A protein sensor array, its preparation method and use

Abstract

The application provides a protein sensing array and a preparation method and application thereof, and the protein sensing array comprises a group of ENSaptamer elements, which is composed of a first DNA-SWCNTs hybrid molecule solution, a second DNA-SWCNTs hybrid molecule solution and a third DNA-SWCNTs hybrid molecule solution; the DNA-SWCNTs hybrid molecule is a single-handed carbon nanotube wound by a single-stranded DNA sequence; the DNA sequence in the first and second DNA-SWCNTs hybrid molecules is shown as SEQ ID NO:1, wherein the chiral index of the single-handed carbon nanotube is (7, 3) and (6, 5) respectively; the DNA sequence in the third DNA-SWCNTs hybrid molecule is shown as SEQ ID NO:2, and the chiral index of the single-handed carbon nanotube in the third DNA-SWCNTs hybrid molecule is (8, 3).

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biochemical sensing, and particularly relates to a protein sensing array and a preparation method and application thereof. BACKGROUND

[0002] Accurate identification of proteins is crucial for proteomics research, clinical diagnosis and biomedical research. Sensitive, convenient and accurate protein detection methods provide an important tool for the development of these fields. However, due to the structural diversity and complexity of the target analyte, protein detection is a challenging problem. At present, the most widely used protein detection method is enzyme-linked immunosorbent assay (ELISA). In this system, the capture antibody fixed on the surface binds to the antigen through the "lock and key" method, and another enzyme-coupled antibody reacts with a chromogenic substrate or a fluorescent substrate to produce a detectable signal. Although this method has high sensitivity, due to its high production cost, instability and defects in quantification, the application of this method is limited.

[0003] The "chemical nose" method provides an alternative solution for using a unique analyte / receptor binding detection method. In this strategy, a sensor array with selective receptors is used instead of "lock and key" specific recognition for analyte detection. In the strategy, the array is able to present chemical diversity in order to react differently to a variety of different analytes. In the past few years, this method has been widely used for the detection of a variety of analytes, including metal ions, volatile reagents, aromatic amines, amino acids, proteins and bacteria, etc.

[0004] CN110632149B discloses an electrochemical sensor for alpha-fetoprotein detection and a preparation method and application thereof. The electrochemical sensor takes a glassy carbon electrode as a substrate, the glassy carbon electrode is modified with a carbon nanotube-alpha-fetoprotein aptamer complex, the carbon nanotube is a carbon nanotube with active and inactive ends on the surface, the alpha-fetoprotein aptamer is connected to the active end of the carbon nanotube to form a carbon nanotube-alpha-fetoprotein aptamer complex; the glassy carbon electrode modified with the carbon nanotube-alpha-fetoprotein aptamer complex captures alpha-fetoprotein, thereby realizing the detection of alpha-fetoprotein.

[0005] CN103336112A discloses a detection method for human immunoglobulin E (hIgE) with high sensitivity, fast and accurate, which can realize fast determination, and the method is realized by constructing a carbon nanotube micro-cantilever beam biosensor. The biosensor comprises a support, a base material, a carbon nanotube, and a pickup circuit, and a layer of nucleic acid aptamer is further modified on the carbon nanotube. First, a detection probe containing hIgE nucleic acid aptamer is made on the carbon nanotube micro-cantilever beam, and during detection, the detection probe is placed in the sample to be detected, and the hIgE in the sample to be detected reacts specifically with the nucleic acid aptamer on the detection probe to form a complex and adhere to the micro-cantilever beam; the mass change of the complex on the micro-cantilever causes the deflection displacement or resonance frequency change of the micro-cantilever beam, and according to the relationship between the two changes and the positive correlation between the mass of the complex and the concentration of hIgE in the sample to be detected, the detection of hIgE is realized.

[0006] However, the existing carbon nanotube-based electrochemical sensor can only detect a limited type of protein, and therefore, it is of important application value to develop a carbon nanotube-based electrochemical sensor which can be widely used and detect a plurality of types of proteins. SUMMARY

[0007] In view of the deficiencies of the prior art, the purpose of the present application is to provide a protein sensing array and a preparation method and application thereof. The present application utilizes the non-specific weak interaction between carbon nanotubes and biological targets to obtain a better sensing probe group, and further establishes a pattern recognition strategy for a plurality of proteins through data analysis. The protein sensing array can simultaneously detect a plurality of analytes, and has high sensitivity and selectivity.

[0008] To achieve the purpose of the present application, the following technical solutions are adopted:

[0009] In a first aspect, the present application provides a protein sensing array, which comprises a group of ENSaptamer elements, and the ENSaptamer elements are composed of a first DNA-SWCNTs hybrid molecule solution, a second DNA-SWCNTs hybrid molecule solution and a third DNA-SWCNTs hybrid molecule solution;

[0010] The DNA-SWCNTs hybrid molecule is a single-handed carbon nanotube wound with a single-stranded DNA sequence;

[0011] The sequence of the DNA in the first DNA-SWCNTs hybrid molecule is shown in SEQ ID NO: 1, and the chiral index of the single-handed carbon nanotube in the first DNA-SWCNTs hybrid molecule is (7, 3);

[0012] The sequence of the DNA in the second DNA-SWCNTs hybrid molecule is shown as SEQ ID NO: 1, and the chiral index of the single-chiral carbon nanotube in the second DNA-SWCNTs hybrid molecule is (6, 5).

[0013] The sequence of the DNA in the third DNA-SWCNTs hybrid molecule is shown as SEQ ID NO: 2, and the chiral index of the single-chiral carbon nanotube in the third DNA-SWCNTs hybrid molecule is (8, 3).

[0014] Preferably, the OD value of the first DNA-SWCNTs hybrid molecule in the first DNA-SWCNTs hybrid molecule solution is 1.0-1.5; and the solvent is water. 1020nm

[0015] Preferably, the OD value of the second DNA-SWCNTs hybrid molecule in the second DNA-SWCNTs hybrid molecule solution is 1.0-1.5; and the solvent is water. 990nm

[0016] Preferably, the OD value of the third DNA-SWCNTs hybrid molecule in the third DNA-SWCNTs hybrid molecule solution is 1.0-1.5; and the solvent is water. 978nm

[0017] Preferably, the first DNA-SWCNTs hybrid molecule, the second DNA-SWCNTs hybrid molecule and the third DNA-SWCNTs hybrid molecule are prepared by a method comprising the following steps:

[0018] (a) preparing a solution one of dextran, polyethylene glycol and water, and standing for stratification to obtain a blank upper phase and a blank lower phase respectively;

[0019] (b) preparing a solution two of single-walled carbon nanotubes, DNA, NaCl and water, ultrasonically dispersing the solution two in an ice water bath, and then centrifuging to collect the supernatant to obtain a DNA-SWCNTs dispersion liquid;

[0020] (c) preparing a solution three of dextran, polyethylene glycol, water and the obtained DNA-SWCNTs dispersion liquid; adding a regulator to the solution three to control the distribution of DNA-SWCNTs in the system, and selecting a corresponding upper phase or lower phase for subsequent separation to obtain the first DNA-SWCNTs hybrid molecule, the second DNA-SWCNTs hybrid molecule and the third DNA-SWCNTs hybrid molecule respectively.

[0021] ​​​The application adopts a double water phase separation method to successfully separate three DNA-SWCNTs hybrid molecules with specific recognition effect, the DNA-SWCNTs hybrid molecules have a high surface charge density on the surface, and the DNA has a large wrapping degree and adsorption strength on the SWCNT surface.

[0022] Preferably, in step (a), the concentration of dextran in the solution one is 8-10%, and the concentration of polyethylene glycol is 7-10%.

[0023] Preferably, in step (a), the molecular weight of the dextran is 200-250kDa, and the molecular weight of the polyethylene glycol is 1-1.5kDa.

[0024] Preferably, in step (a), the standing time is 8-12h, and the standing temperature is 20-25℃.

[0025] Preferably, in step (b), the concentration of single-walled carbon nanotubes in the solution two is 0.8-1.2mg / mL, the concentration of DNA is 1.5-2.5μg / μL, and the concentration of NaCl is 25-35mM.

[0026] Preferably, in step (b), the ultrasonic dispersion time is 2-3h.

[0027] In the application, in step (b), the centrifugation time is 90-100min, and the centrifugal force is 11000-12000g.

[0028] Preferably, in step (c), the concentration of dextran in the solution three is 8-10%, the concentration of polyethylene glycol is 7-10%, and the volume of the DNA-SWCNTs dispersion liquid is 25-30% of the total volume of the solution three.

[0029] Preferably, in step (c), the adjusting agent is selected from polyvinylpyrrolidone, PBS or polyethylene glycol.

[0030] Preferably, the molecular weight of the polyvinylpyrrolidone is 8-10kDa.

[0031] Preferably, the molecular weight of the polyethylene glycol is 1-1.5kDa.

[0032] After the first DNA-SWCNTs hybrid molecule, the second DNA-SWCNTs hybrid molecule and the third DNA-SWCNTs hybrid molecule are separated in the application, a step of purifying the obtained DNA-SWCNTs hybrid molecules by sodium thiocyanate is further included.

[0033] In the present application, the step of impurity removal and purification comprises: vortex mixing the sodium thiocyanate aqueous solution with the DNA-SWCNTs hybrid molecule solution respectively, collecting the precipitate by centrifugation, resuspending to obtain the corresponding aqueous solution, and then performing ultrafiltration.

[0034] In the present application, the concentration of sodium thiocyanate in the sodium thiocyanate aqueous solution is 1-6M.

[0035] In the present application, the volume ratio of the sodium thiocyanate aqueous solution to the DNA-SWCNTs hybrid molecule solution is 1:(0.8-1.2).

[0036] In the present application, the centrifugal force of the centrifugation is 16000-17000g, and the centrifugation time is 25-30min.

[0037] In the present application, the molecular cut-off volume of the ultrafiltration membrane used in the ultrafiltration is 3000Da or more.

[0038] Preferably, the separation step for separating the first DNA-SWCNTs hybrid molecule and the second DNA-SWCNTs hybrid molecule is:

[0039] (1) adding a regulator to solution three, vortexing and centrifuging the mixed system in turn, and collecting the upper phase one and the lower phase one in the solution respectively; separating the first DNA-SWCNTs hybrid molecule from the upper phase one;

[0040] (2) adding a regulator and a blank upper phase to the lower phase one, vortexing and centrifuging the mixed system in turn, and collecting the lower phase two in the solution;

[0041] (3) repeating step (2) until the lower phase eight is obtained;

[0042] (4) adding a regulator and a blank upper phase to the lower phase eight, vortexing and centrifuging the mixed system in turn, and collecting the lower phase nine in the solution;

[0043] (5) adding a regulator and a blank upper phase to the lower phase nine, vortexing and centrifuging the mixed system in turn, and collecting the lower phase ten in the solution;

[0044] (6) repeating step (5) until the upper phase eleven is obtained, and separating the second DNA-SWCNTs hybrid molecule from the upper phase eleven.

[0045] Preferably, in step (1), the volume ratio of solution three to the regulator is 610:(0.45-0.55).

[0046] Preferably, in step (1), the regulator is a polyvinylpyrrolidone solution with a concentration of 8-12%.

[0047] Preferably, in step (2), the volume ratio of the lower phase one, the adjusting agent and the blank upper phase is 1: (0.006-0.007): (0.8-1.2).

[0048] Preferably, in step (2), the adjusting agent is a polyvinylpyrrolidone solution with a concentration of 18-22%.

[0049] Preferably, in step (4), the volume ratio of the lower phase eight, the adjusting agent and the blank upper phase is 1: (0.003-0.004): (0.8-1.2).

[0050] Preferably, in step (4), the adjusting agent is a polyvinylpyrrolidone solution with a concentration of 18-22%.

[0051] Preferably, in step (5), the volume ratio of the lower phase nine, the adjusting agent and the blank upper phase is 1: (0.003-0.004): (0.8-1.2).

[0052] Preferably, in step (5), the adjusting agent is a polyvinylpyrrolidone solution with a concentration of 8-12%.

[0053] Preferably, the separation step for separating the third DNA-SWCNTs hybrid molecules is:

[0054] (1) adding an adjusting agent to the solution three, and vortexing and centrifuging the mixed system in sequence to collect the lower phase one in the solution;

[0055] (2) adding an adjusting agent and a blank upper phase to the lower phase one, and vortexing and centrifuging the mixed system in sequence to collect the upper phase two in the solution;

[0056] (3) standing the obtained upper phase two, centrifuging to obtain the upper phase three, and separating the third DNA-SWCNTs hybrid molecules from the upper phase three.

[0057] Preferably, in step (1), the volume ratio of the solution three and the adjusting agent is 610: (0.8-1.2).

[0058] Preferably, in step (1), the adjusting agent is a polyvinylpyrrolidone solution with a concentration of 18-22%.

[0059] Preferably, in step (2), the volume ratio of the lower phase one, the polyethylene glycol solution and the blank upper phase is 1: (0.025-0.035): (0.8-1.2).

[0060] Preferably, in step (2), the concentration of polyethylene glycol in the polyethylene glycol solution is 55-65%.

[0061] In a second aspect, the present application provides a method for using the protein sensor array of the first aspect, the method comprising:

[0062] (1) respectively determining the near-infrared fluorescence intensity of the first DNA-SWCNTs hybrid molecule solution, the second DNA-SWCNTs hybrid molecule solution and the third DNA-SWCNTs hybrid molecule solution;

[0063] (2) adding the protein to be detected to the first DNA-SWCNTs hybrid molecule solution, the second DNA-SWCNTs hybrid molecule solution and the third DNA-SWCNTs hybrid molecule solution, respectively determining the near-infrared fluorescence intensity of the solution after adding the protein to be detected; and determining the near-infrared fluorescence intensity of the standard protein by the same method;

[0064] (3) determining the type of the protein to be detected according to the corresponding near-infrared fluorescence intensity changes of the standard protein and the protein to be detected.

[0065] The present application adopts the separated DNA-SWCNTs hybrid molecules with specific recognition to construct a protein sensor array, and the protein sensor array has high detection accuracy and can accurately detect multiple samples.

[0066] Preferably, in steps (1) and (2), when determining the near-infrared fluorescence intensity, the excitation wavelength is 512 nm, 575 nm, 679 nm, and the emission wavelength is 1020 nm, 1002 nm, 9700 nm.

[0067] Preferably, the final concentration of the protein to be detected in the detection system is 100-1000 nM.

[0068] In a third aspect, the present application provides the use of the protein sensor array of the first aspect in protein detection.

[0069] The numerical range of the present application includes not only the point values listed above, but also any point values between the above numerical ranges that are not listed, and the present application does not exhaustively list the specific point values included in the range for the sake of brevity and simplicity.

[0070] Compared with the prior art, the present application has the following beneficial effects:

[0071] (1) The present application successfully separates three DNA-SWCNTs hybrid molecules with specific recognition by using the aqueous two-phase separation method, and the DNA-SWCNTs hybrid molecules have a high surface charge density on the surface, and the DNA has a large wrapping degree and adsorption strength on the surface of the SWCNT;

[0072] (2) This invention constructs a protein sensor array using isolated DNA-SWCNT hybrid molecules with specific recognition capabilities. The sensitivity / selectivity of the protein sensor array can be easily altered by changing the surface chemistry, for example, by using different DNA sequences. The protein sensor array has high detection accuracy and can accurately detect a variety of samples. Attached Figure Description

[0073] Figure 1 This is the UV-Vis-NIR absorption spectrum of the DNA-SWCNTs dispersion;

[0074] Figure 2 It is the half-peak width of the maximum absorption peak in the DNA-SWCNTs dispersion;

[0075] Figure 3 This is a diagram showing the maximum absorption wavelengths of SWCNTs;

[0076] Figure 4 It is the ratio of the absorption intensity of the SWCNTs dispersion at 990 nm to the absorption intensity at 910 nm;

[0077] Figure 5 The effect of PVP on the partitioning of F14-SWCNTs in the aqueous two-phase system;

[0078] Figure 6 It is the UV-Vis-Nir spectrum of the upper and lower phases corresponding to the two-phase aqueous partition;

[0079] Figure 7 These are the test results corresponding to the upper and lower phases in a two-phase aqueous partition.

[0080] Figure 8 It is a three-dimensional fluorescence spectrum;

[0081] Figure 9 These are transmission electron microscope (TEM) images of SWCNTs before and after separation;

[0082] Figure 10 The results are the reaction results of different concentrations of NaSCN with SWCNT(6,5);

[0083] Figure 11 It is a sensing mechanism for detecting proteins using single-walled carbon nanotubes;

[0084] Figure 12 The effects of eight proteins on the fluorescence of single-walled carbon nanotubes of different chiralities;

[0085] Figure 13 This involves optimizing protein detection concentrations (0-1000 nM).

[0086] Figure 14 This involves optimizing the concentration of protein for detection (0-50 nM).

[0087] Figure 15 It optimizes the time required for protein detection;

[0088] Figure 16 It is a three-dimensional spectrum of 8 protein classifications;

[0089] Figure 17 This is a re-verification of the accuracy of the protein;

[0090] Figure 18 These are fluorescence patterns showing the interaction between different single-chiral SWCNTs and proteins. Detailed Implementation

[0091] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0092] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0093] Example 1

[0094] In this embodiment, CoMoCAT-SWCNTs with an average diameter of approximately 0.78 nm were used as raw materials. Short-chain DNA was wrapped around dispersed carbon nanotubes. In a two-phase solution of polyethylene glycol (PEG) and dextran (DX), by precisely controlling the concentration of PVP surfactant, a continuous separation strategy was adopted to achieve high-purity single chiral separation.

[0095] 1.1 Experimental Materials and Reagents

[0096] 1.1.1 The materials and reagents used in the experiment are shown in Table 1.

[0097] Table 1

[0098] Reagent name Specification Manufacturer Single-walled carbon nanotubes (SWCNTs) (6,5)≥93％ Sigma-Aldrich (Shanghai) Trading Co., Ltd. Polyethylene glycol (PEG) MW = 1500 Alfa Aesar (China) Chemical Co., Ltd. Dextran (Dex) MW = 250000 Alfa Aesar (China) Chemical Co., Ltd. Polyvinylpyrrolidone (PVP) MW = 10000 Sigma-Aldrich (Shanghai) Trading Co., Ltd. DNA PAGE purification Biolab Scientific Co., Ltd.

[0099] The DNA sequences are shown in Table 2.

[0100] Table 2

[0101] Number Sequence F1 (SEQ ID NO: 1) TTTCCCTTTCCCCCC F2 (SEQ ID NO: 3) TTTCCCCCCCCCTTT F3 (SEQ ID NO: 4) CCCCCCTTTCCCCCC F10 (SEQ ID NO: 5) TTATATTATATT F11 (SEQ ID NO: 6) (GC) 20 ]] F13 (SEQ ID NO: 7) ATTATTATTATT F14 (SEQ ID NO: 2) TCTCCCTCTCCCTCT

[0102] (GC) 20 The corresponding sequence is: GCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGC.

[0103] 1.2 Experimental Methods

[0104] 1.2.1 Preparation of Aqueous Two-Phase Systems

[0105] First, weigh 8g of DX (250kDa) and add 32g of water to prepare a 20% DX solution. Then, weigh 24g of PEG (1.5kDa) and add 16g of water to prepare a 60% PEG solution. Next, pipette 6mL of the 20% DX solution, 1.7mL of the 60% PEG solution, and 4.54mL of H2O, and mix vigorously. Let the mixture stand overnight at room temperature (20°C) to separate the phases. Then, separately remove the upper and lower phases to obtain a blank upper phase and a blank lower phase, and store them in a refrigerator to prevent bacterial growth until needed.

[0106] 1.2.2 Preparation of DNA-SWCNTs Dispersion

[0107] Accurately weigh 1.0 mg SWCNTs into a 2 mL round-bottom microcentrifuge tube, add 200 μL of 10 μg / μL DNA solution, 100 μL of 300 mM NaCl solution and 700 μL of water, mix well and place in an ice-water bath to disperse using an ultrasonic cell disruptor, and sonicate continuously at 18 W for 2 h.

[0108] The prepared samples were transferred to conical microcentrifuge tubes and centrifuged in a benchtop high-speed centrifuge for 90 min at 12000 g and 20 °C to remove undispersed amorphous carbon and other impurities. After centrifugation, the supernatant in the centrifuge tubes was collected as the sample, namely DNA-SWCNTs dispersion (5CT10-SG65i), for subsequent experiments.

[0109] 1.2.3 Screening Study of Unichiral Carbon Nanotubes

[0110] First, single-walled carbon nanotubes (SUVs) were mixed with an aqueous two-phase system. Depending on the distribution of the SUVs, appropriate regulators such as PVP or PBS were sometimes added to control the distribution of DNA-SUVs within the system. The appropriate upper or lower phase was then selected for subsequent separation. After screening through more than ten DNA strands, three types of single-chiral SUVs were obtained, with chiral indices of (6,5), (8,3), and (7,3), respectively.

[0111] By referring to the table, we can see that: the characteristic peaks λ11 and λ22 of (6,5) are 975nm and 567nm; the characteristic peaks λ11 and λ22 of (8,3) are 952nm and 663nm; and the characteristic peaks λ11 and λ22 of (7,3) are 992nm and 505nm. The specific separation steps are shown in the table below.

[0112] The separation steps for TCTCCCTCTCCCTCT-SG65i to purify (7,3) and (6,5) are shown in Table 3.

[0113] Table 3

[0114]

[0115] The separation steps for purifying (8,3) TTTCCCTTTCCCCCC-SG65i are shown in Table 4.

[0116] Table 4

[0117]

[0118] In Table 3, the first DNA-SWCNT hybrid molecule obtained in step 2 is the first phase. Residual PVP in the first phase is removed using sodium thiocyanate, followed by ultrafiltration to obtain a pure first DNA-SWCNT hybrid molecule. In Table 3, the second DNA-SWCNT hybrid molecule obtained in step 12 is the second phase. Residual PVP in the second phase is removed using sodium thiocyanate, followed by ultrafiltration to obtain a pure second DNA-SWCNT hybrid molecule. In Table 4, the third phase obtained in step 4 is the third DNA-SWCNT hybrid molecule. Residual PVP in the third phase is removed using sodium thiocyanate, followed by ultrafiltration to obtain a pure third DNA-SWCNT hybrid molecule.

[0119] Dilute the upper and lower phase solutions in each centrifuge tube 10 times, pipette 100 μL, and measure the UV-Vis-NIR spectrum of the sample.

[0120] 1.2.5 Enlarged Volume

[0121] Aqueous two-phase separation is a readily feasible method for increasing the concentration and overall volume of SWCNTs, both in principle and experimentally. Therefore, this embodiment expands the extraction process tenfold compared to previous methods. The specific process is as follows:

[0122] The purification steps for (6,5) are shown in Table 5.

[0123] Table 5

[0124]

[0125] The purification steps for (7,3) are shown in Table 6.

[0126] Table 6

[0127]

[0128] The purification steps for (8,3) are shown in Table 7.

[0129] Table 7

[0130]

[0131] 1.2.6 Redispersion of Single-Chiral Carbon Nanotubes

[0132] Accurately weigh a certain amount of sodium thiocyanate, dissolve it in water to prepare a series of NaSCN solutions with concentrations of 1M, 2M, 3M, 4M, and 6M, and vortex to mix them thoroughly.

[0133] Take five centrifuge tubes, add 250 μL of chiral carbon nanotubes and 250 μL of NaSCN solution to each, vortex to mix, and centrifuge in a benchtop high-speed centrifuge for 30 min at 17000 g and 4 °C. Remove the supernatant, add 250 μL of water, and take 100 μL of the solution into a cuvette. Detect the near-infrared fluorescence of each chiral carbon nanotube at its excitation wavelength.

[0134] 1.3 Results and Discussion

[0135] 1.3.1 Preparation and Characterization of DNA-SWCNT Dispersion

[0136] Figure 1 This is the UV-Vis-NIR absorption spectrum of the DNA-SWCNTs dispersion, where the concentration of the DNA-SWCNTs dispersion is 10 μg / mL. The DNA-SWCNT dispersion exhibits many different UV absorption peaks. The highest peak values ​​for SWCNT(6,5) are observed at 575 nm and 990 nm, representing the λ22 and λ11 absorption peaks, respectively, indicating the highest content of SWCNT(6,5) in this DNA-SWCNT dispersion system. According to previous literature, the quality of the SWCNT dispersion can be evaluated based on the UV-Vis-NIR absorption spectrum, primarily including: 1. the full width at half maximum (FWHM) of the absorption peaks; 2. the maximum absorption wavelength; and 3. the ratio of the maximum absorption peak intensity to the background absorption at 910 nm.

[0137] The absorption peak width of the SWCNT dispersion is limited because the electronic coupling between SWCNTs is relatively large, which prevents the fine structure in the spectrum from being displayed. Figure 2 It is the half-width at half maximum (WHM) of the maximum absorption peak in the DNA-SWCNTs dispersion. Figure 2 In the table, F1, F2, F3, F10, F11, F13, and F14 represent the sequence designations of the DNA. See Table 2 for the specific sequences. Figure 2It can be seen that in the single-stranded DNA-SWCNT dispersion, the half-width of the absorption peak of the (TCTCCC)2TCT-SWCNT dispersion is smaller than that of other DNA-SWCNT dispersions, indicating that the monodisperse content of SWCNT in the (TCTCCC)2TCT-SWCNT dispersion is relatively large.

[0138] The UV absorption spectrum of the DNA-SWCNTs dispersion showed A990 = 0.776. Based on previous literature stating A990 = 1 and CSWCNT = 13 μg / mL, the concentration of SWCNTs is approximately 1 mg / mL. This is consistent with the previously set target concentration, indicating that the SWCNTs have been completely dispersed by the DNA. This is consistent with the absence of precipitate at the bottom of the centrifuge tube after sonication and centrifugation.

[0139] The absorption peak width of the SWCNTs dispersion is due to the large electronic coupling between SWCNTs, which prevents the fine structure in the spectrum from being displayed. Figure 2 This is the full width at half maximum (FWHM) of the maximum absorption peak in the DNA-SWCNT dispersion. The figure shows that different single-stranded DNAs have different dispersibility. Since the monodisperse content of SWCNTs in the DNA-SWCNT dispersion affects the absorption wavelength of the SWCNT dispersion, analyzing the absorption wavelength of the SWCNT dispersion allows for comparison of the monodisperse content of SWCNTs in different SWCNT dispersions. Figure 3 This is a graph showing the maximum absorption wavelength of the SWCNT(6,5) dispersion. Figure 3 As can be seen, compared with the absorption wavelength of other DNA-SWCNTs dispersions, the maximum absorption wavelength of (TCTCCC)2TCT-SWCNTs is smaller, indicating that the monodisperse content of SWCNTs in the F14-SWCNTs dispersion is the highest.

[0140] Since the ratio of the absorption peak intensity at 990 nm to the absorption intensity at 910 nm of the SWCNTs dispersion is related to the monodisperse content of SWCNTs, this ratio will change with the monodisperse content of SWCNTs in the SWCNTs dispersion. Figure 4 This is the ratio of the absorption peak intensity of the SWCNTs dispersion at 990 nm to the absorption peak intensity at 910 nm. From... Figure 4 As can be seen, the ratio of the absorption intensity at 990 nm to that at 910 nm of the (TCTCCC)2TCT-SWCNTs dispersion is higher than that of other single-stranded DNA-SWCNTs dispersions, indicating that the monodisperse content of SWCNTs in the single-stranded (TCTCCC)2TCT-SWCNTs dispersion is greater.

[0141] In summary, the analysis of the three influencing factors above shows that the F14 sequence has better dispersibility of SWCNTs compared to other single-stranded DNA sequences, and the dispersion quality of DNA-SWCNTs dispersions with specific recognition function is better.

[0142] 1.3.2 DNA-SWCNTs Isolation Process

[0143] The concept of identifying SWCNTs through specific DNA sequences was first proposed in 2009, meaning that specific DNA sequences can form ordered structures on the chiral side of specific SWCNTs. This concept explains most IEX data and is consistent with many observations. However, ATP-based isolation has produced many results that cannot be simply explained by this concept. Sometimes, in previously developed PEG / PAM systems, a single sequence allows for the purification of two species through multi-stage extraction. One example is the purification of (8,4) and (7,4) from (GT)20SWCNTs, another is the purification of left-handed and right-handed (6,5) from TTATATTAT-SWCNTs, and new results from the purification of three SWCNTs in a PEG 1.5kDa / DX 250kDa system from a single dispersion further illustrate this point.

[0144] In this embodiment, single-walled carbon nanotubes (SUVs) were first mixed with an aqueous two-phase system. Depending on the distribution of the SUVs, appropriate regulators such as PVP or PBS were sometimes added to the system to control the distribution of DNA-SUVs. The appropriate upper or lower phase was then selected for subsequent separation. Three types of single-chiral SUVs were obtained through screening of more than ten DNA strands.

[0145] Figure 5 The effect of PVP on the distribution of F14-SWCNTs in an aqueous two-phase system is shown in the figure. The (TCTCCC)2TCT-SG65i dispersion is affected by the PVP concentration, resulting in its distribution across the upper and lower phases in the aqueous two-phase system. Figure 5 As shown in the figure, the volumes of the upper and lower phases in the aqueous two-phase system PEG1.5kDa / DX 250kDa remain relatively stable, with minimal trapping at the phase interface. The UV-Vis-Nir spectrum obtained after diluting the upper phase 10-fold is shown below. Figure 6 As shown, Figure 6 The UV-Vis-Nir spectra of the upper and lower phases corresponding to the aqueous two-phase partition are shown. According to the table, the characteristic peaks λ11 and λ22 of (7,3) are 992 nm and 505 nm, respectively; and the characteristic peaks λ11 and λ22 of (6,5) are 975 nm and 567 nm, respectively. Because the DNA is wrapped around the carbon nanotubes, the measured λ11 and λ22 of the chiral carbon nanotubes show a certain degree of redshift. Figure 5It can be seen that phase 1T (upper phase 1) is (7,3) and its color is pink; phase 9B (lower phase 9) is (6,5) and its color is purple.

[0146] Figure 7 These are the test results corresponding to the upper and lower phases in a two-phase aqueous partition. Figure 7 In Figure a, PVP affects the partitioning of F1-SWCNTs dispersion in an aqueous two-phase system; in Figure b, UV-Vis-Nir spectra of the upper and lower phases corresponding to the partitioning in the aqueous two-phase system; and in Figure c, the one-dimensional fluorescence spectrum of (8,3). Relatively pure (8,3) can be obtained from the TTTCCCTTTCCCCCC-SG65i dispersion. Figure 7 It can be seen that (8,3) is enriched in the 1T phase and contains a small amount of other chiral carbon nanotubes. The upper phase is purified a second time to obtain relatively pure (8,3). According to the table, the characteristic peaks λ11 and λ22 of (8,3) are 952 nm and 663 nm, respectively. Since the DNA is wrapped around the carbon nanotubes, the chiral carbon nanotubes λ11 and λ22 will show a certain degree of red shift.

[0147] The three-dimensional fluorescence spectra of (6,5), (7,3), and (8,3) in the near-infrared spectral region are shown below. Figure 8 As shown. The excitation wavelength range is 400-800 nm. When the excitation wavelength is 575 nm, (6,5)-SWCNTs exhibit a strong fluorescence emission peak at 1000 nm. When the excitation wavelength is 512 nm, (7,3)-SWCNTs exhibit a strong fluorescence emission peak at 1020 nm. When the excitation wavelength is 679 nm, (8,3)-SWCNTs exhibit a strong fluorescence emission peak at 980 nm.

[0148] In addition, electron microscopy was performed on both unisolated and purified SWCNTs and isolated and purified (6,5) saturates, and the results are as follows: Figure 9 As shown, Figure 9 These are transmission electron microscopy (TEM) images of SWCNTs before and after separation. Image a shows unseparated SWCNTs, and image b shows (6,5) SWCNTs. The surface of the SWCNTs is not uniformly coated with DNA; the smaller bundles of SWCNTs are coated with a thicker layer of DNA. This indicates that in the DNA-SWCNT dispersion, most SWCNTs are dispersed in bundles. The separated and purified (6,5) SWCNTs appear as monodisperse SWCNTs coexisting with the smaller bundles in TEM.

[0149] 1.3.3 Study on the chiral recognition mechanism of DNA for SWCNTs

[0150] Based on the solvation energy distribution of DNA-SWCNT dispersions. For a given DNA sequence, the solvation energy distribution of each DNA-(n,m) hybrid may revolve around an average rather than a fixed value, because the encapsulation structure of the DNA on the SWCNTs may adopt multiple conformations. The average solvation energy values ​​of different (n,m) species within a synthetic mixture of the same DNA encapsulation are expected to have some diffusion. Chirally ordered coating structures are expected to produce a narrower solvation energy distribution, and therefore are less likely to overlap with other species, making them easier to purify from the mixture.

[0151] If all solvation peaks are located on the right side of the window (the more hydrophilic side), then all species will remain in the more hydrophilic bottom phase (lower phase). Conversely, if all peaks are located on the left side of the window (the more hydrophobic side), then all SWCNTs will remain in the less hydrophilic top phase (upper phase). If a peak spans the ATP separation window, the corresponding species will be allocated between the top and bottom phases. For the PEG 1.5kDa / DX 250kDa system, in most cases, the initial distribution is located on the right side of the separation window, meaning that all SWCNTs remain in the bottom phase after being loaded into the ATP system. By adding a modifier like PVP, the initial distribution as a whole is dragged to the left (the more hydrophobic side). Ideally, since the amount of PVP added is small, its effect on the relative positions of different species is negligible. As the PVP content gradually increases, SWCNTs are sequentially brought to the top phase until only the last pure single-chiral carbon nanotubes or carbon-free nanotubes remain in the bottom phase. This completes a typical ordination experiment.

[0152] 1.3.4 Effect of NaSCN concentration on the precipitation of single-chiral carbon nanotubes

[0153] Different changes occurred in the chiral carbon nanotube solution after adding different amounts of NaSCN solution, such as... Figure 10 As shown, Figure 10 Figure a shows the effect of different concentrations of NaSCN on the precipitation of SWCNT(6,5), and figure b shows the fluorescence spectra of NaSCN precipitates (6,5) at different concentrations. In figure a, from left to right, the final concentrations of NaSCN added are 0.5M, 1M, 1.5M, 2M, and 3M. As shown in the figure, with increasing NaSCN concentration, more and more carbon nanotubes precipitate. When the NaSCN concentration is 0.5M, the solution is light purple, indicating that there are still a small amount of single-chiral carbon nanotubes in the solution. Afterward, the fluorescence intensity gradually decreases with increasing NaSCN concentration. Therefore, 1M of NaSCN is the optimal concentration for carbon nanotube precipitation.

[0154] Single-walled carbon nanotubes were resuspended, and the three types of single-chiral carbon nanotubes were characterized by UV-Vis-Nir and near-infrared fluorescence spectra. The aqueous two-phase in the carbon nanotube dispersion was washed away at a concentration of 1M NaSCN. The carbon nanotubes were slightly lost after water redispersement, but the overall dispersibility was good.

[0155] In this embodiment, various DNA-SWCNT dispersions were prepared using ultrasonic dispersion, and their dispersibility was characterized using ultraviolet and near-infrared spectroscopy. Sequence screening was also performed. The main conclusions are as follows:

[0156] (1) DNA sequences with specific recognition function have better dispersion quality for SWCNTs, that is, the monodisperse content of SWCNTs in the dispersion of these SWCNTs is high.

[0157] (2) The surface of DNA-SWCNT hybrid molecules with specific recognition function has a high surface charge density, and DNA has a large degree of encapsulation and adsorption strength on the SWCNT surface.

[0158] (3) The two-phase aqueous separation method can easily increase the concentration and overall volume of SWCNTs, which is feasible in principle and experiment.

[0159] (4) When the amount of NaSCN solution added is 1M, it is the optimal concentration for carbon nanotube precipitation and can be well redispersed in water.

[0160] Example 2

[0161] This embodiment develops a protein detection sensor. The method of this embodiment is based on an idea similar to an artificial nose and can simultaneously detect multiple analytes. The protein detection sensor can increase the sensitivity and selectivity of the analytical method. DNA is adsorbed onto single-walled carbon nanotubes, and proteins are adsorbed onto the carbon nanotubes, changing the corona structure on the nanotube surface and transforming the protein's structural conformation into a fluorescence change in the carbon nanotubes.

[0162] 2.1 Experimental Materials and Reagents

[0163] 2.1.1 The experimental materials and reagents are shown in Table 8.

[0164] Table 8

[0165]

[0166]

[0167] 2.2 Experimental Methods

[0168] 2.2.1 Construction of Sensor Array

[0169] The sensor array consists of three near-infrared fluorescence sensors based on SWCNTs. Using specific sensors reduces the need to consider the responses of background sensors in the final analysis. Furthermore, the chemical complexity of the analyte makes predicting overall performance difficult; increasing the number of sensors is beneficial for predicting overall performance. To achieve spectral multiplexing, SWCNTs with non-overlapping fluorescence emissions are required.

[0170] Since the fluorescence emission wavelength of SWCNTs is structure-dependent, multiple SWCNT sensors with different spectra can be set in the near-infrared range. A major advantage of these sensors is that their sensitivity / selectivity can be easily improved by changing the surface chemistry, for example, by using different DNA sequences to modify the sensitivity / selectivity of SWCNT sensors.

[0171] The extracted single-chiral carbon nanotubes were used as protein binders in this work. When proteins were added to the single-chiral carbon nanotube solution, they bound to the surface of the chiral carbon nanotubes to form DNA-nanoconjugates. When eight proteins were added to three types of chiral carbon nanotubes, all fluorescence signals gradually stabilized as the protein concentration increased.

[0172] Eight proteins were prepared into 5 μM solutions with water. 5 μL of each protein solution was added to a 95 μL carbon nanotube, shaken well, and allowed to stand until the final protein concentration was 500 nM. A blank solution containing 5 μL of water was used as a control. Near-infrared fluorescence intensity of the samples was measured at emission wavelengths of 1020 nm, 1002 nm, and 9700 nm under excitation wavelengths of 512 nm, 575 nm, and 679 nm.

[0173] 2.2.2 Optimization of protein detection concentration

[0174] Weigh a certain amount of protein, dissolve it in water to prepare a series of protein solutions with concentrations of 100nM, 1μM, 10μM, and 20μM, add them according to the above method, and vortex to mix them thoroughly.

[0175] Take eight centrifuge tubes and add 95 μL of chiral carbon nanotubes and 5 μL of protein solution to each, respectively, to achieve final protein concentrations of 5 nM, 50 nM, 500 nM, and 1 μM. Let them stand for 60 min. Take one of the above centrifuge tubes and add 95 μL of chiral carbon nanotubes and 5 μL of water as a blank control. Add 100 μL of this solution to a cuvette and detect its near-infrared fluorescence at the emission wavelength.

[0176] 2.2.3 Stability test for protein detection

[0177] Weigh a certain amount of protein, dissolve it in water to prepare a protein solution with a concentration of 5 μM, shake well and let stand. Take 8 centrifuge tubes and add 95 μL of single-chiral carbon nanotubes and 5 μL of protein solution to make the final protein concentration 500 nM. Near-infrared fluorescence detection was performed at intervals of 5 min, 10 min, 15 min, 30 min and 60 min.

[0178] 2.2.4 Repeatability Experiments for Protein Detection

[0179] Weigh a certain amount of protein, dissolve it in water to prepare a 5 μM protein solution, shake well and let stand. Take 6 centrifuge tubes and add 95 μL of single-chiral carbon nanotubes and 5 μL of the same protein solution to each tube, so that the final protein concentration is 500 nM. Take one of the above centrifuge tubes and add 95 μL of single-chiral carbon nanotubes and 5 μL of water as a blank control. Take 100 μL and add it to a cuvette to detect its near-infrared fluorescence at the emission wavelength.

[0180] 2.2.5 Validation experiment for the accuracy of protein detection

[0181] Weigh a certain amount of protein, dissolve it in water to prepare a protein solution with a concentration of 5 μM, shake well and let stand. Take 3 centrifuge tubes and add 95 μL of (6,5), (7,3) and (8,3) single-chiral carbon nanotubes and 5 μL of protein solution respectively. The final concentration of protein is 500 nM, which is one set of experiments. Repeat the experiment with different protein solutions twenty times.

[0182] 2.2.6 Characterization by near-infrared fluorescence microscopy

[0183] Weigh a certain amount of protein, dissolve it in water to prepare a 5 μM protein solution, shake well and let stand. Taking (6,5) chiral carbon nanotubes as an example, take 8 centrifuge tubes, add 95 μL of chiral carbon nanotubes and 5 μL of protein solution to each, vortex to mix thoroughly, take one of the above centrifuge tubes and add 95 μL of chiral carbon nanotubes and 5 μL of water as a blank control, place the 9 centrifuge tubes side by side in the center of the stage, and photograph their fluorescence intensity at an excitation wavelength of 808 nm. (7,3) and (8,3) chiral carbon nanotubes were photographed according to the above experimental method.

[0184] 2.3 Results and Discussion

[0185] 2.3.1 Sensor Detection Mechanism

[0186] The single-walled carbon nanotube (SWCNT) protein sensor array designed and fabricated in this embodiment consists of three functionalized single-chiral carbon nanotubes. To achieve spectral multiplexing, non-overlapping fluorescence emission SWCNTs are required. Currently, only a few cases demonstrate sensing using purified SWCNTs. Since the fluorescence emission wavelength of SWCNTs is structure-dependent, multiple SWCNT sensors with different spectra can be configured in the near-infrared range. A major advantage of these sensors is that their sensitivity / selectivity can be easily altered by changing the surface chemistry, for example, by using different DNA sequences. The sensing mechanism for protein detection using single-walled carbon nanotubes is as follows: Figure 11 As shown, the optical characteristics of SWCNTs in solution are related to their structure and surrounding environment. When molecular recognition elements bind to the surface of single-walled carbon nanotubes, they either change the emission wavelength or quench fluorescence, depending on the nature of the interaction. By utilizing the non-specific weak interactions between carbon nanotubes and biological targets, a good group of sensing probes can be obtained. Further data analysis can then be used to establish pattern recognition strategies for various proteins (such as bovine serum albumin, hemoglobin, and transferrin). Figure 12 The effects of eight proteins on the fluorescence of single-walled carbon nanotubes with different chiralities were investigated. Figure 12 In (6,5), (8,3) and (7,3), the coordinates of the tilted axes are, in order, carbon nanotubes (DNA-wrapped carbon nanotubes), ALBU, BSA, CC, FIB, GLO, HH, HRP, and TRF.

[0187] 2.3.2 Optimization of protein detection concentration

[0188] A series of protein solutions of different concentrations were prepared and added to (6,5)SWCNTs. As the concentration of the added protein solution gradually increased from 5 nM, 50 nM, 500 nM, to 1000 nM, the near-infrared fluorescence intensity gradually stabilized. Figure 13-14 As shown in the figure, the fluorescence intensity changes drastically with protein concentration in the range of 0-500 nM, and gradually stabilizes when the protein concentration is 500 nM.

[0189] 2.3.3 Stability test for protein detection

[0190] Add 5 μL of 10 mM protein solution to 95 μL (6,5) SWCNT and mix thoroughly to achieve a final protein concentration of 500 nM. Measure the near-infrared fluorescence intensity of the sample three times at intervals of 5 min, 10 min, 15 min, and 30 min. It was found that the fluorescence intensity of the sample tended to stabilize after 30 min. The results are as follows: Figure 15 As shown, Figure 15 It optimizes the time required for protein detection.

[0191] 2.3.4 Repeatability Experiments for Protein Detection

[0192] This embodiment constructed a set of ENSaptamer elements to recognize eight proteins (all 500 nM), based on the criterion that they have relatively uniformly distributed β values. The selected proteins were ALBU, BSA, GLO, Fib, Hb, CC, HRP, and TRF, which have different molecular weights, isoelectric points (pi), and oligomeric states. Initially, these proteins were tested on the SWCNTs-ENSaptamer sensing platform with ENSaptamer-3, a subset of three elements containing (6,5), (8,3), and (7,3). Due to the interaction with the single-chiral carbon nanotubes and DNA probes, the presence of different proteins produced different fluorescence responses. Figure 12 Fluorescence enhancement was observed for most probes (all unstructured probes and some structured probes), indicating that the release of bound DNA strands into solution was a result of protein competitive substitution. Some structured probes caused a slight decrease in fluorescence intensity, possibly due to protein perturbation of the DNA structure, making the probes more readily accessible to the carbon nanotubes. In this embodiment, a protein concentration of 500 nM was selected as the optimized concentration, which produced a sufficiently distinct signal response in the constructed sensing array.

[0193] Quantitative analysis of the synthesized fluorescence response patterns was performed using LDA (a powerful statistical technique). Six replicates were performed on each protein sample. LDA was applied to the raw data, generating three canonical factors (90.6%, 7.7%, and 1.7%), representing linear combinations of the fluorescence response matrix (3 SWCNTs - ENSaptamers × 8 proteins × 6 replicates). A 3D plot (e.g., using the two most significant discriminant factors) was generated. Figure 16 As shown in the diagram, each point represents the response pattern of a single protein sample to the sensor array. Importantly, 48 typical fluorescence response patterns (8 proteins × 6 replicates) were clustered into 8 distinct groups. The 8 protein targets were clearly identified using pattern recognition with ENSaptamer-3.

[0194] 2.4.5 Validation experiment for the accuracy of protein detection

[0195] To verify the accuracy of protein detection, we randomly generated 20 sets of data under the same conditions. We then performed LDA analysis on the original 48 sets of data using SYSTAT 12.0, dividing the data into 8 distinct intervals, represented by a cluster dendrogram, as shown below. Figure 17 As shown, Figure 17 The accuracy of the protein detection was re-validated. The 20 random data sets were well distributed into the corresponding intervals, indicating that the protein detection was accurate.

[0196] 2.4.6 Characterization by near-infrared fluorescence microscopy

[0197] This embodiment also included fluorescence microscopy characterization. The control and identification samples were placed side-by-side, and NIR fluorescence imaging was performed using an NIR-II series 900 / 1700 (nip) equipped with an 808 nm laser source. Changes in fluorescence signal were evaluated using (I-I0) / I0, where I and I0 represent the maximum fluorescence intensity of the sample. Results are as follows: Figure 18 As shown, Figure 18 These are fluorescence patterns showing the interactions between different single-chiral SWCNTs and proteins. Figure 18 In the figure, a shows the near-infrared fluorescence microscopy characterization of the interaction between different single-chiral SWCNTs and proteins, b shows the fluorescence change of each single-chiral SWCNT for 8 proteins, and c shows the fluorescence change of each protein for three single-chiral SWCNTs. It can be clearly seen that the intensity of fluorescence is basically consistent with the change of fluorescence signal.

[0198] In summary, this invention uses CoMoCAT-SWCNTs with an average diameter of approximately 0.78 nm as raw materials, employs short-chain DNA wrapped around dispersed carbon nanotubes, and achieves high-purity single-chiral separation in a two-phase solution of polyethylene glycol (PEG) and dextran (DX) by precisely controlling the concentration of PVP surfactant and using a continuous separation strategy. The purified DNA-SWCNTs hybrid molecules with non-overlapping fluorescence emission were obtained through purification and screening. This invention utilizes the screened DNA-SWCNTs hybrid molecules to construct a protein sensing array. This protein sensing array can simultaneously detect multiple analytes and exhibits high sensitivity and selectivity. Experimental verification has shown that the protein sensing array can clearly identify different proteins.

[0199] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A protein sensing array, characterized in that, The protein sensing array includes a set of ENSaptamer elements, which are composed of a first DNA-SWCNT hybrid molecular solution, a second DNA-SWCNT hybrid molecular solution, and a third DNA-SWCNT hybrid molecular solution. The DNA-SWCNTs hybrid molecule is a single-stranded DNA sequence wrapped around a single-chiral carbon nanotube; The DNA sequence in the first DNA-SWCNTs hybrid molecule is shown in SEQ ID NO:2, and the chiral index of the single-chiral carbon nanotubes in the first DNA-SWCNTs hybrid molecule is (7,3). The DNA sequence in the second DNA-SWCNTs hybrid molecule is shown in SEQ ID NO:2, and the chiral index of the single-chiral carbon nanotubes in the second DNA-SWCNTs hybrid molecule is (6,5). The DNA sequence in the third DNA-SWCNTs hybrid molecule is shown in SEQ ID NO:1, and the chiral index of the single-chiral carbon nanotubes in the third DNA-SWCNTs hybrid molecule is (8,3).

2. The protein sensing array according to claim 1, characterized in that, OD of the first DNA-SWCNT hybrid molecule in the first DNA-SWCNT hybrid molecule solution 1020 nm Value is 1.0-1.5; solvent is water; OD of the second DNA-SWCNT hybrid molecule in the solution of the second DNA-SWCNT hybrid molecule 990 nm Value is 1.0-1.5; solvent is water; The OD of the third DNA-SWCNTs hybrid molecules in the solution of the third DNA-SWCNTs hybrid molecules 978nm Value is 1.0-1.5; solvent is water.

3. The protein sensing array according to claim 1 or 2, characterized in that, The first, second, and third DNA-SWCNT hybrid molecules were prepared using a method comprising the following steps: (a) Prepare a solution by mixing dextran, polyethylene glycol and water, allow it to stand and separate into layers, and obtain the blank upper phase and blank lower phase respectively; (b) Prepare a solution two by mixing single-walled carbon nanotubes, DNA, NaCl and water. Disperse the solution two by sonication in an ice-water bath and then collect the supernatant by centrifugation to obtain a DNA-SWCNTs dispersion. (c) Prepare solution three by mixing dextran, polyethylene glycol, water and the obtained DNA-SWCNTs dispersion; add a regulator to solution three to regulate the distribution of DNA-SWCNTs in the system, select the appropriate upper or lower phase for subsequent separation, and obtain the first DNA-SWCNTs hybrid molecule, the second DNA-SWCNTs hybrid molecule and the third DNA-SWCNTs hybrid molecule respectively.

4. The protein sensing array according to claim 3, characterized in that, In step (a), the concentration of dextran in solution one is 8-10%, and the concentration of polyethylene glycol is 7-10%.

5. The protein sensing array according to claim 4, characterized in that, In step (a), the molecular weight of the dextran is 200-250 kDa, and the molecular weight of the polyethylene glycol is 1-1.5 kDa.

6. The protein sensing array according to claim 3, characterized in that, In step (a), the settling time is 8-12 h and the settling temperature is 20-25℃.

7. The protein sensing array according to claim 3, characterized in that, In step (b), the concentration of single-walled carbon nanotubes in solution two is 0.8-1.2 mg / mL, the concentration of DNA is 1.5-2.5 μg / μL, and the concentration of NaCl is 25-35 mM.

8. The protein sensing array according to claim 3, characterized in that, In step (b), the ultrasonic dispersion time is 2-3 hours.

9. The protein sensing array according to claim 3, characterized in that, In step (c), the concentration of dextran in solution three is 8-10%, the concentration of polyethylene glycol is 7-10%, and the volume of the DNA-SWCNTs dispersion is 25-30% of the total volume of solution three.

10. The protein sensing array according to claim 3, characterized in that, In step (c), the modifier is selected from polyvinylpyrrolidone, PBS, or polyethylene glycol.

11. The protein sensing array according to claim 10, characterized in that, The molecular weight of the polyvinylpyrrolidone is 8-10 kDa.

12. The protein sensing array according to claim 10, characterized in that, The polyethylene glycol has a molecular weight of 1-1.5 kDa.

13. The protein sensing array according to claim 3, characterized in that, The separation steps for separating the first DNA-SWCNT hybrid molecule and the second DNA-SWCNT hybrid molecule are as follows: (1) Add a regulator to solution three, vortex and centrifuge the mixed system in sequence, and collect the upper phase one and lower phase one in the solution respectively; separate the first DNA-SWCNT hybrid molecule from the upper phase one; (2) Add a regulator and a blank upper phase to the lower phase I, and vortex and centrifuge the mixed system in sequence to collect the lower phase II in the solution; (3) Repeat step (2) until the lower phase 8 is obtained; (4) Add a regulator and a blank upper phase to the lower phase 8, and vortex and centrifuge the mixed system in sequence to collect the lower phase 9 in the solution; (5) Add a regulator and a blank upper phase to the lower phase nine, and vortex and centrifuge the mixed system in sequence to collect the lower phase ten in the solution; (6) Repeat step (5) until upper phase eleven is obtained, from which the second DNA-SWCNT hybrid molecule is isolated.

14. The protein sensing array according to claim 13, characterized in that, In step (1), the volume ratio of the solution to the regulator is 610:(0.45-0.55).

15. The protein sensing array according to claim 13, characterized in that, In step (1), the regulator is a polyvinylpyrrolidone solution with a concentration of 8-12%.

16. The protein sensing array according to claim 13, characterized in that, In step (2), the volume ratio of the lower phase I, the regulator, and the blank upper phase is 1:(0.006-0.007):(0.8-1.2).

17. The protein sensing array according to claim 13, characterized in that, In step (2), the regulator is a polyvinylpyrrolidone solution with a concentration of 18-22%.

18. The protein sensing array according to claim 13, characterized in that, In step (4), the volume ratio of the lower phase 8, the regulator, and the blank upper phase is 1:(0.003-0.004):(0.8-1.2).

19. The protein sensing array according to claim 13, characterized in that, In step (4), the regulator is a polyvinylpyrrolidone solution with a concentration of 18-22%.

20. The protein sensing array according to claim 13, characterized in that, In step (5), the volume ratio of the lower phase 9, the regulator, and the blank upper phase is 1:(0.003-0.004):(0.8-1.2).

21. The protein sensing array according to claim 13, characterized in that, In step (5), the regulator is a polyvinylpyrrolidone solution with a concentration of 8-12%.

22. The protein sensing array according to claim 3, characterized in that, The separation steps for separating third DNA-SWCNT hybrid molecules are as follows: (1) Add a regulator to solution three, and vortex and centrifuge the mixed system in sequence to collect the lower phase one in the solution; (2) Add a regulator and a blank upper phase to the lower phase I, and vortex and centrifuge the mixed system in sequence to collect the upper phase II in the solution; (3) Allow the obtained upper phase two to stand, centrifuge to obtain upper phase three, and separate the third DNA-SWCNT hybrid molecule from the upper phase three.

23. The protein sensing array according to claim 22, characterized in that, In step (1), the volume ratio of the solution to the regulator is 610:(0.8-1.2).

24. The protein sensing array according to claim 22, characterized in that, In step (1), the regulator is a polyvinylpyrrolidone solution with a concentration of 18-22%.

25. The protein sensing array according to claim 22, characterized in that, In step (2), the volume ratio of the lower phase I, the polyethylene glycol solution, and the blank upper phase is 1:(0.025-0.035):(0.8-1.2).

26. The protein sensing array according to claim 25, characterized in that, In step (2), the concentration of polyethylene glycol in the polyethylene glycol solution is 55-65%.

27. The method of using the protein sensing array according to any one of claims 1-26, characterized in that, The method of use includes: (1) The near-infrared fluorescence intensity of the first DNA-SWCNTs hybrid molecular solution, the second DNA-SWCNTs hybrid molecular solution and the third DNA-SWCNTs hybrid molecular solution were measured respectively; (2) Add the test protein to the first DNA-SWCNTs hybrid molecular solution, the second DNA-SWCNTs hybrid molecular solution and the third DNA-SWCNTs hybrid molecular solution, and measure the near-infrared fluorescence intensity of the solution after adding the test protein; use the same method to measure the near-infrared fluorescence intensity of the standard protein. (3) Determine the type of protein to be tested based on the changes in near-infrared fluorescence intensity of the standard protein and the protein to be tested.

28. The method of using the protein sensing array according to claim 27, characterized in that, In steps (1) and (2), when measuring the near-infrared fluorescence intensity, the excitation wavelengths are 512 nm, 575 nm, and 679 nm, and the emission wavelengths are 1020 nm, 1002 nm, and 9700 nm.

29. The method of using the protein sensing array according to claim 27, characterized in that, The final concentration of the protein to be tested in the detection system is 100-1000 nM.

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