CN116642870A - ROC curve for assisting SERS (surface enhanced Raman Scattering) in identifying CTC (CTC), identifying method and application - Google Patents
ROC curve for assisting SERS (surface enhanced Raman Scattering) in identifying CTC (CTC), identifying method and application Download PDFInfo
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- G—PHYSICS
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
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Abstract
The invention relates to the technical field of early diagnosis of tumors, in particular to a ROC curve for assisting SERS in identifying CTC, an identification method and application. According to the invention, the ROC curve is used for assisting SERS to detect CTC cells for the first time, the SERS signal intensity is utilized to distinguish tumor cells from white blood cells, and the recognition capability and threshold value of the SERS intensity on the tumor cells can be determined by comparing the ROC curve of the SERS intensity of the tumor cells and the white blood cells. The threshold is characterized in that the threshold can be selected according to different purposes of identifying tumor cells, such as performing accurate diagnosis on the tumor cells for gene detection of the tumor cells, or performing preliminary screening on the tumor cells for early warning of tumor recurrence. By means of the unique identification mode, namely preliminary screening and accurate diagnosis of tumor cells, the magnetic SERS nanometer with high capture efficiency has great potential in detecting CTC of real blood.
Description
Technical Field
The invention relates to the technical field of early diagnosis of tumors, in particular to a ROC curve for assisting SERS in identifying CTC, an identification method and application.
Background
Circulating Tumor Cells (CTCs) represent a transitional state of tumor metastasis, which carries rich biological information related to primary tumors and metastases. CTC is used as a noninvasive liquid biopsy mode, and has important clinical significance for in-vitro early diagnosis, drug resistance evaluation, post-healing and survival time judgment and the like of patients. The Surface Enhanced Raman Spectroscopy (SERS) for detecting CTC has the potential advantages of simple operation, no damage, good stability, high sensitivity and the like, and is of great concern. The CTC detection principle is based on the recognition of tumor cells by detecting raman signals of SERS probes targeted to the cell surface after specific binding of the SERS probes to the corresponding receptors on the surface of the tumor cells by their conjugated antibodies. However, leukocytes in the blood carry raman signals due to the non-specific adsorption of SERS probes, which will greatly interfere with CTC recognition. Although some SERS probes made of low adsorption materials reduce binding to normal blood cells, they are not completely eliminated. Therefore, on the premise of unavoidable nonspecific adsorption, how to better distinguish tumor cells from normal blood cells is an important problem to be solved in the field of SERS-based CTC detection.
Subject operating characteristic (ROC) curve analysis is a widely used method for evaluating the performance of diagnostic tests, and ROC curves can be used not only to evaluate the overall diagnostic capacity of a test, but also to determine diagnostic thresholds for corresponding sensitivity and specificity. However, no report on the use of ROC curve for SERS to CTC detection is currently seen.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to use ROC curve in SERS to detect CTC to distinguish CTC from WBC more accurately.
It is another object of the present invention to provide a method of distinguishing CTCs from WBCs, which desirably enables appropriate detection specificity and sensitivity to be obtained from ROC curves.
It is still another object of the present invention to provide a CTC and WBC differentiating system for performing the above differentiating method to achieve the purpose of precisely differentiating CTCs and WBCs.
In order to solve the technical problems and achieve the purposes, the invention provides the following technical scheme:
in a first aspect, the invention provides the use of ROC curves to assist SERS in distinguishing CTCs from WBCs for non-diagnostic purposes.
In a second aspect, the present invention provides a method for distinguishing CTCs from WBCs for non-diagnostic purposes, the method comprising contacting SERS nanoparticles with a solution to be tested comprising CTCs and/or WBCs, detecting SERS signal intensity after incubation of SERS nanoparticle-complex cells, setting a SERS signal intensity threshold, and determining cells corresponding to SERS nanoparticle-complex cells having a SERS signal intensity exceeding the threshold as CTCs and cells corresponding to SERS nanoparticle-complex cells not exceeding the threshold as WBCs.
In an alternative embodiment, the SERS signal intensity threshold is a point value in the range of 0 to 3000a.u. fluorescence intensity.
In alternative embodiments, the SERS nanoparticles consist of a core magnetic particle, a noble metal nanoparticle, a raman signal molecule, a hydrophilic molecule, and a target molecule.
In an alternative embodiment, the core magnetic particles comprise iron nanoparticles, iron oxide nanoparticles, or Fe 3 O 4 At least one of the nanoparticles, preferably Fe 3 O 4 A nanoparticle;
the noble metal nanoparticles comprise at least one of gold particles, silver particles, platinum particles or copper particles, preferably gold particles;
the Raman signal molecule comprises at least one of 4-mercaptobenzoic acid, mercaptopyridine, 4-mercaptoaniline, mercaptonaphthalene, p-fluorothiophenol, rhodamine, crystal violet, alizarin red or Narcissus blue, and preferably 4-mercaptobenzoic acid;
the hydrophilic molecule comprises at least one of polydopamine, bovine serum albumin or polyethylene glycol, preferably polydopamine;
the target molecule comprises at least one of an antibody anti-trop2, anti-EGFR, anti-EpCAM or anti-Her2, preferably anti-trop2.
In an alternative embodiment, the average particle size of the SERS nanoparticles is 100-1000 nm, and the mass percentage of the target molecules in the SERS nanoparticles is 0.01% -1%.
In an alternative embodiment, the SERS nanoparticles are prepared by a process comprising, in Fe 3 O 4 The nano particles are used as cores, gold particles and 4-mercaptobenzoic acid are sequentially loaded in an aqueous solution, and then CH is carried out 3 CH 2 OH and NH 3 ·H 2 And loading polydopamine in an O environment, and finally loading an antibody anti-trop2 in an alkaline solution condition.
In a third aspect, the present invention provides a CTC and WBC differentiation system, the system comprising:
the sample loading module is used for collecting and/or storing samples, wherein the samples comprise known samples for determining the composition of CTC and WBC and/or samples to be tested for determining the composition of unknown CTC and WBC;
the ROC curve construction module is used for detecting the SERS signal intensity of a known sample, drawing a ROC curve with specificity and sensitivity, and outputting a suggested cut-off value according to detection requirements;
the detection module is used for detecting the SERS signal intensity of the sample to be detected and outputting a detection result identifier according to the set cut-off value.
In an alternative embodiment, the cut-off value is a point value in the range of 0 to 3000a.u. fluorescence intensity.
In an alternative embodiment, when the SERS signal intensity of the sample to be detected is greater than the cut-off value, the detection module outputs a result identifier pointing to the CTC; when the SERS signal intensity of the sample to be detected is smaller than or equal to the cut-off value, the detection module outputs a result identifier pointing to the WBC.
According to the invention, the ROC curve is used for assisting SERS to detect CTC cells for the first time, the SERS signal intensity is utilized to distinguish tumor cells from white blood cells, and the recognition capability and threshold value of the SERS intensity on the tumor cells can be determined by comparing the ROC curve of the SERS intensity of the tumor cells and the white blood cells.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a nanoparticle electron microscope (A) Fe according to example 1 of the present invention 3 O 4 NPs,(B)Au NPs,(C)Fe 3 O 4 @Au NPs,(D)Fe 3 O 4 @Au-MBA@PDA NPs；
FIG. 2 is a confocal image of nanoparticles described in example 2 after reaction with green fluorescent donkey anti-rabbit IgG;
FIG. 3 is the capture efficiency of the nanoparticles of example 3 on cancer cells;
FIG. 4 is the SERS intensity after co-incubation of three TNBC cell lines and human normal blood cells WBC, respectively, with the nanoparticles in example 4;
FIG. 5 is a ROC curve comparing the SERS intensities of MDA-MB-231+MDA-MB-468+HCC1806 and WBC cells in example 5.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. The components of the embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.
Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
It should be noted that: like reference numerals and letters denote like items in the following figures, and thus once an item is defined in one figure, no further definition or explanation thereof is necessary in the following figures.
In a specific embodiment, the invention provides for the use of ROC curves to assist SERS in distinguishing CTCs from WBCs for non-diagnostic purposes in a first aspect.
The non-diagnostic purpose includes development of CTC detection preparations, evaluation of tumor efficacy, and the like.
In a second aspect, the present invention provides a method for distinguishing CTCs from WBCs for non-diagnostic purposes, the method comprising contacting SERS nanoparticles with a solution to be tested comprising CTCs and/or WBCs, detecting SERS signal intensity after incubation of SERS nanoparticle-complex cells, setting a SERS signal intensity threshold, and determining cells corresponding to SERS nanoparticle-complex cells having a SERS signal intensity exceeding the threshold as CTCs and cells corresponding to SERS nanoparticle-complex cells not exceeding the threshold as WBCs.
In an alternative embodiment, the SERS signal intensity threshold is a point value in the range of 0 to 3000a.u. fluorescence intensity.
In alternative embodiments, the SERS nanoparticles consist of a core magnetic particle, a noble metal nanoparticle, a raman signal molecule, a hydrophilic molecule, and a target molecule.
In an alternative embodiment, the core magnetic particles comprise iron nanoparticles, iron oxide nanoparticles, or Fe 3 O 4 At least one of the nanoparticles, preferably Fe 3 O 4 A nanoparticle;
the noble metal nanoparticles comprise at least one of gold particles, silver particles, platinum particles or copper particles, preferably gold particles;
the Raman signal molecule comprises at least one of 4-mercaptobenzoic acid, mercaptopyridine, 4-mercaptoaniline, mercaptonaphthalene, p-fluorothiophenol, rhodamine, crystal violet, alizarin red or Narcissus blue, and preferably 4-mercaptobenzoic acid;
the hydrophilic molecule comprises at least one of polydopamine, bovine serum albumin or polyethylene glycol, preferably polydopamine;
the target molecule comprises at least one of an antibody anti-trop2, anti-EGFR, anti-EpCAM or anti-Her2, preferably anti-trop2.
In an alternative embodiment, the average particle size of the SERS nanoparticles is 100-1000 nm, and the mass percentage of the target molecules in the SERS nanoparticles is 0.01% -1%.
In an alternative embodiment, the SERS nanoparticles are prepared by a process comprising, in Fe 3 O 4 The nano particles are used as cores, gold particles and 4-mercaptobenzoic acid are sequentially loaded in an aqueous solution, and then CH is carried out 3 CH 2 OH and NH 3 ·H 2 And loading polydopamine in an O environment, and finally loading an antibody anti-trop2 under the magnet adsorption condition.
In a third aspect, the present invention provides a CTC and WBC differentiation system, the system comprising:
the sample loading module is used for collecting and/or storing samples, wherein the samples comprise known samples for determining the composition of CTC and WBC and/or samples to be tested for determining the composition of unknown CTC and WBC;
the ROC curve construction module is used for detecting the SERS signal intensity of a known sample, drawing a ROC curve with specificity and sensitivity, and outputting a suggested cut-off value according to detection requirements;
the detection module is used for detecting the SERS signal intensity of the sample to be detected and outputting a detection result identifier according to the set cut-off value.
In an alternative embodiment, the cut-off value is a point value in the range of 0 to 3000a.u. fluorescence intensity.
In an alternative embodiment, when the SERS signal intensity of the sample to be detected is greater than the cut-off value, the detection module outputs a result identifier pointing to the CTC; when the SERS signal intensity of the sample to be detected is smaller than or equal to the cut-off value, the detection module outputs a result identifier pointing to the WBC.
In one specific embodiment, the invention discloses a method for identifying Circulating Tumor Cells (CTCs) using ROC curve-assisted SERS techniques. Firstly, a magnetic SERS nanoparticle is prepared, TNBC cells expressing different trop2 are used as model cells, and the nanoparticle can effectively capture trop2 positive tumor cells and endow the tumor cells with Raman signals. For the identification of tumor cells, the threshold value of SERS intensity for identifying tumor cells was determined by ROC curve of TNBC cells (hcc1806+mda-MB-468+mda-MB-231) compared with WBC cell SERS intensity, and the maximum characteristic of the threshold value was that it can be selected according to different purposes of identifying tumor cells, such as performing accurate diagnosis of tumor cells for gene detection of tumor cells or the like (cut-off=281, sensitivity=69.0%, sensitivity=100.0%), or performing preliminary screening of tumor cells for early warning of tumor recurrence or the like (cut-off=206, sensitivity=90%, sensitivity=76%). By means of this unique identification approach, i.e. primary screening and accurate diagnosis of tumor cells, the magnetic SERS nano with high capture efficiency has great potential in detecting CTCs of real blood. The CTC identification method provided by the invention does not need to deliberately avoid the SERS signal of WBC, and provides a new visual angle for CTC identification based on the SERS technology.
Some embodiments of the present invention are described in detail below with reference to the accompanying drawings. The following embodiments and features of the embodiments may be combined with each other without conflict.
Example 1
(1)Fe 3 O 4 Preparation of NPs
0.68g FeCl 3 ·6H 2 O,1.8g CH 3 COONa and 0.75g PEI were added sequentially to 20ml ethylene glycol and then stirred continuously at 60℃until all material was dissolved. Transferring the mixed solution into a reaction kettle, heating to 220 ℃ and reacting for 2 hours, after cooling to room temperature, respectively cleaning 3 times by using ethanol and deionized water, dispersing in 50mL of deionized water, and centrifuging for 5 minutes by 140g of centrifugal force, and taking the supernatant.
(2) Au NPs preparation
10ml 5mM HAuCl 4 ·4H 2 O is added into 40ml of deionized water, heated in an oil bath at 150 ℃, quickly added with 2.3ml of 1% sodium citrate solution (w/w) after boiling, and stopped heating after continuing the reaction for 20min, and cooled to room temperature.
(3)Fe 3 O 4 Preparation of @ Au NPs
After mixing 12ml of Au with 1.6ml of Fe, stirring with a polytetrafluoroethylene rod for 30min, washing with deionized water, and dispersing in 16ml of deionized water.
(4)Fe 3 O 4 Preparation of @ Au-MBA NPs
160ul of 1mM ethanol solution of 4-mercaptobenzoic acid was added to 16ml of Fe 3 O 4 Stirring the mixture in an Au solution for 2 hours by using a polytetrafluoroethylene rod, fully cleaning the mixture by using deionized water, finally dispersing the mixture in 18ml of deionized water solution, and measuring SERS signals by Raman measurement.
(5)Fe 3 O 4 Preparation of @ Au NPs-MBA @ PDA NPs
18ml Fe 3 O 4 @Au-MBA，8mlCH 3 CH 2 OH and 600ul NH 3 ·H 2 After O mixing, stirring with a polytetrafluoroethylene rod for 20min, then slowly adding 2ml of dopamine solution (40 mg/ml), after 5 hours, thoroughly washing with deionized water, and dissolving in 8ml of deionized water.
(6)Fe 3 O 4 Preparation of @ Au-MBA @ PDA-anti-trop2 NPs
Taking 4ml of Fe 3 O 4 At Au-MBA@PDA, magnet adsorption, decanting the supernatant, then adding 4ml Tris-HCl solution (10 mM, pH=8.5), then adding 40ug anti-trop2 antibody, stirring at room temperature for 12h, PBS washing 3 times, finally dispersing in 4ml PBS solution.
Example 2
To demonstrate that the targeting antibody anti-trop2 is linked to Fe 3 O 4 Surface of @ Au NPs-MBA @ PDA NPs, alex488-labeled donkey anti-rubbit IgG and Fe 3 O 4 After 30min incubation of @ Au-MBA @ PDA-anti-trop2 NPs, PBS was washed 3 times and imaged in confocal mode, it can be seen from FIG. 2 that the green fluorescence of Alex488 on the nanoparticle surface was clearly visible, indicating successful coupling of anti-trop2 antibody to Fe 3 O 4 @ Au-MBA @ PDA.
Example 3
Fe is added to 3 O 4 After co-incubation of Au-MBA@PDA-anti-trop2 (146 ug/ml) with triple negative breast cancer cell lines HCC1806 (ATCC CRL-2335), MDA-MB-468 (ATCC HTB-132), MDA-MB-231 (ATCC CRM-HTB-26) and trop2 negative white blood cell WBC (cells) expressing different levels of trop2, respectively, they were captured with magnets, washed thoroughly with PBS, and the captured cells were counted on a cell counter. As shown in fig. 3, the capture efficiency of the magnetic SERS nanoprobe for triple negative breast cancer cells HCC1806, MDA-MB-468 and MDA-MB-468 was 97%,74% and 30%, respectively, indicating that the capture efficiency of the nanoprobe was reduced with a reduction in the trop2 expression level, whereas the capture efficiency for WBC was only 10%, which is statistically different from the other three triple negative breast cancer cells, indicating that the nanoprobe was effective in capturing trop2 positive tumor cells.
Example 4
Fe is added to 3 O 4 After incubation of Au-MBA@PDA-anti-trop2 (146 ug/ml) with HCC1806 MDA-MB-468, MDA-MB-468 and WBC, respectively, these four cells were fixed on a glass slide, 100 cells were randomly selected for each cell, and the SERS intensity of each cell was measured with a Raman instrument. As shown in FIG. 4, the average SERS signal intensity of 4 cells showed an increasing trend with increasing levels of trop2, and the SERS intensity between different cell lines was fluctuated within a certain range, resulting in a certain degree of overlapping of the cell SERS intensity ranges between different cell lines, especially for white blood cells, which were overlapped with MDA-MB-231 which had low expression of trop2 and MDA-MB-468 which had low expression of trop2, so that it was difficult to identify white blood cells and tumor cells by directly measuring the SERS signal intensity of a certain cell.
Example 5
ROC curves comparing the SERS intensities of TNBC cells (hcc1806+mda-MB-468+mda-MB-231) with WBC cells were plotted by SPSS software, with auc=0.913, indicating good recognition of tumor cells by SERS intensities (as shown in fig. 5).
Example 6
Table 1 lists the partial thresholds for SERS intensities for discriminating tumor cells from white blood cells obtained by analyzing the ROC curve in example 5. As can be seen from Table one, each threshold has its own sensitivity and specificity. Furthermore, sensitivity and specificity are characterized by a decrease in one as the other increases. In practice, the threshold value should be selected based on the purpose of the study and the balance of sensitivity and specificity. For example, when a subsequent gene detection is required for tumor cells, it is necessary to accurately distinguish tumor cells from leukocytes. The principle of "misdiagnosis as far as possible within the allowable range of the missed diagnosis rate" should be followed, that is, sensitivity should be considered while specificity is improved as much as possible. Cut-off was 281, specificity was 100% (misdiagnosis rate 0), sensitivity was 69% (missed diagnosis rate 31%). If the purpose of detection is to perform preliminary screening on tumor cells, for example, when monitoring tumor recurrence of a cancer patient, we can perform preliminary screening on CTCs in blood and early warning on the tumor recurrence in time. The principle of "avoiding missed diagnosis as far as possible within the allowable range of the misdiagnosis rate" should be followed, namely that the specificity is considered while the sensitivity is improved as much as possible. When the cut-off value is 206, the sensitivity is 90% (10% missed diagnosis rate), and the specificity is 76% (24% misdiagnosis rate).
TABLE 1 partial threshold for SERS intensity for discriminating tumor cells from white blood cells
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (10)
- Roc curves assist the use of SERS in distinguishing CTCs from WBCs for non-diagnostic purposes.
- 2. A method for distinguishing CTC and WBC for non-diagnosis purpose is characterized in that SERS nano particles are contacted with a solution to be detected containing CTC and/or WBC, the SERS signal intensity of SERS nano particle composite cells is detected after incubation, SERS signal intensity threshold is set, and cells corresponding to SERS nano particle composite cells with SERS signal intensity exceeding the threshold are judged to be CTC, and corresponding cells not exceeding the threshold are judged to be WBC.
- 3. The method of claim 2, wherein the SERS signal intensity threshold is a point value in the range of 0 to 3000a.u. of fluorescence intensity.
- 4. The method of claim 2, wherein the SERS nanoparticles consist of core magnetic particles, noble metal nanoparticles, raman signal molecules, hydrophilic molecules, and target molecules.
- 5. The method of claim 4, wherein the core magnetic particles comprise iron nanoparticles, iron oxide nanoparticles, or Fe 3 O 4 At least one of the nanoparticles, preferably Fe 3 O 4 A nanoparticle;the noble metal nanoparticles comprise at least one of gold particles, silver particles, platinum particles or copper particles, preferably gold particles;the Raman signal molecule comprises at least one of 4-mercaptobenzoic acid, mercaptopyridine, 4-mercaptoaniline, mercaptonaphthalene, p-fluorothiophenol, rhodamine, crystal violet, alizarin red or Narcissus blue, and preferably 4-mercaptobenzoic acid;the hydrophilic molecule comprises at least one of polydopamine, bovine serum albumin or polyethylene glycol, preferably polydopamine;the target molecule comprises at least one of an antibody anti-trop2, anti-EGFR, anti-EpCAM or anti-Her2, preferably anti-trop2.
- 6. The method according to claim 4 or 5, wherein the average particle size of the SERS nanoparticles is 100-1000 nm, and the mass percentage of the target molecules in the SERS nanoparticles is 0.01% -1%.
- 7. The method of claim 6, wherein the SERS nanoparticle is prepared by a method comprising, in Fe 3 O 4 The nano particles are used as cores, gold particles and 4-mercaptobenzoic acid are sequentially loaded in an aqueous solution, and then CH is carried out 3 CH 2 OH and NH 3 ·H 2 And loading polydopamine in an O environment, and finally loading an antibody anti-trop2 in an alkaline solution condition.
- Ctc and WBC differentiation system, characterized in that it comprises:the sample loading module is used for collecting and/or storing samples, wherein the samples comprise known samples for determining the composition of CTC and WBC and/or samples to be tested for determining the composition of unknown CTC and WBC;the ROC curve construction module is used for detecting the SERS signal intensity of a known sample, drawing a ROC curve with specificity and sensitivity, and outputting a suggested cut-off value according to detection requirements;the detection module is used for detecting the SERS signal intensity of the sample to be detected and outputting a detection result identifier according to the set cut-off value.
- 9. The system of claim 8, wherein the cut-off value is a point value in the range of 0 to 3000a.u. fluorescence intensity.
- 10. The system according to claim 8 or 9, wherein the detection module outputs a result identifier pointing to CTC when SERS signal intensity of the sample to be detected is greater than cut-off value; when the SERS signal intensity of the sample to be detected is smaller than or equal to the cut-off value, the detection module outputs a result identifier pointing to the WBC.
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