A method to avoid the influence of cations on carbon nanotube fluorescence sensors, and a gel-based dopamine sensor based on Al-(AT)15-SWCNTs complex and its application.

Abstract

This invention discloses a method to avoid the influence of cations on carbon nanotube fluorescent sensors, and a gel-based dopamine sensor based on Al-(AT)15-SWCNTs complex and its application. The method to avoid the influence of cations on carbon nanotube fluorescent sensors first involves preparing a single-stranded DNA sequence (AT). 15 Modified single-walled carbon nanotube solution ((AT) 15 -SWCNTs); then (AT) 15 The SWCNTs solution and agarose gel were mixed at a 1:1 volume ratio and allowed to settle at (AT). 15 -SWCNTs solidify to obtain a gel-based dopamine sensor; the gel-based dopamine sensor is placed in an Al(NO₃)₃ solution for at least one hour, and then washed by immersing it in an HCl solution with pH ≤ 3.7 to remove residual free Al from the gel. 3+ The ions ultimately yield Al−(AT). 15 A gel-based dopamine sensor for ssDNA-SWCNT complexes. Trivalent aluminum ions pre-occupy the interaction sites of phosphate groups to prevent further interactions between other cations and phosphate groups. Agarose gel-based ssDNA-SWCNTs avoid cation-induced aggregation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biosensors, and particularly relates to a method for avoiding the influence of cations on a carbon nanotube fluorescent sensor and an Al-(AT) 15 -SWCNTs composite gel-based dopamine sensor and application.

[0002] The present application belongs to the 4.4.3 special fermentation products and biological process equipment biosensor under the 4.4 biological manufacturing industry key direction in the 4.4 biological industry in the strategic new industry catalogue. BACKGROUND

[0003] Single-stranded Deoxyribonucleic Acid (ssDNA) modified Single-walled Carbon Nanotubes (SWCNTs) fluorescent sensors (ssDNA-SWCNTs) have shown great potential for detecting biomarkers, including glucose, neurotransmitters (dopamine, serotonin, norepinephrine), nitric oxide, chemotherapeutic agents, and lipids, with working wavelengths in the near-infrared region. The low detection limit, biotransparency, and photostability of ssDNA-SWCNT fluorescence provide the possibility of realizing non-invasive in vivo continuous monitoring. However, body fluids are a complex chemical environment containing various ions, which can cause the precipitation of oligonucleotide-suspended single-walled carbon nanotubes in the aqueous phase, thereby quenching the fluorescence of single-walled carbon nanotubes. In addition to the direct aggregation of single-walled carbon nanotubes, salt-single-stranded DNA interactions can also have a significant impact on the fluorescence of single-stranded DNA-single-walled carbon nanotubes, such as the adjustment of peak intensity and position. The mechanism behind this fluorescence change can be caused by a change in the wrapping conformation, resulting in differences in water, oxygen molecule interactions, or changes in electronic interactions between single-stranded DNA and single-walled carbon nanotubes. Water shielding and deoxygenation can be attributed to the increase in DNA coverage of single-walled carbon nanotubes, which can be attributed to the screening or neutralization of negatively charged phosphate groups. Since most single-walled carbon nanotube-based optical sensors rely on intensity changes or wavelength shifts, shielding salt-ssDNA interactions can help reduce noise and stabilize the sensing ability.(GT) 15 The ssDNA sequence shows selective response to dopamine. However, according to literature and experimental experience, (AT) 15 DNA-wrapped SWCNTs sensors have higher initial fluorescence intensity, and their response to neurotransmitters ((I-I0) / I0) is lower than other DNA, such as(GT) 15 DNA. Because (AT) 15The compact conformation of DNA is a considerable advantage. To reduce the effect of salt on fluorescence, Gillen et al. reported the use of locked nucleic acid nucleotides (LNAs) at several positions of the oligonucleotide. The results showed that, compared with traditional oligonucleotides, the salt-induced wavelength shift was reduced and the wrapped conformation and signal stability were increased when using LNAs. However, due to the negatively charged phosphate group, the interaction between the cations present in the biological fluids and the oligonucleotide is inevitable. The conformation changes induced by monovalent and divalent cations are related to the salt concentration, so the effect on fluorescence is dynamic, which can lead to unpredictable results. SUMMARY

[0004] In view of the above problems existing in the prior art, the present application provides a method for avoiding the influence of cations on a carbon nanotube fluorescence sensor, a gel-based dopamine sensor and application.

[0005] The purpose of the present application is achieved in the following manner:

[0006] A method for avoiding the influence of cations on a carbon nanotube fluorescence sensor, comprising the steps of,

[0007] (1) preparing a single-stranded DNA sequence (AT) 15 modified single-walled carbon nanotube solution ((AT) 15 -SWCNTs);

[0008] (2) preparing a gel-based dopamine sensor of (AT) 15 -SWCNTs

[0009] Prepare 1-4% agarose gel with deionized water, mix (AT) 15 -SWCNTs solution and agarose gel at a volume ratio of 1:1, and wait until (AT) 15 -SWCNTs solidifies into a solid to obtain a gel-based dopamine sensor;

[0010] (3) preparing a gel-based dopamine sensor of Al-(AT) 15 -SWCNTs complex

[0011] Put the gel-based dopamine sensor obtained in step (2) into an Al(NO3)3 solution for at least one hour, and immerse the treated gel-based dopamine sensor in an HCl solution with pH ≤ 3.7 to wash off the free Al 3+ ions remaining in the gel, and finally obtain a gel-based dopamine sensor of Al-(AT) 15 -SWCNTs complex.

[0012] Step (1) prepares (AT) 15The specific process of the SWCNTs solution is as follows,

[0013] a. Dissolve ssDNA in deionized water to adjust the concentration to 1 mg / ml to obtain an ssDNA solution, wherein the ssDNA is (AT) 15 ;

[0014] b. Mix the ssDNA solution and the SWCNT powder according to a mass ratio of 1:1, and then place them in a water bath cup of an ultrasonic disperser, so as to "wrap" the ssDNA on the surface of the SWCNTs and realize the dispersion of the SWCNTs;

[0015] c. Treat the dispersed mixture solution on a centrifuge, and reserve 80% of the supernatant;

[0016] d. Centrifuge the supernatant by using an ultrafiltration membrane to remove all free ssDNA sequences in the supernatant;

[0017] e. Dilute the ssDNA-CNTs suspension to obtain an (AT) 15 -SWCNTs solution.

[0018] The resistivity of the deionized water in step (1) is 18.2 MΩ·cm.

[0019] The gel-based dopamine sensor treated by the high-concentration Al(NO3)3 solution is repeatedly immersed in an HCl solution for washing 4 times.

[0020] A gel-based dopamine sensor of an Al-(AT) 15 -SWCNTs composite is prepared by the above method.

[0021] The gel-based dopamine sensor of the Al-(AT) 15 -SWCNTs composite is used for detecting biomarkers in a body fluid.

[0022] The body fluid is any one of urine, blood or cerebrospinal fluid.

[0023] Compared with the prior art, the present application uses trivalent aluminum ions to preoccupy the interaction sites of the phosphate groups to prevent further interaction between other cations and the phosphate groups. Compared with the ssDNA-SWCNT suspension in water, the ssDNA-SWCNT based on the agarose gel can avoid cation-induced aggregation. The aluminum pretreatment process quenches the (AT) 15The initial fluorescence of the SWCNT gel-based dopamine sensor is reduced, but the sensor exhibits a greater dopamine intensity response in an acidic environment. Therefore, the aluminum pretreatment process is expected to be an important method for enhancing the stability of ion-induced perturbations of gel-based single-stranded DNA suspended single-walled carbon nanotubes, thereby facilitating the application of single-stranded DNA-wrapped single-walled carbon nanotube sensors in in vitro and in vivo analyte detection. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a preparation flowchart of the present application.

[0025] Figure 2 is (AT) 15 Response graph of the SWCNT gel-based dopamine sensor to cations. Left graph: untreated sensor (AT) 15 -SWCNT, right graph: aluminum nitrate-treated sensor Al-(AT) 15 -SWCNT.

[0026] Figure 3 is (AT) 15 Response graph of the SWCNT gel-based dopamine sensor to cations. Left graph: untreated sensor (AT) 15 -SWCNT, right graph: aluminum nitrate-treated sensor Al-(AT) 15 -SWCNT. DETAILED DESCRIPTION

[0027] As Figure 1 shown, a method for avoiding the influence of cations on a carbon nanotube fluorescence sensor includes the following steps:

[0028] (1) Prepare a single-stranded DNA sequence (AT) 15 modified single-walled carbon nanotube solution ((AT) 15 -SWCNTs);

[0029] (2) Prepare (AT) 15 -SWCNTs gel

[0030] Because in an aqueous phase, the addition of cations can cause ssDNA-SWCNTs to aggregate, thereby producing a precipitate. In order to avoid the aggregation of ssDNA-SWCNTs during the aluminum ion treatment process, we uniformly dispersed (AT) 15 -SWCNTs into an original concentration of 1-4% agarose gel at a volume ratio of 1:1, with a 2% concentration being optimal. First, 2% agarose gel was prepared with deionized water, and then (AT) 15 -SWCNTs solution and agarose gel were mixed at a volume ratio of 1:1, and waited until (AT)15 The SWCNTs are solidified to become a solid, and a gel-based dopamine sensor is obtained. The gel-based dopamine sensor is preferably treated in deionized water for 4 hours to stabilize the fluorescence;

[0031] (3) preparing Al-(AT) 15 The gel-based dopamine sensor of the SWCNTs composite

[0032] The gel-based dopamine sensor obtained in step (2) is placed in an Al(NO3)3 solution for more than 1 hour, preferably an Al(NO3)3 solution with a concentration of 1 M. The treated gel-based dopamine sensor is immersed in an HCl solution with a pH of less than or equal to 3.7 for washing, so as to remove free Al 3+ ions remaining in the gel. Preferably, the washing is repeated 4 times, and finally an Al-(AT) 15 SWCNTs composite gel-based dopamine sensor is obtained.

[0033] Step (1) preparing (AT) 15 The specific process of the SWCNTs solution is as follows:

[0034] a. Dissolve ssDNA in deionized water, preferably deionized water with a resistivity of 18.2 MΩ·cm, and adjust to a suitable concentration to obtain an ssDNA solution. The ssDNA is (AT) 15 ;

[0035] b. Mix the ssDNA solution and SWCNT powder according to a mass ratio of 1:1, and then place them in a water bath cup of an ultrasonic disperser. The ssDNA is "wound" on the surface of the SWCNTs to realize the dispersion of the SWCNTs;

[0036] c. Treat the dispersed mixture solution on a centrifuge, and reserve 80% of the supernatant;

[0037] d. Centrifuge the supernatant using an ultrafiltration membrane to remove all free ssDNA sequences in the supernatant;

[0038] e. Dilute the ssDNA-CNTs suspension to obtain an (AT) 15 SWCNTs solution.

[0039] An Al-(AT) 15 SWCNTs composite gel-based dopamine sensor is obtained by the above method.

[0040] The Al-(AT) 15 SWCNTs composite gel-based dopamine sensor is used for detecting biomarkers in a body fluid, which is any one of urine, blood, or cerebrospinal fluid.

[0041] Example 1

[0042] A method for avoiding the influence of cations on a carbon nanotube fluorescent sensor, comprising the following steps:

[0043] (1) Preparation of single-stranded DNA sequence (AT) 15 Modified single-walled carbon nanotube solution (AT) 15 -SWCNTs

[0044] a. Dissolve ssDNA in deionized water with a resistivity of 18.2 MΩ·cm, adjust to a concentration of 100 μM, ssDNA is (AT) 15 ;

[0045] b. Weigh SWCNT to 1 mg / mL, mix the ssDNA solution and SWCNT powder with a mass ratio of about 1:1, and then put them into the water bath cup of the ultrasonic disperser, control the temperature at 4°C, and use the water bath cup model cuphorn, Q700, QSonica in this example, set the amplitude to 1%, and work for 90 minutes, so that the ssDNA is "wrapped" on the surface of the SWCNTs, achieving the dispersion of the SWCNTs;

[0046] c. Treat the dispersed mixture solution on the centrifuge, and reserve 80% of the supernatant, and use the centrifuge model Eppendorf 5424R in this example, centrifuge at 4°C for 4h;

[0047] d. Centrifuge the supernatant using an ultrafiltration membrane to remove all free ssDNA sequences in the supernatant, and use Amicon Ultra 0.5 100 kDa centrifugal filter in this example.

[0048] e. Dilute the ssDNA-CNTs suspension, and finally obtain (AT) 15 -SWCNTs solution, the absorbance of the ssDNA-SWCNTs suspension at 632 nm is 0.742.

[0049] (2) Preparation of (AT) 15 -SWCNTs gel

[0050] First, prepare a 2% agarose gel with deionized water, then mix the (AT) 15 -SWCNTs solution and the agarose gel at a volume ratio of 1:1, take 190 μl of the gel mixture and add it to a culture dish, and wait until the (AT) 15 -SWCNTs solidifies into a solid to obtain a gel-based dopamine sensor.

[0051] (3) Preparation of Al-(AT)15 Gel-based dopamine sensor of SWCNTs composite

[0052] The gel-based dopamine sensor obtained in step (2) was treated in 1 M Al(NO3)3 solution for 2 hours, then the residual Al(NO3)3 solution was removed, and the treated gel-based dopamine sensor was washed in HCl solution with pH=3.7 to remove free Al 3+ ions in the gel, and the washing was repeated 4 times, and finally Al-(AT) 15 -SWCNTs composite was obtained.

[0053] Comparative test 1

[0054] The 18 gel-based dopamine sensors of (AT) 15 -SWCNTs prepared according to the above method were randomly divided into 6 groups, and the 6 groups of sensors were respectively immersed in 1 M NaCl, KCl, MgCl2, CaCl2, Al(NO3)3 and Nd(NO3)3 solutions for 2 hours, and the fluorescence intensity of each sensor excited by 655 nm laser was observed by fluorescence microscope; then the sensors were washed with deionized water, and the fluorescence intensity of the washed sensors excited by 655 nm laser was observed again by fluorescence microscope. As shown in Figure 2 , the test results show that:

[0055] 1) The fluorescence intensity of the sensors treated with NaCl and KCl was enhanced, but after washing, the fluorescence intensity was reduced to a lower level than before adding the salt;

[0056] 2) The sensors treated with MgCl2 and CaCl2 showed higher fluorescence intensity than the sensors treated with NaCl and KCl, and after washing, the intensity of the sensors treated with MgCl2 decreased slightly, and the intensity of the sensors treated with CaCl2 decreased to the same intensity as before adding the salt;

[0057] 3) The fluorescence of the sensors treated with Al(NO3)3 was quenched and could not be recovered after washing; the fluorescence of the sensors treated with Nd(NO3)3 was enhanced, and after washing, the fluorescence intensity of the sensors was further enhanced.

[0058] It is concluded that the (AT) 15 -SWCNTs gel-based dopamine sensor treated with trivalent aluminum ions has more stable properties compared with the pre-treatment with other cations.

[0059] Comparative test 2

[0060] Na+, K+, Ca2+ and Mg2+ ions are the main cations in body fluids such as blood, cerebrospinal fluid and urine. NaCl solution with a concentration of 150 mM, KCl solution with a concentration of 10 mM, CaCl2 solution with a concentration of 5 mM and MgCl2 solution with a concentration of 2 mM were prepared to simulate the composition of body fluids, and in order to avoid the hydrolysis reaction of aluminum ions during the experiment, thereby polymerizing with dopamine, the pH values of all the salts were adjusted to 4.5 by using HCl.

[0061] 15 untreated raw (AT) 15 The (AT) Figure 3 As shown in the figure, the test results show that the untreated (AT) 15 The SWCNT sensor shows different degrees of response to salt solution, and the response to dopamine also appears inconsistent, which leads to the fact that the untreated raw (AT) 15 The SWCNT gel-based dopamine sensor cannot effectively reflect the response to the concentration of dopamine.

[0062] It is concluded that the (AT) 15-SWCNT gel-based dopamine sensor without aluminum ion treatment is easily affected by cations, and the (AT) 15-SWCNT gel-based dopamine sensor with aluminum ion treatment is not affected by cations

[0063] The disturbance of the SWCNT near-infrared signal caused by the interaction between the cations and the negatively charged phosphate backbone of the ssDNA-SWCNT sensor limits the detection of dopamine in body fluids.

[0064] Compared with the ssDNA-SWCNT suspension in water, the ssDNA-SWCNT based on agarose gel can avoid cation-induced aggregation.

[0065] The present application uses trivalent aluminum ions to pre-occupy the interaction sites of phosphate groups to prevent further interaction between other cations and phosphate groups. The aluminum pretreatment process quenches the (AT) 15The initial fluorescence of the SWCNT gel-based dopamine sensor is reduced by the presence of Al, but results in the sensor exhibiting a greater dopamine intensity response in acidic environments. Thus, the Al pretreatment process is expected to be an important method to enhance the stability of ion-induced perturbations of gel-based single-stranded DNA suspended single-walled carbon nanotubes, thereby facilitating the application of single-stranded DNA-wrapped single-walled carbon nanotube sensors in in vitro and in vivo analyte detection.

[0066] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several changes and improvements without departing from the overall concept of the present application, and these should also be considered as the protection scope of the present application.

Claims

1. A method for avoiding the influence of cations on carbon nanotube fluorescent sensors, characterized by: The method comprises the following steps, (1) Preparation of single-stranded DNA sequence (AT) 15 Modified single-walled carbon nanotube solution ((AT) 15 -SWCNTs); (2) Preparation (AT) 15 -SWCNTs gel Prepare 1-4% agarose gel with deionized water, mix (AT) 15 - Mix SWCNTs solution and agarose gel with 1:1 volume ratio, wait until (AT) 15 - SWCNTs solidify into solid, get gel-based dopamine sensor; (3) Preparation of Al-(AT) 15 Gel-based dopamine sensor of SWCNTs composite The gel-based dopamine sensor obtained from step (2) was put into an Al(N03)3 solution for at least one hour, and the treated gel-based dopamine sensor was washed in an HC1 solution with pH < 3.7 to remove free Al 3+ ions in the gel, and finally an Al-(AT) 15 -SWCNTs composite gel-based dopamine sensor was obtained. 2.The method of claim 1, wherein the carbon nanotube fluorescent sensor is not affected by cations. Step (1) Preparation of (AT) 15 The detailed procedure for the preparation of the SWCNTs solution is as follows, a. Dissolve ssDNA in deionized water, adjust the concentration to 1 mg / ml to obtain ssDNA solution, the ssDNA is (AT) 15 ; b. The ssDNA solution and the SWCNT powder are mixed in a mass ratio of 1:1 and then placed in a water bath cup of an ultrasonic disperser to "wrap" the ssDNA on the surface of the SWCNT, so that the SWCNTs are dispersed; c. The dispersed mixture solution is treated on a centrifuge, and 80% of supernatant is reserved; d. The supernatant is centrifuged by using an ultrafiltration membrane to remove all free ssDNA sequences in the supernatant. e. Dilute the ssDNA-CNTs suspension to obtain (AT) 15 - SWCNTs solution. 3.The method of claim 1, wherein the carbon nanotube fluorescent sensor is not affected by cations. The resistivity of the deionized water in step (1) is 18.2 MΩ·cm.

4. The method of claim 1, wherein the carbon nanotube fluorescent sensor is free from the influence of cations. The gel-based dopamine sensor treated by the high-concentration Al(NO3)3 solution in step (3) is repeatedly immersed in an HCl solution for washing 4 times.

5. An Al-(AT) 15 A gel-based dopamine sensor of Al-(AT) The method is prepared by any one of claims 1-4.

6. The Al-(AT) 15 Use of a gel-based dopamine sensor of SWCNTs composite according to claim 5, characterized in that: For detecting dopamine in a body fluid.

7. The Al-(AT) composite of claim 6. 15 Use of a gel-based dopamine sensor of SWCNTs composite, characterized in that: The body fluid is any one of urine, blood or cerebrospinal fluid.

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