Functionalized modified nanoneedle chip and preparation method and application thereof

Abstract

The invention discloses a functional modified nanoneedle chip as well as a preparation method and application thereof, and relates to the technical field of biological chips. Specifically, the functional modified nanoneedle chip disclosed by the invention is obtained by sequentially modifying a nanoneedle chip with NHS-biotin, streptavidin and biotin-poly (T), wherein the length of the poly (T) of the biotin-poly (T) is 20 to 30 nt. According to the method, a three-step modification method of NHS-biotin, streptavidin and biotin-poly (T) is adopted, a functionalized modified nanoneedle chip treatment system is optimized, the extraction efficiency of 106-level cell RNA is larger than or equal to 85%, the cDNA concentration reaches 780 ng / mu L or above and is improved by 27% compared with that of an unmodified nanoneedle group, the problems that 106-level cell RNA capture is insufficient and time consumption is long are effectively solved, and the method has important application prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biochips, in particular to a functionalized nano-needle chip and a preparation method and application thereof. BACKGROUND

[0002] The functionalized nano-needle chip is a microchip based on silicon-based materials and having a high-density nano-scale needle structure on the surface. Biological molecules such as nucleic acids and proteins can be fixed on the surface through chemical modification (such as amino modification), so as to realize efficient capture and detection of target substances in biological samples. Due to the structural specificity, large specific surface area and good biocompatibility, the chip is widely used in fields such as molecular diagnosis, cell biology research and gene expression analysis, and shows important potential in precise medical scenarios such as tumor cell detection, circulating tumor cell (CTC) analysis and single-cell sequencing.

[0003] In related technologies, although the functionalized nano-needle chip has advantages in micro-sample detection, there are still some defects in processing “10 6 cell quantities” (such as puncture-enriched cells or cultured cell lines commonly seen in clinics), such as low RNA capture efficiency, poor repeatability, complex operation process and mismatched reagent system. The surface active site density of the traditional functionalized nano-needle chip is low (<8×10³ / mm²), which is easy to be saturated under 10 6 cell quantities, resulting in an RNA capture rate of only 60%~70%, unstable detection of low-abundance genes (such as CD79A / CD79B) and a coefficient of variation of Ct value often greater than 5%. In addition, when the functionalized nano-needle chip is used for detection, multiple operations such as cell lysis, RNA extraction and cDNA synthesis need to be performed separately, and the whole process takes more than 2 hours, which cannot meet the requirement of rapid detection. In addition, the conventional reverse transcription and qPCR reagents are designed for μg-level RNA, while the RNA amount of 10 6 cell is only 5~10 ng, and direct use of the conventional system easily leads to reagent waste or insufficient template, further reducing the detection repeatability.

[0004] Therefore, it is urgent to develop a functionalized nano-needle chip and a preparation method thereof to improve the RNA capture rate in 10 6 cell quantities and optimize the repeatability and accuracy of detection. SUMMARY

[0005] The first aspect of the present application aims to provide a functionalized nano-needle chip.

[0006] The second aspect of the present application aims to provide a preparation method of the functionalized nano-needle chip.

[0007] A third aspect of the present invention is to provide an RNA capture method.

[0008] The fourth aspect of this invention is to provide a method for synthesizing cDNA.

[0009] The fifth aspect of the present invention is to provide a qPCR detection device.

[0010] The sixth aspect of this invention aims to provide the application of functionalized modified nanoneedle chips in RNA capture, cDNA synthesis, or qPCR detection for non-disease purposes.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a functionalized modified nanoneedle chip, which is obtained by sequentially modifying a nanoneedle chip with NHS-biotin, streptavidin and biotin-poly(T); The length of the poly(T) in the biotin-poly(T) is 20~30nt.

[0012] In some embodiments of the present invention, the area of ​​the nanoneedle chip is 0.2~1 cm².

[0013] In some embodiments of the present invention, the surface amino density of the nanoneedle chip is ≥2×10⁻⁶. 4 Pieces / mm²

[0014] In some embodiments of the present invention, the surface amino density of the nanoneedle chip is less than 1 × 10⁻⁶. 6 Pieces / mm²

[0015] A second aspect of the present invention provides a method for preparing a functionalized modified nanoneedle chip as described in the first aspect, comprising the following steps: S1. Under light-protected conditions, the nanoneedle chip is placed in the NHS-biotin dilution solution, incubated, and washed to obtain a biotin-modified nanoneedle chip. S2. The biotin-modified nanoneedle chip is placed in the streptavidin dilution solution, incubated, and washed to obtain the biotin-streptavidin modified nanoneedle chip. S3. The biotin-streptavidin modified nanoneedle chip is placed in the biotin-poly(T) dilution solution, incubated, and washed to obtain the final product.

[0016] In some embodiments of the present invention, the concentration of the NHS-biotin diluent is 0.05~0.3 mg / mL.

[0017] In some embodiments of the present invention, the concentration of the streptavidin diluent is 0.2~0.4 mg / mL.

[0018] In some embodiments of the present invention, the concentration of the biotin-poly(T) diluent is 10-20 μM.

[0019] In some embodiments of the present invention, in step S1, the incubation time is 0.5 to 2 hours.

[0020] In some embodiments of the present invention, in step S2, the incubation time is 1 to 3 hours.

[0021] In some embodiments of the present invention, in step S3, the incubation time is 0.5 to 2 hours.

[0022] In some embodiments of the present invention, the solvents for the NHS-biotin diluent and the streptavidin diluent are independently selected from phosphate buffer or Tris-HCl buffer.

[0023] In some embodiments of the present invention, the pH value of the phosphate buffer solution is 7.3 to 7.6.

[0024] In some embodiments of the present invention, the pH value of the Tris-HCl buffer solution is 7.4 to 7.6.

[0025] In some embodiments of the present invention, the solvent for the biotin-poly(T) diluent is water.

[0026] A third aspect of the present invention provides an RNA capture method comprising capturing RNA using a functionalized nanoneedle chip as described in the first aspect.

[0027] In some embodiments of the present invention, the capture includes: contacting the functionalized modified nanoneedle chip with cells or a suspension containing cells, then adding a sodium dodecyl sulfate solution, and washing to obtain the product.

[0028] In some embodiments of the present invention, the amount of cells is 1 × 10⁻⁶. 6 ~1×10 8 indivual.

[0029] In some embodiments of the present invention, the cell concentration of the cell-containing suspension is 1 × 10⁻⁶. 4 ~1×10 5 per μL.

[0030] In some embodiments of the present invention, the volume ratio of the sodium dodecyl sulfate solution to the cell-containing suspension is 1:0.8~1.2.

[0031] In some embodiments of the present invention, the mass fraction of the sodium dodecyl sulfate solution is 0.02% to 0.08%.

[0032] A fourth aspect of the present invention provides a cDNA synthesis method comprising reverse transcription using RNA captured by a functionalized nanoneedle chip as described in the first aspect as a template.

[0033] In some embodiments of the present invention, the incubation temperature for reverse transcription is 50±2℃, and the incubation time is 4~8h.

[0034] In some embodiments of the present invention, the incubation process further includes an inactivation treatment.

[0035] In some embodiments of the present invention, the inactivation temperature is 85±2℃ and the time is 20~40s.

[0036] A fifth aspect of the present invention provides a qPCR detection device, comprising: An RNA capture system, wherein the RNA capture system operates the RNA capture method as described in the third aspect; A reverse transcription system, wherein the reverse transcription system operates the cDNA synthesis method as described in the fourth aspect; And qPCR detection system.

[0037] A sixth aspect of the invention provides the application of functionalized modified nanoneedle chips as described in the first aspect in RNA capture, cDNA synthesis, or qPCR detection for non-disease purposes.

[0038] The functionalized modified nanoneedle chip, its preparation method, and its application according to embodiments of the present invention have at least the following beneficial effects: (1) The functionalized modified nanoneedle chip of the present invention can be used for direct capture in live cells, achieving high efficiency and cost reduction. The qPCR detection method based on the functionalized modified nanoneedle chip of the present invention does not require cell lysis or rely on the multi-step process of traditional RNA extraction and purification kits. It can directly capture target RNA in live cells, saving reagent costs (the cost per detection is 30% lower than the magnetic bead method) and reducing time costs (the entire process takes 65 minutes, 46% shorter than the 120 minutes of the traditional centrifuge column method); simultaneously achieving 10 6 The cell RNA extraction efficiency is ≥85%, and the cDNA concentration reaches over 780 ng / μL, which is 27% higher than that of the unmodified nanoneedle group, effectively solving the problem of 10 6 The problem of insufficient RNA capture at the cellular level.

[0039] (2) It helps to preserve cell viability and supports dynamic detection. After the functionalized modified nanoneedle chip of the present invention completes RNA capture in cells, the cells can remain viable and continue to be cultured, breaking through the limitations of traditional "destructive RNA extraction" on subsequent cell research, and providing the possibility for dynamic monitoring of cell function (such as analysis of temporal changes in gene expression under drug action).

[0040] (3) The detection background of the present invention is clean, and the specificity and repeatability are excellent. When the mRNA captured by the functionalized modified nanoneedle chip of the present invention is used for subsequent PCR amplification, it will not be affected by the chemical components in the lysis buffer and purification reagent. Furthermore, due to the specific binding based on the Poly (A) tail, the non-specific interference of other biomolecules such as rRNA is greatly reduced, resulting in a cleaner detection background. The specific detection rate of target genes (such as CD79A / CD79B) is 100% (no false positives in A549 cells), and the coefficient of variation of Ct value is ≤0.5% (far lower than the industry average of 5%), meeting the stringent requirements of clinical testing for accuracy and repeatability.

[0041] (4) Convenient operation and strong stability: No separate elution step is required, and the chip can be stored at 4℃ for 1 week; the reagent dosage is precisely matched 10 6 RNA quantity in cells (to avoid reagent waste); based on the present invention 10 6 The cell mass validation experiment showed that the cell mass gradient, specificity, and reproducibility results were stable and reproducible.

[0042] Other features and advantages of the present invention will be set forth in the following description. Attached Figure Description

[0043] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a schematic diagram of the modification process for the functionalized nanoneedle chip of the present invention; Figure 2 This is a schematic diagram of molecular binding during the functionalization modification process of the nanoneedle chip of the present invention; Figure 3 The method of Embodiment 1 of the present invention is applied to different 10 6 Results of qPCR amplification at the level of cells. Detailed Implementation

[0044] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0045] The terms "preferred," "more preferably," etc., used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0046] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0047] In the description of this invention, the reference term "and / or" includes all and any combination of one or more of the associated listed items.

[0048] In the description of this invention, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0049] In the embodiments of the present invention, Raji cells were purchased from Shanghai Aoneng Biotechnology Co., Ltd.

[0050] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0051] I. Fabrication of Functionalized Nanoneedle Chips Example 1: This embodiment provides a method for preparing functionalized modified nanoneedle chips. A schematic diagram of the specific modification process is shown below. Figure 1 As shown, it specifically includes the following content.

[0052] (1) Biotin modification: Take a silicon-based nanoneedle chip (area 0.5 cm², surface amino density ≥ 2 × 10⁻⁶). 4 pcs / mm 2 Place the mixture in a light-protected reaction dish, add 80 μL of 1 μg / mL NHS-biotin dilution solution, and incubate at room temperature in the dark for 1 hour to activate the amino groups and construct a sufficient biotin layer.

[0053] After incubation, discard the diluent and wash three times with PBS (5 minutes each time, 150 μL each time) to remove unbound NHS-biotin (to avoid subsequent non-specific adsorption) and set aside.

[0054] The preparation method of NHS-Biotin diluent is as follows: Take NHS-Biotin (purchased from Thermo Fisher Scientific), dilute it 1000 times with sterile PBS (pH 7.4) to obtain NHS-Biotin diluent with a final concentration of 1 μg / mL, and use it immediately (activity maintained >90%).

[0055] (2) Biotin-streptavidin modification: Take the modified nanoneedle chip and add 80 μL of streptavidin dilution solution with a final concentration of 10 μg / mL. Incubate at room temperature for 2 hours. After incubation, discard the dilution solution and wash with PBS 3 times (5 minutes each time, 150 μL each time) to remove free streptavidin and obtain biotin-streptavidin modified silicon-based nanoneedle chip.

[0056] The preparation method of streptavidin dilution is as follows: Take streptavidin (purchased from Thermo Fisher Scientific), dilute it 400 times with sterile PBS (pH 7.4) to obtain streptavidin dilution with a final concentration of 10 μg / mL, and store it at 4℃ for later use.

[0057] By constructing a biotin-streptavidin linker layer, the binding site density was ensured to be ≥1.2×10⁻⁶. 4 pcs / mm 2 Validation using fluorescent labeling showed that the binding efficiency of streptavidin to biotin was consistently ≥95% under these conditions.

[0058] (3) Biotin-poly(T) modification: Obtain the biotin-streptavidin-modified functionalized nanoneedle chip described above, add 80 μL of biotin-poly(T) diluent (enough to cover the chip), and incubate at room temperature for 1 hour. Discard the diluent, wash three times with PBS (5 minutes each time, 150 μL per wash), and then immerse the chip in PBS and store at 4°C for later use.

[0059] The preparation method of the biotin-poly(T) diluent is as follows: Biotin-poly(T) (nucleotide length 25 nt, custom-synthesized by Sangon Biotech (Shanghai) Co., Ltd.) is diluted 100-fold with DEPC water to obtain a final concentration of 10 μM. After preparation, it is aliquoted into 50 μL tubes for single use, avoiding repeated freeze-thaw cycles that could lead to primer degradation, and stored at -20℃ for later use.

[0060] Streptavidin has four biotin-binding sites, which can simultaneously bind NHS-biotin (the biotin that is linked to the chip substrate) and biotin-poly(T) (the biotin that captures the primer). There is no competition between the two; streptavidin acts as a "bridge" connecting the two layers. Figure 2 This is a schematic diagram of molecular binding during the functionalization process of nanoneedle chips, demonstrating the mode in which streptavidin binds simultaneously to both the lower NHS-bio and the upper bio-poly(T).

[0061] II. qPCR Detection Method Based on Functionalized Nanoneedle Chips Example 2: This embodiment uses 10 6 Using cells as the detection target, this paper provides a qPCR detection method based on the above-mentioned functionalized modified nanoneedle chip, which specifically includes the following contents.

[0062] (1) Sample loading and RNA capture: First, take the modified chip (0.5 cm) obtained in Example 1 above. 2 Place the chip in a 48-well plate with the chip facing up, then add an appropriate amount of cell suspension. Place the 48-well plate containing the cell suspension into a centrifuge (Thermo Sorvall ST 16R) and centrifuge at 500×g for 10 minutes.

[0063] The amount of cell suspension added should be based on the "cell quantity - volume" relationship, i.e., 100 μL corresponds to 1.2 × 10⁻⁶ cells / mL. 6 -2×10 6 10 cells, 300 μL corresponds to 5 × 10 6 -8×10 6Cells are individually spaced to ensure uniform spreading on the chip surface without stacking, thus avoiding localized capture saturation. Low-speed centrifugation helps promote sufficient contact between cells and biotin-poly(T), increasing RNA capture rate to ≥85%.

[0064] (2) Chip cleaning and RNA release: After centrifugation, discard the cell suspension in the wells, add an equal volume of 0.05% SDS solution, and wash for 3 minutes (to remove 10...). 6 Remove residual proteins and cell membrane debris from the cells, then wash twice with PBS for 3 minutes each time to remove SDS and avoid inhibiting reverse transcriptase. After washing, use sterile forceps to remove the chip and place it into a 0.2 mL RNase-free EP tube for later use.

[0065] RNA adsorbed on the chip surface can be used directly as a reverse transcription template without elution, thus avoiding trace loss of cellular RNA.

[0066] (3) cDNA synthesis: The reaction system was prepared according to the instructions for the Novizan R433 reverse transcription kit. The specific reaction system is shown in Table 1. Table 1:

[0067] After gently mixing the prepared mixture by pipetting, centrifuge at 5000 rpm for 10 seconds (ensuring the liquid pools at the bottom of the tube), and then place it in a PCR instrument and react according to the following program: incubate at 50°C for 5 minutes, then incubate at 85°C for 5 seconds (to inactivate reverse transcriptase). After the reaction is complete, place the obtained cDNA product on ice for later use (use within 1 hour to avoid degradation).

[0068] (4) Preparation and amplification of qPCR reaction system: In this example, CD79A / CD79B was used as the detection gene and GAPDH as the internal reference gene for qPCR detection. A 20 μL system (Novizan Q713 premixed solution, stored in SYBR Green protected from light) was prepared on ice. The qPCR reaction system is shown in Table 2. Table 2:

[0069] The primer sequence information is shown in Table 3.

[0070] Table 3:

[0071] After mixing the prepared reaction mixture, centrifuge at 5000 rpm for 10 seconds (to remove air bubbles). Then, add the reaction solution to an eight-tube qPCR array (free from nuclease contamination), place it in a Cooler Master Q225 Plus-400 qPCR instrument, and set the program: Pre-denaturation: 95℃, 30 seconds; PCR cycles (40 cycles): 95℃ for 10 seconds (denaturation); 60℃ for 30 seconds (annealing, fluorescence acquisition, primer specificity for target genes such as CD79A / CD79B); Melting curve: 60℃→95℃ (heating rate 0.1℃ / second, default instrument settings, to verify amplification specificity).

[0072] III. Different 10 6 qPCR detection results of cell quantity This section sets up different concentration gradients of cells, using the method of Example 2 to respectively treat 1×10 6 5×10 6 8×10 6 1×10 7 qPCR was performed on Raji cells, as detailed below.

[0073] Example 3: This embodiment provides a method based on 1×10 6 The qPCR detection method for individual cells adopted the method in Example 2, wherein the amount of Raji cells used was 1×10⁻⁶. 6 The sample loading and RNA capture steps are as follows: First, take the modified chip (0.5 cm) obtained in Example 1 above. 2 Place the chip in a 48-well plate with the front side facing up, then add 300 μL of cell suspension (containing approximately 5 × 10⁻⁶ cells). 6 (10 cells), then place the 48-well plate containing the cell suspension into a centrifuge (Thermo Sorvall ST 16R) and centrifuge at 500×g for 10 minutes.

[0074] The remaining conditions are the same as in Example 2.

[0075] Example 4: This embodiment provides a method based on 5×10 6 The qPCR detection method for individual cells differs from Example 3 in that the amount of Raji cells used is 5 × 10⁻⁶. 6 The remaining conditions and steps are the same as in Example 3.

[0076] Example 5: This embodiment provides a method based on 8×10 6 The qPCR detection method for individual cells differs from Example 3 in that the amount of Raji cells used is 8 × 10⁸. 6 The remaining conditions and steps are the same as in Example 3.

[0077] Example 6: This embodiment provides a method based on 1×10 7 The qPCR detection method for individual cells differs from Example 3 in that the amount of Raji cells used is 1×10⁻⁶. 7 The remaining conditions and steps are the same as in Example 3.

[0078] The qPCR detection results of Examples 3-6 above are shown in Table 4 and Figure 3 As shown.

[0079] Table 4:

[0080] Test results show that for 1×10 6 ~1×10 7 Cells of different grades were detected using the nanoneedles and method of this invention. The Ct values ​​were stable (without significant fluctuations), the coefficient of variation was <0.05%, and the relative fold increase remained between 0.34 and 0.65, indicating that the nanoneedles and detection method of this invention are effective for different grades of cells. 6 High adaptability of cell mass; of which 5×10 6 The lowest Ct value (optimal capture efficiency) was found in individual cells, at 10. 6 Recommended detection amount for cells at the grade level.

[0081] IV. qPCR Detection Results Based on Different Methods This section tested the impact of different detection methods on qPCR detection results, including Example 4 (5×10⁻⁶). 6 The detection methods (for cells), conventional qPCR detection methods, and detection methods based on unmodified nanoneedles are detailed below: Comparative Example 1: This comparative example provides a conventional 5×10 6 The qPCR detection method for Raji cells is as follows: (1) RNA extraction: The Vazyme Ultrafast Single-Column RNA Extraction Kit for Animal Cells (Catalog No.: RC102) was used. This kit is based on silica membrane purification technology and uses a proprietary lysis buffer to achieve one-step lysis and column loading, taking only 5 minutes and requiring no toxic reagents such as β-mercaptoethanol. The specific operation steps are as follows: S1, take approximately 5 × 10 6 After centrifuging and collecting the cell pellet, add the kit's proprietary lysis buffer (5 × 10⁶ cells per 5 × 10⁶ cells as per the instructions). 6 Add 100 μL of lysis buffer to each cell, vortex thoroughly to mix, and incubate at room temperature for 1 minute to complete cell lysis. S2. Transfer the lysate to the silica-based adsorption column provided with the kit, centrifuge at 12000×g for 1 minute to allow the RNA to be specifically adsorbed onto the column membrane, achieving "one-step lysate loading". S3. Add the washing buffer provided with the kit (anhydrous ethanol has been added according to the instructions before use) to the adsorption column, centrifuge at 12000×g for 30 seconds, discard the waste liquid, and repeat the washing once to remove impurities such as proteins. S4. Add 50 μL of RNase-free water to the center of the adsorption column membrane, let stand at room temperature for 1 minute, centrifuge at 12000×g for 1 minute, and collect the eluent as the purified total RNA.

[0082] (2) cDNA synthesis and qPCR detection Using the extracted total RNA as a template, the reverse transcription kit (purchased from Novizan R433) and qPCR premix (purchased from Novizan Q713) were used to perform the detection according to the same cDNA synthesis and qPCR amplification procedures as in Example 2, ensuring consistency of experimental conditions.

[0083] Comparative Example 2: This comparative example provides a qPCR detection method based on unmodified nanoneedles. The only difference between the unmodified nanoneedles used and those in Example 1 is that the three-layer modification process of NHS-biotin modification, streptavidin linkage, and biotin-poly(T) fixation was not performed.

[0084] All other conditions, such as RNA capture of Raji cells, cDNA synthesis, qPCR detection, as well as cell processing volume, subsequent reagents and reaction procedures, were the same as in Example 4.

[0085] Table 5 compares the qPCR results obtained by the methods in Example 4 and Comparative Examples 1-2. Figure 3 As shown.

[0086] Table 5:

[0087] Based on the above test results, it can be seen that the functionalized modified nanoneedle chip of the present invention has excellent mRNA capture efficiency. The present invention achieves specific capture of mRNA through three-step modification of NHS-biotin, streptavidin and biotin-poly(T) (the test confirms that the proportion of mRNA in the captured nucleic acid is ≥90%), which can meet the core requirement of "targeted detection of mRNA" in gene expression analysis.

[0088] Compared to Comparative Example 1, this invention employs a "direct live cell capture - no elution required" mode. The transcription template is mRNA naturally bound to the chip surface, eliminating the need for a concentration step. The detected data more accurately reflects the "in-situ capture efficiency." Furthermore, using the functionalized nanoneedle chip of this invention for mRNA capture and reverse transcription is time-efficient (65 minutes) and eliminates the need for elution, effectively avoiding trace RNA loss during elution (e.g., the elution recovery rate of Comparative Example 1 is approximately 92%, while the capture recovery rate of this invention is ≥98%). In contrast, the method in Comparative Example 1 requires four steps: "cell lysis - silica matrix column adsorption - washing - elution concentration," with concentration steps during elution, resulting in complex operation, low recovery rate, and long processing time (120 minutes).

[0089] Compared with Comparative Example 2 (unmodified nanoneedles), the qPCR detection results of the functionalized modified nanoneedle chip based on the present invention showed that the GAPDH Ct value decreased from 24.75 to 19.307, and the CD79A Ct value decreased from 29.769 to 22.817, indicating that the three-step modification of NHS-biotin-streptavidin-biotin-poly(T) can significantly improve the mRNA capture efficiency; at the same time, the coefficient of variation of the Ct value of the present invention is 0.026%, which is significantly lower than the industry standard of 5%, and can meet the relevant application requirements.

[0090] Furthermore, in the detection of low-abundance genes (such as CD79B), the present invention used the above method to detect 10 batches of low-abundance samples. The results showed that all samples were detected using the method of the present invention, with a detection rate of 100%. In contrast, using the method of Comparative Example 1, one batch was not detected, with a detection rate of 90%. This indicates that the detection method of the present invention has good stability and reliability.

[0091] IV. Specific Detection Results This section uses the methods of Example 4 and Comparative Example 1 to detect 5×10 6CD79A / CD79B expression in Raji cells (B cells, expressing CD79A / CD79B) and A549 cells (lung adenocarcinoma cells, not expressing CD79A / CD79B).

[0092] The test results are shown in Table 6.

[0093] Table 6:

[0094] The test results show that the nanoneedles of this invention are effective at 10 6 The target gene (CD79A / CD79B in Raji cells) can be specifically detected in the target cells, and the relative fold increase of the target gene in A549 cells is close to 0, indicating that the detection specificity is consistent with that of traditional kits and there are no false positives, which shows great application potential.

[0095] In summary, this invention provides a functionalized modified nanoneedle chip, its preparation method, and its application. The functionalized modified nanoneedle chip of this invention has at least the following beneficial effects: (1) It can be used for direct capture of live cells, achieving high efficiency and cost reduction. The qPCR detection method based on the functionalized modified nanoneedle chip of the present invention does not require cell lysis or the multi-step process of traditional RNA extraction and purification kits. It can directly capture target RNA in the live cell state, saving reagent costs (the cost of a single detection is reduced by 30% compared with the magnetic bead method) and shortening the time cost (the whole process takes 65 minutes, which is 46% shorter than the 120 minutes of the traditional centrifuge column method); at the same time, it can achieve 10 6 The cell RNA extraction efficiency is ≥85%, and the cDNA concentration reaches over 780 ng / μL, which is 27% higher than that of the unmodified nanoneedle group, effectively solving the problem of 10 6 The problem of insufficient RNA capture at the cellular level.

[0096] (2) It helps to preserve cell viability and supports dynamic detection. After the functionalized modified nanoneedle chip of the present invention completes RNA capture in cells, the cells can remain viable and continue to be cultured, breaking through the limitations of traditional "destructive RNA extraction" on subsequent cell research, and providing the possibility for dynamic monitoring of cell function (such as analysis of temporal changes in gene expression under drug action).

[0097] (3) The detection background of the present invention is clean, and the specificity and repeatability are excellent. When the mRNA captured by the functionalized modified nanoneedle chip of the present invention is used for subsequent PCR amplification, it will not be affected by the chemical components in the lysis buffer and purification reagent. Furthermore, due to the specific binding based on the Poly (A) tail, the non-specific interference of other biomolecules such as rRNA is greatly reduced, resulting in a cleaner detection background. The specific detection rate of target genes (such as CD79A / CD79B) is 100% (no false positives in A549 cells), and the coefficient of variation of Ct value is ≤0.5% (far lower than the industry average of 5%), meeting the stringent requirements of clinical testing for accuracy and repeatability.

[0098] (4) Convenient operation and strong stability: No separate elution step is required, and the chip can be stored at 4℃ for 1 week; the reagent dosage is precisely matched 10 6 RNA quantity in cells (to avoid reagent waste); based on the present invention 10 6 The cell mass validation experiment showed that the cell mass gradient, specificity, and reproducibility results were stable and reproducible.

[0099] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A functionalized modified nanoneedle chip, characterized in that, The functionalized modified nanoneedle chip is obtained by sequentially modifying the nanoneedle chip with NHS-biotin, streptavidin and biotin-poly (T). The poly (T) of the biotin-poly (T) has a length of 20-30 nt.

2. The functionalized modified nanopin chip of claim 1, wherein, The surface amino density of the nanoneedle chip is ≥2×10 4 mm².

3. A method for preparing a functionalized modified nanoneedle chip according to claim 1 or 2, characterized in that, The method comprises the following steps: S1. Under light-proof conditions, the nanoneedle chip is placed in the NHS-biotin diluent, incubated, and washed to obtain a biotin-modified nanoneedle chip; S2. The biotin-modified nanoneedle chip is placed in the streptavidin diluent, incubated, and washed to obtain a biotin-streptavidin-modified nanoneedle chip; S3. The biotin-streptavidin-modified nanoneedle chip is placed in the biotin-poly (T) diluent, incubated, and washed to obtain the functionalized modified nanoneedle chip.

4. The preparation method according to claim 3, characterized in that, The concentration of the NHS-biotin diluent is 0.05-0.3 mg / mL; The concentration of the streptavidin diluent is 0.2-0.4 mg / mL; The concentration of the biotin-poly (T) diluent is 10-20 μM.

5. The production method according to claim 3 or 4, characterized by, In step S1, the incubation time is 0.5-2 h; In step S2, the incubation time is 1-3 h; In step S3, the incubation time is 0.5-2 h.

6. The preparation method according to claim 5, characterized in that, The solvents of the NHS-biotin diluent and the streptavidin diluent are independently selected from phosphate buffer or Tris-HCl buffer; The solvent of the biotin-poly (T) diluent is water.

7. A method of RNA capture, comprising, The method comprises capturing using the functionalized modified nanoneedle chip of claim 1 or 2; Preferably, the capturing comprises: contacting the functionalized modified nanoneedle chip with a cell or a cell-containing suspension, then adding a sodium dodecyl sulfate solution, and obtaining after washing; Preferably, the amount of cells is 1 x 10 6 ~1 x 10 8 ~1 x 10 Preferably, the cell concentration of the cell-containing suspension is 1 x 10 4 ~1 x 10 5 cells per μL; Preferably, the volume ratio of the sodium dodecyl sulfate solution to the cell-containing suspension is 1:0.8-1.2; Preferably, the mass fraction of the sodium dodecyl sulfate solution is 0.02%-0.08%.

8. A method for cDNA synthesis, characterized by, The method comprises performing reverse transcription using the RNA captured by the functionalized modified nanoneedle chip of claim 1 or 2 as a template; Preferably, the incubation temperature of the reverse transcription is 50±2℃, and the incubation time is 4-8 h; Preferably, the method further comprises inactivation treatment after the incubation.

9. A qPCR detection device, characterized in that, The method comprises: An RNA capturing system, which runs the RNA capturing method of claim 7; A reverse transcription system, which runs the cDNA synthesis method of claim 8; and A qPCR detection system.

10. The functionalized modified nanoneedle chip of claim 1 or 2 is used in RNA capturing, cDNA synthesis or qPCR detection for non-disease purposes.

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