in the compound of the general Formula (2), R3 includes a C1-C6 alkyl group, a C1-C6 alkoxy group, a C6-C 16 aryl group, a C6-C 16 aryloxy group, F, Cl, Br, I, -NH2, -OH, -NO2, -COOH, -C(=O)-C1-C6 alkyl, and any combination thereof; preferably, R3 represents a C1-C3 alkyl group, a C1-C3 alkoxy group, or a C6-C 12 aryl group; more preferably, R3 represents a phenyl group, a benzyl group, or a tolyl group.

[0057] According to another embodiment of the present application, in the compound of the general formula (2), R4and R5each independently include C1-C6 alkyl, C1-C6 alkoxy, F, Cl, Br, I, -NH2, -OH, -NO2, and any combination thereof; preferably R4and R5each independently include C1-C3 alkyl, C1-C3 alkoxy, Cl, -NH2, -OH, -NO2, and any combination thereof; more preferably R4and R5each independently represent Cl or -NH2.

[0058] The above-mentioned organic luminophore compounds can be directly purchased from commercial channels or prepared according to literature methods. For example, the commercial product L012 is used in some embodiments of the present application, which corresponds to the compound of the formula (2) wherein R3is phenyl, R4is -NH2, and R5is Cl. However, the organic luminophore compounds that can be used in the present application are not limited thereto, and other organic luminophore compounds listed above can also be used in the present application and achieve the same or similar technical effects.

[0059] According to one embodiment of the present application, the content of the organic luminophore compound is 0.01-10.0 wt%, or 0.02-9.0 wt%, or 0.03-8.0 wt%, or 0.04-7.0 wt%, or 0.05-6.0 wt%, or 0.06-5.0 wt%, or 0.05-2.0 wt%, or 0.1%-1.5 wt%, or 0.2%-1.2 wt%, or 0.4%-1.0 wt%, or 0.5%-0.8 wt%, based on the total weight of the chemiluminescent composition.

[0060] According to one embodiment of the present application, the metal ions used in the chemiluminescent composition include: Mn 2+ , Co 2 + , Cr 3+ , Fe 2+ , AuCl4 - , Fe 3+ , Ce 3+ , Cu 2+ , Ni 2+ , Pb 2+ , and any combination thereof; preferably include Mn 2+ , Co 2+ , and combinations thereof; most preferably Mn 2+ . These metal ions can be added into the chemiluminescent composition of the present application in the form of conventional soluble salts, such as their corresponding sulfate, phosphate, chloride, nitrate, and the like.

[0061] According to one embodiment of the present application, the content of the cation is 0.01 to 5% by weight, or 0.02 to 4% by weight, or 0.03 to 3% by weight, or 0.04 to 2% by weight, or 0.05 to 1% by weight, or 0.06 to 0.8% by weight, or 0.07 to 0.5% by weight, or 0.08 to 0.2% by weight, based on the total weight of the chemiluminescent composition, based on the solid weight of the salt of the cation.

[0062] According to one embodiment of the present application, the carbonate-based buffer used in the chemiluminescent composition of the present application comprises a carbonate, a bicarbonate, or a mixture thereof, or an aqueous solution containing carbon dioxide and bicarbonate, or an aqueous solution containing carbon dioxide, carbonate, and bicarbonate, preferably wherein the carbon dioxide is saturated in the aqueous solution. According to one embodiment of the present application, the carbonate and bicarbonate each comprise a conventional soluble salt, such as an ammonium salt, a lithium salt, a sodium salt, a potassium salt, an organic amine salt, and the like, preferably a sodium salt or a potassium salt.

[0063] According to another embodiment of the present application, the pH of the chemiluminescent composition of the present application is 6.5 to 8.5, such as 7.0 to 8.0, or 7.2 to 7.5, by employing the carbonate-based buffer. The carbonate in the buffer system is necessarily a mixture of carbonate / bicarbonate in the above pH range, and the above-mentioned pH values can be obtained by employing appropriate ratios of carbonate / bicarbonate / carbon dioxide (carbonic acid) in the solution system.

[0064] According to one embodiment of the present application, the total content of the carbonate and bicarbonate is 36.2 to 70.5% by weight, such as 38 to 68% by weight, or 40 to 65% by weight, or 42 to 63% by weight, or 45 to 61% by weight, or 48 to 60% by weight, or 50 to 58% by weight, or 52 to 56% by weight, or 53 to 54.4% by weight, based on the total weight of the chemiluminescent composition.

[0065] According to one embodiment of the present application, the chemiluminescent composition comprises only the carbonate-based buffer, without using other buffers.

[0066] According to one embodiment of the present application, the content of H2O2 is 3.9 to 75.9% by weight, for example, 5 to 72% by weight, or 8 to 70% by weight, or 10 to 65% by weight, or 15 to 62% by weight, or 20 to 60% by weight, or 25 to 55% by weight, or 30 to 52% by weight, or 35 to 50% by weight, or 40 to 48% by weight, or 42 to 44.1% by weight, based on the total weight of the chemiluminescent composition. The "content of H2O2" as used herein means the net weight of hydrogen peroxide, excluding the amount of water in the hydrogen peroxide solution.

[0067] In the present application, the "total weight of the chemiluminescent composition" means the total weight of the components (i) to (v) and any other optional components, unless otherwise specified, but does not include water and any other solvent.

[0068] According to one embodiment of the present application, the chemiluminescent composition of the present application can be used by dissolving or dispersing all of its components in a solvent, which is preferably water, such as distilled water, heavy distilled water, deionized water, ultrapure water, and the like. According to another embodiment of the present application, the chemiluminescent composition of the present application can be used by loading a part of its components on a solid support, and dissolving or dispersing another part of its components (e.g., one or more of H2O2, carbonate-based buffer, and luminescence initiation time adjuster) in a solvent, which is preferably water, such as distilled water, heavy distilled water, deionized water, ultrapure water, and the like. The amount of the solvent (e.g., water) used can be 80 to 99.8% by weight, for example, 82 to 99.5% by weight, or 85 to 99.2% by weight, or 88 to 99.1% by weight, or 90 to 99.0% by weight, or 92 to 98.9% by weight, or 95 to 98.5% by weight, or 98 to 98.1% by weight, based on the total weight of the chemiluminescent composition and the solvent (e.g., water) as 100% by weight.

[0069] According to one embodiment of the present application, the components of the chemiluminescent composition are each isolated before use, and are mixed with each other to chemically react and emit light when used. In the present application, by using the combination of the above-described components, a significant increase in chemiluminescence intensity can be achieved, for example, for the most preferred embodiment, when Mn 2+With the ion and carbonate-based buffer (achieve a pH value of about 7.2), the CL luminescence intensity can be improved by up to four orders of magnitude or more relative to the prior art CL system. At the same time, non-transient chemiluminescence can also be achieved, for example, from the starting time of chemiluminescence to the time when the chemiluminescence completely stops, the duration of chemiluminescence can be greater than 10 seconds, or greater than 15 seconds, or greater than 20 seconds, or greater than 25 seconds, or greater than 30 seconds, or greater than 35 seconds, or greater than 40 seconds, or greater than 50 seconds, or greater than 60 seconds, or greater than 80 seconds, or greater than 100 seconds, or greater than 110 seconds, or greater than 120 seconds, or greater than 140 seconds, or greater than 150 seconds, or greater than 180 seconds, or greater than 200 seconds, or greater than 210 seconds, or greater than 220 seconds, or greater than 240 seconds, or greater than 250 seconds, or greater than 260 seconds, or greater than 280 seconds, or greater than 300 seconds, or greater than 320 seconds, or greater than 350 seconds, or greater than 360 seconds, or greater than 380 seconds, or greater than 400 seconds, or greater than 420 seconds, or greater than 450 seconds, or greater than 480 seconds, or greater than 500 seconds, or greater than 550 seconds, or greater than 580 seconds, or greater than 600 seconds, or greater than 620 seconds, or greater than 650 seconds, or up to 680 seconds, or up to 700 seconds, or up to 750 seconds, or up to 800 seconds, or up to 850 seconds, or up to 900 seconds, or up to 950 seconds, or up to 1000 seconds, or up to 1100 seconds, or up to 1200 seconds, or up to 1500 seconds. The long duration of chemiluminescence is very advantageous for automated CL systems.

[0070] According to one embodiment of the present application, a luminescence starting time adjusting agent is further added to the chemiluminescent composition to achieve the purpose of adjusting the luminescence starting time, and the luminescence starting time adjusting agent is selected from: thiourea compounds, C4-C 16 sugar alcohols, C3-C 12 alkyl mono-, di- or triols, C6-C 16 aromatic carboxylic acids, carboxylic anhydrides, carboxylic esters or lactones, sulfone compounds, sulfoxide compounds, piperidine compounds, C6-C 16 aromatic amine compounds, benzoquinone compounds, superoxide dismutase (SOD), C6-C 16 aromatic hydrocarbons, phenazone compounds, C6-C 16 carboxylic acids, carboxylic anhydrides, carboxylic esters or lactones, C6-C 16 cycloalkyl carboxylic acids, carboxylic anhydrides, carboxylic esters or lactones, C6-C16 aromatic carboxylic acids, carboxylic anhydrides, carboxylic esters or lactones, C6-Ci8 16 phenolic compounds, C6-Ci8 16 aromatic amine compounds, amino acids, peptides, and any combination thereof. According to a more specific embodiment of the present application, the luminescence initiation time adjusting agent can be selected from the group consisting of thiourea, ethylenethiourea, aminothiourea, 2-methyl-3- aminothiourea, mannitol, t-butyl alcohol, isopropyl alcohol, benzoic acid, dimethyl sulfoxide (DMSO), tetramethylpiperidine (TEMPO), diphenylamine, p-benzoquinone, superoxide dismutase (SOD), 1,1-diphenyl ethylene, 2,6-di-t-butyl-4-methyl phenol (BHT), t-butyl phenylnitrone (PBN), 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), ascorbic acid, erythorbic acid, ascorbic acid phosphate, ascorbic acid palmitate, ascorbic acid glucoside, gallic acid, pyrogallic acid, catechol, resorcinol, hydroquinone, pyrogallol, phloroglucinol, pyrocatechol, m-dihydroxybenzene, p-dihydroxybenzene, m-diaminobenzene, catechin, dopamine, cysteine, glutathione, and any combination thereof; preferably including thiourea, ethylenethiourea, aminothiourea, 2-methyl-3- aminothiourea, ascorbic acid, gallic acid, pyrogallic acid, catechol, hydroquinone, m-diaminobenzene, p-diaminobenzene, catechin, cysteine, glutathione, and any combination thereof.

[0071] The content of the luminescence initiation time adjusting agent is 0.01 to 6% by weight, or 0.02 to 5% by weight, or 0.04 to 4.5% by weight, or 0.05 to 4% by weight, or 0.08 to 3.5% by weight, or 0.1 to 3.2% by weight, or 0.2 to 3% by weight, or 0.3 to 2.5% by weight, or 0.4 to 2% by weight, or 0.5 to 1.5% by weight, or 0.6 to 1.0% by weight, based on the total weight of the chemiluminescent composition.

[0072] According to one embodiment of the present application, the light emission initiation time of the chemiluminescent composition can be precisely and quantitatively adjusted based on the amount of the light emission initiation time adjusting agent added. The light emission initiation time refers to the time between the time when the CL mixture is obtained by mixing the components including the adjusting agent and the time when the CL mixture starts to emit light. For example, the light emission initiation time can be 1 second to 1000 seconds, or 5 seconds to 900 seconds, or 10 seconds to 800 seconds, or 20 seconds to 700 seconds, or 30 seconds to 600 seconds, or 40 seconds to 550 seconds, or 50 seconds to 500 seconds, or 60 seconds to 450 seconds, or 80 seconds to 400 seconds, or 100 seconds to 350 seconds, or 200 seconds to 300 seconds, or can be within the numerical range formed by any two of the above end values.

[0073] According to another embodiment of the present application, a time-controlled multi-color emission system is established, which comprises using the chemiluminescent composition of the present application, and using multiple organic luminescent compounds therein, between which fluorescent resonance energy transfer (FRET) occurs, including one-step fluorescent resonance energy transfer and multi-step fluorescent resonance energy transfer, and by adjusting the ratio of the multiple organic luminescent compounds, a multi-color CL pattern can be achieved.

[0074] According to another embodiment of the present application, for the case where the chemiluminescent composition is loaded on the surface of a solid carrier, the solid carrier includes graphene oxide, solid paraffin, molecular sieve, zeolite, silicon dioxide, glass, etc. According to another embodiment of the present application, on the surface of the solid carrier, the loading amount of the chemiluminescent composition can be 0.1-15% by weight, for example, 0.2-13.2% by weight, or 0.5-12% by weight, or 0.8-10% by weight, or 1-9% by weight, or 2-8% by weight, or 3-7% by weight, or 4-6% by weight, or 4.5-5% by weight, based on the total weight of the solid carrier and the chemiluminescent composition.

[0075] Examples

[0076] In order to better understand the present application, the present application is further described below in conjunction with examples and drawings. The following examples are only used to further illustrate the present application and cannot be understood as limiting the content of the present application. Any non-essential improvements and adjustments made according to the inventive idea and technical solutions of the present application will be covered within the protection scope of the present application.

[0077] Unless otherwise specified, the reagents used in the following examples are all of analytical purity.

[0078] In the following examples, if it is stated that "the procedure was carried out in the same way as in a certain previous example, with the exception that... ", it is meant that the process conditions and steps recited later are used, while the other process conditions and steps not mentioned are as recited in the cited previous example.

[0079] Materials and reagents

[0080] The organic luminophore compound L012 used in the following examples was purchased from Wako Pure Chemical Industries (Osaka, Japan) and was directly dissolved in ultrapure water to prepare a 4 mM stock solution. Luminol (Sigma-Aldrich) and ABEI (TCI, Japan) were dissolved in 0.01 M NaOH solution to prepare a 4 mM stock solution, respectively. H2O2 was prepared fresh every day with 30% (v / v) H2O2 (Xinke Electrochemical Reagent Factory, Bengbu, China), and all other reagents were purchased from Sinopharm Chemical (Shanghai, China) and were of analytical grade. Ultrapure water was prepared with a Milli-Q system (Millipore, France) and was used throughout.

[0081] Preparation of CO2 / NaHCO3 buffer: In each of the following examples, sodium bicarbonate was dissolved in ultrapure water to obtain a sodium bicarbonate solution at the concentration specified in the example, and CO2 gas was bubbled into the solution for more than 30 minutes before use to obtain a CO2-saturated NaHCO3 buffer.

[0082] In the following examples, the following procedure was used to perform the CL experiments: The CL kinetic curves were determined using a Lumistar Omega microplate CL analyzer, and the stock solution of L-012 in ultrapure water (used directly or quantitatively diluted to obtain the desired concentration) and the aqueous solution of metal sulfate in ultrapure water were added to each micro-well and mixed well. Then 50 μΐ of CO2-saturated NaHCO3 solution in ultrapure water was added to each test well, and optionally a luminescence initiation time adjuster was added to each micro-well, immediately followed by the start of data collection. After 2 seconds of background signal acquisition, the program automatically injected 100 μΐ of H2O2. All experiments were performed at 25 °C.

[0083] CL photographs were taken by a Panasonic Lumix DMCFZ2000 digital camera.

[0084] The CL spectra were measured by a self-made CL spectrum collection system, which was composed of an Acton SP2300i monochromator equipped with a liquid nitrogen-cooled PyLoN 400BR-eXcelon digital charge-coupled device (CCD) (Princeton Instruments, USA). The three-dimensional (3D) CL spectra were plotted by OriginLab.

[0085] Example 1

[0086] In the Example 1, using Lumistar Omega microplate CL analyzer, 25 μΐ of L012 super pure water solution with a concentration of 4 mM and 25 μΐ of manganese sulfate super pure water solution with different concentrations (0.04 mM to 8 mM) were added into each well and mixed well, then 50 μΐ of CO2 saturated NaHCO3 super pure water solution with a concentration of 1 M was added into each test well, and immediately after that, the data collection was started, 2 seconds after the background signal was collected, the program automatically injected 100 μΐ of H2O2 with a concentration of 0.5 M. After all the components were added, the pH value of the system was 7.2, and the whole example was carried out at 25 °C.

[0087] The obtained CL luminescence intensity-time diagram is shown in Figure 1A and Figure 1B . Figure 1A The CL curves shown are in order from low to high peak intensity, experiments using manganese sulfate solution with Mn concentration of 5 μΜ to 100 μΜ, Figure 1B The CL curves shown are in order from high to low peak intensity, experiments using manganese sulfate solution with Mn concentration of 100 μΜ to 1000 μΜ. From Figure 1A and 1B it can be seen that the highest CL luminescence intensity can be achieved when the Mn concentration is 100 μΜ, and the longest CL luminescence duration is achieved when the Mn concentration is 5 μΜ.

[0088] Comparative Example 1

[0089] Comparative Example 1 was carried out in the same way as Example 1, the only difference being that in this comparative example, instead of adding manganese sulfate aqueous solution, the same volume of super pure water was used instead, and its CL curve is shown in Figure 2 From Figure 2 it can be seen that without the use of metal ions, the CL signal is very low (relative intensity value less than 10).

[0090] Comparative Example 2

[0091] Comparative Example 2 was performed in the same manner as Example 1, except that the aqueous solution containing manganese ions was not used, but instead, the same volume and same concentration of aqueous solutions of lead (II) sulfate, zinc (II) sulfate, cadmium (II) sulfate, and mercury sulfate in ultrapure water were used instead. As a result, it was found that in the case of using the above four kinds of metal salts, the CL signal was extremely low (relative intensity value was less than 10).

[0092] Comparative Example 3

[0093] Comparative Example 3 was performed in the same manner as Example 1, except that the aqueous solution of carbonate-based buffer was not used, but instead, the same volume and same concentration of aqueous solution of phosphate-based buffer was used instead, which was also saturated with carbon dioxide, and after mixing, the pH value of the sample was also 7.2. The CL curve thereof is shown in Figure 3 From Figure 3 it can be seen that although the same pH condition and saturated carbon dioxide condition were maintained, the CL signal obtained in this Comparative Example 3 was extremely low (relative intensity value was less than 10).

[0094] From the comparison of the above Example 1 and Comparative Examples 1-3, it can be seen that for the case of the chemiluminescent composition being in an aqueous solution, only when the metal cation specifically defined in the present application is combined with the carbonate-based buffer system, excellent CL intensity and CL luminescence duration effects can be achieved, and in the case of omitting the metal cation, or using other metal cations outside the scope of the present application, or using other buffer systems, even if other process conditions (such as other component concentrations, amounts, pH values, etc.) remain unchanged, the excellent effects as in Example 1 of the present application cannot be achieved, and the luminescence intensity of Comparative Examples 1-3 is extremely low and can be almost ignored.

[0095] Example 2

[0096] In this Example 2, the steps of Example 1 were repeated to synthesize the aqueous solution of the composition, except that a manganese sulfate solution and a cobalt (II) sulfate solution with a concentration of 100 μM were used, and the aqueous solution sample was prepared in a glass vial, and from the time when the sample began to emit light, CL photos of the sample at different times were taken, and the results are listed in Figure 4A and 4B wherein Figure 4A the photos of the sample prepared using manganese sulfate are shown, Figure 4B the photos of the sample prepared using cobalt sulfate are shown.

[0097] From the photos in Figure 4A and Figure 4B it can be seen that using manganese ions can achieve higher luminescence intensity, and using cobalt ions can achieve longer CL luminescence duration.

[0098] Example 3

[0099] In this embodiment, the effect of using thiourea to adjust the luminescence onset time of CL was investigated.

[0100] In Example 3, a Lumistar Omega microplate CL analyzer was used. 25 μl of 4 mM L012 ultrapure aqueous solution and 25 μl of 800 μM manganese sulfate ultrapure aqueous solution were added to each well and mixed thoroughly. Then, 25 μl of CO2-saturated 1 M NaHCO3 ultrapure aqueous solution and 25 μl of thiourea solutions at different concentrations (0, 0.1, 0.2, 0.5, 1, and 2 mg / mL) in ultrapure water were added to each test well. Data collection began immediately, and after acquiring the background signal for 2 seconds, 100 μl of 0.5 M H2O2 was automatically injected. After all components were added, the pH of the system was 7.2, and the entire example was conducted at 25°C.

[0101] The obtained three-dimensional (3D) CL spectrum is shown in Figure 5A The logarithmic graph of the thiourea concentration achieved in each experiment versus the obtained delay time is shown in the figure below. Figure 5B As shown.

[0102] As above Figure 5A As shown, with increasing thiourea concentration, the delay in luminescence onset time gradually increases, ranging from 0 to 83 seconds. The delay time (t...) d log(t) / second) d The value of ( / second) is linearly positively correlated with the logarithm of thiourea concentration (log(thiourea concentration / mg / ml)), such as Figure 5B As shown. Its regression equation is t d =1.5230 + 0.5058 × log c (mg / ml), with a correlation coefficient of 0.996. The time delay (t) d The value per second ( / second) represents the time difference between the arrival of CL intensity at 100 arbitrary units (au) in the system with or without thiourea. Therefore, the CL emission onset time can be precisely controlled using the thiourea concentration. Simultaneously, as the thiourea concentration increases, the peak CL emission value increases slightly until the thiourea concentration reaches 0.2 mg / ml. At this point, further increases in thiourea concentration will cause the peak value to decrease.

[0103] Example 4

[0104] This example 4 uses eight micro-wells in parallel, the same reagents as in example 3 are added in each micro-well, the difference is that different concentrations of thiourea in ultrapure water are used (concentration from 0 mg / mL to 4 mg / mL), H202 is added in all micro-wells at the same time, CL photos of these eight micro-wells at different times are taken, and the results are shown in Figure 6A and Figure 6B These figures show that the CL luminescence of different samples is sequentially adjusted based on the concentration of thiourea, thereby embodying the ability to construct CL patterns and CL codes.

[0105] Example 5

[0106] This example 5 uses a 12-well plate (3x4), and according to the steps described in example 4, CL codes are accurately coded by using different concentrations of thiourea in ultrapure water, and the CL photos and corresponding code information are shown in Figure 7 which shows the CL photos and corresponding codes that change accurately and continuously in about 2 minutes.

[0107] Example 6

[0108] In this example, the effects of using ethylene thiourea, aminothiourea and 2-methyl-3- aminothiourea to adjust the CL luminescence starting time are studied.

[0109] In the example 3, using Lumistar Omega micro-well plate CL analyzer, 25 μl of L012 ultrapure water solution with a concentration of 4 mM and 25 μl of manganese sulfate ultrapure water solution with a concentration of 800 μM are added to each well, and the two are mixed evenly, then 25 μl of CO2 saturated NaHCO3 ultrapure water solution with a concentration of 1 M is added to each test well, and 25 μl of thiourea in ultrapure water with different concentrations (0, 0.1, 0.5, 1, 2 and 2.5 mg / mL) is added, and then the data collection is started immediately, after 2 seconds of background signal acquisition, the program automatically injects 100 μl of H202 with a concentration of 0.5 M. After all components are added, the pH value of the system is 7.2, and the whole example is carried out at 25°C.

[0110] The obtained three-dimensional (3D) CL spectrum is shown in Figures 8A to 8C wherein Figure 8A corresponding to the experiment using ethylene thiourea, Figure 8B corresponding to the experiment using aminothiourea, Figure 8C corresponding to the experiment using 2-methyl-3-aminothiourea.

[0111] From these figures, it can be seen that when using the above-mentioned adjusting agents, the CL luminescence starting time can also be adjusted to different degrees.

[0112] Example 7

[0113] In this example, the effect of thiocyanate on the onset time of CL emission was investigated using luminol and ABEI as the luminescent organic compounds.

[0114] In said Example 7, using a Lumistar Omega microplate CL analyzer, 25 μΐ of a 4 mM solution of luminol or ABEI in ultrapure water and 25 μΐ of a 800 μΜ solution of manganese sulfate in ultrapure water at different concentrations were added to each well and mixed well, then 25 μΐ of a 1 M solution of NaHC03in ultrapure water saturated with C02was added to each test well, and 25 μΐ of a thiocyanate solution in ultrapure water at different concentrations (0.5, 1, 2.5, 5, 10, 15, 17.5, 20, 25 and 30 mg / mL) was added, immediately after which the data collection was started, and after 2 seconds of acquisition of the background signal, the program automatically injected 100 μΐ of a 0.5 M solution of H202. The entire example was carried out at 25 °C.

[0115] The resulting three-dimensional (3D) CL spectra are shown in Figure 9A and Figure 9B where Figure 9A corresponds to the experiment using luminol, Figure 9B corresponds to the experiment using ABEI.

[0116] As can be seen from these figures, for the use of the above organic luminescent compounds, the onset time of CL emission can also be modulated to different degrees with thiocyanate.

[0117] Example 8

[0118] In this example, the multicolor CL by fluorescence resonance energy transfer (FRET) for time modulation was investigated using a variety of different luminescent organic compounds.

[0119] In this Example 8, the procedure of the above Example 3 was repeated, with the difference that in this case two organic luminophores (L012 and Rhodamine B (RhB); or L012 and FL) were used simultaneously or three organic luminophores (L012, RhB and FL) were used. Without wishing to be bound to any particular theory, the above three different formulations established three FRET systems, namely a one-step method from L012 (blue, emission wavelength of about 467 nm) to Rhodamine B (RhB) (red, excitation wavelength of about 550 nm, emission wavelength of about 587 nm) Figure 10A , a one-step method from L012 to fluorescein (FL) (green, excitation wavelength of about 491 nm, emission wavelength of about 523 nm) Figure 10B , and a two-step method from L012 via FL to RhB Figure 10C .

[0120] After optimizing the ratio of L012, FL and RhB, the highest efficiency of FRET was determined as 26.6%, 74.7% and 55.0% for L012-RhB, L012-FL and L012-FL-RhB, respectively.

[0121] As shown in Figure 10D , by adjusting the proportion of FL or RhB, more CL intermediate colors can be generated by mixing the wavelength emission. By using different FRET systems, Figure 7 , the monochrome pattern is successfully converted into a multi-color pattern, as shown in Figure 11A . Different colors of CL emission can also be controlled at the same time Figure 11B , 11C . The emission color and sequence information are actually a double-layer matrix. If we use two colors in a 3x4 well plate without time control, there are 2 12 = 4096 kinds of matrices. If the two colors in the 3x4 well plate are divided into two groups and emitted in sequence, there are (2 12 ) x (2 12 ) = 16,777,216 combinations. In this experiment, 6 colors of CL are divided into 4 groups and emitted in sequence, and the calculation shows that there are about 3.6x10 16 matrices. Therefore, this system has the characteristics of adjustable position, emission time and color, can store a large amount of complex code, and can be used for signal communication and encryption technology by triggering CL reaction in the form of a sequence of multi-color patterns.

[0122] Example 9

[0123] In this example, the chemical luminescence initiation time adjustment effect of the CL system loaded on the solid carrier under the action of thiourea is shown.

[0124] Specifically, 250 μL of polydiallyldimethylammonium chloride (PDDA) (4 wt%, v / v), 40 mL of ultrapure water, 200 μL of a 0.5 M NaOH solution and 500 μL of a 6 mM HAuCl4 solution were mixed and heated to 100°C. After stirring for 1 h, the heat source was removed and naturally cooled to room temperature. Then 200 μL of a 4 mM L012 solution was added, and after stirring at room temperature for 12 h, the resulting lower layer of nanoparticles was re-dispersed in 2 ml of ultrapure water to obtain L012-wrapped gold nanoparticles (GNPs@L012). To 4 ml of a 0.5 mg / ml graphene oxide solution, 5 mM of a 1 mM Mn 2+Mix and stir for 30 min, then add 1 ml of the prepared GNPs@L012, and continue stirring at room temperature for 12 h. After centrifugation at 13000 rpm for 20 min, the resulting lower layer particles are redispersed in 4 ml of ultrapure water to obtain simultaneously immobilized L012 and Mn. 2+ Graphene oxide (Mn) 2+ / GO / GNPs@L012).

[0125] 50 μL of Mn was added to each well using a Lumistar Omega microplate CL analyzer. 2+ / GO / GNPs@L012 dispersion was mixed thoroughly. Then, 40 μl of CO2-saturated 1M NaHCO3 ultrapure water solution was added to each test well, along with 10 μl of thiourea solutions at different concentrations (0.001, 0.01, 0.02, 0.04, 0.1, 0.4, 1, 1.5, 2, 2.5, 3, 3.5, and 4 mg / mL) in ultrapure water. Data collection began immediately, and after acquiring the background signal for 2 seconds, 100 μl of 0.1M H2O2 was automatically injected. The entire experiment was conducted at 25°C. The obtained CL curves are shown below. Figure 12 As shown, it can be seen that for solid-loaded CL materials, the luminescence onset time of CL can also be precisely adjusted by using thiourea.

[0126] Examples 10-15

[0127] In Examples 10-15, all the steps of Example 3 described above were repeated, except that the thiourea solution in Example 3 was replaced with solutions of the luminescence onset time modifier described below at different concentrations (0, 0.1, 0.2, 0.5, 1, and 2 mM) in ultrapure water. The three-dimensional (3D) CL spectra obtained in each of these examples were displayed. Figures 13A to 13F In these figures, the concentrations of various luminescence onset time modifiers gradually increase along the direction of the arrows, representing 0, 0.1, 0.2, 0.5, 1, and 2 mM, respectively. A summary of the specific reagents used in these examples and the accompanying figures showing their experimental results is listed in Table 1 below.

[0128] Table 1: Types of luminescence onset time modifiers used in Examples 10-15 and accompanying figures showing their three-dimensional (3D) CL spectra.

[0129]

[0130] from Figures 13A to 13FAs can be seen from the 3D CL spectra shown in FIGS. 15A and 15B, these reagents can achieve the effect of delaying luminescence to different degrees, and dopamine, gallic acid, pyrogallic acid and ascorbic acid can also achieve the effect of increasing luminescence intensity at the same time with the adjustment of the added concentration.

[0131] Examples 16-18

[0132] In Examples 16-18, all the steps of Example 3 described above were repeated, with the only difference being that the thiourea solution of Example 3 was replaced by solutions of the luminescence initiation time adjusting reagents described below in ultrapure water at different concentrations (0, 0.1, 0.2, 0.5, 1 and 2 mM), and the three-dimensional (3D) CL spectra obtained in each of these examples are shown in FIGS. 14A and 14B. Figures 14A to 14C In these figures, the concentrations of various luminescence initiation time adjusting reagents gradually increase along the direction of the arrows, being 0, 0.1, 0.2, 0.5, 1 and 2 mM, respectively. The types of reagents used in each of these examples and the figures showing the experimental results thereof are summarized in Table 2 below.

[0133] Table 2: Types of luminescence initiation time adjusting reagents used in Examples 16-18 and figures showing the three-dimensional (3D) CL spectra thereof

[0134] Example 16 Example 17 Example 18 Light emission start time adjuster Catechin p-Phenylenediamine p-Dihydroxybenzene Figures showing 3D CL spectra thereof Figure 14A Figure 14B Figure 14C

[0135] As can be seen from the 3D CL spectra shown in FIGS. 15A and 15B, these reagents can achieve the effect of delaying luminescence to different degrees, and dopamine, gallic acid, pyrogallic acid and ascorbic acid can also achieve the effect of increasing luminescence intensity at the same time with the adjustment of the added concentration. Figures 14A to 14C

[0136] Examples 19-20

[0137] In Examples 19-20, all the steps of Example 3 described above were repeated, with the only difference being that the thiourea solution of Example 3 was replaced by solutions of the luminescence initiation time adjusting reagents described below in ultrapure water at different concentrations (0, 2, 4, 8, 40 and 50 mM for cysteine and 0, 1, 5, 10, 20 and 40 mM for glutathione), and the three-dimensional (3D) CL spectra obtained in each of these examples are shown in FIGS. 15A and 15B. Figures 15A to 15B In these figures, the concentrations of various luminescence initiation time adjusting reagents gradually increase along the direction of the arrows, being 0, 2, 4, 8, 40, 50 mM (cysteine, 15A) and 0, 1, 5, 10, 20, 40 mM (glutathione, 15B), respectively. The types of reagents used in each of these examples and the figures showing the experimental results thereof are summarized in Table 3 below.

[0138] Table 3: Types of luminescence initiation time adjusting reagents used in Examples 19-20 and figures showing the three-dimensional (3D) CL spectra thereof

[0139] Example 19 Example 20 Light emission start time adjuster Cysteine Glutathione Figures showing 3D CL spectra thereof Figure 15A Figure 15B ​

[0140] From Figures 15A to 15B As can be seen from the 3D CL spectra shown, these reagents can achieve the effect of delayed luminescence to different degrees. It can be seen that cysteine can achieve a very significant luminescence start time adjustment effect at a concentration range of 40-50 mM, and glutathione can achieve a certain degree of luminescence intensity increase at a concentration range of 2-8 mM.

Claims

1. A chemical method for adjusting the onset time of chemiluminescence, the method comprising: Add a luminescence onset time modifier to the chemiluminescent composition, wherein the luminescence onset time modifier is selected from: thiourea compounds, C6-C6 compounds containing one to four hydroxyl groups. 16 Aromatic carboxylic acids, C6-C 16 Aromatic amine compounds, containing one to six hydroxyl groups in a C6-C group. 16 Phenolic compounds, C6-C6 compounds containing one to six hydroxyl groups. 16 Aromatic amine compounds, amino acids, peptides, ascorbic acid, isoascorbic acid, ascorbic acid phosphate, ascorbate palmitate, ascorbate glucoside, and any combination thereof; The chemiluminescent composition comprises: (i) a luminescent compound, said luminescent compound including an organic luminescent compound, said organic luminescent compound including fluorescein-type luminescent compounds, rhodamine-type luminescent compounds, compounds of formula (1) or salts thereof, and compounds of formula (2) or salts thereof. Equation (1) In equation (1), ring A represents C6-C 14 Fang Huan, R1 and R2 independently represent hydrogen, terminal groups substituted with amino groups, or unsubstituted straight or branched chains (C1-C2). 30 )alkyl, provided that the NR1R2 has at least one NH2 end group; Equation (2) In formula (2), R3, R4, and R5 independently represent C1-C6 alkyl, C1-C6 alkoxy, and C6-C6 alkyl groups. 16 Aryl, C6-C 16 Aryloxy groups, F, Cl, Br, I, -NH2, -OH, -NO2, -COOH, -C(=O)-C1-C6 alkyl groups, and any combination thereof; (ii) Metal ions, said metal ions including: Mn 2+ Co 2+ , and their combinations; (iii) H2O2; and (iv) Carbonate-based buffers; The chemiluminescent composition is in an aqueous solution or at least partially loaded on the surface of a solid carrier.

2. The method as described in claim 1, characterized in that, The luminescence initiation time modifier is selected from: thiourea, ethylene thiourea, aminothiourea, 2-methyl-3-aminothiourea, ascorbic acid, isoascorbic acid, ascorbic acid phosphate, ascorbate glucoside, gallic acid, pyrogallol, catechol, resorcinol, hydroquinone, pyrogallol, pyrogallol, tetraphenylethylene, o-phenylenediamine, p-phenylenediamine, m-phenylenediamine, catechin, dopamine, cysteine, glutathione, and any combination thereof.

3. The method as described in claim 1, characterized in that, The method includes generating patterns composed of different chemiluminescences by adding different amounts or different types of the luminescence onset time modifier to multiple portions of the chemiluminescent composition; or By adding the luminescence onset time modifier to multiple portions of the chemiluminescent composition containing different luminescent compounds, patterns composed of different chemiluminescences are generated.

4. The method as described in claim 1, characterized in that, The metal ion is Mn. 2+ .

5. The method as described in claim 1, characterized in that, The carbonate-based buffer includes carbonates, bicarbonates, or mixtures thereof, or an aqueous solution containing carbon dioxide and bicarbonates, or an aqueous solution containing carbon dioxide, carbonates, and bicarbonates simultaneously. The carbonates and bicarbonates include ammonium salts, lithium salts, sodium salts, potassium salts, and organic amine salts, respectively.

6. A chemical method for generating chemiluminescence of multiple colors and adjusting the onset time of chemiluminescence, the method comprising using the method of claim 1, wherein the chemiluminescent composition comprises multiple luminescent compounds, and fluorescence resonance energy transfer occurs between the multiple luminescent compounds, thereby generating chemiluminescence of multiple colors.

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