Preparation method of biological sample for diamond near-surface quantum sensing

Abstract

The invention provides a preparation method of a biological sample for diamond near-surface quantum sensing, and belongs to the field of quantum sensing detection. The method comprises the steps that a single-layer DNA origami structure is prepared, the single-layer DNA origami structure is provided with a pre-designed biological sample binding site, and the biological sample binding site is coupled with a biological sample through an interaction molecule pair; after the surface of the diamond is subjected to planarization pretreatment through plasma etching, nitrogen ions are injected to form an NV color center, and the diamond containing the NV color center is obtained; in a buffer solution environment containing cations, a solution containing the single-layer DNA origami structure and a to-be-detected biological sample solution are sequentially added to diamond for incubation, and the biological sample for diamond near-surface quantum sensing is obtained. The sample preparation method provides a reliable and efficient sample preparation basis for high-sensitivity and high-specificity single molecule / single particle biological sample detection based on diamond near-surface quantum sensing.

Description

Technical Field

[0001] This application relates to the field of quantum sensing and detection, and more particularly to a method for preparing biological samples for near-surface quantum sensing of diamond. Background Technology

[0002] Quantum sensing technology based on diamond nitrogen-vacancy (NV) centers possesses characteristics such as single-molecule / single-spin sensitivity, nanoscale spatial resolution, low background noise, strong material resolution, and good biocompatibility under room temperature and atmospheric conditions. It is particularly suitable for high-sensitivity, high-resolution, and high-specificity detection and research in biomedical systems. This technology has already achieved the detection of multiple physical quantities such as magnetism, temperature, and charge at the single-molecule, single-cell, and tissue scales, demonstrating enormous application potential. As an emerging magnetic resonance imaging technique, NV center technology advances the detection targets from billions of molecules to the single-molecule scale and improves imaging resolution to the nanometer level. Combined with the inherent specificity of magnetic signals, it has become an ideal tool for conducting single-molecule and nanoscale research, opening up new avenues for the structural and functional study of biomacromolecules and biomagnetic materials.

[0003] However, existing sample preparation methods for achieving single-molecule magnetic resonance detection typically rely on randomly spreading or chemically cross-linking analyte molecules onto a diamond surface. This approach only indirectly controls the spacing by adjusting the total number of molecules, resulting in significant randomness. This method not only suffers from poor sample preparation controllability but also low single-molecule detection efficiency, hindering the further promotion and application of this technology. Therefore, developing a controllable and efficient single-molecule preparation strategy is of great significance. Summary of the Invention

[0004] In view of this, in order to at least partially solve at least one of the aforementioned technical problems, this application provides a method for preparing a biological sample for near-surface quantum sensing of diamond, comprising the following steps S1 to S3.

[0005] Step S1: Prepare a monolayer DNA origami structure. The monolayer DNA origami structure has pre-designed biological sample binding sites, which are coupled to biological samples through interacting molecule pairs.

[0006] Step S2: After planarizing the diamond surface using plasma etching, nitrogen ions are injected to form NV color centers, resulting in diamond containing NV color centers.

[0007] Step S3: In a cation-containing buffer environment, the solution containing the monolayer DNA origami structure and the biological sample solution to be tested are added to the diamond and incubated to fix the biological sample at the biological sample binding site, thereby obtaining a biological sample for diamond near-surface quantum sensing.

[0008] According to the implementation scheme of this application, by employing a monolayer DNA origami structure with pre-designed biological sample binding sites, combined with plasma etching planarization pretreatment of the diamond surface, and incubation and fixation in a cation-containing buffer environment, the DNA origami structure is more complete and denser when bound to the diamond surface. This enables the controllable, orderly, and high-density fixation of biological samples on the near-surface of diamond with nanometer-level precision, providing a reliable and efficient sample preparation basis for highly sensitive and specific single-molecule / single-particle detection based on near-surface quantum sensing of diamond. Attached Figure Description

[0009] Figure 1 This is a schematic diagram of the process for preparing biological samples for near-surface quantum sensing of diamond, as described in this application.

[0010] Figure 2A This is a schematic diagram of the triangular biotin-streptavidin interaction monolayer DNA origami structure of this application.

[0011] Figure 2B This is a schematic diagram of the rectangular biotin-streptavidin interaction monolayer DNA origami structure of this application.

[0012] Figure 3A This is a schematic diagram of the triangular DNA complementary strand monolayer DNA origami structure of this application.

[0013] Figure 3B This is a schematic diagram of the rectangular DNA complementary strand monolayer DNA origami structure of this application.

[0014] Figure 4 This is an atomic force microscopy (AMF) characterization image of the biotin-streptavidin interaction monolayer DNA origami prepared onto a diamond surface in Example 1 of this application.

[0015] Figure 5 This is a schematic diagram illustrating the fabrication of a molecular / spin array onto a diamond surface using DNA origami technology, as described in this application.

[0016] Figure 6 This is an atomic force microscopy characterization of a single-layer DNA origami with a rectangular structure of this application bound to a diamond surface with a second cation buffer containing 1 mM nickel ions.

[0017] Figure 7 This is an atomic force microscopy characterization of the biotin-streptavidin-interacting monolayer DNA origami coupled with ferritin molecules on a diamond surface, as described in Example 1 of this application.

[0018] The meanings of the markings in the attached diagram are as follows:

[0019] 1. Diamond quantum sensor; 2. NV color center near the surface of diamond; 3. Triangular single-layer DNA origami structure; 4. Rectangular single-layer DNA origami structure; 5. Biotin; 6. Streptavidin; 7. Biomolecule / nanoparticle to be measured; 8. Short DNA strand extending from the end of the staple DNA strand; 9. DNA strand complementary to the extended portion of the staple DNA strand. Detailed Implementation

[0020] The embodiments of this application will now be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of this application. In the following detailed description, numerous specific details are set forth to provide a thorough understanding of the embodiments of this application for ease of explanation. However, it will be apparent that one or more embodiments may be implemented without these specific details. Furthermore, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessarily obscuring the concepts of this application.

[0021] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. The term "comprising" as used herein indicates the presence of features, steps, or operations, but does not exclude the presence or addition of one or more other features.

[0022] When using expressions such as "at least one of A, B, and C," they should generally be interpreted in accordance with the meaning commonly understood by a person skilled in the art.

[0023] In this application, the term "DNA origami structure" refers to a structure obtained through DNA origami technology, which is a technique that uses DNA molecules as materials and precisely designs base sequences to enable them to self-assemble into a pre-defined two-dimensional or three-dimensional nanostructure.

[0024] Figure 1 This is a schematic diagram of the process for preparing biological samples for near-surface quantum sensing of diamond, as described in this application.

[0025] Specifically, according to one embodiment of this application, a method for preparing a biological sample for near-surface quantum sensing of diamond is provided, such as... Figure 1 As shown, it includes the following steps S1 to S3.

[0026] Step S1: Prepare a monolayer DNA origami structure. The monolayer DNA origami structure has pre-designed biological sample binding sites, which are coupled to biological samples through interacting molecule pairs.

[0027] Step S2: After planarizing the diamond surface using plasma etching, nitrogen ions are injected to form NV color centers, resulting in diamond containing NV color centers.

[0028] Step S3: In a cation-containing buffer environment, the solution containing the monolayer DNA origami structure and the biological sample solution to be tested are added to the diamond and incubated to fix the biological sample at the biological sample binding site, thereby obtaining a biological sample for diamond near-surface quantum sensing.

[0029] According to the implementation scheme of this application, by employing a monolayer DNA origami structure with pre-designed biological sample binding sites, combined with plasma etching planarization pretreatment of the diamond surface, and incubation and fixation in a cation-containing buffer environment, the DNA origami structure is more complete and denser when bound to the diamond surface. This enables the controllable, orderly, and high-density fixation of biological samples on the near-surface of diamond with nanometer-level precision, providing a reliable and efficient sample preparation basis for highly sensitive and specific single-molecule / single-particle detection based on near-surface quantum sensing of diamond.

[0030] In one or more embodiments of this application, the biological sample includes biological macromolecules and nanoparticles, such as proteins, nucleic acids, magnetic nanoparticles, other functionalized nanoparticles, etc.

[0031] According to the embodiments of this application, in step S1, the interacting molecule pairs are coupled through biotin-streptavidin or base complementary pairing.

[0032] According to the implementation scheme of this application, the biotin-streptavidin interaction has a high affinity, while the complementary base pairing provides a highly programmable sequence specificity. Both can ensure that the biomolecule or nanoparticle to be tested is precisely anchored at a pre-designed position on the DNA origami structure. By introducing the biotin-streptavidin or complementary base pairing biomolecule recognition and binding mechanism, the nanometer-level precision controllable and orderly fixation of biological samples at preset sites on a single layer of DNA origami is achieved.

[0033] According to the implementation scheme of this application, in step S1, the distance between the binding sites of the biological sample is 10~60nm.

[0034] According to the embodiments of this application, the distance between binding sites of biological samples can be 10nm, 20nm, 30nm, 40nm, 50nm, or 60nm, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be within the range of any two values, such as 10~20nm, 15~25nm, etc.

[0035] According to the implementation scheme of this application, by reasonably setting the spatial arrangement spacing of biological sample binding sites, the array formed by the biological samples fixed on DNA origami is matched with the detection characteristics of the near-surface NV centers of diamond (the typical detection range of NV centers is 10-20 nm). The upper limit of the distance range ensures that a single NV center has a high probability of detecting a signal, thus ensuring the detectability of the signal; the lower limit of the distance range can effectively avoid the superposition interference of signals from multiple neighboring sites on a single NV center, ensuring the singularity and clarity of the detection signal source. This allows the biological sample array obtained based on this sample preparation method to improve the overall detection efficiency by increasing the site density, and to ensure that a high signal-to-noise ratio single molecule / single particle signal is obtained at each detection point. Thus, the resolution and reliability of subsequent near-surface diamond quantum sensing measurements are directly optimized and guaranteed in the spatial arrangement dimension.

[0036] According to the implementation scheme of this application, in step S1, the monolayer DNA origami can expose the preset biological sample binding sites more fully and consistently, thereby achieving more efficient and reliable binding with the biological sample to be tested both spatially and biologically. The monolayer DNA origami structure can also reduce the spatial steric hindrance brought by the DNA origami structure itself, allowing the biological sample (such as protein, magnetic nanoparticle) fixed on it to be closer to the diamond surface and enter the optimal detection range (10~20nm) of the near-surface NV color center, thereby enhancing the signal strength and detection sensitivity of quantum sensing. At the biological binding level, all binding sites designed on the monolayer origami are located on the same exposed two-dimensional plane, with a more regular structure, ensuring that each site has consistent reactivity, realizing the fixation of biological samples on the diamond surface with nanometer-level precision, thereby improving the detection sensitivity and accuracy of single-molecule quantum sensing.

[0037] In one or more embodiments of this application, the monolayer DNA origami structure is a rectangular or triangular structure, such as... Figure 2A and Figure 2B ,or Figure 3A and Figure 3B As shown.

[0038] Figure 2A This is a schematic diagram of the triangular biotin-streptavidin interaction monolayer DNA origami structure of this application; Figure 2B This is a schematic diagram of the rectangular biotin-streptavidin interaction monolayer DNA origami structure of this application.

[0039] Figure 3A This is a schematic diagram of the triangular DNA complementary strand monolayer DNA origami structure of this application; Figure 3B This is a schematic diagram of the rectangular DNA complementary strand monolayer DNA origami structure of this application.

[0040] In one or more embodiments of this application, the rectangular monolayer DNA origami structure exhibits excellent spreadability on the planarized diamond surface. Its high theoretical occupancy is beneficial for arranging more biological sample binding sites within a unit area, thereby increasing the sample throughput of subsequent quantum sensing measurements. The triangular monolayer DNA origami structure, on the other hand, possesses excellent geometric stability and can achieve a more complete and regular binding morphology on the planarized diamond surface. This further ensures the consistency and reliability of the spatial position of each structural unit and the preset biological sample binding site, providing a reproducible basis for single-molecule / single-particle detection. The rectangular and triangular monolayer DNA origami structures achieve controllable fixation of biological samples on the near-surface of diamond with both high density and high order, thereby enhancing the practicality and controllability of the biological sample preparation method as a whole.

[0041] In one or more embodiments of this application, the monolayer DNA origami structure further includes a stapled DNA strand containing a fluorescent tag, the stapled DNA strand containing the fluorescent tag being used to identify the monolayer DNA origami structure.

[0042] For example, a stapled DNA strand containing a fluorescent tag can be placed near the center of a triangular DNA origami structure.

[0043] For example, stapled DNA strands with fluorescent tags are placed at each of the four corners of a rectangular DNA origami structure.

[0044] In one or more embodiments of this application, by introducing fluorescent markers into the staple chains of DNA origami, a clear optical identifier is provided for the monolayer DNA origami structure on the diamond surface of the NV color center. This enables the rapid and non-destructive localization and identification of the specific distribution area of ​​the target DNA origami structure using conventional fluorescence microscopy imaging techniques in subsequent measurement steps such as optically detected magnetic resonance (ODMR) or scanning detection based on diamond quantum sensors. This simplifies the process of finding specific targets on complex sample surfaces and avoids blind, large-scale scanning searches in the early stages of atomic force microscopy characterization or quantum sensing measurement. As a result, the localization efficiency and overall measurement throughput of the entire process from sample preparation to final signal detection are improved.

[0045] According to the embodiments of this application, step S2 further includes cleaning the diamond with a strong oxidizing solution, wherein the strong oxidizing solution is a piranha solution or a tri-acid mixture. The piranha solution is a mixture of 95%~98% by mass concentrated sulfuric acid and 30% by mass hydrogen peroxide in a volume ratio of 3:1. The tri-acid mixture is a mixture of 95%~98% by mass concentrated sulfuric acid, 70%~72% by mass perchloric acid, and 65%~68% by mass concentrated nitric acid in a volume ratio of 1:1:1.

[0046] In one or more embodiments of this application, in step S2, the diamond is cleaned with a strong oxidizing solution by boiling at 100~280°C for 2~4 hours.

[0047] In one or more embodiments of this application, cleaning diamond with piranha solution or a mixed solution of concentrated sulfuric acid, perchloric acid, and concentrated nitric acid can effectively remove organic pollutants, particulate matter, metallic impurities, and amorphous carbon layers that may remain on the diamond surface during pre-processing such as plasma etching, ion implantation, and annealing, as well as during the use of diamond. This results in a clean and highly active hydrophilic surface, which serves as a prerequisite for the subsequent stable and uniform adsorption of monolayer DNA origami, and reduces non-specific adsorption.

[0048] In one or more embodiments of this application, step S2 further includes carboxylation or amination of the cleaned diamond.

[0049] In one or more embodiments of this application, carboxylation of diamond can make the diamond surface negatively charged, while amination can make the diamond surface positively charged, thereby improving the adsorption efficiency of negatively charged DNA origami on the surface through electrostatic interaction.

[0050] Furthermore, carboxylation or amination of the cleaned diamond can combine with the cationic buffer environment to form a synergistic effect. That is, through the combined action of specific surface charges and cations in the buffer, the binding strength and uniformity between the DNA origami and the diamond surface can be optimized. This further ensures that the monolayer DNA origami structure can be completely, flatly and firmly fixed to the near surface, laying a solid and controllable interfacial chemical foundation for the nano-precision ordered array fixation of biological samples at preset binding sites.

[0051] According to the embodiments of this application, in step S3, the cation buffer includes a first cation solution and a second cation solution; the first cation solution is a 10-15 mM magnesium ion solution; the second cation solution includes at least one of 0.5-5 mM nickel ions, 5-250 mM magnesium ions, 100-400 mM sodium ions, 100-400 mM potassium ions, and 2-10 mM calcium ions.

[0052] According to the embodiments of this application, the concentration of magnesium ion solution in the first cation solution can be 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, or 15 mM, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be within the range of any two values, such as 10–13 mM, 13–14 mM, etc.

[0053] According to the embodiments of this application, the concentration of nickel ions in the second cation solution can be 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, or 5 mM, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be within the range of any two values, such as 1–3 mM, 2–4 mM, etc.

[0054] According to the embodiments of this application, the concentration of magnesium ions in the second cation solution can be 5 mM, 50 mM, 150 mM, or 250 mM, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be within the range of any two values, such as 5~50 mM, 150~250 mM, etc.

[0055] According to the embodiments of this application, the concentration of sodium ions in the second cation solution can be 100 mM, 200 mM, 300 mM, or 400 mM, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be within the range of any two values, such as 100–300 mM, 200–400 mM, etc.

[0056] According to the embodiments of this application, the concentration of potassium ions in the second cation solution can be 100 mM, 200 mM, 300 mM, or 400 mM, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be within the range of any two values, such as 100–300 mM, 200–400 mM, etc.

[0057] According to the embodiments of this application, the concentration of calcium ions in the second cation solution can be 2 mM, 4 mM, 6 mM, 8 mM, or 10 mM, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be within the range of any two values, such as 2–5 mM, 7–10 mM, etc.

[0058] According to the implementation scheme of this application, the first cation solution neutralizes the negatively charged phosphate groups on the DNA backbone with magnesium ions, maintaining the structural stability of the DNA origami and promoting its initial electrostatic attraction with the negatively charged diamond surface. The second cation solution can enhance the binding force between the DNA origami and the diamond surface that has undergone different chemical modifications (such as carboxylation, amination, or no modification) by introducing divalent cations with high charge density such as nickel ions or adjusting the total concentration and ratio of monovalent and divalent cations during the incubation and fixation stages. This can more effectively shield the diamond surface charge and form ion bridges, thereby ensuring that the monolayer DNA origami structure can be laid flat and fixed on the near-surface of the diamond with high fidelity, high binding strength, and high density, avoiding detachment during subsequent cleaning or measurement. This lays a reliable interface foundation for the final precise positioning of biological samples at preset sites and subsequent high-sensitivity quantum sensing measurements.

[0059] According to the embodiments of this application, it also includes magnetic resonance labeling of the biological sample to be tested, as illustrated in the example below. Figure 5 As shown. Figure 5 This is a schematic diagram illustrating the fabrication of a molecular / spin array onto a diamond surface using DNA origami technology, as described in this application.

[0060] Specifically, for example, in cases where the biological sample to be tested includes proteins and / or nucleic acids, an electron spin tag is introduced into the biological sample to be tested through site-specific modification.

[0061] Specifically, for example, when the biological sample to be tested includes proteins and / or nucleic acids, isotopic nuclear spin tags are introduced during protein expression and / or nucleic acid synthesis.

[0062] According to the implementation scheme of this application, for some biomolecules that do not possess strong magnetic or magnetic resonance signals (such as most proteins and nucleic acids), biological samples that are fixed to the near-surface of diamond by the above preparation method can generate a clear magnetic signal that can be detected by NV color centers, thereby transforming the advantages of precise positioning and orderly arrangement into practical and effective single-molecule magnetic resonance detection capabilities.

[0063] Furthermore, by site-specific modification of proteins or nucleic acids to introduce electron spin tags (such as paramagnetic metal ions or nitrile radicals), or by introducing isotopic nuclear spin tags during protein expression / nucleic acid synthesis, these two highly specific and universal labeling strategies—modifying electron spin tags and introducing isotopic nuclear spin tags—can be adapted to the needs of different biological sample characteristics and different quantum sensing detection modes.

[0064] The following specific embodiments will further explain the solution of this application. Unless otherwise specified, all reagents used are commercially available, and all test or experimental methods used are conventional experimental methods in the art. Furthermore, unless otherwise specified, "parts" and "%" refer to mass measurements.

[0065] Example 1

[0066] Designing single-molecule / single-particle binding sites on origami: Biotin-streptavidin binding sites are designed near the midpoint of the three outer edges of a triangular, single-layer DNA origami structure 3. For example... Figure 2A As shown, biotinylated stapled DNA strands were used at these sites, and then streptavidin 6 was used as a bridge to bind biotinylated biomolecules or nanoparticles 7 to a triangular monolayer DNA origami structure 3.

[0067] To prepare triangular DNA origami samples approximately 100 nanometers in size: 1. Mix the template long-chain DNA solution, the staple short-chain DNA solution, and the buffer solution. The concentration of the template long-chain DNA in this mixture is 10 nM, the concentration of the staple short-chain DNA solution is 100 nM, and the buffer solution is a TAE solution with an additional 12.5 mM magnesium chloride. 2. In a PCR instrument, slowly anneal the mixture by gradually decreasing the temperature from 90℃ to 4℃ at a rate of -0.5℃ / 30s to achieve accurate complementary pairing of DNA strands. 3. Filter the annealed solution sample using a 100 kDa ultrafiltration tube to remove unbound staple DNA. Centrifuge at 2500g for 10 min, repeating this centrifugation step twice.

[0068] To bind streptavidin molecules to biotin-labeled sites on DNA origami: After preparing the DNA origami, add 1 μL of 0.6 mg / mL streptavidin solution to approximately 50 μL of DNA origami solution, react at room temperature for 1 h, and then centrifuge at 2500g for 10 min using a 100 kDa ultrafiltration tube to remove unbound streptavidin. This centrifugation step is performed twice.

[0069] Plasma etching, ion implantation, and cleaning of diamond: The diamond was placed in an inductively coupled plasma reactive ion etching (ICP) system (Plasma-Therm Versaline ICP RIE, Plasma-Therm, USA). The ICP power was set to 400 W, the substrate bias RF power to 250 W, the argon flow rate to 250 mL / min, the chlorine flow rate to 40 mL / min, and the pressure to 8 mTorr (1 Pa) for 30 minutes. Subsequently, a 700 W plasma power, a 100 W substrate bias RF power, an oxygen flow rate to 30 mL / min, and a pressure to 10 mTorr (1.3 Pa) were used for 25 minutes. These two steps etched approximately 2 μm and 4 μm of subsurface polishing damage layers, respectively. Vacuum annealing was performed in a tube furnace (Thermo Fisher Scientific, USA) to remove residual subsurface damage from the previous step. High-purity (>99.5%) alumina ceramic tubes were used, and the annealing was performed at a pressure between 1 × 10⁻⁶. -7 To (1×10) -5 (Pa) and 2×10 -6 To (3×10) -4 Between (Pa), proceed in the following order, starting from room temperature.

[0070] The temperature is raised to 100℃ at a constant rate over 1 hour, and then maintained at this temperature for 11 hours.

[0071] The temperature is raised to 400℃ at a constant rate over 4 hours, and then maintained at this temperature for 8 hours.

[0072] Raise the temperature to 800℃ at a constant rate over 6-12 hours, and maintain this temperature for 8 hours.

[0073] Raise the temperature to 1200℃ at a constant rate over 6-12 hours, and maintain this temperature for 2 hours.

[0074] Cool to room temperature.

[0075] Tri-acid cleaning to remove amorphous carbon: Mix 71.0% perchloric acid, 68.0% concentrated nitric acid, and 98.0% concentrated sulfuric acid in a volume ratio of 1:1:1 to obtain a tri-acid mixed solution. Then place the diamond into the solution and boil at 185°C for 4 hours or overnight.

[0076] For diamond samples, the surface was first etched to 6 micrometers using plasma etching to make the diamond smoother. Then, a high-energy nitrogen ion beam was used to bombard the diamond surface, introducing nitrogen atoms into the diamond lattice. This was followed by a process involving high-temperature annealing at 1000℃ to form diamond NV color centers. The implantation dose was 1×10⁻⁶.9 cm -2 The injected energy was 4 keV. The diamond was then immersed in a piranha solution and boiled at 185°C for 3 hours.

[0077] The test sample was prepared onto diamond: 2.5 μL of the prepared streptavidin-modified DNA origami sample solution was dropped onto the diamond surface, and after standing in a humid chamber for 5 min, it was washed four times with 20 μL of TAE buffer with an additional concentration of 12.5 mM magnesium chloride. Then, 20 μL of biotin-modified biomolecule or nanoparticle solution was dropped onto the diamond, and the mixture was reacted in a humid chamber for 20 min. For gas phase sample preparation, the diamond was washed three times with 20 μL of deionized water and dried with nitrogen gas before subsequent atomic force microscopy characterization and near-surface diamond detection under gas phase conditions. For liquid phase sample preparation, the sample was washed three times with 20 μL of TAE buffer with an additional concentration of 12.5 mM magnesium chloride, and then kept in this liquid environment until subsequent atomic force microscopy characterization and near-surface diamond detection under liquid phase conditions.

[0078] Atomic Force Microscopy (AFM) Characterization: Instrument model: BrukerDimension® Icon TM For gas phase characterization, a ScanAsyst-Air AFM probe and fast scan gas phase measurement mode were selected; for liquid phase characterization, an SNL-10 AFM probe and fast scan liquid phase measurement mode were selected. The results are as follows: Figure 4 , Figure 6 , Figure 7 As shown.

[0079] Figure 4 This is an atomic force microscopy characterization image of the biotin-streptavidin-interacting monolayer DNA origami prepared onto a diamond surface, as described in Example 1 of this application.

[0080] according to Figure 4 It can be seen that the triangular single-layer DNA origami structure 3 was successfully prepared on the surface of the diamond quantum sensor 1, and the streptavidin 6 site can be clearly seen. The regular shape and well-defined outline of the approximately triangular structure did not show large-area aggregation or serious overlap, which is conducive to achieving spatial separation of biological sample binding sites on the diamond surface, avoiding non-specific aggregation, and helping to ensure the independence of the signal source in subsequent single-molecule detection.

[0081] Figure 6 Atomic force microscopy characterization of the rectangular DNA origami structure prepared on a diamond surface with the addition of 1 mM nickel ions.

[0082] according to Figure 6 It can be seen that the rectangular monolayer DNA origami structure 4 was successfully prepared on the surface of the diamond quantum sensor 1, and the density is very high, almost 100% covering the diamond, which is beneficial to increase the density of the biological sample to be tested and improve the efficiency of subsequent diamond near-surface detection.

[0083] Figure 7 This is an atomic force microscopy characterization of the biotin-streptavidin-interacting monolayer DNA origami coupled with ferritin molecules on a diamond surface, as described in Example 1 of this application.

[0084] according to Figure 7 As can be seen, the triangular single-layer DNA origami structure 3 was successfully prepared on the surface of the diamond quantum sensor 1. The streptavidin 6 site and the biomolecule to be tested 7 (ferritin) can be clearly seen. Moreover, the ferritin to be tested did not show obvious aggregation, which ensured the orderliness and single-molecule nature of the signal source during subsequent detection.

[0085] Near-surface detection: The aforementioned diamond sample was placed on an optically probed magnetic resonance (ODMR) confocal experimental platform for near-surface detection.

[0086] Example 2

[0087] The same preparation method as in Example 1 was used, except that the binding sites of the biological samples were connected using complementary base pairing binding strands, such as... Figure 3A As shown, this eliminates the need for the step of binding streptavidin molecules to biotin-labeled sites on DNA origami.

[0088] Design appropriate complementary DNA binding sites (one at the midpoint of each outer side of a triangle, for a total of 3): The staple DNA strand at each site contains an extended 30 bases, and subsequent biomolecules or nanoparticles containing complementary DNA strands bind to the DNA origami through base complementarity pairing.

[0089] Prepare the sample on diamond: Add 2.5 μL of the prepared DNA origami sample solution with DNA strands extended at specific locations to the diamond surface. After standing in a humid chamber for 5 min, wash four times with 20 μL of TAE buffer with an additional concentration of 12.5 mM magnesium chloride. Then, add 20 μL of complementary DNA strand modified biomolecules or nanoparticles to the diamond and react in a humid chamber for 20 min. After that, depending on the actual use scenario, perform the following operations: wash the diamond 3-5 times with 5-20 μL of deionized water and dry it with nitrogen gas before subsequent characterization and detection in a gas phase environment; or wash three times with 20 μL of TAE buffer with an additional concentration of 12.5 mM magnesium chloride, and then keep the sample in this liquid environment until subsequent characterization and detection in a liquid phase environment.

[0090] Example 3

[0091] The same preparation method as in Example 1 was used, except for the following steps:

[0092] Designing molecular or spin arrays: such as Figure 5 As shown, by utilizing the programmable nature of DNA origami, 10×10 spin or molecular binding sites are designed on a rectangular monolayer DNA origami structure to form an array. The stapled DNA strands at the corresponding positions are extended by about 30 bases for binding to complementary DNA strands containing spin tags or to test biomolecules connected to complementary DNA strands.

[0093] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of this application. It should be understood that the above descriptions are merely specific embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for preparing biological samples for near-surface quantum sensing of diamond, characterized in that, include: Prepare a monolayer DNA origami structure, wherein the monolayer DNA origami structure has a pre-designed biological sample binding site, wherein the biological sample binding site is coupled to a biological sample through interacting molecule pairs; After planarizing the diamond surface using plasma etching, nitrogen ions are injected to form NV color centers, resulting in diamond containing NV color centers. In a cationic buffer environment, a solution containing the monolayer DNA origami structure and a biological sample solution to be tested are added sequentially to the diamond containing NV color centers for incubation, so as to fix the biological sample at the biological sample binding site and obtain a biological sample for diamond near-surface quantum sensing.

2. The method according to claim 1, characterized in that, The interacting molecular pairs are coupled through biotin-streptavidin or base complementary pairing.

3. The method according to claim 1, characterized in that, The distance between the binding sites of the biological samples is 10~60nm.

4. The method according to claim 1, characterized in that, The cation buffer solution includes a first cation solution and a second cation solution; The first cation solution is a 10-15 mM magnesium ion solution; The second cation solution includes at least one of the following: 0.5-5 mM nickel ions, 5-250 mM magnesium ions, 100-400 mM sodium ions, 100-400 mM potassium ions, and 2-10 mM calcium ions.

5. The method according to claim 1, characterized in that, The single-layer DNA origami structure is a rectangular or triangular structure.

6. The method according to claim 1, characterized in that, Also includes: The diamond containing the NV color center is cleaned using a strong oxidizing solution, wherein the strong oxidizing solution is a piranha solution or a mixture of three acids, the mixture of three acids comprising 95%~98% by mass of concentrated sulfuric acid, 70%~72% by mass of perchloric acid and 65%~68% by mass of concentrated nitric acid.

7. The method according to claim 6, characterized in that, Also includes: The cleaned diamond containing NV color centers is then subjected to carboxylation or amination treatment.

8. The method according to claim 1, characterized in that, The monolayer DNA origami structure also includes a stapled DNA strand containing a fluorescent tag, which is used to identify the monolayer DNA origami structure.

9. The method according to claim 1, characterized in that, Also includes: The biological sample to be tested was labeled with magnetic resonance.

10. The method according to claim 9, characterized in that, The magnetic resonance labeling method includes any of the following: (1) In the case that the biological sample to be tested includes proteins and / or nucleic acids, an electron spin tag is introduced into the biological sample to be tested through site-directed modification; (2) In the case where the biological sample to be tested includes proteins and / or nucleic acids, an isotopic nuclear spin tag is introduced during the expression of the protein and / or the synthesis of the nucleic acid.

Images