CN113173996A - Aggregation-induced emission peptide assembly, preparation method, detection method and application thereof - Google Patents

Aggregation-induced emission peptide assembly, preparation method, detection method and application thereof Download PDF

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CN113173996A
CN113173996A CN202110453458.2A CN202110453458A CN113173996A CN 113173996 A CN113173996 A CN 113173996A CN 202110453458 A CN202110453458 A CN 202110453458A CN 113173996 A CN113173996 A CN 113173996A
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bacterial endotoxin
black phosphorus
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peptide
targeting peptide
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CN113173996B (en
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刘珍珍
苏宁
谢胜芬
陈小君
冯家祺
陈牡丹
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GUANGDONG MEDICAL DEVICES QUALITY SURVEILLANCE AND TEST INSTITUTE
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Abstract

The invention discloses an aggregation-induced emission peptide assembly, which comprises a black phosphorus nanosheet and an aggregation-induced fluorescence molecule modified bacterial endotoxin targeting peptide, wherein the surface of the black phosphorus nanosheet is coated with polydopamine, and the aggregation-induced fluorescence molecule modified bacterial endotoxin targeting peptide is stacked on the surface of the black phosphorus nanosheet through pi-pi conjugated electrostatic interaction in a solution state. The peptide assembly material with the aggregation-induced emission effect is used as a fluorescence start sensor for detecting bacterial endotoxin, the design thought is novel, the peptide assembly material has specificity and high sensitivity to the bacterial endotoxin, the detection limit to the bacterial endotoxin is improved, the detection process is simple, and the test result is stable. The invention also discloses a preparation method, a detection method and application of the aggregation-induced emission peptide assembly.

Description

Aggregation-induced emission peptide assembly, preparation method, detection method and application thereof
Technical Field
The invention relates to the field of biomedical engineering materials, in particular to an aggregation-induced emission peptide assembly, a preparation method, a detection method and application thereof.
Background
Endotoxin is a generic term for toxic substances present in gram-negative bacteria, and is a toxin released from the cell wall components of various gram-negative bacteria after lysis. Endotoxin is not a protein and is therefore very heat resistant and will not be destroyed even when heated at a high temperature of 100 ℃ for 1 hour, and its biological activity can only be destroyed by heating at a temperature of 160 ℃ for 2 to 4 hours, or by boiling with strong alkali, strong acid or strong oxidant for 30 minutes.
Endotoxemia can occur when a large number of gram-negative pathogenic bacteria die in the focus or blood stream and a large amount of released endotoxin enters the blood. For example, when penicillin comes out, doctors have used penicillin in large dose to treat serious meningitis patients caused by Neisseria, and while killing pathogenic bacteria, the killed pathogenic bacteria disintegrate and release a large amount of endotoxin into blood, so that organ failure is caused, and endotoxin shock is caused to cause death of the patients. It has been reported in the literature that about 70 million patients in the united states suffer from a range of medical health problems caused by bacterial endotoxin each year and cause about 25 million deaths (Expert rev. anti-infection. ther.2011,9,507). Meanwhile, endotoxemia becomes one of the leading causes of death in hospitals and accounts for about 30-50% of the total mortality (Br J Med Pract,2008,1, 7-12). Studies have shown that injections, dialysis and drinking water contamination can also lead to endotoxin infections, for example dialysis patients typically use 2-3 w liters of dialysate per year, however the risk of infection inflammation of the dialysate due to endotoxin contamination is greatly increased (anal. biochem.2015,470, 71-77). The detection of endotoxin in clinical injections is particularly necessary in view of the significant threat of endotoxin to the health of clinical patients.
Currently, most of the methods for detecting endotoxin in the market use enzymatic lysis. However, the measurement result of the method is easily affected by temperature, pH and various interference factors in the sample, and the method has the disadvantages of complicated test steps, large sample reserve, more controlled conditions (PDA J.pharm.Sci.Technol.2012,66,542 and 546) and can not meet the requirement of detecting bacterial endotoxin under the state of sub-nanomole or even picomole. Therefore, the development of specific, sensitive, convenient bacterial endotoxin sensors has become of paramount importance. The earliest reports on endotoxin detection sensors were based on functionalized polyacetylene liposomes, however only high concentrations of endotoxin samples above 100 μ M were detected (J.Am.chem.Soc.2004,126, 5038-5039). Recently endotoxin sensors based on gold nanoparticles (Nano res.2012,5, 486-. However, these sensors are still not stable and sensitive enough to meet the requirement of detecting bacterial endotoxin in sub-nanomolar or even picomolar state.
Disclosure of Invention
The invention aims to overcome at least one defect (deficiency) of the prior art and provides an aggregation-induced emission peptide assembly, a preparation method, a detection method and application thereof.
The technical scheme adopted by the invention is that,
an aggregation-induced emission peptide assembly comprises a black phosphorus nanosheet and an aggregation-induced fluorescence molecule modified bacterial endotoxin targeting peptide, wherein the surface of the black phosphorus nanosheet is coated with polydopamine, and the aggregation-induced fluorescence molecule modified bacterial endotoxin targeting peptide is stacked on the surface of the black phosphorus nanosheet through pi-pi conjugated electrostatic interaction in a solution state.
In the technical scheme, the black phosphorus nanosheet has the characteristics of excellent surface activity, tunable band gap, high carrier mobility, mild on/off ratio, good biocompatibility, biodegradability and the like, has remarkable photostability and relative pH tolerance, and shows high fluorescence efficiency; in biological imaging, most fluorescent dyes are designed to accumulate in a specific organelle, but traditional dyes will quench fluorescence due to concentration quenching effects if they accumulate too much at a specific location, the aggregation-induced emission (AIE) effect has the advantages of no self-quenching, good light stability, photoresponse to an analyte and the like, and the luminescent agent (AIEgens) with the AIE characteristic has excellent performance, is reflected by high brightness, light stability in an aggregation state and low fluorescence in a molecular dissolution state, even when a large amount of AIEgens are aggregated, can generate enhanced fluorescence which is resistant to photobleaching, and is connected with polypeptide which can specifically bind to bacterial endotoxin, the modified polypeptide can be combined with the bacterial endotoxin and shows the concentration of the bacterial endotoxin through fluorescence, even, the type of the targeting peptide can be changed, and the detection of other toxins, biomarkers and even pathogenic bacteria can be realized.
Therefore, the black phosphorus nanosheet coated with the polydopamine on the surface can generate pi-pi conjugated electrostatic interaction with the bacterial endotoxin targeting peptide modified by aggregation-induced fluorescence molecules, so that a large number of the black phosphorus nanosheets are stacked to generate fluorescence signals, and the negative correlation between the fluorescence intensity and the bacterial endotoxin content is further realized. When bacterial endotoxin exists in a detection sample, the bacterial targeting peptide is separated from the black phosphorus nanosheet and is combined with the bacterial endotoxin, the competitive combination action of the target and a receptor enables the fluorescence to be linearly weakened, compared with the existing forward fluorescence detection mode that the fluorescence intensity is in positive correlation with the bacterial endotoxin concentration, the reverse fluorescence detection mode has more obvious fluorescence intensity change, so that when the peptide assembly is used as a fluorescence sensor, the lowest detection limit can reach subnanomolar or even picomolar level, when the bacterial endotoxin with high concentration level exists in the sample, the change of the fluorescence can be directly observed even through naked eyes, the test result is stable, the influence of other interference factors such as the temperature and the pH of the sample is not easy, and the inaccurate detection result caused by too short or too long detection time can be avoided.
Preferably, the aggregation-inducing fluorescent molecule is a tetraphenylethylene derivative.
More preferably, the tetraphenylethylene derivative is tetracarboxy tetraphenylethylene (TPE- (COOH)4)。
In the technical scheme, the tetraphenylethylene and the derivatives thereof belong to one of AIEgenes, can generate AIE effect and have excellent AIE characteristic, and can easily realize self-assembly and charge conversion through a benzene ring conjugated framework and an easily-modified ionic functional group structure, so that the operation of preparing the peptide assembly by modification is simpler.
A method for preparing an aggregation-induced emission peptide assembly, comprising the steps of:
s1 preparation of the black phosphorus nanosheet coated with the polydopamine comprises the following specific steps:
a1, fully dispersing blocky black phosphorus in anhydrous 1-methyl-2-pyrrolidone, carrying out ultrasonic crushing at 15-25 ℃ for 6-9 h, then centrifuging at 2000-4000 rpm for 20-30 min, and collecting brown supernatant;
a2, centrifuging the brown supernatant prepared in the step A1 at a rotating speed of 9000-12000 rpm for 15-30 min to obtain a flaky black phosphorus precipitate, freeze-drying the flaky black phosphorus precipitate, performing ultrasonic dispersion on the flaky black phosphorus precipitate for 0.5-1 h to uniformly disperse the flaky black phosphorus precipitate in absolute ethyl alcohol, adding a dopamine hydrochloride aqueous solution and a sodium hydroxide aqueous solution, stirring for 2-5 h in the dark, centrifuging, washing with pure water for multiple times, and freeze-drying to obtain a dopamine-coated black phosphorus nanosheet;
s2 preparation of bacterial endotoxin targeting peptide modified by aggregation-induced fluorescence molecule: dissolving a tetraphenylethylene derivative in a PBS phosphate buffer solution, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, stirring for 3-5 h, adding bacterial endotoxin targeting peptide, reacting for 10-15 h, dialyzing in a dark place, and freeze-drying to obtain the tetraphenylethylene modified bacterial endotoxin targeting peptide;
preparation of S3 peptide assembly: and (3) dissolving the tetraphenylethylene modified bacterial endotoxin targeting peptide prepared in the step (S2) in a PBS phosphate buffer solution, adding the polydopamine coated black phosphorus nanosheet prepared in the step (S1), and stirring for 0.25-1 h to obtain the aggregation-induced fluorescence molecule modified bacterial endotoxin targeting peptide in a solution state, wherein the bacterial endotoxin targeting peptide is assembled into a peptide assembly of the polydopamine coated black phosphorus nanosheet.
Preferably, in the step A1, the mass fraction of the blocky black phosphorus dispersed in the anhydrous 1-methyl-2-pyrrolidone solution is 0.25-0.5 mg/ml.
Preferably, in the step A2, the mass ratio of the freeze-dried flaky black phosphorus precipitate to the added dopamine hydrochloride is 1: 1-2, and the concentration ratio of the dopamine hydrochloride aqueous solution to the sodium hydroxide aqueous solution is 1: 1-2.
Preferably, in step S2, the mole ratio of the tetraphenylethylene derivative, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and bacterial endotoxin targeting peptide is 1: 1-4.
Preferably, in step S2, the molecular weight cut off by dialysis is 1000-2000 Da.
Preferably, in step S3, the concentration of the tetraphenylethylene-modified bacterial endotoxin targeting peptide prepared in step S2 dissolved in PBS phosphate buffer solution is 1 μ M to 5 μ M, and the ratio of the concentration of the tetraphenylethylene-modified bacterial endotoxin targeting peptide to the mass of the added polydopamine-coated black phosphorus nanosheets is 1 μ M:25 μ g to 50 μ g.
Preferably, in step a1, the ultrasonication is performed with a 650W ultrasonicator, and the ultrasonicator sets a single sonication time of 10s and a single gap time of 5 s.
Preferably, the steps S1, S2 and S3 are all carried out at room temperature, and the room temperature is 5-40 ℃.
Preferably, in the step a2, the preparation method of the absolute ethyl alcohol adopted is as follows: adding 1-2 g of calcium hydride into 500ml of ethanol, stirring and drying for 6-24 h, and distilling at normal pressure to obtain the absolute ethanol.
In the technical scheme, the cell endotoxin targeting peptide and the tetraphenylethylene derivative are bonded to form the tetraphenylethylene modified bacterial endotoxin targeting peptide, and the peptide assembly is formed to be used as a fluorescence sensor of the aggregation-induced emission peptide assembly through electrostatic interaction between pi-pi conjugation and a poly dopamine coated black phosphorus nanosheet in a solution state.
In addition, in the technical scheme, the release strategy triggered by the combination of the targeting peptide is combined with AIE, so that a good research idea is provided for constructing biomolecule detection platforms such as protein, DNA and aptamers, and the detection of the substances can be realized by changing the type of the targeting peptide and selecting the targeting peptide capable of being combined with other toxins, biomarkers and even pathogenic bacteria.
A method for detecting bacterial endotoxin by using the aggregation-induced emission peptide assembly or the aggregation-induced emission peptide assembly prepared by the method as a fluorescence sensor specifically comprises the following steps:
l1 stacking the bacterial endotoxin targeting peptide modified by the aggregated fluorescent molecule on the surface of the black phosphorus nanosheet to generate a fluorescent signal, wherein the fluorescent sensor is in an on state;
l2 mixing the aggregation-induced emission peptide assembly with a detection sample, and allowing the bacterial endotoxin targeting peptide modified by the aggregated fluorescent molecule to break loose electrostatic interaction and be competitively combined with the bacterial endotoxin in the detection sample;
l3 gathers the bacterial endotoxin targeting peptide that the fluorescence molecule modified and combines with the bacterial endotoxin in the testing sample, constantly breaks away from black phosphorus nanometer slice surface, and fluorescence signal weakens gradually, and fluorescence sensor is in the closed condition.
In the technical scheme, when the aggregation-induced emission peptide assembly is used as a fluorescence sensor to detect the endotoxin in the cells, the operation process is simple, no redundant reagent is needed to be added, a large amount of medical wastes are not easy to generate in the detection process, the environment is protected, energy is saved, the competitive binding action of a target of targeting peptide of the endotoxin with high specificity and sensitivity to the endotoxin and a receptor is utilized, the fluorescence intensity of the fluorescence sensor and the endotoxin concentration are in a negative correlation relationship, the change of the fluorescence intensity is used as a signal molecule to detect the content of the bacterial endotoxin in a detection sample, the detection limit of the bacterial endotoxin concentration in the sample is improved to a subnanomolar or even picomolar level, the aggregated fluorescence molecule is not self-quenched, the light stability is good, and the interference of a plurality of factors such as temperature, reaction time, reaction temperature and the like can be eliminated, the detection result is more stable.
The aggregation-induced emission peptide assembly fluorescence sensor or the preparation method is applied to detection of bacterial endotoxin and gram-negative bacteria viable bacteria.
In the technical scheme, the aggregation-induced emission peptide assembly in a solution state can be used as a bacterial endotoxin fluorescence sensor in clinical common injections (including normal saline, blood, albumin injection, insulin and other common injections) and used for products such as detection kits, indicators and the like, has good market transformation prospects, and in addition, as bacterial endotoxin mainly exists on the surface of the outer membrane of gram-negative bacteria, some detection methods only aim at the detection of the bacterial endotoxin but cannot detect the gram-negative bacteria, the aggregation-induced emission peptide assembly is functionally expanded, can be used for detecting the viable bacteria of the gram-negative bacteria, and is vital for the detection of the bacterial endotoxin.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention combines the release strategy triggered by the combination of the targeting peptide with the aggregation-induced emission effect, provides a good research idea for constructing biomolecule detection platforms such as protein, DNA, aptamers and the like, and can realize the detection of the substances by changing the type of the targeting peptide and selecting the targeting peptide capable of being combined with other toxins, biomarkers and even pathogenic bacteria;
(2) compared with the traditional method for detecting bacterial endotoxin by an enzymatic cracking method, the method adopts the change of fluorescence intensity as a signal molecule to measure the content of the bacterial endotoxin in a detection sample, has simple detection process and stable test result, and is not easily influenced by other interference factors such as sample temperature, pH and the like;
(3) when the aggregation-induced emission peptide assembly is used as a fluorescence sensor, the lowest detection limit can reach sub-nanomolar or even picomolar level, when bacterial endotoxin with high concentration level exists in a sample, the change of fluorescence can be directly observed even by naked eyes, and the test result is stable;
(4) the preparation method of the aggregation-induced emission peptide assembly is simple in steps, easily-obtained cell targeting peptide is adopted, and other reagents used in the preparation method are easy to prepare, so that the peptide assembly is easier to prepare;
(5) the aggregation-induced emission peptide assembly can be used as a bacterial endotoxin fluorescence sensor in clinical common injection, is used for products such as detection kits and indicators, has good market transformation prospect, and also has functional expansion on the fluorescence sensor, so that the aggregation-induced emission peptide assembly can be used for detecting gram-negative bacteria viable bacteria, expands the detection types and plays a vital role in bacterial endotoxin detection.
Drawings
FIG. 1 is a high-power transmission electron microscope observation of the morphology change of black phosphorus nanosheets and polydopamine-coated black phosphorus nanosheets.
FIG. 2 shows the near infrared spectroscopic analysis of tetraphenylethylene derivatives, bacterial endotoxin targeting peptides and tetraphenylethylene derivative modified bacterial endotoxin targeting peptides.
FIG. 3 is a calibration curve of fluorescence titration spectra and fluorescence intensity variation of aggregation-induced emission peptide assemblies in bacterial endotoxin solutions of different concentrations.
FIG. 4 is a graph comparing the fluorescence sensitivity of aggregation inducing luminescent peptide assemblies and aggregation inducing fluorescent molecule modified bacterial endotoxin targeting peptides in bacterial endotoxin solutions at a concentration of 10 nM.
FIG. 5 is a graph comparing the relative fluorescence intensity of aggregation-inducing luminescent peptide assemblies in bacterial endotoxin solutions of equivalent concentrations containing different interferents.
FIG. 6 is a graph showing the comparison of the relative fluorescence intensity of the aggregation-induced emission peptide assemblies in the detection of different gram-negative bacteria.
Detailed Description
The drawings are only for purposes of illustration and are not to be construed as limiting the invention. For a better understanding of the following embodiments, certain features of the drawings may be omitted, enlarged or reduced, and do not represent the size of an actual product; it will be understood by those skilled in the art that certain well-known structures in the drawings and descriptions thereof may be omitted.
The preparation of absolute ethanol described in the following examples follows the following operating steps: adding 1-2 g of calcium hydride into 500ml of ethanol, stirring and drying for 6-24 h, and distilling at normal pressure to obtain the absolute ethanol.
The following examples describe anhydrous 1-methyl-2-pyrrolidone available from Shanghai Allantin reagent, Inc. at a purity of 99.5%.
The ultrasonication apparatus described in the following examples is Ningbo Xinzhi Biotech GmbH, model JY 92-IIDN.
The tetraphenylethylene derivative described in the following examples is tetracarboxy tetraphenylethylene (TPE- (COOH)4) From Saian Rexi Biotech Ltd, with a purity of 99%.
The bacterial endotoxin targeting peptides described in the examples below were purchased from Gill Biochemical Co., Ltd, Shanghai, under the sequence KKNYSSSISSIHCRKRKRK-NH2, with a purity of > 98%.
The PBS phosphate buffer solution described in the examples below was 0.01M in concentration and 7.4 in pH.
Example 1
The embodiment provides a black phosphorus nanosheet coated with polydopamine, and the preparation method specifically comprises the following steps:
a1, putting blocky black phosphorus into 20ml of anhydrous 1-methyl-2-pyrrolidone, carrying out ultrasonic treatment for 0.5h until the blocky black phosphorus is fully dispersed, putting the blocky black phosphorus into an ice water bath, maintaining the temperature at 15 ℃, carrying out ultrasonic crushing for 6h by adopting a 650w ultrasonic crusher, setting the single ultrasonic time to be 10s and the single gap time to be 5s, then carrying out centrifugation for 20min at 2000rpm to remove the black phosphorus particles which are not peeled off, collecting brown supernatant, and storing at low temperature;
a2 centrifuging the brown supernatant prepared in the step A1 at 9000rpm for 15min to remove 1-methyl-2-pyrrolidone, collecting the stripped flaky black phosphorus precipitate, freeze-drying the flaky black phosphorus precipitate, ultrasonically dispersing the flaky black phosphorus precipitate for 0.5-1 h to uniformly disperse the flaky black phosphorus precipitate in 5ml of absolute ethyl alcohol, respectively adding 10 mu L of dopamine hydrochloride aqueous solution and 10 mu L of sodium hydroxide aqueous solution, stirring for 2h in the dark, centrifuging, washing with pure water for multiple times, and freeze-drying to obtain the dopamine-coated black phosphorus nanosheet.
In the step A1, the mass fraction of the blocky black phosphorus dispersed in the anhydrous 1-methyl-2-pyrrolidone is 0.25 mg/ml;
in step A2, the mass ratio of the flake black phosphorus precipitate after freeze drying to the added dopamine hydrochloride is 1:1, and the concentration of the added dopamine hydrochloride aqueous solution and sodium hydroxide aqueous solution is 1: 1.
Example 2
The embodiment provides a black phosphorus nanosheet coated with polydopamine, and the preparation method specifically comprises the following steps:
a1, putting blocky black phosphorus in 20ml of anhydrous 1-methyl-2-pyrrolidone, carrying out ultrasonic treatment for 1h until the blocky black phosphorus is fully dispersed, putting the blocky black phosphorus in an ice water bath, maintaining the temperature at 25 ℃, carrying out ultrasonic crushing for 9h by adopting a 650w ultrasonic crusher, setting the single ultrasonic time to be 10s and the single gap time to be 5s, then carrying out centrifugation for 30min at the rotating speed of 4000rpm to remove the black phosphorus particles which are not peeled off, collecting brown supernatant, and storing at low temperature;
a2, centrifuging the brown supernatant prepared in the step A1 at 12000rpm for 30min to remove 1-methyl-2-pyrrolidone, collecting the stripped flaky black phosphorus precipitate, freeze-drying the flaky black phosphorus precipitate, ultrasonically dispersing the flaky black phosphorus precipitate for 1h to uniformly disperse the flaky black phosphorus precipitate in 10ml of absolute ethyl alcohol, respectively adding 50 mu L of dopamine hydrochloride aqueous solution and 50 mu L of sodium hydroxide aqueous solution, stirring for 5h in a dark place, centrifuging, washing with pure water for multiple times, and freeze-drying to obtain the dopamine-coated black phosphorus nanosheet.
In the step A1, the mass fraction of the blocky black phosphorus dispersed in the anhydrous 1-methyl-2-pyrrolidone is 0.5 mg/mL;
in step A2, the mass ratio of the flake black phosphorus precipitate after freeze drying to the added dopamine hydrochloride is 1:2, and the concentration of the added dopamine hydrochloride aqueous solution and sodium hydroxide aqueous solution is 1: 2.
Example 3
The embodiment provides a bacterial endotoxin targeting peptide modified by aggregation-induced fluorescence molecules, which is prepared by the following specific steps:
dissolving a certain mass of tetraphenylethylene derivative in 5-10 ml of PBS phosphate buffer solution at room temperature, adding a certain amount of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, stirring for 3-5 h, adding bacterial endotoxin targeting peptide, reacting for 10-15 h, dialyzing by a dialysis bag in a dark place, and freeze-drying to obtain the tetraphenylethylene modified bacterial endotoxin targeting peptide.
The mole ratio of the tetraphenylethylene derivative, the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, the N-hydroxysuccinimide and the bacterial endotoxin targeting peptide is 1:2:2: 2; the intercepted molecular weight of the dialysis bag is 1000-2000 Da.
Example 4
The embodiment provides a bacterial endotoxin targeting peptide modified by aggregation-induced fluorescence molecules, which is prepared by the following specific steps:
dissolving a certain mass of tetraphenylethylene derivative in 5-10 ml of PBS phosphate buffer solution at room temperature, adding a certain amount of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, stirring for 3-5 h, adding bacterial endotoxin targeting peptide, reacting for 10-15 h, dialyzing by a dialysis bag in a dark place, and freeze-drying to obtain the tetraphenylethylene modified bacterial endotoxin targeting peptide.
The mole ratio of the tetraphenylethylene derivative, the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, the N-hydroxysuccinimide and the bacterial endotoxin targeting peptide is 1:4:4: 4; the dialysis bag retained a molecular weight of 2000 Da.
Example 5
The embodiment provides an aggregation-induced emission peptide assembly, which is prepared by using the poly-dopamine-coated black phosphorus nanosheet of embodiment 3 and the aggregation-induced fluorescence molecule-modified bacterial endotoxin targeting peptide of embodiment 1, and specifically includes the following steps:
dissolving the tetraphenylethylene-modified bacterial endotoxin targeting peptide prepared in the embodiment 3 in 10ml of PBS phosphate buffer solution, adding the polydopamine-coated black phosphorus nanosheet prepared in the embodiment 1, and stirring for 0.25-1 h to obtain the aggregation-induced fluorescence molecule-modified bacterial endotoxin targeting peptide in a solution state, wherein the bacterial endotoxin targeting peptide is assembled into a peptide assembly of the polydopamine-coated black phosphorus nanosheet.
In this example, the concentration of the tetraphenylethylene-modified bacterial endotoxin targeting peptide was 1 μ M, and the ratio of the concentration of the tetraphenylethylene-modified bacterial endotoxin targeting peptide to the mass of the added polydopamine-coated black phosphorus nanosheets was 1 μ M:25 μ g.
Example 6
The embodiment provides an aggregation-induced emission peptide assembly, which is prepared by using the poly-dopamine-coated black phosphorus nanosheet of embodiment 3 and the aggregation-induced fluorescence molecule-modified bacterial endotoxin targeting peptide of embodiment 1, and specifically includes the following steps:
dissolving the tetraphenylethylene-modified bacterial endotoxin targeting peptide prepared in the embodiment 3 in 10ml of PBS phosphate buffer solution, adding the polydopamine-coated black phosphorus nanosheet prepared in the embodiment 1, and stirring for 0.25-1 h to obtain the aggregation-induced fluorescence molecule-modified bacterial endotoxin targeting peptide in a solution state, wherein the bacterial endotoxin targeting peptide is assembled into a peptide assembly of the polydopamine-coated black phosphorus nanosheet.
In this example, the concentration of the tetraphenylethylene-modified bacterial endotoxin targeting peptide was 5 μ M, and the ratio of the concentration of the tetraphenylethylene-modified bacterial endotoxin targeting peptide to the mass of the added polydopamine-coated black phosphorus nanosheets was 1 μ M:50 μ g.
Example 7
The embodiment is to determine the prepared black phosphorus nanosheet coated with the polydopamine, and the specific determination method comprises the following steps:
weighing 2mg of the polydopamine-coated black phosphorus nanosheet (BP @ PDA) of example 1, dispersing in 1ml of pure water, slowly dropping 200. mu.l of the dispersion on a special copper net for a high power transmission electron microscope, observing by using the high power transmission electron microscope after natural drying, and simultaneously determining the particle size by using a Malvern particle sizer.
2mg of ordinary black phosphorus nanosheets (BP NSs) were weighed and assayed by the same method as described above.
The test result is shown in fig. 1, wherein fig. 1A is the appearance of a common black phosphorus nanosheet displayed under a high-power transmission electron microscope, fig. 1B is the appearance of a polydopamine-coated black phosphorus nanosheet displayed under a high-power transmission electron microscope, fig. 1C is the particle size of the common black phosphorus nanosheet determined by a malvern particle sizer, and fig. 1D is the particle size of the polydopamine-coated black phosphorus nanosheet determined by a malvern particle sizer.
Comparing fig. 1B with fig. 1A, it can be seen that the poly-dopamine coated black phosphorus nanosheet of fig. 1B has a dense PDA coating on its surface compared to the ordinary black phosphorus nanosheet of fig. 1A. As can be seen from fig. 1C, the average particle size of the ordinary black phosphorus nanosheet is only 260.1nm, and the particle size dispersion PDI is 0.333, while as can be seen from fig. 1D, the average particle size of the black phosphorus nanosheet coated with polydopamine is 480nm, and the particle size dispersion PDI is 0.445, and a comparison of the average particle size and the particle size dispersion PDI with the polydopamine shows that the particle size of the black phosphorus nanosheet coated with polydopamine in example 1 is significantly increased, and the dispersion degree is smaller, which indicates that the black phosphorus nanosheet coated with polydopamine is successfully prepared.
Example 8
In this example, the specific determination method for the prepared tetraphenylethylene derivative-modified bacterial endotoxin targeting peptide is as follows:
the bacterial endotoxin targeting Peptide (TPE-Peptide), the tetraphenylethylene derivative (TPE derivative) and the bacterial endotoxin targeting Peptide modified by the tetraphenylethylene derivative prepared in example 3 were respectively subjected to infrared spectroscopic characterization by a potassium bromide tabletting method.
The characterization and determination results are shown in FIG. 2, and TPE-Peptide and TPE derivative appear at 650cm-1～900cm-1Characteristic absorption peak of benzene ring, and endotoxin-targeting peptide at 1642cm-1And 1550cm-1And the strong amido characteristic absorption peak shows that the TPE derivative and the bacterial endotoxin targeting peptide successfully react to finally obtain the tetraphenylethylene derivative modified bacterial endotoxin targeting peptide.
Example 9
In this embodiment, the validity of the aggregation-induced emission peptide assembly as a fluorescence sensor in detecting bacterial endotoxin is verified, and the specific verification experimental method includes:
after the aggregation-induced emission peptide assembly in the solution state in example 6 is mixed with 0-10 μ M of bacterial endotoxin aqueous solutions with different concentrations, the fluorescence intensity of the mixture is measured by a fluorescence spectrophotometer under the irradiation of an excitation wave of 360nm, and when the aggregation-induced emission effect is weakened, the bacterial endotoxin can be effectively detected.
The verified results are shown in FIG. 3, in which FIG. 3A is the fluorescence titration spectrum of the aggregation-induced emission peptide assembly under different concentrations of the bacterial endotoxin in water, and FIG. 3B is the calibration curve of the fluorescence variation of the aggregation-induced emission peptide assembly under different concentrations of the bacterial endotoxin in water, wherein F0The fluorescence intensity of the fluorescence sensor at 495nm under the condition without bacterial endotoxin, F is the fluorescence intensity of the fluorescence sensor when different concentrations of bacterial endotoxin exist, F/F0Representing the relative fluorescence intensity of the fluorescence sensor in the presence of different concentrations of bacterial endotoxin.
As shown in fig. 3A, when no bacterial endotoxin is added, the fluorescence intensity of the fluorescence sensor is the maximum, so that no bacterial endotoxin exists, and the bacterial endotoxin targeting peptide modified by aggregation-induced fluorescence molecules is aggregated in a large amount through electrostatic interaction between pi-pi conjugation and the black phosphorus nanosheet coated with polydopamine, so that an aggregation-induced luminescence effect is generated and strong fluorescence is obtained; when the concentration of the bacterial endotoxin aqueous solution added to the fluorescence sensor is gradually increased, a large amount of bacterial endotoxin targeting peptides modified by aggregation-induced fluorescence molecules are combined with bacterial endotoxin to generate competitive mutual reaction, so that the modified bacterial endotoxin targeting peptides are continuously separated from the surface of a polydopamine-coated black phosphorus nanosheet, the aggregation-induced luminescence effect is correspondingly weakened, and the fluorescence intensity is gradually reduced; when the endotoxin concentration of the added bacteria was increased to 10. mu.M, the fluorescence intensity of the fluorescence sensor was the weakest.
FIG. 3B is a graph showing a standard curve according to the linear relationship between the concentration of the added bacterial endotoxin solution and the fluorescence intensity of the fluorescence sensor, wherein R is2The correlation coefficient is 0.99847, and it can be seen that the bacterial endotoxin concentration and the relative fluorescence intensity of the fluorescence sensor are in a negative correlation relationship, and as the bacterial endotoxin concentration increases, the relative fluorescence intensity of the fluorescence sensor correspondingly and stably decreases.
In addition, in the experimental process, the fact that the fluorescence intensity of the sensor reaches a saturation state within 3min is found to be about several times faster than the measuring time (about 1h) of the traditional enzyme cracking method, meanwhile, the sensor still keeps a stable state at the temperature of 25 ℃, special storage and strict temperature requirements are not needed, the detection time is short, and the test result is more stable.
Example 10
In this embodiment, the detection effect of the aggregation-induced emission peptide assembly as a fluorescence sensor under low-concentration bacterial endotoxin is verified, and the specific verification experimental method includes:
the concentration of the aggregation-inducing luminescent peptide assembly in the solution state of example 6 and the concentration of the aggregation-inducing fluorescent molecule-modified bacterial endotoxin targeting peptide solution of example 4 were adjusted to be the same, and the adjusted concentrations were mixed with a bacterial endotoxin solution having a concentration of 10nM, respectively, and the change in fluorescence intensity before and after mixing of bacterial endotoxin with the solutions of example 6 and example 4 was measured by a fluorescence spectrophotometer under irradiation of an excitation wave of 360 nM.
The results of the validation experiment are shown in FIG. 4, where F0The fluorescence intensity of the fluorescence sensor or the modified targeting peptide at 495nm under the condition without bacterial endotoxin, F is the fluorescence intensity of the fluorescence sensor or the modified targeting peptide under the condition with bacterial endotoxin, and F/F0Represents the relative fluorescence intensity of the fluorescence sensor or modified targeting peptide in the presence of different concentrations of bacterial endotoxin, all data are presented as mean ± standard deviation, and differences between experimental groups were analyzed using One way ANOVA method in GraphPad software (inc., La Jolla, CA, USA). The significance of the experimental results was determined from the P values, where P < 0.001, representing significant differences.
As shown in fig. 4, the relative fluorescence intensities of the aggregation-induced luminescent Peptide assemblies (TPE-Peptide/BP @ PDA) before and after the addition of bacterial endotoxin at a concentration of 10nm were about 1.0 and 0.9, and the significance was ap, indicating that the fluorescence intensities thereof had significant changes before and after the addition of bacterial endotoxin; the fluorescence intensity of the aggregation-induced fluorescence molecule modified bacterial endotoxin targeting Peptide (TPE-Peptide) before and after the bacterial endotoxin with the concentration of 10nm is added is weaker and has no obvious change,
as can be seen from the above experimental results, the aggregation-induced emission peptide assembly in example 6 can be used for detecting bacterial endotoxin at low concentration, and has an obvious change and is easy to detect because the aggregation-induced emission peptide assembly is formed by stacking bacterial endotoxin targeting peptides modified by aggregation-induced fluorescence molecules on the poly-dopamine-coated black phosphorus nanosheets through pi-pi conjugated electrostatic interaction, and shows a strong fluorescence intensity before adding bacterial endotoxin, and after adding bacterial endotoxin, the targeting peptides are separated from the black phosphorus nanosheets and combined with the black phosphorus nanosheets to weaken the fluorescence intensity, so that the fluorescence intensity and the bacterial endotoxin content show an inverse proportion of negative feedback, and the fluorescence intensity can change significantly. Therefore, the aggregation-induced emission peptide assembly of the fluorescence sensor has a negative correlation between the fluorescence intensity and the content of bacterial endotoxin in the presence of the polydopamine-coated black phosphorus nanosheet, and the detection principle has an important application prospect for a reagent with low-concentration bacterial endotoxin.
Example 11
In this embodiment, the effect of aggregation-induced emission peptide assembly as a fluorescence sensor on the influence of interferents on endotoxin is evaluated by selecting interferents affecting endotoxin measured by a conventional enzymatic hydrolysis method, such as water, ethylenediaminetetraacetic acid, citrate, glucose, bovine serum albumin, and phosphate, and the specific evaluation experiment method is as follows:
the 6 kinds of interferents were mixed with bacterial endotoxin solutions of the same concentration, and the mixture was added to the aggregation-induced emission peptide assembly of example 5 in a solution state, and the fluorescence intensity at 495nm was measured using a fluorescence spectrophotometer. Meanwhile, a sample to which only an interfering substance was added was set as a control group to which the interfering substance was added in the same concentration as described above to the aggregation-inducing luminescent peptide assembly of example 5 in a solution state, and the fluorescence intensity at 495nm was measured by a fluorescence spectrophotometer as well.
The results of the evaluation are shown in FIG. 5, in which F0The fluorescence intensity of the fluorescence sensor at 495nm under the condition without bacterial endotoxin, F is the fluorescence intensity of the fluorescence sensor in the presence of bacterial endotoxin, F0and/F is the relative fluorescence intensity of the fluorescence sensor in the presence of bacterial endotoxin.
As can be seen from FIG. 5, the control group to which only the interferent was added had a relatively weak fluorescence intensity, compared to the samples to which the interferent and bacterial endotoxin were added, which significantly increased the relative fluorescence intensity, but the fluorescence intensity was not affected by the presence of the interferent. Therefore, even when the fluorescence sensor provided by the invention is used for detection, the fluorescence sensor still has high selectivity on bacterial endotoxin even if potential interferents exist, and the test effect can still be stable and good.
Example 12
Although the above examples demonstrate that the aggregation-inducing luminescent peptide assembly of the present invention can be used as a fluorescence sensor for detecting bacterial endotoxin, bacterial endotoxin is mainly present on the outer membrane surface of gram-negative bacteria, and therefore, the present example further expands the evaluation of the detection performance of the fluorescence sensor on the viable gram-negative bacteria.
The gram-negative bacteria used for detection in this example were all klebsiella, pseudomonas aeruginosa, escherichia coli, and salmonella, which were purchased from the Guangdong province culture Collection.
The specific detection experimental method comprises the following steps:
culturing the above strains at 37 deg.C to logarithmic growth phase, centrifuging to remove culture medium, diluting to 1.0 × 104CFU/mL to obtain bacterial suspension, diluting 100 μ L of bacterial suspension to 2mL, adding the aggregation-induced emission peptide assembly prepared in example 5, incubating for 1min, and measuring the fluorescence intensity at 495nm under the irradiation of 360nm excitation wave by using a fluorescence spectrophotometer, wherein the fluorescence intensity is recorded as F1。
The experimental results of the detection are shown in FIG. 6, wherein F0Fluorescence intensity at 495nm for the fluorescence sensor in the absence of bacterial endotoxin, F0/F1Relative fluorescence intensity in the presence of various gram-negative bacteria.
As can be seen from FIG. 6, the relative fluorescence intensity of the fluorescence sensor is significantly increased after gram-negative bacteria are added, and the experimental result shows that the aggregation-induced emission peptide assembly of the invention can be used as a fluorescence sensor for detecting the existence of various gram-negative bacteria, and the functional expansion advantage is crucial to endotoxin detection.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the technical solutions of the present invention, and are not intended to limit the specific embodiments of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention claims should be included in the protection scope of the present invention claims.

Claims (10)

1. The aggregation-induced emission peptide assembly is characterized by comprising a black phosphorus nanosheet and an aggregation-induced fluorescence molecule modified bacterial endotoxin targeting peptide, wherein the surface of the black phosphorus nanosheet is coated with polydopamine, and the aggregation-induced fluorescence molecule modified bacterial endotoxin targeting peptide is stacked on the surface of the black phosphorus nanosheet through pi-pi conjugated electrostatic interaction in a solution state.
2. The assembly of claim 1, wherein the aggregation-inducing fluorescent molecule is a tetraphenylethylene derivative.
3. A method for preparing an aggregation-induced emission peptide assembly, comprising the steps of:
s1 preparation of the black phosphorus nanosheet coated with the polydopamine comprises the following specific steps:
a1, fully dispersing blocky black phosphorus in anhydrous 1-methyl-2-pyrrolidone, carrying out ultrasonic crushing at 15-25 ℃ for 6-9 h, then centrifuging at 2000-4000 rpm for 20-30 min, and collecting brown supernatant;
a2, centrifuging the brown supernatant prepared in the step A1 at a rotating speed of 9000-12000 rpm for 15-30 min to obtain a flaky black phosphorus precipitate, freeze-drying the flaky black phosphorus precipitate, performing ultrasonic dispersion on the flaky black phosphorus precipitate for 0.5-1 h to uniformly disperse the flaky black phosphorus precipitate in absolute ethyl alcohol, adding a dopamine hydrochloride aqueous solution and a sodium hydroxide aqueous solution, stirring for 2-5 h in the dark, centrifuging, washing with pure water for multiple times, and freeze-drying to obtain a dopamine-coated black phosphorus nanosheet;
s2 preparation of bacterial endotoxin targeting peptide modified by aggregation-induced fluorescence molecule: dissolving a tetraphenylethylene derivative in a PBS phosphate buffer solution, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, stirring for 3-5 h, adding bacterial endotoxin targeting peptide, reacting for 10-15 h, dialyzing in a dark place, and freeze-drying to obtain the tetraphenylethylene modified bacterial endotoxin targeting peptide;
preparation of S3 peptide assembly: and (3) dissolving the tetraphenylethylene modified bacterial endotoxin targeting peptide prepared in the step (S2) in a PBS phosphate buffer solution, adding the polydopamine coated black phosphorus nanosheet prepared in the step (S1), and stirring for 0.25-1 h to obtain the aggregation-induced fluorescence molecule modified bacterial endotoxin targeting peptide in a solution state, wherein the bacterial endotoxin targeting peptide is assembled into a peptide assembly of the polydopamine coated black phosphorus nanosheet.
4. The preparation method according to claim 3, wherein in the step A1, the mass fraction of the blocky black phosphorus dispersed in the anhydrous 1-methyl-2-pyrrolidone solution is 0.25-0.5 mg/ml.
5. The preparation method according to claim 3, wherein in the step A2, the mass ratio of the freeze-dried flaky black phosphorus precipitate to the added dopamine hydrochloride is 1: 1-2, and the concentration ratio of the dopamine hydrochloride aqueous solution to the sodium hydroxide aqueous solution is 1: 1-2.
6. The method according to claim 3, wherein in step S2, the mole ratio of the tetraphenylethylene derivative, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and bacterial endotoxin-targeting peptide is 1:1 to 4.
7. The method according to claim 3, wherein the cut-off molecular weight of dialysis is 1000 to 2000Da in step S2.
8. The preparation method according to claim 3, wherein in step S3, the concentration of the tetraphenylethylene-modified bacterial endotoxin targeting peptide prepared in step S2 dissolved in PBS phosphate buffer solution is 1-5 μ M, and the ratio of the concentration of the tetraphenylethylene-modified bacterial endotoxin targeting peptide to the mass of the added polydopamine-coated black phosphorus nanosheets is 1 μ M: 25-50 μ g.
9. A method for detecting bacterial endotoxin by using the aggregation-inducing luminescent peptide assembly of claim 1 or 2 or the aggregation-inducing luminescent peptide assembly prepared by the method of any one of claims 3 to 8 as a fluorescence sensor, comprising the steps of:
l1 stacking the bacterial endotoxin targeting peptide modified by the aggregated fluorescent molecule on the surface of the black phosphorus nanosheet to generate a fluorescent signal, wherein the fluorescent sensor is in an on state;
l2 mixing the aggregation-induced emission peptide assembly with a detection sample, and allowing the bacterial endotoxin targeting peptide modified by the aggregated fluorescent molecule to break loose electrostatic interaction and be competitively combined with the bacterial endotoxin in the detection sample;
l3 gathers the bacterial endotoxin targeting peptide that the fluorescence molecule modified and combines with the bacterial endotoxin in the testing sample, constantly breaks away from black phosphorus nanometer slice surface, and fluorescence signal weakens gradually, and fluorescence sensor is in the closed condition.
10. Use of an aggregation-inducing luminescent peptide assembly according to claim 1 or 2 or a method according to any one of claims 3 to 8 for the detection of bacterial endotoxin and live gram-negative bacteria.
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