A probe and detection method for specifically detecting type I collagen

Abstract

The present invention provides a probe and a detection method for specifically detecting type I collagen, wherein the probe comprises a polypeptide and a lanthanide ion, wherein the polypeptide is an X-targeted polypeptide, wherein X is 5-carboxyl-1,10-phenanthroline, and its name is Phen, and 5-carboxyl-1,10-phenanthroline is combined with the lanthanide ion through electrostatic interaction. The probe can specifically detect type I collagen, and the detection method is simple and efficient. The probe has targeting, a stable fluorescence signal and lifespan, can avoid the problems of photobleaching and unstable emission peak, and has great potential in the accurate diagnosis of collagen-related diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of protein detection, and in particular to a probe and a detection method for specifically detecting type I collagen. Background Art

[0002] Rapid developments in the field of collagen detection are significantly deepening our understanding of tissue structure and disease. Conventional collagen detection methods face challenges in terms of sensitivity and specificity, especially when distinguishing different types of collagen or denatured collagen. In order to develop peptides that can specifically bind to specific types of collagen, the accuracy of detection has been significantly improved by fluorescently labeling peptides. However, traditional organic fluorescent dyes are limited by photobleaching and unstable emission peaks. Summary of the invention

[0003] In view of this, the present invention aims to propose a probe and a detection method for specifically detecting type I collagen. The probe can specifically detect type I collagen, and the detection method is simple and efficient. The probe is targeted, has a stable fluorescence signal and lifetime, can avoid the problems of photobleaching and unstable emission peak, and has great potential in the accurate diagnosis of collagen-related diseases.

[0004] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0005] A probe for specifically detecting type I collagen comprises a polypeptide and a lanthanide ion. The polypeptide is an X-targeting polypeptide, X is 5-carboxyl-1,10-phenanthroline, and its name is Phen. 5-carboxyl-1,10-phenanthroline is combined with the lanthanide ion through electrostatic interaction.

[0006] 5-Carboxyl-1,10-phenanthroline and lanthanide elements (such as Tb 3+ 、Eu 3+ ) form a highly absorbable complex when combined with other drugs, which has high stability and is not easily degraded in the human microenvironment.

[0007] Preferably, the PI value of the targeting polypeptide suitable for the human physiological environment is in the range of 4.0 to 9.0.

[0008] The PI value is the isoelectric point. There is no charge near the isoelectric point. If it is too much higher or lower than the isoelectric point, the polypeptide will precipitate. The coagulation of the polypeptide can be affected by adjusting the pH value. If the PI value is close to the physiological environment, it means that it can be better applied in the physiological environment; if it is higher or lower than the physiological environment, the application is limited. Therefore, under the pH value conditions that meet the human body, the PI value has a specific response between 4.0 and 9.0.

[0009] Preferably, the targeting polypeptide includes AHVWMQAP, (GOP)n , where n is any integer greater than 6.

[0010] GOP is the abbreviation of Gly-Hyp-Pro, and its Chinese name is Glycine-Hydroxyproline-Proline (GOP) n The Chinese name of the sequence can be written as (Glycine-Hydroxyproline-Proline) n .

[0011] Preferably, the polypeptide is Phen-(GOP) 10 Or Phen-AHVWMQAP.

[0012] Preferably, the lanthanide ion is Tb 3+ Ion or Eu 3+ ion.

[0013] Preferably, the lanthanide ion is Tb 3+ ions, the probe is Tb-[Phen-(GOP) 10 ] 3 or Tb-[Phen-AHVWMQAP] 4 .

[0014] Preferably, the lanthanide ion is Eu 3+ ions, the probe is Eu-[Phen-(GOP) 10 ] 3 or Eu-[Phen-AHVWMQAP] 4 .

[0015] The present invention also provides a method for preparing a probe for specifically detecting type I collagen as described in any one of the above items, the method comprising: solid phase synthesis of an X-targeting polypeptide; and chelating lanthanide ions to the X-targeting polypeptide.

[0016] Specifically, the specific method of solid phase synthesis is:

[0017] ① Add Rink ammonia resin into a reactor with a sieve plate and use dichloromethane to swell the resin;

[0018] ② Removal of the N-terminal Fmoc protecting group by piperidine / N,N-dimethylformamide (DMF) solution;

[0019] ③ Dissolve the amino acid with Fmoc protection at the N-terminus, HOBt and HBTU in DMF, activate at low temperature for 20 min, then drop DIEA into the solution, mix the solution and add it into the reactor for reaction for 0.5-3 h;

[0020] ④ Add acetic anhydride to the reactor, and the reaction is complete by color development. The resin is washed three times with DMF and DCM respectively;

[0021] ⑤ The reaction solution is added to icy ether to precipitate the polypeptide. The precipitate is collected by centrifugation, and an excess of icy ether is added to precipitate again and the precipitate is collected by centrifugation. The precipitate is washed with icy ether for 2-5 times and then air-dried to obtain a crude peptide; the crude peptide is purified by reverse phase liquid chromatography to obtain a pure peptide.

[0022] The method for chelating lanthanide ions to the X-targeting polypeptide is:

[0023] The X-targeting peptide is dissolved in DMSO to prepare a solution, and then mixed with lanthanide ions and stirred for 1-3 hours to obtain a lanthanide-assisted peptide probe.

[0024] The present invention also provides a method for specifically detecting type I collagen, the method comprising the following steps:

[0025] 1) Prepare type I collagen solution with PBS solution;

[0026] 2) Add the protein solution to a 96-well plate and incubate at 4°C to allow complete protein binding;

[0027] 3) Pour off the protein solution, add the above-mentioned probe into the well plate, and continue incubating at 4°C;

[0028] 4) Wash with PBS solution three times, 3-5 min each time; measure its fluorescence intensity on Infinite M200 multi-function microplate reader, and repeat three times for each measurement.

[0029] Preferably, the incubation time in step 2) is 3 to 5 hours, and the incubation time in step 3) is 2 to 4 hours.

[0030] Compared with the prior art, the probe and detection method for specific detection of type I collagen described in the present invention have the following advantages:

[0031] The probe for specifically detecting type I collagen of the present invention has specificity and improves the detection accuracy; and the probe is simple to prepare, convenient to detect and has strong binding ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0033] Figure 1 Characterization of the fluorescence spectrum and binding ratio of the probe for specific detection of type I collagen according to the present invention;

[0034] Figure 2 UV absorption spectrum of the probe for specific detection of type I collagen;

[0035] Figure 3 Circular dichroism spectrum of the probe for specific detection of type I collagen;

[0036] Figure 4 To characterize the targeting of probes for specific detection of type I collagen;

[0037] Figure 5 Characterization of the linear correlation of the probe for specific detection of type I collagen;

[0038] Figure 6 Magnetic resonance imaging of a probe specifically detecting type I collagen. DETAILED DESCRIPTION

[0039] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0040] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] Example 1 Tb-[Phen-AHVWMQAP] 4 Preparation of probes

[0042] The targeting peptide in this example is: AHVWMQAP, the sequence number is SEQ NO.1, the PI value is 6.79; the peptide is Phen-AHVWMQAP; the probe is Tb-[Phen-AHVWMQAP] 4 , the response pH value of the probe is close to the normal physiological environment of the human body.

[0043] 1. Preparation of peptides by solid phase synthesis

[0044] (1) Add 0.345 mmol / g Rink ammonia resin into a reactor with a sieve plate and use 5 mL of dichloromethane to swell the resin;

[0045] (2) The N-terminal Fmoc protecting group was removed by 20% piperidine / N,N-dimethylformamide (DMF) solution, and the complete removal of the protecting group was detected by colorimetric reaction;

[0046] (3) The amino acid (5 equiv) with Fmoc protection at the N-terminus, HOBt (5 equiv) and HBTU (5 equiv) were dissolved in DMF and activated at low temperature for 20 min. DIEA (8 equiv) was then added dropwise to the solution. The mixed solution was added to the reactor and reacted for 0.5-3 h.

[0047] (4) After the reaction, the reaction solution was withdrawn from the reactor, and the resin was washed three times with 10 mL of DMF and DCM respectively. The condensation of amino acids was detected by colorimetric reaction. The resin was treated with 20% piperidine / DMF solution for three times, for 5 min, 5 min and 15 min respectively. The resin was washed three times with 10 mL of DMF and DCM respectively. The removal of the protecting group was detected by colorimetric reaction.

[0048] (5) Repeat steps (3) and (4) and use Fmoc-protected amino acids P, A, Q, M, W, V, H and A in sequence until the target peptide is synthesized; connect Phen (8 equiv), activate at low temperature for 20 min, add 25% acetic anhydride to the reactor, and detect the completion of the reaction by colorimetric reaction. Wash the resin three times with 10 mL DMF and DCM respectively;

[0049] (6) The resin was washed three times with DCM and methanol respectively. The resin was drained and a cutting solution (TFA:TIS:water = 95:2.5:2.5) was added and reacted for 3 h.

[0050] (7) The reaction solution was added to icy ether to precipitate the peptide. The precipitate was collected by centrifugation, dissolved with a small amount of TFA, and precipitated again by adding excess icy ether and centrifuged. The precipitate was washed twice with icy ether and air-dried to obtain a crude peptide. The crude peptide was purified by reverse phase liquid chromatography to obtain a pure peptide.

[0051] 2. Probe Preparation

[0052] The above synthesized peptide was dissolved in DMSO to prepare a 0.5 mM solution, and 5 mM TbCl 3 .6H 2 O was mixed and stirred for 1-3 hours, and the dialysis bag was dialyzed for 48 hours, and the dialysate was changed every 4 hours. During the dialysis process, the materials were combined by electrostatic interaction until they were stable, and the probe Tb-[Phen-AHVWMQAP] was obtained. 4 .

[0053] 3. Characterization of probes

[0054] Pair probe Tb-[Phen-AHVWMQAP] 4 Fluorescence spectra and binding ratio characterization were performed and the fluorescence spectra were recorded by a RF6000 fluorescence spectrophotometer (Shimadzu SSL, Kyoto, Japan).

[0055] The results are as follows Figure 1 As shown, Tb-[Phen-AHVWMQAP] 4Fluorescence spectrum results: the left figure shows four different spectral bands in the wavelength range of 630-710 nm. These bands come from the transition of the 5D0 excited state to the 7FJ energy level (J=1-4). There is an obvious fluorescence emission peak at 700 nm, which is the transition from 5D0 to 7F4. The right figure shows the binding ratio of the polypeptide Phen-AHVWMQAP to Tb. The titration experiment was used to study the changes in the intensity of the fluorescence emission peak with the increase in the molar fraction of the polypeptide and the lanthanide ion. The right figure shows that the intensity of the fluorescence emission peak is the largest when the molar fraction is 0.8, and the optimal binding molar ratio of Tb to Phen-AHVWMQAP is calculated to be 1:4.

[0056] In addition, the probe prepared in this embodiment also has a stable fluorescence signal and lifespan, which can avoid the problems of photobleaching and unstable emission peak.

[0057] Example 2 Tb-[Phen-(GOP) 10 ] 3 Preparation

[0058] The targeting polypeptide of this embodiment is: (GOP) 10 , sequence number is SEQ NO.2, PI value is 5.52; polypeptide is Phen-(GOP) 10 ; The probe is Tb-[Phen-(GOP) 10 ] 4 , the response pH value of the probe is close to the normal physiological environment of the human body.

[0059] 1. Preparation of peptides by solid phase synthesis

[0060] The polypeptide was prepared by the solid phase synthesis method in the same manner as in Example 1, wherein in step (5), Fmoc-protected P, O and G were used in sequence for a total of 10 cycles until the polypeptide of the target sequence was synthesized.

[0061] 2. Probe Preparation

[0062] The above synthesized peptide was dissolved in DMSO to prepare a 0.5 mM solution, and 5 mM TbCl 3 .6H 2 O was mixed and stirred for 1-3 hours, and the dialysis bag was dialyzed for 48 hours, and the dialysate was changed every 4 hours. During the dialysis process, the materials were combined by electrostatic interaction until they were stable, and the probe Tb-[Phen-(GOP) 10 ] 3 .

[0063] The probe prepared in this embodiment also has a stable fluorescence signal and lifetime, and can avoid the problems of photobleaching and unstable emission peak.

[0064] Example 3 Eu-[Phen-(GOP)10 ] 3 Preparation

[0065] 1. Preparation of peptides by solid phase synthesis

[0066] Phen-(GOP) 10 The preparation method is the same as Example 2.

[0067] 2. Probe Preparation

[0068] The above synthesized peptide was dissolved in DMSO to prepare a 0.5 mM solution, and 5 mM EuCl 3 .6H 2 O was mixed and stirred for 1-3 hours, and the dialysis bag was dialyzed for 48 hours, and the dialysate was changed every 4 hours. During the dialysis process, the materials were combined by electrostatic interaction until they were stable, and the probe Eu-[Phen-(GOP) 10 ] 3 .

[0069] The probe prepared in this embodiment also has a stable fluorescence signal and lifespan, and can avoid the problems of photobleaching and unstable emission peak. The response pH value of the probe is close to the normal physiological environment of the human body.

[0070] Example 4 Eu-[Phen-AHVWMQAP] 4 Preparation

[0071] 1. Preparation of peptides by solid phase synthesis

[0072] The preparation method of Phen-AHVWMQAP is the same as that in Example 1.

[0073] 2. Probe Preparation

[0074] The above synthesized peptide was dissolved in DMSO to prepare a 0.5 mM solution, and 5 mM EuCl 3 .6H 2 O was mixed and stirred for 1-3 hours, and the dialysis bag was dialyzed for 48 hours, and the dialysate was changed every 4 hours. During the dialysis process, the materials were combined by electrostatic interaction until they were stable, and the probe Eu-[Phen-AHVWMQAP] was obtained. 4 .

[0075] The probe prepared in this embodiment also has a stable fluorescence signal and lifespan, and can avoid the problems of photobleaching and unstable emission peak. The response pH value of the probe is close to the normal physiological environment of the human body.

[0076] Example 5 UV absorption and circular dichroism characterization of the probe

[0077] Tb-[Phen-(GOP) prepared in Example 2 10 ] 3For example, the peptide Phen-(GOP) before Tb addition 10 And the peptide Phen-(GOP) after adding Tb 10 UV-visible absorption spectra and circular dichroism spectra were characterized. The UV-visible absorption spectra were recorded by a UV-1750 spectrophotometer (Shimadzu Corporation, Kyoto, Japan); the circular dichroism spectra were recorded by an Avivmodel 400 circular dichroism spectrometer (Applied Photophysics Ltd, UK) UV-1750, and the temperature of the cuvette was controlled by a Peltier temperature controller.

[0078] Figure 2 Phen-(GOP) 10 Peptide probe plus Tb 3+ UV absorption peaks before and after; Figure 3 Phen-(GOP) 10 Peptide probe plus Tb 3+ The circular dichroism spectra before and after the addition of lanthanide ions to the peptide showed that the spectral peaks did not change significantly, indicating that the stability of the peptide was not affected by the lanthanide ions and the original properties of the peptide were retained.

[0079] In addition, the performance of the probes prepared in Example 1 and Examples 3-4 is close to that of the probe prepared in Example 2, and the original performance of the polypeptide can also be guaranteed.

[0080] Example 6 Specific detection of type I collagen

[0081] Tb-[Phen-AHVWMQAP] prepared in Example 1 4 For example.

[0082] PBS solution was prepared with type I collagen, bovine serum albumin, human serum albumin, hemoglobin, trypsin and pepsin. 100 μL of protein solution was added to 96-well plates and incubated at 4°C for 5 h to allow complete protein binding. The protein solution was poured off and 100 μL of probe Tb-[Phen-AHVWMQAP] 4 Add to the wells and continue incubation at 4°C for 4 hours. Wash with PBS solution three times, 3-5 minutes each time. Measure its fluorescence intensity (ex: 280nm, em: 545 nm) on the Infinite M200 multifunctional microplate reader (TECAN, Switzerland). Repeat each measurement three times.

[0083] The results are as follows Figure 4As shown, from left to right are type I collagen, bovine serum albumin, trypsin, pepsin, human serum albumin and hemoglobin. The results show that: the bovine serum albumin signal is very weak, indicating that the lanthanide auxiliary polypeptide probe has a very weak ability to bind to bovine serum albumin; the trypsin, pepsin, human serum albumin and hemoglobin signals are also very weak, indicating that the lanthanide auxiliary polypeptide probe basically does not bind to bovine serum albumin, human serum albumin, hemoglobin, trypsin and pepsin; the type I collagen signal is significantly stronger than bovine serum albumin, trypsin, pepsin, human serum albumin and hemoglobin, indicating that the probe prepared by the present invention can specifically recognize type I collagen.

[0084] In addition, the probes prepared in Examples 2-4 have similar performance to that of the probe prepared in Example 1 and can also specifically recognize type I collagen.

[0085] Example 7 Characterization of probe linear correlation

[0086] Tb-[Phen-AHVWMQAP] prepared in Example 1 4 For example.

[0087] In the quantitative detection of type I collagen, the probe Tb-[Phen-AHVWMQAP] 4 Mix with different concentrations of target type I collagen (0.5, 5, 10, 20, 30, 60, 80, 100 μM) and incubate at 4°C for 2-5 h. Pour off the protein solution and wash with PBS solution 3 times, 3-5 min each time. Measure its fluorescence intensity (ex: 300 nm, em: 700 nm) on Infinite M200 multifunctional microplate reader (TECAN, Switzerland). Repeat three times for each measurement.

[0088] The results are as follows Figure 5 Shown: The concentration of target type I collagen and the probe Tb-[Phen-AHVWMQAP] at the maximum fluorescence emission wavelength of 700 nm. 4 The fluorescence intensity of the probe Tb-[Phen-AHVWMQAP] 4 The proportion of binding to the target type I collagen increased, resulting in the probe Tb-[Phen-AHVWMQAP] 4 The fluorescence intensity of the probe Tb-[Phen-AHVWMQAP] 4 The detection of target type I collagen has a wide linear range (0.5-100 μM) and a low detection limit (113 nM), which indicates that the probe has strong response ability and detection sensitivity for type I collagen.

[0089] Example 8 Magnetic Resonance Imaging of Probe

[0090] Eu-[Phen-AHVWMQAP] prepared in Example 4 4 As an example, a 4T1 xenograft mouse model was imaged.

[0091] 4T1 cells and VitroGel were evenly mixed at a volume ratio of 2:1. 150 μL of cell suspension (containing about 1×10 6 The 4T1 tumor-bearing mouse model was obtained after a two-week growth period. The mice were anesthetized by intraperitoneal injection of pentobarbital (10% w / v), and a 1 mg / mL PBS solution of the peptide probe was injected into the nude mice through the tail vein, and the magnetic resonance imaging results were collected. Figure 6 As shown, the arrow indicates the location of the tumor.

[0092] Type I collagen is the collagen most widely distributed in tissues. The probe for specifically detecting type I collagen prepared in the present invention can be used for diagnostic technology to reconstruct images of tumor sites in rats.

[0093] Comparative Example 1 X Different

[0094] Compared with Example 1, the difference is that X is different. X is 2,9-dicarboxy-1,10-phenanthroline. The prepared fluorescent probe has low fluorescence efficiency and photobleaching phenomenon. It has low specificity for type I collagen and has no obvious fluorescence response.

[0095] Comparative Example 2 Lanthanide ions are different (non-Tb 3+ Ion or Eu 3+ ion)

[0096] Compared with Example 1, the difference is that the lanthanide ions are different. This comparative example uses La 3+ and Ce 3+ The probes were prepared, and the fluorescence signals of the two probes obtained showed obvious quenching response, making it impossible to carry out the subsequent collagen recognition experiment.

[0097] Comparative Example 3 Different types of targeting polypeptides (non-AHVWMQAP or (GOP) 10 )

[0098] Compared with Example 1, the difference is that the types of targeting polypeptides are different. In this comparative example, polypeptides with different PI values, such as SSKPAKRVRVRARK (PI=12.48), AATSSSKQKRSRKK (PI=12.03), YKGSNRGTKRKRKN (PI=11.75), and SSTSTTAKRKL (PI=11.17) (produced by Shanghai Chupeptide Biotechnology Co., Ltd.), are used to replace and prepare probes. It is found that the four probes obtained by using targeting polypeptides with high PI values ​​have basically no fluorescence emission signals and no specific response to multiple proteins, and cannot be used as probes for identifying type I collagen.

[0099] By comparing the above examples and comparative examples, it can be seen that the probe prepared by the present invention can be used to specifically detect type I collagen, and the response effect is good, and the detection method is simple and efficient. In addition, it can be seen from comparative examples 1-3 that the specificity of the probe is closely related to X, the targeting polypeptide and the lanthanide ion. The electronegativity of Eu and Tb is 1.2, and their ability to attract electrons when forming chemical bonds with other compounds is comparable. 5-Carboxyl-1,10-phenanthroline is a metal chelator, and the exposed functional group - COOH can bind to Eu (4f 7 6s 2 ) and Tb (4f 9 6s 2 )'s spin orbit overlaps, the binding energy is the lowest, and the probe formed has high stability. The fluorescent probe constructed by 5-carboxyl-1,10-phenanthroline and polypeptide selected in the invention combines with rare earth ion Eu 3+ and Tb 3+ When bound, the offset directions of their electron pairs are similar, so they have strong specific recognition.

[0100] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A probe for specifically detecting type I collagen, Features: The probe includes a polypeptide and a lanthanide ion, wherein the polypeptide is an X-targeting polypeptide, X is 5-carboxyl-1,10-phenanthroline, and its name is Phen, and 5-carboxyl-1,10-phenanthroline is combined with the lanthanide ion through electrostatic interaction; Among them, the polypeptide is Phen-(GOP) 10 or Phen-AHVWMQAP; lanthanide ion is Tb 3+ Ion or Eu 3+ ion.

2. The probe for specifically detecting type I collagen according to claim 1, Features: The PI value range of targeted peptides suitable for the human physiological environment is 4.0~9.

0.

3. The probe for specifically detecting type I collagen according to claim 1, Features: The lanthanide ion is Tb 3+ ions, the probe is Tb-[Phen-(GOP) 10 ] 3 or Tb-[Phen-AHVWMQAP] 4 .

4. The probe for specifically detecting type I collagen according to claim 1, Features: The lanthanide ion is Eu 3+ ions, the probe is Eu-[Phen-(GOP) 10 ] 3 or Eu-[Phen-AHVWMQAP] 4 .

5. A method for preparing a probe for specifically detecting type I collagen according to any one of claims 1 to 4, Features: The method comprises: solid phase synthesis of X-targeting polypeptide; chelating lanthanide ions to the X-targeting polypeptide.

6. A method for specifically detecting type I collagen, Features: The method comprises the following steps: 1) Prepare type I collagen solution with PBS solution; 2) Add the protein solution to a 96-well plate and incubate at 4°C to allow complete protein binding; 3) Pour off the protein solution, add the probe according to any one of claims 1 to 4 into the well plate, and continue incubating at 4°C; 4) Wash with PBS solution three times, 3-5 min each time; measure its fluorescence intensity on Infinite M200 multi-function microplate reader, and repeat three times for each measurement.

7. The method for specifically detecting type I collagen according to claim 6, Features: The incubation time for step 2) is 3 to 5 hours, and the incubation time for step 3) is 2 to 4 hours.

Images