CN113075272B

CN113075272B - Novel photo-induced electrochemical biosensor constructed based on carbon, nitrogen and nitrogen

Info

Publication number: CN113075272B
Application number: CN202110357645.0A
Authority: CN
Inventors: 朱明慧; 杨湉; 丁乔; 袁亚利
Original assignee: Chongqing Chaoyang Middle School; Southwest University
Current assignee: Chongqing Chaoyang Middle School; Southwest University
Priority date: 2021-04-01
Filing date: 2021-04-01
Publication date: 2022-07-22
Anticipated expiration: 2041-04-01
Also published as: CN113075272A

Abstract

The invention relates to a novel photo-induced electrochemical biosensor constructed based on carbon, nitrogen and nitrogen, and belongs to the technical field of sensing detection. Firstly, dripping C on the surface of clean GCE₃N₅Obtaining an initial PEC signal; then gold nanoparticles are electrodeposited; then, double-stranded DNA formed by hybridization of SP and TP of which the MB and SH are respectively modified by 5 'and 3' is assembled on the modified interface through Au-S bonds; then adding MiRNA182-5p and single-stranded RS, displacing SP in TP-SP by the target, and hybridizing the MiRNA182-5p and TP; then the RS replaces MiRNA182-5p, and circulation of the target object is realized. The cycle amplification reaction based on strand displacement has the advantages of self-priming property, high sensitivity and high accuracy, and realizes the sensitive detection of MiRNA182-5 p.

Description

Novel photo-induced electrochemical biosensor constructed based on carbon, nitrogen and nitrogen

Technical Field

The invention belongs to the technical field of sensing detection, and particularly relates to a novel photo-electrochemical biosensor constructed on the basis of carbon, nitrogen and nitrogen.

Background

In recent years, sensitive detection of various disease markers has been a focus of attention in basic research and clinical diagnosis. MiRNAs are endogenous non-coding small molecule single-stranded RNAs with about 22 nucleotides, can control the activity of more than 50 percent of human coding genes, and play a vital role in regulation and control in biological processes such as biological development, cell differentiation, apoptosis, proliferation, immunity and the like. Meanwhile; abnormal expression of mirnas in human tissues and blood is closely related to the development, progression and therapeutic response of cancer. miRNA can be used as a biomarker for cancer diagnosis and prediction, and also can be used as a potential action target of a new drug, so that the realization of the miRNA has important significance for the detection of the miRNA.

The biosensor is a novel device which is sensitive to target biological substances (such as proteins, cells, small molecules and the like) and can convert the concentration of the target biological substances into detectable signals (such as optical signals, electric signals and the like) so as to realize the rapid measurement of the physics, chemistry and biomass of the target biological substances. Currently, various sensing technologies based on electrochemistry, optics (fluorescence, chemiluminescence, surface plasmon resonance, raman spectroscopy) and mechanics have been developed. The photoelectrochemical sensing is taken as one of biosensors, and can adopt light as an excitation source and an electric signal as a reading signal; meanwhile, the excitation source and the detection signal have different energy forms, so the method has the characteristics of quick response, good selectivity, high sensitivity, simple operation and the like. In order to effectively improve the photocurrent response signal and further improve the sensitivity, various amplification strategies are generally adopted, such as energy transfer, DNA cycling, multiple signal output, high photoelectric conversion efficiency photoelectric material synthesis, electron donor/acceptor participation, and the like.

Graphitic carbon nitride (g-C)₃N₄) As a conjugated polymer semiconductor, the conjugated polymer semiconductor has an excellent electronic structure, but the performance of the conjugated polymer semiconductor is limited by factors such as low photocatalytic efficiency, insufficient light absorption, poor electric activity and the like. By mixing g-C₃N₄The smallest monomer of the framework (2,5, 8-triamino-s-heptazine) is subjected to further calcination to give C₃N₅The absorption of visible light can be widened, and the photocurrent signal can be effectively improved.

Therefore, further investigation is required to find C₃N₅A novel Photoelectrochemical (PEC) biosensor with an electrode surface for driving the target MiRNA182-5p to perform strand displacement cyclic amplification is constructed for a substrate, so that the detection of the target MiRNA182-5p is effectively realized.

Disclosure of Invention

An object of the present invention is to provide a photoelectrically active material C₃N₅The preparation method of (1); the second purpose of the invention is to provide a photoelectric active material C₃N₅(ii) a Another object of the present invention is to provide a photo-electrically active material C₃N₅Application in photo-induced electrochemical biosensors.

In order to achieve the purpose, the invention provides the following technical scheme:

1. photoelectric active material C₃N₅The preparation method comprises the following steps:

(1) dispersing calcined melamine yellow powder into deionized water to form suspension, refluxing to remove impurities, centrifugally washing, and drying at room temperature to obtain a white product, namely 2,5, 8-triamino-s-heptazine;

(2) dispersing the 2,5, 8-triamino-s-heptazine prepared in the step (1) into hydrazine hydrate, sealing the mixture in a high-pressure kettle, taking out the mixture, heating the mixture for 24 to 36 hours at the temperature of 140 ℃, and cooling the mixture to obtain light yellow suspension;

(3) adjusting the pH value of the light yellow suspension liquid in the step (2) to 1-2 by using acid, and filtering to obtain filtrate and solid residue respectively;

(4) adjusting the pH of the obtained filtrate to 7.5-8.5 by using alkali, filtering to obtain a solid I, dissolving the obtained solid residue by using acid, adjusting the pH to 7.5-8.5 by using alkali, and filtering to obtain a solid II;

(5) combining the solid I and the solid II and repeatedly carrying out the following steps: dissolving with acid, adjusting the pH value to 7.5-8.5 with alkali, and filtering;

(6) washing the solid obtained after filtering in the step (5) with deionized water and ethanol in sequence, drying in vacuum, heating to 245 ℃ at the heating rate of 2-4 ℃/min, and keeping the temperature for 2h to obtain a yellow product C₃N₅。

Preferably, the calcination in step (1) is calcination at a temperature of 425 ℃ for 12 h.

Preferably, the mass volume fraction of the hydrazine hydrate in the step (2) is 55%, and the mass volume ratio of the 2,5, 8-triamino-s-heptazine to the hydrazine hydrate is 1.6:15, g: ml.

Preferably, in the step (3), the step (4) and the step (5), the acid is hydrochloric acid with the mass volume fraction of 10%, and the alkali is sodium hydroxide solution with the mass volume fraction of 10%.

2. The photoelectric active material C prepared by the preparation method₃N₅。

3. The above-mentioned photoelectric active material C₃N₅Application in photo-induced electrochemical biosensors.

Preferably, the detection target of the photoelectrochemical biosensor is MiRNA182-5p, and the nucleotide sequence of the MiRNA182-5p is shown in SEQ ID NO. 8.

4. A method of constructing a photo-electrochemical biosensor, the method comprising the steps of:

(1) dropping the photoelectric active material C on the surface of the pretreated glassy carbon electrode₃N₅Continuously assembling gold nanoparticles through electrodeposition to obtain a modified electrode;

(2) assembling the double-stranded DNA on the interface of the modified electrode in the step (1) through an Au-S bond;

(3) and (3) blocking the nonspecific adsorption sites of the product in the step (2) by using mercaptohexanol with the mass fraction of 0.5-1%, blocking for 40min or more, and then rinsing with deionized water to obtain the photo-induced electrochemical biosensor.

Preferably, the diameter of the glassy carbon electrode is 4 mm.

Preferably, the double-stranded DNA is formed by hybridization of the substrate strands SP and TP;

3 'and 5' of the substrate chain SP respectively modify sensitizers of methylene blue and sulfydryl;

the nucleotide sequence of the TP is shown as SEQ ID NO.1, and the nucleotide sequence of the substrate chain SP is shown as SEQ ID NO. 2.

The invention is based on C with high photoelectric conversion efficiency₃N₅For the sensitive interface, a novel PEC biosensor is constructed by combining strand displacement cyclic amplification to detect MiRNA182-5 p. Firstly, dripping photoelectric material C on clean GCE surface₃N₅An initial photocurrent signal is obtained. Gold nanoparticles were then assembled by electrodeposition. Then, double-stranded DNA (TP-SP) formed by hybridizing substrate chains SP and TP of 5' modified sensitizers, namely Methylene Blue (MB) and Sulfydryl (SH) respectively are assembled on the modified interface through Au-S bonds. The photocurrent signal was significantly enhanced due to the sensitization of MB. Then, the target MiRNA182-5p and single-stranded DNA (RS) are added, and the target first replaces SP in the double-stranded DNA (TP-SP) on the electrode surface to allow the MiRNA182-5p to hybridize with TP, so that the photocurrent signal is reduced. Then the RS replaces MiRNA182-5p, and the recycling of MiRNA182-5p is realized. The cycle amplification reaction based on strand displacement has self-initiation property and high efficiencyThe sensitivity and the accuracy of the sensor are improved effectively, and the detection of the MiRNA182-5p is successfully realized.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objectives and other advantages of the invention may be realized and attained by the means of the instrumentalities and combinations particularly pointed out hereinafter.

Drawings

For a better understanding of the objects, aspects and advantages of the present invention, reference will now be made to the following detailed description taken in conjunction with the accompanying drawings in which:

FIG. 1 shows a photo-electrically active material C₃N₅The reaction process (A) and the flow chart (B) for preparing the photo-electrochemical biosensor;

FIG. 2 shows a photo-electrically active material C prepared in example 1₃N₅A TEM image of (D);

FIG. 3 shows the signal change (A) and the detection result (B) of MiRNA182-5p for different incubation times of the Photoelectrochemical (PEC) biosensor prepared in example 2;

FIG. 4 shows the selectivity (A) and stability (B) measurements of the photo-electrochemical (PEC) biosensor prepared in example 2.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention. It should be noted that, in the following embodiments, features in the embodiments may be combined with each other without conflict.

The TP, SP, RS, miRNA-21, miRNA-141, miRNA-122, miRNA-126, and miRNA182-5p referred to in the examples below were all artificially synthesized and provided by Biotechnology, Inc. (Shanghai).

Wherein the nucleotide sequences of TP, SP, RS, miRNA-21, miRNA-141, miRNA-122, miRNA-126 and miRNA182-5p are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 8:

TP：5'-GTA GCA TAA GGT GTA GTG GAGA AGT GTG AGT TCT ACC ATT GCC AAA TTT T-SH-3'(SEQ ID NO.1)；

SP：5'-GGT AGA ACT CAC ACT TCT CAC TCC TAA ACA CTC CTT TTT-MB-3'(SEQ ID NO.2)；

RS：5'-TCA CAC TCA AGA GTC TCC ACT ACA CCT TAT GCT AC-3'(SEQ ID NO.3)；

miRNA-21：5'-UAG CUU AUC AGA CUG AUG UUG A-3'(SEQ ID NO.4)；

miRNA-141：5'-UAA CAC UGU CUG GUA AAG AUG G-3'(SEQ ID NO.5)；

miRNA-122：5'-UGGAGUGUGACAAUGGUGUUUG-3'(SEQ ID NO.6)；

miRNA-126：5'-CAU UAU UAC UUU UGG UAC-3'(SEQ ID NO.7)；

miRNA182-5p：5'-UUU GGC AAU GGU AGA ACU CAC ACU-3'(SEQ ID NO.8)。

example 1

Preparing a photoelectric active material C₃N₅The reaction formula is shown as A in figure 1, and the specific method is as follows:

(1) 5.0g of melamine was calcined in a muffle furnace at 425 ℃ for 12h to give a pale yellow powder;

(2) then dispersing the mixture into deionized water to obtain a suspension, refluxing for several hours to remove unreacted melamine and other impurities, centrifugally washing, and drying at room temperature to obtain a white product (2,5, 8-triamino-s-heptazine);

(3) continuously dispersing 1.6g of 2,5, 8-triamino-s-heptazine in 15mL of 55% hydrazine hydrate solution, sealing in a 25mL autoclave, heating in an oven at 140 ℃ for 24h, and cooling to obtain a light yellow suspension;

(4) the pale yellow suspension was transferred to a 100mL beaker and 10% hydrochloric acid was addedAdjusting the pH value to be between 1 and 2, and then filtering to remove unreacted substances to obtain a solid residue containing the 2,5, 8-triamino-s-heptazine. The filtrate was added with 10% sodium hydroxide solution, maintaining the pH between 7.5 and 8.5. The solid was then dissolved in hydrochloric acid and reprecipitated in sodium hydroxide, this process being repeated three times. And finally, washing the obtained solid for multiple times by using deionized water and ethanol, and drying in vacuum. Finally, heating the dried product to 245 ℃, wherein the heating rate is 2 ℃, and preserving the heat for 2 hours to obtain C₃N₅The TEM image is shown in FIG. 2.

Example 2

The opto-electronically active material C prepared in example 1 was used₃N₅The photo-induced electrochemical biosensor is prepared by the following steps as shown in the B in the figure 1:

(1) the photoelectric active material C prepared in example 1 was dropped on the surface of a glassy carbon electrode (GCE, Φ ═ 4mm) pretreated (polished with 0.3 μm and 0.05 μm aluminum oxide powders in this order, rinsed with ultrapure water, ultrasonically washed with ultrapure water, ethanol, and ultrapure water, and then dried at room temperature, respectively)₃N₅After drying, carrying out electrodeposition assembly on gold nanoparticles (AuNPs) by cyclic voltammetry (deposition time is 30s, and deposition potential is-0.2V), thereby obtaining an AuNPs modified electrode;

(2) assembling double-stranded DNA on the interface of the modified electrode in the step (1) through Au-S bonds (wherein the double-stranded DNA is formed by hybridizing a substrate chain SP and a TP, the nucleotide sequence of the TP is shown in SEQ ID NO.1, 5 'and 3' of the substrate chain SP respectively modify sensitizers Methylene Blue (MB) and Sulfydryl (SH), and the nucleotide sequences of the Sulfydryl (SH) are shown in SEQ ID NO. 2);

(3) and (3) blocking the nonspecific adsorption sites of the product assembled in the step (2) by using mercaptohexanol (HT) with the mass fraction of 1%, blocking for 40min, then using deionized water for trickle washing to obtain the photo-electrochemical biosensor, and storing at 4 ℃ for later use.

Example 3

The detection application of the photo-electrochemical (PEC) biosensor prepared in example 2 is mainly reflected in:

1. optimized selection of detection conditions:

firstly, respectively incubating the photo-electrochemical (PEC) biosensors prepared in 6 embodiments 2 at a target MiRNA-182-5p concentration of 1nmol/L for different times (20min, 40min, 60min, 80min, 100min and 120min), and rinsing with deionized water after incubation is finished;

secondly, a three-electrode system is adopted, GCE is used as a working electrode, a platinum wire electrode is used as a counter electrode, a saturated calomel electrode (saturated KCl solution) is used as a reference electrode, and H is contained in the solution₂O₂The working electrode was subjected to an off-on-off (5s-10s-5s) PEC scan in PBS (1) at a scan voltage range of 0V (vs. Ag/AgCl), and the PEC response values from the stable scans were recorded.

With increasing incubation time of target and RS, the signal of the photo-electrochemical (PEC) biosensor gradually increased until the signal reached a steady state at 80min, with specific results as shown in fig. 3 a.

2. The photo-electrochemical (PEC) biosensor prepared in example 2 was used to detect MiRNA182-5 p:

(1) preparation of double-stranded DNA (TP-SP): mixing 4. mu. mol/L of the single-stranded DNA TP with 4. mu. mol/L of the single-stranded DNA SP labeled with MB, and hybridizing at 37 ℃ for 2 hours to form double-stranded DNA (TP-SP);

(2) 20 mu L of double-stranded DNA (TP-SP) is dripped on the surface of the Photoelectrochemical (PEC) biosensor prepared in the embodiment 2, incubated for 12h at 4 ℃, and then washed in deionized water;

(3) continuously dropwise adding a sealant HT, reacting for 40min, and then rinsing with deionized water;

(4) adding the product after the washing into a mixture of a target MiRNA182-5p and a single-stranded RS of 2 mu mol/L, incubating for 80min, washing in deionized water, and performing PEC signal detection (likewise, a three-electrode system is adopted, wherein GCE is used as a working electrode, a platinum wire electrode is used as a counter electrode, a saturated calomel electrode (saturated KCl solution) is used as a reference electrode, and 50 mu L H is contained in the mixture₂O₂PBS (pH 7.4, from 0.1mol/L K)₂HPO₄，0.1mol/L NaH₂PO₄And 0.1mol/L KCl) was added to the sample to prepare a solution, and the working electrode was subjected to off-on-off (5s-10 s-5)s) PEC scan, scan voltage range 0V (vs. ag/AgCl), and record PEC response values from stable scans).

The PEC signal of the sensor decreases with the increase of the concentration of MiRNA182-5p and is proportional to the logarithm of the concentration of MiRNA182-5p, and the linear response ranges from 10fmol/L to 10nmol/L, and the specific result is shown as B in FIG. 3.

3. Selective detection of the photo-electrochemical (PEC) biosensor prepared in example 2:

(1) dropwise adding 20 mu L of double-stranded DNA (TP-SP) to the surface of the Photoelectrochemical (PEC) biosensor prepared in the example 2, incubating for 12h at 4 ℃, and then washing with deionized water;

(2) then, dropwise adding a sealant HT, reacting for 40min, and then using deionized water for rinsing;

(3) respectively incubating the products washed in the step (2) in the mixture of the interferents miRNA-21, miRNA-141, miRNA-122 and miRNA-126 with the same concentration, the blank pattern, the target MiRNA182-5p and the single-stranded RS with a certain concentration for 80min, washing in deionized water, and detecting PEC signals (likewise, a three-electrode system, namely GCE as a working electrode, a platinum wire electrode as a counter electrode, a saturated calomel electrode (saturated KCl solution) as a reference electrode, and adding 50 mu L H of the reference electrode₂O₂PBS (pH 7.4, from 0.1mol/L K)₂HPO₄，0.1mol/L NaH₂PO₄And 0.1mol/L KCl) were scanned off-on-off (5s-10s-5s) PEC at a scanning voltage range of 0V (vs. ag/AgCl), and PEC response values from the stable scans were recorded.

The PEC signals of the interfering miRNAs were measured separately, and the measurements were repeated three times, and the PEC signal intensities were averaged three times, and the specific results are shown in fig. 4 a, which shows that the PEC response value of the sensor to the target miRNAs 182-5p was lower even when the interfering miRNAs were at a concentration higher than that of the target miRNAs 182-5p, indicating that the Photoelectrochemical (PEC) biosensor prepared according to the present invention has good selectivity.

4. Stability testing of the photo-electrochemical (PEC) biosensor prepared in example 2:

(1) 20 mu L of double-stranded DNA (TP-SP) is dripped on the surface of the Photoelectrochemical (PEC) biosensor prepared in the example 2, and the mixture is incubated at the temperature of 4 ℃ for 12 hours and then washed by deionized water;

(2) then, dripping a sealant HT, reacting for 40min, and washing with deionized water;

(3) then adding target MiRNA182-5p with different concentrations (1fmol/L,10fmol/L,100fmol/L,1pmol/L,10pmol/L,100pmol/L,1nmol/L) and 2 μmol/L single-stranded RS mixture, incubating for 80min, washing with ionized water, and performing PEC signal detection (likewise adopting three-electrode system: GCE as working electrode, platinum wire electrode as counter electrode, saturated calomel electrode (saturated KCl solution) as reference electrode, in the presence of 50 μ L H₂O₂PBS (pH 7.4, from 0.1mol/L K)₂HPO₄，0.1mol/L NaH₂PO₄And 0.1mol/L KCl) were subjected to an off-on-off (5s-10s-5s) PEC scan for 8 cycles at a scan voltage ranging from 0V (vs. Ag/AgCl) and the response value of the PEC obtained from the stable scan was recorded), and as a result, as shown in B in FIG. 4, it can be seen that the Photoelectrochemical (PEC) biosensor prepared according to the present invention has good stability in detecting the target MiRNA182-5 p.

Finally, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

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Claims

1. A method of constructing a photo-electrochemical biosensor, comprising the steps of:

(1) dripping a photoelectric active material C on the surface of the pretreated glassy carbon electrode₃N₅Continuously assembling gold nanoparticles through electrodeposition to obtain a modified electrode;

(2) assembling double-stranded DNA on the interface of the modified electrode in the step (1) through Au-S bond;

(3) blocking the non-specific adsorption sites of the product in the step (2) by mercaptohexanol with the mass fraction of 0.5-1%, blocking for more than 40min, and then rinsing with deionized water to obtain the photo-induced electrochemical biosensor;

the photoelectric active material C in the step (1)₃N₅The preparation method comprises the following steps:

a. dispersing calcined melamine yellow powder into deionized water to form suspension, refluxing to remove impurities, centrifugally washing, and drying at room temperature to obtain a white product, namely 2,5, 8-triamino-s-heptazine;

b. dispersing the 2,5, 8-triamino-s-heptazine prepared in the step a into hydrazine hydrate, sealing the mixture in a high-pressure kettle, taking out the mixture, heating the mixture at the temperature of 140 ℃ for 24 to 36 hours, and cooling the mixture to obtain light yellow suspension;

c. adjusting the pH of the light yellow suspension liquid in the step b to be 1-2 by using acid, and filtering to respectively obtain filtrate and solid residue;

d. adjusting the pH of the obtained filtrate to 7.5-8.5 by using alkali, filtering to obtain a solid I, dissolving the obtained solid residue by using acid, adjusting the pH to 7.5-8.5 by using alkali, and filtering to obtain a solid II;

e. combining the solid I and the solid II and repeatedly carrying out the following steps: dissolving with acid, adjusting the pH value to 7.5-8.5 with alkali, and filtering;

f. e, washing the solid obtained after filtering in the step e with deionized water and ethanol in sequence, drying in vacuum, heating at a heating rate of 2-4 ℃/min, and keeping the temperature for 2h to obtain a yellow product C₃N₅；

The double-stranded DNA in the step (2) is formed by hybridizing a substrate chain SP and a substrate chain TP, wherein 5 'and 3' of the substrate chain SP respectively modify sensitizers of methylene blue and sulfydryl, the nucleotide sequence of the TP is shown as SEQ ID NO.1, and the nucleotide sequence of the substrate chain SP is shown as SEQ ID NO. 2.

2. The method of claim 1, wherein the calcining in step a is at a temperature of 425 ℃ for 12 h.

3. The method for constructing a hydrazine hydrate according to claim 1, wherein the mass volume fraction of the hydrazine hydrate in the step b is 55%, and the mass volume ratio of the 2,5, 8-triamino-s-heptazine to the hydrazine hydrate is 1.6:15, g: ml.

4. The method for constructing a hollow fiber membrane according to claim 1, wherein the acid in the steps c, d and e is hydrochloric acid with a mass volume fraction of 10%, and the base is sodium hydroxide solution with a mass volume fraction of 10%.

5. The construction method according to claim 1, wherein the detection target of the photoelectrochemical biosensor is MiRNA182-5p, and the nucleotide sequence of MiRNA182-5p is shown in SEQ ID NO. 8.

6. The construction method according to claim 1, wherein the glassy carbon electrode has a diameter of 4 mm.