A logic-gated nanogel probe for live cell typing and a preparation method thereof

Abstract

This invention relates to the field of biodetection technology, providing a logic-gated nanogel probe for live cell typing and its preparation method. The invention uses a thermosensitive polymer nanogel as a carrier, with its surface covalently modified with an antibody that specifically binds to the EGFR L858R mutant protein and a peptide nucleic acid that specifically binds to miR-21. Internally, it encapsulates a self-quenched fluorescent dye. The probe employs an AND logic gating design, triggering gel network dissociation and dye release to generate a fluorescent signal only when it simultaneously recognizes the EGFR L858R protein on the cell surface and captures miR-21 within the cell. This invention solves the problems of high false positive rates in existing single-target detection methods and the inability of traditional liquid biopsy techniques to perform simultaneous genotype-phenotype analysis in situ on live cells. It achieves highly specific and sensitive in situ interpretation of circulating tumor cells, and has significant application value in the field of precision diagnosis and treatment of lung cancer.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, specifically to a logic-gated nanogel probe for live cell typing and its preparation method. Background Technology

[0002] Lung cancer is the leading cause of cancer-related death worldwide, and its treatment has entered the era of precision medicine centered on the detection of driver gene mutations. Specific mutations in genes such as epidermal growth factor receptor (EGFR) (e.g., L858R) are key therapeutic targets for non-small cell lung cancer (NSCLC), directly guiding the clinical application of tyrosine kinase inhibitors (TKIs). Currently, DNA sequencing technologies based on tissue or liquid biopsies (such as next-generation sequencing) are the gold standard for identifying these mutations. However, these methods sequence nucleic acid fragments detached from their native microenvironment, providing only average genotypic information of the cell population. They cannot reveal the spatial heterogeneity and clonal evolution dynamics within the tumor, nor can they correlate specific genotypes with the phenotype and spatial location of living cells at the single-cell level. Circulating tumor cells (CTCs), as intact living cells shed from primary or metastatic tumors and entering the peripheral blood, are a valuable source of information reflecting the real-time biological state of tumors. However, existing CTC detection technologies mainly rely on single surface markers such as epithelial cell adhesion molecules (EpCAM) for enrichment and identification. This not only misses cell subpopulations that have undergone epithelial-mesenchymal transition (EMT), but more importantly, it lacks the ability to perform in situ, multi-parameter molecular phenotypic analysis on captured live cells, especially failing to rapidly and specifically identify the mutation status of their driver genes (such as EGFR L858R).

[0003] Therefore, developing new methods capable of in situ, multiplex molecular verification of live cells and achieving simultaneous genotype-phenotype analysis has become a key bottleneck that urgently needs to be overcome in this field. Logic-gated molecular diagnostics is a cutting-edge direction in biosensing, its core being the integration and analysis of multiple biomarkers using molecular interactions similar to computer logic operations (such as AND, OR, NOT). Compared to traditional single-marker detection, logic-gated technology can effectively integrate multi-dimensional information such as spatial distribution and expression abundance. Therefore, cutting-edge technologies for precise "genotyping" of live cells, and diagnostic strategies inspired by computer science that implement logic-gated operations at the molecular level, provide entirely new ideas for solving the aforementioned challenges. Summary of the Invention

[0004] The purpose of this invention is to address the aforementioned technical problems by providing a logic-gated nanogel probe for live cell typing.

[0005] The nanogel probe of the present invention comprises an environmentally responsive nanocarrier, at least two recognition molecules respectively bound to the carrier, and a signal reporting unit;

[0006] The release of the signal reporting unit is triggered synergistically by the binding of the at least two recognition molecules to their corresponding targets;

[0007] The environmentally responsive nanocarrier is a thermosensitive polymer gel with a low critical solution temperature (LCST).

[0008] The at least two recognition molecules include a protein recognition unit and a nucleic acid recognition unit;

[0009] The protein recognition unit is an antibody or fragment thereof that specifically binds to the EGFR L858R mutant protein, and the nucleic acid recognition unit is a peptide nucleic acid that specifically binds to miR-21;

[0010] The signal reporting unit is an aggregate of fluorescent dye molecules in a self-quenching state;

[0011] The release of the signal reporting unit is triggered by the simultaneous binding of the at least two recognition molecules to their corresponding targets.

[0012] The probe achieves AND logic gating through the network state switching of the temperature-sensitive polymer gel:

[0013] Only when the protein recognition unit and the nucleic acid recognition unit bind to their respective targets and produce a synergistic effect, the thermosensitive polymer gel changes from a physically contracted closed state to a phase transition swelling open state, thereby releasing the physical lock on the signal reporting unit and triggering the output of a fluorescence signal; if only a single target binds, the nanogel remains in a contracted state to suppress signal release.

[0014] This invention also provides a method for preparing a logic-gated nanogel probe for live cell typing, specifically including the following steps:

[0015] (1) Preparation of thermosensitive nanogel core by precipitation polymerization: Under nitrogen protection and at 70 °C, monomer N-isopropylacrylamide, crosslinking agent and comonomer acrylic acid were subjected to free radical polymerization in aqueous solution. After the reaction, the nanogel was purified by dialysis to obtain thermosensitive poly(N-isopropylacrylamide-co-acrylic acid) (PNIPAm-co-AAc).

[0016] (2) Surface functionalization modification: The purified nanogel was diluted with 2-(N-morpholino)ethanesulfonic acid buffer at pH 6.0 to a solid content of 1 mg / mL. 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) at a final concentration of 10 mM were added sequentially. After activating the carboxyl group at room temperature, a monoclonal antibody against human EGFR L858R mutant protein and peptide nucleic acid (PNA) modified with the 5' end amino group were added. The mixture was stirred continuously at room temperature. After the reaction was completed, uncoupled molecules were removed by centrifugation and washing. The functionalized gel was then resuspended in PBS buffer.

[0017] (3) Encapsulation of fluorescent dye and purification of probe: The functionalized gel dispersion was incubated at 4 °C for 1 hour to allow the gel network to fully swell. Then, sulfonated anthocyanin dye Cy5.5 was added and incubated at 4 °C in the dark for 24 hours to allow the dye molecules to fully diffuse and self-quench due to high concentration aggregation. Subsequently, the system was heated to 37 °C and maintained for 1 hour to allow the gel to shrink and physically capture the dye molecules inside the network. Finally, the product was purified using gel chromatography to obtain pure bifunctional fluorescent nanoprobes, which were stored in PBS at 4 °C for later use.

[0018] Furthermore, the concentration of the monomer N-isopropylacrylamide in step (1) is 100 mM.

[0019] Furthermore, the crosslinking agent in step (1) is N,N'-methylenebisacrylamide.

[0020] Furthermore, the concentration of the crosslinking agent N,N'-methylenebisacrylamide in step (1) is 2 mM.

[0021] Furthermore, the concentration of the comonomer acrylic acid in step (1) is 5 mM.

[0022] Furthermore, the final concentration of the monoclonal antibody against the human EGFR L858R mutant protein in step (2) is 50 μg / mL.

[0023] Furthermore, the final concentration of the 5'-terminal amino-modified PNA in step (2) is 20 μM.

[0024] Furthermore, the sequence of the PNA in step (2) is 5'-TCA ACA TCA GTC TGA TAA GCT A-3', which is completely complementary to the sequence of human miR-21 (has-miR-21-5p), and is used to specifically recognize and capture miR-21 in cells.

[0025] Furthermore, the concentration of the sulfonated anthocyanin dye Cy5.5 in step (3) is 1 mM.

[0026] This invention also provides the use of a logic-gated nanogel probe for live cell typing in the preparation of reagents or kits for in vitro differentiation or identification of lung cancer cells:

[0027] Furthermore, the lung cancer cells are intact, living cells;

[0028] Furthermore, the live cells are derived from body fluid samples, which are selected from peripheral blood, pleural effusion, bronchoalveolar lavage fluid, or fine needle aspiration fluid;

[0029] Furthermore, the distinction or identification refers to distinguishing live cells that simultaneously express EGFR L858R mutant protein and miR-21 from a complex cellular background.

[0030] The advantages of this invention are:

[0031] 1. This invention achieves precise integration of a thermosensitive gel carrier, a dual-target recognition element, and a fluorescence signal switch, successfully constructing a smart nanoprobe with extremely low background signal (quenching efficiency >95%) that can be triggered by specific biological signals to release dyes, laying the material foundation for its high signal-to-noise ratio detection in complex biological environments.

[0032] 2. This invention features an AND logic gating design that fundamentally improves detection specificity. The probe generates a strong signal only in cells where both the EGFR L858R mutant protein and miR-21 are present, while in cells with only a single marker, the signal is indistinguishable from the background. This fundamentally eliminates the high false-positive problem caused by non-specific marker expression in traditional single-target probes.

[0033] 3. This invention develops a novel in situ detection method that directly targets and drives gene functional products and relies on the intact microenvironment of living cells. This technology overcomes the limitations of traditional detection methods (such as EpCAM-based CTC capture or ctDNA sequencing) in that they cannot correlate genotype and live cell phenotype at the single-cell level, providing a precise tool for dynamically monitoring drug-sensitive clones in liquid biopsy for lung cancer. Attached Figure Description

[0034] Figure 1 This is a physical characterization diagram of the logic-gated nanogel probe for live cell typing prepared in this invention: Figure 1 (a) is the particle size distribution diagram of the nanogel probe before modification; Figure 1 (b) is the particle size distribution diagram of the modified nanogel probe.

[0035] Figure 2 This is the Zeta potential of the nanogel probe of this invention.

[0036] Figure 3This describes the temperature response performance of the nanogel probe of the present invention.

[0037] Figure 4 This describes the fluorescence quenching performance of the nanogel probe of this invention.

[0038] Figure 5 This describes the logic gating performance of the nanogel probe of this invention. Detailed Implementation

[0039] The technical solutions described in this invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of this invention. Obviously, the embodiments described in this specification are only some feasible technical solutions of this invention. Other implementation methods obtained by those skilled in the art based on the embodiments of this invention without any creative effort should be considered to fall within the scope of protection of this invention.

[0040] Example 1: Preparation of Nanogel Probes

[0041] (1) Preparation of thermosensitive nanogel core by precipitation polymerization: Under nitrogen protection and at 70 °C, 100 mM monomer N-isopropylacrylamide, 2 mM crosslinking agent N,N'-methylenebisacrylamide, and 5 mM comonomer acrylic acid were subjected to free radical polymerization in aqueous solution. After the reaction, the thermosensitive PNIPAAm-co-AAc nanogel was obtained by dialysis purification.

[0042] (2) Surface functionalization modification: The purified nanogel was diluted to a solid content of 1 mg / mL with 0.1 M pH 6.0 2-(N-morpholino)ethanesulfonic acid buffer. EDC (5 mM) and NHS (10 mM) were added sequentially, and the carboxyl groups were activated at room temperature for 15 min. Subsequently, a monoclonal antibody against human EGFR L858R mutant protein (50 μg / mL) and 5'-terminal amino-modified PNA (sequence complementary to miR-21) (20 μM) were added, and the reaction was carried out with gentle stirring at room temperature for 2 h. After the reaction was complete, uncoupled molecules were removed by centrifugation and washing, and the functionalized gel was resuspended in PBS buffer.

[0043] (3) Encapsulation of fluorescent dye and purification of probe: The functionalized gel dispersion was incubated at 4 °C for 1 hour to allow the gel network to fully swell. Then, 1 mM of sulfonated anthocyanin dye Cy5.5 was added, and the mixture was incubated at 4 °C in the dark for 24 h to allow the dye molecules to fully diffuse and self-quench due to high concentration aggregation. Subsequently, the system was heated to 37 °C and held for 1 h to allow the gel to shrink and physically capture the dye molecules inside the network. Finally, the product was purified using gel chromatography to obtain a pure bifunctional fluorescent nanoprobe, which was then stored in PBS at 4 °C for later use.

[0044] Comparative Example 1: Preparation of Single-Target Response Nanogel Probe A

[0045] The difference between this comparative example and Example 1 is that in step (2), only the anti-EGFR L858R antibody is modified, and the 5' end amino-modified PNA is not modified. Other experimental methods and parameters are the same as in Example 1.

[0046] Comparative Example 2: Preparation of Single-Target Response Nanogel Probe B

[0047] The difference between this comparative example and Example 1 is that in step (2), only the 5' end amino-modified PNA is modified, and the anti-EGFR L858R antibody is not modified. Other experimental methods and parameters are the same as in Example 1.

[0048] Comparative Example 3: Preparation of Nanogel Probes Based on the Traditional Marker Epithelial Cell Adhesion Molecule EpCAM

[0049] The difference between this comparative example and Example 1 is that in step (2), the anti-EGFR L858R antibody was replaced with the anti-EpCAM antibody (concentration 50 μg / mL), while the 5' end amino-modified PNA modification was retained. Other experimental methods and parameters are the same as in Example 1.

[0050] Experimental Example 1: Microscopic Characterization of the Nanogel Probe Prepared in Example 1

[0051] The nanogel probe prepared in Example 1 was ultrasonically dispersed in water (concentration of 10 µg / mL), and the particle size and polydispersity index (PDI) of the nanogel probe before and after functionalization were measured by dynamic light scattering (DLS).

[0052] The results are as follows Figure 1 As shown in (a) and (b), the diameter of the unmodified nanogel probe is about 100 nm and the PDI is < 0.1, indicating that the synthesized product has good monodispersity. The diameter of the modified nanogel probe is about 125 nm and the PDI is < 0.15, indicating that the biomolecules have been successfully anchored on the gel surface and the functionalization modification is uniform and controllable, without causing significant aggregation or degradation of the gel.

[0053] Experimental Example 2: Zeta potential characterization of the nanogel probe prepared in Example 1

[0054] To characterize whether the nanogel probes were successfully functionalized, the charge on the probe surface was characterized. The nanogel probes were ultrasonically dispersed in deionized water, and the zeta potential of the probes was evaluated using a zeta potential meter. The results are as follows: Figure 2As shown, the zeta potentials before and after modification are -20.5 mV and -3.1 mV, respectively. This is because the zeta potential of the nanogel probe before modification is negative due to the presence of carboxyl groups, while the zeta potential after functionalization modification approaches neutrality due to the introduction of positively charged PNA. Therefore, the nanogel probe prepared in Example 1 was successfully modified.

[0055] Experimental Example 3: Characterization of the temperature response performance of the nanogel probe prepared in Example 1

[0056] To verify the temperature responsiveness of the nanogel probe prepared in Example 1, its phase transition process was monitored using UV-Vis spectrophotometry. Nanogel dispersions (solid content 0.1 mg / mL) at different temperatures (20-50 °C) were placed in a sample cell for measurement, and their absorbance at a wavelength of 500 nm was measured. The midpoint of the temperature range where absorbance decreases sharply was defined as the lower critical dissolution temperature.

[0057] The results are as follows Figure 3 As shown, the absorbance-temperature curve exhibits a significant steep drop, with the LCST between 32-35 °C. This confirms that the gel has a significant temperature response: at 4 °C (below LCST), it undergoes sufficient hydrophilic swelling, facilitating the diffusion of dye molecules, resulting in high transparency and low absorbance; at 37 °C (above LCST), it undergoes dramatic hydrophobic shrinkage, resulting in lower transparency and higher absorbance, effectively trapping dye molecules within the network.

[0058] Experimental Example 4: Characterization of the fluorescence quenching performance of the nanogel probe prepared in Example 1

[0059] Fluorescence spectrometry was used to measure the fluorescence spectra of a 10 nM free Cy5.5 dye solution and the final prepared nanoprobe dispersion (equivalent dye concentration 10 nM) (excitation wavelength: 678 nm, emission scan range: 690-750 nm). To ensure that the nanogel probe has a low background signal, the fluorescence quenching efficiency (QE) of the probe was calculated using the fluorescence intensity (Ff) of the free dye at the maximum emission peak as a reference, according to the following formula: QE = [1 - (Fp / Ff)] × 100%, where Fp is the fluorescence intensity of the probe at the same wavelength.

[0060] The results are as follows Figure 4As shown, the free Cy5.5 dye exhibits a strong fluorescence intensity (13734 au) at 694 nm. In contrast, the nanogel probe prepared in Example 1 shows an extremely low fluorescence intensity of only 683 au under the same conditions. Calculations show that its fluorescence quenching efficiency is over 95%. This data demonstrates that the dye molecules are successfully encapsulated in the gel network and exist in a highly aggregated quenched state, thus ensuring extremely low background fluorescence in the unactivated state. Therefore, the nanogel probe prepared in Example 1 exhibits a fluorescence signal that is turned off when unactivated.

[0061] Experimental Example 5: Characterization of the logic gating performance of the nanogel probe prepared in Example 1

[0062] To verify the logic-gated performance of the nanogel probes prepared in Example 1, four lung cancer cell models were constructed: Model I (NCI-H1975) served as a double-positive control, simultaneously expressing the EGFR L858R mutant protein and high levels of miR-21; Model II simulated the "mutant protein-only positive" state by inhibiting miR-21 in NCI-H1975 cells with an inhibitor; Model III simulated the "miRNA-only positive" state by transfecting EGFR wild-type A549 cells with a miR-21 mimic; and Model IV (BEAS-2B) served as a double-negative control. The cells were co-incubated with the nanogel probes prepared in Example 1 and Comparative Examples 1-3, respectively. After incubation, the cells were fixed and stained, and images were observed using a confocal microscope. The mean fluorescence intensity (MFI) of single cells was quantitatively analyzed using ImageJ software.

[0063] like Figure 5 As shown, in the double-positive model I, the probe exhibited a strong specific fluorescence signal with a mean fluorescence intensity (MFI) of approximately 25211 au. In contrast, the MFIs of Comparative Examples 1 and 2 were both below 4000 au, indicating that synergistic activation of the dual targets is crucial for achieving high-intensity signal output. Although Comparative Example 3 also detected a strong signal (MFI of 20214 au), its target was a general epithelial biomarker and lacked specificity for driver mutations, thus differing fundamentally from this probe in terms of clinical guidance.

[0064] In Models II and III, the MFI of the probe in Example 1 remained at the background level (<800 au) because the logic gates did not meet the activation condition. However, under the same conditions, Comparative Example 1 (in Model II) and Comparative Example 2 (in Model III) produced false positive signals as high as 17520 au and 16943 au, respectively. This result indicates that single-target detection strategies are highly susceptible to misjudgment due to the presence of a single biomarker. This probe, through its AND logic gate design, elevates the detection standard from "biomarker presence" to "coexistence of functional states," thereby significantly improving specificity.

[0065] Furthermore, all probes exhibited low background signal (MFI < 1000 au) in the negative cell model IV, and no significant response was detected in the cell lysate of Comparative Example 3, further confirming that the function of the probes in Example 1 is strictly dependent on the in-situ microenvironment provided by intact living cells. In summary, the nanogel probes developed in Example 1 can achieve in-situ interpretation of target living cells with ultra-high specificity and high sensitivity.

[0066] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the principle of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A logic-gated nanogel probe for live cell typing, characterized in that, The nanogel probe comprises an environmentally responsive nanocarrier, at least two recognition molecules respectively bound to the carrier, and a signal reporting unit; The environmentally responsive nanocarrier is a thermosensitive polymer gel with a low critical dissolution temperature. The at least two recognition molecules include a protein recognition unit and a nucleic acid recognition unit; The signal reporting unit is an aggregate of fluorescent dye molecules in a self-quenching state; The release of the signal reporting unit is triggered by the simultaneous binding of the at least two recognition molecules to their corresponding targets. The probe achieves AND logic gating through the network state switching of the temperature-sensitive polymer gel: Only when the protein recognition unit and the nucleic acid recognition unit bind to their respective targets and produce a synergistic effect, the thermosensitive polymer gel changes from a physically contracted closed state to a phase transition swelling open state, thereby releasing the physical lock on the signal reporting unit and triggering the output of a fluorescence signal. If only a single target binds, the nanogel remains in a contracted state to suppress signal release.

2. The nanogel probe according to claim 1, characterized in that, The protein recognition unit is an antibody or fragment thereof that specifically binds to the EGFR L858R mutant protein, and the nucleic acid recognition unit is a peptide nucleic acid that specifically binds to miR-21.

3. A method for preparing a logic-gated nanogel probe for live cell typing, characterized in that, The preparation method includes the following steps: (1) Preparation of thermosensitive nanogel core by precipitation polymerization: Under nitrogen protection and 70 °C, monomer N-isopropylacrylamide, 2 mM crosslinking agent N,N'-methylenebisacrylamide and 5 mM comonomer acrylic acid were subjected to free radical polymerization in aqueous solution; after the reaction was completed, the thermosensitive poly(N-isopropylacrylamide-co-acrylic acid) nanogel was obtained by dialysis purification. (2) Surface functionalization modification: The purified nanogel was diluted with 0.1 M pH 6.0 2-(N-morpholino)ethanesulfonic acid buffer to a solid content of 1 mg / mL; 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added sequentially to a final concentration of 5 mM and a final concentration of 10 mM, and the carboxyl group was activated at room temperature for 15 min; then, a monoclonal antibody against human EGFRL858R mutant protein and a peptide nucleic acid modified with the 5' end amino group were added, and the reaction was carried out with gentle stirring at room temperature for 2 h; after the reaction was completed, the uncoupled molecules were removed by centrifugation and washing, and the functionalized gel was resuspended in PBS buffer; (3) Encapsulation of fluorescent dye and purification of probe: The above functionalized gel dispersion was incubated at 4 °C for 1 h to allow the gel network to fully swell. Then, sulfonated anthocyanin dye Cy5.5 was added and incubated at 4 °C in the dark for 24 h to allow the dye molecules to fully diffuse and self-quench due to high concentration aggregation. Subsequently, the system was heated to 37 °C and held for 1 h to allow the gel to shrink and physically capture the dye molecules inside the network. Finally, the product was purified using a Sephadex G-50 gel chromatography column to obtain a pure bifunctional fluorescent nanoprobe, which was stored in PBS at 4 °C for later use.

4. The method for preparing the nanogel probe according to claim 3, characterized in that, The concentration of the monomer N-isopropylacrylamide in step (1) is 100 mM.

5. The method for preparing the nanogel probe according to claim 3, characterized in that, The final concentration of the monoclonal antibody against the human EGFR L858R mutant protein in step (2) is 50 μg / mL, and the final concentration of the 5' amino-modified peptide nucleic acid is 20 μM.

6. The method for preparing the nanogel probe according to claim 3, characterized in that, The peptide nucleic acid sequence in step (2) is 5'-TCA ACA TCA GTC TGA TAA GCT A-3', which is used to specifically recognize and capture miR-21 in cells.

7. The method for preparing the nanogel probe according to claim 3, characterized in that, The concentration of the sulfonated anthocyanin dye Cy5.5 in step (3) is 1 mM.

8. The use of a nanogel probe according to any one of claims 1-2 in the preparation of a reagent or kit for in vitro differentiation or identification of lung cancer cells, characterized in that, The lung cancer cells are live cells derived from a body fluid sample.

9. The use according to claim 8, characterized in that, The body fluid samples were selected from peripheral blood, pleural effusion, bronchoalveolar lavage fluid, or fine needle aspiration fluid.

10. The use according to claim 8, characterized in that, The distinction or identification refers to the process of differentiating live cells from a complex cellular background that simultaneously express both the EGFR L858R mutant protein and miR-21.

Images