CN115420779A - Pt NPs@TPB NCs/dissolved O2 ternary electrochemiluminescence biosensor, preparation method and application - Google Patents

Abstract

The invention provides a biosensor based on a Pt NPs@TPB NCs/dissolved O ₂ ternary electrochemiluminescence system, a preparation method and an application. The ternary electrochemiluminescence system includes: tetraphenyl-1,3-butadiene nanocrystalline TPB NCs and endogenous co-reactant dissolved O ₂ and co-reaction accelerator platinum nanoparticles Pt NPs dissolved O ₂ Constituted Pt NPs@TPB NCs/dissolved O ₂ ECL signal system; the platinum nanoparticles wrap tetraphenyl-1,3-butadiene nanocrystal Pt NPs@TPB NCs as ECL luminescent material. The luminous system of the present invention exhibits ultra-high ECL luminous efficiency. The new ECL biosensor based on this luminescent system is used for the application detection of tumor marker microRNA-21, which has the advantages of simple operation, fast response, detection range of 100aM to 100pM, and detection limit as low as 83.8aM.

Description

Preparation of Pt NPs@TPB NCs/dissolved O2 ternary electrochemiluminescence biosensor Method and application

technical field

The invention relates to the field of electrochemiluminescence and the field of biological analysis and detection, in particular to a biosensor based on a Pt NPs@TPB NCs/dissolved O ₂ ternary electrochemiluminescence system, a preparation method and an application thereof.

Background technique

Electrochemiluminescence (ECL ⁾ , a promising analytical technique inspired by electrochemical processes, has been gradually applied in environmental monitoring2, ^{food safety3} and The field of clinical diagnosis ⁴ . With the development of aqueous ECL ^{luminophores5-8} , the rise of aggregation-induced emission ( ^AIE ) ECL research9,10 opened the door to strong ECL emission from organic molecules. AIE molecules in the free state have almost no obvious ECL signal, while in the aggregated state, non-radiative transitions in AIE molecules are suppressed through various mechanisms (such as coordination-induced enhancement of ECL ¹¹ , ion-induced AIE self-assembly ¹² , etc.), Enhances the radiative transition, thereby enhancing its ECL signal.

However, the ECL signals of AIE nanomaterials reported so far are all derived from the interaction between ECL luminophores and exogenous coreactive reagents (such as peroxodisulfate (S ₂ O ₈ ^2- ), tripropylamine (TPrA), etc. role, the addition of co-reaction reagents increases the complexity of the ECL reaction process in aqueous solution ¹³ . Compared with these exogenous co-reactants, dissolved _O2 as an endogenous co _- reactant has the advantage of being stable and non-toxic, and is a promising co-reactant. Due to the low reactivity of oxygen radicals (ROS, such as O ₂ ^·- and OH ^· ), no AIE nanomaterial ECL luminescence system using dissolved O ₂ as a co-reactant has been reported.

Studies have shown that microRNA plays a key role in the pathogenesis, occurrence and development of cancer. Therefore, efficient and ultra-sensitive detection of the content of microRNA in human cancer cells is of great significance for the early diagnosis and treatment of cancer. However, the current microRNA detection technology (Northern blot hybridization detection, reverse transcription polymerase chain reaction (RT-PCR), etc.) is difficult to meet the rapid and ultra-sensitive detection of microRNA due to the shortcomings of relatively low sensitivity and complicated primer design. Therefore, establishing a new method for the analysis of cancer marker 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.

Contents of the invention

In view of this, in order to overcome the deficiencies of the prior art, the present invention proposes a tetraphenyl-1,3-butadiene nanocrystal (Pt NPs@TPB NCs)/dissolved O ₂ wrapped by platinum nanoparticles (PtNPs) Ternary ECL system in which PtNPs have a co-reaction promoting effect on dissolved oxygen. Compared with the state-of-the-art Ru(bpy) ₃ ²⁺ /dissolved O ₂ system, Pt NPs@TPB NCs exhibit ultrahigh ECL luminescence efficiency. And based on the Pt NPs@TPB NCs/dissolved O ₂ ternary ECL system, combined with the target-induced DNA walker, a novel ultrasensitive ECL biosensor was constructed, which can be used for the detection of microRNA-21 in cancer cells.

The invention provides a ternary electrochemiluminescence system of Pt NPs@TPB NCs/dissolved O ₂ , the ternary electrochemiluminescence system comprises: tetraphenyl-1,3-butadiene nanocrystal TPB NCs and Pt NPs@TPB NCs/dissolved O ₂ ECL signal system composed of endogenous co-reactant dissolved O ₂ and O ₂ co-reaction accelerator platinum nanoparticles Pt NPs;

The platinum nanoparticles wrap tetraphenyl-1,3-butadiene nanocrystal Pt NPs@TPB NCs as ECL luminescent material.

Further, the co-reaction accelerator Pt NPs is prepared using sodium citrate as a protecting agent.

The present invention also provides a method for constructing the above-mentioned ternary electrochemiluminescent system, the method comprising the steps of:

A. Preparation of tetraphenyl-1,3-butadiene nanocrystalline Pt NPs@TPB NCs wrapped in platinum nanoparticles as ECL luminescent material;

B. The Pt NPs@TPB NCs solution was coated on the surface of the glassy carbon electrode GCE, and after drying to form a film, the electrode was placed in air-saturated PBS to obtain the ECL response of Pt NPs@TPB NCs/GCE.

Further, the preparation of the Pt NPs@TPB NCs includes steps:

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

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

A.3. Centrifuge the solution after it turns dark brown, and dissolve the precipitate in deionized water to obtain a Pt NPs@TPBNCs solution.

Further, the preparation steps of the TPB NCs include:

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

A.1.2 Inject the TPB solution dropwise into the Poloxamer 188 solution for ultrasonic treatment;

A.1.3 After removing THF, the solution was centrifuged and washed repeatedly with deionized water, centrifuged to obtain TPB NCs precipitate, which was dissolved in deionized water to obtain the TPB NCs.

The present invention also provides a biosensor based on the above-mentioned ternary electrochemiluminescent system, the biosensor comprising:

I. The above-mentioned ternary electrochemiluminescence system, and GCE is dripped with hexanethiol to block non-specific sites;

Ⅱ. Nucleotide capture probes modified to GCE;

III. Analyte-induced DNA walking amplifiers.

The present invention also provides a method for preparing the above-mentioned ternary electrochemiluminescence biosensor, the method comprising the steps of:

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

(2) Modify the capture probe with amino groups onto GCE and incubate overnight at 4°C;

(3) After washing with deionized water, the modified GCE was added dropwise with hexanethiol to block non-specific sites to obtain a biosensor;

(4) The analyte-induced DNA walker amplification product solution was incubated on the prepared biosensor, and after washing with PBS, the ECL signal of the biosensor was measured in an air-saturated PBS solution.

Further, the preparation steps of the DNA walker amplification product solution induced by the analyte are:

(a.) DNA hairpin probe HP is formed into a stem-loop structure, while substrate DNA SD and ferrocene-modified DNA probe P-Fc generate duplex structure SD/P-Fc;

(b.) The carboxyl-coated magnetic beads MBs were activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and mixed with amino-labeled HP and SD /P-Fc and N-hydroxysuccinimide (NHS) solutions were mixed, and the HP and SD/P-Fc were cross-linked through amide bonds to assemble DNA walkers on MBs;

(c.) After magnetic separation, the DNA walker was redispersed in the reaction solution containing different concentrations of target miRNA-21 and Pb ²⁺ and reacted at 30°C for 2 hours;

(d.) The SD/P-Fc-containing supernatant was collected by magnetic separation as a DNA walker amplification product solution.

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

Further, the application includes the steps of:

a. Incubating the DNA walker amplification reaction induced by miRNA-21 in the cell lysate into the biosensor;

b. Detect ECL signal;

c. When the ECL response of the sensor decreases slightly with the increase of the number of cells to be tested, the expression of miRNA-21 in the cell is low; the ECL signal decreases significantly, indicating that the expression of miRNA-21 in the cell is high.

Compared with prior art, the beneficial effect of the present invention is:

(1) The present invention adopts endogenous ECL co-reaction reagent to dissolve O for the first time As the ECL co _- reaction reagent of aggregation-induced emission (AIE) nanomaterials, it replaces the traditional toxic amine co-reaction reagent, which is beneficial to environmental protection, At the same time, the addition of exogenous co-reaction reagents is avoided, and the experimental operation is simplified.

(2) The present invention proposes tetraphenyl-1,3-butadiene nanocrystals (Pt NPs@TPBNCs) wrapped with platinum nanoparticles as an ECL luminescent material, which exhibits ultra-high ECL luminous efficiency. The obvious co-reaction promotion of dissolved O ₂ by PtNPs in the NPs@TPB NCs/dissolved O ₂ ternary ECL system.

(3) The present invention provides a strong ECL signal based on the Pt NPs@TPB NCs/dissolved O ₂ ternary ECL system, combined with the target-induced DNA walker to achieve efficient conversion of the target microRNA-21, and constructs a new type of ECL The biosensing platform is expected to be applied to the clinical detection of tumor marker microRNA-21. It has the advantages of simple operation, fast response, detection range of 100aM to 100pM, and detection limit as low as 83.8aM.

Description of drawings

Figure 1: CV curves and corresponding ECL signals of TPB NCs/GCE in air-saturated PBS;

A. When scanning TPB NCs/GCE in air-saturated PBS solution, a weak _O2 reduction peak appeared around -0.6V, B. A weak ECL signal of TPB NCs was observed at +1.25V (5a.u. ).

Figure 2: A.CV 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 Curves and B. Corresponding ECL curves.

Figure 3: a.Pt NPs@TPB NCs/GCE in air-saturated PBS, b.Pt NPs@TPB NCs/GCE in _N2 -saturated PBS, c.Pt NPs@TPB NCs/GCE in 30U mL ^-1 A. CV curves and B. corresponding ECL curves of SOD in air-saturated PBS, or d. Pt NPs@TPBNCs/GCE in air-saturated PBS containing 150 nM L-cys.

Figure 4: A. ECL curves of biosensors incubated with different concentrations of miRNA-21 (from ag: 100aM, 1fM, 10fM, 100fM, 1pM, 10pM or 100pM); B. Between ΔI _ECL intensity and logarithm of miRNA-21 concentration Calibration curve; C. The specificity of testing the ECL biosensor under different interfering miRNAs; (error bars, SD, n=3); D. The biosensor of the present invention was continuously scanned for 14 cycles when the miRNA-21 concentration was 100pM stability.

Figure 5: Detection of miRNA-21 in Hela and MCF-7 with different cell numbers, a. 10 cells, b. 10 ² cells, c. 10 ³ cells, d. 10 ⁴ cells, Or e. The number of cells is 10 ⁵ .

Figure 6: Morphology of prepared Pt nanomaterials, where A. Scale bar: 1 cm = 10 nm, B. Scale bar: 1 cm = 5 nm.

Figure 7: The technical roadmap of the present invention: (A) Schematic diagram of the synthesis of Pt NPs@TPB NCs; (B) construction of "signal off" ECL biosensing platform; (C) DNA walker amplification induced by the target to be tested The rationale for the strategy.

detailed description

Example ₁ : ECL signal of TPB NCs/dissolved O system

1. Preparation of TPB NCs

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 TPB solution (1 mg/mL) was injected dropwise into 10 mL of Poloxamer 188 (Pluronic F68) solution (10 mg/mL) for 30 minutes of sonication. Then, after removing THF, the solution was centrifuged and washed repeatedly with deionized water, and centrifuged to obtain a precipitate of TPB NCs, which was dissolved in 1 mL of deionized water to obtain TPB NCs (1 mg/mL).

2. Detection process

Firstly, the glassy carbon electrode (GCE, Φ=4mm) was polished on the suede with 0.3 and 0.05 μm alumina powder, and then ultrasonically cleaned alternately in ultrapure water and ethanol, and dried with nitrogen gas for later use. Then, 5 μL of TPB NCs solution was coated on the surface of GCE and dried in a 37 °C incubator to form a film. Subsequently, the modified electrode was placed in air-saturated 0.1M pH7.4PBS and the ECL signal of the modified electrode was measured with an MPI-EII electrochemiluminescence detector, with a scanning potential of -1V to 1.3V and a scanning rate of 0.3V/s . The amplifier gain is set to 10×.

When scanning TPB NCs/GCE in air-saturated PBS solution, a weak _O2 reduction peak appeared around -0.6V (Fig. 1A), and a weak ECL signal of TPB NCs was observed at +1.25V (5a.u. , Figure 1B), which indicates that TPB NCs as ECL emitters 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 excited states of TPB NCs (TPB NCs ^* ) to obtain ECL Signal.

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

The co-reaction acceleration of nanomaterials can be summarized into two aspects. One is the enrichment of dissolved oxygen (O ₂ ) by nanomaterials, and the other is the ability of the material surface to promote dissolved O ₂ to generate reactive oxygen species (ROS). Therefore, by preparing different PtNPs and measuring the electrochemical and ECL signals of TPB NCs with different Pt NPs as co-reaction accelerators, the correlation between the co-reaction accelerating ability and the surface properties of Pt NPs was investigated. And the corresponding morphology of the as-prepared Pt NPs was characterized by transmission electron microscopy (TEM).

Preparation of different Pt nanoparticles.

(1) Pt NPs-1 prepared Pt NPs without protective agent by using NaBH ₄ as reducing agent.

First, 5 mL of 0.01 M freshly prepared NaBH ₄ solution was injected dropwise into 4 mL of 2 mM H ₂ PtCl ₆ aqueous solution and vigorously stirred for 20 min. After centrifugation, the precipitate was alternately washed with absolute ethanol and deionized water, and then dissolved in deionized water for later use.

(2) Pt NPs-2 Pt NPs with cetyltrimethylammonium bromide (CTAB) as protective agent.

Heat the 150 mM CTAB and 1.5 mM _K2PtCl4 solution at 50 °C for ₅ min to mix well. Then, after injecting ice-cold NaBH ₄ (1 mL, 30 mM), the beaker was covered with plastic wrap, and the H ₂ in the beaker was released through a needle on the film. The needle was then 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 a supernatant, and then centrifuged at 12000 rpm for 10 minutes to obtain a precipitate.

(3) Pt NPs-3 Pt NPs with 3-thiophene malonate (TA) as protective agent

Add 1 mL of 0.038M H ₂ PtCl ₆ aqueous solution to 50 mL of deionized water. Then, 0.5 mL of 0.3 M TA was added dropwise to the above solution, and the resulting solution was heated at 100 °C for 20 min to obtain a dark brown solution.

(4) Pt NPs-4 Pt NPs with sodium citrate as protective agent.

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 for 30 minutes in the dark. Then inject 0.2 mL of 50 mM NaBH4 solution into the above solution within ₂ min. When the color of the solution changed from light yellow to brown, the solution was stirred for 1 hour in the dark to obtain Pt NPs-4.

(5) Pt NPs-5 Pt NPs with glucose as protective agent.

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

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

Firstly, the glassy carbon electrode (GCE, Φ=4mm) was polished on the suede with 0.3 and 0.05 μm alumina powder, and then ultrasonically cleaned alternately in ultrapure water and ethanol, and dried with nitrogen gas for later use. Then, 5 μL of TPB NCs solution was coated on the GCE surface, and after drying in a 37 °C incubator, Pt NPs-1 was continued to be drip-coated and dried in a 37 °C incubator to form a film. Subsequently, the modified electrode was placed in 0.1M pH 7.4 PBS and the ECL signal of the modified electrode was measured with an MPI-E electrochemiluminescence detector, with a scanning potential of -1V to 1.3V and a scanning rate of 0.3V/s. The amplifier gain was set to 10× (the modification and detection process of Pt NPs-2～Pt NPs-5 were consistent with the above). The result is as follows:

Compared with the ECL signal of Pt NPs-1 without protecting agent (curve a, Figure 2B), Pt NPs-2 using cetyltrimethylammonium bromide (CTAB) as protecting agent showed weaker The ECL signal (curve b, Fig. 2B), which may be attributed to the incomplete exposure of catalytic sites on the surface of PtNPs-2 would affect the catalytic performance. Notably, 3-thiophenemalonic acid (TA)-stabilized Pt NPs (Pt NPs-3) showed the weakest ECL response among all Pt NPs, which was attributed to the amorphous Pt NPs-3 having low catalytic performance. In addition, we also synthesized different morphologies of PtNPs (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 exhibit strong reduction currents for dissolved O starting from about −0.6 V (curves d and e). Meanwhile, Pt NPs-4 as a co-reaction promoter showed the highest ECL signal, which was attributed to the large specific surface area of Pt NPs with uniform particle size for enriching oxygen and excellent catalytic activity to generate a large amount of ROS. Therefore, the surface catalytic sites and particle size of Pt NPs can strongly affect the generation of _ROS in dissolved O. Therefore, sodium citrate-reduced Pt NPs-4 was used to synthesize Pt NPs@TPB NCs for further performance studies. The morphology of the prepared Pt nanomaterials (PtNPs-4) is shown in Fig. 6.

Example 3: ECL enhancement mechanism of Pt NPs@TPB NCs

1. Preparation of Pt NPs@TPB NCs

Redisperse 1 mL of TPB NCs (1 mg/mL) into 9 mL of deionized water to obtain a homogeneous TPB NCs solution, then inject 0.25 mL of sodium citrate (40 mM) and 375 μL of ₁ % _H2PtCl6 aqueous solution into the above solution and stir for 60 min . 0.5 mL of freshly prepared ice-cold NaBH ₄ solution (30 mM) was added rapidly with continuous stirring. Finally, the solution color turned to 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.

2. CV and ECL responses of Pt NPs@TPB NCs/GCE in air-saturated PBS solution

After 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 began to show a strong cathodic reduction peak current at about -0.604V, and a strong ECL emission (17340a.u.) appeared at 1.25V, that is, the ECL signal of Pt NPs@TPB NCs increased by several thousand relative to TPB NCs times, which was attributed to the introduction of Pt NPs on TPB NCs as co-reaction promoters. To further evaluate the ECL enhancement effect of Pt NPs on TPB NCs/dissolved O ₂ system, the CV and ECL responses of Pt NPs@TPB NCs/GCE were measured in nitrogen (N ₂ ) saturated PBS. As shown in curve b of Figure 3A, due to the lack of dissolved _O2 as a co-reactant, the current intensity decreased rapidly, accompanied by a barely visible ECL signal (curve b, Figure 3B). Subsequently, superoxide dismutase (SOD) and L-cysteine (L-cys) were used as effective sources of superoxide (O ₂ ^·- ) and hydroxyl radical (OH ^· ) and O ₂ ^·- scavengers to study the ECL-enhancing effect of multiple ROS in the ternary ECL system. As shown in Fig. 3B, the ECL signal of Pt NPs@TPB NCs/GCE in PBS solution containing L-cys (curve c) decreased more than that of the same modified electrode scan in PBS solution containing SOD (curve d). , which indicated that both OH ^· and O ₂ ^·- could react with TPB NCs ^·+ to generate TPB NCs ^* , which was used for ECL enhancement of Pt NPs@TPB NCs/dissolved O ² system.

Here, based on the above experimental results, the high ECL signal of the Pt NPs@TPB NCs/dissolved O _ternary ECL system is attributed to the generation of a large number of ROS and the fast electron transfer between TPB NCs and ROS, and the corresponding mechanism is shown below.

O ₂ ^·- +H ₂ O ₂ →O ₂ +OH ^- +OH ^· [3]

TPB NCs-e ^- → TPB NCs ⁺ [4]

TPB NCs+O ₂ ^·- →TPB NCs ^·- +O ₂ [5]

TPB NCs ^·- +OH →TPB NCs ^* +OH ^- [6]

TPB NCs ⁺ +TPB NCs ^- →TPB NCs ^* +TPB NCs [7]

TPB NCs ^* →TPB NCs+hv [8]

Example 4: Performance of ECL biosensors

1. Preparation of biosensors:

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

2. Target-induced DNA walker amplification:

4 μM DNA hairpin probe (HP) was annealed at 95°C for 5 minutes, then slowly cooled to room temperature to form a stem-loop structure. At the same time, with the concentration ratio of substrate DNA (SD) and ferrocene-modified DNA probe (P-Fc) kept at 1:1, a 4 μM DNA duplex structure (SD /P-Fc), and then slowly cooled to room temperature. Subsequently, 40 μL of LEDC (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 Carboxyl groups of magnetic beads (MBs). Subsequently, 40 μL of NHS (N-hydroxysuccinimide, 0.05M), 50 μL of HP, and 50 μL of SD/P-Fc were mixed with the above solution and shaken overnight at 4 °C to cross-link the above DNA on MBs via amide bonds Assemble the DNA walker. After magnetic separation, the DNA walker was redispersed in 100 μL of reaction solution containing different concentrations of target miRNA-21 (100aM-100pM) and 100 μM Pb ²⁺ , and reacted at 30°C for 2 hours. Finally, the supernatant containing S1/P-Fc was collected by magnetic separation as a product solution for future use.

Three, the performance of the ECL biosensor of the present invention

The product solution was incubated on the prepared biosensor for 2 hours. After washing with PBS, the ECL signal of the biosensor was measured in 0.1 M pH 7.4 PBS with a scan potential ranging from −1 V to 1.3 V at a scan rate of 0.3 V/s. The amplification gain is set to 10×.

Under optimal experimental conditions (HP and SD/P-Fc concentration ratio 1:2, incubation time between CP and P-Fc-containing product solution for 2 h), different Concentration of miRNA-21 produced by S1/P-Fc solution. As shown in Figure 4A, the ECL response gradually decreased with increasing miRNA-21 concentration. In addition, there is a good linear relationship between the change of ECL intensity (ΔI _ECL =I ₀ -I) and the logarithm of miRNA-21 concentration (Fig. 4B), and the linear regression equation is expressed as ΔI=8583.86+2032.52lg c (R= 0.998), the limit of detection (LOD) was as low as 83.8aM (S/N=3). In addition, using other miRNAs (miRNA-141, miRNA-199a, miRNA-155) as interference, the specificity of this ECL biosensor was investigated by comparing the ECL signal of miRNA-21 with the interfering substances under the same conditions. As shown in Figure 4C, although the interferor concentration (100 pM) was 10-fold higher than that of miRNA-21 (10 pM), the interfering miRNA exhibited a slight change in ECL response compared with the blank control. However, when the target miRNA-21 was added to the above interferents, the ECL signal decreased significantly, which was basically consistent with 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 continuous scanning for 14 cycles (Fig. 4D), which showed no obvious fluctuation (RSD = 1.51%), which indicated the good stability of the proposed biosensor.

Example 5: Analysis of miRNA-21 in cancer cells

First, 10 ⁶ cervical cancer cells (HeLa) and human breast cancer cells (MCF-7) were collected respectively by a cell counter, and treated with an RNA extraction kit to obtain cell lysates. The practicability of this method in cancer cell analysis was investigated by testing the expression of miRNA-21 in HeLa and MCF-7 cell lysates.

The two cell lysates were diluted to sample solutions with different cell numbers (10 ² -10 ⁶ ). The sample solution was replaced with the standard miRNA-21 solution, and after the target-induced DNA walker amplification reaction, it was incubated into the biosensor, and its ECL signal was detected. As shown in Fig. 5, when the number of HeLa cells increased from ¹⁰ cells to 105 cells, the ECL response of the sensor decreased slightly, which meant that miRNA-21 was underexpressed in HeLa cells. In MCF-7 cells, as the number of cells increased from 10 to 10 ⁵ , the ECL signal decreased significantly, indicating that miRNA-21 was highly expressed in MCF-7 cells, which was very consistent with the research results reported in the literature. The above results show that the method proposed in the present invention has great feasibility in monitoring the expression of miRNA biomarkers in cancer cells.

The present in situ reduced Pt NPs on TPB NCs not only provide a large number of active sites for _O2 to in situ generate ROS to react with TPB NCs to obtain strong ECL emission as a "signal enhancement" state, but also through the Pt-N The bond is used to immobilize the capture probe (CP DNA) for further hybridization of the ferrocene-labeled product DNA (P-Fc). Subsequently, the target-induced DNA walker amplification process is performed in homogeneous phase. As shown in Figure 1C, hairpins (HP), substrate DNA (SD), and ferrocene-labeled DNA strands (P-Fc) were assembled on magnetic beads (MBs) in scale to obtain DNA walkers. In the presence of target miRNA-21, HP on MBs can hybridize with miRNA-21 to form a partial duplex structure, in which the single-stranded part acts as a DNA swing arm. The target-induced DNA walker is assisted by Pb ²⁺ DNase. Moreover, SD DNA is specifically recognized by the DNA swing arm containing the DNA sequence of the enzyme chain, and is cut by Pb ²⁺ DNase, releasing the DNA swing arm to participate in another cutting process and producing a large amount of S1/P-Fc at the same time. After magnetic separation, the product solution containing S1/P-Fc was incubated on the biosensor, and P-Fc was captured to the electrode surface through the hybridization of P-Fc and CP DNA. Due to the strong quenching effect of ferrocene, the Reduced ECL signal, referred to as the "signal quenched" state. Notably, based on the synergistic effect of Pt NPs@TPB NCs co-reaction acceleration and crystallization-induced enhancement of ECL emission and target-induced DNA walker amplification strategy, the present invention developed an ultrasensitive ECL biosensing platform capable of realizing Ultrasensitive detection of miRNA-21 with a detection range of 100aM to 100pM and a detection limit of 83.8aM. Therefore, this method shows great potential in biological analysis and clinical diagnosis.

Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and not 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 it can be described in terms of form and Various changes may be made in the details without departing from the scope of the invention defined by the claims.

references:

¹ Dong, JR; Lu, YX; Xu, Y.; Chen, FF; Yang, JM;

² Han, ZG; Yang, ZF; Sun, HS; Xu, YL; Ma, XF; Shan, DL; Chen, J.; Insoluble organic molecules for ultrasensitive aqueous-phase detection. Angew. Chem. Int. Ed. 2019, 58, 5915–5919.

³ Zhang, RR; Gan, XT; Xu, JJ; Pan, QF; Liu, H.; ,2022,370,131353.

⁴ Chen, MM; Xu, CH; Zhao, W.; Chen, HY; Xu, JJ Single cell imaging of electrochemiluminescence-driven photodynamic therapy. Angew. Chem. Int. Ed. 2022, 61, e202117401.

⁵ Xiong, XY; Xiong, CY; Gao, Y.; Xiao, Y.; Chen, MM; Wen, W.; multiple convertible resonance energy transfersystem.Anal.Chem.,2022,94,7861-7867.

⁶ Yang, XL; Wei, YX; Wang, ZM; Wang, JX; Qi, HL; Gao, Q.; Zhang, CX Highly efficient electrogenerated chemiluminescence quenching on lipid-coated multifunctional magnetic nanoparticles for the determination of proteases. 2022, 94, 2305-2312.

⁷ Gao, N.; Zeng, H.; Wang, XF; Zhang, Y.; Zhang, S.; Cui, RW; Ed., 2022, 61, e202204485.

⁸ Zhao, YR; Bouffier, L.; Xu, GB; Loget, G.; Sojic, N. Chem. Sci., 2022, 13, 2528-2550.

⁹ Wei, X.; Zhu, MJ; Cheng, Z.; Lee, M.; Yan, H.; , 58, 3162–3166.

¹⁰ Gao, XW; Zhao, HM; Wang, DY; Xu, YQ; Zhang, B.; Zou, GZ Selectively lighting up singlet oxygen via aggregation-induced electrochemiluminescence energy transfer.

¹¹ Wang, XF; Liu, HW; Jiang, JX; Qian, MP; Qi, HL; Gao, Q.; Zhang, CX Highly efficient aggregation-induced enhanced electrochemiluminescence of cyanophenyl-functionalized tetraphenylethene and its application in biothiolsanalysis. 2022,94,5441-5449.

¹² Gao, XW; Zhao, HM; Wang, DY; Xu, YQ; Zhang, B.; Zou, GZ Selectively lighting up singlet oxygen via aggregation-induced electrochemiluminescence energy transfer.

¹³ Zheng, HZ; Zu, YBEmission of tris(2,2′-bipyridine) ruthenium(ii) by coreactant electrogenerated chemiluminescence: from O ₂ -insensitive to highly O ₂ -sensitive.J.Phys.Chem.B 2005,109,12049– 12053.

Claims (10)

1. A ternary electrochemiluminescence system of Pt NPs@TPB NCs/dissolved O ₂ , characterized in that the ternary electrochemiluminescence system comprises: tetraphenyl-1,3-butadiene nanocrystals Pt NPs@TPB NCs/dissolved O ₂ ECL signal system composed of TPB NCs and endogenous co-reactant dissolved O ₂ and co-reaction accelerator platinum nanoparticles Pt NPs for dissolved O ₂ ; The platinum nanoparticles wrap tetraphenyl-1,3-butadiene nanocrystal Pt NPs@TPB NCs as ECL luminescent material. 2. The ternary electrochemiluminescence system according to claim 1, wherein the co-reaction accelerator PtNPs is prepared by using sodium citrate as a protective agent. 3. The construction method of the ternary electrochemiluminescent system according to claim 1, wherein the method comprises the steps of: A. Preparation of tetraphenyl-1,3-butadiene nanocrystalline Pt NPs@TPB NCs wrapped in platinum nanoparticles as ECL luminescent material; B. The Pt NPs@TPB NCs solution was coated on the surface of the glassy carbon electrode GCE, and after drying to form a film, the electrode was placed in an air-saturated PBS solution to obtain the ECL response of the Pt NPs@TPB NCs/GCE. 4. The construction method of the ternary electrochemiluminescence system according to claim 3, wherein the preparation of the PtNPs@TPB NCs comprises the steps of: A.1. Disperse TPB NCs into deionized water to obtain a homogeneous TPB NCs solution, A.2. Inject sodium citrate and H ₂ PtCl ₆ aqueous solution into the TPB NCs solution and stir, and quickly add ice-cold NaBH ₄ solution under continuous stirring; A.3. Centrifuge the solution after it turns dark brown, and dissolve the precipitate in deionized water to obtain a Pt NPs@TPB NCs solution. 5. The construction method of the ternary electrochemiluminescent system according to claim 4, wherein the preparation step of the TPBNCs comprises: A.1.1 Dissolve tetraphenyl-1,3-butadiene powder in tetrahydrofuran to obtain TPB solution; A.1.2 Inject the TPB solution dropwise into the Poloxamer 188 solution for ultrasonic treatment; A.1.3 After removing THF, the solution was centrifuged and washed repeatedly with deionized water, centrifuged to obtain TPB NCs precipitate, which was dissolved in deionized water to obtain the TPB NCs. 6. based on the biosensor of the ternary electrochemiluminescent system described in claim 1, it is characterized in that, described biosensor comprises: 1. the ternary electrochemiluminescence system described in claim 2, and GCE drips hexanethiol blocking non-specific site; Ⅱ. Nucleotide capture probes modified to GCE; III. Analyte-induced DNA walking amplifiers. 7. according to the preparation method of the biosensor of ternary electrochemiluminescence system described in claim 6, it is characterized in that, described method comprises the steps: (1) Apply the Pt NPs@TPB NCs solution on the GCE surface and dry it in an incubator at 37 °C; (2) Modify the capture probe with amino groups onto GCE and incubate overnight at 4°C; (3) After washing with deionized water, the modified GCE was added dropwise with hexanethiol to block non-specific sites to obtain a biosensor; (4) The analyte-induced DNA walker amplification product solution was incubated on the prepared biosensor, and after washing with PBS, the ECL signal of the biosensor was measured in an air-saturated PBS solution. 8. according to the preparation method of the biosensor of ternary electrochemiluminescence system described in claim 7, it is characterized in that, the preparation step of the DNA walker amplification product solution that described analyte induces is: (a.) The DNA hairpin probe HP is formed into 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 were activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC, and mixed with amino-labeled HP and SD/P- Fc and N-hydroxysuccinimide NHS solution were mixed, shaken at 4°C overnight, and the HP and SD/P-Fc were cross-linked through amide bonds to assemble DNA walkers on MBs; (c.) After magnetic separation, the DNA walker was redispersed in the reaction solution containing different concentrations of target miRNA-21 and Pb ²⁺ and reacted at 30°C for 2 hours; (d.) The SD/P-Fc-containing supernatant was collected by magnetic separation as a DNA walker amplification product solution. 9. The application of the biosensor according to claim 6, wherein the application is to detect the expression level of miRNA-21 in cells. 10. The application of the biosensor according to claim 9, wherein the application comprises the steps of: a. Incubating the DNA walker amplification reaction induced by miRNA-21 in the cell lysate into the biosensor; b. Detect ECL signal; c. When the ECL response of the sensor decreases slightly with the increase of the number of cells to be tested, the expression of miRNA-21 in the cell is low; the ECL signal decreases significantly, indicating that the expression of miRNA-21 in the cell is high.

Images