Neurotransmitter sensing array and preparation method and application thereof

Abstract

The invention relates to a neurotransmitter sensing array as well as a preparation method and application thereof. The neurotransmitter sensing array comprises a single-walled carbon nanotube connected with single-stranded DNA (deoxyribonucleic acid), and the single-walled carbon nanotubes are (8, 4)-SWCNT (single-walled carbon nanotubes). A fluorescent sensing array based on (8, 4)-ssDNA-SWCNT is constructed, three non-specific DNA chains are selected as sensing elements, and a neurotransmitter is identified by outputting near-infrared fluorescent signals of the (8, 4)-ssDNA-SWCNT.

Description

Technical Field

[0001] This invention relates to the field of biochemical sensing technology, and in particular to a neurotransmitter sensing array, its preparation method, and its application. Background Technology

[0002] Neurotransmitters play a crucial role in maintaining physiological processes, including regulating metabolism, participating in neuromodulation, and influencing organ function. Under normal physiological conditions, neurotransmitters are essential for brain function, controlling learning, memory, sleep, and mood, and regulating blood pressure, heart rate, and muscle contraction. Serotonin, in particular, plays a significant role in various diseases and may affect the cardiovascular system or trigger diabetes. Therefore, there is an urgent need to develop powerful recognition and sensing tools to gain a deeper understanding of the roles of neurotransmitters in brain physiological and pathological processes.

[0003] Array sensing, as an emerging molecular recognition strategy, relies on non-specific recognition capabilities rather than traditional "lock and key" interactions. Chemical array ("nose / tongue") strategies offer a feasible approach for large-scale analyte identification. However, due to the structural and reactivity similarities among various neurotransmitters, developing a reliable platform for the simultaneous detection of multiple neurotransmitters under physiological conditions remains a significant challenge. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a neurotransmitter sensing array, its preparation method, and its application. A fluorescence sensing array based on (8,4)-ssDNA-SWCNT is constructed, and three non-specific DNA strands (F14, (GT)10, and (TCG)4TC) are selected as sensing elements. Neurotransmitters are identified by outputting near-infrared fluorescence signals from (8,4)-ssDNA-SWCNT.

[0005] To achieve this objective, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a neurotransmitter sensing array, the neurotransmitter sensing array comprising single-walled carbon nanotubes connected to single-stranded DNA;

[0007] The single-walled carbon nanotubes are (8,4)-SWCNTs.

[0008] In this invention, a neurotransmitter sensing array is prepared by linking (8,4)-SWCNTs to single-stranded DNA. The interaction between single-stranded DNA and SWCNTs is mainly influenced by factors such as conformational matching, van der Waals forces, and electrostatic attraction of the single-stranded DNA. The addition of specific neurotransmitters will interact with neurotransmitters in different ssDNA-SWCNTs solutions, thereby producing unique fluorescence responses. Based on these fluorescence responses, linear discriminant analysis (LDA) can be used to effectively distinguish and detect multiple neurotransmitters.

[0009] Preferably, the nucleic acid sequence of the single-stranded DNA includes any one or a combination of at least two of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0010] SEQ ID NO:1:

[0011] TCTCCCTCTCCCTCT.

[0012] SEQ ID NO:2:

[0013] GTGTGTGTGTGTGTGTGT.

[0014] SEQ ID NO:3:

[0015] TCGTCGTCGTCGTC.

[0016] In this invention, the use of the above-mentioned single-stranded DNA can significantly improve the accuracy of detection and enhance the differentiation between different neurotransmitters.

[0017] In a second aspect, the present invention provides a method for preparing a neurotransmitter sensing array according to the first aspect, the method comprising: preparing (8,4)-SWCNT, and then ligating (8,4)-SWCNT with single-stranded DNA to obtain the array.

[0018] Preferably, when the nucleic acid sequence of the single-stranded DNA is SEQ ID NO:1, the preparation method includes:

[0019] (1) Mix diethanol solution, polyethylene glycol solution and deionized water to obtain solution one. After incubation, obtain upper and lower phases. Place single-walled carbon nanotubes and single-stranded DNA in a solvent to obtain solution two. After sonication and centrifugation, collect the supernatant. Mix the supernatant with the upper phase to obtain solution three. Add polyvinylpyrrolidone solution and mix to obtain upper and lower solutions.

[0020] (2) After mixing the lower layer solution, upper phase and polyvinylpyrrolidone solution obtained in step (1), the blue upper layer solution was collected, and after adding a precipitant, (8,4)-SWCNT was prepared by dispersing in water.

[0021] (3) Add cleaning agent to clean, centrifuge to obtain precipitate, add deionized water, single-stranded DNA and sodium chloride to obtain solution IV, remove free single-stranded DNA, and sonicate to obtain the final product.

[0022] Preferably, in step (1), the first solution comprises an 8-12% diethanol solution and a 6-10% polyethylene glycol solution. The 8-12% concentration can be, for example, 8%, 9%, 10%, 11%, or 12%. The 6-10% concentration can be, for example, 6%, 7%, 8%, 9%, or 10%.

[0023] Preferably, in step (1), the incubation temperature is 20-30℃ and the incubation time is 5-10 days. The 20-30℃ can be, for example, 20℃, 22℃, 24℃, 26℃, 28℃, or 30℃. The 5-10 days can be, for example, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days.

[0024] Preferably, in step (1), the solvent includes a NaCl solution.

[0025] Preferably, in step (1), the second solvent comprises single-walled carbon nanotubes with a concentration of 0.5-3 mg / mL, single-stranded DNA with a concentration of 0.5-3 mg / mL, and NaCl with a concentration of 20-40 mM. The 20-40 mM concentration can be, for example, 20 mM, 22 mM, 24 mM, 26 mM, 28 mM, 30 mM, 32 mM, 34 mM, 36 mM, 38 mM, or 40 mM. The 0.5-3 mg / mL concentration can be, for example, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, or 3 mg / mL.

[0026] Preferably, in step (1), the volume ratio of the supernatant to the upper phase in solution three is 1:(0.5-2). The (0.5-2) can be, for example, 0.5, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8 or 2, etc.

[0027] Preferably, in step (1), the concentration of polyvinylpyrrolidone in the polyvinylpyrrolidone solution is 5-15%. The 5-15% can be, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%.

[0028] Preferably, in step (1), the volume ratio of the solution tri and the polyvinylpyrrolidone solution is 1:(0.5-2). The (0.5-2) can be, for example, 0.5, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8 or 2, etc.

[0029] Preferably, in step (2), the volume ratio of the lower layer solution, the upper layer phase, and the polyvinylpyrrolidone solution is (130-180): (130-180):1. The (130-180) can be, for example, 130, 140, 150, 160, 170, or 180.

[0030] Preferably, in step (2), the concentration of polyvinylpyrrolidone in the polyvinylpyrrolidone solution is 25-35%. The 25-35% can be, for example, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35%, etc.

[0031] Preferably, in step (2), the precipitant includes sodium thiocyanate.

[0032] Preferably, in step (3), the solution comprises 1%-3% SDC-(8,4)-SWCNT, a final concentration of 1-3 μg / μL single-stranded DNA, and 25-35 mM NaCl. The 25-35 mM concentration can be, for example, 25 mM, 26 mM, 28 mM, 30 mM, 32 mM, 34 mM, or 35 mM. The 1%-3% concentration can be, for example, 1%, 1.5%, 2%, 2.5%, or 3%. The 1-3 μg / μL concentration can be, for example, 1 μg / μL, 1.5 μg / μL, 2 μg / μL, 2.5 μg / μL, or 3 μg / μL.

[0033] Preferably, when the nucleic acid sequence of the single-stranded DNA is SEQ ID NO:2 and / or SEQ ID NO:3, the preparation method includes: replacing the nucleic acid sequence in the neurotransmitter sensing array linked to SEQ ID NO:1.

[0034] Preferably, the substitution includes: adjusting the absorbance of the neurotransmitter sensing array connected to SEQ ID NO:1, mixing it with sodium deoxycholate solution to obtain solution five, adding a precipitant and centrifuging to obtain a precipitate, and resuspending it to obtain the neurotransmitter sensing array connected to SEQ ID NO:2 and / or SEQ ID NO:3.

[0035] Preferably, the solution five comprises (8,4)-SWCNT linked to SEQ ID NO:1 with an absorbance of 2-4 at 1134 nm and sodium deoxycholate with a concentration of 0.5-2%. The 0.5-2% concentration can be, for example, 0.5%, 0.6%, 0.8%, 1.0%, 1.2%, 1.4%, 1.6%, 1.8%, or 2.0%. The absorbance of 2-4 can be, for example, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, or 4.

[0036] Preferably, the precipitant comprises sodium thiocyanate.

[0037] Preferably, the cleaning agent comprises methanol and / or isopropanol.

[0038] Thirdly, the present invention provides a method for qualitative analysis and quantitative detection of neurotransmitters, wherein the neurotransmitter sensing array described in the first aspect is mixed with the neurotransmitter to obtain a mixed solution, which is then incubated in a solvent and subjected to near-infrared fluorescence detection.

[0039] Preferably, the volume ratio of the neurotransmitter to the neurotransmitter sensing array is 1:(0.5-5). The (0.5-5) can be, for example, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5.

[0040] Preferably, the concentration of the neurotransmitter is 5-50 μM, for example, it can be 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM or 50 μM.

[0041] Preferably, the solvent includes a PBS solution with a concentration of 5-20 mM, such as 5 mM, 10 mM, 15 mM or 20 mM.

[0042] Preferably, the volume ratio of the mixed solution to the solvent is 1:(5-15). The (5-15) can be, for example, 5, 6, 8, 10, 12, 14, or 15.

[0043] Compared with the prior art, the present invention has at least the following beneficial effects:

[0044] 1. This invention constructs a fluorescence sensing array using (8,4)-ssDNA-SWCNTs and three non-specific ssDNA sequences (F14, (GT)10, (TCG)4TC). The interaction between ssDNA and SWCNTs is mainly influenced by factors such as conformational matching of ssDNA, van der Waals forces, and charge attraction. The addition of specific neurotransmitters will interact with neurotransmitters in different ssDNA-SWCNTs solutions, thereby producing unique fluorescence responses. Based on these fluorescence responses, linear discriminant analysis (LDA) can be used to effectively distinguish and detect multiple neurotransmitters.

[0045] 2. The fluorescence sensing array constructed in this invention can accurately identify high concentrations of neurotransmitters, and the proposed strategy can provide different response curves for single neurotransmitters and mixtures of neurotransmitters. Attached Figure Description

[0046] Figure 1 The diagram shows the principle of a fluorescent sensing array for neurotransmitter pattern recognition based on (8,4) chiral carbon nanotubes. Figure a is a schematic diagram of the principle, figure b is a schematic diagram of the fluorescence signal change value, figure c is a diagram of the original data of the fluorescence signal change value, and figure d is a pattern recognition diagram.

[0047] Figure 2 The following are fluorescence spectra of the (8,4)-ssDNA-SWCNT sensor array for detecting neurotransmitters. Figure a shows the fluorescence spectrum of ssDNA with F14, Figure b shows the fluorescence spectrum of ssDNA with (GT)10, and Figure c shows the fluorescence spectrum of ssDNA with (TCG)4TC and F14.

[0048] Figure 3 The figures are characterization diagrams of (8,4) chiral carbon nanotube materials. Figure A is the UV-Vis-NIR absorption diagram, Figure B is the NIR fluorescence spectrum, and Figure C is the NIR emission diagram.

[0049] Figure 4 The images show near-infrared fluorescence camera characterizations of the (8,4)-F14-SWCNT sensor array. Image A shows the near-infrared fluorescence camera images of F14 with different neurotransmitters, and Image B shows the grayscale images of F14 with different neurotransmitters.

[0050] Figure 5 The images show near-infrared fluorescence camera characterizations of the (8,4)-(GT)10-SWCNT sensor array. Image A shows near-infrared fluorescence camera images of different neurotransmitters in the (GT)10 pair, and image B shows grayscale images of different neurotransmitters in the (GT)10 pair.

[0051] Figure 6The images show near-infrared fluorescence camera characterizations of the (8,4)-(TCG)4TC-SWCNT sensor array. Image A shows the near-infrared fluorescence camera images of (TCG)4TC for different neurotransmitters, and Image B shows the grayscale images of (TCG)4TC for different neurotransmitters.

[0052] Figure 7 The figures show the qualitative analysis results of different neurotransmitters. Figure A shows the fluorescence signal changes of seven different neurotransmitters detected by three sequences, Figure B shows the LDA pattern recognition data, and Figure C shows the fluorescence signal change heatmap.

[0053] Figure 8 The figures show the qualitative analysis results of the mixture of two neurotransmitters. Figure A shows the fluorescence signal changes of the two neurotransmitters detected by three sequences, Figure B shows the LDA pattern recognition data, and Figure C shows the fluorescence signal change heatmap.

[0054] Figure 9 Figure A shows the qualitative analysis results of the mixture of three neurotransmitters. Figure B is a schematic diagram of the fluorescence signal changes of the three sequences detecting the mixture of three neurotransmitters. Figure C is a schematic diagram of LDA pattern recognition data. Figure C is a heatmap of fluorescence signal changes.

[0055] Figure 10 Figure 1 shows the quantitative detection results of serotonin by the (8,4)-F14-SWCNT sensor array. Figure 2a shows the near-infrared fluorescence of serotonin at different concentrations detected by the F14 sequence, and Figure 3b shows the linearity within the detection range.

[0056] Figure 11 Figure 1 shows the simulated serum sample detection results of the (8,4)-F14-SWCNT sensor array. Figure 2a shows the concentration optimization for dopamine detection, Figure 3b shows the concentration optimization for serotonin detection, and Figure 4c shows the system detection time optimization.

[0057] Figure 12 The results show the effects of different parameters on the (8,4)-F14-SWCNT sensor array. Figure A is a schematic diagram of the fluorescence signal changes of seven different neurotransmitters detected by three sequences in simulated serum. Figure B is a diagram of LDA pattern recognition data. Figure C is a heatmap of fluorescence signal changes.

[0058] Figure 13 The image shows the detection results for different single-stranded DNAs. Detailed Implementation

[0059] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0060] The sources of reagents used in the following examples are:

[0061] Single-walled carbon nanotubes: Sigma-Aldrich (Shanghai);

[0062] Polyethylene glycol: Alfa Essar (China) Chemical Co., Ltd.;

[0063] Dextran: Sigma-Aldrich (Shanghai);

[0064] Polyvinylpyrrolidone: Sigma-Aldrich (Shanghai).

[0065] Example 1

[0066] This embodiment describes the fabrication of a neurotransmitter sensing array.

[0067] (1) Sorting of (8,4)-SWCNT

[0068] First, a solution of 12 mL of 20 wt% diethylene glycol (DX) and 3.44 mL of 60 wt% polyethylene glycol (PEG) was prepared and mixed with 9 mL of deionized water to form a polymer aqueous two-phase (ATP) stock solution. This solution was then vigorously vortexed and incubated at 25°C for 7 days to promote phase separation. Finally, the upper and lower phases were carefully extracted and stored separately.

[0069] Next, a DNA-SWCNT dispersion was prepared. F14 DNA, (GT)10 DNA, and (TCG)4TC DNA were sonicated with SWCNTs in a solution containing 30 mM NaCl. The concentration of SWCNTs was 1 mg / mL, and the concentrations of F14 DNA, (GT)10 DNA, and (TCG)4TC DNA were each 2 mg / mL. The mixture was then centrifuged to remove impurities, and the supernatant was collected for subsequent ATP separation. The nucleic acid sequence of F14 DNA is shown in SEQ ID NO:1, the nucleic acid sequence of (GT)10 DNA is shown in SEQ ID NO:2, and the nucleic acid sequence of (TCG)4TC DNA is shown in SEQ ID NO:3.

[0070] The separation process employed the scalable ATPE method. 180 μL of L14-SWCNTs dispersion, 180 μL of (GT)10-SWCNTs dispersion, and 180 μL of (TCG)4TC-SWCNTs dispersion were each mixed with 300 μL of the upper phase, followed by vortexing and centrifugation to form an ATP system. Subsequently, 1.9 μL of polyvinylpyrrolidone (PVP) (10 wt%) was added to the ATP system. After thorough vortexing and gentle centrifugation, the resulting solution exhibited a pale pink upper layer (labeled 1T) and a black lower layer. Next, 300 μL of the collected black lower layer was mixed with 300 μL of blank upper phase and 2 μL of PVP (30 wt%), followed by thorough vortexing and gentle centrifugation to obtain a blue upper layer rich in (8,4)-SWCNTs (labeled 2T). Finally, 2T was extracted and precipitated using 2.0 M sodium thiocyanate (NaSCN) solution. Three (8,4)-SWCNTs were obtained by sorting with F14 DNA, (GT)10 DNA and (TCG)4TC DNA, respectively.

[0071] (2) Substitution of nucleic acid sequences

[0072] The three types of (8,4)-SWCNT were diluted in deionized water, and their absorbance at the E11 transition (1134 nm) was adjusted to 4.0. 100 µL of SWCNT solution was mixed with 100 µL of 2% sodium deoxycholate (SDC) to promote the exchange of ssDNA with SDC. After adding 200 µL of 2.0 M sodium thiocyanate (NaSCN) solution, SWCNT precipitation was induced. The mixture was centrifuged at 17,000 g for 3 minutes, the supernatant was discarded, and the precipitate was resuspended in 100 µL of deionized water.

[0073] (3) Cleaning

[0074] To remove residual DOC, 180 µL of methanol was slowly added, followed by 400 µL of isopropanol. The mixture was centrifuged again at 17,000 g for 3 minutes, and the supernatant was discarded. Finally, 20 µL of 10.0 mg / mL F14 DNA, 10.0 mg / mL (GT)10 DNA, and 10.0 mg / mL (TCG)4TC DNA, along with 100 µL of deionized water, were added, and the NaCl concentration was adjusted to 30 mM. Three rounds of ultrafiltration were performed using a 30 kDa cutting membrane to remove the free target DNA sequence. The resulting precipitate was sonicated in an ice bath for 10 minutes to obtain three functionalized F14-ssDNA-(8,4)-SWCNT, (GT)10-ssDNA-(8,4)-SWCNT, and (TCG)4TC-ssDNA-(8,4)-SWCNT.

[0075] Example 2

[0076] This embodiment performs neurotransmitter detection.

[0077] To detect seven different neurotransmitters (serotonin (5-HT), dopamine (DA), adrenaline (Ep), acetylcholine (Ach), glutamate (Glu), glycine (Gly), and histamine (HA)), 5 μL of F14-ssDNA-(8,4)-SWCNT, 5 μL of (GT)10-ssDNA-(8,4)-SWCNT, and 5 μL of (TCG)4TC-ssDNA-(8,4)-SWCNT were incubated with 5 μL of different concentrations of neurotransmitter solutions (5 μM, 25 μM, and 50 μM) in 90 μL of 10 mM pH 7.4 PBS buffer. After vortexing to homogenize, near-infrared fluorescence detection was performed. Fluorescence emission spectra from 900 to 1300 nm were captured using an excitation wavelength of 596 nm, and the optimal fluorescence intensity was recorded at 1134 nm.

[0078] Using the LDA linear discriminant analysis method, the fluorescence response matrices (3 ssDNA-(8,4)-SWCNTs × 7 neurotransmitters × 6 repeats) at three neurotransmitter concentrations of 5.0 μM, 25 μM, and 50 μM were processed and analyzed to obtain fluorescence pattern diagrams of different analytes, and corresponding classification diagrams were drawn using Origin 2021 software.

[0079] The schematic diagram of the specific detection process is as follows: Figure 1 As shown in Figure a, a fluorescence sensing array was constructed using (8,4)-ssDNA-SWCNTs and three non-specific ssDNA sequences (F14, (GT)10, and (TCG)4TC). The interaction between ssDNA and SWCNTs is mainly influenced by factors such as conformational matching, van der Waals forces, and electrostatic attraction of ssDNA. The analytes, including neurotransmitters, interact with these ssDNA sequences through hydrogen bonds, electrostatic forces, and van der Waals forces, leading to a decrease or increase in the near-infrared fluorescence signal of SWCNTs. The study found that the addition of specific neurotransmitters interacts with neurotransmitters in different ssDNA-SWCNTs solutions, resulting in unique fluorescence responses. Based on these fluorescence responses, linear discriminant analysis (LDA) can effectively distinguish and detect multiple neurotransmitters. We developed a neurotransmitter detection sensing array based on chiral SWCNTs, such as... Figure 1 As shown, DNA separation was first performed in an aqueous two-phase system using polyethylene glycol (PEG, molecular weight 1.5 kDa) and dihydroxyglucosamine (DX, molecular weight 250 kDa) via DNA dispersion technology. Figure 1 As shown in Figure a, relatively pure (8,4)-SWCNTs were obtained. Subsequently, (8,4)-ssDNA-SWCNTs and ssDNA sequences with three non-specific sequences (F14, (GT)10, and (TCG)4TC) were constructed into a fluorescence sensing array. The interaction between ssDNA and SWCNTs is mainly influenced by factors such as conformational matching, van der Waals forces, and electrostatic attraction of ssDNA. The analyte neurotransmitters interact with these ssDNA sequences through hydrogen bonds, electrostatic forces, and van der Waals forces, leading to a decrease or increase in the near-infrared fluorescence signal of SWCNTs, such as... Figure 1 As shown in Figure c. The study found that the addition of specific neurotransmitters interacts with neurotransmitters in different ssDNA-SWCNT solutions, resulting in unique fluorescent responses. Based on these fluorescent responses, linear discriminant analysis (LDA) can effectively distinguish and detect various neurotransmitters, such as... Figure 1 As shown in d.

[0080] Specific test results are as follows: Figure 2 Figure a shows the effect of seven neurotransmitters on the fluorescence intensity of (8,4)-F14-SWCNT at an emission wavelength of 1134 nm. 5-HT significantly reduced the fluorescence intensity, while DA and Ep showed a clear trend of increasing fluorescence intensity. Figure 2 As shown in b, seven neurotransmitter pairs (8,4)-(GT) are displayed. 10 The fluorescence intensity response of -SWCNT at the same emission wavelength showed that DA, Ep, and HA significantly enhanced the fluorescence intensity, with DA showing the most significant enhancement. Figure 2 As shown in c, the fluorescence intensity of seven neurotransmitters at the emission wavelength of (8,4)-(TCG)4TC-SWCNT at 1134 nm is displayed, indicating that DA and Ep significantly enhance the fluorescence intensity of (8,4)-(TCG)4TC-SWCNT.

[0081] and with Figure 3 As shown, Figure 3 Characterization of the proposed SWCNT material, Figure 3 The ultraviolet-visible-near-infrared absorption spectrum shown in Figure a is... Figure 3 Figure b shows the near-infrared fluorescence emission spectrum. Figure 3 Figure c shows the near-infrared two-dimensional excitation emission spectrum.

[0082] Example 3

[0083] This embodiment investigates the effects of different neurotransmitters on (8,4)-ssDNA-SWCNT.

[0084] To more directly compare the effects of different neurotransmitters on (8,4)-ssDNA-SWCNT, we characterized the changes in near-infrared fluorescence intensity. 5-HT, Ach, DA, Ep, Glu, Gly, and HA were added to (8,4)-SWCNT samples of three different ssDNAs, with a final concentration of 50 μM for each. Using water as a control, changes in fluorescence intensity were observed using a near-infrared fluorescence microscope at an excitation wavelength of 808 nm, demonstrating the changes in fluorescence intensity resulting from the interaction between (8,4)-F14-SWCNT and the seven neurotransmitters.

[0085] like Figure 4 As shown in Figures A and B, the results indicate that F14 has the most significant quenching effect on (8,4)-F14-SWCNT, consistent with the change in the relative fluorescence signal of (8,4)-F14-SWCNT. Figure 5 As shown in Figures A and B, the fluorescence intensity changes of (8,4)-(GT)10-SWCNT interacting with seven neurotransmitters show that DA and Ep significantly enhance the fluorescence signal. Figure 6 As shown in Figures A and B, the interactions between (8,4)-(TCG)4TC-SWCNT and seven neurotransmitters are revealed. DA, Ep, and HA show the most significant enhancement effects on the fluorescence signal, consistent with the relative fluorescence signal changes of (8,4)-(TCG)4TC-SWCNT. Combined with the near-infrared fluorescence intensity variation characterization, this indicates that the interactions between the seven neurotransmitters and (8,4)-ssDNA-SWCNT exhibit stable and reliable fluorescence signal changes.

[0086] Example 4

[0087] This embodiment investigates the discriminative capability of the (8,4)-ssDNA-SWCNT sensor array.

[0088] Seven widely used neurotransmitters (5-HT, dopamine (DA), adrenaline (Ep), acetylcholine (Ach), glutamate (Glu), glycine (Gly), and histamine (HA)) were selected as analytes. The fluorescence signal change value (I-I0 / I0) was defined as the response signal, where I0 and I represent the near-infrared fluorescence emission intensity with and without the neurotransmitter, respectively. Fluorescence responses were measured using concentrations of the neurotransmitter at 5 μM, 25 μM, and 30 μM. F14, (GT)10, and (TCG)4TC exhibited different affinities for the neurotransmitter.

[0089] like Figure 7As shown in Figures A, B, and C, the (8,4)-ssDNA-SWCNT sensor array exhibited different fluorescence responses to seven neurotransmitters at 50 μM. Linear discriminant analysis (LDA) was performed on the dataset (3 sensing elements × 7 neurotransmitters × 6 replicates) using discriminant factors, generating a two-dimensional canonical score map with a 96.9% confidence level. This visually divided the seven neurotransmitters into seven non-overlapping groups, and the heatmap showed significant differences, indicating its ability to completely distinguish neurotransmitters. Further evaluation of its qualitative ability involved testing with different concentrations of neurotransmitters. LDA analysis showed that only high-concentration analytes (50 µM) could be effectively distinguished, indicating limited sensitivity to neurotransmitters.

[0090] like Figure 8 As shown in Figures A, B, and C, in addition to successfully detecting seven neurotransmitters, we further investigated the differentiation of neurotransmitter mixtures. To demonstrate the capability of the sensing strategy, the sensor detected mixtures of two neurotransmitters with different mixing ratios (100% 5-HT + 0% DA, 80% 5-HT + 20% DA, 60% 5-HT + 40% DA, 40% 5-HT + 60% DA, 20% 5-HT + 80% DA, 0% 5-HT + 100% DA), as shown in Figures A, B, and C. Figure 3 As shown, the six neurotransmitter samples are clearly distributed in six independent clusters in the typical scoring map, and the cross-validation accuracy of the identification is 100%. The differences can also be clearly seen in the heatmap.

[0091] like Figure 9 Figures A, B, and C show the LDA results of samples containing three neurotransmitters (20% 5-HT + 40% DA + 40% Ep, 40% 5-HT + 40% DA + 20% Ep, 30% 5-HT + 10% DA + 60% Ep, 50% 5-HT + 30% DA + 20% Ep, 60% 5-HT + 30% DA + 10% Ep, and 80% 5-HT + 10% DA + 10% Ep). No overlap was observed, and this clear distinction indicates that the (8,4)-ssDNA-SWCNT sensor array can accurately identify high concentrations of neurotransmitters. Furthermore, the proposed strategy can provide different response curves for individual neurotransmitters and mixtures of neurotransmitters.

[0092] Example 5

[0093] This embodiment explores the qualitative capabilities of the (8,4)-ssDNA-SWCNT sensor array.

[0094] Studies have shown that our (8,4)-ssDNA-SWCNT probe can be used for qualitative analysis of neurotransmitters. Serotonin (5-HT), a key neurotransmitter, regulates various physiological processes, and imbalances in its levels may lead to neurodegenerative diseases, depression, diabetes, and serotonin syndrome. Furthermore, 5-HT acts as a growth factor for tumor cells, influencing various stages of tumor development. Measuring 5-HT concentrations in blood and serum can aid in the early diagnosis of related diseases.

[0095] The results showed that (8,4)-F14-SWCNT exhibited good detection ability for 5-HT. Figure 10 As shown in Figure a, near-infrared fluorescence spectral data at different 5-HT concentrations are presented; to accurately determine the 5-HT concentration, a calibration curve was established under optimized conditions, as shown in Figure a. Figure 10 As shown in Figure b, (8,4)-F14-SWCNT exhibits excellent linearity for the detection of 5-HT in the range of 50 nM to 50 µM, with an R² of 0.997.

[0096] Example 6

[0097] This embodiment investigates the discriminative power of the (8,4)-ssDNA-SWCNT sensor array.

[0098] The performance of the sensing system was further evaluated to distinguish neurotransmitters in real human serum. To this end, the sensor array system was applied to human serum samples spiked with seven relevant proteins to obtain reliable discrimination capabilities of the sensor. However, in the current situation, such as... Figure 11 As shown in Figures a, b, and c, each relevant neurotransmitter involved in the serum sample matrix produced a distinct response, and all seven neurotransmitters achieved 100% identification accuracy, as illustrated by a heatmap of the response patterns of neurotransmitters (50 μM) in human serum using a sensor array. Therefore, these results suggest that this (8,4)-F14-SWCNT-based fluorescent nanosensor may be applicable to distinguishing neurotransmitters in real biological samples.

[0099] Example 7

[0100] This embodiment explores the effects of single-stranded DNA concentration and DNA sequence on the results.

[0101] (1) Different single-stranded DNA concentrations

[0102] This embodiment explores the effect of different single-stranded DNA concentrations on the DNA sequence effect.

[0103] like Figure 12As shown, F14-SWCNT exhibited significant fluorescence quenching upon the addition of 5-HT, indicating a strong selective interaction between F14-SWCNT and 5-HT, demonstrating its potential as a 5-HT recognition element. In contrast, (GT)10-SWCNT showed significant fluorescence enhancement in the presence of DA and Ep, indicating that this sequence has high sensitivity to catecholamine neurotransmitters. Furthermore, (TCG)4TC-SWCNT showed significant fluorescence enhancement in the presence of DA, Ep, and HA, indicating that this sequence also has good responsiveness to structurally diverse neurotransmitters.

[0104] (2) Different DNA sequences

[0105] This embodiment investigated the effect of different DNA sequences on the results. The specific DNA sequences used are shown in Table 1.

[0106] Table 1

[0107]

[0108] like Figure 13 As shown, F14, (GT)10, and (TCG)4TC have different responses to the three different neurotransmitters. A1, A2, (AC)6, (GT)15, and (AT)15 have small responses and are therefore not used. (GA)15, (G3T)7, (CCG)5CC, and (G2T)10 have low replacement efficiency and unstable replacement products.

[0109] In summary, this invention utilizes DNA dispersion technology to separate (8,4)-SWCNTs in an aqueous two-phase system using polyethylene glycol (PEG, molecular weight 1.5 kDa) and dihydroxyglucosamine (DX, molecular weight 250 kDa) to obtain relatively pure (8,4)-SWCNTs. Subsequently, (8,4)-ssDNA-SWCNTs and ssDNA sequences with three non-specific sequences (F14, (GT)10, (TCG)4TC) are constructed into a fluorescent sensing array. This fluorescent sensing array can effectively distinguish and detect multiple neurotransmitters.

[0110] 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 neurotransmitter sensing array, characterized in that, The neurotransmitter sensing array includes single-walled carbon nanotubes connected to single-stranded DNA. The single-walled carbon nanotubes are (8,4)-SWCNTs.

2. The neurotransmitter sensing array according to claim 1, characterized in that, The nucleic acid sequence of the single-stranded DNA includes any one or a combination of at least two of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:

3.

3. A method for preparing a neurotransmitter sensing array according to claim 1 or 2, characterized in that, The preparation method includes: preparing (8,4)-SWCNT, and then ligating (8,4)-SWCNT with single-stranded DNA.

4. The method for preparing the neurotransmitter sensing array according to claim 3, characterized in that, When the nucleic acid sequence of the single-stranded DNA is SEQ ID NO:1, the preparation method includes: (1) Mix diethanol solution, polyethylene glycol solution and deionized water to obtain solution one. After incubation, obtain upper and lower phases. Place single-walled carbon nanotubes and single-stranded DNA in a solvent to obtain solution two. After sonication and centrifugation, collect the supernatant. Mix the supernatant with the upper phase to obtain solution three. Add polyvinylpyrrolidone solution and mix to obtain upper and lower solutions. (2) After mixing the lower layer solution, upper phase and polyvinylpyrrolidone solution obtained in step (1), the blue upper layer solution was collected, and after adding a precipitant, (8,4)-SWCNT was prepared by dispersing in water. (3) Add cleaning agent to clean, centrifuge to obtain precipitate, add deionized water, single-stranded DNA and sodium chloride to obtain solution IV, remove free single-stranded DNA, and sonicate to obtain the final product.

5. The method for preparing the neurotransmitter sensing array according to claim 4, characterized in that, In step (1), the first solution includes an 8-12% diethanol solution and a 6-10% polyethylene glycol solution; Preferably, in step (1), the incubation temperature is 20-30℃ and the incubation time is 5-10 days; Preferably, in step (1), the solvent includes a NaCl solution; Preferably, in step (1), the solvent two includes single-walled carbon nanotubes with a concentration of 0.5-3 mg / mL, single-stranded DNA with a concentration of 0.5-3 mg / mL, and NaCl with a concentration of 20-40 mM; Preferably, in step (1), the volume ratio of the supernatant to the upper phase in solution three is 1:(0.5-2); Preferably, in step (1), the concentration of polyvinylpyrrolidone in the polyvinylpyrrolidone solution is 5-15%; Preferably, in step (1), the volume ratio of the solution tri and the polyvinylpyrrolidone solution is 1:(0.5-2).

6. The method for preparing a neurotransmitter sensing array according to any one of claims 3-5, characterized in that, In step (2), the volume ratio of the lower layer solution, the upper layer phase and the polyvinylpyrrolidone solution is (130-180):(130-180):1; Preferably, in step (2), the concentration of polyvinylpyrrolidone in the polyvinylpyrrolidone solution is 25-35%; Preferably, in step (2), the precipitant includes sodium thiocyanate; Preferably, in step (3), the solution includes SDC-(8,4)-SWCNT at a concentration of 1%-3%, single-stranded DNA at a concentration of 1-3 μg / μL, and NaCl at a concentration of 25-35mM.

7. The method for preparing a neurotransmitter sensing array according to any one of claims 4-6, characterized in that, When the nucleic acid sequence of the single-stranded DNA is SEQ ID NO:2 and / or SEQ ID NO:3, the preparation method includes: replacing the nucleic acid sequence in the neurotransmitter sensing array linked to SEQ ID NO:1 to obtain the DNA. Preferably, the substitution includes: adjusting the absorbance of the neurotransmitter sensing array connected to SEQ ID NO:1, mixing it with sodium deoxycholate solution to obtain solution five, adding a precipitant and centrifuging to obtain a precipitate, and resuspending it to obtain the neurotransmitter sensing array connected to SEQ ID NO:2 and / or SEQ ID NO:3; Preferably, the solution five comprises (8,4)-SWCNT linked to SEQ ID NO:1 with an absorbance of 2-4 at 1134 nm and sodium deoxycholate with a concentration of 0.5-2%; Preferably, the precipitant comprises sodium thiocyanate; Preferably, the cleaning agent comprises methanol and / or isopropanol.

8. A method for qualitative analysis and quantitative detection of neurotransmitters, characterized in that, The method includes mixing the neurotransmitter sensing array according to claim 1 or 2 with a neurotransmitter to obtain a mixed solution, incubating it in a solvent, and performing near-infrared fluorescence detection.

9. The method for qualitative analysis and quantitative detection of neurotransmitters according to claim 8, characterized in that, The volume ratio of the neurotransmitter to the neurotransmitter sensing array is 1:(0.5-5); Preferably, the concentration of the neurotransmitter is 5-50 μM.

10. The method for qualitative analysis and quantitative detection of neurotransmitters according to claim 8 or 9, characterized in that, The solvent includes a PBS solution with a concentration of 5-20 mM; Preferably, the volume ratio of the mixed solution to the solvent is 1:(5-15).

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