Compound for detecting target analyte, preparation method thereof and application thereof in detecting target analyte
阅读说明：本技术 用于检测目标分析物的化合物、其制备方法及其检测目标分析物的应用 (Compound for detecting target analyte, preparation method thereof and application thereof in detecting target analyte ) 是由 唐本忠 周成成 许文涵 于 2019-06-06 设计创作，主要内容包括：本发明提供了用于检测目标分析物的化合物、其制备方法及其在荧光探针、传感器、检测试剂盒等中的应用,所述化合物由下式I表示：<Image he="216" wi="700" file="DDA0002087018230000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>B<Sup>+</Sup>表示带正电荷的基团,各个R1独立地表示氢或者使得所述化合物的正辛醇/水分配系数值在2.0以上的疏水基团,条件是至少一个R1是使得所述化合物的正辛醇/水分配系数值在2.0以上的疏水基团,n表示1-4中的整数,m表示1-100中的整数；以及X<Sup>-</Sup>为抗衡阴离子。所述化合物能够准确快速地鉴定各种目标分析物。(The present invention provides a compound for detecting a target analyte, which is represented by the following formula I: B + represents a positively charged group, each R1 independently represents hydrogen orA hydrophobic group such that the compound has an n-octanol/water partition coefficient value of 2.0 or more, with the proviso that at least one R1 is a hydrophobic group such that the compound has an n-octanol/water partition coefficient value of 2.0 or more, n represents an integer from 1 to 4, and m represents an integer from 1 to 100; and X ‑ Are counter anions. The compounds enable accurate and rapid identification of various analytes of interest.)
1. A compound for detecting a target analyte, characterized by the following formula I:
wherein the content of the first and second substances,
l represents a flexible chain linking group,
B +represents a group having a positive charge,
each R1 independently represents hydrogen or a hydrophobic group such that the compound has an n-octanol/water partition coefficient value of 2.0 or more, provided that at least one R1 is a hydrophobic group such that the compound has an n-octanol/water partition coefficient value of 2.0 or more,
n represents an integer of 1 to 4,
m represents an integer of 1 to 100; and
X -are counter anions.
2. A method for preparing a compound for detecting a target analyte, comprising the steps of:
reacting a compound of formula III with a compound of formula IV in the presence of a catalyst and an organic solvent to obtain a compound of formula V, and
reacting a compound of formula V with B- (R1) in the presence of a catalyst and an organic solvent nReacting the compound represented thereby to obtain a compound of formula I;
wherein C is a nucleophilic group, X is a halogen, preferably Br,
b is an organic precursor group capable of carrying a positive charge, preferably an amine or amino group,
n is an integer of 1 to 3,
B +represents a positively charged group;
each R1 independently represents hydrogen or a hydrophobic group such that the compound has an n-octanol/water partition coefficient value of 2.0 or more, with the proviso that at least one R1 is a hydrophobic group such that the compound has an n-octanol/water partition coefficient value of 2.0 or more and
m represents an integer of 1 to 100.
3. The method of claim 2, wherein the step of removing the substrate comprises removing the substrate from the substrate
C may be selected from-OH, -SH, NH 2Or a substituted amine group, preferably-OH,
optionally, the catalyst is selected from a metal carbonate catalyst, a metal hydroxide catalyst, a metal alkoxide catalyst, or any combination thereof, preferably a metal carbonate catalyst (e.g., sodium carbonate, potassium carbonate), more preferably K 2CO 3，
Preferably, the organic solvent is selected from any one of aromatic hydrocarbon solvents, aliphatic hydrocarbon solvents, oxygen-containing heterocyclic solvents, alcohol solvents, ketone solvents, ester solvents, ether reagents, and mixtures thereof, and more preferably from any one of tetrahydrofuran, acetone, ethanol, methanol, ethyl acetate, diethyl ether, and mixtures thereof.
4. The compound of claim 1 or the method of any one of claims 2-3, wherein the compound is represented by formula II below,
wherein R2, R3, R4 and R5 may be the same or different and are independently selected from hydrogen, hydroxyl, substituted or unsubstituted hydroxyalkyl, amino, substituted or unsubstituted alkylamino, substituted or unsubstituted alkyl, substituted or unsubstituted unsaturated hydrocarbon, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl, or combinations thereof, and in the case of substitution, at least one hydrogen of hydroxyalkyl, alkylamino, alkyl, unsaturated hydrocarbon, cycloalkyl, heteroalkyl, aryl and heteroaryl is selected from halogen, hydroxyl, aldehyde, carboxyl, amino, C2-C18 alkenyl optionally substituted with one or more C6-C18 aromatic cycloalkyl groups or an aromatic heterocyclic group of ring carbon atoms 5-18, and an aromatic heterocyclic group optionally substituted with one or more C6-C18 aromatic cycloalkyl groups or ring carbon atoms 5-18 is selected from the group consisting of halogen, hydroxyl, aldehyde, carboxyl, amino, and aromatic heterocyclic groups of ring carbon atoms 5-18 Substituted C2-C18 alkynyl, C1-C18 alkyl optionally substituted with one or more C6-C18 aromatic cyclic hydrocarbon groups or cyclic aromatic heterocyclic groups of ring carbon atoms 5-18, aromatic cyclic hydrocarbon groups of ring carbon atoms 6-18, cyclic aromatic heterocyclic groups of ring carbon atoms 5-18, mercapto, cyano and nitro;
l represents a substituted or unsubstituted alkoxy linking group or a substituted or unsubstituted alkyl chain linking group;
each R1 is the same or different and is independently a substituted or unsubstituted C1-C18 alkyl group, an aromatic cyclic hydrocarbon group having 6-18 ring-forming carbon atoms, or a substituted or unsubstituted cyclic hydrocarbon group having 6-18 ring-forming carbon atoms, and in the case of substitution, at least one hydrogen of the C1-C18 alkyl group, the aromatic cyclic hydrocarbon group, and the cyclic hydrocarbon group is selected from a halogen atom, a hydroxyl group, an aldehyde group, a carboxyl group, an amino group, a C2-C18 alkenyl group optionally substituted with one or more C6-C18 aromatic cyclic hydrocarbon groups or an aromatic heterocyclic group having 5-18 ring-forming carbon atoms, a C2-C18 alkynyl group optionally substituted with one or more C6-C18 aromatic cyclic hydrocarbon groups or an aromatic heterocyclic group having 5-18 ring-forming carbon atoms, a C1-C18 alkyl group optionally substituted with one or more C6-C18 aromatic cyclic hydrocarbon groups or an aromatic heterocyclic group having 5-18 ring-forming carbon atoms, At least one of an aromatic cyclic hydrocarbon group having 6 to 18 ring-forming carbon atoms, an aromatic heterocyclic group having 5 to 18 ring-forming carbon atoms, a mercapto group, a cyano group and a nitro group;
n represents a number of 3, and n represents a number of,
m is an integer of 1 to 100, and
X -are counter anions.
5. A compound according to any one of claims 1 and 4 or a process according to any one of claims 2 to 4, characterised in that each R1 is the same and represents methyl, ethyl, propyl, n-butyl, hydroxy-substituted butyl, heptyl, phenyl, phenylalkyl, cyclohexyl or alkyl-substituted cyclohexyl.
6. A compound according to claim 1 or a method according to any one of claims 2 to 3, characterised in that
wherein the aggregation-inducing luminescent group may be substituted or unsubstituted, and in the case of substitution, the substituent may be one or more, and is selected from at least one of a halogen atom, a hydroxyl group, an aldehyde group, a carboxyl group, an amino group, a C2-C18 alkenyl group optionally substituted with one or more C6-C18 aromatic cyclic hydrocarbon groups or an aromatic heterocyclic group of ring-forming carbon atoms 5 to 18, a C2-C18 alkynyl group optionally substituted with one or more C6-C18 aromatic cyclic hydrocarbon groups or an aromatic heterocyclic group of ring-forming carbon atoms 5 to 18, a C1-C18 alkyl group optionally substituted with one or more C6-C18 aromatic cyclic hydrocarbon groups or an aromatic heterocyclic group of ring-forming carbon atoms 5 to 18, an aromatic cyclic hydrocarbon group of ring-forming carbon atoms 6 to 18, an aromatic heterocyclic group of ring-forming carbon atoms 5 to 18, a mercapto group, a cyano group and a nitro group.
7. The compound according to any one of claims 1 and 3 to 6 or the method according to any one of claims 2 to 6, wherein each R1 independently represents a hydrophobic group such that the compound has an n-octanol/water partition coefficient value in the range of 3.0 to 7.0.
8. The compound according to any one of claims 1 and 3 to 7 or the method according to any one of claims 2 to 7, characterized in that the compound is selected from at least one of the following:
9. a fluorescent probe for detecting a target analyte, characterized by comprising a compound according to any one of claims 1 and 4 to 8.
10. A sensor for detecting a target analyte, characterized by comprising an array of the fluorescent probes of claim 9.
11. A kit for detecting a target analyte, characterized by comprising the sensor of claim 10.
12. A method of detecting a target analyte in a sample, comprising the steps of:
(1) providing a sample comprising an analyte of interest; and
(2) adding the sample to the fluorescent probe array of the sensor of claim 10; and
(3) the fluorescent signals from each fluorescent probe were collected and subjected to statistical data analysis.
13. A method of making a sensor for detecting a target analyte in a sample, comprising the steps of: disposing the fluorescent probes of claim 9 in an array on a substrate.
14. The fluorescent probe according to claim 9, the sensor according to claim 10 or the kit according to claim 11, characterized in comprising at least three different compounds according to claim 1.
15. The fluorescent probe according to claim 14, the sensor according to claim 14 or the kit according to claim 14, characterized in that the differences are selected from the group consisting of different fluorescence response signals, different values of n-octanol/water partitioning coefficient, different hydrophobicity, different electrostatic properties, different aggregation states, different interactions with target analytes or any combination thereof.
16. The fluorescent probe according to claim 9, the sensor according to claim 10 or the kit according to claim 11, characterized by comprising: at least one of said compounds having an n-octanol/water partition coefficient value of 3 to 5, at least one of said compounds having an n-octanol/water partition coefficient value of 5 to 6, and at least one of said compounds having an n-octanol/water partition coefficient value of 6 or more.
17. The compound according to any one of claims 1 and 3 to 8, the method according to any one of claims 2 to 8, the fluorescent probe according to claim 9, the sensor according to claim 10, the kit according to claim 11, or the method according to claim 12 or 13, characterized in that the target analyte is selected from a microorganism, such as a bacterium or a virus, in particular a gram-negative bacterium, a gram-positive bacterium or a fungus; a cell; a protein; a nucleic acid; a metabolite; at least one of a biomarker and any mixture thereof.
Technical Field
The invention relates to the field of functional materials, in particular to a compound for detecting a target analyte, a preparation method thereof and application thereof in detecting the target analyte, for example, the compound is used for preparing a fluorescent probe, a biosensor and a kit; applications for detecting target analytes such as microorganisms (e.g., rapid detection and identification of pathogenic bacteria) and methods of making fluorescent probes, biosensors, and kits
Background
Many microorganisms (such as bacteria or viruses, in particular gram-negative bacteria, gram-positive bacteria or fungi), cells, proteins, nucleic acids, metabolites and/or biomarkers are closely associated with human health and death and are therefore of vital importance for the detection and/or identification of these substances, especially of pathogenic bacteria.
Currently, there are up to 9 billion annual cases of infection, with 200 tens of thousands of children dying from serious pathogenic infections. To ensure effective treatment, pathogenic bacteria must first be identified quickly and reliably. Methods for identifying microorganisms so far include plate culture, microscopic examination, and techniques for detecting genes and immunological features of microorganisms. However, the disadvantages of these techniques themselves limit their widespread use. For example, the plating method takes a long time, usually requiring 24 hours or more; pathogenic bacteria with similar sizes and shapes are difficult to distinguish through microscopic examination; detection of genetic and immunological features of pathogenic bacteria requires advanced technologies such as Polymerase Chain Reaction (PCR), gene chips, and specific immuno arrays. These techniques are complex and require expensive instrumentation. Moreover, after a complicated multi-step treatment, a false positive result is inevitably obtained. Even for some advanced identification methods, such as automated biochemical instruments and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), which are often used in hospitals and other authorities, the accuracy of identification is only 90-95% and still takes several hours to obtain results. Lack of timely and reliable pathogenic bacteria information not only causes the use of a sub-prescription and delays the illness condition, but also the indiscriminate use of antibiotics can generate selective pressure on pathogenic bacteria, and further aggravate the drug resistance of bacteria. As predicted by the american Centers for Disease Control (CDC), up to 1000 million deaths worldwide will occur annually due to antibiotic-resistant infections by 2050. Therefore, there is an urgent need to develop a simple and accurate method for identifying pathogenic bacteria. The method has important significance for timely finding the source of infectious diseases, guiding the use of antibiotics and slowing down drug resistance.
The fluorescent probe is an ideal tool for identifying pathogenic bacteria due to the advantages of high response speed, high sensitivity, simplicity and the like. Several biochemical sensors have been developed that identify pathogenic bacteria based on fluorescent signals. However, the ubiquitous presence of aggregation-induced fluorescence quenching (ACQ) effects in conventional fluorescent molecules forces their use at very low working concentrations, greatly limiting the extent to which fluorescent probes label biological analytes, resulting in a significant decrease in detection sensitivity. Furthermore, this ACQ effect of traditional fluorescent molecules forces them to be detected in a fluorescent "off" manner. Inevitably, the emission intensity of fluorescent molecules is often influenced by some external factors such as H 2O, air and the like, and further reduces the sensitivity and accuracy of identification. To overcome this problem, researchers have designed specific quenchers to reduce the emission intensity of fluorescent probes and then used this system of probes with weak emission to identify biological analytes in a "light-up" fashion. While the approach of employing quenchers is effective, it complicates the sensor and increases cost.
Unlike conventional fluorescent probes, aggregation-induced emission (AIE) materials are characterized by no or weak luminescence upon dissolution, but strong emission upon aggregation. This feature gives AIE molecules detection in a "lighted" manner, which makes them highly resistant to external factors and photobleaching. Moreover, the AIE compound probe has the advantages of low fluorescence background and no washing, can greatly improve the detection sensitivity and accuracy, and can well meet the requirements of an ideal fluorescence sensor.
There remains a need for improved AIE compounds and probes therefor.
Disclosure of Invention
The present invention provides novel AIE compounds for detecting target analytes, methods for their preparation, and their use for detecting target analytes. The compound can be used for preparing various fluorescent probes, sensor arrays and kits for detecting various target analytes, particularly can realize accurate, rapid, simple and reliable identification on pathogenic bacteria, and can even efficiently distinguish normal bacteria from drug-resistant bacteria.
Specifically, the present invention provides:
a compound for detecting a target analyte, characterized by the following formula I:
wherein the content of the first and second substances,
l represents a flexible chain linking group,
B +represents a group having a positive charge,
each R1 independently represents hydrogen or a hydrophobic group such that the compound has an n-octanol/water partition coefficient value of 2.0 or more, provided that at least one R1 is a hydrophobic group such that the compound has an n-octanol/water partition coefficient value of 2.0 or more,
n represents an integer of 1 to 3,
m represents an integer of 1 to 100; and
X -are counter anions.
A method for preparing a compound for detecting a target analyte, comprising the steps of:
reacting a compound of formula III with a compound of formula IV in the presence of a catalyst and an organic solvent to obtain a compound of formula V, and
in the presence of a catalyst and an organic solvent, converting the compound of formula V into a compound of formula VCompound and B- (R1) nReacting the compound represented thereby to obtain a compound of formula I;
wherein C is a nucleophilic group, which may be selected from-OH, -SH or-NH 2Substituted amine groups, preferably-OH, X is halogen, preferably Br,
b is an organic precursor group capable of carrying a positive charge, preferably an amine or amino group,
n is an integer of 1 to 3,
B +represents a positively charged group;
each R1 independently represents hydrogen or a hydrophobic group such that the compound has an n-octanol/water partition coefficient value of 2.0 or more, with the proviso that at least one R1 is a hydrophobic group such that the compound has an n-octanol/water partition coefficient value of 2.0 or more and
m represents an integer of 1 to 100.
Preferably, the catalyst is selected from a metal carbonate catalyst, a metal hydroxide catalyst, a metal alkoxide catalyst or any combination thereof, preferably a metal carbonate catalyst (sodium carbonate, potassium carbonate), more preferably K 2CO 3Or any combination thereof,
preferably, the organic solvent is selected from aromatic hydrocarbon solvents, aliphatic hydrocarbon solvents, oxygen-containing heterocyclic solvents, alcohol solvents, ketone solvents, ester solvents, and ether reagents, and more preferably tetrahydrofuran, acetone, ethanol, methanol, ethyl acetate, and diethyl ether.
Preferably, the compound is represented by the following formula II,
wherein R2, R3, R4 and R5 may be the same or different and are independently selected from hydrogen, hydroxyl, substituted or unsubstituted hydroxyalkyl, amino, substituted or unsubstituted alkylamino, substituted or unsubstituted alkyl, substituted or unsubstituted unsaturated hydrocarbon, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl, or combinations thereof, and in the case of substitution, at least one hydrogen of hydroxyalkyl, alkylamino, alkyl, unsaturated hydrocarbon, cycloalkyl, heteroalkyl, aryl and heteroaryl is selected from halogen, hydroxyl, aldehyde, carboxyl, amino, C2-C18 alkenyl optionally substituted with one or more C6-C18 aromatic cycloalkyl groups or an aromatic heterocyclic group of ring carbon atoms 5-18, and an aromatic heterocyclic group optionally substituted with one or more C6-C18 aromatic cycloalkyl groups or ring carbon atoms 5-18 is selected from the group consisting of halogen, hydroxyl, aldehyde, carboxyl, amino, and aromatic heterocyclic groups of ring carbon atoms 5-18 Substituted C2-C18 alkynyl, C1-C18 alkyl optionally substituted with one or more C6-C18 aromatic cyclic hydrocarbon groups or cyclic aromatic heterocyclic groups of ring carbon atoms 5-18, aromatic cyclic hydrocarbon groups of ring carbon atoms 6-18, cyclic aromatic heterocyclic groups of ring carbon atoms 5-18, mercapto, cyano and nitro;
l represents an alkoxy chain linking group, an alkyl chain linking group, a substituted or unsubstituted alkoxy chain linking group, or a substituted or unsubstituted alkyl chain linking group;
each R1 is the same or different and is independently a substituted or unsubstituted C1-C18 alkyl group, an aromatic cyclic hydrocarbon group having 6-18 ring-forming carbon atoms, or a substituted or unsubstituted cyclic hydrocarbon group having 6-18 ring-forming carbon atoms, and in the case of substitution, at least one hydrogen of the C1-C18 alkyl group, the aromatic cyclic hydrocarbon group, and the cyclic hydrocarbon group is selected from a halogen atom, a hydroxyl group, an aldehyde group, a carboxyl group, an amino group, a C2-C18 alkenyl group optionally substituted with one or more C6-C18 aromatic cyclic hydrocarbon groups or an aromatic heterocyclic group having 5-18 ring-forming carbon atoms, a C2-C18 alkynyl group optionally substituted with one or more C6-C18 aromatic cyclic hydrocarbon groups or an aromatic heterocyclic group having 5-18 ring-forming carbon atoms, a C1-C18 alkyl group optionally substituted with one or more C6-C18 aromatic cyclic hydrocarbon groups or an aromatic heterocyclic group having 5-18 ring-forming carbon atoms, At least one of an aromatic cyclic hydrocarbon group having 6 to 18 ring-forming carbon atoms, an aromatic heterocyclic group having 5 to 18 ring-forming carbon atoms, a mercapto group, a cyano group and a nitro group;
n represents a number of 3, and n represents a number of,
m is an integer of 1 to 100, and
X -are counter anions.
Preferably, each R1 is the same and represents methyl, ethyl, propyl, n-butyl, hydroxy-substituted butyl, heptyl, phenyl or cyclohexyl.
Preferably, the first and second electrodes are formed of a metal,
wherein the aggregation-inducing luminescent group may be substituted or unsubstituted, and in the case of substitution, the substituent may be one or more, and is selected from at least one of a halogen atom, a hydroxyl group, an aldehyde group, a carboxyl group, an amino group, a C2-C18 alkenyl group optionally substituted with one or more C6-C18 aromatic cyclic hydrocarbon groups or an aromatic heterocyclic group of ring-forming
Preferably, each R1 independently represents a hydrophobic group such that the compound has an n-octanol/water partition coefficient value in the range of 3.0 to 7.0.
Preferably, the compound is selected from at least one of the following:
a fluorescent probe for detecting a target analyte comprising a compound as described in any of the above.
A sensor for detecting a target analyte comprising an array of the above-described fluorescent probes.
A kit for detecting a target analyte comprising the sensor described above.
A method of detecting a target analyte in a sample, comprising the steps of:
providing a sample comprising an analyte of interest; and
adding the sample to the fluorescent probe array of the sensor; and
the fluorescent signals from each fluorescent probe were collected and subjected to statistical data analysis.
A method of making a sensor for detecting a target analyte in a sample, comprising the steps of: the fluorescent probes described above are arranged in an array on a substrate.
Preferably, the fluorescent probe, the sensor or the kit comprises at least three different compounds of any of the above.
Preferably, the differences are selected from the group consisting of different fluorescence response signals, different values of n-octanol/water partition coefficients, different hydrophobicity, different electrostatic properties, different aggregation states, different interactions with target analytes, or any combination thereof.
Preferably, said fluorescent probe, said sensor or said kit comprises: at least one of said compounds having an n-octanol/water partition coefficient value of 3 to 5, at least one of said compounds having an n-octanol/water partition coefficient value of 5 to 6, and at least one of said compounds having an n-octanol/water partition coefficient value of 6 or more.
Preferably, the target analyte is selected from a microorganism, such as a bacterium or a virus, in particular a gram-negative bacterium, a gram-positive bacterium or a fungus; a cell; a protein; a nucleic acid; a metabolite; at least one of a biomarker and any mixture thereof.
Drawings
FIG. 1 shows a) the normalized absorption spectrum of 20 μ M TPE-ARs in DMSO and the normalized emission spectrum of 200 μ M TPE-ARs in an organic solvent/water mixed system with a water content of 96%, excitation wavelength: 340 nm; b) and d) -i) emission spectra of 200. mu. MTPE-ARs in organic solvent/water mixed systems with different water contents, excitation wavelength: 340 nm; c) TPE-ARs relative emission intensity (I/I) 0) A graph relating water content; the TPE-ARs adopt an organic solvent/water mixed system which comprises a methanol/water mixture: TPE-AMe, TPE-APrA and TPE-ABn; acetonitrile/water mixture: TPE-AEt, TPE-ABu, and TPE-ACH; DMSO/water mixture: TPE-AHex, where vol% represents vol%.
FIG. 2 shows the particle size distribution of TPE-ARs in an organic solvent/water mixed system with a water content of 96% (methanol/water mixture: TPE-AMe, TPE-APrA and TPE-ABn; acetonitrile/water mixture: TPE-AEt, TPE-ABu and TPE-ACH; DMSO/water mixture: TPE-AHex); TPE-
FIG. 3 shows the TPE-ARs fluorescence intensity as a function of concentration in PBS.
FIG. 4 shows the cryo-scanning electron micrographs of a) -c) TPE-AMe, TPE-AEt and TPE-APrA in PBS; d) -g) cryo-TEM images of TPE-ABu, TPE-ACH, TPE-ABn and TPE-AHex in PBS. The concentration is 200. mu.M.
FIG. 5 shows a) fluorescence spectra of 20 μ M TPE-APrA in PBS and microbial suspension; b) zeta potential results of 7 pathogenic bacteria before and after incubation with 20 mu M TPE-ARs; c) laser confocal images of 7 pathogens incubated with 20 μ M AIE molecules (TPE-APrA, TPE-ACH, and TPE-AHex) for 15 minutes, respectively, with excitation wavelengths: 405nm, emission wavelength range: 430 nm and 500 nm.
FIG. 6 shows the fluorescence response (CTPE-
FIG. 7 shows a) fluorescence spectra of 20 μ M TPE-AHex in PBS and pathogen suspensions; b) the profile of TPE-ARs fluorescence intensity as a function of concentration in PBS; c) particle size distribution of TPE-ABu and TPE-AHex at concentrations of 20. mu.M and 200. mu.M in PBS; d) and e) cryo-transmission electron micrographs of 20 μ M TPE-ABu and TPE-AHex in PBS.
FIG. 8 shows a) a plot of the fluorescence response of TPE-APrA, TPE-ACH and TPE-AHex (combination AB1C) sensor arrays to 7 microorganisms (transformed from FIG. 7), b) a two-dimensional standard score difference plot (■ represents the centroid of each group) obtained by LDA analysis of the fluorescence response plots in panel a).
FIG. 9 shows a two-dimensional standard score differential plot of all fluorescence sensor arrays (except TPE-APrA, TPE-ACH and TPE-AHex sensor arrays) constructed from three groups of TPE-ARs molecules against 7 pathogens (■ represents the centroid of each group).
FIG. 10 shows) three-dimensional standard score plots (■ represents the centroid of each group) for the TPE-APrA, TPE-ACH and TPE-AHex sensor arrays for 7 pathogens, b) the centroid coordinates of the 7 pathogens in the three-dimensional standard score plots.
FIG. 11 shows a) fluorescence response profiles of TPE-APrA, TPE-ACH and TPE-AHex (combination AB1C) sensor arrays for 8 microorganism mixtures; each value is the average of six independent tests, and the error bars represent the standard deviation of six measurements; the excitation wavelength is 340nm, and the emission wavelength is 470 nm; i is 0And I is the fluorescence intensity of TPE-ARs before and after the addition of pathogenic bacteria, b) a two-dimensional standard score discrimination chart (■ represents the centroid of each group) obtained by means of the fluorescence response chart in LDA analysis chart a).
FIG. 12 shows TPE-AMe 1H NMR spectrum (400MHz, DMSO-d 6).
FIG. 13 showsFrom TPE-AMe 13C NMR spectrum (100MHz, DMSO-d 6).
FIG. 14 shows TPE-AMe +The MALDI-TOF high-resolution mass spectrogram.
FIG. 15 shows TPE-AEt 1H NMR spectrum (400MHz, DMSO-d 6).
FIG. 16 shows TPE-AEt 13C NMR spectrum (100MHz, DMSO-d 6).
FIG. 17 shows TPE-AEt +The MALDI-TOF high-resolution mass spectrogram.
FIG. 18 shows TPE-APrA 1H NMR spectrum (400MHz, DMSO-d 6).
FIG. 19 shows TPE-APrA 13C NMR spectrum (100MHz, DMSO-d 6).
FIG. 20 shows TPE-APrA +The MALDI-TOF high-resolution mass spectrogram.
FIG. 21 shows TPE-ABu 1H NMR spectrum (400MHz, DMSO-d 6).
FIG. 22 shows TPE-ABu 13C NMR spectrum (100MHz, DMSO-d 6).
FIG. 23 shows TPE-ABu +The MALDI-TOF high-resolution mass spectrogram.
FIG. 24 shows TPE-ACH 1H NMR spectrum (400MHz, DMSO-d 6).
FIG. 25 shows TPE-ACH 13C NMR spectrum (100MHz, DMSO-d 6).
FIG. 26 shows TPE-ACH +The MALDI-TOF high-resolution mass spectrogram.
FIG. 27 shows TPE-ABn 1H NMR spectrum (400MHz, Methanol-d 4).
FIG. 28 shows TPE-ABn 13C NMR spectrum (100MHz, DMSO-d 6).
FIG. 29 shows TPE-ABn +The MALDI-TOF high-resolution mass spectrogram.
FIG. 30 shows TPE-AHex 1H NMR spectrum (400MHz, DMSO-d 6).
FIG. 31 shows TPE-AHex 13C NMR spectrum (100MHz, DMSO-d 6).
FIG. 32 shows TPE-AHex +The MALDI-TOF high-resolution mass spectrogram.
FIG. 33 shows a schematic diagram of preparing a sensor array and using the sensor array to detect a target analyte according to one embodiment of the invention.
Detailed Description
Embodiments of the present invention are described in detail below. The embodiments described below are exemplary only, are intended to illustrate the invention, and should not be construed as limiting the invention. The embodiments are not specified to specific techniques or conditions, according to the techniques or conditions described in the literature in the field or according to the product description. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
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 found in the descriptions of "Organic Chemistry", Thomas Sorrell, University Science Books, Sausaltito: 1999, and "March's Advanced 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.
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.
In the various parts of this specification, substituents of the disclosed compounds are disclosed in terms of group type or range. It is specifically intended that the invention includes each and every independent subcombination of the various members of these groups and ranges. For example, the term "C1-18 alkyl" includes methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl.
In each of the sections of the invention, linking substituents such as L are described. Where the structure clearly requires a linking group, the markush variables listed for that group are understood to be linking groups. For example, if the structure requires a linking group and the markush group definition for the variable recites "alkyl" or "aromatic group," it is understood that the "alkyl" or "aryl" represents an attached alkylene group or arylene group, respectively.
The term "hydrocarbon group" as used herein includes aromatic hydrocarbon groups and aliphatic hydrocarbon groups. Aliphatic hydrocarbon groups include "alkyl" or "alkyl group", alkenyl and alkynyl groups, which may be saturated or unsaturated, straight or branched chain divalent hydrocarbon groups. The hydrocarbyl group may be optionally substituted with one or more substituents described herein. In one embodiment of the invention, the alkyl group contains 1 to 18 carbon atoms. In another embodiment, the alkyl group contains 1 to 12 carbon atoms; in yet another embodiment, the alkyl group contains 1 to 6 carbon atoms; in yet another embodiment, the alkyl group contains 1 to 4 carbon atoms; in yet another embodiment, the alkyl group contains 1 to 3 carbon atoms.
Examples of alkyl groups include, but are not limited to, C1-12 alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, n-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2, 3-dimethyl-2-butyl, 3, 3-dimethyl-2-butyl, n-heptyl, n-octyl, and the like.
The term "alkenyl" denotes a straight or branched chain monovalent hydrocarbon radical of a carbon atom having at least one site of unsaturation, i.e., a carbon-carbon sp2 double bond, wherein the alkenyl radical is optionally substituted with one or more substituents as described herein, including the positioning of "cis" and "tan", or the positioning of "E" and "Z". In one embodiment, the alkenyl group contains 2 to 8 carbon atoms; in another embodiment, the alkenyl group contains 2 to 6 carbon atoms; in yet another embodiment, the alkenyl group contains 2 to 4 carbon atoms. Examples of alkenyl groups include, but are not limited to, vinyl, allyl, and the like.
The term "alkynyl" denotes a straight or branched chain monovalent hydrocarbon radical of a carbon atom having at least one site of unsaturation, i.e., a carbon-carbon sp triple bond, wherein the alkynyl radical is optionally substituted with one or more substituents described herein. 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.
The term "carboxy", whether used alone or in combination with other terms, such as "carboxyalkyl", denotes-CO 2H; the term "carbonyl", whether used alone or in combination with other terms, such as "aminocarbonyl" or "acyloxy", denotes- (C ═ O) -.
The terms "halogen" and "halo" refer to fluorine (F), chlorine (Cl), bromine (Br), or iodine (I).
The term "aromatic group" includes groups in which two hydrogen atoms are removed from the aromatic ring so that they are directly attached to other groups. Preferably, the aromatic group has at least one heteroatom in the ring-forming atoms, such as N, O or S.
The term "aromatic cycloalkyl" includes monocyclic, bicyclic and tricyclic aryl groups in which at least one ring system is aromatic and in which each ring system contains 6 to 18 atoms. The aryl group is typically, but not necessarily, attached to the parent molecule through an aromatic ring of the aryl group. The term "aryl" may be used interchangeably with the terms "aromatic ring" or "aromatic ring". Examples of the aryl group may include phenyl, biphenyl, naphthyl, and anthracene. The aryl group is optionally substituted with one or more substituents described herein.
In the present invention, the substituent may be selected from at least one of a halogen atom, a hydroxyl group, an aldehyde group, a carboxyl group, an amino group, a C2-C18 alkenyl group optionally substituted with one or more C6-C18 aromatic cyclic hydrocarbon groups or an aromatic heterocyclic group of ring-forming
Examples of the aromatic cyclic hydrocarbon group and the aromatic heterocyclic group include, for example, phenyl, naphthyl, anthryl, phenanthryl, tetracenyl, pyrenyl, benzo [ c ] phenanthryl, benzophenanthryl, fluorenyl, benzofluorenyl, dibenzofluorenyl, biphenyl, terphenyl, quaterphenyl, fluoranthenyl, pyrrolyl, pyrazinyl, pyridyl, pyrimidinyl, triazinyl, indolyl, isoindolyl, imidazolyl, furyl, benzofuryl, isobenzofuryl, dibenzofuryl, dibenzothienyl, quinolyl, isoquinolyl, quinoxalyl, carbazolyl, phenanthridinyl, acridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxazinyl, oxazolyl, oxadiazolyl, furazanyl, thienyl, benzothienyl, dihydroacridinyl, azacarbazolyl, quinazolinyl and the like.
Examples of the substituent include:
in recent years, the AIE molecules are used for constructing a fluorescent sensor array, and pathogenic bacteria are identified by means of a mathematical statistical method, but the accuracy and the simplicity of the method cannot meet the requirements, and the identification accuracy of unknown samples is only 91.7-93.75%. In order to improve the identification accuracy, the AIE molecular design should be optimized to expand the difference between the pathogenic bacteria fluorescence response signals; in addition, the simplicity of the sensor array should be maintained while improving accuracy.
In the present invention, it is considered that the difference in the characteristic fluorescence response of pathogenic bacteria is amplified based on multivalent interactions, considering that hydrophobic residues exist on the surface of pathogenic bacteria in addition to negatively charged groups. Thus, the present invention designs and synthesizes a series of AIE molecules, especially a series of Tetraphenylethylene (TPE) -based AIE molecules (TPE-ARs). These AIE molecules have a positive charge and different hydrophobic groups, have precisely adjusted hydropathic and hydrophobic properties, and have an n-octanol/water partition coefficient (logP) of 2 or more, preferably 3 to 7, more preferably 3.426 to 6.071, to thereby regulate the electrostatic and hydrophobic interactions between the AIE molecules and pathogenic bacteria. In addition, flexible linking chains (preferably alkoxy chains) may be introduced to improve the water solubility of TPE-ARs and the flexibility when interacting with pathogenic bacteria. Based on the AIE molecules, the invention successfully constructs a plurality of fluorescence sensor arrays, realizes the rapid and accurate identification of a plurality of pathogenic bacteria by means of Linear Discriminant Analysis (LDA), and even can efficiently distinguish normal bacteria and drug-resistant bacteria. .
For example, a series of sensor arrays constructed by the AIE molecule TPE-ARs designed by the invention can be used for quickly and reliably detecting and distinguishing pathogenic bacteria. Each sensor array can be composed of three TPE-ARs with significant fluorescence response differences, balancing fluorescence response diversity and fluorescence sensor simplicity. Each TPE-AR has quaternary ammonium salts with obvious hydrophobicity difference, so that the electrostatic and hydrophobic effects between AIE molecules and pathogenic bacteria can be regulated. Meanwhile, TPE-ARs also present various aggregation behaviors, and further enrich the multivalent interaction with different pathogenic bacteria. Due to the different multivalent interactions between the TPE-ARs and the pathogenic bacteria, each sensor array can provide a characteristic fluorescence response spectrum for different pathogenic bacteria. The fluorescence spectrum obtained by LDA recognition through a mathematical statistical method realizes effective identification of various pathogenic bacteria, even normal bacteria and drug-resistant bacteria can be distinguished, and the accuracy rate is close to 100%. In addition, the sensor array is also suitable for identifying mixtures comprising two or more pathogenic bacteria. Moreover, the sensor arrays have the advantages of rapidness, high flux, no washing and the like, and have great potential of providing timely and reliable pathogenic bacteria information for clinic.
In an embodiment, the invention also provides a method for constructing a fluorescent sensor array for rapid and accurate detection and differentiation of pathogenic bacteria, wherein the sensor array comprises a fluorescent probe with an n-octanol/water partition coefficient (logP) value of 3.0-6.0.
In one aspect, the present invention provides a compound for detecting a target analyte, represented by formula I below:
wherein the content of the first and second substances,
l represents a flexible chain linking group,
B +represents a group having a positive charge,
each R1 independently represents hydrogen or a hydrophobic group such that the compound has an n-octanol/water partition coefficient value of 2.0 or more, provided that at least one R1 is a hydrophobic group such that the compound has an n-octanol/water partition coefficient value of 2.0 or more,
n represents an integer of 1 to 3,
m represents an integer of 1 to 100; and
X -to counter anions, e.g. Br -。
In another aspect, the invention also provides a method of preparing a compound for detecting a target analyte, comprising the steps of:
reacting a compound of formula III with a compound of formula IV in the presence of a catalyst and an organic solvent to obtain a compound of formula V, and
reacting a compound of formula V with B- (R1) in the presence of a catalyst and an organic solvent nReacting the compound represented thereby to obtain a compound of formula I;
wherein C is a nucleophilic group selected from the group consisting of-OH, -SH, -NH 2Substituted amine groups, preferably-OH, X is halogen, preferably Br,
b is an organic precursor group capable of carrying a positive charge, preferably an amine or amino group,
n is an integer of 1 to 3,
B +represents a positively charged group;
each R1 independently represents hydrogen or a hydrophobic group such that the compound has an n-octanol/water partition coefficient value of 2.0 or more, with the proviso that at least one R1 is a hydrophobic group such that the compound has an n-octanol/water partition coefficient value of 2.0 or more and
m represents an integer of 1 to 100.
Preferably, the catalyst is selected from a metal carbonate catalyst, a metal hydroxide catalyst, a metal alkoxide catalyst or any combination thereof, preferably a metal carbonate catalyst (sodium carbonate, potassium carbonate), more preferably K 2CO 3，
Preferably, the organic solvent is selected from aromatic hydrocarbon solvents, aliphatic hydrocarbon solvents, oxygen-containing heterocyclic solvents, alcohol solvents, ketone solvents, ester solvents, ether reagents, more preferably tetrahydrofuran, acetone, ethanol, methanol, ethyl acetate or diethyl ether.
Preferably, the compound is represented by the following formula II,
wherein R2, R3, R4 and R5 may be the same or different and are independently selected from hydrogen, hydroxyl, substituted or unsubstituted hydroxyalkyl, amino, substituted or unsubstituted alkylamino, substituted or unsubstituted alkyl, substituted or unsubstituted unsaturated hydrocarbon, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl, or combinations thereof, and in the case of substitution, at least one hydrogen of hydroxyalkyl, alkylamino, alkyl, unsaturated hydrocarbon, cycloalkyl, heteroalkyl, aryl and heteroaryl is selected from halogen, hydroxyl, aldehyde, carboxyl, amino, C2-C18 alkenyl optionally substituted with one or more C6-C18 aromatic cycloalkyl groups or an aromatic heterocyclic group of ring carbon atoms 5-18, and an aromatic heterocyclic group optionally substituted with one or more C6-C18 aromatic cycloalkyl groups or ring carbon atoms 5-18 is selected from the group consisting of halogen, hydroxyl, aldehyde, carboxyl, amino, and aromatic heterocyclic groups of ring carbon atoms 5-18 Substituted C2-C18 alkynyl, C1-C18 alkyl optionally substituted with one or more C6-C18 aromatic cyclic hydrocarbon groups or cyclic aromatic heterocyclic groups of ring carbon atoms 5-18, aromatic cyclic hydrocarbon groups of ring carbon atoms 6-18, cyclic aromatic heterocyclic groups of ring carbon atoms 5-18, mercapto, cyano and nitro;
l represents an alkoxy chain linking group, an alkyl chain linking group, a substituted or unsubstituted alkoxy chain linking group, a substituted or unsubstituted alkyl chain linking group;
each R1 is the same or different and is independently a substituted or unsubstituted C1-C18 alkyl group, an aromatic cyclic hydrocarbon group having 6-18 ring-forming carbon atoms, or a substituted or unsubstituted cyclic hydrocarbon group having 6-18 ring-forming carbon atoms, and in the case of substitution, at least one hydrogen of the C1-C18 alkyl group, the aromatic cyclic hydrocarbon group, and the cyclic hydrocarbon group is selected from a halogen atom, a hydroxyl group, an aldehyde group, a carboxyl group, an amino group, a C2-C18 alkenyl group optionally substituted with one or more C6-C18 aromatic cyclic hydrocarbon groups or an aromatic heterocyclic group having 5-18 ring-forming carbon atoms, a C2-C18 alkynyl group optionally substituted with one or more C6-C18 aromatic cyclic hydrocarbon groups or an aromatic heterocyclic group having 5-18 ring-forming carbon atoms, a C1-C18 alkyl group optionally substituted with one or more C6-C18 aromatic cyclic hydrocarbon groups or an aromatic heterocyclic group having 5-18 ring-forming carbon atoms, At least one of an aromatic cyclic hydrocarbon group having 6 to 18 ring-forming carbon atoms, an aromatic heterocyclic group having 5 to 18 ring-forming carbon atoms, a mercapto group, a cyano group and a nitro group;
n represents a number of 3, and n represents a number of,
m is an integer of 1 to 100, and
X -are counter anions.
Preferably, each R1 is the same and represents methyl, ethyl, propyl, n-butyl, hydroxy-substituted butyl, heptyl, phenyl or cyclohexyl.
Preferably, the first and second electrodes are formed of a metal, at least one selected from the group consisting of:
the aggregation-inducing luminescent groups described above may be substituted with one or more substituents described herein.
Preferably, each R1 independently represents a hydrophobic group such that the compound has an n-octanol/water partition coefficient value in the range of 3.0 to 7.0.
Preferably, the compound is selected from at least one of the following:
in another aspect, the invention provides a fluorescent probe for detecting a target analyte, comprising a compound as described in any of the above.
In another aspect, the invention provides a sensor for detecting a target analyte, comprising an array of fluorescent probes as described above.
In another aspect, the invention provides a kit for detecting a target analyte, comprising a sensor as described above. The kit may also contain sample tubes and processing solutions, such as phosphate buffered saline.
In another aspect, the present invention provides a method of detecting a target analyte in a sample, comprising the steps of:
providing a sample comprising an analyte of interest; and
adding the sample to the fluorescent probe array of the sensor; and
the fluorescent signals from each fluorescent probe were collected and subjected to statistical data analysis.
The sample may be a plasma sample, urine sample, soil sample, water sample, food or beverage containing a pathogen.
Fig. 33 shows a schematic of a method of making a sensor using AIE molecules and detecting a target analyte using a sensor array. As shown in this figure, a plurality (3 or more) of different AIE molecules can be used as fluorescent probes and then applied in an array on a substrate to form a sensor array. A target analyte (e.g., a pathogen) is then applied to the sensor array. And detecting corresponding signals of fluorescence emitted by the sensor array by using a fluorescence device (a fluorescence spectrometer or a microplate reader), and performing data analysis on the signals. The data analysis results are shown in a two-dimensional standard score difference plot.
For example, the data analysis may include Linear Discriminant Analysis (LDA), a mathematical statistical method widely used for pattern recognition, which may be used to analyze pathogen fluorescence response patterns generated by sensor arrays. Through LDA analysis, the pathogenic bacteria fluorescence response map can be converted into a two-dimensional standard score distinguishing map. If the detection scores of the target analytes are well able to form independent clusters and are completely separated from each other, the properties of the different target analytes can be identified and even assigned, classified, characterized, or even quantified. Particularly, the probe can realize efficient identification on pathogenic bacteria, and more importantly, can distinguish normal bacteria from drug-resistant bacteria, such as normal escherichia coli (e.coli) and ampicillin-resistant bacteria (e.colir), and normal staphylococcus aureus (s.aureus) and penicillin-resistant bacteria (s.aureus), which are important for effective treatment.
The authentication capabilities of the sensor array can then also be verified by the LDA's cross-validation to determine the discrimination accuracy and authentication accuracy.
In yet another aspect, the present invention is a method of making a sensor for detecting a target analyte in a sample, comprising the steps of: the fluorescent probes described above are arranged in an array on a substrate. The array may be a 3 × 1 array, a 3 × 2 array, a 3 × 3 array, a 3 × 4 array, or the like
Preferably, the fluorescent probe, the sensor or the kit comprises at least three different compounds of any of the above.
Preferably, the fluorescent probes or sensor arrays described above can be prepared by selecting appropriate compounds based on differences in fluorescence response signals, differences in n-octanol/water partition coefficient values, differences in hydrophobicity, differences in electrostatic properties, differences in aggregation state, differences in interaction with target analytes, or any combination thereof.
For example, a fluorescent probe, sensor or kit comprises: at least one of said compounds having an n-octanol/water partition coefficient value of 3 to 5, at least one of said compounds having an n-octanol/water partition coefficient value of 5 to 6, and at least one of said compounds having an n-octanol/water partition coefficient value of 6 or more.
Preferably, the target analyte is selected from a microorganism, such as a bacterium or a virus, in particular a gram-negative bacterium, a gram-positive bacterium or a fungus; a cell; a protein; a nucleic acid; a metabolite; at least one of a biomarker and any mixture thereof.
Examples of the present invention
The following examples are provided to illustrate the invention and to assist those skilled in the art in understanding the invention. However, the following examples of the present invention should not be construed to unduly limit the present invention. Variations and modifications to the discussed examples may occur to those of ordinary skill in the art without departing from the scope of the discovery.
Synthesis of compounds
In the examples, typical compound synthetic routes are as follows:
synthetic TPE-AMe
N, N, N-trimethyl-2- (2- (4- (1,2, 2-triphenylvinyl) phenoxy) ethoxy) ethane-1-ammonium bromide (TPE-AMe) is synthesized. First, 4- (1,2, 2-triphenylvinyl) phenol was synthesized: benzophenone (1.82g, 10mmol), 4-hydrobenzophenone (1.98g, 10mol) and zinc powder (2.60g, 40mmol) were placed in a two-necked round bottom flask. The flask was evacuated and purged with nitrogen three times. Under a nitrogen atmosphere, 70mL of dry tetrahydrofuran was added, followed by the slow addition of 2.2mL of titanium tetrachloride (20mmol) in a dry ice acetone bath with stirring. The reaction mixture was heated to reflux under nitrogen overnight. After cooling to room temperature, 50mL of dilute hydrochloric acid (1M) was added to the mixture, and the mixture was extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate and filtered. After evaporation of the solvent, the crude product was purified by column chromatography on silica gel using n-hexane/ethyl acetate (40: 1) as eluent. A white powder was obtained in 52% yield. Then, (2- (4- (2- (2-bromoethoxy) ethoxy) phenyl) ethylene-1, 1, 2-triyl) triphenyl was synthesized: a two-necked round-bottomed flask was charged with 4- (1,2, 2-triphenylvinyl) phenol (1.74g, 5mmol), dibromoethyl ether (1.39g, 6mmol) and potassium carbonate (1.38g, 10 mmol). 30mL of acetone was added under nitrogen. The reaction mixture was heated to reflux overnight. After evaporation of the solvent, the crude product was purified by silica gel column chromatography using hexane/dichloromethane (20: 1) as eluent to give a colorless oil in 65% yield. And finally synthesizing TPE-AMe: a round-bottom flask was charged with (2- (4- (2- (2-bromoethoxy) ethoxy) phenyl) ethylene-1, 1, 2-triyl) triphenyl (2mmol) and trimethylamine (10mmol), and heated under reflux in ethanol (20mL) for 24 hours. The solvent was removed in vacuo and the crude product was repeatedly dissolved with a small amount of methanol, then an excess of tetrahydrofuran was added to precipitate a white powder, which was dried to give TPE-AMe in 82% yield.
FIG. 12 shows TPE-AMe 1H NMR spectrum (400MHz, DMSO-d 6).
FIG. 13 shows TPE-AMe 13C NMR spectrum (100MHz, DMSO-d 6).
FIG. 14 shows TPE-AMe +The MALDI-TOF high-resolution mass spectrogram.
Synthetic TPE-AEt
Synthesizing N-ethyl-N, N-dimethyl-2- (2- (4- (1,2, 2-triphenyl vinyl) phenoxy) ethoxy) ethane-1-ammonium bromide (TPE-AEt). The synthesis steps of the intermediate products of 4- (1,2, 2-triphenylvinyl) phenol and (2- (4- (2- (2-bromoethoxy) ethoxy) phenyl) ethylene-1, 1, 2-triyl) triphenyl in the first two steps are the same as those of TPE-AMe. Synthetic TPE-AEt: a round-bottom flask was charged with (2- (4- (2- (2-bromoethoxy) ethoxy) phenyl) ethylene-1, 1, 2-triyl) triphenyl (2mmol) and N, N-dimethylethylamine (10mmol), and heated under reflux in ethanol (20mL) for 24 hours. The solvent was removed in vacuo and the crude product was repeatedly dissolved with a small amount of methanol, then an excess of tetrahydrofuran was added to precipitate a white powder, which was dried to give TPE-AEt in 84% yield.
FIG. 15 shows TPE-AEt 1H NMR spectrum (400MHz, DMSO-d 6).
FIG. 16 shows TPE-AEt 13C NMR spectrum (100MHz, DMSO-d 6).
FIG. 17 shows TPE-AEt +The MALDI-TOF high-resolution mass spectrogram.
Synthetic TPE-APrA
Synthesizing 3-hydroxy-N, N-dimethyl-N- (2- (2- (4- (1,2, 2-triphenyl vinyl) phenoxy) ethyoxyl) ethyl) propyl-1-ammonium bromide (TPE-APrA). The intermediate product of the first two steps is TPE-AMe. Synthesizing TPE-APrA: a round-bottom flask was charged with (2- (4- (2- (2-bromoethoxy) ethoxy) phenyl) ethylene-1, 1, 2-triyl) triphenyl (2mmol) and 2- (dimethylamino) ethan-1-ol (10mmol), and heated under reflux in ethanol (20mL) for 24 hours. The solvent was removed in vacuo, the crude product was repeatedly dissolved with a small amount of methanol, and then an excess of tetrahydrofuran was added to precipitate a white powder, which was dried to give TPE-APrA in 86% yield.
FIG. 18 shows TPE-APrA 1H NMR spectrum (400MHz, DMSO-d 6).
FIG. 19 shows TPE-APrA 13C NMR spectrum (100MHz, DMSO-d 6).
FIG. 20 shows TPE-APrA +The MALDI-TOF high-resolution mass spectrogram.
Synthetic TPE-ABu
N, N-dimethyl-N- (2- (2- (4- (1,2, 2-triphenylvinyl) phenoxy) ethoxy) ethyl) butane-1-ammonium bromide (TPE-ABu) is synthesized. The intermediate product of the first two steps is TPE-AMe. Synthetic TPE-ABu: a round-bottom flask was charged with (2- (4- (2- (2-bromoethoxy) ethoxy) phenyl) ethylene-1, 1, 2-triyl) triphenyl (2mmol) and N, N-dimethylbut-1-amine (10mmol), and heated under reflux in ethanol (20mL) for 24 hours. The solvent was removed in vacuo and the crude product was repeatedly dissolved with a small amount of methanol, then an excess of tetrahydrofuran was added to precipitate a white powder, which was dried to give TPE-ABu in 89% yield.
FIG. 21 shows TPE-ABu 1H NMR spectrum (400MHz, DMSO-d 6).
FIG. 22 shows TPE-ABu 13C NMR spectrum (100MHz, DMSO-d 6).
FIG. 23 shows TPE-ABu +The MALDI-TOF high-resolution mass spectrogram.
Synthetic TPE-ACH
N, N-dimethyl-N- (2- (2- (4- (1,2, 2-triphenylvinyl) phenoxy) ethoxy) ethyl) cyclohexane ammonium bromide (TPE-ACH) is synthesized. The intermediate product of the first two steps is TPE-AMe. Synthesizing TPE-ACH: a round-bottom flask was charged with (2- (4- (2- (2-bromoethoxy) ethoxy) phenyl) ethylene-1, 1, 2-triyl) triphenyl (2mmol) and N, N-dimethylcyclohexane (10mmol), and heated under reflux in ethanol (20mL) for 24 hours. The solvent was removed in vacuo and the crude product was repeatedly dissolved with a small amount of methanol, then an excess of tetrahydrofuran was added to precipitate a white powder, which was dried to give TPE-ACH in 84% yield.
FIG. 24 shows TPE-ACH 1H NMR spectrum (400MHz, DMSO-d 6).
FIG. 25 shows TPE-ACH 13C NMR spectrum (100MHz, DMSO-d 6).
FIG. 26 shows TPE-ACH +The MALDI-TOF high-resolution mass spectrogram.
Synthetic TPE-ABn
Synthesizing N-benzyl-N, N-dimethyl-2- (2- (4- (1,2, 2-triphenyl vinyl) phenoxy) ethoxy) ethane-1-ammonium bromide (TPE-ABn). The intermediate product of the first two steps is TPE-AMe. Synthesizing TPE-ABn: a round-bottom flask was charged with (2- (4- (2- (2-bromoethoxy) ethoxy) phenyl) ethylene-1, 1, 2-triyl) triphenyl (2mmol) and N, N-dimethyl-1-phenyl (10mmol), and heated under reflux in ethanol (20mL) for 24 hours. The solvent was removed in vacuo, the crude product was repeatedly dissolved with a small amount of methanol, and then an excess of tetrahydrofuran was added to precipitate a white powder, which was dried to give TPE-ABn in 65% yield.
FIG. 27 shows TPE-ABn 1H NMR spectrum (400MHz, Methanol-d 4).
FIG. 28 shows TPE-ABn 13C NMR spectrum (100MHz, DMSO-d 6).
FIG. 29 shows TPE-ABn +The MALDI-TOF high-resolution mass spectrogram.
Synthetic TPE-AHex
N, N-dimethyl-N- (2- (2- (4- (1,2, 2-triphenylvinyl) phenoxy) ethoxy) ethyl) hexane-1-ammonium bromide (TPE-AHex) was synthesized. The intermediate product of the first two steps is TPE-AMe. Synthesizing TPE-ABn: a round-bottom flask was charged with (2- (4- (2- (2-bromoethoxy) ethoxy) phenyl) ethylene-1, 1, 2-triyl) triphenyl (2mmol) and N, N-dimethylhex-1-amine (10mmol), and heated to reflux in ethanol (20mL) for 24 hours. The solvent was removed in vacuo and the crude product was repeatedly dissolved with a small amount of methanol, then an excess of tetrahydrofuran was added to precipitate a white powder which was dried to give TPE-AHex in 89% yield.
FIG. 30 shows TPE-AHex 1H NMR spectrum (400MHz, DMSO-d 6).
FIG. 31 shows TPE-AHex 13C NMR spectrum (100MH, DMSO-d 6).
FIG. 32 shows TPE-AHex +The MALDI-TOF high-resolution mass spectrogram. .
Physical Property characterization of Compounds
As described above, 7 TPE-ARs (TPE-AMe, TPE-AEt, TPE-APrA, TPE-ABu, TPE-ACH, TPE-ABn and TPE-AHex) were prepared by a simple synthetic route with considerable productivity. Their photophysical properties were studied and are summarized in table 1. As shown in figure 1a, the main absorption peak of these AIE molecules in DMSO is at 313 or 314 nm. Their AIE properties were then investigated by varying the water content of the organic solvent/water mixed system. Taking TPE-AHex as an example (FIG. 1b), TPE-AHex emits weak fluorescence in a DMSO/water mixed system with a water content of 0-90 vol%. By further increasing the water content to 96%, a strong emission peak at 465nm was observed, the difference being clearly observable by the naked eye (as shown by the interpolated plot in FIG. 1 b). Similarly, at relatively high water content, the remaining 6 TPE-ARs also exhibited significantly enhanced fluorescence emission (FIGS. 1c-1i), with typical AIE properties. The dynamic light scattering results confirm that this phenomenon is mainly due to the formation of aggregates (fig. 2). The maximum emission wavelength of these TPE-ARs aggregates was around 470nm (FIG. 1 a). Therefore, in the present invention, the functionalized modified TPE has no significant effect on its maximum absorption and emission wavelengths, which will facilitate pathogen detection based on fluorescence intensity quantification. To optimize working concentrations of TPE-ARs, the Critical Aggregation Concentration (CAC) of these AIE molecules in PBS was determined by measuring the change in fluorescence intensity of these AIE molecules as a function of concentration. As shown in FIG. 3, the CAC of TPE-AMe, TPE-AEt, TPE-APrA, TPE-Abu, TPE-ACH, TPE-ABn, and TPE-AHex in PBS were determined to be 42.5, 79.2, 47.4, 44.4, 80.2, 76.5, and 65.3 μ M, respectively. Above CAC, these AIE molecules form diverse aggregate morphologies (fig. 4). To achieve high detection sensitivity, AIE molecular PBS solution with weak background fluorescence concentration of 20 μ M was used for subsequent pathogen identification test.
Table 1 photophysical, hydrophilic-hydrophobic and aggregation properties of TPE-ARs.
a) Absorption maximum in DMSO, b) emission maximum in the aggregate state, c) α AIEI aggregate/I solution; d) quantum yield of TPE-ARs in solid state (measured by integrating sphere method); e) ClogP is defined as the calculated logP (n-octanol/water partition coefficient), and the ClogP value is estimated by the software chem biodraw 14.0.
Sensor array preparation and microbial detection
7 microorganisms were selected as the study model. Among them, staphylococcus aureus (s.aureus), penicillin-resistant s.aureus (abbreviated as s.aureus) R) And enterococcus faecalis (e.faecalis) as gram positive bacteria; coli (e.coli), ampicillin-resistant e.coli (ampicillin-resistant e.coli, abbreviated e.coli) R) And pseudomonas aeruginosa (pnosa) is a gram-negative bacterium; candida albicans (c. albicans) is a fungus. The addition of these pathogens to 7 TPE-ARs solutions gave significantly different fluorescence intensities but did not have a significant effect on their maximum emission wavelength, which greatly facilitated the identification of the pathogens. Using TPE-APrA as an example (FIG. 5a), with gram-positive bacteria S RTPE-APrA shows different fluorescence intensities after the respective incubation of gram-negative bacteria E.coli and fungi C.albicans, and follows the rule C.albicans>S.aureus R>E.coli. This means that TPE-APrA has different binding strength with the three pathogens. At the same time, no significant change in zeta potential of the pathogenic bacteria occurred after addition of TPE-ARs (FIG. 5b), suggesting that these AIE molecules either inserted into the membrane of the pathogenic bacteria or entered the cytoplasm. This result was further confirmed by laser Confocal (CLSM) imaging (fig. 5 c). From the imaging results of TPE-APrA, TPE-ACH and TPE-AHex and 7 pathogenic bacteria, the AIE molecules can effectively stain the pathogenic bacteria and present various fluorescent responses. Different fluorescent signals can be given under the action of the same pathogenic bacteria and different AIE molecules and under the action of different pathogenic bacteria and the same AIE molecule, and therefore the premise is provided for successfully constructing a fluorescent sensor array for identifying the pathogenic bacteria.
In order to verify the capability of identifying the pathogenic bacteria of 7 TPE-ARs, a simple high-throughput technology of a microplate reader is used for recording the fluorescence intensity of the TPE-ARs at 470nm after the pathogenic bacteria are added, and the excitation wavelength is 340 nm. The fluorescence intensity of TPE-ARs in PBS was used as a control. Relative fluorescence intensity (I-I) of TPE-ARs before and after addition of pathogenic bacteria 0)/I 0Was used to characterize the fluorescent response of each AIE molecule to the 7 pathogenic bacteria selected. As shown in FIG. 7, 7 TPE-ARs with fine-tuned hydrophibicity and ClogP ranging from 3.426 to 6.071 exhibited diverse fluorescence response signals to the selected 7 pathogens due to the different multivalent interactions between TPE-ARs and pathogens. The ability of these AIE molecules to identify pathogenic bacteria is well documented.
Based on the fluorescence response profile, the 7 TPE-ARs could be divided into A, B, C three groups as the ClogP value increased (FIG. 6). The color depth of the drawn circle indicates the relative fluorescence intensity magnitude. The darker the circle color, the greater the relative fluorescence intensity. For group A, which contains TPE-AMe, TPE-AEt and TPE-APrA (3< ClogP <5), TPE-ARs respond strongly to gram-positive bacteria and fungi fluorescence, but relatively weakly to gram-negative bacteria; this result is in full agreement with the CLSM imaging results (fig. 5 c). For the group B, the ClogP value is 5-6, the group B comprises TPE-Abu, TPE-ACH and TPE-ABn, the AIE molecules of the group B show similar fluorescence response to the three types of bacteria, and the group B can be further divided into B1 (the TPE-ACH has larger fluorescence intensity change) and B2 (the TPE-ABu and the TPE-ABn have smaller fluorescence intensity change) according to the fluorescence response intensity. Group C contains TPE-AHex with ClogP >6, which is very opposite to group A, and has strong fluorescence response to gram-negative bacteria and relatively weak response to gram-positive bacteria and fungi. Based on these fluorescent signal responses, it can be concluded that as the ClogP value of TPE-ARs increases, the affinity of TPE-ARs to gram-positive bacteria and fungi gradually decreases and the affinity to gram-negative bacteria gradually increases, suggesting that hydrophobic effects play a more important role for gram-negative bacteria than gram-positive bacteria and fungi. It is also noteworthy that, although TPE-ARs exhibit similar fluorescence signals to the same pathogen in the same group, their extent is still different, indicating subtle differences in the weak interactions between TPE-ARs and pathogens.
Interestingly, TPE-ABu and TPE-AHex showed reduced fluorescence after incubation with gram-positive bacteria and fungi, while fluorescence remained unchanged or increased after incubation with gram-negative bacteria (FIGS. 6 and 7 a). This phenomenon is clearly different from the fluorescent "lighting" behavior of other TPE-ARs on pathogenic bacteria. To understand this particular phenomenon, the fluorescence intensity of 7 TPE-ARs below their CAC was carefully studied. It was found that TPE-ABu and TPE-AHex exhibited moderate fluorescence intensity compared to the other 5 AIE molecules (FIG. 7 b). This means that below the CAC, TPE-ABu and TPE-AHex have formed large, loose pre-micelles, as evidenced by Dynamic Light Scattering (DLS) and cryo-TEM results (FIGS. 7c-7 e). This phenomenon is very similar to the aggregation behaviour of oligomeric surfactants. TEM images show that below CAC, TPE-ABu and TPE-AHex assemble to form ribbon and platelet aggregates, respectively (FIGS. 7d and 7 e). The formation of these pre-micelles may be attributed to TPE-ABu and TPE-AHex containing relatively long hydrophobic chains, thereby providing relatively strong hydrophobic interactions. Based on the Restricted Intramolecular Movement (RIM) mechanism of the AIE molecule, assuming that the interaction between TPE-ARs and pathogenic bacteria is weaker than the interaction of TPE-ARs themselves in pre-micellar aggregates, the fluorescence intensity of TPE-ARs may decrease after incubation with pathogenic bacteria. Generally, gram-positive bacteria and fungi have relatively loose and porous cell walls, and thus the weak interaction between such pathogenic bacteria and the TPE-AR is not effective in limiting the intramolecular movement of the TPE-AR, resulting in TPE-AR having lower fluorescence intensity than its pre-aggregate. In contrast, for gram-negative bacteria, whose cell wall consists of a phospholipid outer membrane and a thin cross-linked peptidoglycan layer, the strong hydrophobic interaction between TPE-ARs and the phospholipid membrane of such bacteria effectively limits the intramolecular movement of TPE groups, resulting in an increase in fluorescence. The diverse aggregation behavior of TPE-ARs further enriches the multivalent interactions between TPE-ARs and pathogenic bacteria.
Based on the mathematically optimized combination of three sets of TPE-ARs with different fluorescence responses (AB1C, AB2C, AB1B2, B1B2C) with fluorescence response diversity and fluorescence sensor simplicity, 17 fluorescence sensor arrays each consisting of 3 TPE-ARs were constructed (Table 2).
TABLE 2 Classification accuracy and Cross-validation accuracy of constructed fluorescence sensor arrays
Further reduction in the number of AIE molecules was shown to reduce the accuracy of the identification. To verify the identification ability of the constructed sensor arrays, Linear Discriminant Analysis (LDA) was chosen to analyze the pathogen fluorescence response profiles generated by the sensor arrays. Taking the sensor array (combination AB1C) constructed by TPE-APrA, TPE-ACH and TPE-AHex as an example, the fluorescence response map of pathogenic bacteria (figure 8a) can be converted into a two-dimensional standard score discrimination map (figure 8b) by LDA analysis. The 7 pathogens well formed 7 groups and were completely separated from each other. Interestingly, the location of these pathogens in the standard differential map is clearly disease dependentThe type of original bacteria, gram-negative bacteria, are located on the left, while gram-positive bacteria and fungi are located on the right. Through the cross validation of LDA, the distinguishing accuracy reaches 100%, and the constructed sensor array is proved to realize efficient identification on pathogenic bacteria, and more importantly, the sensor array can distinguish normal bacteria and drug-resistant bacteria, such as normal escherichia coli (E.coli) and ampicillin-resistant strains (E.coli) R) The differentiation of (1) between normal Staphylococcus aureus (S.aureus) and penicillin-resistant strains (S.aureus) R) This will be critical to effective treatment.
The identification ability of 17 fluorescent sensor arrays was cross-validated by LDA (fig. 9 and table 2), wherein the discrimination accuracy of 14 sensor arrays for 7 pathogens was 100% and the identification accuracy was close to 100%.
Based on the above results, it can be found that TPE-ARs having a shorter hydrophobic chain or a weaker hydrophobicity, i.e. 3< ClogP <5, bind more selectively to gram-positive bacteria and fungi; TPE-ARs with long hydrophobic chains or strong hydrophobicity (ClogP >6) have stronger affinity to gram-negative bacteria. TPE-ARs with moderate hydrophobicity (5< ClogP <6) have similar affinities for three species of bacteria. Three groups of AIE molecules with different hydrophilicity and hydrophobicity are optimized and combined to successfully construct a high-efficiency fluorescent sensor array. In contrast, the introduction of benzene ring decreased the fluorescence signal response of the pathogen, thus decreasing the sensitivity and accuracy of identification, as the 3 sensor arrays (TPE-AMe, TPE-ABn and TPE-AHex; TPE-AEt, TPE-ACH and TPE-ABn; and TPE-ACH, TPE-ABn and TPE-AHex) comprising the AIE molecule TPE-ABn had a lower discrimination and identification accuracy (Table 2). This is probably due to the relatively large steric hindrance of the benzene ring, which would prevent the interaction of the TPE groups with pathogenic bacteria. In summary, the positive charge, hydrophobic substituent and aggregation behavior of TPE-ARs promote multivalent interactions with pathogenic bacteria, thereby broadening the difference between the fluorescence response signals of pathogenic bacteria.
Further to verify the ability of these sensor arrays to identify unknown samples, we randomly selected 14 samples from 7 pathogen models. TPE-APrA, TPE-ACH and TPE-AHex (combination AB1C) constructed sensor arrays as verification representatives. The fluorescence response spectra of the 14 samples generated by the sensor array were converted to standard scores using the discriminant function established for the training samples shown in fig. 9. As shown in fig. 10, mahalanobis distances from the sample to be measured to the centroids of seven groups of training samples are calculated in the three-dimensional standard score difference map. The minimum mahalanobis distance determines the sample's attribution. In this way, 14 randomly selected samples were fully identified with 100% accuracy, confirming the high reliability of the constructed fluorescence sensor array (table 3).
TABLE 3 identification of TPE-APrA, TPE-ACH and TPE-AHex sensor arrays on 14 randomly selected microbiological samples.
In practice, clinical diagnostics often encounter complex mixed samples. Therefore, TPE-APrA, TPE-ACH and TPE-AHex (combination AB1C) constructed sensor arrays were also used as representatives to distinguish and identify mixed pathogens. Similarly, by converting the fluorescence response spectrum of the mixed bacteria generated by the sensor array into a two-dimensional standard score differential map by means of Linear Discriminant Analysis (LDA), 8 mixed bacteria can well form 8 groups and are completely separated from each other, and the differential accuracy reaches 100% (FIG. 11). Based on the established discriminant function, 8 randomly selected pooled samples were also fully characterized (100% accuracy) (table 4).
TABLE 4 identification of 8 randomly selected microbial blends by TPE-APrA, TPE-ACH and TPE-AHex sensor arrays.
It is to be understood that the above embodiments are merely exemplary embodiments that have been employed to illustrate the principles of the present disclosure, which, however, is not to be taken as limiting the disclosure. It will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the disclosure, and these are to be considered as the scope of the disclosure.