Pt NPs@TPB NCs / Dissolved O 2 Ternary electrochemiluminescence biosensor, preparation method and application

Abstract

The present invention provides a biosensor, a preparation method and an application based on a ternary electrochemiluminescence (ECL) system of Pt NPs@TPB NCs / dissolved O2. The ternary ECL system includes: a Pt NPs@TPB NCs / dissolved O2 ECL signal system composed of tetraphenyl-1,3-butadiene nanocrystals (TPB NCs), an endogenous coreactant dissolved O2, and a coreactant promoter for dissolved O2, platinum nanoparticles (Pt NPs); the platinum nanoparticles encapsulating tetraphenyl-1,3-butadiene nanocrystals (Pt NPs@TPB NCs) serve as the ECL luminescent material. The luminescent system of the present invention exhibits ultra-high ECL luminescence efficiency. A novel ECL biosensor constructed based on this luminescent system is used for the detection application of the tumor marker microRNA-21, and has the advantages of simple operation, rapid reaction, a detection range from 100 aM to 100 pM, and a detection limit as low as 83.8 aM.

Description

Technical Field

[0001] The present invention relates to the fields of electrochemiluminescence and bioanalysis detection, and particularly relates to a biosensor based on a ternary electrochemiluminescence system of Pt NPs@TPB NCs / dissolved O 2 , a preparation method thereof, and an application thereof. Background Technique

[0002] Electrochemiluminescence (ECL) is a promising analytical technique excited by an electrochemical process. Due to its high controllability, low background signal, and high sensitivity 1 , it has been gradually applied to environmental monitoring 2 , food safety 3 , and the field of clinical diagnosis 4 . With the development of aqueous-phase ECL luminophores 5-8 , the rise of aggregation-induced emission (AIE) ECL research 9,10 has opened the door for strong ECL emission of organic molecules. Free AIE molecules hardly have obvious ECL signals, while in the aggregated state, through various mechanisms (such as coordination-induced enhanced ECL 11 , ion-induced AIE self-assembly 12 , etc.), the non-radiative transitions in AIE molecules are inhibited, and the radiative transitions are enhanced, thereby improving their ECL signals.

[0003] However, the ECL signals of the reported AIE nanomaterials currently all originate from the interaction between the ECL luminophore and exogenous coreactants (such as persulfate (S 2 O 8 2- ), tripropylamine (TPrA), etc.). The addition of exogenous coreactants increases the complexity of the ECL reaction process in aqueous solutions 13 . Compared with these exogenous coreactants, dissolved O 2 has the advantages of stability and non-toxicity as an endogenous coreactant and is a promising coreactant. However, due to the fact that dissolved O 2 faces the dilemma of low reaction activity of generating reactive oxygen species (ROS, such as O 2 •- and OH • ), there has been no reported ECL luminescence system of AIE nanomaterials using dissolved O 2 as a coreactant.

[0004] Studies have shown that microRNA plays a key role in the pathogenesis, occurrence, and development of cancer. Therefore, highly efficient and ultrasensitive detection of the content of microRNA in human cancer cells is of great significance for the early diagnosis and treatment of cancer. However, current microRNA detection technologies (such as Northern blot hybridization detection, reverse transcription polymerase chain reaction (RT-PCR), etc.) are difficult to meet the requirements of rapid and ultrasensitive detection of microRNA due to relatively low sensitivity, complex primer design, and other disadvantages. Therefore, establishing a new method for analyzing cancer biomarker microRNA with high sensitivity and rapid response for early screening, clinical diagnosis, and treatment of diseases has important clinical value and social significance, and is the direction of unremitting efforts of those skilled in the art. Summary of the Invention

[0005] In view of this, in order to overcome the deficiencies of the prior art, the present invention proposes a ternary electrochemiluminescence (ECL) system of tetraphenyl-1,3-butadiene nanocrystals (Pt NPs@TPB NCs) wrapped by platinum nanoparticles (PtNPs) / dissolved O 2 where PtNPs have a co-reactant promoting effect on dissolved oxygen. Compared with the existing Ru(bpy) 3 2+ / dissolved O 2 system, Pt NPs@TPB NCs exhibit ultra-high ECL luminescence efficiency. And based on this Pt NPs@TPB NCs / dissolved O 2 ternary ECL system, combined with a target-induced DNA walker, the present invention constructs a novel ultrasensitive ECL biosensor for the detection of microRNA-21 in cancer cells.

[0006] The present invention provides a ternary electrochemiluminescence system of Pt NPs@TPB NCs / dissolved O 2 The ternary electrochemiluminescence system includes: a Pt NPs@TPB NCs / dissolved O 2 ECL signal system composed of tetraphenyl-1,3-butadiene nanocrystals TPB NCs, an endogenous co-reactant dissolved O 2 and a co-reactant promoter platinum nanoparticles Pt NPs of dissolved O 2 ;

[0007] The platinum nanoparticle-wrapped tetraphenyl-1,3-butadiene nanocrystals Pt NPs@TPB NCs serve as the ECL luminescent material.

[0008] Furthermore, the co-reactant promoter Pt NPs are prepared using sodium citrate as a protective agent.

[0009] The present invention also provides a method for constructing the above ternary electrochemiluminescence system, and the method includes the steps:

[0010] A. Prepare platinum nanoparticle-coated tetraphenyl-1,3-butadiene nanocrystals Pt NPs@TPB NCs as the ECL luminescent material;

[0011] B. Coat the Pt NPs@TPB NCs solution on the surface of a glassy carbon electrode GCE, and after drying to form a film, place the electrode in air-saturated PBS to obtain the ECL response of Pt NPs@TPB NCs / GCE.

[0012] Furthermore, the preparation of the Pt NPs@TPB NCs includes the steps:

[0013] A.1. Disperse the TPB NCs into deionized water to obtain a uniform TPB NCs solution,

[0014] A.2. Inject an aqueous solution of sodium citrate and H 2 PtCl 6 into the TPB NCs solution and stir, and quickly add ice-cold NaBH 4 solution under continuous stirring;

[0015] A.3. After the solution color turns dark brown, centrifuge, and dissolve the precipitate in deionized water to obtain the Pt NPs@TPBNCs solution.

[0016] Furthermore, the preparation steps of the TPB NCs include:

[0017] A.1.1 Dissolve tetraphenyl-1,3-butadiene powder in tetrahydrofuran to obtain a TPB solution;

[0018] A.1.2 Dropwise inject the TPB solution into the poloxamer 188 solution and perform ultrasonic treatment;

[0019] A.1.3 After removing THF, centrifuge the solution and wash it repeatedly with deionized water, and then centrifuge to obtain the TPB NCs precipitate, and dissolve it in deionized water to obtain the TPB NCs.

[0020] The present invention also provides a biosensor based on the above ternary electrochemiluminescence system, and the biosensor includes:

[0021] I. The above ternary electrochemiluminescence system, and non-specific sites are blocked by dropping hexanethiol on the GCE;

[0022] II. A nucleotide capture probe modified on the GCE;

[0023] III. A DNA walking amplifier induced by the analyte.

[0024] The present invention also provides a method for preparing the above-mentioned ternary electrochemiluminescence biosensor, and the method includes the steps:

[0025] (1) Coat the Pt NPs@TPB NCs solution on the surface of the GCE and dry it in an incubator at 37 °C;

[0026] (2) Modify the capture probe with an amino group onto the GCE and incubate it overnight at 4 °C;

[0027] (3) After rinsing with deionized water, drop thiolhexane onto the modified GCE to block non-specific sites and obtain the biosensor;

[0028] (4) Incubate the DNA walker amplification product solution induced by the analyte on the prepared biosensor, wash it with PBS, and measure the ECL signal of the biosensor in an air-saturated PBS solution.

[0029] Furthermore, the preparation steps of the DNA walker amplification product solution induced by the analyte are:

[0030] (a.) Form a stem-loop structure of the DNA hairpin probe HP, and at the same time generate a double-stranded structure SD / P-Fc of the substrate DNA SD and the ferrocene-modified DNA probe P-Fc;

[0031] (b.) Activate the carboxyl-coated magnetic beads MBs with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and mix them with the HP and SD / P-Fc labeled with an amino group and N-hydroxysuccinimide (NHS) solution, and crosslink the HP and SD / P-Fc onto the MBs through amide bonds to assemble the DNA walker;

[0032] (c.) After magnetic separation, redisperse the DNA walker in a reaction solution containing different concentrations of the target miRNA-21 and Pb 2+ and react at 30 °C for 2 hours;

[0033] (d.) Collect the supernatant containing SD / P-Fc by magnetic separation as the DNA walker amplification product solution.

[0034] The present invention also provides the application of the above-mentioned biosensor, and the application is to detect the expression level of miRNA-21 in cells.

[0035] Furthermore, the application includes the steps:

[0036] a. Incubate the miRNA-21-induced DNA walker amplification reaction in the cell lysate into the biosensor;

[0037] b. Detect the ECL signal;

[0038] c. When the number of cells to be detected increases, if the ECL response of the sensor slightly decreases, it indicates that miRNA-21 is lowly expressed in the cells; if the ECL signal significantly decreases, it indicates that miRNA-21 is highly expressed in the cells.

[0039] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0040] (1) The present invention first uses an endogenous ECL coreactant to dissolve O 2 As an ECL coreactant for aggregation-induced emission (AIE) nanomaterials, it replaces the traditional amine coreactants with relatively high toxicity, which is beneficial to environmental protection. At the same time, the addition of exogenous coreactants is avoided, simplifying the experimental operation.

[0041] (2) The present invention proposes using tetraphenyl-1,3-butadiene nanocrystals encapsulated with platinum nanoparticles (Pt NPs@TPBNCs) as the ECL luminescent material, which exhibits ultra-high ECL luminescence efficiency. At the same time, the co-reaction promoting effect of Pt NPs on dissolved O 2 in the ternary ECL system of Pt NPs@TPB NCs / dissolved O 2 was explored.

[0042] (3) Based on the strong ECL signal provided by the ternary ECL system of Pt NPs@TPB NCs / dissolved O 2 and combined with the target-induced DNA walker to achieve the efficient conversion of the target microRNA-21, a novel ECL biosensing platform was constructed, which is expected to be applied to the clinical detection of the tumor marker microRNA-21. It has the advantages of simple operation, rapid reaction, a detection range of 100 aM to 100 pM, and a detection limit as low as 83.8 aM. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 : CV curve and corresponding ECL signal of TPB NCs / GCE in air-saturated PBS;

[0044] A. When scanning TPB NCs / GCE in an air-saturated PBS solution, a weak O 2 reduction peak appears at about -0.6 V, B. A weak ECL signal (5 a.u.) of TPB NCs is observed at +1.25 V.

[0045] Figure 2: A. CV curves and B. corresponding ECL curves of TPB NCs modified electrodes with a. Pt NPs-1, b. Pt NPs-2, c. Pt NPs-3, d. Pt NPs-4 or e. Pt NFs as co-reaction promoters.

[0046] Figure 3 : A. CV curves and B. corresponding ECL curves of a. Pt NPs@TPB NCs / GCE in air-saturated PBS, b. Pt NPs@TPB NCs / GCE in N 2 -saturated PBS, c. Pt NPs@TPB NCs / GCE in air-saturated PBS containing 30 U mL -1 SOD, or d. Pt NPs@TPB NCs / GCE in air-saturated PBS containing 150 nM L-cys.

[0047] Figure 4 : A. ECL curves of biosensors incubated with different concentrations of miRNA-21 (from a-g: 100 aM, 1 fM, 10 fM, 100 fM, 1 pM, 10 pM or 100 pM); B. Δ I ECL calibration curve between intensity and logarithm of miRNA-21 concentration; C. specificity of the tested ECL biosensor under different interfering miRNAs; (error bars, SD, n = 3); D. stability of the biosensor of the present invention at a miRNA-21 concentration of 100 pM for 14 consecutive scan cycles.

[0048] Figure 5 : Detection of miRNA-21 in different cell numbers of Hela and MCF-7, a. 10 cells, b. 10 2 cells, c. 10 3 cells, d. 10 4 cells, or e. 10 5 cells.

[0049] Figure 6 : Morphology of the prepared Pt nanomaterials, where A. scale bar: 1 cm = 10 nm, B. scale bar: 1 cm = 5 nm.

[0050] Figure 7:Technical roadmap of the present invention: (A) Schematic diagram of the synthesis of Pt NPs@TPB NCs; (B) Construction of a "signal-off" ECL biosensing platform; (C) Principle of the DNA walker amplification strategy induced by the target analyte to be detected. Detailed implementation manners

[0051] Example 1: ECL signal of the TPB NCs / dissolved O 2 system

[0052] I. Preparation of TPB NCs

[0053] First, 5 mg of tetraphenyl-1,3-butadiene (TPB) powder was dissolved in 5 mL of tetrahydrofuran to obtain a 1 mg / mL TPB solution. Subsequently, 1 mL of the TPB solution (1 mg / mL) was dropwise injected into 10 mL of a poloxamer 188 (Pluronic F68) solution (10 mg / mL) and sonicated for 30 minutes. Then, after removing THF, the solution was centrifuged and repeatedly washed with deionized water and centrifuged to obtain a TPB NCs precipitate, which was dissolved in 1 mL of deionized water to obtain TPB NCs (1 mg / mL).

[0054] II. Detection process

[0055] First, the glassy carbon electrode (GCE, Φ = 4 mm) was polished with 0.3 and 0.05 μm alumina powders on suede, and alternately sonicated and cleaned in ultrapure water and ethanol, and dried with nitrogen for later use. Then, 5 μL of the TPB NCs solution was coated on the surface of the GCE and dried to form a film in an incubator at 37 °C. Subsequently, the modified electrode was placed in air-saturated 0.1 M PBS at pH 7.4 and the ECL signal of the modified electrode was measured using an MPI-EII type electrochemiluminescence detector. The scanning potential was from -1 V to 1.3 V, and the scanning rate was 0.3 V / s. The amplifier gain was set to 10×.

[0056] When scanning the TPB NCs / GCE in an air-saturated PBS solution, a weak O 2 reduction peak appeared at about -0.6 V (Figure 1A), and a weak ECL signal (5 a.u., Figure 1B) of TPB NCs was observed at +1.25 V, indicating that TPB NCs as an ECL emitter can lose electrons on the electrode surface to generate TPB NCs radical cations (TPB NCs •+ ), which react with a small amount of ROS to generate the excited state of TPB NCs (TPB NCs * ) to obtain an ECL signal.

[0057] Example 2: Co-reaction acceleration performance of different Pt NPs on TPB NCs.

[0058] The co-reaction acceleration of nanomaterials is summarized in two aspects. One is the enrichment of nanomaterials for dissolved oxygen (O 2 ), and the other is the ability of the material surface to promote the generation of reactive oxygen species (ROS) from dissolved O 2 . Therefore, by preparing different Pt NPs and measuring the electrochemical and ECL signals of TPB NCs with different Pt NPs as co-reaction promoters, the correlation between the co-reaction acceleration ability and the surface properties of Pt NPs was studied. And the corresponding morphology of the prepared Pt NPs was characterized by transmission electron microscopy (TEM).

[0059] Preparation of different Pt nanoparticles.

[0060] (1) Pt NPs-1: Pt NPs without a protective agent were prepared using NaBH 4 as a reducing agent.

[0061] First, 5 mL of freshly prepared 0.01 M NaBH 4 solution was gradually added dropwise to 4 mL of 2 mM H 2 PtCl 6 aqueous solution, and the mixture was vigorously stirred for 20 minutes. After centrifugation, the precipitate was washed alternately with absolute ethanol and deionized water, and then dissolved in deionized water for standby.

[0062] (2) Pt NPs-2: Pt NPs with cetyltrimethylammonium bromide (CTAB) as a protective agent.

[0063] 150 mM CTAB and 1.5 mM K 2 PtCl 4 solutions were heated at 50 °C for 5 minutes to mix evenly. Then, after injecting ice-cold NaBH 4 (1 mL, 30 mM), the beaker was covered with plastic wrap, and the H 2 inside the beaker was released through the needle on the film. Subsequently, the needle was blocked with plastic wrap, and the solution was kept at 50 °C for 6 hours. Finally, the solution was centrifuged at 3000 rpm for 30 minutes to obtain the supernatant, and then centrifuged at 12000 rpm for 10 minutes to obtain the precipitate.

[0064] (3) Pt NPs-3: Pt NPs with 3-thiophene malonic acid (TA) as a protective agent

[0065] 1 mL of 0.038 M H 2 PtCl 6The aqueous solution was added to 50 mL of deionized water. Then, 0.5 mL of 0.3 M TA was dropped into the above solution, and the resulting solution was heated at 100 °C for 20 minutes to obtain a dark brown solution.

[0066] (4) Pt NPs-4: Pt NPs with sodium citrate as the protective agent.

[0067] First, 1 mL of H 2 PtCl 6 (16 mM) aqueous solution and 1 mL of 40 mM sodium citrate solution were diluted to 38 mL with deionized water and stirred in the dark for 30 minutes. Then, 0.2 mL of 50 mM NaBH 4 solution was injected into the above solution. When the color of the solution changed from light yellow to brown, the solution was stirred in the dark for another 1 hour to obtain Pt NPs-4.

[0068] (5) Pt NPs-5: Pt NPs with glucose as the protective agent.

[0069] 0.8 g of glucose and 0.2 g of ascorbic acid were dissolved in 30 mL of deionized water by heating in an 80 °C water bath. Subsequently, 1 mL of H 2 PtCl 6 (20 mM) was added to the solution and stirred for 5 minutes to form a Pt NPs sol. Then, the solution was placed in a 50 °C water bath for about 15 minutes while adding 0.3 mL of H 2 PtCl 6 (20 mM) to obtain Pt nanoflowers (Pt NPs-5).

[0070] Effects of different Pt NPs on the CV and ECL signals of TPB NCs.

[0071] First, the glassy carbon electrode (GCE, Φ = 4 mm) was polished with 0.3 and 0.05 μm alumina powder on suede, and then alternately ultrasonically cleaned in ultrapure water and ethanol and dried with nitrogen for use. Then, 5 μL of TPB NCs solution was coated on the surface of the GCE, dried in an incubator at 37 o °C, and then Pt NPs-1 was dropwise coated and dried in an incubator at 37 oIt was dried to form a film in an incubator at C. Subsequently, the modified electrode was placed in 0.1 M PBS with pH 7.4, and the ECL signal of the modified electrode was measured using an MPI-E type electrochemiluminescence detector. The scanning potential was from -1 V to 1.3 V, and the scanning rate was 0.3 V / s. The amplifier gain was set to 10× (the modification and detection processes of Pt NPs-2 to Pt NPs-5 were the same as above). The results are as follows:

[0072] Compared with the ECL signal of Pt NPs-1 without a protective agent (curve a, Figure 2B), Pt NPs-2 using cetyltrimethylammonium bromide (CTAB) as a protective agent showed a weaker ECL signal (curve b, Figure 2B), which may be attributed to the incomplete exposure of the catalytic sites on the surface of Pt NPs-2, affecting the catalytic performance. It is worth noting that 3-thiophene malonic acid (TA)-stabilized Pt NPs (Pt NPs-3) showed the weakest ECL response among all Pt NPs, which was attributed to the low catalytic performance of the amorphous Pt NPs-3. In addition, we also synthesized different morphologies of Pt NPs (Pt NPs-4 and Pt NFs) using sodium citrate and glucose as protective agents. As shown in Figure 2A, both Pt NPs-4 and Pt NFs showed a strong reduction current of dissolved O 2 starting from about -0.6 V (curves d and e). At the same time, Pt NPs-4 as a co-reaction promoter showed the highest ECL signal, which was attributed to the large specific surface area of the uniformly sized Pt NPs to enrich oxygen and excellent catalytic activity to generate a large amount of ROS. Therefore, the surface catalytic sites and particle size of Pt NPs strongly affect the generation of ROS in dissolved O 2 Thus, Pt NPs-4 reduced by sodium citrate was used to synthesize Pt NPs@TPB NCs for further performance studies. The morphology of the prepared Pt nanomaterial (Pt NPs-4) is as Figure 6 shown.

[0073] Example 3: ECL Enhancement Mechanism of Pt NPs@TPB NCs

[0074] I. Preparation of Pt NPs@TPB NCs

[0075] 1 mL of TPB NCs (1 mg / mL) was redispersed in 9 mL of deionized water to obtain a uniform TPB NCs solution, and then 0.25 mL of sodium citrate (40 mM) and 375 μL of 1% H 2 PtCl 6An aqueous solution was injected into the above solution and stirred for 60 minutes. Under continuous stirring, 0.5 mL of freshly prepared ice-cold NaBH 4 solution (30 mM) was added rapidly. Finally, the solution color turned dark brown, indicating the in-situ reduction of PtNPs onto TPB NCs. After centrifugation, the precipitate was dissolved in 2 mL of deionized water to obtain a Pt NPs@TPB NCs (0.5 mg / mL) solution for further use.

[0076] II. CV and ECL responses of Pt NPs@TPB NCs / GCE in air-saturated PBS solution

[0077] After the in-situ generation of Pt NPs on TPB NCs, the CV and ECL responses of Pt NPs@TPBNCs / GCE were investigated in air-saturated PBS solution. Pt NPs@TPB NCs showed a strong cathodic reduction peak current starting at approximately -0.604 V and a strong ECL emission (17340 a.u.) at 1.25 V. That is, the ECL signal of Pt NPs@TPB NCs increased by thousands of times relative to TPB NCs, which was attributed to the introduction of Pt NPs as a co-reactant promoter on TPB NCs. To further evaluate the ECL enhancement effect of PtNPs on the TPB NCs / dissolved O 2 system, the CV and ECL responses of Pt NPs@TPBNCs / GCE were measured in nitrogen (N 2 )-saturated PBS. As shown by curve b in Figure 3 Figure A, due to the lack of dissolved O 2 as a co-reactant reagent, the current intensity decreased rapidly and was accompanied by an almost invisible ECL signal (curve b, Figure 3B). Subsequently, superoxide dismutase (SOD) and L-cysteine (L-cys) were used as effective scavengers of superoxide (O 2 •- ) and hydroxyl radical (OH • ), respectively, to study the ECL enhancement of multiple ROS in the ternary ECL system. As shown in 2 •- Figure B, the ECL signal of Pt NPs@TPB NCs / GCE in PBS solution containing L-cys (curve c) decreased more during the scan of the same modified electrode than that in PBS solution containing SOD (curve d), indicating that both OH Figure 3 and O • and O 2 •- can react with TPB NCs •+ to generate TPB NCs *, for the ECL enhancement of the Pt NPs@TPB NCs / dissolved O 2 system.

[0078] Herein, based on the above experimental results, the high ECL signal of the Pt NPs@TPB NCs / dissolved O 2 ternary ECL system is attributed to the generation of a large amount of ROS and the rapid electron transfer between TPB NCs and ROS, and the corresponding mechanism is shown as follows.

[0079]

[0080] Example 4: Performance of the ECL biosensor

[0081] I. Preparation of the biosensor:

[0082] First, 5 μL of the Pt NPs@TPB NCs solution was coated on the surface of the GCE and dried in an incubator at 37 o °C. Subsequently, 10 μL of the capture probe (CP DNA, 2 uM) with an amino group (NH 2 ) was modified onto the GCE and incubated overnight at 4 ºC. After rinsing with deionized water, 10 μL of hexanethiol (HT, 5 mM) was added dropwise to the modified GCE for 1 hour to block non-specific sites, and the biosensor was reserved for use.

[0083] II. Target-induced DNA walker amplification:

[0084] The 4 μM DNA hairpin probe (HP) was annealed at 95 °C for 5 minutes and then slowly cooled to room temperature to form a stem-loop structure. At the same time, with the concentration ratio of the substrate DNA (SD) and the ferrocene-modified DNA probe (P-Fc) maintained at 1:1, a 4 μM DNA duplex structure (SD / P-Fc) was generated by heating to 85 °C for 10 minutes and then slowly cooled to room temperature. Subsequently, 40 μL of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 0.2 M) was added to 20 μL of magnetic beads MB (5 mg / mL) and stirred for 30 minutes to activate the carboxyl groups of the magnetic beads (MBs). Subsequently, 40 μL of NHS (N-hydroxysuccinimide, 0.05 M), 50 μL of HP, and 50 μL of SD / P-Fc were mixed with the above solution and shaken overnight at 4°C to assemble the DNA walker by crosslinking the above DNA onto the MBs through amide bonds. After magnetic separation, the DNA walker was redispersed in 100 μL containing different concentrations of the target miRNA-21 (100 aM - 100 pM) and 100 μM Pb 2+In the reaction solution, react at 30 °C for 2 hours. Finally, collect the supernatant containing S1 / P-Fc by magnetic separation as the product solution for standby.

[0085] III. Performance of the ECL biosensor of the present invention

[0086] Incubate the product solution on the prepared biosensor for 2 hours. After washing with PBS, measure the ECL signal of the biosensor in 0.1 M pH 7.4 PBS, with the scanning potential from -1 V to 1.3 V and the scanning rate of 0.3 V / s. The amplification gain is set to 10×.

[0087] Under the optimal experimental conditions (the concentration ratio of HP and SD / P-Fc is 1:2, and the incubation time between CP and the product solution containing P-Fc is 2 hours), S1 / P-Fc solutions generated by incubating different concentrations of miRNA-21 from 100 aM to 100 pM on the biosensor were used. As shown in Figure 4A, the ECL response gradually decreases with the increase in the concentration of miRNA-21. In addition, there is a good linear relationship between the change in ECL intensity (Δ I ECL = I 0 - I ) and the logarithm of the miRNA-21 concentration (Figure 4B). The linear regression equation is expressed as Δ I = 8583.86 + 2032.52 lg c ( R = 0.998), and the limit of detection (LOD) is as low as 83.8 aM ( S / N = 3). In addition, using other miRNAs (miRNA-141, miRNA-199a, miRNA-155) as interference, the specificity of the ECL biosensor was studied by comparing the ECL signals of miRNA-21 and the interfering substances under the same conditions. As shown in Figure 4C, although the concentration of the interfering substance (100 pM) is 10 times higher than that of miRNA-21 (10 pM), compared with the blank control, the interfering miRNA shows a slight change in the ECL response. However, when the target miRNA-21 is added to the above interfering substances, the ECL signal significantly decreases, which is basically the same as the ECL signal obtained by incubating miRNA-21 alone. The above results demonstrate the outstanding specificity of the proposed ECL biosensor for the target miRNA-21. In addition, to further evaluate the stability of the biosensor, the ECL signal was measured by continuously scanning 14 cycles (Figure 4D), and no obvious fluctuation was shown (RSD = 1.51%), indicating that the proposed biosensor has good stability.

[0088] Example 5: Analysis of miRNA-21 in cancer cells

[0089] First, 10 cervical cancer cells (HeLa) and human breast cancer cells (MCF-7) were separately taken by a cell counter, and cell lysates were obtained by treatment with an RNA extraction kit. The practicability of this method in cancer cell analysis was studied by testing the expression of miRNA-21 in HeLa and MCF-7 cell lysates. 6 The two cell lysates were respectively diluted into sample solutions with different cell numbers (10

[0090] ~10 2 ). The sample solutions were used to replace the standard miRNA-21 solution, and after the target-induced DNA walker amplification reaction, they were incubated into the biosensor, and their ECL signals were detected. As 6 shown, when the cell number of HeLa increased from 10 cells to 10 Figure 5 , the ECL response of the sensor decreased slightly, which means that miRNA-21 is lowly expressed in HeLa cells. In MCF-7 cells, as the cell number increased from 10 to 10 5 , the ECL signal decreased significantly, indicating that miRNA-21 is highly expressed in MCF-7 cells, which is very consistent with the research results reported in the literature. The above results show that the method proposed by the present invention has great feasibility in monitoring the expression of cancer cell miRNA biomarkers. 5

[0091] The Pt NPs in-situ reduced on TPB NCs of the present invention not only provide a large number of active sites for O 2 , in-situ generate ROS to react with TPB NCs to obtain strong ECL emission as the "signal enhancement" state, but also are used to immobilize the capture probe (CP DNA) through Pt-N bonds to further hybridize with the ferrocene-labeled product DNA (P-Fc). Subsequently, the target-induced DNA walker amplification process is carried out in homogeneous phase. As Figure 7 shown in C, the hairpin (HP), substrate DNA (SD), and ferrocene-labeled DNA strand (P-Fc) were assembled on magnetic beads (MBs) in proportion to obtain a DNA walker. In the presence of the target miRNA-21, HP on the MBs can hybridize with miRNA-21 to form a partial double-stranded structure, where the single-stranded part serves as a DNA swinging arm. The target-induced DNA walker starts to operate with the assistance of Pb 2+ DNAzyme. And, the SD DNA is specifically recognized by the DNA swinging arm containing the enzyme strand DNA sequence and is cleaved by Pb 2+ ​DNAzyme cleavage releases the DNA arm to participate in another cleavage process, and a large amount of S1 / P-Fc is generated simultaneously. After magnetic separation, the product solution containing S1 / P-Fc is incubated on the biosensor, and P-Fc is captured on the electrode surface through the hybridization of P-Fc with CPDNA. Due to the strong quenching effect of ferrocene, a decreased ECL signal is presented, which is regarded as the "signal quenching" state. It should be noted that based on the synergistic effect of the co-reaction acceleration and crystallization-induced enhanced ECL emission in Pt NPs@TPB NCs and the target-induced DNA walker amplification strategy, the present invention develops a ultrasensitive ECL biosensing platform, which can achieve the ultrasensitive detection of miRNA-21 with a detection range from 100 aM to 100 pM and a detection limit of 83.8 aM. Therefore, this method shows great potential in biological analysis and clinical diagnosis.

[0092] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

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Claims

1. A ternary electrochemiluminescence system of Pt NPs@TPB NCs / dissolved O 2 ​ It is characterized in that The ternary electrochemiluminescence system includes: tetraphenyl-1,3-butadiene nanocrystals TPB NCs and endogenous coreactant dissolved O 2 and dissolved O 2 The coreactant promoter platinum nanoparticles Pt NPs that make up the Pt NPs@TPB NCs / dissolved O 2 ECL signal system; the platinum nanoparticles encapsulated tetraphenyl-1,3-butadiene nanocrystals Pt NPs@TPB NCs are used as the ECL luminescent material, and the coreactant promoter Pt NPs are prepared with sodium citrate as the protective agent; The preparation method of Pt NPs@TPB NCs comprises the following steps: A.

1. Disperse TPB NCs into deionized water to obtain a uniform TPB NCs solution. A.

2. Inject sodium citrate and H 2 PtCl 6 aqueous solution into the TPB NCs solution and stir. Rapidly add ice-cold NaBH 4 solution under continuous stirring; A.

3. After the solution color turns dark brown, centrifuge it, and dissolve the precipitate in deionized water to obtain the Pt NPs@TPB NCs solution.

2. According to the construction method of the ternary electrochemiluminescence system described in claim 1, It is characterized in that the method comprises the steps of: A. Prepare the platinum nanoparticles encapsulated tetraphenyl-1,3-butadiene nanocrystals Pt NPs@TPB NCs as the ECL luminescent material; B. Coat the Pt NPs@TPB NCs solution on the surface of the glassy carbon electrode GCE, and after drying to form a film, place the electrode in an air-saturated PBS solution to obtain the ECL response of Pt NPs@TPB NCs / GCE.

3. According to the construction method of the ternary electrochemiluminescence system described in claim 2, It is characterized in that the preparation steps of the TPBNCs include: A.1.1 Dissolve the tetraphenyl-1,3-butadiene powder in tetrahydrofuran to obtain a TPB solution; A.1.2 Dropwise inject the TPB solution into the poloxamer 188 solution and perform ultrasonic treatment; A.1.3 After removing THF, centrifuge the solution and wash it repeatedly with deionized water, and then centrifuge to obtain the TPB NCs precipitate, and dissolve it in deionized water to obtain the TPB NCs.

4. The biosensor based on the ternary electrochemiluminescence system described in claim 1, It is characterized in that the biosensor comprises: I. The ternary electrochemiluminescence system described in claim 1, and hexanethiol is dropped on the GCE to block non-specific sites; II. The nucleotide capture probe modified onto the GCE; III. The DNA walker amplifier induced by the analyte.

5. According to the preparation method of the biosensor of the ternary electrochemiluminescence system described in claim 4, It is characterized in that the method comprises the steps of: (1) Coat the Pt NPs@TPB NCs solution on the surface of the GCE and dry it in an incubator at 37 °C; (2) Modify the capture probe with an amino group onto the GCE and incubate it overnight at 4 °C; (3) After rinsing with deionized water, drop hexanethiol on the modified GCE to block non-specific sites to obtain the biosensor; (4) Incubate the DNA walker amplified product solution induced by the analyte on the prepared biosensor, wash it with PBS, and measure the ECL signal of the biosensor in an air-saturated PBS solution.

6. According to the preparation method of the biosensor of the ternary electrochemiluminescence system described in claim 5, It is characterized in that the preparation steps of the DNA walker amplified product solution induced by the analyte are: (a.)The DNA hairpin probe HP forms a stem-loop structure, while the substrate DNA SD and the ferrocene-modified DNA probe P-Fc form a duplex structure SD / P-Fc; (b.)The carboxyl-coated magnetic beads MBs are activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC and mixed with HP labeled with amino group, SD / P-Fc, and N-hydroxysuccinimide NHS solution. They are shaken overnight at 4 °C, and the HP and SD / P-Fc are cross-linked on the MBs through amide bonds to assemble the DNA walker; (c.)After magnetic separation, the DNA walker was redispersed in the reaction solution containing different concentrations of the target miRNA-21 and Pb 2+ and reacted at 30 °C for 2 h; (d.)The supernatant containing SD / P-Fc is collected by magnetic separation as the DNA walker amplification product solution.

7. The application of the biosensor according to claim 4, wherein, the application is to detect the expression level of miRNA-21 in cells.

8. The application of the biosensor according to claim 7, wherein, the application comprises the steps of: a. Incubating the miRNA-21-induced DNA walker amplification reaction in the cell lysate into the biosensor; b. Detecting the ECL signal; c. When the ECL response of the sensor slightly decreases with the increase in the number of cells to be detected, it indicates that miRNA-21 is lowly expressed in cells; when the ECL signal significantly decreases, it indicates that miRNA-21 is highly expressed in cells.

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