CN110146566B - Modified electrode, combined product, electrochemiluminescence biosensor and application thereof - Google Patents
Modified electrode, combined product, electrochemiluminescence biosensor and application thereof Download PDFInfo
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Abstract
The invention relates to a modified electrode, a combined product, an electrochemiluminescence biosensor and application thereof. The modified electrode comprises an electrode and a luminophor modified on the electrode; the luminophores are predominantly carboxyl-functionalized poly [ (9, 9-dioctylfluorene-2, 7-diyl) -co- (1, 4-benzo- {2, 1', 3} -thiadiazole) ] dots. The electrochemiluminescence biosensor has no co-reaction reagent, can overcome the defects of insufficient system stability, measurement error, lack of reproducibility in detection and the like in the conventional ECL biosensor determination in the presence of the co-reaction reagent, constructs a double signal amplification electrochemiluminescence strategy without a co-reaction reagent, has an ideal ECL signal response value and excellent ECL luminescence efficiency, has excellent high sensitivity, specific selectivity and stability, and provides a new method for detecting nucleic acid.
Description
Technical Field
The invention relates to the field of biosensors, in particular to a modified electrode, a combined product, an electrochemiluminescence biosensor and application thereof.
Background
MicroRNAs (miRNAs) are endogenous, non-protein-coding, short (usually about 19-25 bases) and single-stranded RNAs that have been shown to be important regulators in several biological processes such as hematopoietic function, cell proliferation and apoptosis. Research shows that miRNA can be used as ideal biomarker candidate, and the abnormal expression of miRNA is closely related to the occurrence of various cancers. Therefore, there is an urgent need to develop highly sensitive, highly specific and reliable techniques for detecting mirnas.
Electrochemiluminescence (ECL) has not only the advantages of electrochemical methods, such as simplicity and stability of the device, but also a broader dynamic range and higher sensitivity than conventional Chemiluminescence (CL). And thus have been widely used for environmental analysis, biochemical detection and clinical detection. Generally, ECL assays are performed in the substantial presence of co-reactive reagents. However, the addition of exogenous co-reactive reagents to the detection solution can affect the environment of the test solution, causing the ECL system to lack sufficient stability, resulting in measurement errors. Furthermore, while the drawbacks of adding an exogenous co-reactant can also be avoided by using dissolved oxygen as a co-reactant, in the case of dissolved oxygen as a co-reactant, an unknown concentration of dissolved oxygen would result in unavoidable errors and lack of reproducibility in the detection.
Therefore, it is of great practical interest to develop a sensitive, simple ECL system for the detection of miRNAs with high ECL emission efficiency without any co-reactive reagents.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first objective of the present invention is to provide a modified electrode modified with a conjugated polymer dot emitter, which is a surface functionalized electrode and has the advantages of high charge carrier mobility, fast radiance, etc.
The second purpose of the invention is to provide a combination product comprising the modified electrode, wherein the combination product is designed based on a co-reactive reagent-free dual signal amplification Electrochemiluminescence (ECL) strategy, and can be used for constructing a co-reactive reagent-free dual signal amplification electrochemiluminescence biosensor.
The third objective of the present invention is to provide an electrochemiluminescence biosensor assembled from the above-mentioned combination product, wherein the electrochemiluminescence biosensor is an ECL biosensor without co-reaction reagent, and combines with a biped DNA walker (walker) dual amplification strategy, so as to overcome the defects of insufficient system stability, measurement error, lack of reproducibility in detection, etc. in the existing ECL biosensor measurement performed in the presence of co-reaction reagent, and simultaneously have a relatively ideal ECL signal response value and excellent ECL luminescence efficiency.
The fourth purpose of the invention is to provide the application of the electrochemiluminescence biosensor in detecting nucleic acid, especially miRNA.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
the modified electrode comprises an electrode and a luminous body modified on the electrode.
The luminophores are predominantly carboxyl-functionalized poly [ (9, 9-dioctylfluorene-2, 7-diyl) -co- (1, 4-benzo- {2, 1', 3} -thiadiazole) ] dots.
Optionally, the electrode is selected from a glassy carbon electrode, a gold electrode.
Optionally, the electrode is selected from a glassy carbon electrode.
As an embodiment, the PFBT-COOH sites of the present invention are poly (styrene-maleic anhydride) (PSMA) functionalized PFBT sites.
The modified electrode takes the conjugated polymer and the conjugated polymer point as luminophors, particularly PFBT and carboxyl functionalized PFBT-COOH points, the polymer and the polymer point have various outstanding characteristics of high charge carrier mobility, high radiation rate, easy surface functionalization, good light stability and the like, and the use of the material provides a good platform for constructing an ECL biosensor without a co-reaction reagent type and expands the application of the conjugated polymer point in clinical analysis.
In the present invention, the PFBT-COOH sites not only exhibit higher ECL strength, but also exhibit more excellent film forming ability and stability. Using N without any co-reactants2Under the condition of removing dissolved oxygen, when the voltage of a photomultiplier tube (PMT) is set to be 800V and the amplification level is set to be 3, the ECL intensity of a PFBT-COOH point can reach 18,000a.u., and the signal intensity is about 5 times higher than the ECL signal response value of a PS-COOH-co-PFO point and 24 times higher than the pure PFBTECL signal response value. Therefore, the PFBT-COOH point provides a more ideal choice for the construction of an ECL sensing platform without a co-reaction reagent.
According to another object of the present invention, there is provided a combination product comprising the above-mentioned modified electrode, DNA S1, DNAs 2; DNA hairpin H1 and DNA hairpin H2.
The DNA S1 and the DNA S2 can be in complementary pairing, and a quencher is coupled to the DNA S2.
The DNA hairpin H1 and the DNA hairpin H2 can be assembled under the catalysis of nucleic acid to be detected to form a biped DNA walker; the double-foot part of the double-foot DNA walker can be complementarily paired with the DNA S1, and an excision enzyme site of DNA exonuclease is reserved on the paired DNA S1.
The combination product does not include a co-reactant.
Optionally, the combination further comprises the exonuclease.
Optionally, the exonuclease is exonuclease iii.
Optionally, the quencher is selected from a black hole quencher, ferrocene.
Optionally, the quencher is a black hole quencher.
In the invention, DNA exonuclease, especially exonuclease III (Exo III), can perform two-step amplification on ECL signal intensity, so that the electrochemiluminescence biosensor obtains obviously enhanced ECL signals, and a double amplification strategy can endow the sensor with better signal amplification capability.
According to another object of the present invention, there is provided a method for detecting a nucleic acid, which comprises detecting a nucleic acid to be detected using the above-mentioned combination product.
Optionally, the method of detecting nucleic acids comprises a method of assembling an electrochemiluminescence biosensor:
a) assembling DNA S1 by using the modified electrode as a matrix, and introducing DNA S2 which is complementarily paired with the DNA S1;
b) under the catalysis of the nucleic acid to be detected, assembling DNA hairpins H1 and H2 to generate a biped DNA walker;
c) co-incubating the biped DNA walker and the DNA exonuclease with the electrode obtained in step a);
wherein the steps a) and b) are not in sequence.
Optionally, the DNA S1 is assembled on the modified electrode in step a) in the presence of a cross-linking agent; the cross-linking agent is EDC and NHS.
Optionally, the preparation method of the modified electrode comprises:
and (3) dripping the dispersion liquid of the luminophor on the surface of the electrode, and drying to form a film to obtain the modified electrode.
Optionally, the dispersion medium of the dispersion is selected from redistilled water or ultrapure water.
Optionally, the concentration of the luminophore in the dispersion is 0.2 mg-mL-1～0.4mg·mL-1。
Optionally, pretreatment of the electrode is further included before the luminophor modifies the electrode.
Optionally, the pre-processing comprises: the electrode was polished at least 2 times and then ultrasonically cleaned in the presence of a cleaning agent.
Optionally, the polishing is aluminum oxide polishing; the alumina has a particle size of not more than 0.30 μm, and the particle size decreases as the number of polishing times increases.
Optionally, the cleaning agent comprises ethanol and water.
Optionally, the power of the ultrasound is 160-200W, and the ultrasound time is 2-5 minutes.
Optionally, the preparation process of the biped DNA walker comprises:
heating the DNA hairpin H1 and the DNA hairpin H2 to 90-100 ℃ for 5-10 minutes, preferably to 95 ℃ for 5 minutes, and slowly cooling to room temperature to form a hairpin structure;
and mixing the target, the DNA hairpin H1 and the DNA hairpin H2 to obtain a mixture, and incubating the mixture at 25-65 ℃ for 60-120 minutes, preferably at 37 ℃ for 90 minutes to obtain the biped DNA walker.
Optionally, the incubation time of the biped DNA walker and the DNA exonuclease on the electrode is 1-3 hours, preferably 2 hours.
As an embodiment, DNA S1 was assembled with luminophore-modified electrodes as a matrix, without any co-reagents added to obtain a strong ECL signal; then, a Black Hole Quencher (BHQ) -labeled DNA S2(BHQ-S2) was introduced by hybridization with the DNA S1. Due to the efficient quenching effect of BHQ on PFBT-COOH signals, ECL signals reach a 'signal off' state, thereby realizing the signal conversion function.
As an embodiment, a biped DNA walker produced by assembly of the target-catalyzed hairpin and an exodna enzyme are incubated onto the electrodes simultaneously. In one aspect, BHQ-S2 was replaced by a biped DNA walker for release from the electrode surface. On the other hand, exodnases can digest blunt or recessed 3' ends in double stranded DNA (dsdna). With the help of DNA exonuclease, the released DNA walker replaces another BHQ-S2 to realize the circulation process of the DNA walker. As expected, the ECL signal switches to a state of "signal on". Due to the combination of high luminescence efficiency of conjugated polymer dot luminophores, high quenching efficiency of BHQ and double amplification strategies, the biosensor of the proposed ECL 'off-on' model realizes ultra-sensitive detection of target substances.
Optionally, the nucleic acid is RNA, preferably miRNAs.
Optionally, the nucleic acid is miRNAs-155.
Alternatively, the sequence of the DNA S1 is shown as SEQ ID NO. 1;
the sequence of the DNA S2 is shown as SEQ ID NO. 2;
the sequence of the miRNAs-155 is shown as SEQ ID NO. 3;
the sequence of H1 is shown as SEQ ID NO. 4, and the sequence of H2 is shown as SEQ ID NO. 5.
Optionally, the linear range of the detection method is 10amol · L-1～5pmol·L-1The detection limit is 3.3 amol.L-1。
According to another important object of the present invention, there is provided an electrochemiluminescence biosensor assembled by the above-mentioned assembly method.
The electrochemiluminescence biosensor provided by the invention utilizes conjugated polymer dots as luminophors to construct a co-reaction-free reagent type ECL biosensor, and realizes the cyclic process of obtaining a strong ECL signal, quenching the signal and recovering the signal under the condition of not adding any co-reaction reagent, namely the cyclic switching of the 'signal on' state- 'signal off' state- 'signal on' state of the ECL signal, and the sensing of the ECL signal is completed.
Compared with the prior art, the beneficial effects of the invention include but are not limited to:
(1) the modified electrode provided by the invention is a surface functionalized electrode and has the advantages of high charge carrier mobility, high radiation rate and the like.
(2) The combined product provided by the invention is designed based on a dual-signal amplification Electrochemiluminescence (ECL) strategy of a co-reaction-free reagent type, and can be used for constructing a co-reaction-free reagent type dual-signal amplification electrochemiluminescence biosensor.
(3) The electrochemiluminescence biosensor provided by the invention does not use a coreactant, combines a biped DNA walker (walker) and an enzyme trigger double amplification strategy, can overcome the defects of insufficient system stability, measurement error, lack of reproducibility in detection and the like in the conventional ECL biosensor measurement in the presence of a coreactant reagent, constructs a double signal amplification electrochemiluminescence strategy without a coreactant reagent, and has an ideal ECL signal response value and excellent ECL luminescence efficiency.
(4) The electrochemiluminescence biosensor provided by the invention has excellent high sensitivity, lower detection limit, specific selectivity and stability when being used for detecting miRNA, and the linear range of the miRNA detection is 10 amol.L-1～5pmol·L-1The detection limit is 3.3 amol.L-1Provides a new method for the detection of miRNA.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a process for constructing an electrochemiluminescence biosensor according to an embodiment of the present invention;
FIG. 2 is the result of non-denaturing polyacrylamide gel electrophoresis analysis of the biped DNA walker according to one embodiment of the present invention;
FIG. 3 shows the results of characterization of cyclic voltammetry (A) and ECL response (B) for an electrochemiluminescence biosensor according to an embodiment of the present invention;
in fig. 3A, a curve a represents a redox curve of a bare electrode, a curve b represents a redox curve of a PFBT-COOH point modified electrode, a curve c represents a redox curve after DNAS1 is incubated, a curve d represents a redox curve after HT blocking, a curve e represents a redox curve after BHQ-S2 is introduced, and a curve f represents a redox curve after a DNA walker and ExoIII are incubated on the surface of the electrode at the same time;
in FIG. 3B, curve a shows the ECL signal response curve of a bare electrode, curve B shows the ECL signal response curve of a PFBT-COOH point modified electrode, curve c shows the ECL signal response curve after DNAS1 is incubated and sealed by HT, curve d shows the ECL signal response curve after BHQ-S2 is introduced, and curve e shows the ECL signal response curve after a DNA walker and ExoIII are incubated on the surface of the electrode at the same time;
FIG. 4 shows ECL signal response results of an electrochemiluminescence biosensor to different concentrations of a target according to an embodiment of the present invention; wherein, FIG. 4A is ECL response curve, curves a-i correspond to target concentration of 0, 10 amol. L respectively-1、100amol·L-1、1fmol·L-1、5fmol·L-1、50fmol·L-1、100fmol·L-1、1pmol·L-1And 5 pmol. L-1The response curve below; FIG. 4B is a calibration curve;
FIG. 5 shows ECL signal response results of an electrochemiluminescence biosensor without exonuclease for different concentrations of target in one embodiment of the invention compared to a biosensor with a trigger enzyme; wherein, FIG. 5A is ECL response curve, curves a-h correspond to target concentration of 0.5 fmol.L-1、1fmol·L-1、5fmol·L-1、10fmol·L-1、50fmol·L-1、100fmol·L-1、0.5pmol·L-1And 5 pmol. L-1The response curve below; FIG. 5B is a calibration curve; FIGS. 5C and 5D show the concentration of the target substance at 5 fmol. L, respectively-1And 1 pmol. L-1When the comparison results were responded with ECL signals with and without exonuclease;
FIG. 6 shows the results of selectivity (A) and stability (B) of an electrochemiluminescence biosensor according to an embodiment of the present invention; wherein, in FIG. 6A, a is blank control, and b-g are each 100 fmol.L-1miRNA-21, 100 fmol. L of-1miRNA-141 of (1), 100 fmol. L-1miRNA-126, 100 fmol.L of-1Let-7a, 1 fmol. L-1The result of ECL signal response of the mixture of miRNA-155, and interfering substance of (a);
FIG. 7 shows ECL signal response results of an electrochemiluminescence biosensor in different cell lysates according to an embodiment of the present invention; wherein a is blank control, b-e are cell concentrations of 10 and 10 respectively2、103、104The result of (1).
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Wherein: n- (3- (dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide sodium salt (NHS) and Tetrahydrofuran (THF) were purchased from Sigma Aldrich, Inc. (St.Louis, USA);
poly [ (9, 9-dioctylfluorene-2, 7-diyl) -co- (1, 4-benzo- {2, 1', 3} -thiadiazole) ] (PFBT) was purchased from american polymer sources;
poly (styrene-maleic anhydride) (PSMA) was purchased from alatin limited (shanghai, china);
all of the DNA oligonucleotides and exonucleases III (Exo III) in the present invention were synthesized and purified by Biotechnology engineering (Shanghai) Ltd;
phosphate Buffered Saline (PBS) (pH 7.4, 0.10 mol. L)-1) From H3PO4And NaOH. By dissolving potassium ferricyanide and potassium ferrocyanide with PBS buffer, a ferricyanide solution (Fe (CN))6 3-/4-，5mmol·L-1，pH 7.4)。
The characterization instrument information used in the present invention is as follows:
ECL measurement was performed using an MPI-A ECL analyzer (Siamey electronics technologies, Inc., Siamen, China), with a scanning potential set to 0 to 1.25V and a photomultiplier set to 800V;
electrochemical measurements were performed using CHI600D electrochemical workstation (shanghai chen hua limited instrument, shanghai, china);
transmission Electron Microscopy (TEM) was used to characterize the morphology of the material.
Experimental example 1 Synthesis of PFBT-COOH Point
First, PFBT and PSMA were dissolved in THF to prepare a solution having a concentration of 1.0 mg. multidot.mL-1The stock solution of (1). Then, 4.0mL of the prepared PFBT and 800. mu.L of PSMA were addedMixed and sonicated to form a homogeneous mixed solution. Subsequently, 10mL of redistilled water was poured into the above solution and stirred overnight. Finally, THF was removed by partial vacuum evaporation to obtain PFBT-COOH spots, which were stored away from light.
EXAMPLE 1 preparation of biosensor
Preparation of biped DNA walker
The biped DNA walker is synthesized by a target-catalyzed hairpin assembly process (CHA).
Heating the DNA hairpin H1 and the DNA hairpin H2 to 95 ℃, keeping the reaction for 5min, and then slowly cooling to room temperature to form a hairpin structure;
10 mu L of target miRNA-155 with different concentrations and 5 mu L of 10 mu mol.L-1DNA hairpin H1 was added to 5. mu.L of 10. mu. mol. L-1DNA hairpin H2; the mixture was then incubated at 37 ℃ for 90 min. Obtaining the biped DNA walker.
Construction of the biosensor
Figure 1 illustrates the stepwise construction and assembly process of an ECL biosensor.
a) Polishing a glassy carbon electrode (GCE, phi is 4.0mm) by using 0.30 and 0.05 mu m of alumina respectively, and then carrying out ultrasonic cleaning by using ethanol and secondary distilled water sequentially;
b) dripping 10 mu L of redistilled water dispersion liquid of PFBT-COOH points on the surface of a glassy carbon electrode, drying in the air to form a film, and immersing the modified electrode into a cross-linking agent (0.02 g.mL) at room temperature-1EDC and 0.01 g/mL-1NHS) for 30 min;
c) the concentration of 10. mu.L was adjusted to 10. mu. mol. L-1The DNA S1 of (1) was connected to a PFBT-COOH spot-modified electrode having an active carboxyl group, and incubated overnight at 4 ℃; at room temperature with 1.0 mmol. multidot.L-1After blocking Hexanethiol (HT) for 40min, BHQ-S2 complex (10. mu.L, 10. mu. mol. L) was added-1) Incubating at 37 ℃ for 2h on the decorated electrode;
d) and dripping 10 mu L of the prepared biped DNA walker and 5 mu L of Exo III onto the surface of the modified electrode, and incubating for 2h to obtain the electrochemiluminescence biosensor.
Comparative example 1 ECL biosensor without using DNA exonuclease
The procedure for constructing the ECL biosensor in this comparative example is substantially the same as in example 1 except that no exonuclease is used.
Experimental example 2 characterization of biped DNA walker
The biped DNA walker synthesized by target-catalyzed hairpin assembly was analyzed by polyacrylamide gel electrophoresis (PAGE).
The prepared DNA strand was dropped into a freshly prepared polyacrylamide gel (16%) well, and then subjected to electrophoresis in a 1 XTBE buffer at a potential of 120V for 120 min. After staining with Ethidium Bromide (EB), photographic imaging was performed with a biogel imaging system. As shown in FIG. 2, the missing band in lane 1 corresponds to miRNA-155 (1.0. mu. mol. L)-1) Because it has few nucleotide bases and is not easily stained. Lanes 2 and 3 show the results corresponding to H2 (1.0. mu. mol. L)-1) And H1 (1.0. mu. mol. L)-1) Different strips of (2). Two independent bands (1.0. mu. mol. L) appear in lane 4-1H2 and 1.0. mu. mol. L-1H1) indicating that H2 and H1 can coexist stably in the solution. As expected, when miRNA-155 (1.0. mu. mol. L) was added-1)，H1(1.0μmol·L-1) And H2 (1.0. mu. mol. L)-1) The presence of a band of distinct DNA complexes at the top of lane 5 demonstrates the successful production of a bipedal DNA walker by the target-catalyzed hairpin assembly process.
Experimental example 3 electrochemical characterization and ECL Signal characterization of biosensors
Electrochemical characterization
At 5.0 mmol. multidot.L-1Of [ Fe (CN) ]6]3-/4-Cyclic Voltammograms (CVs) were measured to characterize the stepwise construction process of the biosensor prepared in example 1.
As shown in fig. 3A, CVs of the bare electrode showed reversible redox peaks (curve a). The redox peak current of the PFBT-COOH spot modified electrode decreased (curve b) due to the modified film on the electrode surface blocking the electron transfer. Due to the property of DNA strands to block electron transfer, a significantly reduced redox peak current was detected after incubation of DNA S1 on the prepared electrode (curve c). After blocking with HT, the redox peak current slightly increased (curve d). When BHQ-S2 was introduced, the peak current decreased slightly again (curve e), indicating successful introduction of BHQ-S2. Finally, after the biped DNA walker and ExoIII were incubated simultaneously on the electrode surface, the redox current increased significantly (curve f) because on the one hand BHQ-S2 was released from the electrode surface by the biped DNA walker displacement, while DNAs1 was sheared with the aid of ExoIII, allowing the DNA walker to release and freely displace another BHQ-S2, eventually leaving the DNA strands entirely free from the electrode.
ECL Signal characterization
FIG. 3B shows an electrochemiluminescence biosensor prepared in example 1 in PBS (0.10 mol. L)-1pH 7.4) the ECL signal response curve of the process was constructed stepwise. As shown in fig. 3B, the bare electrode showed almost no ECL signal response in PBS (curve a), while the PFBT-COOH spot modified electrode (curve B) showed a strong ECL signal response, with response values close to 18000a.u., indicating that the PFBT-COOH spot had strong ECL luminescence efficiency. After incubation of the electrodes with DNA S1 and further blocking with HT (curve c), the ECL signal response decreased slightly. When BHQ-S2 was introduced (curve d), the ECL signal response decreased significantly due to the efficient ECL quenching effect of BHQ on PFBT-COOH point. However, when the DNA walker and Exo III were incubated onto the electrode surface simultaneously (curve e), BHQ-S2 was displaced from the electrode surface by the biped DNA walker, allowing the quenched ECL signal to return to the "on" state.
Experimental example 4 ECL biosensor for ECL detection of miRNA
Example 1 response of the prepared ECL biosensor to the target miRNA-155
The response of the ECL biosensor prepared in example 1 to the target miRNA-155 was tested with miRNA-155 as the target. The test results are shown in fig. 4.
As can be seen in FIG. 4A, the anodal ECL signal was from 10 amol.L depending on the miRNA-155 concentration-1To 5 pmol. L-1Is increased. When the signal-to-noise ratio (S/N) is 3, the detection limit is calculated to be 3.3 amol.L-1Wherein, curve a is no targetECL response when present.
FIG. 4B shows that the measured ECL signal has a good positive correlation with the logarithm of the miRNA-155 concentration, the linear equation is that I is 45813.8+2573.6lgc, and the correlation coefficient R is20.9938. Here, I represents the ECL signal intensity and c represents the concentration of miRNA-155.
Response of ECL biosensor prepared in comparative example 1 to target miRNA-155
The biosensor prepared in comparative example 1 was tested for response to the target miRNA-155, using miRNA-155 as the target. The test results are shown in fig. 5.
As shown in FIG. 5A, in the absence of ExoIII, the ECL intensity was from 0.5 fmol.L with miRNA-155 concentration-1Increasing to 5 pmol. L-1Gradually increased, and the detection limit is 0.17fmol & L when the signal-to-noise ratio (S/N) is 3-1. The limit of detection was increased by about 50-fold compared to the two-step amplification in the presence of Exo III in example 1 (fig. 4A). FIGS. 5C and 5D show a comparison of ECL signal intensity in the presence and absence of ExoIII in the presence of the same concentration of miRNA-155. From the results, it can be found that the biosensor in the comparative example, in which secondary amplification was not performed using ExoIII, exhibited a relatively low ECL signal. However, when two-step amplification was performed using ExoIII, the biosensor obtained significantly enhanced ECL signal, indicating that the dual amplification strategy of the present invention can confer superior signal amplification capability to the sensor.
The invention is compared with the methods already reported in the prior art
The results of comparing the method for detecting a target object provided by the present invention with the methods reported in the prior art are shown in table 1. From the results in table 1, it can be found that the detection of the ECL biosensor miRNA provided by the present invention shows higher sensitivity and lower detection limit, which is attributed to the following reasons: (1) PFBT-COOH points have excellent luminous efficiency; (2) resonance energy transfer from the excited state of PFBT-COOH to BHQ ensures that BHQ has high quenching efficiency on ECL signals of PFBT-COOH points and has low background signals; (3) the combination of the target catalyzes hairpin assembly and bipedal DNA walker to achieve dual signal amplification.
TABLE 1 comparison of ECL biosensors of the present invention with other strategies for detecting miRNA
| Detection method | Linear range | Detection limit | Literature reference |
| Fluorescence | 1fmol·L-1～100nmol·L-1 | 1fmol· |
1 |
| Electrochemistry method | 5fmol·L-1～5pmol·L-1 | 10fmol· |
2 |
| ECL | 1fmol·L-1～50pmol·L-1 | 0.33fmol· |
3 |
| ECL | 100amol·L-1～1nmol·L-1 | 29.5amol· |
4 |
| ECL | 10amol·L-1～5pmol·L-1 | 3.3amol·L-1 | The invention |
The literature is listed below:
1.Yin,B.C.；Liu,Y.Q.；Ye,B.C.Sensitive Detection of MicroRNA in ComplexBiological Samples via Enzymatic Signal Amplification Using DNA PolymeraseCoupled with Nicking Endonuclease.Anal.Chem.2013,85,11487-11493.
2.Koo,K.M.；Carrascosa,L.G.；Shiddiky,M.J.A.；Trau,M.Poly(A)Extensionsof MiRNAs for Amplification-Free Electrochemical Detection on Screen-PrintedGold Electrodes.Anal.Chem.2016,88,2000-2005.
3.Zhang,P.；Lin,Z.F.；Zhuo,Y.；Yuan,R.；Chai,Y.Q.Dual microRNAs-FueledDNA Nanogears:A Case of Regenerated Strategy for MultipleElectrochemiluminescence Detection of MicroRNAs with SingleLuminophore.Anal.Chem.2017,89,1338-1345.
4.Liu,J.L.；Tang,Z.L.；Zhang,J.Q.；Chai,Y.Q.；Zhuo,Y.；Yuan,R.Morphology-Controlled 9,10-Diphenylanthracene Nanoblocks as ElectrochemiluminescenceEmitters for MicroRNA Detection with One-Step DNA WalkerAmplification.Anal.Chem.2018,90,5298-5305.
experimental example 5 selectivity and stability of ECL biosensor
Selectivity of ECL biosensor
Common short single stranded RNAs (including miRNA-21, miRNA-141, miRNA-126, and let-7a) were selected as potential interfering substances to study the selectivity of the ECL biosensors provided herein.
As shown in FIG. 6A, the proposed biosensor adds miRNA-21(100 fmol. L) separately-1)、miRNA-141(100fmol·L-1)、miRNA-126(100fmol·L-1) And let-7a (100 fmol. L)-1) Time of flightThe ECL signal intensity of (a) was consistent with that of the blank control. However, when at lower concentrations (1 fmol. L)-1) When miRNA-155 is tested, the ECL signal intensity is obviously enhanced. In addition, after adding the mixture of miRNA-155 and interfering substances, the obtained ECL signal intensity and concentration are 1fmol L-1There was no significant difference in response of miRNA-155 compared to the response. The results show that the ECL biosensor provided by the invention can specifically detect miRNA-155.
Stability of ECL biosensor
At a concentration of 10 fmol. L-1In the presence of miRNA-155, the stability of the biosensor was assessed by continuous scanning. As shown in fig. 6B, the ECL signal intensity showed no significant fluctuation with a Relative Standard Deviation (RSD) of 0.04% for 16 scans at continuous cycling potentials, indicating that the ECL biosensor provided by the present invention has satisfactory stability.
Experimental example 6 application of ECL biosensor
The utility of the biosensor was evaluated by studying the expression of miRNA-155 in cell lysates of human cervical cancer cells (Hela) and breast cancer cells (MCF-7).
As shown in fig. 7, the blank experiment showed very weak ECL signal emission. And the ECL signal intensity is increased along with the increase of the cell number of the Hela, which indicates that the miRNA-155 has certain expression in the Hela cells. At the same time, as the concentration of MCF-7 increased from 10 to 104The ECL signal intensity of each cell is also obviously increased, which indicates that the miRNA-155 is over-expressed in the MCF-7. The results show that the ECL biosensor provided by the invention provides a new method for detecting miRNA.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> Min Hospital in sandlot dam area of Chongqing city, ninth Min Hospital of Chongqing city, university of southwest
<120> modified electrode, combined product, electrochemiluminescence biosensor and application thereof
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Claims (14)
1. A method for detecting nucleic acid, which comprises detecting nucleic acid to be detected by using the following combination product;
the combination product comprises:
modifying the electrode; DNA S1, DNA S2; DNA hairpin H1 and DNA hairpin H2;
the DNA S1 and the DNA S2 can be complementarily paired, and a quenching agent is coupled on the DNA S2;
the DNA hairpin H1 and the DNA hairpin H2 can be assembled under the catalysis of nucleic acid to be detected to form a biped DNA walker; the double-foot part of the double-foot DNA walker can be complementarily paired with the DNA S1, and an enzyme cutting site of DNA exonuclease is reserved on the paired DNA S1;
the combination product does not include a co-reactant;
the sequence of the DNAS1 is shown as SEQ ID NO. 1;
the sequence of the DNAS2 is shown as SEQ ID NO. 2;
the nucleic acid is miRNAs-155, and the sequence of the nucleic acid is shown as SEQ ID NO. 3;
the sequence of H1 is shown as SEQ ID NO. 4, and the sequence of H2 is shown as SEQ ID NO. 5.
2. The method of claim 1, wherein the combination product further comprises the exonuclease;
the DNA exonuclease is exonuclease III;
the quenching agent is selected from a black hole quenching agent and ferrocene.
3. The method of claim 2, wherein the quencher is a black hole quencher.
4. The method of claim 1, wherein the modified electrode comprises an electrode and a luminophore modified on the electrode;
the luminophores are mainly carboxyl functionalized poly [ (9, 9-dioctylfluorene-2, 7-diyl) -co- (1, 4-benzo- {2, 1', 3} -thiadiazole) ] dots;
the electrode is selected from a glassy carbon electrode and a gold electrode.
5. The method of claim 4, wherein the electrode is selected from a glassy carbon electrode.
6. The method of claim 1, comprising the assembly method of an electrochemiluminescence biosensor:
a) assembling DNA S1 by using the modified electrode as a matrix, and introducing DNA S2 which is complementarily paired with the DNA S1;
b) under the catalysis of the nucleic acid to be detected, assembling DNA hairpins H1 and H2 to generate a biped DNA walker;
c) co-incubating the biped DNA walker and the DNA exonuclease with the electrode obtained in step a);
wherein the steps a) and b) are not in sequence.
7. The method according to claim 6, wherein the DNA S1 is assembled on the modified electrode in step a) in the presence of a cross-linking agent; the cross-linking agent is EDC and NHS.
8. The method of claim 6, wherein the modified electrode is prepared by a method comprising:
and (3) dripping the dispersion liquid of the luminophor on the surface of the electrode, and drying to form a film to obtain the modified electrode.
9. The method according to claim 8, wherein a dispersion medium of the dispersion liquid is selected from the group consisting of redistilled water and ultrapure water; the concentration of the luminophor in the dispersion liquid is 0.2 mg/mL-1～0.4mg·mL-1。
10. The method according to claim 6, wherein the preparation process of the biped DNA walker comprises:
mixing the target, the DNA hairpin H1 and the DNA hairpin H2 to obtain a mixture, and incubating the mixture at 25-65 ℃ for 60-120 minutes.
11. The method according to claim 10, wherein the mixture is incubated at 37 ℃ for 90 minutes to obtain the biped DNA walker.
12. The method according to claim 6, wherein the incubation time of the biped DNA walker and the DNA exonuclease on the electrodes is 1-3 hours.
13. The method of claim 12, wherein the biped DNA walker and the DNA exonuclease are incubated on the electrodes for 2 hours.
14. An electrochemiluminescence biosensor assembled by the assembly method as defined in any one of claims 6, 7, 10 to 13.
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| CN110596082B (en) * | 2019-10-17 | 2020-07-24 | 苏州大学 | Probe for detecting trace uranyl ions and portable EC L detector based on probe |
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| CN112007173B (en) * | 2020-08-21 | 2021-07-30 | 上海交通大学 | Application of fluorescent conjugated polymer nano probe in peripheral nerve imaging |
| CN112432980B (en) * | 2020-12-09 | 2021-11-02 | 江南大学 | Pathogen electrochemical detection method based on DNA walker and nanoflower structure |
| CN113219031A (en) * | 2021-03-15 | 2021-08-06 | 皖南医学院 | DNA biped walker signal amplifier, nano-electrode based biosensor and methods of use and applications thereof |
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