Method for preventing carbon nanotube fluorescence sensor from being influenced by cations, gel-based dopamine sensor of Al-(AT) 15-SWCNTs compound and application of gel-based dopamine sensor

Abstract

The invention discloses a method for preventing a carbon nanotube fluorescence sensor from being influenced by cations, a gel-based dopamine sensor of an Al-(AT) 15-SWCNTs compound and application of the gel-based dopamine sensor, the method for preventing the carbon nanotube fluorescence sensor from being influenced by the cations comprises the following steps: firstly, preparing a single-stranded DNA sequence (AT) 15 modified single-walled carbon nanotube solution ((AT) 15-SWCNTs); then, mixing the (AT) 15-SWCNTs solution and agarose gel according to a volume ratio of 1: 1, and waiting until the (AT) 15-SWCNTs are solidified into a solid, so as to obtain a gel-based dopamine sensor; and putting the gel-based dopamine sensor into an Al (NO) solution for at least one hour, soaking the treated gel-based dopamine sensor into an HCl solution with the pH value of less than or equal to 3.7, and washing to remove residual free Al < 3 + > ions in gel, thereby finally obtaining the gel-based dopamine sensor of the Al-(AT) 15-SWCNTs compound. Trivalent aluminum ions are used for occupying interaction sites of phosphate groups in advance, so that further interaction between other cations and the phosphate groups is prevented. The ssDNA-SWCNT based on the agarose gel can be used for avoiding the aggregation induced by cations.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensors, and particularly relates to a method for preventing carbon nanotube fluorescent sensors from being affected by cations and an Al-(AT) 15 -SWCNTs composite gel-based dopamine sensor and applications.

[0002] The present invention belongs to the category of strategic emerging industries, 4.4 of the bio-industry, 4.4.3 of the key direction of the bio-manufacturing industry, special fermentation products and bioprocess equipment biosensors. Background Art

[0003] Single-stranded deoxyribonucleic acid (ssDNA)-modified single-walled carbon nanotubes (SWCNTs) fluorescence sensors (ssDNA-SWCNTs) operate in the near-infrared region and have shown great potential for detecting biomarkers, including glucose, neurotransmitters (dopamine, serotonin, norepinephrine), nitric oxide, chemotherapeutic agents, and lipids. The low detection limit, biotransparency, and photostability of ssDNA-SWCNT fluorescence provide the possibility of achieving non-invasive in vivo continuous monitoring. However, body fluids are a complex chemical environment containing a variety of ions that may cause oligonucleotide-suspended SWCNTs to precipitate in the aqueous phase, thereby quenching the fluorescence of SWCNTs. In addition to the direct aggregation of SWCNTs, salt-ssDNA interactions can also have a significant impact on the fluorescence of ssDNA-SWCNTs, such as the modulation of peak intensity and position. The mechanism behind this fluorescence change could be caused by changes in wrapping conformation, resulting in differences in interactions with water, oxygen molecules, or changes in electronic interactions between ssDNA and SWNTs. Water shielding and oxygen removal can be attributed to increased coverage of SWNTs by DNA, which can be attributed to screening or neutralization of negatively charged phosphate groups. Since most SWNT-based optical sensors rely on intensity changes or wavelength shifts, shielding salt-ssDNA interactions could help reduce noise and stabilize sensing capabilities. (GT) 15 ssDNA sequences show selective response to dopamine. However, according to literature and experimental experience, (AT) 15 The DNA-wrapped SWCNTs sensor has a higher initial fluorescence intensity, and its response to neurotransmitters ((I-I0) / I0) is lower than that of other DNAs, 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 (LNA) at several positions of oligonucleotides. The results showed that the salt-induced wavelength shift was reduced and the stability of the wrapped conformation and signal was increased when using LNA compared to conventional oligonucleotides. However, due to the negatively charged phosphate groups, the interaction between cations present in biological fluids and oligonucleotides is inevitable. The conformational changes induced by monovalent and divalent cations are salt concentration-dependent, so the effect on fluorescence is dynamically changing, which may lead to unpredictable results. Summary of the invention

[0004] In view of the above problems existing in the prior art, the present invention provides a method for preventing a carbon nanotube fluorescent sensor from being affected by cations, a gel-based dopamine sensor and applications.

[0005] The object of the present invention is achieved in the following manner: A method for preventing a carbon nanotube fluorescent sensor from being affected by cations 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 and mix (AT) 15 - SWCNTs solution and agarose gel were mixed in a volume ratio of 1:1 and waited until (AT) 15 -SWCNTs solidify and become solid, obtaining a gel-based dopamine sensor; (3) Preparation of Al-(AT) 15 -SWCNTs composite gel-based dopamine sensor The gel-based dopamine sensor obtained in step (2) was placed in an Al(NO3)3 solution for at least one hour, and the treated gel-based dopamine sensor was washed in a HCl solution with a pH value of ≤ 3.7 to remove the free Al remaining in the gel. 3+ ions, and finally Al-(AT) 15 -SWCNTs composite gel-based dopamine sensor.

[0006] Step (1) Preparation (AT) 15 The specific process of SWCNTs solution is as follows, a. Dissolve ssDNA in deionized water and adjust the concentration to 1 mg / ml to obtain a ssDNA solution, wherein the ssDNA is (AT) 15; b. Mix the ssDNA solution and SWCNT powder in a mass ratio of 1:1 and put them into the water bath cup of the ultrasonic disperser to "wrap" the ssDNA around the surface of the SWCNTs to achieve dispersion of the SWCNTs; c. Process the dispersed mixture solution in a centrifuge and retain 80% of the supernatant; d. centrifuging the supernatant 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.

[0007] The resistivity of the deionized water in step (1) is 18.2 MΩ·cm.

[0008] Step (3) After being treated with the high concentration Al(NO3)3 solution, the gel-based dopamine sensor was repeatedly immersed in the HCl solution for washing 4 times.

[0009] A kind of Al-(AT) 15 -SWCNTs composite gel-based dopamine sensor prepared by the above method.

[0010] Al-(AT) 15 -SWCNTs composite gel-based dopamine sensor for detecting biomarkers in body fluids.

[0011] The body fluid is any one of urine, blood or cerebrospinal fluid.

[0012] Compared with the prior art, the present invention uses trivalent aluminum ions to pre-occupy the interaction sites of the phosphate groups to prevent further interactions between other cations and the phosphate groups. Compared with the ssDNA-SWCNT suspension in water, the ssDNA-SWCNT based on agarose gel can avoid cation-induced aggregation. The aluminum pretreatment process quenches (AT) 15 -SWCNT gel-based dopamine sensor, but resulted in the sensor showing a greater dopamine intensity response in an acidic environment. Therefore, the aluminum pretreatment process is expected to be an important method to enhance the stability of gel-based ssDNA-suspended SWCNTs against ion-induced perturbations, thereby promoting the application of ssDNA-wrapped SWCNT sensors in in vitro and in vivo analyte detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is a preparation flow chart of the present invention.

[0014] Figure 2 Yes (AT) 15-Response diagram of SWCNTs gel-based dopamine sensor to cations. Left: Untreated sensor (AT) 15 -SWCNT, right: Sensor Al-(AT) treated with aluminum nitrate 15 -SWCNT.

[0015] Figure 3 Yes (AT) 15 -Response of SWCNTs gel-based dopamine sensor to dopamine in a cationic environment. Left: Untreated sensor (AT) 15 -SWCNT, right: Sensor Al-(AT) treated with aluminum nitrate 15 -SWCNT. DETAILED DESCRIPTION

[0016] like Figure 1 As shown, a method for preventing a carbon nanotube fluorescent sensor from being affected by cations 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 Because in the aqueous phase, the addition of cations will cause ssDNA-SWCNTs to aggregate and thus produce precipitation. In order to avoid the aggregation of ssDNA-SWCNTs during aluminum ion treatment, we added (AT) 15 -SWCNTs were uniformly dispersed in agarose gel with an initial concentration of 1-4% at a volume ratio of 1:1, with 2% being the optimal concentration. First, 2% agarose gel was prepared with deionized water, and then (AT) 15 -SWCNTs solution and agarose gel were mixed in a volume ratio of 1:1 and waited until (AT) 15 -SWCNTs solidify and become solid to obtain a gel-based dopamine sensor. The gel-based dopamine sensor is preferably treated in deionized water for 4 hours to stabilize the fluorescence; (3) Preparation of Al-(AT) 15 -SWCNTs composite gel-based dopamine sensor The gel-based dopamine sensor obtained in step (2) is placed in an Al(NO3)3 solution for more than 1 hour, preferably a 1 M Al(NO3)3 solution, and the treated gel-based dopamine sensor is washed in a HCl solution with a pH of ≤ 3.7 to remove the free Al remaining in the gel. 3+ ions, it is best to repeat the washing 4 times, and finally obtain Al-(AT) 15-SWCNTs composite gel-based dopamine sensor.

[0017] Step (1) Preparation (AT) 15 The specific process of SWCNTs solution is as follows: 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 a ssDNA solution, wherein the ssDNA is (AT) 15 ; b. Mix the ssDNA solution and SWCNT powder in a mass ratio of 1:1 and put them into the water bath cup of the ultrasonic disperser to "wrap" the ssDNA around the surface of the SWCNTs to achieve dispersion of the SWCNTs; c. Process the dispersed mixture solution in a centrifuge and retain 80% of the supernatant; d. centrifuging the supernatant 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.

[0018] A kind of Al-(AT) 15 -SWCNTs composite gel-based dopamine sensor prepared by the above method.

[0019] The Al-(AT) 15 -SWCNTs composite gel-based dopamine sensor is used to detect biomarkers in body fluids, which are any of urine, blood or cerebrospinal fluid.

[0020] Example 1 A method for preventing a carbon nanotube fluorescent sensor from being affected by cations comprises the following steps: (1) Preparation of single-stranded DNA sequence (AT) 15 Modified single-walled carbon nanotube solution (AT) 15 -SWCNTs) a. Dissolve ssDNA in deionized water with a resistivity of 18.2 MΩ·cm and adjust the concentration to 100 μM. ssDNA is (AT) 15 ; b. Weigh SWCNT to 1 mg / mL, mix the ssDNA solution and SWCNT powder in a mass ratio of about 1:1, and put them into the water bath cup of the ultrasonic disperser. The temperature is controlled at 4 ° C. The water bath cup used in this example is cuphorn, Q700, QSonica. The amplitude is set to 1%, the working time is 90 minutes, and the ssDNA is "entangled" on the surface of SWCNT to achieve the dispersion of SWCNTs; c. The dispersed mixture solution was processed in a centrifuge and 80% of the supernatant was retained. In this example, the centrifuge model used was Eppendorf 5424R and centrifuged at 4°C for 4h; d. Centrifuge the supernatant using an ultrafiltration membrane to remove all free ssDNA sequences in the supernatant. In this example, an Amicon Ultra 0.5 100 kDa centrifugal filter was used.

[0021] e. Dilute the ssDNA-CNTs suspension and finally obtain (AT) 15 -SWCNTs solution and the absorbance of ssDNA-SWCNTs suspension at 632 nm was 0.742.

[0022] (2) Preparation (AT) 15 -SWCNTs gel First, prepare 2% agarose gel with deionized water, and then (AT) 15 -SWCNTs solution and agarose gel were mixed in a volume ratio of 1:1, 190 μl of the gel mixture was added to the culture dish, and waited until (AT) 15 -SWCNTs solidify into a solid, obtaining a gel-based dopamine sensor.

[0023] (3) Preparation of Al-(AT) 15 -SWCNTs composite gel-based dopamine sensor The gel-based dopamine sensor obtained in step (2) was placed in a 1 M Al(NO3)3 solution for 2 hours, and then the remaining Al(NO3)3 solution was removed. The treated gel-based dopamine sensor was immersed in a pH = 3.7 HCl solution for washing to remove the free Al remaining in the gel. 3+ ions, and washed repeatedly for 4 times, finally obtaining Al-(AT) 15 -SWCNTs composite gel-based dopamine sensor.

[0024] Comparative test 1 The 18 (AT) prepared according to the above method 15-SWCNTs agarose gel-based dopamine sensors were randomly divided into 6 groups. The 6 groups of sensors were immersed in 1M NaCl, KCl, MgCl2, CaCl2, Al(NO3)3 and Nd(NO3)3 solutions for 2 hours, and the fluorescence intensity of each sensor was observed under 655nm laser excitation using a fluorescence microscope. Then they were washed with deionized water, and the fluorescence intensity of the washed sensors was observed again under 655nm laser excitation using a fluorescence microscope. Taking the fluorescence response of (7,6) chiral carbon nanotubes as an example, Figure 2 As shown, the test results show: 1) The fluorescence intensity of the sensor treated with NaCl and KCl increased, but after washing, its fluorescence intensity decreased to a lower level than before the addition of salt; 2) Sensors treated with MgCl2 and CaCl2 showed higher fluorescence intensity than those treated with NaCl and KCl. After washing, the intensity of the sensor treated with MgCl2 decreased slightly, while the intensity of the sensor treated with CaCl2 decreased to the same intensity as before the addition of salt; 3) The fluorescence of the sensor treated with Al(NO3)3 was quenched and could not be restored after washing; the fluorescence of the sensor treated with Nd(NO3)3 was enhanced, and after washing, the fluorescence intensity of the sensor was further enhanced.

[0025] It is concluded that compared with pretreatment with other cations, the trivalent aluminum ion-treated (AT) 15 -SWCNTs gel-based dopamine sensor is more stable.

[0026] Comparative test 2 Na⁺, K⁺, Ca2⁺ and Mg2⁺ ions are the main cations present in body fluids such as blood, cerebrospinal fluid and urine. 150mM NaCl solution, 10mM KCl solution, 5mM CaCl2 solution and 2mM MgCl2 solution were prepared to simulate the composition of body fluids. In order to avoid the hydrolysis reaction of aluminum ions during the experiment and thus polymerization with dopamine, we used HCl to adjust the pH value of all salts to 4.5.

[0027] 15 unprocessed original (AT) 15-SWCNTs gel-based dopamine sensors were randomly divided into 5 groups, and HCl, 150mM NaCl solution, 10mM KCl solution, 5mM CaCl2 solution, and 2mMMgCl2 solution with a pH of 4.5 were added respectively. The fluorescence intensity of each sensor excited by a 655nm laser was observed using a fluorescence microscope. Then dopamine hydrochloride (dopamine adjusted with HCl at pH 4.5) was added to a final concentration of 1μM, and the fluorescence intensity of each sensor excited by a 655nm laser was observed using a fluorescence microscope. Taking the fluorescence response of (7,6) chiral carbon nanotubes as an example, Figure 3 As shown, the test results show that: untreated (AT) 15 -SWCNTs sensors showed different degrees of response to salt solutions, and thus inconsistent responses to dopamine, resulting in the untreated original (AT) 15 -SWCNTs gel-based dopamine sensors cannot effectively reflect the response to dopamine concentration.

[0028] Conclusion: (AT)15-SWCNTs gel-based dopamine sensor that has not been treated with aluminum ions is easily affected by cations, while (AT)15-SWCNTs gel-based dopamine sensor that has been treated with aluminum ions is not affected by cations. The perturbation 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.

[0029] Compared with ssDNA-SWCNT suspension in water, agarose gel-based ssDNA-SWCNT can avoid cation-induced aggregation.

[0030] The present invention uses trivalent aluminum ions to pre-occupy the interaction sites of the phosphate groups to prevent further interactions between other cations and the phosphate groups. The aluminum pretreatment process quenches (AT) 15 -SWCNT gel-based dopamine sensor, but resulted in the sensor showing a greater dopamine intensity response in an acidic environment. Therefore, the aluminum pretreatment process is expected to be an important method to enhance the stability of gel-based ssDNA-suspended SWCNTs against ion-induced perturbations, thereby promoting the application of ssDNA-wrapped SWCNT sensors in in vitro and in vivo analyte detection.

[0031] The above is only a preferred embodiment of the present invention. It should be pointed out that for those skilled in the art, several changes and improvements can be made without departing from the overall concept of the present invention, which should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preventing a carbon nanotube fluorescent sensor from being affected by cations, characterized in that: The following steps are included: (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 and mix (AT) 15 -SWCNTs solution and agarose gel were mixed in a volume ratio of 1:1 and waited until (AT) 15 -SWCNTs solidify and become solid, obtaining a gel-based dopamine sensor; (3) Preparation of Al-(AT) 15 -SWCNTs composite gel-based dopamine sensor The gel-based dopamine sensor obtained in step (2) was placed in an Al(NO3)3 solution for at least one hour, and the treated gel-based dopamine sensor was immersed in a HCl solution with a pH value of ≤ 3.7 for washing to remove the free Al remaining in the gel. 3+ ions, and finally Al-(AT) 15 -SWCNTs composite gel-based dopamine sensor.

2. A method for preventing a carbon nanotube fluorescent sensor from being affected by cations according to claim 1, characterized in that: Step (1) Preparation (AT) 15 The specific process of SWCNTs solution is as follows, a. Dissolve ssDNA in deionized water and adjust the concentration to 1 mg / ml to obtain a ssDNA solution, wherein the ssDNA is (AT) 15 ; b. Mix the ssDNA solution and SWCNT powder in a mass ratio of 1:1 and place them in a water bath cup of an ultrasonic disperser to "wrap" the ssDNA around the SWCNT surface to achieve dispersion of the SWCNTs; c. Process the dispersed mixture solution in a centrifuge and retain 80% of the supernatant; d. centrifuging the supernatant 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 for preventing a carbon nanotube fluorescent sensor from being affected by cations according to claim 1, characterized in that: The resistivity of the deionized water in step (1) is 18.2 MΩ·cm.

4. The method for preventing a carbon nanotube fluorescent sensor from being affected by cations according to claim 1, characterized in that: Step (3) After being treated with the high concentration Al(NO3)3 solution, the gel-based dopamine sensor was repeatedly immersed in the HCl solution for washing 4 times.

5. A kind of Al-(AT) 15 -SWCNTs composite gel-based dopamine sensor, characterized by: Prepared by the method described in any of claims 1-4.

6. Al-(AT) as claimed in claim 5 15 -SWCNTs composite gel-based dopamine sensor for detecting biomarkers in body fluids.

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

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