Single magnetic microsphere analysis method based on single particle inductively coupled plasma mass spectrometry

Abstract

The purpose of this invention is to establish a single-particle inductively coupled plasma mass spectrometry (ICP-MS) method for analyzing single magnetic microspheres, which can effectively avoid signal fluctuations caused by errors during absolute quantification, achieving stable and accurate quantification of two prostate cancer biomarkers. The principle of this invention is: using... + Fe 58 The frequency signals of isotopes are used to unify the number of magnetic microspheres, and the results are obtained by evaluating the frequency signals of individual magnetic microspheres. + Au 197 and + Pt 194 The intensity distribution, calculated through average intensity or fitted intensity, yields stable analytical results unaffected by changes in the number of magnetic microspheres detected. This is achieved using the intensity distribution on a single magnetic microsphere. + Au 197 and + Pt 194 The linear relationship between intensity and tPSA and fPSA demonstrates that this analytical method enables the simultaneous detection of two prostate cancer biomarkers and can be applied to the simultaneous analysis of the concentrations of the two biomarkers in the serum of patients with prostate disease.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of analytical chemistry detection, and particularly relates to a single particle inductively coupled plasma mass spectrometry (SP-ICPMS) based analysis method. BACKGROUND

[0002] The rapid development of multi-component analysis in the field of precision medicine provides a strong guarantee for the effective diagnosis of malignant tumors. Absolute quantification strategy can directly measure the expression of total probe amount directly related to the sample concentration. When combined with multiplexed bioassay, absolute quantification greatly promotes personalized treatment of cancer prognosis. However, there is still a challenge that the total amount of probes is affected by errors in the analysis strategy, which may cause fluctuations in the detection signal. Therefore, it is still a growing demand to build a stable data acquisition and processing method. Single particle inductively coupled plasma mass spectrometry (SP-ICPMS) is widely used in single cell analysis, medical detection and other fields due to its strong element resolution and high throughput. SP-ICPMS collects and records the transient signal generated by a single nanoparticle through time-resolved technology, and provides results including nanoparticle fitting intensity, intensity distribution, signal frequency, particle number concentration, mass concentration, etc. through analysis of nanoparticle intensity distribution information and frequency information. Among them, the analysis of fitting intensity is completed by Gaussian or Poisson fitting of intensity distribution. The fitting intensity obtained in SP-ICPMS detection is a relatively independent variable, which is not affected by the total amount of nanoparticles in the detection or the occasional abnormal transient signal in the detection, and is expected to avoid the fluctuations of the total amount of probes caused by errors in absolute quantification, and improve the stability of analysis and detection. SUMMARY

[0003] The purpose of the present application is to use the fitting calculation or average value processing method of intensity distribution of SP-ICPMS to establish a single magnetic microsphere analysis method which can effectively avoid signal instability or fluctuations in the detection process, and realize stable and accurate quantitative detection of analytes. The method takes magnetic microspheres as the capture center, takes the detection of two cancer markers related to prostate cancer as the analysis model, uses sandwich immunization to capture total prostate specific antigen (tPSA), free prostate specific antigen (fPSA), tPSA antibody modified gold nanoparticles (AuNPs) and fPSA antibody modified platinum nanoparticles (PtNPs) respectively, and performs SP-ICPMS analysis on the magnetic microspheres capturing nanoparticles. The fitting intensity of the magnetic microspheres is used as the detection signal of the tPSA and fPSA, and the fitting intensity of the magnetic microspheres is used as the detection signal of the tPSA and fPSA. + Fe 58The frequency signal of the isotopes is uniform to the number of the magnetic microspheres, and the intensity distribution is Gaussian fitted and the average value is calculated to obtain the intensity of the single magnetic microsphere + Au 197 and + Pt 194 The intensity is not affected by the number of the magnetic microspheres, and the signal fluctuation of the probe signal in the absolute quantification caused by the different number of the magnetic microspheres is well avoided. The linear relationship between the intensity of the single magnetic microsphere and tPSA and fPSA is utilized to realize the simultaneous detection of the two cancer markers, and is applied to the simultaneous analysis of the concentrations of the two markers in the serum of the patients with prostate diseases. + Au 197 and + Pt 194 The linear relationship between the intensity of the single magnetic microsphere and tPSA and fPSA is utilized to realize the simultaneous detection of the two cancer markers, and is applied to the simultaneous analysis of the concentrations of the two markers in the serum of the patients with prostate diseases. BRIEF DESCRIPTION OF DRAWINGS

[0004] Figure 1 It is the mechanism diagram of the single magnetic microsphere analysis method based on SP-ICPMS in the application.

[0005] Figure 2 It is the intensity distribution of the single magnetic microsphere and the stability in the intensity signal acquisition in the analysis method of the application. + Fe 58 The signal of the isotopes in the SP-ICPMS and the linear relationship with the number of the magnetic microspheres in the detection.

[0006] Figure 3 It is the intensity distribution of the single magnetic microsphere and the stability in the intensity signal acquisition in the analysis method of the application. + Au 197 and + Pt 194 The intensity distribution of the single magnetic microsphere and the stability in the intensity signal acquisition in the analysis method of the application.

[0007] Figure 4 It is the linear relationship and selectivity in the simultaneous detection of the two prostate-related antigens in the analysis method of the application.

[0008] Figure 5 It is the accuracy control in the detection of the serum samples of the patients in the analysis method of the application.

[0009] Figure 6 It is the application effect in the recovery of the human serum in the analysis method of the application. DETAILED DESCRIPTION

[0010] To achieve the above object, the application provides the following technical scheme (the experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents and the like used in the following examples can be obtained from commercial channels unless otherwise specified. The water used in the following examples is ultrapure water treated by a Milli-Q ultrapure water purification system).

[0011] Preparation of 31 nm AuNPs

[0012] a1 Take 2 mL of 1% chloroauric acid (w / v) into 60 mL of ultrapure water, mix in a three-neck flask with magnetic stirrer, heat to boiling in a heating mantle and keep refluxing for 15 min;

[0013] a2 Add 1.2 mL of 1% sodium citrate reducing agent, keep refluxing for 20 min with stirring;

[0014] a3 Turn off the heating mantle temperature control system, keep stirring with the stirrer, slowly cool the solution to room temperature, pour the solution into a 100 mL stock bottle, and dilute to 100 mL.

[0015] Preparation of 36 nm PtNPs

[0016] b1 Mix 3.6 mL of 0.2% chloroplatinic acid with ultrapure water in a three-neck flask, heat to boiling in a heating mantle and keep for 15 min;

[0017] b2 Then add 1.1 mL of 1% sodium citrate reducing agent mixed with 0.05% citric acid, followed by 0.55 mL of 1% sodium citrate reducing agent mixed with 0.05% citric acid and 0.08-0.11% KBH4, keep refluxing for 10 min;

[0018] b3 Turn off the heating mantle heating system, slowly cool the mixed solution to room temperature, take out the first-stage seed nanoparticle solution and dilute to volume;

[0019] b4 Mix 56 mL of ultrapure water with 2 mL of the first-stage seed solution, add 1.8 mL of 1% chloroplatinic acid solution, then add 1 mL of 1% sodium citrate reducing agent mixed with 1.25% ascorbic acid solution;

[0020] b5 Slowly heat to boiling, keep refluxing for 15 min, take out the second-stage seed solution and dilute to volume;

[0021] b6 Mix 56 mL of ultrapure water with 12 mL of the second-stage seed solution, add 1.8 mL of 1% chloroplatinic acid solution, then add 1 mL of 1% sodium citrate reducing agent mixed with 1.25% ascorbic acid solution;

[0022] b7 Slowly heat to boiling, keep for 15 min, take out the 36 nm Pt nanoparticle solution and dilute to volume for standby.

[0023] Activation of magnetic microspheres

[0024] c1 Commercial magnetic microspheres (MyOneTM Carboxylated magnetic beads, 1 µm) were activated in 20 µg units;

[0025] c2 After washing 20 µg magnetic microspheres with 25 mM pH 6.5 morpholine ethanesulfonic acid buffer (MES buffer), 20 µL of 150 mg / mL EDC (CAS: 1892-57-5) and 20 µL of 25 mg / mL (CAS: 106627-54-7) were added for activation (25 minutes);

[0026] c3 After continuing to wash once with MES buffer, the activated magnetic microspheres were coated with 30 µg tPSA antibody solution, and the coupling reaction was completed at 25 degrees for 6 hours to label the antibody on the magnetic microspheres;

[0027] c4 The labeled magnetic microspheres were added to a PBS solution containing 10% bovine serum albumin (BSA) for blocking (60 minutes), so that the final concentration of BSA was 1%. After washing three times with a PBS buffer solution containing 1% BSA, the antibody-labeled magnetic microspheres were prepared.

[0028] Labeling of metal nanoparticles

[0029] d1 Take 1 mL AuNPs, first add 100 µL of pH 9.0 borate buffer solution, adjust the pH to about 8.0-8.4, so that the nanoparticle pH is near the isoelectric point of the antibody (no charge itself), and mix well with vortex;

[0030] d2 Add 30 µg of tPSA reporter antibody for electrostatic adsorption labeling. The sample is vortexed and wrapped at room temperature (25 degrees Celsius) for 60 minutes;

[0031] d3 Add 130 uL of blocking agent 10% BSA solution, block at room temperature (25°C) for 60 min;

[0032] d4 After the reaction, the solution is centrifuged at 4 degrees Celsius (9000 rpm) for 18 minutes, and after the supernatant is removed, the concentrated solution is dissolved in a PBS buffer solution containing 1% BSA (0.5 mL);

[0033] d5 The method of labeling fPSA reporter antibody with PtNPs is similar to that of AuNPs;

[0034] d6 Mix the two kinds of labeled nanoparticle reporter probes with each other and vortex well. Finally, the volume of the mixed probe is 1 mL.

[0035] Immune response

[0036] e1 Add 5 μg antibody-labeled magnetic microspheres, 60 μL sample, and 25 μL mixed probe into a 200 μL centrifuge tube, respectively;

[0037] e2 After the solution is stirred uniformly, incubate at 37 o C for 2 h to perform immunoreaction.

[0038] Preparation of detection sample

[0039] f1 After reaction, wash the magnetic microsphere sample twice with PBS containing 1% BSA and 0.05% Tween-20, and resuspend in 100 μL PBS containing 1% BSA.

[0040] f2 After mixing, take an appropriate amount of mixed solution, add to a 4 mL centrifuge tube, dilute the sample with 4 mL ultrapure water, mix by vortex, and determine the obtained solution by SP-ICPMS.

[0041] SP-ICPMS single magnetic microsphere analysis

[0042] g1 Insert the suction pump tube of ICPMS into the diluted solution to collect signals, with a scanning time of 10 s and a scanning speed of 200 μs / time.

[0043] g2 Use + Fe 58 isotope to calibrate and unify the number of magnetic microspheres. If the number of magnetic microspheres in single detection differs too much, the dilution multiple in the preparation process of the detection sample should be adjusted in time to control a reasonable number of magnetic microspheres (500-600 times of + Fe 58 signal number in single determination);

[0044] g3 Use the fitting intensity of + Au 197 on a single magnetic microsphere to establish concentration analysis of tPSA, and use the fitting intensity of + Pt 194 on a single magnetic microsphere to establish concentration analysis of fPSA.

[0045] The present application is further described below in conjunction with the drawings of the specification, but the analysis method of the present application is not limited to the following examples.

[0046] Example 1, + Fe 58 isotope is used to determine the number of magnetic microspheres in SP-ICPMS;

[0047] In this invention, the counting of magnetic microspheres and maintaining a consistent number of them during detection are crucial analytical techniques. First, ICP-MS spectral scanning of Fe isotopes is performed. [The remaining text appears to be incomplete and requires further context.] Figure 2 (a) It can be seen that, + Fe 58 The isotopes are well distinguishable from each other, and there is no interference from isotopic signal peaks. Figure 2 (b) and (c) show that the magnetic microspheres detected by SP-ICPMS... + Fe 58 The signal is significantly stronger than the background signal, with each signal exceeding the background representing one detected magnetic microsphere. Furthermore, the number of signals is linearly related to the number of detected magnetic microspheres. Figure 2 (d) Therefore, SP-ICPMS can be used to detect... + Fe 58 The number of magnetic microspheres is used to reflect the number of magnetic microspheres, thus standardizing the number of magnetic microspheres in a single measurement.

[0048] Example 2: Elimination of signal fluctuations by the analysis method of the present invention;

[0049] In absolute quantification, the number of probes reported is directly related to the signal generated by the probes, and the total probe concentration is inevitably affected by operational errors, leading to fluctuations in probe signal readings. To evaluate the stability of data collection and processing for the single magnetic microsphere analysis method, the examples varied the number of magnetic beads in each measurement, and the Au or Pt intensity distribution on the individual magnetic beads was determined by SP-ICPMS. Figure 3 (a) and (b)). The intensity distribution was calculated to obtain the average content of Au or Pt on a single magnetic microsphere. Figure 3 (c) The results show that the Au or Pt intensity on each magnetic microsphere remains stable regardless of the number of magnetic microspheres. This is because, during the test, the change in the number of magnetic microspheres has little effect on the number of PtNPs and AuNPs on a single magnetic microsphere. The evaluation of the Au or Pt intensity on a single magnetic microsphere can effectively avoid the influence of absolute quantitative error on signal stability.

[0050] Example 3: The analytical method of the present invention simultaneously detects two prostate-related markers in patient serum;

[0051] This embodiment investigates the detection performance of SP-ICPMS-based single magnetic microsphere analysis method for tPSA and fPSA in human serum;

[0052] 1. Linearity of single magnetic microsphere analysis method for fPSA detection

[0053] like Figure 4(a) As shown in Figure, according to the proportional relationship between the fPSA concentration and the signal of Pt on a single magnetic microsphere, a linear relationship of 0.05-100 ng / mL was obtained, and the linear correlation coefficient was 0.998. The detection limit of fPSA was 0.012 ng / mL;

[0054] 2. Linear relationship of the single magnetic microsphere analysis method for tPSA detection

[0055] As shown in Figure Figure 4 (b) As shown in Figure, according to the proportional relationship between the tPSA concentration and the signal of Au on a single magnetic microsphere, a linear relationship of 0.05-100 ng / mL was obtained, and the linear correlation coefficient was 0.985. The detection limit of fPSA was 0.021 ng / mL;

[0056] In the anti-interference test, 50 ng / mL was used as the concentration of fPSA or tPSA sample in the interference experiment, and the interference antigens and concentrations used were alpha fetoprotein (AFP) 500 ng / mL, carcinoembryonic antigen (CEA) 500 ng / mL, human immunoglobulin G (IgG) 500 ng / mL, carbohydrate antigen 19-9 (CA199) 500 U / mL, carbohydrate antigen 12-5 (CA125) 500 U / mL, and carbohydrate antigen 15-3 (CA153) 500 U / mL. The results are shown in Figure Figure 4 (c) As shown in Figure, in addition to tPSA or fPSA causing changes in the intensity of Au or Pt on a single magnetic microsphere, the intensity of the remaining interference substances was comparable to that of the blank, proving that the specificity of the established nanoanalysis method for tPSA and fPSA detection was considerable.

[0057] Example 4, Investigation of the detection of tPSA and fPSA in actual blood samples by the analysis method of the present application and the investigation of the recovery of the addition of standard;

[0058] 1. Serum collection

[0059] The serum used in the application of the analysis method of the present application was obtained from Mianyang Central Hospital;

[0060] 2. Blood sample detection

[0061] Before analyzing 15 serum samples using the method of this invention, the tPSA and fPSA levels in each serum sample were obtained by hospital testing (ARCHITECT i2000, Abbott). 60 μL of serum, 5 μg of magnetic microspheres, and 25 μL of the mixed probe were added to a 200 μL centrifuge tube and mixed thoroughly. The immunoreaction was carried out at 37°C. o The reaction was completed at C for 2 hours. The analytical procedure, detected using nanoscale analysis methods, was consistent with the description in the specific implementation method. Results + Fe 58 like Figure 5 As shown in (a) and (b), within the linear range of fPSA and tPSA detection in the method of the invention, the biomarker content measured by SP-ICPMS is in good agreement with the values ​​obtained by the hospital, and the correlation coefficients of fPSA and tPSA detection are 0.984 and 0.995, respectively.

[0062] 3. Spiked recycling

[0063] The spiked recovery experiment was divided into two groups, with tPSA and fPSA spiked separately (at concentrations of 5, 10, and 20 ng / mL, with equal volumes of spiked serum). The spiked serum was analyzed using a single magnetic microsphere assay. Results are as follows: Figure 6 As shown, the established method can achieve a recovery rate of 85%-119%, proving that the analytical method of the present invention has the ability to analyze and detect actual samples.

Claims

1. A method for single magnetic microsphere analysis based on single particle inductively coupled plasma mass spectrometry, characterized in that: With magnetic microspheres as the reaction center, the target and probe are enriched on the surface of the magnetic microspheres through characteristic recognition; in single particle inductively coupled plasma mass spectrometry detection, the collected + Fe 58 The frequency signal is used to unify the number of magnetic microspheres, and by calculating the intensity distribution of the probe on the magnetic microspheres, the intensity of the single magnetic microsphere is obtained + Au 197 And + Pt 194 The intensity is little affected by the change of the number of magnetic microspheres in the reaction center, which can well avoid the fluctuation of the signal in absolute quantification due to the different number of magnetic microspheres; the intensity distribution is averaged to evaluate the probe signal intensity of single magnetic microsphere, or the signal distribution is fitted and the intensity obtained by fitting is used as the representative value; the single particle inductively coupled plasma mass spectrometry detection conditions are scanning time of 10 s and scanning speed of 200 µs / time.

2. The method of analysis of claim 1, wherein, The detection device used in the single magnetic microsphere analysis method is single particle inductively coupled plasma mass spectrometry or inductively coupled plasma-time of flight mass spectrometry.

3. The method of analysis of claim 1, wherein, The analysis method is suitable for reaction centers with probe enrichment capacity, and the reaction centers are silica microspheres, polystyrene microspheres and magnetic microspheres; and the metal nanoparticles used by the probe are gold nanoparticles and platinum nanoparticles.

4. The method of analysis of claim 1, wherein, The single magnetic microsphere analysis method based on single particle inductively coupled plasma mass spectrometry detects antigen protein analytes, nucleic acids or enzyme analytes.

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