Method for elution of nucleic acid probes from a biological sample and use thereof

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a method for eluting nucleic acid probes from biological samples, comprising the following steps: (1) obtaining a biological sample containing nucleic acid probes, and (2) immersing the biological sample in an elution solution containing quaternary phosphonium salt to elute the nucleic acid probes. This invention can achieve rapid and complete dissociation and elution of nucleic acid probes, thereby obtaining excellent multi-round staining cycle efficiency.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for eluting nucleic acid probes from biological samples, which is particularly suitable for high-throughput, multi-round labeling or spatial transcriptome analysis of thick tissue samples. Background Technology

[0002] To systematically study the molecular architecture of isolated biological tissues at high resolution and multiple scales, particularly to elucidate the spatial heterogeneity of gene expression, multi-round nucleic acid staining techniques have emerged. This technique, by cyclically performing nucleic acid hybridization, imaging, and signal removal on the same biological sample, can detect dozens or even hundreds of different nucleic acid targets in a single sample, providing a revolutionary tool for spatial transcriptomics and biological research. Among these methods, hybridization chain reaction (HCR) and SABER are widely used for in situ nucleic acid imaging of tissue samples due to their strong signal amplification capabilities and multiplexing capabilities. However, the core challenge of these multi-round nucleic acid staining techniques lies in how to efficiently and thoroughly remove the nucleic acid probe signals from the previous round of hybridization while maintaining the integrity of the sample's tissue morphology, nuclear staining characteristics, and the covalent structure of endogenous nucleic acid molecules, thus ensuring the specificity of subsequent rounds of hybridization and the registration accuracy of the imaging.

[0003] Various signal removal methods have been proposed in existing multi-round nucleic acid staining techniques, but all have significant drawbacks. DNase digestion utilizes DNase to degrade the DNA probe used in the previous hybridization round; however, this method is time-consuming, typically requiring several hours or even overnight incubation, severely limiting experimental throughput. Furthermore, DNase, as a large protein molecule, is difficult to diffuse into thick tissue samples, resulting in ineffective removal of deep-layer signals. More critically, DNase treatment degrades genomic DNA, causing a sharp weakening or disappearance of nuclear staining signals in the second and subsequent rounds, making it difficult to meet the high-precision registration requirements of multi-round imaging. Moreover, DNase treatment in conventional aqueous environments lacks effective inhibition of RNases, easily leading to the degradation of endogenous RNA in the sample, making it impossible to detect RNA targets in subsequent rounds.

[0004] Photoquenching utilizes strong light to photobleach the fluorescent groups from the previous hybridization round, thereby eliminating the signal. However, this method only quenches the fluorescence signal and cannot remove the nucleic acid probe itself; the probe molecules remain in the sample and may interfere with subsequent hybridization rounds. Modern commercially available fluorescent dyes are highly photostable, making complete quenching difficult. For thick tissue samples, light cannot penetrate evenly to the deeper layers, resulting in residual signals in the deeper layers. Because the probe is not removed, this method is limited by the number of orthogonal spectral channels and cannot achieve truly unlimited staining rounds.

[0005] Cleavable probe methods incorporate cleavable linkers (such as disulfide bonds) during probe design, releasing the fluorescent group after imaging by cleaving the linkers with a reducing agent. However, this method also fails to remove the nucleic acid portion of the probe, only removing the fluorescent group. Limited by the number of orthogonal chemical channels, designing multiple orthogonal cleavable groups presents chemical complexity and is difficult to extend indefinitely. Furthermore, the cleavage efficiency is limited, often failing to achieve complete removal, resulting in signal residue. While high-concentration formamide elution can be used as a nucleic acid denaturant to disrupt probe-target hybridization, its elution efficiency is insufficient, often requiring prolonged or multiple treatments to partially remove the signal, and it easily leads to tissue shrinkage and RNA loss.

[0006] In summary, existing multi-round nucleic acid staining techniques lack a method that can rapidly, thoroughly, and non-destructively remove nucleic acid hybridization signals, support unlimited rounds of staining, and is particularly suitable for thick tissue samples. Summary of the Invention

[0007] The purpose of this invention is to provide an elution method that can completely remove nucleic acid hybridization probe signals from biological samples in a short time. This method can maintain the integrity of the tissue morphology of biological samples and the covalent structure of endogenous nucleic acid molecules (including DNA and RNA). It has efficient diffusion ability during tissue penetration, can act uniformly inside thick tissue samples, does not interfere with the efficiency of subsequent hybridization cycles after signal removal, supports multiple rounds of cyclic staining, and does not introduce the risk of RNA degradation.

[0008] On the one hand, this invention proposes a method for eluting nucleic acid probes from biological samples, comprising the following steps: (1) Obtain biological samples containing nucleic acid probes. (2) Immerse the biological sample in an elution solution containing quaternary phosphonium salt to elute the nucleic acid probe.

[0009] In some embodiments, the concentration of quaternary phosphonium salt in the eluent is greater than 25 w / v, preferably 25 w / v%-60 w / v, for example 30 w / v%, 35 w / v%, 40 w / v%, 45 w / v%, 50 w / v% or 55 w / v.

[0010] In some embodiments, the elution time in step (2) is 10 min or more, preferably 15 min or more, for example 15-60 min, preferably 15-40 min. In some embodiments, the elution time is 15 min or 30 min.

[0011] In some embodiments, the elution temperature in step (2) is 4-65°C, preferably 25-55°C, and more preferably 30-50°C. For example, in some embodiments, the elution temperature is 37°C.

[0012] In some embodiments, the eluent further comprises an amide compound.

[0013] In some embodiments, the amide compound is selected from formamide, acetamide, acrylamide, N,N-dimethylformamide, N,N-dimethylacetamide, benzamide, N,N-dimethylbenzamide, phenylacetamide, pyridineamide, nicotinamide, isonicotinamide, N-methylnicotinamide, or combinations thereof.

[0014] In some embodiments, the concentration of the amide compound in the eluent is 1-35 w / w, preferably 5-25 w / w.

[0015] In some embodiments, the eluent further comprises a buffer salt and / or a surfactant that promotes the dissolution of the quaternary phosphonium salt.

[0016] In some embodiments, the biological sample containing the nucleic acid probe is a biological sample that has undergone nucleic acid probe-based in situ hybridization treatment.

[0017] In some implementations, the nucleic acid probe-based in situ hybridization is selected from fluorescence in situ hybridization (FISH), single-molecule fluorescence in situ hybridization (smFISH), fluorescence in situ hybridization combined with hybridization chain reaction (HCR-FISH), fluorescence in situ hybridization combined with rolling circle amplification (RCA-FISH), SABER-FISH, clampFISH, or Π-FISH.

[0018] In some embodiments, the biological sample is a cell or tissue.

[0019] In some embodiments, the biological sample is preferably a tissue sample with a thickness of 2 μm-50 mm.

[0020] On the other hand, the present invention also proposes a kit for multi-round in situ hybridization, comprising a target nucleic acid-specific recognition probe and an elution buffer containing quaternary phosphonium salt.

[0021] In some embodiments, the concentration of quaternary phosphonium salt in the eluent is greater than 25 w / v, preferably 25 w / v%-60 w / v, for example 30 w / v%, 35 w / v%, 40 w / v%, 45 w / v%, 50 w / v% or 55 w / v.

[0022] In some embodiments, the eluent further comprises an amide compound.

[0023] In some embodiments, the amide compound is selected from formamide, acetamide, acrylamide, N,N-dimethylformamide, N,N-dimethylacetamide, benzamide, N,N-dimethylbenzamide, phenylacetamide, pyridineamide, nicotinamide, isonicotinamide, N-methylnicotinamide, or combinations thereof.

[0024] In some embodiments, the concentration of the amide compound in the eluent is 1-35 w / w, preferably 5-25 w / w.

[0025] In some embodiments, the eluent further comprises a buffer salt, a surfactant that promotes the dissolution of the quaternary phosphonium salt, and / or a preservative.

[0026] In another aspect, the present invention also proposes the application of the kit in nucleic acid fluorescence detection or nucleic acid fluorescence imaging for non-diagnostic purposes.

[0027] Compared with the prior art, the present invention has the following main technical advantages: The present invention provides a method for eluting nucleic acid hybridization probes from biological samples. Utilizing the small molecule properties of substances such as quaternary phosphonium salts and their strong electrostatic and hydrogen-bonding interactions with the phosphate backbone of nucleic acids, this method efficiently disrupts the secondary structural stability of nucleic acid hybridization complexes within a short time, achieving rapid and complete probe dissociation and elution. This results in excellent multi-round staining cycle efficiency while avoiding the drawbacks of large molecules like DNases that struggle to penetrate thick tissues. Since quaternary phosphonium salts function solely through charge, hydrogen bond competition, and conformational interference, without involving covalent bond breakage or enzymatic degradation, and simultaneously inhibiting nuclease activity, they effectively protect the covalent integrity of genomic DNA and RNA molecules in the sample, maintaining the staining patterns of nuclear dyes such as DAPI, and ensuring accurate registration and cell segmentation in multi-round imaging. Because this method does not rely on photoquenching of fluorescent groups or chemical cleavage of cleavable linkers, it completely elutes the hybridization probes, thus avoiding the limitations of probe orthogonality in nucleic acid methods. This allows for a convenient and low-loss achievement of a total number of target nucleic acid labeling types exceeding the orthogonal limit in multiple staining cycles.

[0028] This method will enable multi-round nucleic acid staining technology to truly achieve high-throughput, multi-round spatial transcriptomics analysis of thick tissue samples, making it possible to fully reconstruct multidimensional molecular structures using the same biological sample. Attached Figure Description

[0029] The following figures are intended only to illustrate and explain the present invention and do not limit the scope of the invention. Wherein: Figure 1 Images of HCR fluorescence signals in the cerebral cortex after treatment with different concentrations of methyltriphenylphosphonium bromide; Figure 2Statistical analysis of HCR fluorescence intensity in the cerebral cortex after elution with different concentrations of methyltriphenylphosphonium bromide; Figure 3 Images of HCR fluorescence signals in the cerebral cortex after treatment with 25% methyltriphenylphosphonium bromide at different elution times; Figure 4 Images showing the elution effect of HCR fluorescence signals in different elution systems; Figure 5 This invention compares the elution effect of the eluent of this invention with that of conventional reagents on the elution of HCR signals. Figure 6 The fluorescence comparison images show the HCR signal of mouse cerebral cortex before, after, and after re-staining secondary probes. Figure 7 Comparison of HCR signals before and after DNase I elution and after counterstaining; Figure 8 Comparison of HCR signals before and after elution with 50% formamide and after counterstaining; Figure 9 Comparison of HCR signals before and after elution and after counterstaining in the composite solution; Figure 10 This is a diagram showing the results of multiple rounds of elution and counterstaining. The length of the scale bar marker in each micrograph is set to 100 μm. Detailed Implementation

[0030] The present invention will be further described below with reference to embodiments. The description of the embodiments below is only for the purpose of helping to understand the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principle of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

[0031] In this specification, the reference to "one embodiment" means that the specific features, parameters, steps, etc., described in that embodiment are included in at least one embodiment according to the present invention. Therefore, the use of terms such as "according to one embodiment of the present invention" or "in one embodiment" in this specification does not specifically refer to the same embodiment, and the use of terms such as "in another embodiment," "different embodiments of the present invention," or "other embodiments of the present invention" does not specifically mean that the mentioned features can only be included in specific different embodiments. Those skilled in the art should understand that the specific features, parameters, steps, etc., disclosed in one or more embodiments of this specification can be combined in any suitable manner.

[0032] The present invention proposes a method for eluting nucleic acid probes from biological samples, comprising the following steps: (1) Obtain biological samples containing nucleic acid probes. (2) Immerse the biological sample in an elution solution containing quaternary phosphonium salt to elute the nucleic acid probe.

[0033] In some embodiments of the present invention, the hydrocarbon group of the quaternary phosphonium salt is selected from substituted or unsubstituted aryl, alkyl, cycloalkyl, and alkenyl groups. In some embodiments, the hydrocarbon group has 1-20 carbon atoms, preferably 1-10.

[0034] In some embodiments, the aryl group can be phenyl, methylphenyl, ethylphenyl, etc., the alkyl group can be methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, hexadecyl, etc., the cycloalkyl group can be cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc., and the alkenyl group can be allyl, butenyl, pentenyl, etc.

[0035] In some embodiments, the quaternary phosphonium salt is selected from one or more of tetraphenylphosphonium salt, alkyltriphenylphosphonium salt, dialkyldiphenylphosphonium salt, trialkylphenylphosphonium salt, or tetraalkylphosphonium salt.

[0036] In some embodiments, the quaternary phosphonium salt is a phosphonium halide.

[0037] In some embodiments, the quaternary phosphonium salt is preferably tetraphenylphosphonium chloride, methyltriphenylphosphonium bromide, methyltriphenylphosphonium chloride, heptyltriphenylphosphonium bromide, tetrabutylphosphonium chloride, hydroxymethyltriphenylphosphonium bromide, propyltriphenylphosphonium bromide, cyclopropyltriphenylphosphonium bromide, benzyltriethylphosphonium bromide, bromomethyltriphenylphosphonium bromide, ethyltriphenylphosphonium iodide, tributylhexylphosphonium bromide, allyltributylphosphonium bromide, etc.

[0038] In the embodiments of this invention, various quaternary phosphonium salts with different substituents were selected. The results showed that the selected quaternary phosphonium salts all achieved good elution effects, indicating that the substituents do not significantly affect the elution effect. For some quaternary phosphonium salts with poor solubility in water, the solubility can be improved by adding surfactants, and the elution effect can also be improved by increasing the elution temperature or elution time.

[0039] In this invention, mass-volume percentage concentration or mass-volume concentration refers to the number of grams of solute contained in 100 ml of solution, which can be expressed as w / v%. For example, a mass-volume percentage concentration of 1% means that 100 ml of solution contains 1 gram of solute.

[0040] In this invention, volume percentage concentration or volume concentration refers to the percentage of solute volume to the total solution volume, which can be expressed as v / v.

[0041] In this invention, mass percentage concentration or mass concentration refers to the percentage of solute mass to the total mass of the solution, which can be expressed as w / w.

[0042] The elution buffer of this invention can effectively elute nucleic acid hybridization probes from biological samples without significantly affecting the morphological integrity of the samples or the covalent integrity of nucleic acid molecules. Effective elution means that after treatment with the elution buffer, no identifiable nucleic acid hybridization signal from the previous round can be detected under a fluorescence microscope. Specifically, for FISH methods involving signal amplification, such as HCR, the same amplified probe cannot detect the nucleic acid hybridization signal remaining from the previous round under a fluorescence microscope. Therefore, the method of this invention is particularly suitable for multi-round nucleic acid hybridization imaging.

[0043] Example 1: Concentration gradient and elution time verification of HCR fluorescence signal elution efficiency Experimental samples: Several 24-week-old C57BL / 6J mice were selected, and their brain tissue was dissected after cardiac perfusion. Coronal sections of brain tissue with a thickness of 100 μm were prepared for later use.

[0044] Experimental reagents: Hybridization Buffer I: 10 mL of formamide (Aladdin, catalog number: F120616-250 mL), 10 mL of 20×SSC stock solution (Sangon Biotech, catalog number: B548109-0200), 300 μL of 1M citric acid stock solution (pH 6.0), 200 μL of 10% Tween-20 stock solution (Aladdin, catalog number: T108669), 100 μL of 5 mg / mL heparin stock solution (Aladdin, catalog number: H758140), 6 mL of 50% dextran sulfate stock solution (Yuanye Biotechnology, catalog number: S14047), 800 μL of 50×Denhardt's stock solution (Aladdin, catalog number: D120788), with the remainder being DEPC. Add purified water (Sangon Biotech, catalog number: B501005-0500, hereinafter the same) to a total of 40 mL.

[0045] Hybridization buffer II: 10 mL of 20×SSC stock solution, 200 μL of 10% Tween-20 stock solution, 6 mL of 50% dextran sulfate stock solution, and the remainder is DEPC pure water to make up to 40 mL.

[0046] Washing buffer: 5×SSCT (10ml 20xSSC plus 400ul 10%Tween20, bring to a final volume of 40mL).

[0047] The HCR-specific recognition probes and HCR fluorescent amplification probes for SST mRNA detection are shown in Table 1.

[0048] Table 1. SST mRNA-specific recognition probe sequences and HCR fluorescent amplification probes SEQ ID NO SST mRNA specific recognition probe sequence 1 CCAGTTCCTGTTTCCCGGTGGCAGCTAGAAGAGTCTTCCTTTACG 2 AATCCTCGGGCTCCAGGGCATCATTTAGAAGAGTCTTCCTTTACG 3 AGAAGAAGTTCTTGCAGCCAGCTTTTAGAAGAGTCTTCCTTTACG 4 GGAAGAGATATGGGGTTTGGGGGAGTAGAAGAGTCTTCCTTTACG 5 ACAGTCTTCAATTTCTAATGCAGGGTAGAAGAGTCTTCCTTTACG 6 GAGGAGGGCAGCAAACGGAACCAGAGACTTCTGCAGAAACTGACG 7 GAGGAGGGCAGCAAACGGAACTGTCTGGTTGGGCTCGGACAGCAG 8 GAGGAGGGCAGCAAACGGAAGTTCCCGGGGTGCCATTGCTGGGTT 9 GAGGAGGGCAGCAAACGGAAGGATCAGAGGTCTGGCTAGGACAAC 10 GAGGAGGGCAGCAAACGGAAAAGTTGAGCATCGGGGGCCAGGAGT HCR fluorescent amplification probe (containing Cy5 fluorescent dye) 11 CGTAAAGGAAGACTCTTCCCGTTTGCTGCCCTCCTCGCATTCTTTCTTGAGGAGGGCAGCAAACGGGAAGAG 12 GAGGAGGGCAGCAAACGGGAAGAGTCTTCCTTTACGCTCTTCCCGTTT GCTGCCCTCCTCAAGAAAGAATGC Experimental instruments and analysis software: constant temperature controlled incubator, laser confocal scanning microscope, Imaris fluorescence image analysis software.

[0049] Experimental methods: 1. Preprocessing of brain tissue sections Take a 100 μm coronal section of mouse brain tissue after preparation, and rinse it three times with PBS buffer at room temperature, with each rinse lasting 5 min, to fully remove residual impurities in the tissue.

[0050] 2. HCR in situ hybridization staining Add hybridization buffer I containing SST mRNA-specific detection probes to completely immerse the slides in the probe hybridization system; incubate at 37°C in the dark for 12 h; after incubation, rinse three times with washing buffer at room temperature for 5 min each time; then add hybridization buffer II containing HCR fluorescent amplification probes and incubate at room temperature in the dark for 12 h; after incubation, rinse three times with washing buffer at room temperature for 5 min each time, and then rinse twice with PBS buffer at room temperature for 5 min each time to complete the HCR standard staining procedure.

[0051] 3. HCR fluorescence signal elution 3.1 Prepare aqueous solutions of methyltriphenylphosphonium bromide (Aladdin, catalog number: 1779-49-3, hereinafter the same) with the following mass-volume percentage concentration gradients: 5%, 10%, 25%, and 50%, and set up a control group (untreated group) without elution buffer treatment. Add 200 μL of the corresponding concentration of methyltriphenylphosphonium bromide solution (treated group) to each group of samples and incubate at different temperatures for 15 min.

[0052] 3.2 After the treatment was completed, the PBS buffer was rinsed twice at room temperature for 5 minutes each time.

[0053] 3.3 With fixed laser confocal microscopy imaging parameters, in situ fluorescence imaging was performed on brain tissue sections from all groups. The imaging results for each group were analyzed using Imaris image analysis software.

[0054] The study found that the HCR signal (i.e., the nucleic acid probe) could be eluted when incubated at room temperature (25°C), but a longer elution time was required when incubated at 4°C. As the incubation temperature increased, for example to 40°C or 50°C, the required elution time gradually decreased. In multi-round in situ hybridization experiments, considering the impact on the analyte in tissues, it is recommended not to exceed 65°C. In subsequent embodiments, unless otherwise specified, the present invention generally involves incubation at 37°C.

[0055] Figure 1 shows the HCR signal distribution of mouse cerebral cortex after elution with different concentrations of methyltriphenylphosphonium bromide. The results indicate that as the concentration of the eluent increases, the fluorescent spots in the brain tissue gradually become sparse, and the background fluorescence significantly decreases; in the 25% and 50% concentration groups, the cerebral cortex region is almost entirely negative. Further quantitative statistical analysis (…) Figure 2 The elution effect of methyltriphenylphosphonium bromide on HCR signals is concentration-dependent. At a concentration of 5%, the average fluorescence intensity was not significantly different from the control group; however, when the concentration increased to 10% or higher, the average fluorescence intensity decreased significantly. Specifically, the mean fluorescence intensity of the 25% and 50% concentration groups had decreased to baseline levels, with no significant difference between them. These qualitative and quantitative results jointly confirm that methyltriphenylphosphonium bromide concentrations of 25% and higher can achieve efficient and thorough elution of HCR signals from the cerebral cortex.

[0056] 3.4 Based on the above results, a 25% methyltriphenylphosphonium bromide solution was selected as the eluent to further investigate the effect of elution time on the signal. The specific steps are as follows: Elution time gradients of 1 min, 10 min, and 15 min were set; the reaction system was eluted under light-protected conditions at a constant temperature of 37°C. After elution, the system was rinsed twice with PBS buffer at room temperature, each rinse lasting 5 min.

[0057] 3.5 The Imaris image analysis software was used to analyze the results of each component image, such as... Figure 3 As shown: 1) When the elution time is 1 min, a very small amount of weak fluorescent signal residue is occasionally seen in the field of view; 2) When the elution time was extended to 10 min, the residual fluorescence signal in the field of view was further reduced, but there were still some scattered and weak fluorescence spots visible. 3) When the elution time reaches 15 min, almost no obvious fluorescent signal residue can be observed in the field of view, the background is clean, and the elution effect is optimal.

[0058] The results showed that 25% methyltriphenylphosphonium bromide solution for 15 min could effectively elute 100 μm mouse cerebral cortex HCR probes.

[0059] Example 2: Validation of different elution systems Based on the processing system described in Example 1 above, to further verify the broad applicability of this method, this example adds amide compounds commonly used as eluents to the eluent to obtain a composite eluent. The content of the amide compound in the eluent can be 1-30 w / w%, for example, 5 w / w%, 10 w / w%, 15 w / w%, or 25 w / w.

[0060] This embodiment provides the following elution system: 1) Single-component eluent: Only 25% tetraphenylphosphonium chloride (Aladdin, catalog number: T162792-5g), 25% methyltriphenylphosphonium chloride (Aladdin, catalog number: M102707-5g), and 50% tetrabutylphosphonium chloride (Aladdin, catalog number: T101355-5g) were used as eluents (all are mass-volume percentage concentrations); 2) Composite component system: Four different composite eluents were prepared using methyltriphenylphosphonium bromide and amides: Composite eluent 1: 50 w / v% methyltriphenylphosphonium bromide + 25 w / w% nicotinamide (Aladdin, catalog number: 98-92-0); Composite eluent 2: 50 w / v% methyltriphenylphosphonium bromide + 25 w / w% formamide; Composite eluent 3: 50 w / v% methyltriphenylphosphonium bromide + 25 w / w% isonicotinamide (Aladdin, catalog number: 1453-82-3); Compound eluent 4: 50 w / v% methyltriphenylphosphonium bromide + 25 w / w% acetamide (Aladdin, catalog number: 60-35-5).

[0061] The specific processing method is as follows: The samples to be eluted are placed in solutions containing a single elution buffer or a combination elution buffer and incubated at 37°C for 15 min in the dark; then rinsed twice with PBS buffer for 5 min each time, and the residual HCR fluorescence signal intensity is detected by fluorescence microscopy and image analysis system.

[0062] Figure 4 As shown, no obvious specific fluorescence signal was observed in the cerebral cortex region of any of the groups (including single-component eluents and four compound eluents).

[0063] The above results demonstrate that methyltriphenylphosphonium bromide exhibits good compatibility with nicotinamide, formamide, isonicotinamide, and acetamide. Regardless of the addition of these amide auxiliaries, near-complete elution of the HCR fluorescence signal can be achieved, validating the broad applicability of this elution method in terms of component selection and the stability of the results.

[0064] Furthermore, this invention also selected propyltriphenylphosphonium bromide, cyclopropyltriphenylphosphonium bromide, heptapropyltriphenylphosphonium bromide, benzyltriethylphosphonium bromide, hydroxymethyltriphenylphosphonium bromide, bromomethyltriphenylphosphonium bromide, ethyltriphenylphosphonium iodide, tributylhexylphosphonium bromide, and allyltributylphosphonium bromide as eluent components. The results showed that all of the above-mentioned quaternary phosphonium salts could effectively elute HCR fluorescence signals. For quaternary phosphonium salts that are not easily soluble in water, cationic or nonionic surfactants can be added to improve their solubility. Example 3 Comparison of elution effects of different eluents on HCR signal

[0065] To compare the technical performance of the eluent described in this invention with existing conventional eluents, this embodiment conducts parallel tests with several conventional eluents as comparisons (elution conditions: incubation at 37°C for 15 min in the dark): The eluent of this invention is 50 w / v% methyltriphenylphosphonium bromide; The standard eluents were 10 v / v% Triton X-100 (SIGMA, catalog number: T8787), 10 v / v% Tween-20, 8 w / v% sodium dodecyl sulfate SDS (Aladdin, catalog number: S108349), and 50 w / v% choline chloride (Aladdin, catalog number: C108896-25g) (all analytical grade, with sterile, enzyme-free water as the solvent).

[0066] Figure 5 As shown, after treatment with conventional eluents, HCR-specific fluorescent spots are still widely distributed in the field of view, indicating that conventional eluents cannot wash away the specific fluorescent signal; while after treatment with the eluent of the present invention, no HCR-specific fluorescent signal is identifiable in the field of view.

[0067] This embodiment demonstrates through comparative experiments that conventional eluents in the prior art (such as detergents and strong ionic agents) cannot effectively remove the stable hybridization signal formed by HCR, while the methyltriphenylphosphonium bromide reagent described in this invention exhibits "complete elution" capability, providing a key solution that existing technologies cannot solve for cyclic detection or multiple rounds of reuse in HCR technology. In addition to choline chloride, this invention also selected other quaternary ammonium salts such as tetrabutylammonium chloride and trimethylphenylammonium chloride as eluents. The results showed that, under the same conditions, although the elution effect of these quaternary ammonium salts was better than the aforementioned conventional eluents, it still lagged behind the quaternary phosphonium salt eluent of this invention. Example 4: Confirmation of target sites after one round of HCR mRNA staining

[0068] To verify that the target of the substance removed by the elution operation after the first round of standard in situ hybridization staining of HCR mRNA in mouse cerebral cortex tissue is a primary probe, and to clarify the elution target by combining the experimental phenomenon that "the secondary probe directly amplifies after elution and shows negative and no staining signal".

[0069] 1. Elution treatment 100 μm sections of mouse cerebral cortex that have undergone standard first-round staining were placed in a hybridization cassette and immersed in elution buffer preheated to 37°C (elution buffer containing 50 w / v% methyltriphenylphosphonium bromide, or a combined elution buffer containing 50 w / v% methyltriphenylphosphonium bromide and 20 w / w% formamide). Elution was carried out at 37°C for 30 min to ensure sufficient contact between the elution buffer and the cerebral cortex tissue, allowing potential elution targets to fully dissociate from the target mRNA. After elution, the tissue was washed three times with RNase-free PBS buffer for 5 min each time to remove residual elution buffer and dissociated material, and fluorescence images were acquired. 2. Target elution validation (direct secondary probe amplification detection)

[0070] Based on the principle of HCR hybridization (secondary probes can only specifically bind to primary probes and cannot directly bind to target mRNA in the mouse cerebral cortex), to verify that the elution operation removes the primary probe, the eluted group was directly subjected to secondary probe amplification treatment. The residual status of the primary probe was determined by the difference in staining signal. The specific operation is as follows: After the brain slices that have completed the first round of standard staining are washed with PBS, hybridization buffer II containing HCR fluorescent amplification probes are added and incubated at room temperature in the dark for 12 h. After incubation, the slices are washed 3 times at room temperature with washing buffer for 5 min each time, and then washed 2 times at room temperature with PBS buffer for 5 min each time to complete the HCR secondary probe staining process and acquire fluorescence images.

[0071] 3. Fluorescence Imaging Analysis The imaging analysis results are shown in Figure 6: 1) Before elution: After HCR amplification, mouse cerebral cortex slices showed dense and high-intensity fluorescent signals, indicating that the probe had successfully hybridized and amplified.

[0072] 2) After elution: After treatment with the eluent described in this invention, the background of the field of view is dark and there is no obvious residual fluorescence, which proves that the eluent has a highly efficient and thorough removal ability.

[0073] 3) Counterstaining of secondary probes after elution: When using a single-component elution buffer, fluorescence signals occasionally appeared. Further research results showed that under the elution conditions described in this example, the primary probe was eluted from the target but may have formed non-specific bindings at other sites in the tissue, failing to be completely eluted. Increasing the elution time (e.g., 40 or 50 minutes) or eluting twice eliminated the fluorescence signal. When using a combined elution buffer, no significant fluorescence signal was observed in the field of view under the elution conditions described in this example.

[0074] The above results confirm that the elution solution of the present invention causes the primary probe to dissociate from the target mRNA in the mouse cerebral cortex and be completely removed, resulting in the secondary probe having no binding site and being unable to initiate the amplification reaction. The elution effect of the composite elution solution on the primary probe is better than that of the single-component elution solution. Example 5: Validation of Sample Elution-Rehybridization Multiplexing for Adapted Multi-Round HCR Detection

[0075] The difference between this embodiment and Example 4 is that: during the HCR hybridization reaction, target nucleic acid detection, DAPI nuclear staining, and oligo (dT) total mRNA staining were performed simultaneously on the samples; after the first round of staining, the samples were treated with three elution methods: 15 U / mL DNase I, 50 w / w% formamide, and a combined elution buffer (50 w / v% methyltriphenylphosphonium bromide + 20 w / w% formamide), all under the condition of incubation at 37°C for 30 min; after elution, the samples were washed three times with PBS for 10 min each time. The samples were then counterstained again for target nucleic acid, DAPI nuclear staining, and oligo (dT) to evaluate the effects of different elution methods on counterstaining efficiency, fluorescence imaging quality, and signal retention.

[0076] based on Figure 7 , Figure 8 , Figure 9 Fluorescence imaging results showed: 1) After elution, the composite eluent eluted more thoroughly, and the original fluorescence signal was basically removed; the 50 w / w% formamide was not eluted cleanly, and there was fluorescence residue, and the background was not completely removed; DNaseI was eluted cleanly, and there was no obvious residual signal.

[0077] 2) Re-dyeing effect: Samples eluted and counterstained with DNase I: The quality of fluorescence imaging after counterstaining was generally poor. The DAPI signal in the cell nucleus was weak, the nuclear outline was not clear enough, and it was slightly deformed. The signal intensity of oligo (dT) was insufficient, the signal recovery after counterstaining was generally poor, and some non-target areas had residual weak fluorescence, resulting in unsatisfactory results.

[0078] Samples eluted with 50% formamide and then counterstained: After counterstaining, the background fluorescence is high, the DAPI staining is blurred, the oligo (dT) signal is partially diffuse, the target signal is weakened, and the imaging clarity is low.

[0079] Samples eluted and counterstained with the composite elution buffer of this embodiment: The fluorescence imaging quality after counterstaining is excellent, with complete and clear DAPI staining outlines; oligo (dT) exhibits specific punctate fluorescence with a clean background; the target signal is well preserved and the signal intensity is stable.

[0080] In summary, the composite elution solution outperforms the 50 w / w% formamide elution group and the DNaseI elution group in terms of overall counterstaining effect, signal specificity, and imaging quality. Therefore, it can provide an efficient, mild, and highly compatible HCR signal elution solution. Example 6: Validation of continuous elution-counterstained detection in multiple rounds of HCR-in situ hybridization

[0081] This embodiment is based on the HCR hybridization-elution-counterstaining technology system established in Examples 1-5 above. Using the composite elution buffer of this invention (50 w / v% methyltriphenylphosphonium bromide + 20 w / w% formamide), multiple rounds of continuous targeted detection were carried out on 100 μm mouse brain slices to verify the feasibility, signal stability and spatial localization consistency of the elution-counterstaining system for multi-round cyclic HCR-fluorescence in situ hybridization. The HCR hybridization process for each round was the same as in Example 1, and the elution condition was incubation at 37°C for 30 min.

[0082] This embodiment sequentially performs four independent rounds of HCR-in situ hybridization and target staining, elution, and counterstaining procedures, and then detects the following: Round 1: Foxp2, Satb2; Round 2: Fezf2, Slc17a7; Round 3: VIP, Pvalb; Round 4: Npy, Gad1; Each round of hybridization simultaneously completes the target mRNA-specific fluorescent labeling. After the fluorescence signal from the previous round is completely removed by the compound elution buffer, the next round of HCR hybridization and staining is carried out without damaging the brain slice tissue structure and cell spatial position. Finally, the fluorescence signals from the four rounds of detection are spatially registered and superimposed to achieve visualization analysis of multi-gene co-localization.

[0083] Elution-counterstaining effect and imaging results are as follows: Figure 10 : The targets Foxp2, Satb2, Fezf2, Slc17a7, Vip, Pvalb, Npy, and Gad1, detected sequentially in four rounds, all exhibited clear and sharp specific punctate fluorescence signals; there was no signal attenuation issue caused by multiple rounds of cycling. After superimposing and integrating the fluorescence signals from the four rounds (top image), the cellular spatial distribution and co-expression patterns of the eight target genes in mouse brain slices can be clearly displayed; the locations of positive cells detected in different rounds are precisely matched, with no cell displacement or localization deviation, achieving the construction of a multi-gene in situ atlas at the single-cell level.

[0084] As verified by this embodiment, the composite elution-counterstaining system provided by the present invention can support at least 4 or more consecutive HCR-in situ hybridization cycles. It has the advantages of thorough elution, controllable background, minimal sample damage, stable signal over multiple cycles, and accurate spatial localization. It solves the technical defects of traditional formamide and DNaseI elution systems that cannot perform multi-cycle detection well, and provides a stable and reproducible technical solution for the analysis of multi-gene in situ maps of single cells in brain tissue.

[0085] Those skilled in the art will understand that, in order to improve the stability, solubility, and other properties of quaternary phosphonium salts, the eluent may also contain various additives, such as buffer salts, surfactants, and preservatives. Buffer salts may be selected from one or more of phosphates, Tris-HCl, bicarbonates, borates, MES, and HEPES. Surfactants may be cationic or nonionic surfactants. For quaternary phosphonium salts that are not readily soluble in water, the addition of surfactants can improve their solubility. The preservatives are selected from one or more of sodium azide and Proclin 300. The properties of these additives are well known to those skilled in the art and will not be described in detail here.

[0086] The elution buffer in this invention can be prepared as a kit for nucleic acid detection or nucleic acid imaging, either alone or together with these auxiliary agents, nucleic acid probes, hybridization buffers, and other components. These components can be stored in several separate reagent bottles.

[0087] The method of this invention solves the problems of low signal removal efficiency, poor tissue penetration, and poor nucleic acid retention in existing multi-round nucleic acid staining techniques, and provides a powerful tool for fields such as multi-round in situ hybridization, three-dimensional spatial transcriptomics analysis, and pathological diagnosis.

[0088] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for elution of a nucleic acid probe in a biological sample, characterized by, Includes the following steps: (1) Obtain biological samples containing nucleic acid probes. (2) Immerse the biological sample in an elution solution containing quaternary phosphonium salt to elute the nucleic acid probe.

2. The elution method of claim 1, wherein, The elution time in step (2) is more than 10 minutes.

3. The elution method of claim 1, wherein, The concentration of quaternary phosphonium salt in the eluent is greater than 25 w / v.

4. The elution method of claim 1, wherein, The elution temperature in step (2) is 4-65℃.

5. The elution method of claim 1, wherein, The eluent further contains amide compounds.

6. The elution method of claim 5, wherein, The amide compounds are selected from formamide, acetamide, acrylamide, N,N-dimethylformamide, N,N-dimethylacetamide, benzamide, N,N-dimethylbenzamide, phenylacetamide, pyridineamide, nicotinamide, isonicotinamide, N-methylnicotinamide, or combinations thereof.

7. The elution method of claim 5, wherein, The concentration of amide compounds in the eluent is 1-35 w / w.

8. The elution method of claim 1, wherein, The biological sample is a cell or tissue.

9. A kit for multi-round in situ hybridization, characterized by, It includes nucleic acid probes that specifically recognize target nucleic acids and elution buffers containing quaternary phosphonium salts.

10. The kit according to claim 9, wherein, The concentration of quaternary phosphonium salt in the eluent is greater than 25 w / v.

11. The kit according to claim 9, wherein, The eluent further contains amide compounds.

12. The kit according to claim 11, wherein, The amide compounds are selected from formamide, acetamide, acrylamide, N,N-dimethylformamide, N,N-dimethylacetamide, benzamide, N,N-dimethylbenzamide, phenylacetamide, pyridineamide, nicotinamide, isonicotinamide, N-methylnicotinamide, or combinations thereof.

13. The kit according to claim 11, wherein, The concentration of amide compounds in the eluent is 1-35 w / w.

14. The use of the kit according to any one of claims 9-13 in nucleic acid fluorescence detection or nucleic acid fluorescence imaging for non-diagnostic purposes.

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