Cell induction detection kit based on nanoparticles and application

Abstract

The invention provides a cell induction detection kit based on nanoparticles and application thereof, the kit is composed of a cell induction sensor, a recognition element of the cell induction sensor is an AS1411 aptamer, and an indicator is AuNPs. The cell induction detection kit based on the nanoparticles is applied to cancer disease detection.

Description

Technical Field

[0001] The present invention relates to a kit, and more particularly to a cell sensing detection kit based on nanoparticles and its application. Background Art

[0002] Lung cancer is one of the most common tumors worldwide and the leading cause of cancer-related death in humans. According to cancer statistics, lung cancer has a high incidence and mortality rate. Lung cancer can be divided into two main types: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Among them, NSCLC is the most common, accounting for about 85%. If it is diagnosed in stage I when NSCLC is diagnosed, the 5-year survival rate will reach 60%. This highlights the urgent need for sensitive, accurate and dynamic early diagnosis methods, which are essential to provide doctors with valuable insights to evaluate treatment outcomes. Therefore, developing sensitive and specific detection methods for NSCLC is a promising strategy for early diagnosis of cancer.

[0003] Nucleic acid aptamers, also known as chemical antibodies or aptamers, are single-stranded DNA or RNA molecules with a length of about 15-100 nucleotides. Aptamers are screened from random libraries synthesized by the ligand exponential enrichment (SELEX) system. Aptamers, as a new type of molecular probe, can bind to various target molecules, such as small molecules, proteins or cells, by folding themselves to form unique three-dimensional structures (such as hairpins and G-quadruplexes). Compared with antibodies, aptamers are derived from in vitro screening and have the advantages of easy modification, high affinity and specificity, stability and low immunogenicity. Currently, aptamers are being developed as probes for detecting a variety of targets, such as cancer cells, pathogens and metal ions. The integration of nanomaterials with aptamer molecules as recognition elements has become a prominent research focus. Gold nanoparticles (AuNPs) are often used as ideal indicator materials in detection sensors due to their excellent biocompatibility and optical properties. The surface of AuNPs can adsorb biomolecules through interactions such as electrostatic and hydrophobic forces, thereby enhancing their stability in solution without significantly changing the structure or function of the adsorbed biomolecules. Therefore, sensors of AuNPs combined with biomolecules have been widely used in the fields of medicine, food safety and environmental monitoring. Nucleolar protein is a marker overexpressed on the surface of cancer cell membranes, but not expressed in normal cell membranes. This feature makes nucleolar protein a potential marker for cancer diagnosis. The adsorption of nucleolar protein-specific aptamer AS1411 on the surface of gold nanoparticles enables the development of non-invasive, label-free and sensitive cell-sensing sensors, which helps in the early diagnosis and treatment of NSCLC.

[0004] At present, there are reports on the use of nucleic acid aptamers to detect tumor cells in peripheral blood. However, most detection methods are mainly based on enrichment. Enrichment methods are prone to target loss and are also easily affected by large-volume cells. Therefore, researchers are in urgent need of proposing a new method to solve the problems of low traditional enrichment efficiency and target loss, and to detect tumor cells quickly, sensitively, and specifically. Summary of the invention

[0005] Aiming at the problems existing in the traditional methods introduced in the background technology. The present application establishes a simple and rapid method for detecting cancer by cell sensing based on nanoparticles. The cell sensing sensor uses AS1411 aptamer as a recognition element and AuNPs as an indicator. First, AuNPs were prepared, and the preparation conditions were optimized, such as: the amount of trisodium citrate, the concentration of NaCl, the interaction time between NaCl and AuNPs, the concentration of AS1411 aptamer and AuNPs, and the interaction time. Under the optimal conditions, there is a linear relationship between the concentration of A549 cells and the absorbance of the A695 / A520 ratio value. The analysis signal of the cell sensing sensor was compared with the analysis signal of the normal control cells. The kit designed for the cell sensing sensor includes AuNPs, NaCl, and AS1411 aptamer.

[0006] Among them, the preparation method of the cell sensing detection kit based on nanoparticles comprises the following steps:

[0007] 1) Preparation of AuNPs; AuNPs were prepared under optimized conditions.

[0008] 2) Characterization by transmission electron microscopy, dynamic light scattering, ultraviolet spectroscopy, etc.;

[0009] 3) Optimize the NaCl concentration, the interaction time between NaCl and AuNPs, the concentration of AS1411 aptamer and AuNPs, and the interaction time.

[0010] The beneficial technical effects of the present invention are: ① The cell sensing sensor constructed by the present invention has a simple detection process and does not require enrichment and washing steps; ② The technical solution of the present invention solves the problems of cumbersome operation process, low efficiency of enriching tumor cells, and low sensitivity of existing detection technologies; ③ The technical solution of the present invention can be applied to whole blood samples; ④ The present invention provides a new strategy for early detection of malignant development and metastasis of lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 A diagram showing the cell sensing element of the present invention;

[0012] Figure 2The NaCl concentration, the interaction time between NaCl and AuNPs, the concentrations of AS1411 aptamer and AuNPs, and the interaction time were optimized.

[0013] Figure 3 Schematic diagram of the preparation method of the cell sensing detection kit of nanoparticles according to the present invention;

[0014] Figure 4 Cell sensing sensors are used in quantitative detection and specific analysis of tumor cells;

[0015] Figure 5 Tumor cells spiked into whole blood samples were comparatively analyzed using the cell sensing sensor. DETAILED DESCRIPTION

[0016] Example 1 Investigation of the preparation method of the kit

[0017] 1. Preparation of gold nanoparticles (AuNPs):

[0018] 500 μL of 1% HAuCl4·3H2O was added to 50 mL of deionized water (ddH2O) and heated to boiling under vigorous stirring. Subsequently, 1 mL of 1% trisodium citrate was introduced into the mixture. The solution was stirred continuously for 6 minutes, during which time the solution turned wine red. After stirring, the solution was allowed to cool to room temperature and then stored in a glass bottle at 4°C.

[0019] 2. Evidence analysis of cell-sensing sensor elements

[0020] The resulting AuNPs were characterized by obtaining UV-Vis spectra in the range of 400–800 nm using a SpectraMax i3x spectrophotometer. The boiling time was varied from 2 to 10 min, resulting in a gradual shift of the maximum absorption peak of the gold nanoparticles to shorter wavelengths (see Figure 1 A). When the boiling time exceeds 4 minutes, the maximum absorption peak of the AuNP solution at 520nm remains unchanged, indicating that the size and morphology of the AuNP particles tend to be stable. The synthesized AuNPs were characterized using UV spectral response scanning, dynamic light scattering, and transmission electron microscopy. UV spectral response scanning of AuNPs ( Figure 1 B) shows that there is a maximum absorption peak at 520nm and no other peaks, indicating that the size distribution of AuNPs is uniform. Dynamic light scattering measurements show that the diameter of AuNP particles is 25nm ( Figure 1 C). In addition, transmission electron microscopy analysis ( Figure 1 D) Confirms that the prepared AuNPs are spherical and uniform in size. Overall, our data demonstrate the successful synthesis of AuNPs.

[0021] 3.1 Optimize the NaCl concentration of the component:

[0022] To achieve the desired detection performance, the NaCl concentration and the interaction time between NaCl and AuNPs were optimized. For the NaCl concentration, 100 μL of AuNP solution was first placed in a 2 mL centrifuge tube, and then different final concentrations of NaCl (10, 20, 30, 40, 50, 60, 70, and 80 mM) were added. The mixture was then incubated at room temperature for 15 min. Figure 2 As shown in Figure A, within the NaCl concentration range of 10 to 40 mM, the A695 / A520 absorbance ratio of the AuNP solution remained relatively low, indicating that the AuNPs were dispersed in the solution. However, compared with the lower concentrations of 10 to 40 mM, a significant increase in the A695 / A520 absorbance was observed when the NaCl concentration reached 50 mM. In addition, as the NaCl concentration continued to rise above 50 mM, there was no significant increase in the A695 / A520 absorbance, indicating that the AuNPs had completely aggregated. Therefore, the optimal NaCl concentration was determined to be 50 mM.

[0023] 3.2 Optimization of NaCl-AuNP interaction time:

[0024] The optimal reaction time for NaCl-AuNP interaction was evaluated at intervals of 2, 4, 6, 8, 10, 12, and 14 min. We fixed the NaCl concentration at 50 mM and optimized the interaction time between NaCl and AuNPs ( Figure 2 B). There was no significant difference in A695 / A520 absorbance between 2 and 14 minutes of interaction time. Therefore, 2 minutes of interaction time was determined to be the optimal time for the interaction between NaCl and gold nanoparticles.

[0025] 3.3 Optimizing the concentration of AS1411 aptamer:

[0026] Nucleic acid aptamers as biological probes can be adsorbed to the surface of AuNPs, effectively preventing them from aggregating in NaCl solutions. We further optimized the concentration of the aptamer and the interaction time between the aptamer and AuNPs. A 100 μL solution of gold nanoparticles (AuNPs) was aliquoted into several 2 mL centrifuge tubes, to which the aptamers were added to reach final concentrations of 20, 60, 100, 140, 180, 220, 260, and 300 nM, respectively. The solution was incubated at room temperature for 60 min. After this incubation, a 50 mM sodium chloride (NaCl) solution was introduced, and the A695 / A520 values ​​were measured after another 15 min. The optimal concentration of the aptamer was determined by comparing the A695 / A520 values ​​of each sample. The optimal NaCl concentration and duration were used to increase the concentration of the AS1411 aptamer and its interaction time with AuNPs within the aptamer sensor platform, aiming to prevent particle aggregation in the AuNPs solution. As Figure 2 As shown in Figure C, the A695 / A520 absorbance of the AuNP solution gradually decreased when the AS1411 aptamer concentration was in the range of 20 to 220 nM. However, when the AS1411 aptamer concentration increased from 260 nM to 300 nM, the A695 / A520 absorbance did not decrease significantly. These findings indicate that AuNPs were completely encapsulated by the AS1411 aptamer at a concentration of 260 nM. Therefore, the final concentration of the aptamer was determined to be 260 nM.

[0027] 3.4 Aptamer-AuNP interaction time:

[0028] The optimal reaction time was determined by evaluating the A695 / A520 values ​​of various aptamers interacting with the AuNP solution at different time intervals of 5, 10, 15, 20, 25, 30, and 35 min. The interaction time of AS1411 aptamer with AuNPs was shown in Figure 2. Figure 2 D. Within 15 min of interaction time, the A695 / A520 absorbance value of the AuNP solution decreased significantly, and within the interaction time range of 15 to 35 min, there was no further change in absorbance. Therefore, 15 min was determined to be the optimal interaction time between AS1411 aptamer and AuNP.

[0029] Example 2 Activity Study of the Kit

[0030] 1. Analysis of detection principle

[0031] Under normal conditions, gold nanoparticles remain well dispersed in the solution, appearing wine red with a maximum absorption peak at 520 nm. After the addition of NaCl, the negative charge in the solution is neutralized, destroying the charge stability and causing the gold nanoparticles to aggregate, resulting in the solution changing from wine red to blue. However, the presence of the AS1411 aptamer allows interaction with AuNPs, promotes its adsorption on the AuNP surface, and provides a protective function against aggregation in NaCl solutions. When A549 cells are introduced into the system, the AS1411 aptamer, which has a stronger affinity for these cells, undergoes a conformational change, inducing the separation of the aptamer from the AuNPs. Ultimately, the AS1411 aptamer forms a stable G-quadruplex structure and binds to the A549 cells, causing the AuNPs to aggregate, followed by a change in solution color to blue (as shown in Figure 2). Figure 3 shown).

[0032] 2. Quantitative detection

[0033] Dilute the cell suspension to the desired concentration, ranging from 10 1 Up to 10 7 cells / mL. First, 100 μL of gold nanoparticle (AuNP) solution was mixed with 300 nM aptamer and incubated in a 2 mL centrifuge tube at room temperature for 30 minutes. Subsequently, samples with different cell concentrations were added to the centrifuge tubes and incubated at room temperature for 60 minutes. Finally, 75 mL of sodium chloride solution was introduced into the reaction system and incubated for another 15 minutes. The UV-visible absorption spectrum was recorded in the wavelength range of 400 to 600 nm. The standard curve for quantitative detection of cell sensing was drawn using OriginPro software, with the vertical coordinate being A695 / A520 and the horizontal coordinate being the cell concentration. Figure 4 A describes the described cell sensing with different concentrations of cancer cells (100-10 7 The curve relationship between the number of cells / mL and the logarithm of the absorbance. As the concentration of A549 cells increases, the A695 / A520 value gradually increases. Specifically, in the concentration range of 1 to 105 cells / mL, the corresponding A695 / A520 value increases steadily. In contrast, when the A549 cell concentration is 10 5 Up to 10 7 The A695 / A520 values ​​remained relatively constant when the concentration of A549 cells / mL was within the range of 101 to 106 cells / mL, indicating that the assay reached a steady state. In addition, the A695 / A520 values ​​showed a strong linear correlation with the logarithm of the corresponding A549 cell concentration in the range of 101 to 106 cells / mL. The linear regression equation was expressed as y = 0.1323x + 0.252 (R 2 =0.991), where x and y represent the logarithm of A549 cell concentration and the corresponding A695 / A520 value, respectively ( Figure 4B). The limit of detection (LOD) of the aptamer sensor platform targeting A549 cells was calculated to be 7 cells / mL according to the formula of 3 standard deviations divided by the slope of the calibration curve. These results indicate that the cell-sensing sensor platform has stable detection performance in the blood environment.

[0034] 3. Specificity Experiment of Sensing Platform

[0035] To verify the high specificity of our cell-based sensor, we also used other cell lines, including Beas-2b and HUVEC. Figure 4 As shown in C, a mixture of A549 tumor cells and various normal cells produced the highest signal on our aptasensor platform, while the presence of interfering cell populations produced relatively low signals, similar to those observed with blank or normal cells. These findings highlight the high specificity of our cell-sensing sensors.

[0036] 4. Cell sensing sensor detection in whole blood samples

[0037] A549 cells at the indicated concentrations were introduced into whole blood samples from healthy donors and subsequently lysed with red blood cell lysate. Centrifugation was performed at 500 g for 10 minutes. After three washes and additional centrifugation, the resulting cell pellet was detected by the constructed cell-sensing sensor. In order to evaluate the detection performance of the constructed cell-sensing sensor in complex biological samples, we introduced a series of A549 cell concentrations as mimics of circulating tumor cells (CTCs) into fresh blood samples obtained from healthy donors. Subsequently, we performed quantitative measurements. Our results showed that there was no significant change in the A695 / A520 values ​​between PBS buffer and blood samples containing the same number of cancer cells ( Figure 5 ). In addition, the recovery rates ranged from 96.89% to 106.42%, and the average relative standard deviation (RSD) was less than 5%. These results indicate that the cell-sensing sensor exhibits effective and stable detection capabilities in the blood environment.

Claims

1. A cell sensing detection kit based on nanoparticles, the kit comprising a cell sensing sensor, characterized in that: The recognition element of the cell sensing sensor is the AS1411 aptamer, and the indicator is AuNPs.

2. The nanoparticle-based cell sensing detection kit according to claim 1, characterized in that: The preparation method of the AuNPs indicator is: 1) Add 500 μL of 1% HAuCl4·3H2O to 50 mL of deionized water (ddH2O) and heat to boiling under vigorous stirring to obtain a mixed solution; 2) 1 mL of 1% trisodium citrate was introduced into the mixed solution and stirred continuously for 6 minutes, during which time the solution turned wine red; 3) Allow the mixed solution to cool to room temperature and then store it in a glass bottle at 4°C to obtain the AuNPs indicator.

3. The nanoparticle-based cell sensing detection kit according to claim 1, characterized in that: The final concentration of the AS1411 aptamer was determined to be 260 nM.

4. Use of the nanoparticle-based cell sensing detection kit according to claims 1-3 in cancer disease detection.

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