An upconversion fluorescence test kit for detecting NSCLC marker CYFRA 21-1, preparation method and application thereof

Abstract

The present invention discloses an upconversion fluorescence test kit for detecting NSCLC marker CYFRA 21-1, a preparation method and an application. The kit includes carboxyl magnetic beads, UCNPs, Ab1, Ab2, BIBA, HEMA, CuBr2, ME6TREN, AA, BSA, NHS, EDC, and PBS buffer. The present invention uses ATRP as a signal amplification strategy and UCNPs as a signal unit. First, Ab2 is connected to the initiator BIBA, and UCNPs are connected to the monomer HEMA. Subsequently, a fluorescence test kit is constructed based on magnetic beads. Ab1 is connected to the carboxyl magnetic beads, and then CYFRA 21-1 antigen specifically recognizes Ab1, and Ab2 specifically recognizes CYFRA 21-1 to form a sandwich structure, and the initiator BIBA is introduced into the system. After adding the connected UCNPs / HEMA, signal amplification is achieved through ATRP polymerization reaction. The kit of the present invention has a wider detection range and a lower LOD, and has good selectivity, reproducibility and stability, and is expected to be used for the detection of CYFRA 21-1.

Description

Technical Field

[0001] The invention relates to an up-conversion fluorescence test kit for detecting NSCLC marker CYFRA21-1 constructed based on ATRP signal amplification strategy, a preparation method and application, and belongs to the technical field of bioanalysis. Background Art

[0002] Lung cancer pathology is divided into small cell lung cancer (small cell lung cancer) and non-small cell lung cancer (non-small cell lung cancer). Early diagnosis of lung cancer is the key to improving the survival rate and prognosis of lung cancer patients. Serum markers are of great significance in the early diagnosis of lung cancer. The markers used for screening and evaluation of treatment effects of small cell lung cancer (SCLC) mainly include neuron-specific enolase (NSE), cancer embryonic antigen (CEA), squamous cell carcinoma antigen (SCC), etc., and CYFRA21-1 (soluble fragment of cytokeratin 19) is the most valuable serum tumor marker for non-small cell lung cancer, especially for the early diagnosis, efficacy observation, and prognosis monitoring of patients with squamous cell carcinoma. Therefore, the development of a stable and efficient method for detecting the content of CYFRA21-1 is expected to improve the early diagnosis rate of potential patients with lung cancer.

[0003] At present, the traditional methods for detecting serum markers include radioimmunoassay, enzyme-linked immunosorbent assay, chemiluminescence immunoassay, etc., but due to high cost, complex operation and low sensitivity, they are difficult to be used in actual detection. In recent years, with the rapid development of biomedical technology, fluorescent labeling technology has been widely used in the field of biological detection due to its high sensitivity and simple operation. Traditional fluorescent molecules are down-conversion luminescence, such as fluorescent proteins, quantum dots and organic fluorescent dyes. However, these fluorescent molecules are often accompanied by overlapping excitation and absorption spectra, resulting in obvious photobleaching and light flickering during the application process, and cannot be well applied to the analysis of complex samples. Upconversion materials (UCNPs) are a new type of nanoluminescent material with a different luminescence mechanism from traditional dyes, belonging to anti-Stokes luminescence. UCNPs have excellent optical properties, such as good photostability, low biological toxicity, high luminescence intensity, not easy to bleach, long fluorescence lifetime, etc., but the method based on the traditional target-signal 1:1 ratio limits the sensitivity and applicability of fluorescent biological probes. ATRP reaction is a process of dynamic growth of biomolecules. It uses polymerization to aggregate signal molecules at biorecognition sites to form long-chain polymers, amplifying the recognition process of a single biomolecule by hundreds of times through chain growth, thereby effectively amplifying the biodetection signal. Therefore, the present invention adopts the ATRP signal amplification strategy to improve the sensitivity of the fluorescence test kit. Summary of the invention

[0004] In view of the deficiencies in the prior art, the purpose of the present invention is to provide an upconversion fluorescence test kit for detecting NSCLC marker CYFRA21-1 based on ATRP signal amplification strategy, a preparation method and application, which is simple to operate, and the signal is amplified exponentially, thereby improving the sensitivity of detection, and at the same time has good stability, selectivity and reproducibility.

[0005] In order to achieve the above object, one of the technical solutions of the present invention is:

[0006] An upconversion fluorescence test kit for detecting NSCLC marker CYFRA 21-1 based on ATRP signal amplification strategy, comprising the following raw materials: carboxyl magnetic beads, UCNPs, Ab 1 ,Ab 2 , BIBA, HEMA, CuBr 2 、ME 6 TREN, AA, BSA, NHS, EDC, PBS buffer.

[0007] Furthermore, some raw materials need to be prepared into solutions when used, among which the concentration of UCNPs solution is 5 mg / mL, Ab 1 The solution concentration was 1 μg / mL, Ab 2 The solution concentration was 4 μg / mL, the BIBA solution concentration was 30 mM, the HEMA solution concentration was 60 mM, and the CuBr 2 / ME 6 CuBr in TREN solution 2 and ME 6 The concentration of TREN was 10 mM, the concentration of AA solution was 2 mM, the concentration of BSA solution was 10 mg / mL, the concentration of NHS solution was 50 mM, and the concentration of EDC solution was 200 mM.

[0008] Furthermore, the UCNPs are NaYF 4 :Er,Yb.

[0009] One of the technical solutions of the present invention is: a method for detecting the NSCLC marker CYFRA 21-1, comprising the following steps:

[0010] (1) After activating BIBA, connect Ab 2 , to obtain Ab 2 -BIBA;

[0011] (2) After activating UCNPs, HEMA was connected to obtain UCNPs / HEMA;

[0012] (3) Activate carboxyl magnetic beads and then connect Ab 1 , incubation;

[0013] (4) unreacted activated sites on the magnetic beads after blocking step (3);

[0014] (5) adding the sample to be tested and incubating;

[0015] (6) Add the Ab connected in step (1) 2 -BIBA, incubation;

[0016] (7) Add the UCNPs / HEMA and CuBr connected in step (2) 2 / ME 6 TREN solution, AA solution, incubation;

[0017] (8) The magnetic beads washed in step (7) are dispersed in PBS buffer and the emission spectrum is tested.

[0018] Further, the specific method is:

[0019] (1) Connecting the initiator BIBA and Ab in the ATRP reaction 2

[0020] ① Take 250 μL of BIBA solution, add 250 μL of NHS solution and EDC solution respectively, and place in a shaker to shake overnight;

[0021] ② Add 250 μL of Ab to the solution in step ① above. 2 The solution was placed in a shaker and shaken for 2 hours to obtain Ab 2 -BIBA solution;

[0022] (2) Connecting UCNPs and HEMA monomers

[0023] ① Take 250 μL of UCNPs solution, add 250 μL of NHS solution and EDC solution respectively, and shake in a shaker to react overnight;

[0024] ② Add 250 μL of HEMA solution to the solution in step ① above, place in a shaker and shake for 2 h to obtain a UCNPs / HEMA solution;

[0025] (3) Modification of Ab 1

[0026] ① Take 20 μL of carboxyl magnetic bead solution, add PBS buffer, wash, magnetically separate, remove the supernatant, disperse the washed magnetic beads in PBS buffer, add 20 μL of NHS solution and EDC solution respectively, and shake in a shaker to react overnight;

[0027] ② The reaction solution of step ① was magnetically separated, the supernatant was removed, PBS buffer was added for washing, and after washing, it was dispersed in PBS buffer, and then 20 μL Ab was added 1 The solution was placed in a shaker and shaken for 1 h;

[0028] (4) Blocking site

[0029] The reaction solution of step (3) was magnetically separated, the supernatant was removed, PBS buffer was added for washing, and after washing, it was dispersed in PBS buffer, and then 20 μL of BSA solution was added, and the mixture was placed in a shaker for shaking for 0.5 h;

[0030] (5) Modification of target

[0031] The sealed reaction solution of step (4) was magnetically separated, the supernatant was removed, PBS buffer was added for washing, and after washing, it was dispersed in PBS buffer, 20 μL of the solution to be tested was added, and the mixture was placed in a shaker for shaking for 1 h;

[0032] (6) Modification of Ab 2

[0033] The reaction solution of step (5) was magnetically separated, the supernatant was removed, PBS buffer was added for washing, and after washing, it was dispersed in PBS buffer and the Ab connected in step (1) was added. 2 -20 μL of BIBA solution was placed in a shaker and shaken for 1 h;

[0034] (7) ATRP reaction

[0035] The reaction solution of step (6) was magnetically separated, the supernatant was removed, PBS buffer was added for washing, and after washing, it was dispersed in PBS buffer, and CuBr 2 / ME 6 20 μL each of TREN solution, UCNPs / HEMA solution, and AA solution were placed in a shaker and shaken for 75 min;

[0036] (8) Fluorescence detection

[0037] The reaction solution obtained in step (7) was magnetically separated, the supernatant was removed, PBS buffer was added for washing, and after washing, the solution was dispersed in PBS buffer and the fluorescence intensity was measured by a fluorescence spectrophotometer.

[0038] Furthermore, the temperature of the shaking reaction is 36-39°C.

[0039] Furthermore, in step (8), the emission spectrum is tested under an excitation light of 980 nm.

[0040] One of the technical solutions of the present invention is: an application of the up-conversion fluorescence test kit in the detection of NSCLC marker CYFRA21-1.

[0041] One of the technical solutions of the present invention is: an application of the up-conversion fluorescence test kit in the preparation of a reagent for detecting the NSCLC marker CYFRA21-1.

[0042] The schematic diagram of the detection method of the present invention is as follows Figure 1 shown.

[0043] Beneficial effects of the present invention:

[0044] 1. The present invention adopts the atom transfer radical polymerization (ATRP) strategy and uses long-chain polymers to amplify the fluorescence signal, avoiding the use of nanomaterials and biological enzymes (complicated synthesis and purification steps and easily affected by the external environment and temperature, etc.) in the currently commonly used signal amplification strategies. The signal is amplified exponentially, improving the sensitivity, stability and reproducibility of the detection.

[0045] 2. The present invention uses upconversion nanomaterials (UCNPs) as fluorescent materials, avoiding the problems of poor photostability, easy bleaching, and high biological toxicity of traditional fluorescent materials. UCNPs are excited by infrared light, so they have strong tissue penetration ability, can reduce background fluorescence, and improve detection sensitivity.

[0046] 3. The present invention adopts ATRP as a signal amplification strategy and uses UCNPs as signal units. First, CYFRA21-1 labeled antibody (Ab 2 ) was connected to the initiator (BIBA), and UCNPs were connected to the monomer HEMA. Then, a fluorescent test kit was constructed based on magnetic beads, and CYFRA 21-1 was coated with antibody (Ab 1 ) is connected to carboxyl magnetic beads, and then the CYFRA 21-1 antigen-specific recognition Ab 1 , Ab 2 The specific recognition of CYFRA 21-1 forms a sandwich structure, and the initiator BIBA is introduced into the system. After adding the connected UCNPs / HEMA, the signal is amplified by ATRP polymerization reaction. The results show that within the range of 1pg / mL to 100μg / mL, the fluorescence intensity and the logarithm of the CYFRA 21-1 concentration show a good linear relationship, and the linear equation is F(au)=3719.885log[C CYFRA21-1 μg / mL]+27204.65(R 2 =0.9982), and the detection limit of the fluorescence test kit was calculated to be 3.87×10 -5Compared with the existing method for detecting CYFRA 21-1, the present invention has a wider detection range and a lower LOD, and has good selectivity, reproducibility and stability, and is expected to be used for the detection of CYFRA 21-1. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Schematic diagram of the principle of the detection method of the present invention.

[0048] Figure 2 (A) is the fluorescence spectra of the fluorescence test kit under different construction conditions.

[0049] Figure 2 (B) is the laser confocal scanning image in the presence of the target CYFRA21-1.

[0050] Figure 2 (C) is the laser confocal scanning image in the absence of the target CYFRA21-1.

[0051] Figure 3 (A) is the HRTEM scanning spectrum in the presence of the target compound CYFRA21-1.

[0052] Figure 3 (B) is the HRTEM scanning spectrum in the absence of the target compound CYFRA21-1.

[0053] Figure 3 (C) is the AFM scanning spectrum in the presence of the target compound CYFRA21-1.

[0054] Figure 3 (D) is the AFM scanning spectrum in the absence of the target compound CYFRA21-1.

[0055] Figure 4 (A) HRTEM scanning spectrum of UCNPs-OA.

[0056] Figure 4 (B) UCNPs surface (OA, BF 4 - , PAA, HEMA) test results of DLS and Zeta potential.

[0057] Figure 4 (C) UCNPs surface (OA, BF 4 - , PAA, HEMA).

[0058] Figure 5 (A) is the infrared test result after the surface of UCNPs was coated with OA.

[0059] Figure 5 (B) UCNPs surface is BF 4 - Infrared test results after coating.

[0060] Figure 5 (C) is the infrared test result after the surface of UCNPs was coated with PAA.

[0061] Figure 5 (D) is the infrared test result after the surface of UCNPs was coated with HEMA.

[0062] Figure 6 (A) is the optimization result of ATRP reaction initiator BIBA concentration.

[0063] Figure 6 (B) Optimization results of monomer HEMA concentration in ATRP reaction.

[0064] Figure 6 (C) ATRP reaction time optimization results.

[0065] Figure 6 (D) is the UCNPs concentration optimization result.

[0066] Figure 7 (A) Fluorescence spectra of different CYFRA 21-1 concentrations.

[0067] Figure 7 (B) is the linear relationship between fluorescence intensity and CYFRA 21-1 concentration.

[0068] Figure 8 (A) is a study on the selectivity of the fluorescent test kit of the present invention for different serum proteins (BSA, CTnI, CEA, CYFA 21-1, all at a concentration of 0.1 μg / mL).

[0069] Figure 8 (B) Comparison of fluorescence intensity of different concentrations of CYFRA 21-1 in 5% and 10% NHS and 0.1 M PBS buffer system. DETAILED DESCRIPTION

[0070] The specific implementation modes of the present invention are further described in detail below in conjunction with the embodiments.

[0071] Example 1

[0072] An upconversion fluorescence test kit for detecting NSCLC marker CYFRA21-1 based on ATRP signal amplification strategy, comprising the following raw materials: carboxyl magnetic beads (MBs) (70102-5, Suzhou Beaver Biomedical Engineering Co., Ltd.), upconversion nanomaterials (UCNPs) (NaYF4 :Er,Yb), CYFRA 21-1 coated antibody (Ab 1 )(CY11N005, Beijing Zhongkai Health Technology Co., Ltd.), CYFRA 21-1 labeled antibody (Ab 2 )(CY11N007, Beijing Zhongkai Health Technology Co., Ltd.), 2-bromo-2-methylpropionic acid (BIBA), hydroxyethyl methacrylate (HEMA), CuBr 2 , tris-(N,N-dimethylaminoethyl)amine (Me 6 TREN), ascorbic acid (AA), bovine serum albumin (BSA), N-hydroxysuccinimide (NHS), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and PBS buffer.

[0073] Some raw materials need to be prepared into solutions when used, among which the concentration of UCNPs solution is 5 mg / mL, Ab 1 The solution concentration was 1 μg / mL, Ab 2 The solution concentration was 4 μg / mL, the BIBA solution concentration was 30 mM, the HEMA solution concentration was 60 mM, and the CuBr 2 / ME 6 CuBr in TREN solution 2 and ME 6 The concentration of TREN was 10 mM, the concentration of AA solution was 2 mM, the concentration of BSA solution was 10 mg / mL, the concentration of NHS solution was 50 mM, the concentration of EDC solution was 200 mM, the concentration of PBS buffer was 0.1 M, and the pH was 7.4.

[0074] Example 2

[0075] A method for detecting NSCLC marker CYFRA 21-1 comprises the following steps:

[0076] (1) Connecting the initiator BIBA and Ab in the ATRP reaction 2

[0077] ① Take 250 μL of BIBA solution (30 mM), add 250 μL of NHS solution (50 mM) and EDC solution (200 mM), respectively, and place in a shaker at 37°C for overnight reaction;

[0078] ② Add 250 μL of Ab to the solution in step ① above. 2 The solution (4 μg / mL) was placed in a shaker at 37°C for 2 h to obtain Ab 2 -BIBA solution;

[0079] (2) Connecting UCNPs and HEMA monomers

[0080] ① Take 250 μL of UCNPs solution (5 mg / mL), add 250 μL of NHS solution (50 mM) and EDC solution (200 mM), and shake in a shaker at 37 °C overnight;

[0081] ② Add 250 μL HEMA solution (60 mM) to the solution in step ① above, place in a shaker at 37°C and shake for 2 h to obtain a UCNPs / HEMA solution;

[0082] (3) Modification of Ab 1

[0083] ① Take 20 μL of carboxyl magnetic bead solution (10 mg / mL), add 180 μL of PBS buffer, wash, separate by magnetic force, remove the supernatant, add 200 μL of PBS buffer to wash, remove the supernatant, disperse the washed magnetic beads in 140 μL PBS buffer, add 20 μL of NHS solution (50 mM) and EDC solution (200 mM), respectively, and place in a shaker at 37°C for overnight reaction;

[0084] ② The reaction solution of step ① was separated by magnetic separation, the supernatant was removed, 200 μL PBS buffer was added and washed twice, and then dispersed in 180 μL PBS buffer, and then 20 μL Ab was added. 1 The solution (1 μg / mL) was placed in a shaker at 37 °C for 1 h;

[0085] (4) Blocking site

[0086] The reaction solution of step (3) was magnetically separated, the supernatant was removed, 200 μL PBS buffer was added and washed twice, and then dispersed in 180 μL PBS buffer, and then 20 μL BSA solution (10 mg / mL) was added, and the mixture was placed in a shaker at 37°C for 0.5 h;

[0087] (5) Modification of target

[0088] The sealed reaction solution of step (4) was magnetically separated, the supernatant was removed, 200 μL PBS buffer was added and washed twice, and then dispersed in 180 μL PBS buffer, 20 μL of the test solution (containing the target CYFRA21-1) was added, and the mixture was placed in a shaker at 37°C and shaken for 1 h;

[0089] (6) Modification of Ab 2

[0090] The reaction solution of step (5) was magnetically separated, the supernatant was removed, and 200 μL PBS buffer was added to repeat the washing twice to remove the non-specifically bound CYFRA 21-1 protein. After washing, it was dispersed in 180 μL PBS buffer, and then the Ab connected in step (1) was added. 2 -20 μL of BIBA solution was placed in a shaker at 37°C for 1 h;

[0091] (7) ATRP reaction

[0092] The reaction solution of step (6) was magnetically separated, the supernatant was removed, and 200 μL PBS buffer was added to repeat the washing twice. After washing, it was dispersed in 140 μL PBS buffer, and CuBr 2 / ME 6 20 μL of TREN solution, UCNPs / HEMA solution, and AA solution (2 mM) were placed in a shaker at 37 °C for 75 min;

[0093] (8) Fluorescence detection

[0094] The reaction solution obtained in step (7) was magnetically separated, the supernatant was removed, 200 μL PBS buffer was added and washed twice, and then dispersed in 500 μL PBS buffer. The fluorescence intensity of the mixed solution was measured by a fluorescence spectrophotometer with an excitation light of 980 nm. The CYFRA 21-1 concentration was calculated based on the fluorescence intensity.

[0095] Example 3: Feasibility Verification

[0096] In order to explore the feasibility of the kit to detect CYFRA 21-1, this experiment compared the fluorescence intensity detected by the kit under different construction conditions. Figure 2 As shown in (A), Ab was not added during the construction process. 1 (curve b), CYFRA 21-1 (curve c), Ab 2 (curve d), ATRP solution (CuBr 2 / ME 6 TREN solution, UCNPs / HEMA solution, AA solution (curve e), UCNPs / HEMA (curve f) did not show strong fluorescence intensity, while the experimental group Ab 1 / CYFRA 21-1 / Ab 2 / BIBA / UCNPs / HEMA (curve a) showed a strong fluorescence intensity at a wavelength of about 540nm, indicating that it is feasible to construct an upconversion fluorescence test kit to detect CYFRA 21-1. In addition, the laser confocal scanning spectrum ( Figure 2 B and Figure 2C) clearly shows that the fluorescence signal can only be observed when the target CYFRA 21-1 is present. In addition, from the HRTEM scanning spectrum, when CYFRA21-1 is present ( Figure 3 A), it can be clearly seen that a large number of UCNPs aggregate on the surface of the magnetic beads after modification, while when CYFRA21-1 is not added ( Figure 3 B) Only the rough surface of the magnetic beads can be seen. In addition, the AFM scanning spectrum shows that CYFRA21-1 exists ( Figure 3 C) compared with no CYFRA21-1 ( Figure 3 D) The height of the magnetic beads increased by about 344.1 nm.

[0097] The above results indicate that it is feasible to construct an upconversion fluorescence test kit for detecting CYFRA 21-1 based on the fluorescence biosensing strategy based on the stable luminescence characteristics of UCNPs and combined with the ATRP efficient polymerization method technology.

[0098] Example 4: Characterization

[0099] In the design process of the fluorescence test kit, the fluorescence properties of the luminescent material, the size and morphology of the material, and whether the luminescent material can be successfully modified have a crucial influence on the detection performance. The size of the synthesized upconversion material is an important indicator affecting the connection efficiency. Generally speaking, the smaller the particle size of UCNPs, the higher the connection efficiency. However, as the particle size decreases, the luminescence efficiency will decrease. Therefore, ensuring that the upconversion material has the smallest possible particle size and has high luminescence properties is a prerequisite for ensuring the performance of the upconversion fluorescence test kit. Using a transmission electron scanning microscope to directly observe the morphology and particle size information of UCNPs is an ideal method, such as Figure 4 As shown in A, NaYF synthesized by thermal decomposition 4 : The particle size of Yb, Er is about 26±3nm, the size is uniform, and the morphology is approximately hexagonal. In addition, the particle size data is consistent with the test results of DLS ( Figure 4 B).

[0100] To ensure that the upconversion material has good luminescence performance at this particle size, the BF modified in sequence was tested on the steady-state transient fluorescence spectrometer FLS 1000. 4 - , polyacrylic acid (PAA) and HEMA after the fluorescence lifetime of UCNPs. The test results are as follows Figure 4 As shown in Figure C, the lifetime of the final product UCNPs / HEMA is 218 μs. Compared with the unmodified OA-UCNPs, there is almost no loss. Such luminescence performance is sufficient for designing upconversion luminescent bioprobes, and the surface modification has no significant effect on the luminescence performance of the material.

[0101] At the same time, the particle size and Zeta potential changes of UCNPs during the transformation process were tested to verify the results of surface modification. Figure 4 As shown in B. After UCNPs are coated with PAA and connected to the monomer HEMA of ATRP reaction through esterification reaction, the particle size reaches the maximum, and the DLS test result is about 45nm, which meets the use requirements of fluorescent probes. When different compounds are coated on the surface of UCNPs, the zeta potential will be significantly different, such as Figure 4 B. The infrared spectra of different surface coatings are shown in Figure 5 As shown, we can see that each coating (OA, BF 4 - , PAA, HEMA).

[0102] The combined changes in particle size, potential and infrared spectrum indicate that the surface modification of UCNPs is successful.

[0103] Example 5: Optimization of detection conditions

[0104] In order to achieve the best performance of the upconversion fluorescence test kit for CYFRA 21-1 detection, the present invention optimizes the relevant experimental conditions.

[0105] (1) Optimization of BIBA and HEMA concentrations

[0106] Initiator BIBA and monomer HEMA play a vital role in ATRP polymerization reaction and directly affect the fluorescence intensity. For example, low concentration of initiator is conducive to controlling the polymerization process, but too low concentration will lead to a decrease in polymerization rate. Monomer concentration can directly affect the polymerization reaction rate. In order to explore the influence of the concentration of initiator BIBA and monomer HEMA, other conditions were fixed and a series of concentration gradients of initiator BIBA and monomer HEMA were set to construct the kit. The results are shown in Figure 2. Figure 6 A and Figure 6 As shown in B. Figure 6 As can be seen from A, the fluorescence intensity increases with the increase of BIBA concentration until it reaches a maximum value at 30mM. After that, the fluorescence intensity decreases with the increase of concentration. Because at the beginning, when the concentration of the initiator BIBA increases, more free radicals combine with HEMA, resulting in an increase in the fluorescence signal. However, as the initiator concentration increases, the rate of free radical polymerization gradually decreases, resulting in a decrease in fluorescence intensity. Figure 6 As can be seen from B, the fluorescence intensity increases with the increase of HEMA concentration between 10 and 60 mM, but there is no more change after 60 mM. Therefore, the optimal concentrations of the initiator BIBA and the monomer HEMA are 30 mM and 60 mM, respectively.

[0107] (2) Optimization of ATRP reaction time

[0108] At the same time, the fluorescence intensity is related to the polymerization reaction time of ATRP. Since there are a large number of carboxyl sites on the surface of UCNPs, the extension of the polymerization time can bind more signal molecules to the active sites and enhance the fluorescence signal. Figure 6 As shown in Figure C, the fluorescence intensity increases continuously with time until it gradually stabilizes at 75 min, which indicates that the maximum amount of signal molecules accumulated on the surface of the magnetic beads is 75 min, so the optimal time for ATRP polymerization is 75 min.

[0109] (3) UCNPs concentration optimization

[0110] In the process of constructing the fluorescence kit, the fluorescence intensity value is directly affected by the concentration of the upconversion material (UCNPs). Therefore, in order to explore the effect of UCNPs concentration, other conditions were fixed and a series of UCNPs with a concentration gradient were set to construct the kit. Figure 6 As shown in D, when the UCNPs concentration is within 5 mg / mL, the fluorescence intensity increases with the increase of UCNPs concentration. After that, the UCNPs concentration continues to increase, and the fluorescence intensity gradually stabilizes without obvious changes. This shows that at the selected initiator concentration, the binding sites of UCNPs have been exhausted. Therefore, 5 mg / mL of UCNPs is the optimal concentration for constructing the kit of the present invention.

[0111] Example 6: Analytical Performance

[0112] Under the optimal experimental conditions, the present invention studies the response of different CYFRA21-1 concentrations to fluorescence. Figure 7 As shown, within the range of 1 pg / mL to 100 μg / mL, the fluorescence intensity and the logarithm of the CYFRA21-1 concentration showed a good linear relationship, and the linear equation was F(au)=3719.885log[C CYFRA21-1 μg / mL]+27204.65(R 2 =0.9982), and the detection limit of the kit was calculated to be 3.87×10 -5 ng / mL, in the process of constructing the kit, the ATRP polymerization reaction makes more upconversion material (UCNPs) molecules aggregate on the surface of the magnetic beads to form long-chain polymers, and the signal amplification process is realized by chain growth, which significantly reduces the detection limit of the kit and improves the detection sensitivity. Compared with the related performance of fluorescent biosensors reported in previous literature, the kit of the present invention has a wider detection range and higher sensitivity (Table 1), which shows that the upconversion fluorescence test kit constructed based on the ATRP signal amplification strategy can achieve high-sensitivity detection of CYFRA21-1.

[0113] Table 1 Comparison of the method of the present invention with other methods for detecting biomarkers

[0114]

[0115] Example 7: Selectivity, stability and reproducibility

[0116] In order to explore the selectivity of the fluorescent test kit of the present invention for detecting CYFRA 21-1, under the same experimental conditions, the fluorescence responses of BSA, cardiac troponin I (CTnI), CYFRA 21-1 and carcinoembryonic antigen (CEA) (all at a concentration of 0.1 μg / mL) were measured, and a blank group (Blank) was set up. Figure 8 As can be seen from A, the fluorescence test kit has a high fluorescence intensity signal only for CYFRA21-1, which is the result of specific recognition of antigen and antibody. By comparison, it can be concluded that the fluorescence test kit of the present invention has a high selectivity.

[0117] At the same time, in order to ensure the reliability of the detection results of CYFRA 21-1 by the fluorescence test kit of the present invention, based on the intra-group and inter-group experimental analysis (n=5), the reproducibility of the fluorescence test kit of the present invention was studied. The RSD within the group and between the groups were 3.64% and 4.05%, respectively, indicating that the prepared fluorescence test kit has good reproducibility. In addition, the stability analysis of the fluorescence test kit of the present invention was carried out. Under the same conditions, six fully modified magnetic beads were prepared, and fluorescence detection was performed immediately after preparation, the results were recorded, and then fluorescence detection was performed after storage in a 4°C refrigerator for three weeks. It was found that the fluorescence signal could still reach 94.83% of the initial fluorescence signal, indicating that the upconversion fluorescence test kit has good storage stability.

[0118] Example 8: Serum sample analysis

[0119] In order to explore the feasibility of upconversion fluorescence test kit in practical application, this study analyzed serum samples of the fluorescence test kit. Different concentrations of CYFRA 21-1 (10fg / mL, 10pg / mL, 10ng / mL) were added to 5% and 10% (v / v) normal human serum (NHS) samples. Then the fluorescence signal intensity in 5% normal human serum and 10% normal human serum was detected, and the results of different concentrations of CYFRA 21-1 in normal human serum were compared with the fluorescence signals obtained at the same concentration in 0.1M PBS buffer (pH 7.4). The results are shown in Figure 2. Figure 8As shown in Figure B, the fluorescence signal intensity of different concentrations of CYFRA 21-1 in a 5% normal human serum system is 87.1%, 95.6%, and 97.2% of the fluorescence signal intensity in a 0.1M PBS buffer (pH 7.4) system. The fluorescence signal intensity of different concentrations of CYFRA 21-1 in a 10% normal human serum system is 93.9%, 91.0%, and 98.8% of the fluorescence signal intensity in a 0.1M PBS buffer (pH 7.4). Therefore, the up-conversion fluorescence test kit of the present invention has good anti-interference ability in complex human serum samples and has good clinical application prospects.

[0120] In order to verify the practicality of the fluorescent test kit of the present invention in detecting CYFRA21-1 antigen in complex biological samples, this experiment tested 6 clinical serum samples from the Third Affiliated Hospital of Henan University of Traditional Chinese Medicine. This experiment compared the analysis results of magnetic particle chemiluminescence (MPCA) and the analysis results of the fluorescent test kit. The results showed (Table 2) that the relative error and relative standard deviation of the method of the present invention and the MPCA detection results were less than 5.02% and 3.32%, respectively, indicating that the fluorescent test kit of the present invention has good detection ability in clinical trials.

[0121] Table 2 Comparison of the test results of the present invention with the clinical test method (MPCA)

[0122]

Claims

1. An upconversion fluorescence test kit for detecting non-small cell lung cancer (NSCLC) marker CYFRA 21-1 based on ATRP signal amplification strategy. It is characterized in that Including the following raw materials: carboxyl magnetic beads, upconversion nanomaterials UCNPs, CYFRA21-1 coated antibody Ab 1 、CYFRA 21-1 labeled antibody Ab 2 , 2-bromo-2-methylpropionic acid BIBA, hydroxyethyl methacrylate HEMA, CuBr 2 , tris-(N,N-dimethylaminoethyl)amine ME 6 TREN, ascorbic acid AA; the UCNPs are NaYF 4 :Er,Yb.

2. The upconversion fluorescence test kit for detecting the non-small cell lung cancer (NSCLC) marker CYFRA 21-1 constructed based on the ATRP signal amplification strategy according to claim 1, It is characterized in that It also includes bovine serum albumin BSA, N-hydroxysuccinimide NHS, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDC, and PBS buffer.

3. The upconversion fluorescence test kit for detecting the non-small cell lung cancer (NSCLC) marker CYFRA 21-1 constructed based on the ATRP signal amplification strategy according to claim 1 or 2, It is characterized in that Some raw materials need to be prepared into solutions when used, among which the concentration of UCNPs solution is 5 mg / mL, Ab 1 The solution concentration was 1 μg / mL, Ab 2 The solution concentration was 4 μg / mL, the BIBA solution concentration was 30 mM, the HEMA solution concentration was 60 mM, and the CuBr 2 / ME 6 CuBr in TREN solution 2 and ME 6 The concentration of TREN was 10 mM, the concentration of AA solution was 2 mM, the concentration of BSA solution was 10 mg / mL, the concentration of NHS solution was 50 mM, and the concentration of EDC solution was 200 mM.

4. Use of the upconversion fluorescence test kit according to claim 1 in preparing a reagent for detecting CYFRA21-1, a marker of non-small cell lung cancer (NSCLC).

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