CN114349736A - Compound and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biology, and discloses a compound as shown in the following formula (1) and a double-aggregation induced luminescence fluorescent agent system comprising the compound:

the invention also discloses the use of the compound of the invention for dyeing microorganisms, and the use of the double aggregation-induced emission fluorescent luminescent agent system of the invention for monitoring the metabolic state of microorganisms and microorganismsThe morphology and metabolic state of biological membranes.

Description

Compound and application thereof

Technical Field

The present invention relates to a compound, the use of the compound for staining microorganisms, a method for staining microorganisms, a dual aggregation induced emission fluorescent emitter (AIEgen) system comprising the compound, and a method for monitoring the metabolic state of microorganisms, quantitatively monitoring the activity of microorganisms, monitoring the morphology and metabolic state of biofilms of microorganisms using the dual aggregation induced emission fluorescent emitter (AIEgen) system.

Background

Microorganisms are the oldest and most abundant population on earth. Since their appearance in the world 40 billion years ago, they have dominated the planet in all respects. Their total number is estimated to be in the trillion categories, some of which have been acclimated to humans and used in our daily lives. However, the pathogenic microorganisms therein are always threatening our human lives. Human development appears to be a history of struggling with microorganisms. Due to the fact that ubiquitous microorganisms are too small to observe, only 1400 pathogens have been discovered and understood to date. This dilemma places fear, panic and a tremendous burden on society when epidemics occur. For example, black death is one of the most fatal epidemic diseases in human history, causing 0.75-2 million deaths in continental europe and north africa. Until 1894, the chief culprit pathogen Yersinia pestis was discovered, and the severe situation was well controlled. Therefore, it is very important to develop a method for detecting, identifying and quantitatively determining microorganisms. In addition, microorganisms often form biofilms to survive and adapt in various environments. In the microbial world, the pattern of aggregation is common but also complex and lacks sufficient understanding. To date, in situ visualization of biofilm formation is an urgent need.

In clinical and research, there are several methods commonly used for detection of microorganisms, such as gram staining, plate counting, Minimum Inhibitory Concentration (MIC) test, and electron microscopy (SEM and TEM). Gram stain is the most widely used method for distinguishing gram-positive and gram-negative bacteria, but it requires a complicated procedure including steps of washing and counterstaining, and it can only roughly classify bacteria into two major categories regardless of live/dead states. The plate counting method has been developed to quantify viable bacteria by spreading a diluted bacterial suspension on appropriate agar, but requires a long culture time of 24 hours or more and can only accurately culture a small fraction of microorganisms on appropriate medium agar. MIC is the most common method of characterizing the antimicrobial ability of drugs and biomaterials. However, MIC results are based on macroscopic turbidity readings, which are not sensitive enough and may in practice generate systematic errors. The use of SEM and TEM enables researchers to observe more detailed microbial structures, but these techniques require complex sample preparation. It should be further noted that none of these four methods belong to in situ characterization techniques, and it is difficult to satisfy real-time monitoring of dynamic microorganisms. Therefore, further technical innovation is needed to realize in-situ visualization of microorganisms.

Fluorescence techniques have many advantages, such as high sensitivity, nanoscale resolution, and in situ/in situ visualization properties. Therefore, fluorescent probes can well meet all these requirements for microbial research in the biological and medical fields. However, the perfect use of conventional fluorescent probes in biological systems is limited by the inherent drawbacks of aggregation-induced quenching (ACQ). Most hydrophobic ACQ emitters can form aggregates in water, and strong pi-pi interactions can lead to partial or complete fluorescence quenching. In addition, this drawback is exacerbated in complex biological systems and prevents these traditional fluorescent probes from being incorporated into biofilm monitoring. Since the biofilm contains a large number of bacteria, sufficient fluorescent molecules are needed to match the number of bacteria, and ACQ emitters do not work efficiently at such high concentrations. To avoid ACQ effects, few and weak fluorescent probes are the only choice, even though they can only apply incomplete information of the measured biofilm. In addition, traditional fluorescent probes invariably suffer from their toxicity, such as PI and SYTO9 (commonly used in commercial bacterial imaging kits). To reduce interference with high toxicity, these probes must be rinsed from the sample prior to imaging. The assisted flush procedure appeared to be completely incapable of biofilm observation. In addition to the considerable spectral overlap, the paired SYTO9 and PI introduce inevitable systematic errors into the results. Therefore, there is an urgent need for a biocompatible system for fluorescent microbial imaging with wash-free and minimal spectral overlap characteristics.

It is known that aggregation-induced emission fluorescent light emitting agents (aiegens) as a novel fluorescent material can completely compensate for all the above-mentioned drawbacks.

The working mechanism of AIEgen is based on confinement of intramolecular motion (RIM), which includes confinement of intramolecular rotation (RIR) and confinement of intramolecular vibration (RIV). AIEgen has many advantages such as tunable chemical structure and biocompatibility, turn-on characteristics and tunable emission spectra. For the first component, the near-infrared AIEgen probe can be easily designed to have broad-spectrum targeting ability, thereby giving this component the ability to stain all microorganisms with high efficiency. Then, by modulating the donor-pi-acceptor structure, a red emitting AIEgen can be obtained. Red emission is very important in biological applications because it has less autofluorescence and higher resolution. Furthermore, deeper penetration depths can be achieved using red-emitting AIE probes. Next, two positive charges are introduced into the structure with a short carbon chain to achieve both universal targeting ability and good biocompatibility. For the traditional fluorescent probe development, due to the complex chemical structure, the complex synthetic route and the complex purification procedure, the design of the near-infrared probe is troublesome. To achieve minimal spectral overlap, the second component should be sufficiently blue. However, ultraviolet light is harmful to biological samples, so blue AIEgen was chosen. Tetraphenylethylene derivatives containing boronic acid structure (TPE-2BA) are widely reported AIEgen, which have broad-spectrum targeting ability and can selectively image dead microorganisms.

Therefore, there is a need in the art for a biocompatible system that can stain microorganisms, enable in situ visualization of microorganisms, and enable visualization of biofilms.

Disclosure of Invention

In view of the above, the present invention provides a compound having a spectroscopic staining ability for microorganisms, and its application in staining microorganisms, and provides a biocompatible system capable of visualizing a biofilm, and its application in dynamic monitoring of a biofilm.

The invention successfully designs and synthesizes the compound shown in the formula (1), and the compound has specific AIE characteristics and good biocompatibility. The broad-spectrum staining ability of the compounds facilitates in situ and real-time imaging of microorganisms in both live and dead states. By referring to TPE-2BA, which is suitable for dead microorganism staining only, the present invention further provides a two-component AIEgen system for the detection of microbial activity with minimal spectral overlap. In addition, the system is successfully applied to monitoring the metabolic state of microorganisms, quantitatively detecting the activity of the microorganisms, and monitoring the form and metabolic state of biofilms of the microorganisms. Numerous experiments have demonstrated that this two-component AIEgen system has great potential in the detection, visualization and quantitative analysis of microorganisms.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other embodiments can be obtained according to the drawings without inventive labor.

FIG. 1 shows the design principle and synthetic route (A) of water-soluble NIR AIEgen DCQA; fluorescence spectra (B) of DCQA in DMSO/toluene mixed solvents of different toluene contents (volume fraction%); maximum and relative intensities (α) of PL_AIE＝I/I₀) Diagram (C) of DMSO/toluene mixed solvent with DCQA, wherein I₀Is f_tPL intensity at 0%; concentration of 10 × 10^{- 6}M，λ_ex519nm, (inset: fluorescent microscope image of DCQA powder,. lambda.)_ex＝510-550nm，λ_em＝590-800nm)。

Figure 2 shows bacterial staining properties of DCQA. (A-C) shows CLSM images of live (upper panel) and dead (lower panel) microorganisms stained with DCQA, excitation wavelength: 561nm, emission range: 590-759 nm. (A) The method comprises the following steps Aureus, 5 μm scale bar. (B) The method comprises the following steps Coli, scale bar 5 μm; (C) the method comprises the following steps C. albicans, scale bar 10 μm. (D-F) shows the in situ emission spectrum of DCQA generated by CLSM. (D) The method comprises the following steps (S.aureus.; (E) the method comprises the following steps Coli.; (F) the method comprises the following steps C. albicans, excitation wavelength 561nm, emission range 530-780 nm, concentration of DCQA 10 μ M.

FIG. 3 shows a CLSM image of a mixture of dead/live microbial cells co-stained with DCQA and TPE-2 BA. (A) The method comprises the following steps Gram-positive bacteria, left: s.aureus, right: b.sublitis, scale bar 5 μm. (B) The method comprises the following steps Gram-negative bacteria, left e.coli, right s.marcocens, scale bar 5 μm. (C) The method comprises the following steps Fungi, c.albicans on the left, s.cerevisiae on the right, with a scale bar of 10 μm. Excitation wavelengths of 561nm (DCQA) and 405nm (TPE-2 BA). Emission ranges of 590-759nm (DCQA) and 410-580nm (TPE-2 BA). The concentrations of DCQA and TPE-2BA were 10. mu.M and 50. mu.M, respectively.

FIG. 4 shows the results of a quantitative study of the mixture of DCQA and TPE-2 BA. (A) Coli suspensions were analyzed for relative activity in a fluorescence microplate reader. Integrated intensities of blue (460 ± 5nm) and red (730 ± 5nm) emissions of suspensions excited at 310 ± 10nm and 519 ± 10nm were obtained, and for each live/dead e.coli ratio, the red/blue fluorescence ratio (ratio R/B) was calculated. Each point represents the average of four measurements. The line is a least squares fit of the relationship between the percentage of viable bacteria (x) and the ratio R/b (y). (B) Coli suspensions were analyzed for relative activity. Coli was stained with DCQA and TPE-2BA at different given percentages. The activity of e.coli suspensions was calculated by CLSM images of each given percentage of live e.coli. (C) Statistical analysis was performed by flow cytometry on e.coli stained with TPE-2BA and DCQA mixtures at different live/dead ratios. Lambda for TPE-2BA channel_ex＝405nm,λ_em450 ± 40 nm. λ for DCQA channel_ex＝561nm,λ_em780 ± 60 nm. (D) Coli suspensions were analyzed for relative activity by mean intensity obtained by flow cytometry. The integrated intensities of the blue (450 ± 40nm) and red (780 ± 60nm) emissions of the suspensions excited at 405nm and 561nm were obtained and the red/blue fluorescence ratio (ratio) was calculated for each live/dead eRatio R/B). Each point represents the average of 5000 counts. The line is a least squares fit of the relationship between the percentage of viable bacteria (x) and the ratio R/b (y).

FIG. 5 shows the monitoring of biofilm morphology and metabolic status achieved by the combination of DCQA and TPE-2 BA. CLSM images of microbial biofilms (three days old) stained with DCQA and TPE-2 BA. (A) S. aureus. (B) e.coli. (C) c.albicans. scale bar: 20 μm.

FIG. 6 shows a molecular orbital amplitude plot of the HOMO and LUMO energy levels of DCQA.

FIG. 7 shows the extinction spectrum of DCQA in DMSO (A). (B) Fluorescence decay curve of solid-state DCQA.

FIG. 8 shows FL emission spectra of DCQA-stained microorganisms at different time points. (A) S. aureus (B) e.coli (C) c.albicans. 519nm as excitation wavelength and 540-840 nm as emission range. The concentration of DCQA was 10. mu.M. (D-E) FL intensity at different times. The peak wavelength is 720 nm.

Fig. 9 shows PL intensities of different bacterial components interacting with DCQA. Excitation wavelength 519nm. Emission range 530 nm and 900 nm. The concentration of DCQA was 10. mu.M.

Detailed Description

The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. It is to be understood that the described embodiments are merely a subset of the present invention and not all embodiments. All other embodiments that can be derived by one of ordinary skill in the art from the embodiments disclosed herein are within the scope of the present invention.

Definitions and general terms

Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated by the accompanying structural and chemical formulas. The invention is intended to cover alternatives, modifications and equivalents, which may be included within the scope of the invention as defined by the appended claims. Those skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described herein. In the event that one or more of the incorporated documents, patents, and similar materials differ or contradict this application (including but not limited to defined terminology, application of terminology, described techniques, and the like), this application controls.

It will be further appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications referred to herein are incorporated by reference in their entirety.

The following definitions as used herein should be applied unless otherwise indicated. For the purposes of the present invention, the chemical elements are in accordance with the CAS version of the periodic Table of the elements, and the handbook of chemistry and Physics, 75 th edition, 1994. In addition, general principles of Organic Chemistry can be referred to as described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausaltito: 1999, and "March's sAdvanced Organic Chemistry" by Michael B.Smith and Jerry March, John Wiley & Sons, New York:2007, the entire contents of which are incorporated herein by reference.

The articles "a," "an," and "the" as used herein are intended to include "at least one" or "one or more" unless otherwise indicated or clearly contradicted by context. Thus, as used herein, the articles refer to articles of one or more than one (i.e., at least one) object. For example, "a component" refers to one or more components, i.e., there may be more than one component contemplated for use or use in embodiments of the described embodiments.

The term "comprising" is open-ended, i.e. includes the elements indicated in the present invention, but does not exclude other elements. However, those skilled in the art will understand that in some particular cases embodiments may alternatively be described using the language "consisting essentially of … …" or "consisting of … …".

In addition, unless otherwise explicitly indicated, the descriptions of the terms "… independently" and "… independently" and "… independently" used in the present invention are interchangeable and should be understood in a broad sense to mean that the specific items expressed between the same symbols do not affect each other in different groups or that the specific items expressed between the same symbols in the same groups do not affect each other.

As used herein, the term "λ_ex"refers to the excitation wavelength.

As used herein, the term "λ_em"refers to the emission wavelength.

As used herein, the phrase "aggregation-induced quenching" or "ACQ" refers to a phenomenon in which aggregation of a pi-conjugated fluorophore significantly reduces the fluorescence intensity of the fluorophore. The formation of aggregates is said to "suppress" the luminescence of the fluorophore.

As used herein, the phrase "aggregation-induced emission" or "AIE" refers to a phenomenon in which a compound exhibits significantly enhanced light emission when aggregated in an amorphous or crystalline (solid) state, while exhibiting weak or almost no light emission in a dilute solution.

As used herein, the term "emission intensity" refers to the fluorescence/phosphorescence intensity typically obtained from fluorescence spectrometer or fluorescence microscope measurements;

as used herein, the term "fluorophore" or "fluorine atom" refers to a molecule that exhibits fluorescence.

As used herein, the term "luminescent agent" or "luminophore" refers to a molecule that exhibits luminescence.

As used herein, the term "AIEgen" refers to a molecule that exhibits AIE properties.

As used herein, the term "halogen" refers to fluorine, chlorine, bromine and iodine, "halo" refers to substitution by "halogen".

As used herein, the term "alkyl" refers to a straight or branched chain saturated hydrocarbon group. Examples of alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and z ' -propyl), butyl (e.g., n-butyl, z ' -butyl, sec-butyl, tert-butyl), pentyl (e.g., n-pentyl, z ' -pentyl, -pentyl), hexyl, and the like. In various embodiments, the alkyl group can have 1 to 40 carbon atoms (i.e., C1-40 alkyl), such as 1 to 30 carbon atoms (i.e., C1-30 alkyl). In some embodiments, alkyl groups may have 1 to 6 carbon atoms, and may be referred to as "lower alkyl". Examples of lower alkyl groups include methyl, ethyl, propyl (e.g., n-propyl and z '-propyl), and butyl (e.g., n-butyl, z' -butyl, sec-butyl, tert-butyl). In some embodiments, alkyl groups may be substituted as described herein. An alkyl group is typically not substituted with another alkyl, alkenyl, or alkynyl group.

As used herein, "unsaturated alkyl" refers to a hydrocarbon group containing a double bond, a triple bond, or a pi bond, wherein the hydrocarbon group containing a double bond is an "alkenyl", the hydrocarbon group containing a triple bond is an "alkynyl", and the hydrocarbon group containing a pi bond can form a benzene ring.

As understood by those skilled in the art, the term "alkenyl" refers to a straight or branched chain alkyl group having one or more carbon-carbon double bonds. Examples of alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl, and the like. One or more of the carbon-carbon double bonds may be internal (e.g., in 2-butene) or terminal (e.g., in 1-butene). In various embodiments, alkenyl groups may have 2 to 40 carbon atoms (i.e., C2-40 alkenyl groups), such as 2 to 20 carbon atoms (i.e., C2-20 alkenyl groups). In some embodiments, the alkenyl group may be substituted as described herein. An alkenyl group is typically not substituted with another alkenyl, alkyl, or alkynyl group.

As understood by those skilled in the art, the term "alkynyl" refers to a straight or branched chain alkyl group having one or more carbon-carbon triple bonds. In one embodiment, alkynyl groups contain 2-8 carbon atoms; in another embodiment, alkynyl groups contain 2-6 carbon atoms; in yet another embodiment, alkynyl groups contain 2-4 carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl, propargyl, 1-propynyl, and the like. In some embodiments, alkynyl groups may be substituted as described herein. An alkynyl group is typically not substituted with another alkenyl, alkyl, or alkynyl group.

As understood by those skilled in the art, a "benzene ring" is a structure of a benzene molecule having the formula C₆H₆Which is a planar regular hexagon with one carbon atom at each vertex, one hydrogen atom bonded to each carbon atom, and a carbon-carbon bond in the benzene ring being a unique bond between a single bond and a double bond. In some embodiments, the phenyl ring may be substituted as described herein.

As used herein, the term "heteroalkyl" refers to an alkyl group that includes a heteroatom.

As understood by those skilled in the art, the term "heteroatom" refers to an atom of any element other than carbon or hydrogen, including, but not limited to, oxygen (O), nitrogen (N), sulfur (S), silicon (Si), selenium (Se), and phosphorus (P).

As used herein, the term "cycloalkyl" refers to a saturated hydrocarbon group containing an alicyclic structure, wherein the alicyclic structure has a single cyclic alicyclic ring and a fused cyclic alicyclic ring. The general molecular formula of the cycloalkyl containing 1 alicyclic ring and no substituted alkyl on the ring is-C_nH_2n(n.gtoreq.3). Examples of cycloalkyl groups include cyclopropane, cyclobutane, cyclohexane, and the like.

As used herein, the term "heterocycloalkyl" refers to a cycloalkyl group that contains a heteroatom, where the "heteroatom" is defined above.

As used herein, the term "aryl" refers to an aromatic monocyclic hydrocarbon ring system or a polycyclic ring system in which two or more aromatic hydrocarbon rings are fused (i.e., have a common bond) together, or at least one aromatic monocyclic hydrocarbon ring is an aromatic monocyclic hydrocarbon ring system or a polycyclic ring system. Groups fused to one or more cycloalkyl and/or cycloheteroalkyl rings. The aryl group can have 6 to 24 carbon atoms in its ring system (e.g., a C6-24 aryl group), which can include multiple fused rings. In some embodiments, the polycyclic aryl group can have 8 to 24 carbon atoms. Any suitable ring position of the aryl group can be covalently linked to the defined chemical structure. Examples of the aryl group having only an aromatic carbocyclic ring include phenyl, 1-naphthyl (bicyclic), 2-naphthyl (bicyclic), anthracenyl (tricyclic), phenanthrenyl (tricyclic), pentenyl (pentacyclic)And the like. Examples of polycyclic ring systems in which at least one aromatic carbocyclic ring is fused to one or more cycloalkyl and/or cycloheteroalkyl rings include benzo derivatives of cyclopentane (i.e., indanyl, which is a 5, 6-bicycloalkyl/aryl ring system), cyclohexane (i.e., tetrahydronaphthyl, which is a 6, 6-bicycloalkyl/aryl ring system), imidazoline (i.e., benzimidazolinyl, which is a 5, 6-bicycloalkyl/aryl ring system), and pyran (i.e., chromenyl, which is a 6, 6-bicycloalkyl/aromatic ring system). Other examples of aryl groups include benzodioxanyl, benzodioxazolyl, chromanyl, indolyl, and the like. In some embodiments, aryl groups may be substituted as described herein. In some embodiments, an aryl group may have one or more halogen substituents and may be referred to as a "haloaryl" group. Perhaloaryl, i.e. aryl in which all hydrogen atoms are replaced by halogen atoms (e.g. -C)₆F₅) Included within the definition of "haloaryl". In certain embodiments, an aryl group is substituted with another aryl group and may be referred to as a biaryl group. Each aryl group in the biaryl group may be substituted as disclosed herein.

As used herein, the term "heteroaryl" refers to an aromatic monocyclic ring system comprising at least one ring heteroatom selected from oxygen (O), nitrogen (N), sulfur (S), silicon (Si), and selenium (Se), or multiple rings. A ring system, wherein at least one ring present in the ring system is aromatic and comprises at least one ring heteroatom. Polycyclic heteroaryl groups include those having two or more heteroaryl groups fused together, as well as those having at least one monocyclic heteroaryl group fused to one or more aromatic carbocyclic, non-aromatic carbocyclic and/or non-aromatic cycloheteroalkyl rings. In general, heteroaryl groups can have, for example, 5 to 24 ring atoms and contain 1-5 ring heteroatoms (i.e., 5-20 membered heteroaryl). The heteroaryl group may be attached to the defined chemical structure at any heteroatom or carbon atom that results in a stable structure. Typically, heteroaryl rings do not contain an O-O, S-S or S-O bond. However, one or more of the N or S atoms in the heteroaryl group can be oxidized (e.g., pyridine N-oxide thiophene S-oxide, thiophene S, S-dioxide). Examples of heteroaryl groups include, for example, 5 or 6 membered monocyclic and 5-6 bicyclic ring systems: whereinMay contain O, S, NH, N-alkyl, N-aryl, N- (arylalkyl) (e.g., N-benzyl), SiH₂SiH (alkyl), Si (alkyl)₂SiH (arylalkyl), Si (arylalkyl)₂Or Si (alkyl) (arylalkyl). Examples of such heteroaryl rings include pyrrolyl, furyl, thienyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, isothiazolyl, thiazolyl, thiadiazolyl, isoxazolyl, oxazolyl, oxadiazolyl, 2-methylquinolyl, isoquinolyl, quinoxalinyl, quinazolinyl, benzotriazolyl, benzimidazolyl, benzothiazolyl, benzisothiazole, benzisoxazolyl, benzoxazolyl, cinnolinyl, 1H-indazolyl, 2H-indazolyl, indolizinyl, isobenzofuryl, naphthyridinyl, phthalazinyl, pteridinyl, purinyl, oxazolo, thiazolo, imidazopyridinyl, furopyridyl, thienopyridyl, pyridopyrimidinyl, pyridopyrazinyl, Pyridopyrazinyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, and the like. Other examples of heteroaryl groups include 4,5,6, 7-tetrahydroindolyl, tetrahydroquinolinyl, benzothienopyridyl, benzofuranylpyridyl, and the like. In some embodiments, heteroaryl groups may be substituted as described herein.

As used herein, the term "donor" material refers to an organic material, such as an organic nanoparticle material, having holes as the predominant current or charge carrier.

As used herein, the term "acceptor" material refers to an organic material, such as an organic nanoparticle material, having electrons as the predominant current or charge carrier.

Where a range of values is provided, such as a concentration range, a percentage range, or a ratio range, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the subject matter described. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and such embodiments are also encompassed within the subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the subject matter.

For a better understanding of the present teachings and in no way limiting the scope of the present teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions used in the specification and claims, as well as other numerical values, are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

It should be noted that the terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The foregoing summary, as well as the following detailed description, is intended merely to be illustrative of the invention and is not intended to be in any way limiting. The scope of the invention is to be determined by the appended claims without departing from the spirit and scope of the invention.

In the present invention, the inventors have designed a novel near-infrared AIEgen, which has the following structure (1):

wherein two R are₁Independently selected from H, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkyl-NCS, alkyl-N₃And alkyl-NH₂Wherein two R are₁May be the same or different;

d is

Wherein R is₂Is an alkyl chain having quaternary ammonium end groups;

X^-is a counterion, preferably X is independently selected from F, Cl, Br, I and PF₆。

As understood by those skilled in the art, "counter ion" refers to an ion in the electric double layer having a charge of opposite sign to the surface charge of the colloidal particle. The counter ions mainly play a role in balancing the surface charges of the colloidal particles, so that the double-electric-layer system on the surface of the colloid is in an electrically neutral state. In the present invention, other ions having the same function known to those skilled in the art may be used in addition to the counter ions exemplified above, and the present invention is not further limited thereto.

In some embodiments, the compound is a compound having the structure of formula (2), (3):

in some embodiments, the compound is a compound having the structure of formula (4), (5):

in a specific embodiment, the compound is (E) -4- (2- (7- (diphenylamino) -9-ethyl-9H-carbazol-2-yl) vinyl) -1- (3- (trimethylammonio) propyl) quinoline-1-ammonium bromide (DCQA) having the structure shown in formula (6):

the above-mentioned compound represented by formula (1) has a strong D-a intensity, which can promote Intramolecular Charge Transfer (ICT), resulting in a smaller electronic band gap and a longer absorption/emission wavelength. Besides the electron-withdrawing ability, the introduction of the electron acceptor with positive charge can also enable the fluorophore to have unique biological functions, and have universal microorganism targeting and imaging ability. It was found that the integration of the AIE properties of the above compounds showing large stokes shifts, near infrared emission and two positive charges with a short carbon chain makes the above compounds of formula (1) promising candidates for biological applications.

Accordingly, the present invention provides the use of the above-mentioned compound represented by the formula (1) for staining microorganisms.

The above-mentioned compound represented by formula (1) can interact with various microorganisms in a short time due to general microbial targeting and imaging ability, and has a broad-spectrum staining ability for all microorganisms. In addition, the experimental results also show that it shows good staining ability in both live and dead states and has a suitable staining window for both imaging and high throughput analysis, and thus, it can act as a universal detection probe for various microorganisms.

Further, the present invention provides a method for staining a microorganism, which comprises staining a microorganism with the above-mentioned compound represented by formula (1).

The above-mentioned properties of the above-mentioned compound represented by formula (1) may allow the method for dyeing microorganisms using the above-mentioned compound represented by formula (1) to have advantages of high dyeing efficiency, rapid reaction performance, and short interaction time.

The metabolic state of the microorganism is closely related to the functions, the biofilm forming capability, the secretion of virulence factors, the drug resistance generation and other characteristics of the microorganism. With the change of environment, the metabolic state of the microorganism is also changed. Especially for opportunistic pathogens, changes in their metabolic levels may lead to conditions that are harmful to us. The activity of microorganisms is a key factor in the metabolism of microorganisms. It has been reported that some bacteria sacrifice themselves when faced with a particular antibiotic, and that other bacteria are warned of danger by dispersing their resistance genes. The remaining microbial species will receive the resistance gene and directly acquire resistance to the particular antibiotic. Therefore, monitoring the metabolic state of microorganisms is of great importance, and in this work, factors of the activity of microorganisms are selected as criteria.

As described above, the above compound represented by the formula (1) has a broad-spectrum staining ability for all microorganisms as a novel fluorescent probe. In addition, as known to those skilled in the art, TPE-2BA can be used for monitoring the metabolic state of planktonic microorganisms and biofilms thereof, and is a blue fluorescent probe specially used for dead microorganism imaging, so that a validated dead microorganism AIEgen probe TPE-2BA can be selected as a synergistic metabolic sensor of the above compounds represented by formula (1) so as to be used for monitoring the metabolic state of microorganisms. In addition, the emission peak of TPE-2BA is around 450nm, and the minimum spectral overlap with the compound shown in the formula (1) can be realized, so that the TPE-2BA is a perfect combination of microbial activity monitoring and morphology tracking.

Accordingly, the present invention also provides a two-component aggregation-induced emission fluorescent light emitting agent (AIEgen) system comprising: the compound shown in the formula (1) and TPE-2 BA.

As described above, the two-component aggregation-induced emission fluorescent light emitting agent (AIEgen) system provided by the present invention can be used for monitoring the metabolic state of microorganisms. Accordingly, the present invention also provides a method of monitoring the metabolic state of a microorganism, the method comprising: the metabolic state of the microorganism is monitored using the above-described dual aggregation-induced emission fluorescent light emitter (AIEgen) system.

Specifically, the above-mentioned compound represented by formula (1) in the two-component aggregation-induced emission fluorescent light emitting agent (AIEgen) system, which is imaged in response to the entire microbial cells, can be used to detect the exact number of total microbes, and TPE-2BA responds to the activity of microbes, and thus, the activity of microbes can be accurately calculated by precisely determining the number of dead microbial cells and total microbial cells.

It is known to those skilled in the art that in the development of antibiotics and antimicrobial materials, there is a need to characterize and quantify the killing efficiency of these materials. Therefore, the present invention also applies the two-component aggregation-induced emission fluorescent luminescent agent (AIEgen) system provided by the present invention to a quantitative determination of the activity of microorganisms, and thus provides a method for quantitatively determining the activity of microorganisms, the method comprising: the activity of the microorganism was quantitatively detected using the double aggregation induced luminescence fluorescent luminescent agent (AIEgen) system as described above.

The quantitative capability of the bi-component AIEgen system is tested by three methods, namely a plate reader, a fluorescence microscope/laser confocal microscope and a flow cytometry.

For the microplate reader method, live and dead bacteria were mixed in different ratios to produce a given percentage of live bacteria. The mixture was then stained with DCQA and TPE-2 BA. Finally, peak intensities at different excitation wavelengths were measured using a microplate reader. Since the red/blue fluorescence ratio has a good linear correlation with a given percentage of viable bacteria mixture, the two-component AIEgen system enables an efficient and reliable characterization of the activity of microorganisms by using a microplate reader.

For fluorescence microscopy/confocal laser microscopy, crosstalk can be greatly reduced and systematic errors are minimized due to the minimal spectral overlap between the above compound of formula (1) and TPE-2 BA. The sample preparation process is the same as that of the microplate reader. After staining, the mixture of different percentages of viable bacteria was imaged by confocal laser microscopy through the channel (N) of the above compound represented by formula (1)_{Total number of}) The total bacterial cell count can be obtained by using TPE-2BA channel (N)_{Death by death}) The number of dead cells can be calculated and then the percentage of viable bacteria counted (L%) can be calculated by equation 1:

equation 1:

for flow cytometry, applied to test the staining capacity of the two-component AIEgen system, mixtures of different percentages of live bacteria can be analyzed by reading the fluorescence signal of the labeled sample, so that the activity of the microorganisms can be easily distinguished.

The microbial biofilm is the most common life form of microorganisms in nature, and can realize quick adaptation to the environment, generation of drug resistance and cooperation with other microorganisms in a biofilm state. According to IUPAC, biofilms are aggregates of microorganisms in which cells are often embedded in an autogenous matrix of Extracellular Polymers (EPS) that adhere to each other and/or to surfaces. Therefore, since the biofilm is composed of many substances, its structure is complicated and the study of its structure and relative function becomes extremely difficult. Therefore, there is a strong need for a method that can visualize the structure and function of a biofilm.

As mentioned above, the two-component AIEgen system is very effective for planktonic microorganism imaging and activity recognition, and due to its AIE properties, should work well in biofilms.

Accordingly, the present invention applies the two-component AIEgen system as described above to the monitoring of the morphology and metabolic state of a biofilm of microorganisms and thus proposes a method of monitoring the morphology and metabolic state of a biofilm of microorganisms, said method comprising: the morphology and metabolic state of the biofilm of microorganisms is monitored using the above-described dual aggregation-induced emission fluorescent light emitter (AIEgen) system to visualize the morphology and metabolic state of the biofilm.

In the above two-component AIEgen system, the above compound represented by formula (1) emitting red light is responsible for monitoring morphology based on its broad dyeing property and low toxicity, and two positive charges effectively and firmly impart the general dyeing ability of the above compound represented by formula (1) to negatively charged microorganisms through electrostatic interaction. Furthermore, it is known to those skilled in the art that when the biofilm reaches the mature stage, the core area tends to be anoxic and less nutritious, and therefore more dead cells are inside, and therefore, the blue-emitting AIEgen TPE-2BA is responsible for staining the dead microorganisms. In this case, images of DCQA and TPE-2BA were merged, so that it was more straightforward to observe the morphology and activity of the biofilm simultaneously. Therefore, against the background of the overall biofilm morphology, the distribution of dead cells can be more clearly represented, thereby visualizing the morphology and activity of the biofilm.

In conclusion, the invention successfully designs a novel near infrared AIEgen, (E) -4- (2- (7- (diphenylamino) -9-ethyl-9H-carbazole-2-yl) vinyl) -1- (3- (trimethylamino) propyl) quinoline-1-ammonium bromide (DCQA). DCQA has a broad spectrum of staining abilities for all microorganisms, including gram positive bacteria, gram negative bacteria, and fungi. TPE-2BA is a blue fluorescent probe specifically used for imaging dead microorganisms, and the metabolic state of planktonic microorganisms and their biofilms can be successfully monitored by introducing TPE-2 BA. Thus, a two-component AIEgen system with minimal spectral overlap was designed and manufactured for quantification of microbial activity and monitoring of biofilm formation. In addition, the quantitative capability of this two-component AIEgen system has been investigated by three different most commonly used techniques (i.e., confocal laser microscopy, microplate reader, and flow cytometry). Finally, this two-component AIEgen system successfully imaged three microbial biofilms.

Examples

In this context, unless otherwise specified, the test methods employed are all conventional methods, and, unless otherwise specified, the test materials used in the following examples are all purchased from conventional reagent stores.

1. Experimental part

1.1 instruments and materials

All chemicals and reagents were commercially available and used without further purification.

¹H and¹³c NMR spectra were measured on a Bruker ARX 400NMR spectrometer using CDCl₃And DMSO-d₆As a solvent, tetramethylsilane (TMS; δ ═ 0ppm) was selected as an internal reference. High resolution mass spectra (HR-MS) were obtained on a Finnigan MAT TSQ 7000 mass spectrometer system operating in MALDI-TOF mode. Absorption spectra were measured on a Milton Roy Spectronic 3000Array spectrophotometer. Steady state Photoluminescence (PL) spectra were measured on a Perkin-Elmer spectrofluorometer LS 55. Absolute fluorescence quantum yield is integrated by calibrationBall (Labsphere) measurements. Confocal laser scanning microscope images were collected on a Zeiss laser scanning confocal microscope (LSM 710) and analyzed using ZEN 2009 software (Carl Zeiss).

3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich as received. For bacterial culture, Tryptic Soy Broth (TSB), Luria-Bertani broth (LB), yeast extract-protein D-glucose (YPD), Mueller Hinton broth (MH), and casein hydrolysate were purchased from sorafei international trade limited and all used directly after receiving. Glucose was purchased from Sigma-Aldrich. Crystal violet is available from sorafeo international trade ltd. For cell culture, Dulbecco's Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), penicillin and streptomycin solutions were purchased from Invitrogen.

1.2 Strain information

Staphylococcus aureus (s.aureus, ATCC 6538), bacillus subtilis (ATCC 6633), micrococcus luteus (m.luteus, ATCC 9341), escherichia coli (e.coli, 8099), klebsiella pneumoniae (k.pneumoniae, ATCC 700603), serratia marcescens (s.marcocens, CMCC 41002), candida albicans (c.albicans, ATCC 10231), yeast (s.cerevisiae, ATCC 9763).

1.3 Experimental methods

1.3.1 bacterial culture and staining

Individual bacterial colonies on solid media were transferred to 5mL liquid media and grown at 37 ℃ for 10 h. The concentration of bacteria was determined by measuring the optical density at 600nm (OD600) and then 10 was measured⁹The bacteria of the CFU were transferred to a 1.5mL microcentrifuge tube. The bacteria were harvested by centrifugation at 11700g for 3 minutes. After removing the supernatant, the bacteria were killed with 200 μ L of 75% alcohol (dead bacteria) or treated with an equal amount of PBS (live bacteria) and then washed with PBS. Then, 1mL of the appropriate concentration of dye solution in saline was added to the microcentrifuge tube. After vortexing, the bacteria were incubated in a shaking incubator at 30 ℃ for a designed period of time.

1.3.2 fungal culture and staining

Individual bacterial colonies on solid media were transferred to 5mL liquid media and grown at 25 ℃ for 10 h. The concentration of bacteria was determined by measuring the optical density at 600nm (OD600) and then 10 was measured⁹The bacteria of the CFU were transferred to a 1.5mL microcentrifuge tube. The bacteria were harvested by centrifugation at 11700g for 3 minutes. After removing the supernatant, the bacteria were killed with 200 μ L of 75% alcohol (dead bacteria) or treated with an equal amount of PBS (live bacteria) and then washed with PBS. Then, 1mL of a dye solution of the appropriate concentration in saline was added to the microcentrifuge tube. After vortexing, the bacteria were cultured in a shaking incubator at 25 ℃ for the designed time.

1.3.3 biofilm culture and staining

The overnight microbial culture was subcultured to the relevant medium (s. aureus: tsb. e. coli supplemented with glucose: lb.c. albicans supplemented with casein hydrolysate: YPD broth) at an OD600 of 0.5 and then 2mL of the bacterial/fungal suspension was added to the confocal dish. The dishes were incubated statically at 37 ℃. After 72 hours, the medium was removed and the plates were washed with sterile PBS. Then, 1mL of the probe was added to the petri dish and incubated for the designed time.

1.3.4 quantitative analysis of Escherichia coli with different activities by enzyme-labeling instrument

Individual E.coli colonies on solid medium were transferred to 5mL of liquid medium and grown at 37 ℃ for 10 h. After reaching the logarithmic phase, 500. mu.L of the culture suspension was transferred to a 1.5mL microcentrifuge tube. The bacteria were centrifuged at 11700g for 3 minutes. After removal of the supernatant, the bacteria were killed with 1.5mL of 75% alcohol (dead bacteria) or treated with an equal amount of PBS (live bacteria). Then, live bacteria and dead bacteria were mixed at different ratios (L/D ratios), that is, 0, 10%, 30%, 50%, 70%, 90% and 100%. Next, the bacteria were centrifuged at 11700g for 3 minutes. Then, 200 μ L of a solution in which the dye was dissolved in an appropriate concentration of buffer (pH 10) was added to the microcentrifuge tube. After vortexing, 100 μ L of the suspension was transferred to a 96-well plate and incubated at 37 ℃ for the designed time. Finally, the fluorescence peak was recorded by a microplate reader (Varioskan LUX multimode microplate reader) using a black 96-well plate (Thermo Scientific, opaque, flat-bottomed, non-sterile). The integrated intensities of the blue (460. + -.5 nm) and red (730. + -.5 nm) emissions of the suspensions excited at 310. + -.10 nm and 519. + -.10 nm are obtained and the red/blue fluorescence Ratio (Ratio R/B) for each live/dead Ratio of E.coli is calculated. Each point represents the average of four measurements.

1.3.5 quantitative analysis of bacteria/fungi with different viability by flow cytometry.

Individual E.coli colonies on solid medium were transferred to 5mL of liquid medium and grown at 37 ℃ for 10 h. After a log phase was reached, 500. mu.L of the bacterial suspension was transferred to a 1.5mL microcentrifuge tube. The bacteria/fungi were centrifuged at 11700g for 3 minutes. After removal of the supernatant, the bacteria were killed with 1.5mL of 75% alcohol (dead bacteria) or treated with an equal amount of PBS (live bacteria). The live bacteria/fungi and dead bacteria/fungi were then mixed in different ratios (L/D ratios), i.e., 0, 10%, 30%, 50%, 70%, 90% and 100%. Next, the bacteria/fungi were centrifuged at 11700g for 3 minutes. Then, 2mL of a dye solution (pH 10) dissolved in an appropriate concentration of buffer was added to the microcentrifuge tube. After vortexing, the suspension was incubated at 37 ℃ for the designed time. Finally, bacteria were measured by flow cytometry (BD FACS Ariac III). The integrated intensities of the blue (450. + -.40 nm) and red (780. + -.60 nm) emissions of the suspensions excited at 405nm and 561nm are obtained and the red/blue fluorescence Ratio (Ratio R/B) for each live/dead Ratio of E.coli is calculated. Each point represents the average of 5000 counts.

1.3.6 laser scanning confocal microscope quantitative analysis of bacteria/fungi of different viability

Individual E.coli colonies on solid medium were transferred to 5mL of liquid medium and grown at 37 ℃ for 10 h. After the log phase was reached, 500. mu.L of bacteria were transferred to a 1.5mL microcentrifuge tube. The bacteria were centrifuged at 11700g for 3 minutes using a centrifuge. After removal of the supernatant, the bacteria were killed with 1.5mL of 75% alcohol (dead bacteria) or treated with an equal amount of PBS (live bacteria). Then, live bacteria and dead bacteria were mixed at different ratios (L/D ratios), that is, 0, 10%, 30%, 50%, 70%, 90% and 100%. Next, the bacteria were centrifuged at 11700g for 3 minutes. Then, 100 μ L of a solution in which the dye was dissolved in an appropriate concentration of buffer (pH 10) was added to the microcentrifuge tube. After vortexing, the suspension was incubated at 37 ℃ for the designed time. Finally, confocal fluorescence images were obtained with a Zeiss LSM 800 confocal laser scanning microscope. Different given percentages of live E.coli were stained with DCQA and TPE-2 BA. The activity of the E.coli suspension was calculated from the CLSM image of each given percentage of live E.coli.

1.3.7 Experimental procedures for Minimum Inhibitory Concentration (MIC) testing

Single colonies of s.aureus, e.coli or c.albicans were cultured in Mueller-Hinton broth. After reaching the logarithmic phase, 1X 10⁶mL^-1The cells of (a) were subjected to MIC testing. DCQA and TPE-2BA were diluted by gradient through sterile MH broth. After diluting the probe, a volume of standard inoculum equal to the diluted probe was added to each dilution vessel to achieve a microbial concentration of about 5X 10⁵Cell mL^-1. The 96-well plate was incubated at 37 ℃ for 24 hours in an incubator. After incubation, the dilution series of containers was observed for microbial growth, usually expressed as turbidity. The last tube in the dilution series not shown was recorded as the MIC value for the probe.

Example 1: synthesis of DCQA

The design principle and the synthetic route of DCQA are shown in FIG. 1. Specifically, 7- (diphenylamino) -9-ethyl-9H-carbazole-2-carbaldehyde (0.50g, 1.28mmol) and 1- (3-trimethylaminopropyl) -4-methylquinoline dibromide (0.52g, 1.28mmol) were dissolved in anhydrous ethanol (15 mL). 2 drops of piperidine were added and the solution was refluxed under nitrogen overnight. After cooling to room temperature, the solvent was removed by evaporation under reduced pressure. The residue was purified by neutral alumina column chromatography eluting with a dichloromethane/methanol mixture solvent to afford DCQA as a dark brown crystalline solid. (0.62g, yield: 62%).¹H NMR(400MHz,DMSO-d₆,ppm):δ9.45(d,J＝6.5Hz,1H),9.17(d,J＝8.7Hz,1H),8.64-8.59(m,2H),8.51-8.41(m,2H),8.30-8.26(m,2H),8.17(d,J＝8.1Hz,1H),8.11-8.06(m,2H),7.82(d,J＝8.3Hz,1H),7.33-7.29(m,4H),7.18(s,1H),7.08-7.03(m,6H),6.87(d,J＝8.4Hz,1H),5.03(t,J＝7.3Hz,2H),4.41-4.35(m,2H),3.60-3.56(m,2H),3.08(s,9H),2.45-2.41(m,2H),1.27(t,J＝7.1Hz,3H).¹³C NMR(100MHz,DMSO-d₆,ppm):δ153.27,147.46,147.32,146.70,145.13,141.80,140.12,137.80,135.16,132.09,129.36,128.96,126.48,124.61,123.64,122.84,121.89,120.97,120.00,118.13,117.56,116.65,115.76,109.12,103.98,61.93,53.02,52.34,36.86,23.09,13.56。

The strong D-a intensity in the fluorophore can facilitate Intramolecular Charge Transfer (ICT), resulting in a smaller electronic bandgap and longer absorption/emission wavelength. Besides the electron-withdrawing ability, the introduction of the electron acceptor with positive charge can also enable the fluorophore to have unique biological functions, and have universal microorganism targeting and imaging ability. To validate these hypotheses, DCQA contains quinolinium salts as acceptor, carbazole fragments as donor, and pi-bridge, as shown in fig. 1A. DCQA was synthesized in high yield by a typical Knoevenagel condensation reaction and was well characterized (fig. 1A). Prior to optical studies, Density Functional Theory (DFT) calculations of the new molecule were performed to understand its ICT. As shown in fig. 6, the electron cloud of HOMO of DCQA is mainly located on the carbazole ring, the diphenylamino group, and the carbazole skeleton. LUMO is mainly contributed by the orbital of the acceptor moiety and part of the carbazole ring.

The photophysical properties of DCQA were subsequently studied by ultraviolet-visible absorption and Photoluminescence (PL) spectroscopy. As shown in fig. 7A, DCQA shows maximum absorption at 519nm in dimethyl sulfoxide (DMSO) due to ICT from the carbazole moiety to the quinolinium salt group.

As shown in FIGS. 1B and 1C, the AIE characteristics of DCQA were studied in DMSO/toluene mixed solvents of different toluene contents. The compound shows very weak emission in DMSO, since the energy of the excited state is consumed by the active intramolecular movement through non-radiative pathways. After the toluene was gradually added to the DMSO solution, the fluorescence of the molecule became stronger. Due to the RIM mechanism, the fluorescence intensity of DCQA was 306 times higher at 99% by volume toluene than in pure DMSO solution. These results indicate that DCQA has AIE activity and emits near infrared light at 729 nm. Due to the AIE properties, DCQA showed bright solid state fluorescence at 729nm with a fluorescence quantum yield of 1.2% as determined by integrating sphere (fig. 1C). The above results also show that the emission color of these fluorophores can be extended relatively easily into the near infrared region by adjusting the ICT intensity. Time resolved fluorescence measurements of solid state DCQA showed a lifetime of 1.484ns (fig. 7B). Furthermore, the compounds show a very large stokes shift (Δ ν ═ 300nm), which can be attributed to the excited state intramolecular charge transfer between the electron donor and the electron acceptor within the same dye molecule. A larger stokes shift is advantageous for biological imaging because the interference between excitation and emission is small.

In summary, the integration of large stokes shifts, near infrared emission, and AIE properties of two positive charges with short carbon chains makes DCQA a promising candidate for biological applications.

Example 2: application of DCQA in microbial staining

The microbiological imaging experiments were initially performed by incubating DCQA with three indicated types of strains, s.aureus (for gram positive bacteria), e.coli (for gram negative bacteria), c.albicans (for fungi), followed by CLSM imaging with excitation at 560nm, as shown in fig. 2A-2C. As can be seen from FIGS. 2A-2C, all three microorganisms were efficiently stained by DCQA.

In addition, DCQA showed good staining capacity in both the live and dead states. The incubation time test showed that DCQA can interact with various microorganisms in a short time and a strong signal can be detected in only 5 minutes, as shown in fig. 8. Thereafter, the fluorescence signal did not change much, indicating that DCQA had a suitable staining window for both imaging and high throughput analysis. This demonstrates that DCQA can be used as a universal detection probe for various microorganisms, with high staining efficiency, rapid reactivity and short interaction time.

FIGS. 2D-E show the in situ fluorescence spectra of DCQA obtained by laser confocal microscopy in different microorganisms. DCQA emission from red to near infrared was detected in both live and dead colonies for all s.aureus, e.coli and c.albicans. Although the emission peaks obtained by confocal were blue-shifted, 686, 687 and 690nm, respectively, due to the lower sensitivity and detection limit of confocal detectors compared to PL detectors. This indicates that DCQA has universal staining ability for all microorganisms, high efficiency, and no metabolic selectivity.

In addition, negatively charged dominant bacterial components such as LPS, DNA and RNA were selected to study the binding capacity of DCQA (fig. 9). The results indicate that DCQA can interact with these three negatively charged components due to electrostatic interactions. This is consistent with the phenomenon observed in fluorescence images, i.e., the entire microbial cell is illuminated by DCQA.

Example 3: application of two-component aggregation-induced emission fluorescent luminous agent system in monitoring metabolic state of microorganism

The metabolic state of the microorganism is closely related to the functions, the biofilm forming capability, the secretion of virulence factors, the drug resistance generation and other characteristics of the microorganism. With the change of environment, the metabolic state of the microorganism is also changed. Especially for opportunistic pathogens, changes in their metabolic levels may lead to conditions that are harmful to us. The activity of microorganisms is a key factor in the metabolism of microorganisms. It has been reported that some bacteria sacrifice themselves when faced with a particular antibiotic, and that other bacteria are warned of danger by dispersing their resistance genes. The remaining microbial species will receive the resistance gene and directly acquire resistance to the particular antibiotic. Therefore, it is very important to monitor the metabolic state of the microorganism. In this work, factors of microbial activity were selected as criteria. To achieve minimal spectral overlap with DCQA, the metabolic sensor probe should have a suitable emission spectrum to match DCQA. However, ultraviolet or deep blue light is harmful to biological systems, and therefore the validated dead microorganism AIEgen probe TPE-2BA was chosen as a co-metabolic sensor. The emission peak of TPE-2BA is around 450nm, which makes it a perfect combination of microbial activity monitoring and morphology tracking.

To demonstrate the general ability of the two-component AIEgen system consisting of DCQA and TPE-2BA to efficiently monitor the metabolic state of microorganisms, three indicator strains (s.aureus, e.coli, c.albicans) were used for confocal imaging. In addition, three other types of strains were selected among these three types: subtilis, gram positive, is a bacterium produced by an endospore; marcocens, gram negative, is an indicator strain for air filtration and purification system testing. Cerevisiae, a fungus, is the most common yeast in our daily life.

Three microorganisms were selected, gram positive bacteria, gram negative bacteria and fungi respectively. Within each category, two most commonly studied types of strains were selected, with s.aureus and b.subtilis representing gram-positive bacteria; coli and s. marcocens represent gram-negative bacteria. C. albicans and s. cerevisiae represent fungi.

Each microorganism in the logarithmic growth phase was divided into two groups. One group received 75% ethanol treatment and was effective in killing microorganisms. The other group did not receive any treatment. Then the two groups were mixed with 1: 1 to prepare a sample. Confocal images were taken directly after 30 min incubation with the two-component AIEgen system.

In fig. 3A, the DCQA channel shows that all gram positive bacteria have been stained and imaged. The TPE-2BA channel is effective in indicating dead bacteria. Live and dead bacteria can be easily distinguished by the combined channels, red for live bacteria and blue or white for dead bacteria. As shown in fig. 3B and 3C, the same results were obtained by confocal laser microscopy for gram-negative bacteria and fungi.

These results indicate that a two-component AIEgen system has been successfully designed and manufactured and can be used to efficiently detect microbial activity. In this system, DCQA is imaged in response to the entire microbial cell in order to detect the exact number of total microbes, while TPE-2BA is responsive to the activity of the microbes. The activities of gram-positive bacteria, gram-negative bacteria and fungi can be accurately calculated by accurately determining the number of dead microbial cells and total microbial cells.

The effect of the two-component AIEgen system on bacterial activity was further investigated by MIC analysis. MIC analysis the toxicity of the two-component AIEgen system was further investigated (table 1). The MIC values of DCQA for the three indicator strains, s.aureus, e.coli and c.albicans, exceeded 80uM, eight times their working concentrations. The MIC values of TPE-2BA for the three indicator strains exceeded 400uM, which is eight times that of their working concentrations. The results indicate that the two components are non-toxic to microorganisms and that the two-component AIEgen system is suitable for microbial morphology and activity detection and visualization.

TABLE 1 MIC results of DCQA and TPE-2BA for 3 indicator strains

Example 4: application of double-aggregation induced luminescence fluorescent luminescent agent system in quantitative detection of activity of microorganisms

In addition to the qualitative analysis described above, our two-component AIE system also works well for more meaningful quantitative analyses. In the development of antibiotics and antimicrobial materials, there is a need to characterize and quantify the killing efficiency of these materials. Several methods are commonly used to quantitatively detect the activity of microorganisms. The most commonly used are microplate readers, fluorescence/confocal laser microscopes, flow cytometry and SEM, among which the microplate reader is the most efficient and readily available technology because of its high throughput and high sensitivity; fluorescence microscopy can provide more detailed information about the microorganism, such as morphology and state of membrane integrity; by using the fluorescent probe, the number of live bacteria or dead bacteria can be easily estimated and counted by an image; the remaining methods are not fluorescence based and therefore have lower sensitivity than the former. Furthermore, plate counting can only determine the number of viable microorganisms, whereas SEM requires a fixation process, which limits their application in the characterization of microbial activity, since they are not in situ methods. Therefore, the three most commonly used techniques were chosen to test the quantitative capability of the two-component AIEgen system. Coli was selected as a model microorganism.

An enzyme-labeling instrument:first, microplate readers have been employed due to their high throughput and large sample volume characteristics. Will be alive andcoli are mixed in different proportions to produce a given percentage of viable bacteria. The mixture was then stained with DCQA and TPE-2BA for 30 minutes. Finally, peak intensities at different excitation wavelengths were measured using a microplate reader.

For DCQA, the excitation wavelength is 519 + -10 nm, and the emission peak is 730 + -5 nm. For TPE-2BA, the excitation wavelength is 310 +/-10 nm, and the emission peak is 460 +/-5 nm. It can be observed in fig. 4A that the red/blue fluorescence ratio has a good linear correlation with a given percentage (from 0% to 100%) of live bacterial mixture, indicating that the two-component AIEgen system is able to efficiently and reliably characterize the activity of microorganisms by using a microplate reader.

Laser confocal microscopy:in addition, due to the minimal spectral overlap between DCQA (730nm) and TPE-2BA (460nm), crosstalk has been greatly reduced and systematic errors minimized. The microbial activity discrimination ability of this two-component AIEgen system was then investigated with a confocal laser microscope. The sample preparation process is the same as that of the microplate reader. After 30 minutes of staining, mixtures of different percentages of viable bacteria were imaged by confocal laser microscopy, with over 300 bacterial cells collected from each mixture. As shown in fig. 3, through the DCQA channel (N)_{Total number of}) The total bacterial cell count can be obtained by using TPE-2BA channel (N)_{Death by death}) The number of dead cells can be calculated. The percentage of viable bacteria counted (L%) can then be calculated by equation 1.

Equation 1:

the excitation wavelength of the laser confocal microscope used for DCQA is 560nm, and the emission range is 600-750 nm. For TPE-2BA, 405nm was chosen as the excitation wavelength and 420-500nm was chosen as the emission range. There is no crosstalk between the two channels due to the minimal spectral overlap of the two-component AIEgen system. It can be observed from fig. 4B that the red/blue fluorescence ratio has a good linear correlation with a given percentage (from 0% to 100%) of the live bacterial mixture, indicating that the two-component AIEgen system is able to efficiently and reliably characterize the activity of microorganisms by using a microplate reader.

Flow cytometry:flow cytometry is a powerful technique in the field of biological research that can provide high throughput analysis of a variety of biological samples (e.g., cells and microorganisms). By reading the fluorescence signal of the labeled sample, data can be continuously acquired and analyzed. The activity of the microorganisms can be easily distinguished by flow cytometry. Flow cytometry was then applied to test the staining capacity of the two-component AIEgen system. The sample preparation process is the same as that of a microplate reader method and a laser confocal microscope. After 30 minutes of staining, mixtures of different percentages of viable bacteria were analyzed by flow cytometry, each mixture analyzed 5000 bacterial cells. The histogram and linear fit results are given in fig. 4C and 4D.

Example 5: application of double-aggregation induced luminescence fluorescent luminous agent system in monitoring of form and metabolic state of biological membrane of microorganism

The microbial biofilm is the most common life form of microorganisms in nature, and can realize quick adaptation to the environment, generation of drug resistance and cooperation with other microorganisms in a biofilm state. According to IUPAC, biofilms are aggregates of microorganisms in which cells are often embedded in an autogenous matrix of Extracellular Polymers (EPS) that adhere to each other and/or to surfaces. Therefore, since the biofilm is composed of many substances, its structure is complicated and the study of its structure and relative function becomes extremely difficult. However, there is an urgent need to visualize its structure and function. As indicated above, the two-component AIEgen system is very effective for planktonic microorganism imaging and activity recognition, and due to its AIE properties, it is believed that the system should work well in biofilms. Therefore, three basic microbial biofilms were chosen for illustration, namely s. From the first set of FIGS. 5A-C, DCQA was found to have described morphology, and it is believed that DCQA stains all microbial cells in a biofilm. Mushroom-like morphology has been well stained and revealed by DCQA as found in s. Coli biofilm was characterized by loose morphology and was also well stained and visualized by DCQA (fig. 5B). The thickness was found to be about 40um, slightly thicker than s. This is because the distribution of e.coli bacterial cells is much looser compared to s.aureus biofilms. DCQA successfully demonstrated the differences between the two major bacterial biofilm models. DCQA also successfully imaged c.albicans biofilms with typical morphology (fig. 5C). These results are consistent with the design principle that red-emitting DCQA is responsible for monitoring morphology based on its broad staining properties and low toxicity. The two positive charges effectively and strongly confer DCQA universal staining capability on negatively charged microorganisms through electrostatic interaction. In the second group of FIGS. 5A-C, the metabolic state of each biofilm has been obtained and visualized for blue-emitting AIEgen TPE-2 BA. During the growth of biofilms, there are four stages, namely bacterial adhesion, biofilm formation, biofilm maturation and biofilm diffusion. In mature biofilms, some microbial cells die due to the natural life cycle and the development of biofilm structure and function. When the biofilm reaches the mature stage, the core region tends to be hypoxic and less nutritious, and therefore more dead cells are inside. This phenomenon was significantly manifested in mushroom-like s.aureus biofilms (second panel, fig. 5A). Aureus cells are located in the bottom and core regions of the biofilm, consistent with the description in the classical biofilm model. Coli biofilm in fig. 5B, it looks like an absorbent foam, in which the bacterial cells are randomly dispersed, due to its polysaccharide-rich extracellular matrix. Coli biofilms are in a lower hypoxic environment due to loose packing, and therefore fewer dead cells are found there. For c.albicans biofilms, most dead cells were found at the site of aggregation due to the natural life cycle of fungal cells. In the third group of FIGS. 5A-C, merged images of DCQA and TPE-2BA are presented. And the observation of the form and activity of the biological membrane is more direct. Morphology can be observed directly in 3D merged images, while activity can be reported in color, where blue or white represents dead cells and red represents live cells. Meanwhile, the distribution of dead cells can be more clearly shown by taking the form of the total biomembrane as the background. The orthographic view further demonstrates that the two-component AIEgen system can visualize the morphology and activity of biofilms.

The above examples show that the present invention successfully designs and synthesizes NIR emitting AIEgen, DCQA. The precise molecular design allows DCQA to have specific AIE properties and good biocompatibility. The broad-spectrum staining ability of DCQA facilitates in situ and real-time imaging of gram-positive bacteria, gram-negative bacteria and fungi in both live and dead states. The metabolic status of planktonic microorganisms and their biofilms has been successfully monitored by the introduction of TPE-2BA, a blue fluorescent probe specifically used for imaging dead microorganisms. With the support of TPE-2BA, which is only suitable for dead microorganism staining, we further constructed a two-component AIEgen system for the detection of microbial activity. Furthermore, the system has been successfully applied to monitor the morphology and metabolic state of microbial biofilms. This two-component AIEgen system has proven to have great potential in the detection, visualization and quantitative analysis of microorganisms.

The invention is not to be considered as limited to the specific embodiments shown and described, but is to be understood as being modified in all respects, all changes and equivalents that come within the spirit and scope of the invention.

Claims (10)

1. A compound, wherein the structure of the compound is shown as formula (1):

wherein two R are₁Independently selected from H, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkyl-NCS, alkyl-N₃And alkyl-NH₂Wherein two R are₁May be the same or different;

d is

Wherein R is₂Is an alkyl chain having quaternary ammonium end groups;

X^-is a counterion, preferably X is independently selected from F, Cl, Br, I and PF₆。

2. The compound of claim 1, wherein the structure of the compound is represented by formula (2) or (3):

3. the compound of claim 1, wherein the compound is

4. The compound of claim 1, wherein the compound is

5. Use of a compound according to any one of claims 1 to 4 for staining a microorganism.

6. A method of staining a microorganism, the method comprising: staining said microorganism with a compound according to any one of claims 1-4.

7. A dual-assembly induced-luminescence fluorescent-luminescent agent system, comprising: the compound according to any one of claims 1 to 4 and a tetraphenylethylene derivative containing a boronic acid structure.

8. A method of monitoring the metabolic state of a microorganism, the method comprising: monitoring the metabolic state of said microorganism using the dual aggregation-induced emission fluorescent emitter system of claim 7.

9. A method of quantitatively detecting the activity of a microorganism, the method comprising: quantitatively detecting the activity of said microorganisms using the dual aggregation induced luminescence fluorescent emitter system of claim 7.

10. A method of monitoring the morphological and metabolic state of a biofilm of a microorganism, the method comprising: the use of the dual aggregation-induced emission fluorescent emitter system of claim 7 to visualize the morphology and metabolic state of said biofilm, thereby monitoring the morphology and metabolic state of a biofilm of microorganisms.

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