Hydrogel microneedle, preparation method and biological background interference resisting electrochemical sensing application

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a hydrogel microneedle, a preparation method and biological background interference resisting electrochemical sensing application. The hydrogel microneedle provided by the invention is composed of a hydrogel matrix and a nucleic acid nano reservoir-oxidation signal silence zone electroactive molecule compound, the nucleic acid nano-reservoir is of a stable cross-shaped double-chain structure formed by complementarily pairing four single-chain nucleic acids; the oxidation signal silence zone electroactive molecules are embedded into the nucleic acid nano-reservoir through an intercalation effect. The hydrogel microneedle provided by the invention can be prepared into an electrochemical sensor, and when the hydrogel microneedle is used for directly extracting and detecting tumor interstitial fluid, degradation and dilution of target miRNA can be reduced, a one-to-many responsive release indicator is transferred to the surface of an electrode, negative potential electrochemical oxidation of electroactive molecules is promoted to generate an electric signal without biological background interference, and the electrochemical sensor is used for detecting tumor interstitial fluid. Therefore, the problems of low-abundance marker detection sensitivity and background interference of endogenous biomolecules in a complex physiological matrix are solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to a hydrogel microneedle, a preparation method thereof, and an application in anti-biological background interference electrochemical sensing. Background Art

[0002] Functionalized microneedle sensors with biorecognition elements can specifically capture target biomarkers in tumor interstitial fluid (TIF), and then through electrochemical techniques with high sensitivity and rapid analysis characteristics, achieve accurate and sensitive in-situ analysis of biomarkers in tumor interstitial fluid. This method can directly detect the extracted biomarkers in-situ without additional steps for transferring tumor interstitial fluid, thus avoiding dilution or degradation of biomarkers during the transfer process of tumor interstitial fluid caused by traditional microneedle collection devices. However, this method also has some problems. Since microneedles often have a dense internal structure, this will cause the diffusion of target biomolecules to the transducer surface (i.e., the electrode surface) to slow down compared to small molecules, thereby reducing the probability of analyte capture and the signal intensity generated by detection. In addition, since most electroactive interfering small molecules (such as ascorbic acid, uric acid, dopamine, tryptophan, lysine) in tumor interstitial fluid can generate electrochemical signals with positive oxidation potentials on the working electrode, if the analyte to be measured also generates an electrical signal at a positive redox potential, then the detection signal it generates is easily interfered by the electrochemical signals generated by interfering small molecules, resulting in inaccurate detection results of the analyte, especially for low-abundance analytes. However, most current homogeneous electrochemical studies require applying a positive potential for oxidation, so the accurate detection of biomarkers in tumor interstitial fluid still faces great challenges.

[0003] In summary, it is necessary to propose new methods and strategies to supplement the deficiencies of the existing technology. Summary of the Invention

[0004] The purpose of the present invention is to provide a hydrogel microneedle based on the responsive transfer of electroactive molecules, a preparation method thereof, and an application, which partially solve or alleviate the above deficiencies in the existing technology. The present invention specifically adopts the following technical solutions.

[0005] A hydrogel microneedle with anti-biological background interference, the hydrogel microneedle is composed of a hydrogel matrix and a nucleic acid nanorepository-oxidation signal silent region electroactive molecule complex; the nucleic acid nanorepository is a stable cruciform double-stranded structure formed by complementary pairing of 4 single-stranded nucleic acids; the oxidation signal silent region electroactive molecule includes doxorubicin hydrochloride or methylene blue; the oxidation signal silent region electroactive molecule is embedded in the double-stranded nucleic acid structure of the nucleic acid nanorepository through intercalation.

[0006] As a preference, the oxidation signal silent region electroactive molecule is doxorubicin hydrochloride.

[0007] Furthermore, the hydrogel microneedles have a loose and porous structure; alternatively, the hydrogel matrix coats the nucleic acid reservoir-oxidation signal silencing region molecular electroactive molecule complex.

[0008] Furthermore, the 4 single-stranded nucleic acids have a base complementary pairing structure.

[0009] As a preference, among the 4 single-stranded nucleic acids, the first nucleic acid has a sequence structure that is partially base complementary paired with the second nucleic acid and the fourth nucleic acid; the second nucleic acid also has a sequence structure that is partially base complementary paired with the third nucleic acid; the third nucleic acid also has a sequence structure that is partially base complementary paired with the fourth nucleic acid; any one of the sequences in the cruciform double-stranded structure has a sequence structure for binding to the target nucleic acid.

[0010] Alternatively, as a preference, among the 4 single-stranded nucleic acids, the first nucleic acid is used for complementary pairing with the second nucleic acid and the fourth nucleic acid; the second nucleic acid is also used for complementary pairing with the third nucleic acid; the third nucleic acid is also used for complementary pairing with the fourth nucleic acid.

[0011] Furthermore, the hydrogel matrix includes methacrylated hyaluronic acid and a photoinitiator.

[0012] As a preference, the volume ratio of the methacrylated hyaluronic acid to the photoinitiator is 10:1.

[0013] As a preference, the sequences of the 4 single-stranded nucleic acids are as shown in SEQ ID NO.1-4.

[0014] The preparation method of the above-mentioned hydrogel microneedles includes the following steps: S01: Prepare the nucleic acid nanorepository: Anneal equal volumes of the first nucleic acid, the second nucleic acid, the third nucleic acid, and the fourth nucleic acid at 90-100 °C to form a metastable hairpin structure; then place it at a temperature below 50 °C (preferably 30-40 °C), shake, and carry out a hybridization reaction to form a stable cruciform (double-stranded structure) nucleic acid nanorepository; S02: Prepare the nucleic acid nanorepository-oxidation signal silencing region electroactive molecule complex: Add an oxidation signal silencing region electroactive molecule solution to the cruciform nucleic acid nanorepository, and co-incubate at a temperature below 50 °C (preferably 30-40 °C) to form a nucleic acid nanorepository-oxidation signal silencing region electroactive molecule complex; the oxidation signal silencing region electroactive molecule includes doxorubicin hydrochloride or methylene blue; S03: Mix methacrylated hyaluronic acid and a photoinitiator uniformly to obtain a hydrogel matrix solution containing the photoinitiator; add the nucleic acid nanorepository-oxidation signal silencing region electroactive molecule complex to obtain a mixed solution. Place the mixed solution in a mold which has multiple regularly arranged downwardly concave microneedle cavities of the same size. Concentrate the mixed solution at a temperature below 50 °C (preferably 30 - 40 °C) and fully fill the voids of the mold microneedle cavities, and crosslink under ultraviolet light to obtain patch-shaped hydrogel microneedles.

[0015] Further, the volume ratio of the cross-shaped nucleic acid nanorepository to the oxidation signal silencing region electroactive molecule is 10:1 - 5; the volume ratio of the cross-shaped nucleic acid nanorepository to the oxidation signal silencing region electroactive molecule includes 10:1, 10:2, 10:3, 10:4, or 10:5.

[0016] Further, the volume ratio of the hydrogel matrix solution containing the photoinitiator to the nucleic acid nanorepository-oxidation signal silencing region electroactive molecule complex is 10:1 - 5; the volume ratio of the hydrogel matrix solution containing the photoinitiator to the oxidation signal silencing region electroactive molecule includes 10:1, 10:2, 10:3, 10:4, or 10:5.

[0017] Further, the photoinitiator is lithium phenyl(2,4,6-trimethylbenzoyl)phosphate (LAP).

[0018] As a preference, the volume ratio of the methacrylated hyaluronic acid to the photoinitiator standard solution is 10:1.

[0019] Use of the above hydrogel microneedles in the preparation of an electrochemical sensor for anti-biological background interference.

[0020] Further, the electrochemical sensor is used for in-situ detection of interstitial fluid.

[0021] Beneficial technical effects: The present invention provides a hydrogel microneedle (patch) with anti-biological background interference. The hydrogel microneedle is an electrochemical microneedle that generates a negative potential oxidation signal for anti-interference. Experiments prove that the hydrogel microneedle has the potential to be prepared into an electrochemical nanosensor.

[0022] When using this electrochemical microneedle sensor to directly extract and detect tumor interstitial fluid, it is possible to reduce the degradation and dilution of the target miRNA, achieve "one-to-many" responsive release of the indicator transferred to the electrode surface (i.e., one target triggers the release of multiple doxorubicin molecules), promote the negative potential electrochemical oxidation of the electroactive molecule to generate an electro signal without biological background interference, and thus solve the problems of detection sensitivity of low-abundance markers and background interference of endogenous biomolecules in complex physiological matrices.

[0023] Experimental verification of the present invention shows that when the hydrogel microneedle sensor prepared by the present invention is used to explore the sensing performance for the target nucleic acid (miRNA-21), the hydrogel microneedle sensor exhibits a low detection limit (1.25 pM) and high reproducibility (RSD 1.9%). Compared with qRT-PCR, in differentiating breast cancer patients from healthy patients, the clinical accuracy (AUC value) is significantly improved from 0.73 to 0.92. Brief Description of the Drawings

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to actual scale. Obviously, the following-described drawings are some embodiments of the present invention, and those of ordinary skill in the art can obtain other drawings based on these drawings without creative efforts.

[0025] Figure 1 Schematic diagram of the process for preparing FWJ-DOX-HAMA MNs in one embodiment of the present invention; Figure 2 To verify the construction and disassembly of the cross-shaped nucleic acid nanorepository prepared by the present invention; Figure 3 To study the assembly and target-induced disassembly processes of FWJ prepared by the present invention using the FRET method; Figure 4 To explore the interaction between the electroactive molecule and the FWJ nanorepository using circular dichroism experiments; Figure 5 To study the adsorption and release of MB and DOX on FWJ prepared by the present invention; Figure 6 Detection of the hydrogel microneedle array patch prepared from HAMA in one embodiment of the present invention; Figure 7 To verify the ability of the hydrogel microneedle array patch to extract tumor interstitial fluid; Figure 8 To detect the mechanical properties of the hydrogel microneedle array patch; Figure 9 To verify the sensitivity of the hydrogel microneedle array patch; Figure 10 To detect the specificity and reproducibility of the hydrogel microneedle array patch; Figure 11 To detect the anti-interference of the hydrogel microneedle array patch; Figure 12For the high - repeatability detection of the hydrogel microneedle array patch; Figure 13 Results of comparing the hydrogel microneedle sensor prepared by the present invention with q - PCR; Figure 14 To verify the application of the hydrogel microneedle sensor prepared by the present invention in breast cancer detection; Figure 15 To detect the interstitial fluid of breast cancer patients' tissues using the hydrogel microneedle sensor prepared by the present invention; Figure 16 ROC curve for detecting miRNA using the hydrogel microneedle sensor prepared by the present invention and qPCR method. Detailed embodiments

[0026] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0027] As used herein, "and / or" includes any and all combinations of one or more of the listed related items.

[0028] As used herein, "a plurality of" means two or more, that is, it includes two, three, four, five, etc.

[0029] As used in this specification, the term "about" typically represents + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0030] In this specification, certain embodiments may be disclosed in a format within a certain range. It should be understood that this description of "within a certain range" is only for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub - ranges and individual numerical values within this range. For example, the description of the range 1 - 6 should be regarded as having specifically disclosed sub - ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and individual numbers within this range, such as 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0031] Detailed description of the drawings: Figure 1 a is a schematic diagram for the preparation of microneedles. Figure 1 b is a digital photo of the HAMA microneedle array. Figure 1 c is the SEM image of the HAMA microneedles. Figure 1 d is the three-dimensional confocal image of the HAMA microneedles prepared with the mixed fluorescent molecule Cy5.

[0032] Figure 2 a is a schematic diagram for the formation of FWJ and the addition of miRNA-responsive disassembly. Figure 2 b is the PAGE analysis of the target-induced strand displacement reaction: Lane M: standard DNA; Lane 1: DNAa (2 μM); Lane 2: DNAb (2 μM); Lane 3: DNAc (2 μM); Lane 4: DNAd (2 μM); Lane 5: miRNA-21 (400 nM); Lane 6: FWJ; Lane 7: FWJ + miRNA-21 (400 nM); Lane 8: DNAd + miRNA-21 (400 nM); Lane 9: DNAa + b + c (2 μM).

[0033] Figure 3 a is the fluorescence spectrum of FWJ after reacting with the absence and presence of miRNA-21. Figure 3 b is the time-dependent fluorescence signal of the FWJ nanorepository in the presence of miRNA-21 (1 nM).

[0034] Figure 4 a-b are the circular dichroism spectra of FWJ and FWJ + MB, FWJ and FWJ + DOX respectively. Figure 4 c-d are the time-resolved fluorescence intensity decay curves of FWJ and FWJ + MB, FWJ and FWJ + DOX.

[0035] Figure 5 a is the loading efficiency of FWJ for MB or DOX at different times. Figure 5 b is the release efficiency of MB and DOX at different time points when there is a target in FWJ-MB and FWJ-DOX.

[0036] Figure 6 a-b are the photos of the HAMA solution, HAMA + FWJ + DOX solution and the hydrogel formed by photocrosslinking respectively. Figure 6 c-d are the SEM images of the HAMA hydrogel and HAMA-FWJ-DOX hydrogel (scale bar is 100 μm) respectively. Figure 6 e is the energy dispersive X-ray spectroscopy elemental analysis of the HAMA-FWJ-DOX hydrogel. Figure 6 f is the infrared spectrum of the FWJ-DOX-HAMA hydrogel.

[0037] Figure 7 a is the weight change diagram of the microneedle absorbing PBS at different times; Figure 7 b is the swelling rate change curve of the microneedle absorbing PBS at different times; Figure 7 c - d are the CLSM images of the microneedle absorbing small molecule DOX and large molecule nucleic acid DNA - Cy5 respectively.

[0038] Figure 8 a is the mechanical compression test of the microneedle; Figure 8 b is the skin surface photo of the microneedle pressed into pig skin for 10 min.

[0039] Figure 9 a is the peak current response of the nanosensor to different concentrations of miRNA - 21 (5 pM - 10 nM); Figure 9 b is the linear relationship between the peak current and the logarithm of the miRNA - 21 concentration.

[0040] Figure 10 a is the specificity study of the microneedle sensor for detecting 50 pM miRNA - 21 and 500 pM interfering miRNA; Figure 10 b is the repeatability evaluation of the microneedle sensor for 20 independent detections of miRNA - 21 (concentration of 50 pM).

[0041] Figure 11 a is the anti - interference performance study of the nanosensing system (adding 50 μM DA, AA, UA, Tyr, Trp, L - DOPA in 50 pM miRNA - 21); Figure 11 b is the spiked recovery detection of different concentrations of miRNA - 21 in serum samples (sample 1: 100 pM, sample 2: 50 pM, sample 3: 10 pM).

[0042] Figure 12 a - b are the peak currents of the electrochemical microneedle sensor for detecting miRNA - 21 in cell TIF (10 6 MCF - 10A cells and 10 6 MCF - 7 cells) and the total RNA extracted (n = 5).

[0043] Figure 13 a is the ultraviolet absorption spectrum of the RNA extracted from MCF - 7 cells; Figure 13 b is the qRT - PCR standard curve of miRNA - 21 extracted from different numbers of cells. Figure 14Box plots of the relative expression levels of miRNA-21 detected directly by TIF, extracted miRNA, and qRT-PCR in tumor-bearing mice (n = 3) and healthy mice (n = 3), respectively.

[0044] Figure 15 Heat maps of the expression levels of TIF and extracted miRNA in breast cancer patients and healthy donors plotted by micro-needle sensors and qRT-PCR, respectively.

[0045] Figure 16 ROC curves of breast cancer classification results based on miRNA-21 levels in micro-needle nano-sensors and qRT-PCR, respectively.

[0046] Glossary of terms: The "nucleic acid nano-reservoir" described in the present invention refers to a nano-structure formed by self-assembly of nucleic acids and used as a storage and controlled-release carrier.

[0047] The "electroactive molecule with an oxidized signal silent region" described in the present invention refers to a class of electroactive biomolecules that do not generate their own background interference within the Raman biological "silent region" defined at 1800 - 2800 cm -1 and have characteristic peaks within this region.

[0048] The "electroactive molecule responsive transfer" described in the present invention refers to the fact that the nucleic acid nano-reservoir - electroactive molecule complex with an oxidized signal silent region (FWJ-DOX) contained in the hydrogel micro-needle sensor provided by the present invention can disassemble (also known as responsive disassembly) after capturing the target nucleic acid, so that the electroactive molecule (DOX) is released from the micro-needle and rapidly transferred to the electrode, and a negative potential oxidation signal anti-interference electrochemical detection is realized at the electrode interface.

[0049] Experimental reagents and instruments: 1. Main materials, reagents and equipment Table 1 Experimental reagents Table 2 Nucleic acid sequences involved Table 3 Experimental instruments 2. Oxidation potentials of common electroactive interferents Table 4 Example 1 This example provides an example of the preparation of hydrogel micro-needles.

[0050] 1. Preparation of cross-shaped nucleic acid nano-reservoir (FWJ) The first nucleic acid (DNA a), the second nucleic acid (DNA b), the third nucleic acid (DNA c), and the fourth nucleic acid (DNA d), each at 10 μM, were annealed at 95°C for 5 min to form a metastable hairpin structure, and then stored in a 4°C refrigerator for later use. Subsequently, 10 μL (10 μM) of each of the first nucleic acid (DNA a), the second nucleic acid (DNA b), the third nucleic acid (DNA c), and the fourth nucleic acid (DNA d) were mixed evenly and placed in a thermostatic oscillator for a 2-h hybridization reaction at 37°C to assemble a cross-shaped nucleic acid nanorepository.

[0051] 2. Preparation of cross-shaped nucleic acid nanorepository - electroactive molecule complex 300 μL of an electroactive molecule (10 μM) solution in the oxidation signal silent region was added to the prepared FWJ solution. In this example, the preferred electroactive molecule in the oxidation signal silent region was doxorubicin hydrochloride (DOX), and its preferred volume ratio of FWJ:DOX was 10:1. Incubation was carried out in a shaker (37°C), and the electroactive molecule was embedded in the nucleic acid nanorepository through intercalation to obtain a cross-shaped nucleic acid nanorepository - doxorubicin hydrochloride complex (FWJ-DOX). Fluorescence intensity was detected at time points of 0 min, 30 min, 1 h, 2 h, 4 h, 6 h, and 8 h respectively.

[0052] 3. Preparation of FWJ-DOX hydrogel microneedle array (FWJ-DOX-HAMA MNs) patch An aqueous solution of methacrylated hyaluronic acid (HAMA) was fully mixed with the cross-shaped nucleic acid nanorepository (FWJ-DOX) loaded with DOX, and then poured into a PDMS mold. Subsequently, the mixed hydrogel solution was uniformly filled into the tiny cavities of the mold by low-speed centrifugation. The hydrogel network crosslinking was achieved through a photopolymerization reaction of methacrylate groups induced by ultraviolet light. Finally, after drying at room temperature and demolding, a complete microneedle patch FWJ-DOX-HAMA MNs was obtained (see Figure 1 a). The PDMS mold had multiple regularly arranged downwardly concave microneedle cavities of the same size for the formation of microneedles. Finally, the formed FWJ-DOX hydrogel microneedle array patch was peeled off from the PDMS mold, simply referred to as the hydrogel microneedle patch.

[0053] In some preferred embodiments, the hydrogel microneedle patch showed a 10 × 10 needle tip array, the base of the patch was square, the needle tips of the microneedles were sharp, and the needle tip morphology was uniform and regular ( Figure 1b). The scanning electron microscope (SEM) image of the microneedle array shows that the obtained microneedle array patch has a height of 1000 ± 10 μm, the width of the microneedle base is about 390 ± 10 μm, the inter-needle spacing is about 800 μm, and the sharpness of the needles is well maintained with the pyramid structure ( Figure 1 c). The three-dimensional confocal image shows that the fluorescent molecule Cy5 in the microneedles emits uniform and strong green fluorescence, indicating that the prepared microneedles are uniform ( Figure 1 d). Those skilled in the art can understand that, according to actual needs, the prepared microneedle patch may not be limited to the above morphologies.

[0054] Specific preparation method of hydrogel microneedles: First, vortex and mix the 5% (w / v) HAMA solution and the 0.25% (w / v) initiator LAP standard solution in a ratio of 10:1 to obtain the HAMA solution containing the photoinitiator. Then, mix the HAMA solution containing the photoinitiator with FWJ-DOX evenly, and its preferred ratio is HAMA:FWJ-DOX = 10:1. Centrifuge the above mixed solution to remove air bubbles at a centrifugal force of 3000 rpm for 3 minutes. Then, use a pipette to take out 350 μL of the mixed solution after removing air bubbles into the mold, coat it evenly, and then place the mold in an oven at 40 °C to concentrate for 5 h. Take out the mold, cover the lid, wrap it with sealing film, place it in a special centrifuge tube, and centrifuge with a centrifuge (4000 rpm, 5 min) to fill the voids at the tips of the mold. After centrifugation, take out the mold, add 100 μL of the mixed solution again, concentrate it in an oven at 40 °C for 5 h, and then centrifuge for the second time (4000 rpm, 5 min). Finally, take out the mold after centrifugation, crosslink it under ultraviolet light for 10 min (wavelength is 365 nm), and carefully peel off the cured microneedle patch FWJ-DOX-HAMA MNs from the mold.

[0055] Characterization by laser scanning confocal microscope (CLSM): Use the laser scanning confocal microscope to perform three-dimensional imaging on the microneedles, and evaluate the ability of the microneedles to absorb nucleic acids (20 μM, DNA-Cy5) and small molecules (20 μM, DOX) by taking images.

[0056] Swelling property detection: Record the dry mass (W0) of the microneedles before soaking, and then immerse the microneedles in the agarose gel containing PBS liquid. At time points of 2 min, 4 min, 6 min, 8 min, 10 min, and 15 min, take out the microneedles, remove the moisture from the surface with filter paper, and immediately weigh the mass (W t ). The swelling rate is calculated as follows: Swelling rate (SR) (%) = (W t − W0) / W0× 100%.

[0057] Insertability and recoverability test: Insert the microneedles into fresh porcine skin with thumb force and hold for 10 min. Record the appearance of the porcine skin before insertion and after removal, and then take pictures of the porcine skin at 1 min, 5 min, and 10 min after removal to observe the recovery of the porcine skin.

[0058] Mechanical test and in vitro tumor penetration efficiency test: Test the mechanical properties of the microneedles through a universal testing machine. Apply a force perpendicular to the microneedles using a flat-headed stainless steel cylindrical probe (at a constant speed of 0.05 mm min -1 . Measure the displacement until each tip breaks. To evaluate the ability of the microneedles to insert into tumors in vitro, dissect the mouse tumors, then attach the microneedles to the tumors for 10 min and observe the insertion of the microneedles.

[0059] Example 2 Detect and verify the hydrogel microneedle patch and intermediate products prepared in Example 1.

[0060] 1. Construction and disassembly of the cross-shaped nucleic acid nanorepository (FWJ) The miRNA-21-triggered strand displacement reaction (SDR) disassembly can be applied to target capture, signal transduction, and amplification. The design principle is as Figure 2 shown in a. First, anneal 4 single-stranded nucleic acids DNAa, DNAb, DNAc, and DNAd to form a stable cross-shaped double-stranded structure (FWJ). miRNA-21 can trigger the strand displacement reaction (SDR) to form the d / miRNA-21 structure, resulting in the disassembly of the cross-shaped double-stranded structure. The formation of the strand displacement reaction and the PAGE experimental results of the reaction are as Figure 2 shown in b. Single bands of single-stranded DNAa, DNAb, DNAc, DNAd, and miRNA-21 were observed in lanes 1, 2, 3, 4, and 5, while the FWJ in lane 6 showed a slower migration speed due to the formation of a higher molecular weight. After introducing miRNA-21 (22 bp), the d / miRNA-21 was competitively formed through SDR, resulting in the disassembly of FWJ. A new band was observed in lane 7, and its migration position was consistent with the lagging band d / miRNA-21 in lane 8. The migration behavior of the a + b + c triple-strand mixture in lane 9 was consistent with that of the individual a, b, and c strands, indicating that no hybridization reaction occurred to form double-stranded nucleic acids, proving that the cross-shaped nucleic acid nanorepository was formed in Example 1.

[0061] 2. Use fluorescence resonance energy transfer (FRET) method to study the assembly and target-induced disassembly process of the FWJ nanocarrier The b-chain and d-chain were labeled with the fluorescent molecule Cy5 and the quenching molecule BHQ, respectively. When annealed to form the FWJ double-stranded structure, the proximity of the fluorescent and quenching molecules led to fluorescence quenching, with a fluorescence intensity of 3065 ( Figure 3 a). In the presence of miRNA-21, the d-chain hybridized with miRNA-21, causing the distance between the b-chain and d-chain to be greater than the distance at which FRET can occur, resulting in fluorescence recovery to 8930, demonstrating target-induced separation of the b-chain and d-chain. The FWJ was incubated with a fixed concentration of miRNA-21 for different times to optimize the sensing time of the nucleic acid reservoir. As Figure 3 shown in b, the fluorescence of the FWJ without the addition of the target remained essentially unchanged within 240 min. However, in the presence of the target miRNA-21, the fluorescence intensity of the FWJ increased rapidly within 0 - 120 min and then leveled off and reached a maximum after 120 - 240 min, indicating that the optimal reaction time for the FWJ nanoreactor was 120 min.

[0062] 3. Circular dichroism (CD) was used to investigate the conformational changes of the FWJ nanoreactor before and after binding to electroactive molecules Figure 4 a - b show the circular dichroism spectra of the nucleic acid nanoreactor FWJ and its addition of different electroactive molecules, methylene blue (MB) or doxorubicin hydrochloride (DOX). When only FWJ was present, the curve showed a negative peak at 245 nm and a positive peak at 275 nm. When MB and DOX were intercalated into FWJ, the intensity of the negative peak decreased and the intensity of the positive peak increased significantly, indicating that the intercalative binding of the electroactive molecules caused the elongation of the DNA structure. As Figure 4 shown in c - d, compared with the solutions of MB or DOX alone, after the addition of FWJ, the fluorescence lifetimes of MB or DOX increased from 0.35 nanoseconds (ns) to 0.39 ns and from 1.1 ns to 1.15 ns, respectively, indicating that the addition of double-stranded nucleic acids could effectively prolong the fluorescence lifetimes of MB or DOX. The rigid structure of the nucleic acid increased the anti-rotation inactivation ability of the electroactive molecules, thereby enhancing the radiative properties, and time-resolved fluorescence decay measurements further demonstrated the interaction between DNA and MB or DOX. The above results indicate that the nano-nucleic acid prepared in Example 1 has the ability to capture electroactive molecules as a reservoir and the potential to trigger the release of electroactive molecules in a target-responsive manner.

[0063] MB exhibits characteristic light absorption (λ = 668 nm) and fluorescence emission (500 - 1000 nm) behaviors in the long - wavelength region. When methylene blue forms a complex with the DNA double - helix structure through an intercalation effect, its fluorescence is significantly quenched. This phenomenon results from the energy transfer of the excited state from the methylene blue molecule to the double - helix backbone due to the π - π conjugation between the methylene blue molecule and DNA base pairs. Doxorubicin exhibits strong orange fluorescence in the free state (excitation wavelength 480 nm), while its fluorescence is quenched after it intercalates into the DNA double - strand due to the intramolecular charge - transfer effect. The above changes can be used to analyze the interaction between MB / DOX and DNA. Within the first 6 h, the loading amount of FWJ for MB or DOX increases rapidly ( Figure 5 a). In the presence of the target miRNA - 21, the MB and DOX loaded in the nucleic acid are released, resulting in the recovery of fluorescence intensity ( Figure 5 b). The fluorescence recovery intensity of the miRNA - added group increases sharply within the range of 10 - 180 min. The results show that miRNA - 21 can cause the disassembly of the double - stranded nucleic acid structure, the release of MB and DOX from the double - stranded nucleic acid, leading to the recovery of fluorescence.

[0064] 4. Detection of hydrogel microneedle arrays prepared from methacrylated hyaluronic acid The HAMA solution and the HAMA+FWJ+DOX solution ( Figure 6 a) cross - link under light irradiation to form the HAMA gel and the HAMA+FWJ+DOX gel ( Figure 6 b). The SEM image of the freeze - dried HAMA gel shows that the HAMA hydrogel has a loose flaky structure with uniform pore distribution ( Figure 6 c). After mixing with the responsive nucleic acid FWJ - DOX, the hydrogel still has a loose flaky structure and has no effect on the pore structure ( Figure 6 d). This interpenetrating network structure not only increases the possibility of free diffusion of small molecules within the network but also improves the extraction rate of interstitial fluid samples. Scanning electron microscopy energy - dispersive X - ray spectroscopy (EDS) was used to study the interfacial composition of the mixed responsive nucleic acid microneedles. The elemental analysis results confirm that the MNs contain uniformly distributed C, N, O, and P elements, indicating that the nucleic acid nanorepository is uniformly dispersed in the microneedles ( Figure 6 e). In the FTIR spectrum of FWJ DOX - HAMA, the characteristic doublets at 1620 cm -1 and 1570 cm -1 respectively represent the C = C stretching vibration and the C = O vibration of anthraquinone, which are characteristic of the anthraquinone ring of DOX. The strong absorption peak at 1080 cm -1 originates from the symmetric stretching vibration of PO2 -, proving that FWJ - DOX is successfully doped into the hydrogel network ( Figure 6 f).

[0065] 5. Extraction of Tumor Interstitial Fluid by Hydrogel Microneedle Arrays The extraction of TIF by hydrogel microneedle arrays requires the microneedles to have high swelling ability to extract sufficient sample solution for analysis. Therefore, the swelling ability is a very important parameter for hydrogel microneedles, and the swelling ability of the microneedles determines the amount of interstitial fluid extracted by the microneedles.

[0066] The swelling capacity of the MNs patch was calculated by detecting the mass change of the MNs patch before and after incubation in PBS. As Figure 7 shown in a, the weight of the prepared MNs patch increased with the increase of incubation time to 0.25 g. The swelling rate increased with the increase of incubation time and reached about 100% within 6 min in vitro and tended to be stable within 15 min, mainly due to the high hydrophilicity of HAMA ( Figure 7 b). The MNs patch is similar to a dry sponge and can quickly absorb water. The morphology of the microneedles before and after swelling with DOX small molecules and DNA-Cy5 biomolecules was observed. It can be seen that the microneedles still maintained their complete structure after soaking for 15 min, and the degree of swelling gradually increased, while the tips of the microneedles did not dissolve, showing good swelling performance ( Figure 7 left images in c and d). The uniformly distributed red fluorescence in the confocal images indicates that the microneedles can extract small molecules and nucleic acid molecules in the interstitial fluid and quickly diffuse to the bottom of the microneedles in the microneedles ( Figure 7 right images in c and d), proving that the prepared microneedles have strong tumor tissue sampling ability.

[0067] 6. Detection of Other Properties of FWJ-DOX Hydrogel Microneedle Array Patches The mechanical strength of the microneedle patch was evaluated by a compression test using a universal testing machine to explore whether the prepared hydrogel microneedles have the ability to penetrate tissues. It was found that they can withstand a pressure of 0.2 N / needle ( Figure 8 a). Existing literature shows that the transdermal threshold is 0.1 N / needle, proving that the hydrogel microneedles prepared in Example 1 have the ability to penetrate tissues.

[0068] Furthermore, the actual skin insertion ability of the MNs patch was tested by directly inserting the MNs patch into fresh pig skin. After removing the microneedles 10 min later, 10 × 10 array pinholes were clearly shown on the surface of the pig skin, indicating that the microneedles can easily penetrate the pig skin ( Figure 8 b).

[0069] Example 3 Electrochemical performance evaluation of the hydrogel microneedle patch prepared in Example 1 and actual detection and analysis of tumor interstitial fluid.

[0070] The hydrogel microneedles prepared in Example 1 have the potential to be used as sensors, hereinafter simply referred to as microneedle sensors or microneedle nanosensors.

[0071] 1. Verification of the sensitivity of the microneedle sensor: First, immerse the hydrogel microneedle sensor in agarose containing miRNA-21 (final concentrations of 5 pM, 10 pM, 50 pM, 100 pM, 1 nM, and 10 nM) for 15 min, and then take out the microneedles. Place the microneedle backing on the surface of the screen-printed electrode and react for 2 h before performing electrochemical tests.

[0072] 2. Analysis of the stability of the microneedle sensor: Examine the long-term storage stability of the homogeneous electrochemical biosensor by continuously monitoring the DPV signals at different time points. Test the current response after the microneedle sensor has been placed for 2, 4, 6, 8, and 10 days. Detect the stability of the microneedle sensor at different days by repeating 7 DPV experiments on the same nanosensor for 10 fM miRNA-21 at a voltage of -1 to 0 V.

[0073] 3. Anti-interference study of the microneedle sensor: Immerse the microneedle tips vertically downward in a mixed solution of DA, AA, UA, TYR, TRP, L-DOPA (50 μM), and miRNA-21 (50 pM) for 2 h, then use an electrochemical workstation to measure the DPV signals and record the peak currents.

[0074] 4. Electrochemical detection of mouse tumor interstitial fluid: After applying the microneedle patch to the surface of a mouse tumor for 15 min, take it out. Place the microneedle on the surface of the screen-printed electrode and react for 2 h, then use an electrochemical workstation to measure the DPV signals.

[0075] 5. Electrochemical detection of human tissue interstitial fluid: Apply the microneedle to the surface of a human tissue sample, immerse the microneedle for 15 min and then take it out. Subsequently, place the microneedle backing on the surface of the screen-printed electrode for 2 h, and use an electrochemical workstation to detect the electrochemical signals of the microneedle.

[0076] The experimental results are as follows.

[0077] 1. Under the optimal experimental conditions, the concentration of miRNA-21 was determined using the hydrogel microneedles prepared in Example 1. In the miRNA-21 solution with a concentration range of 5 pM - 10 nM, the peak current of the hydrogel microneedles gradually increased with the increase in the concentration of miRNA-21 ( Figure 9 a), and there was a good linear relationship between the peak current and the concentration of miRNA-21 ( Figure 9b). In addition, the detection limit (LOD) of the hydrogel microneedles is 1.25 pM at a signal-to-noise ratio (S / N) of 3:1, demonstrating their high-sensitivity detection ability for miRNA-21. This proves the potential of the hydrogel microneedles to be prepared as electrochemical nanosensors.

[0078] 2. Using SM-miRNA-21, DM-miRNA-21, TM-miRNA-21, miRNA-141, let-7a, miRNA-141, miRNA-221, and miRNA-1246 as controls, the specificity of the hydrogel microneedles prepared in Example 1 for miRNA-21 was detected, and the obtained results are compared as Figure 10 shown in a. The peak current of miRNA-21 is significantly higher than that of other interfering miRNAs, and the current signal intensity increases by at least 2.2 times. This indicates that the hydrogel microneedles (or microneedle nanosensors) have good sequence specificity and the ability to distinguish target miRNAs from similar miRNAs. The current results of 20 groups of independent microneedle nanosensors detecting 50 pM miRNA-21 are shown in Figure 10 b. The current responses of the microneedle nanosensors have little difference, and the RSD is 1.9%, proving that the sensing system has good repeatability. This is because the selected electrochemically silent region electroactive molecule DOX has the characteristic of anti-interference of negative potential oxidation signals, thus eliminating background interference and improving the repeatability of the sensor. The above results show that microneedle nanosensors have obvious advantages in the detection of miRNA repeatability and accuracy.

[0079] 3. There are many interfering substances in actual biological samples. For example, interstitial fluid contains complex components such as TYR, UA, and AA, and their presence poses a great challenge to achieving accurate detection. To address this problem, further verification was carried out.

[0080] First, DA, AA, UA, TYR, TRP, and L-DOPA were added to the detection system as interfering substances, and the anti-interference performance of the hydrogel microneedles prepared in Example 1 was evaluated separately. As Figure 11 shown in a, compared with the detection results containing only the target miRNA-21, there is no obvious difference in the electrochemical responses of the nanosensing system in different interfering substances, indicating that the interfering substances do not cause obvious interference to the recognition and signal output of the target. Secondly, the system uses human serum to simulate the actual biological sample environment, and the standard addition method is used to further evaluate the anti-interference ability of the nanosensing system. miRNA-21 at 100 pM, 50 pM, and 10 pM was added to the serum diluted 100 times for detection experiments ( Figure 11b), the spiked recovery rate of the hydrogel microneedles for serum samples was between 94.2% and 108.7%. These results indicate that the hydrogel microneedles prepared in Example 1 have good anti-interference ability. This is because after the target reaction, the influence of background interferents was removed to the greatest extent by negative potential detection (i.e., generating an electrical signal at negative potential), achieving near-background-free sensing and a simple purification step.

[0081] 4. By comparing two different methods of directly detecting miRNA from cell TIF and detecting after extracting total RNA, the advantages of the electrochemical microneedle sensor with anti-biological background interference in direct detection in actual biological samples can be further explored. According to Figure 12 The results in a-b show that the levels of miRNA-21 in MCF-7 breast cancer cells detected directly from interstitial fluid and after extracting total RNA are both higher than the miRNA levels detected in MCF-10A normal cells.

[0082] The coefficient of variation (CV) refers to the degree of dispersion of test data from the same batch of test samples. When directly detecting in two kinds of cell lysates, the inter-group coefficients of variation were as low as 7.0% and 4.2% respectively. However, after extracting the RNA of the two kinds of cells, the inter-group coefficients of variation CV of detecting miRNA-21 using hydrogel microneedles were 42.7% and 17.6% respectively. In comparison, the coefficients of variation increased by 6.1 times and 4.2 times. This indicates that the microneedle-assisted nanosensor can directly detect miRNA in interstitial fluid of cells, has good detection performance, and further verifies the potential of the hydrogel microneedles to detect target miRNA in actual biological samples.

[0083] 5. To compare the electrochemical detection strategy proposed in the present invention with the current gold standard for miRNA detection, qRT-PCR, the extracted RNA samples were subjected to qRT-PCR experiments. The total RNA of MCF-7 cells was extracted using a total RNA extraction kit, and the absorbance of the extracted samples was measured using an ultraviolet spectrophotometer to evaluate the quality and concentration of the RNA. The ultraviolet absorption spectrum of the total extracted RNA shows ( Figure 13 a), the ultraviolet absorption spectrum of the total extracted and purified RNA shows an obvious absorption peak at 260 nm, the A260 / A280 ratio is 1.9 (excluding protein contamination), and the spectral baseline from 230 - 300 nm is smooth without miscellaneous peaks, indicating that a high-purity RNA sample was obtained. Subsequently, reverse transcription was performed using the stem-loop primer miR-21-StemLoopPrimer to synthesize cDNA, and qRT-PCR analysis was performed by SYBR Green I fluorescence dye method. A curve was plotted with the cycle threshold (Ct) as a function of the logarithm of the cell number ( Figure 13b), the cycle threshold decreased with the increase in the number of MCF-7 cells, and the coefficient of variation (CV) of the inter-group variation detected by qRT-PCR was as high as 9.3%, indicating significant fluctuations in the detection results. This difference may stem from degradation or loss during the RNA extraction process, directly leading to large fluctuations in the detected miRNA expression levels.

[0084] 6. A mouse model bearing breast cancer tumors was used as the research object to verify the potential application of electrochemical microneedle sensing in breast cancer diagnosis. A mouse xenograft model of breast cancer was successfully constructed based on the 4T1 cell line. Tissue samples were collected when the tumor volume reached 80 - 100 mm 3 . miRNA-21 in the tumor-infiltrating fluid (TIF) of tumor-bearing mice and healthy mice was directly detected using a microneedle sensor. The results showed ( Figure 14 a, b) that the expression level of miRNA-21 in the TIF of the tumor-bearing group was significantly increased by 12-fold compared with that of the healthy control group. When analyzing the extracted and purified TIF samples, although the expression level of miRNA-21 in the tumor-bearing group was still higher than that of the healthy group, the difference was reduced to 9-fold. In addition, the average expression level of miRNA-21 directly detected in the TIF of tumor-bearing mice was 1.3 times that of the extracted and purified TIF, and the data dispersion of the direct detection of TIF was lower ( Figure 14 b), indicating that the traditional RNA extraction process is prone to RNA degradation or loss, thereby expanding the detection error. When detecting the TIF extract by qRT-PCR, the difference in the expression of miRNA-21 between the tumor-bearing group and the healthy group was further reduced, which may be related to the fluctuations in enzyme activity during the reverse transcription and amplification processes. The above results confirmed that the microneedle sensor with effective shielding of matrix interference through negative potential oxidation signal output showed higher detection fidelity, and its direct detection strategy significantly reduced the variability between samples. Therefore, the electrochemical microneedle sensor can achieve accurate analysis of miRNA in biological samples without complex pretreatment, avoiding systematic errors introduced by multi-step operations in traditional methods.

[0085] 7. Hydrogel microneedles were used to detect the clinical TIF samples of breast cancer (BC) patients. A total of 10 breast cancer patients and 10 healthy controls (HD) were collected in the experiment. miRNA in the TIF samples of BC patients and HD was quantitatively analyzed by two methods: microneedle sensing and qRT-PCR. The results showed that the expression level of miRNA-21 in the samples of BC patients was significantly higher than that of the HD group ( Figure 15 a), and this trend was consistent with the results detected by qRT-PCR ( Figure 15 b).

[0086] 8. In the foregoing embodiments, the detection of miRNA expression levels by hydrogel microneedles has been deeply analyzed. The results of evaluating the diagnostic efficacy of microneedle sensors and qRT-PCR methods using the receiver operating characteristic curve (ROC) are asFigure 16 When differentiating between healthy control groups and breast cancer (BC) patients, the area under the ROC curve (AUC) of the microneedle sensor method was 0.92 ( Figure 16 a), significantly higher than the AUC value of 0.73 for the traditional qRT-PCR method ( Figure 16 b). In summary, the nanosensor demonstrated more excellent diagnostic performance compared to qRT-PCR when differentiating breast cancer patients from normal controls, providing a new powerful tool for the diagnosis of breast cancer.

[0087] It should be noted that in this article, the terms "including", "comprising" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not explicitly listed, or further includes elements inherent to such process, method, article or device. Without further limitations, an element defined by the statement "including one..." does not exclude the existence of additional identical elements in the process, method, article or device including that element.

[0088] The embodiments of the present invention have been described above in conjunction with the accompanying drawings. However, the present invention is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the purpose of the present invention and the scope protected by the claims. All of these are within the protection scope of the present invention.

Claims

1. A hydrogel microneedle with resistance to biological background interference, characterized in that: The hydrogel microneedle is composed of a hydrogel matrix and a nucleic acid nanoreservoir-oxidation signal silent zone electroactive molecule complex; the nucleic acid nanoreservoir is a stable cross-shaped double-stranded structure formed by complementary pairing of four single-stranded nucleic acids; the oxidation signal silent zone electroactive molecule includes doxorubicin hydrochloride or methylene blue; the oxidation signal silent zone electroactive molecule is embedded in the nucleic acid nanoreservoir through intercalation.

2. The hydrogel microneedle according to claim 1, characterized in that The hydrogel microneedle has a loose porous structure; or, the hydrogel matrix encapsulates the nucleic acid reservoir-oxidation signal silent zone molecule electroactive molecule complex.

3. The hydrogel microneedle according to claim 1, characterized in that Among the four single-stranded nucleic acids, the first nucleic acid has a sequence structure that is complementary to the bases of the second nucleic acid and the fourth nucleic acid; the second nucleic acid also has a sequence structure that is complementary to the bases of the third nucleic acid; The third nucleic acid also has a sequence structure that is complementary to a portion of the fourth nucleic acid bases; any one sequence in the cross-shaped double-stranded structure has a sequence structure that binds to the target nucleic acid.

4. The hydrogel microneedle according to claim 1, characterized in that The hydrogel matrix includes methacryloyl hyaluronic acid and a photoinitiator.

5. The hydrogel microneedle according to claim 3, characterized in that: The sequences of the four single-stranded nucleic acids are shown in SEQ ID NO.1-4.

6. The method for preparing the hydrogel microneedle according to any one of claims 1 to 5, characterized in that: The following steps are involved: S01: preparing a nucleic acid nanoreservoir: annealing equal volumes of the first nucleic acid, the second nucleic acid, the third nucleic acid and the fourth nucleic acid at 90-100°C to form a metastable hairpin structure; then placing at a temperature below 50°C, shaking, and hybridizing to form a stable cross-shaped nucleic acid nanoreservoir; S02: preparing a nucleic acid nanoreservoir-oxidation signal silent zone electroactive molecule complex: adding an oxidation signal silent zone electroactive molecule solution to the cross-shaped nucleic acid nanoreservoir, and co-incubating at a temperature below 50° C. to form a nucleic acid nanoreservoir-oxidation signal silent zone electroactive molecule complex; the oxidation signal silent zone electroactive molecule comprises doxorubicin hydrochloride or methylene blue; S03: mixing the methacryloyl hyaluronic acid and the photoinitiator uniformly to obtain a hydrogel matrix solution containing the photoinitiator; The nucleic acid nanoreservoir-oxidation signal silent zone electroactive molecule complex is added to obtain a mixed solution, and the mixed solution is placed in a mold having a plurality of downwardly concave microneedle cavities of uniform size and regular arrangement, and concentrated at a temperature below 50°C to allow the mixed solution to fully fill the gaps in the microneedle cavities of the mold, and cross-linked under ultraviolet light to obtain patch-like hydrogel microneedles.

7. The preparation method according to claim 6, characterized in that: The volume ratio of the cross-shaped nucleic acid nanoreservoir to the oxidation signal silent zone electroactive molecule is 10:1-5; or, the volume ratio of the hydrogel matrix solution containing the photoinitiator to the nucleic acid nanoreservoir-oxidation signal silent zone electroactive molecule complex is 10:1-5.

8. The preparation method according to claim 6, characterized in that: The photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate.

9. Use of the hydrogel microneedle according to any one of claims 1 to 5 in preparing an electrochemical sensor resistant to biological background interference.

10. The use according to claim 9, characterized in that The electrochemical sensor is used for in-situ detection of interstitial fluid.

Images