CN110186891B - Polypeptide fluorescent probe specifically combining copper ions and cysteine - Google Patents

Abstract

The invention discloses a method for specifically combining copper ions and cysteinePolypeptide fluorescent probes. The amino acid sequence of the polypeptide is shown as SEQ ID NO: 1 is shown in the specification; modifying the N end of the polypeptide with a fluorescent substance to form a polypeptide fluorescent probe P, wherein the probe can be combined with Cu in a targeted mode²⁺The copper ion detection reagent has the advantages of good selectivity, low toxicity, low detection limit, simple preparation and convenient detection, and provides an efficient detection method for the detection of copper ions in biological and environmental samples; probes P and Cu²⁺The probe can specifically identify Cys, and an efficient detection method is provided for the rapid detection of Cys in biological and environmental samples.

Description

Polypeptide fluorescent probe specifically combining copper ions and cysteine

Technical Field

The invention belongs to the technical field of analysis and detection, and particularly relates to a polypeptide fluorescent probe specifically binding copper ions and cysteine.

Background

Cu²⁺Cys (cysteine) is an indispensable small molecule in human body and participates in regulating various physiological activities in vivo. Cu²⁺Has important effects on gene expression, immunoregulation, hematopoiesis, etc., and appropriate amount of Cu²⁺Can promote physiological process of organism, but when Cu in vivo²⁺When the dosage is excessive, liver aggregation can cause cirrhosis and hepatic ascites, and diseases such as Alzheimer disease, Down syndrome and the like are also related to the imbalance of copper metabolism in the body. Cys plays an important role in biological functions such as protein synthesis and translation modification in the organism. When Cys is insufficient in vivo, it causes symptoms such as hair loss, growth retardation, and muscle weakness, but when Cys is excessive in vivo, it causes neurotoxicity. At the same time, Cu²⁺The great use of Cys in industry and manufacturing industry causes Cu in the environment²⁺And Cys pollution, and the pollutants can be enriched in the human body after the animal plants enter the human body, so that great harm is brought to the life health of people.

Thus developing simple and rapid detection of Cu²⁺The method with Cys has great practical significance for disease diagnosis, environmental monitoring, food and drug analysis and the like. Shen et al have designed and synthesized a novel small molecule chemical sensor with 1, 8-naphthalimide as the fluorophore, the small molecule fluorescent probe has high selectivity to Cys, the fluorescence of the probe is quenched after adding Cys, Cu is added into the system²⁺Then, Cu²⁺Complexation with Cys occurs, and fluorescence of the probe is recovered. Liu et al developed the detection of Cu in a pure water system using phenanthrenequinone compounds as fluorophores²⁺And Cys based on copper ionsIdentification of Cu by strong paramagnetic quenching effect²⁺And the sulfydryl of the Cys can be coordinated with copper ions, and the copper ions are removed from the probe molecules, so that the aim of detecting the Cys is fulfilled. However, the above probes generally have the disadvantages of low sensitivity, high toxicity, complex preparation, poor biocompatibility, high detection limit, and the like.

Therefore, research and development of a method capable of specifically recognizing Cu has been conducted²⁺Cys, and has low toxicity, and the novel polypeptide fluorescent probe with sensitive detection is very important. The polypeptide fluorescent probe P developed by the invention is used for detecting Cu²⁺The method is free from interference of other ions, has the characteristics of high selectivity, good biocompatibility, simple preparation and low detection limit, and can be used for specifically detecting Cu²⁺And the probe is connected with Cu²⁺The formed complex P-Cu probe can specifically detect the content of Cys.

Disclosure of Invention

The invention provides a polypeptide, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO: 1 is shown.

The invention also provides an application of the polypeptide in preparing a polypeptide fluorescent probe.

The invention also provides a method for specifically recognizing Cu²⁺The polypeptide fluorescent probe P is prepared by modifying a luminescent substance to the N end of the polypeptide sequence.

Preferably, the luminescent material is any one of fluorescein compounds, rhodamine compounds, coumarin compounds, quinoline compounds, naphthalimide compounds and polyarylalkene compounds.

Preferably, the luminescent material is dansyl chloride.

The invention also provides a method for specifically recognizing Cu²⁺The polypeptide fluorescent probe P is used for detecting Cu²⁺Application in content, polypeptide fluorescent probe P and Cu²⁺Is bonded by 1:1 coordination, the Cu²⁺Has a minimum detection limit of 52nM, the Cu²⁺The detection method comprises the following steps:

(1) preparation of a standard curve: adding 10mM HEPES buffer solution with pH7.4^-5The polypeptide fluorescent probe P of mol/L is addedCu with the concentration of 0.0, 0.25, 0.5, 0.75, 1.0, 1.25 μ M²⁺And detecting the fluorescence intensity of the solution at a wavelength of 540nm with Cu²⁺The concentration is an abscissa, the fluorescence intensity is an ordinate, and a standard curve is drawn;

(2) cu in the sample²⁺And (3) content determination: adding a sample to be detected with a certain concentration into HEPES buffer solution with the concentration of 10mM and the pH value of 7.4, and detecting the fluorescence intensity of the solution at 540 nm;

(3) calculating Cu in sample to be measured²⁺The content of (A): and (3) substituting the fluorescence intensity value obtained in the step (2) into the standard curve prepared in the step (1), and calculating the content of Cu2+ in the sample to be detected.

The invention also provides a method for specifically recognizing Cu²⁺The polypeptide fluorescent probe P is applied to the preparation of a cell imaging reagent.

The invention also provides a polypeptide fluorescent probe P-Cu for specifically recognizing Cys, wherein the polypeptide fluorescent probe P-Cu is formed by the polypeptide fluorescent probe P and Cu²⁺Formed by 1:1 coordination bonding.

The invention provides an application of a polypeptide fluorescent probe P-Cu for specifically recognizing Cys in detecting the content of Cys, wherein the polypeptide fluorescent probe P-Cu is combined with Cys in a 2:1 coordination mode, the minimum detection limit of Cys is 43nM, and the detection method comprises the following steps:

(1) preparation of a standard curve: adding 10mM HEPES buffer solution with pH7.4^-5Adding Cys with the concentration of 0.0, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 mu M into the polypeptide fluorescent probe P-Cu of mol/L, detecting the fluorescence intensity of the solution at the wavelength of 515nm, and drawing a standard curve by taking the concentration of Cys as an abscissa and the fluorescence intensity as an ordinate;

(2) determination of Cys content in samples: adding a sample to be detected with a certain concentration into HEPES buffer solution with the concentration of 10mM and the pH value of 7.4, and detecting the fluorescence intensity of the solution at 515 nm;

(3) calculating the content of Cys in the sample to be detected: and (3) substituting the fluorescence intensity value obtained in the step (2) into the standard curve prepared in the step (1), and calculating the content of Cys in the sample to be detected.

The invention also provides application of the polypeptide fluorescent probe P-Cu for specifically recognizing Cys in preparation of a cell imaging reagent.

① the polypeptide provided by the invention is directly synthesized by a solid phase method, the preparation is simple, the polypeptide can be used for preparing a polypeptide fluorescent probe, ② the polypeptide fluorescent probe P prepared by the polypeptide takes the polypeptide as a main structural unit, the polypeptide fluorescent probe P has the advantages of good biocompatibility and low toxicity, and ③ the polypeptide probe is used for Cu²⁺④ the polypeptide probe and Cu have better response, no interference of other metal ions in the same main group and strong specificity²⁺The formed P-Cu probe has better response to Cys and is not influenced by other amino acids and S^2-Ion interference and good specificity.

Drawings

FIG. 1 is a structural diagram of a fluorescent polypeptide, wherein (a) is a structural diagram of a polypeptide fluorescent polypeptide probe P and (b) is a structural diagram of a P-Cu probe;

FIG. 2 is a graph of cation recognition spectra of fluorescent polypeptide probe P;

FIG. 3 fluorescent polypeptide probes P vs Cu²⁺A histogram of interference of other metal ions during identification;

FIG. 4 fluorescent polypeptide probes P vs Cu²⁺A plot of the fluorescence titration trend of (a);

FIG. 5 fluorescent polypeptide probes P vs Cu²⁺The detection limit of (2);

FIG. 6 is a diagram showing an amino acid recognition spectrum of a P-Cu probe;

FIG. 7 bar graph of other amino acid interference with Cys recognition by P-Cu probes;

FIG. 8 is a graph of the trend of fluorescence titration of P-Cu probe against Cys;

FIG. 9 detection limit of Cys by P-Cu probe;

FIG. 10 is a graph of different pH responses of a polypeptide fluorescent probe;

FIG. 11 is a photograph of an image of a cell, wherein a is a photograph of a fluorescence field of a HeLa cell incubated for 1 hour with 10. mu. M P added thereto, b is a photograph of a bright field corresponding thereto, and c is a photograph of an overlay of a fluorescence field and a bright field corresponding thereto; d is the addition of 10. mu.M P followed by 10. mu.M Cu to HeLa cells²⁺Incubating for 1h to obtain a fluorescence field image, wherein e is a corresponding bright field image, and f is a corresponding fluorescence field and bright field superposition image; g is in the directionAnd adding 10 mu M of P-Cu into the HeLa cell, then adding 5 mu M of Cys into the HeLa cell, and incubating for 1h to obtain a fluorescence field image, wherein h is a corresponding bright field image, and i is a corresponding fluorescence field and bright field superposition image.

Detailed Description

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments. Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention as defined in the appended claims.

Example 1 polypeptide and fluorescent Probe for polypeptide P-Cu

1.1 preparation method

(1) 100mg Rink ammonia resin was added to a reactor with sieve plate and the resin was swollen with 5mL of dichloromethane; removing the Fmoc protecting group at the N end by using 20% piperidine/N, N-Dimethylformamide (DMF) solution, and detecting complete removal of the protecting group by color reaction;

(2) dissolving 4eq amino acid with Fmoc protection at the N-terminal with 4eq HOBt and 4eq HBTU in DMF, activating at low temperature for 20min, adding 6eq DIEA dropwise into the solution, mixing the solution, adding into a reactor, and reacting for 3 hrs;

(3) after the reaction, the reaction solution was taken out of the reactor, and the resin was washed 3 times with 5mL of DMF and DCM, respectively. The amino acid condensation was complete as detected by chromogenic reaction, and the resin was treated with 20% piperidine/DMF solution 3 times for 5min, 5min and 15min, respectively. Washing the resin with 5mL of DMF and DCM for 3 times respectively, and detecting complete removal of the protecting group through color reaction;

(4) repeating the steps (2) and (3) until synthesizing a polypeptide with a target sequence, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO: 1 is shown.

(5) Dissolving 4eq of dansyl chloride of a fluorescent substance, 4eq of HOBt and 4eq of HBTU in DMF, activating at low temperature for 20min, adding 6eq of DIEA dropwise into the solution, mixing the solution, adding the mixture into a reactor, and reacting for 6 hrs.

(6) After the reaction, the reaction solution was taken out of the reactor, and the resin was washed 3 times with 5mL of DMF and DCM, respectively. The reaction was developed to detect complete condensation of dansyl chloride and the resin was washed with DCM and methanol in turn 3 times. The resin was drained and cleavage solution (TFA: TIS: water: 95: 2.5: 2.5) was added and reacted for 3 hrs;

(7) and (4) adding the reaction liquid obtained in the step (6) into the ethyl acetate to precipitate the polypeptide. Centrifuging to collect precipitate, dissolving the precipitate with a small amount of TFA, adding excessive ethyl glacial ether to precipitate again, centrifuging to collect precipitate, washing the precipitate with ethyl glacial ether for 2 times to obtain crude peptide, purifying the crude peptide by reversed phase liquid chromatography to obtain pure peptide, and lyophilizing to obtain polypeptide fluorescent probe P, wherein the structure is shown in (a) of FIG. 1;

(8) the polypeptide fluorescent probes P and Cu obtained in the step (7)²⁺The polypeptide fluorescent probe P-Cu is formed by mixing the components in a ratio of 1:1, and the structure is shown as (b) in figure 1.

1.2 results

The amino acid sequence of the polypeptide prepared according to the steps is shown as SEQ ID NO: 1, the structure of P of the prepared fluorescent polypeptide probe is shown in (a) of FIG. 1, and the structure of the polypeptide fluorescent probe P-Cu is shown in (b) of FIG. 1.

Example 2 polypeptide fluorescent probes P vs Cu²⁺Specificity study of the assay

2.1 evaluation experiment of cation selectivity of polypeptide fluorescent Probe P

HEPES buffer (10mM, pH7.4) was added to each of the solutions at 10^-5Polypeptide fluorescent probe P of mol/L and 10^-5metal cation (Ag) in mol/L⁺，Al³⁺，Ca²⁺，Cd²⁺，Co²⁺，Cr³⁺，Cu²⁺，Fe³⁺，Hg²⁺，K⁺，Mg²⁺，Mn²⁺，Na⁺，Ni²⁺，Pb²⁺) And detecting the change of the fluorescence emission spectrum of the solution. As a result, only Cu was observed as shown in FIG. 2²⁺In the presence of (1), the fluorescence of the probe P is obviously quenched, and the other 15 metal ions can not quench the fluorescence of the polypeptide fluorescent probe P, so that the polypeptide is required to be fluorescently detectedThe needle P can recognize Cu²⁺。

2.2 polypeptide fluorescent probes P vs Cu2⁺Specific detection study of

HEPES buffer (10mM, pH7.4) was added to each of the solutions at 10^-5Polypeptide fluorescent probe P of mol/L and 10^-5Various metal cations (Ag) in mol/L⁺，Al³⁺，Ca²⁺，Cd²⁺，Co²⁺，Cr³⁺，Cu²⁺，Fe³⁺，Hg²⁺，K⁺，Mg²⁺，Mn²⁺，Na⁺，Ni²⁺，Pb²⁺) Detecting the change of fluorescence emission spectrum of the solution, and adding 10 to the above solutions containing cations respectively^{- 5}mol/L of Cu²⁺And detecting the fluorescence emission spectrum of the solution, and drawing a graph corresponding to the maximum emission wavelength. The results are shown in FIG. 3, Cu despite the presence of other cations²⁺Still can cause the fluorescence quenching of the fluorescent polypeptide probe, which indicates that the polypeptide fluorescent probe P can specifically detect Cu²⁺And is not interfered by other cations.

2.3 polypeptide fluorescent probes P and Cu²⁺Determination of binding ratio

Adding 10 to HEPES buffer (10mM, pH7.4)^-5Adding different concentrations of Cu into the polypeptide fluorescent probe P²⁺(0.0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0eq), detecting the fluorescence emission spectrum change of the solution, and taking the corresponding value at the emission wavelength of 540nm as a fluorescence titration trend chart. The results are shown in FIG. 4, along with Cu²⁺The fluorescence intensity at 540nm decreased with increasing concentration, when 1.0eq of Cu was added²⁺After that, the fluorescence intensity at 540nm did not change any more, indicating that Cu was added²⁺Has reached saturation, i.e. the polypeptide fluorescent probes P and Cu²⁺Combined in a 1:1 stoichiometric ratio.

2.4 polypeptide fluorescent probes P vs Cu²⁺Determination of detection Limit

Firstly, determining the standard deviation sigma under blank conditions by measuring fluorescence data only with the polypeptide fluorescent probe P through 10 times of repeated experiments;

secondly, by successively increasing Cu²⁺Concentration, measuring a series of fluorescence data at an emission wavelength of 540nm, and plotting to obtain a straight line with a linear correlation coefficient of 0.9908, wherein the result is shown in FIG. 5;

and finally, calculating the pair of Cu of the polypeptide fluorescent probe P by using a formula of the limit of detection LOD (long-range of detection) being 3 sigma/k²⁺Where σ is the standard deviation under blank conditions and k is the slope of the curve. Obtaining the P pairs of Cu of the polypeptide fluorescent probes by calculation²⁺The lowest detection limit of (c) was 52 nM.

EXAMPLE 3 specificity study of the polypeptide fluorescent Probe P-Cu for detection of cysteine (Cys)

3.1 Selective evaluation experiment of polypeptide fluorescent Probe P-Cu for amino acid detection

10 was added to each HEPES buffer (10mM, pH7.4)^-5Polypeptide fluorescent probes P-Cu and 10 in mol/L^{- 5}Cysteine (Cys), glutamic acid (Glu), aspartic acid (Asp), asparagine (Asn), phenylalanine (Phe), alanine (Ala), glutamine (Gln), threonine (Thr), serine (Ser), tyrosine (Tyr), lysine (Lys), tryptophan (Trp), proline (Pro), glycine (Gly), valine (Val), methionine (Met), leucine (Leu), isoleucine (Ile), arginine (Arg), histidine (His), Glutathione (GSH) and S in mol/L^2-And detecting the change of the fluorescence emission spectrum of the solution. As a result, as shown in fig. 6, the fluorescence intensity was significantly increased only when Cys was added, but the fluorescence intensity was not significantly changed by any other substance.

3.2 specificity analysis of detection of Cys by polypeptide fluorescent Probe P-Cu

10 was added to each HEPES buffer (10mM, pH7.4)^-5mol/L of P-Cu and 10^-5mol/L of (Glu, Asp, Asn, Phe, Ala, Gln, Thr, Ser, Tyr, Lys, Trp, Pro, Gly, Val, Met, Leu, Ile, Arg, His, GSH and S^2-) Detecting the change of fluorescence emission spectrum of the solution, and adding 10 to the above solutions containing other substances^{- 5}mol/L Cys, detecting the fluorescence of the solutionThe emission spectrum is plotted against the maximum emission wavelength. The results are shown in FIG. 7, and Cys can still cause the recovery of the fluorescent polypeptide probe despite the existence of other amino acids and anions, which indicates that the polypeptide fluorescent probe P-Cu detects Cys without the existence of other amino acids and S^2-Has specificity.

3.3 determination of binding ratio of polypeptide fluorescent Probe P-Cu to Cys

Adding 10 to HEPES buffer (10mM, pH7.4)^-5Adding Cys (0.0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8eq) with different concentrations into the probe of P-Cu in mol/L, detecting the change of the fluorescence emission spectrum of the solution, and taking the corresponding value at the emission wavelength of 515nm to draw a fluorescence titration trend graph. As shown in FIG. 8, the fluorescence intensity at 515nm increased with increasing Cys concentration, and after adding 0.5 equivalent of Cys, the fluorescence intensity at 515nm did not change, indicating that the added Cys was saturated, i.e., the polypeptide fluorescent probe P-Cu was bound to Cys at a stoichiometric ratio of 2: 1.

3.4 determination of Cys detection Limit by polypeptide fluorescent Probe P-Cu

Firstly, measuring fluorescence data only with P-Cu through 10 times of repeatability experiments, and determining standard deviation sigma under blank conditions;

secondly, a straight line with a linear correlation coefficient of 0.9967 can be observed by plotting through measuring serial fluorescence data at the emission wavelength of 515nm by gradually increasing the Cys concentration, and the result is shown in FIG. 9;

and finally, calculating the lowest detection limit of the P-Cu probe for detecting Cys by using a formula of the detection limit LOD being 3 sigma/k, wherein sigma is standard deviation under blank conditions, and k is the slope of the curve. And calculating to obtain the minimum detection limit of the polypeptide fluorescent probe P-Cu to Cys of 43 nM.

Example 4 pH-responsive determination of polypeptide probes

Fluorescence emission spectra of the fluorescent polypeptide probe P, the polypeptide fluorescent probe P-Cu and the polypeptide fluorescent probe P-Cu + Cys were measured in HEPES buffer solutions at different pH values (2, 4, 6, 8, 10, 12), and the maximum fluorescence intensity was plotted. KnotAs shown in FIG. 10, the pH of the system has a wide application range, and can be used for monitoring Cu in the environment²⁺Cys can also be used for Cu in cells²⁺And detecting Cys.

Example 5 cellular imaging of polypeptide probes

(1) Culturing HeLa cells in a DMEM medium, adding 10 mu M of probe P into the cells at 37 ℃, incubating for 1h, then washing for 3 times, observing by a Zeissz upright fluorescence microscope, and imaging in a dark field;

(2) HeLa cells were cultured in DMEM medium, 10. mu.M of probe P was added to the cells at 37 ℃ and 10. mu.M of Cu was added²⁺Incubating for 1h, washing for 3 times, observing through a Zeiss upright fluorescence microscope, and imaging in a dark field;

(3) HeLa cells were cultured in DME medium, 10. mu.M of the probe P-Cu and 5. mu.M of Cys were added to the cells at 37 ℃ and incubated for 1 hour, followed by 3 washes, observation by Zeiss epifluorescence microscope and dark-field imaging.

The cell imaging results are shown in FIG. 11, in which the cells have stronger fluorescence after the probe P is added, and then Cu is added²⁺The cell fluorescence is quenched and finally the Cys cell fluorescence is added for recovery. This indicates that the probe P is able to penetrate live HeLa cells and is able to detect Cu in the cells²⁺And Cys. Wherein a is a fluorescence field image after 10 mu M P is added into the HeLa cells and incubated for 1h, b is a corresponding bright field image, and c is a corresponding fluorescence field and bright field superposition image; d is adding 10 μ M P to HeLa cells and then adding 10 μ M Cu²⁺Incubating for 1h to obtain a fluorescence field image, wherein e is a corresponding bright field image, and f is a corresponding fluorescence field and bright field superposition image; g is a fluorescence field image after 10 mu M of P-Cu is added into the HeLa cell and 5 mu M of Cys is added into the HeLa cell for incubation for 1h, h is a corresponding bright field image, and i is a superposition image of the corresponding fluorescence field and the bright field.

Sequence listing

<110> Lanzhou university

<120> a polypeptide fluorescent probe specifically binding copper ions and cysteine

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>5

<212>PRT

<213> Artificial sequence ()

<400>1

Asp Glu Lys Glu Asp

1 5

Claims (9)

1. A polypeptide, wherein the amino acid sequence of said polypeptide is as set forth in SEQ ID NO: 1 is shown.

2. Cu capable of specifically recognizing²⁺The polypeptide fluorescent probe P, which is characterized by comprising a fluorescent substance and the polypeptide as claimed in claim 1, wherein the fluorescent substance is modified at the N-terminal of the polypeptide.

3. The polypeptide fluorescent probe P as claimed in claim 2, wherein the fluorescent substance is any of fluorescein, rhodamine, coumarin, quinoline, naphthalimide and polyarylalkene.

4. The polypeptide fluorescent probe P as claimed in claim 3, wherein the fluorescent substance is dansyl chloride.

5. The method for detecting Cu by using the polypeptide fluorescent probe P as claimed in claim 2²⁺The application of the content is characterized in that the polypeptide fluorescent probe P and Cu²⁺Is bonded by 1:1 coordination, the Cu²⁺With a minimum detection limit of 52nM, said use comprising the steps of:

(1) preparation of a standard curve: adding 10mM HEPES buffer solution with pH7.4^-5mol/L polypeptide fluorescent probeP, then adding Cu with the concentration of 0.0, 0.25, 0.5, 0.75, 1.0 and 1.25 mu M²⁺And detecting the fluorescence intensity of the solution at a wavelength of 540nm with Cu²⁺The concentration is an abscissa, the fluorescence intensity is an ordinate, and a standard curve is drawn;

(2) cu in the sample²⁺And (3) content determination: adding a sample to be detected with a certain concentration into HEPES buffer solution with the concentration of 10mM and the pH value of 7.4, and detecting the fluorescence intensity of the solution at 540 nm;

(3) calculating Cu in sample to be measured²⁺The content of (A): substituting the fluorescence intensity value obtained in the step (2) into the standard curve prepared in the step (1), and calculating the Cu in the sample to be detected²⁺The content of (a).

6. A method for detecting Cu by using the polypeptide fluorescent probe P as defined in claim 2²⁺The cell imaging agent is characterized in that the polypeptide fluorescent probe P is used for Cu²⁺The lowest detection limit of (c) was 52 nM.

7. The polypeptide fluorescent probe P-Cu for specifically recognizing Cys is characterized in that the polypeptide fluorescent probe P-Cu consists of the polypeptide fluorescent probe P and Cu as claimed in claim 2²⁺A complex formed by 1:1 coordination bonding.

8. The use of the polypeptide fluorescent probe P-Cu of claim 7 for detecting Cys content, wherein the polypeptide fluorescent probe P-Cu is coordinately bound to Cys at a ratio of 2:1, and the minimum detection limit of Cys is 43nM, the use comprising the steps of:

(1) preparation of a standard curve: adding 10mM HEPES buffer solution with pH7.4^-5Adding Cys with the concentration of 0.0, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 mu M into the polypeptide fluorescent probe P-Cu of mol/L, detecting the fluorescence intensity of the solution at the wavelength of 515nm, and drawing a standard curve by taking the concentration of Cys as an abscissa and the fluorescence intensity as an ordinate;

(2) determination of Cys content in samples: adding a sample to be detected with a certain concentration into HEPES buffer solution with the concentration of 10mM and the pH value of 7.4, and detecting the fluorescence intensity of the solution at 515 nm;

(3) calculating the content of Cys in the sample to be detected: and (3) substituting the fluorescence intensity value obtained in the step (2) into the standard curve prepared in the step (1), and calculating the content of Cys in the sample to be detected.

9. The use of the polypeptide fluorescent probe P-Cu as set forth in claim 7 in the preparation of a cell imaging reagent for detecting Cys, wherein the minimum detection limit of Cys by the polypeptide fluorescent probe P-Cu is 43 nM.

Images