Preparation method of microneedle-mediated two-channel electrochemical aptamer sensor

Abstract

The invention discloses a preparation method of a micro-needle mediated two-channel electrochemical aptamer sensor, which comprises the following steps: firstly, constructing an interstitial fluid extraction module based on a hollow micro-needle, the interstitial fluid extraction module comprising a substrate and a hollow micro-needle array arranged on the substrate; a flower-like gold nanostructure is deposited on the surface of DSPCE to obtain a DSPCE-Au electrode, a vancomycin aptamer marked by electrochemical signal molecules and a C-reactive protein aptamer / auxiliary probe hybrid double strand are connected to the DSPCE-Au electrode through Au-S bonds to obtain an electrochemical detection module, and the electrochemical detection module is integrated with a mesenchymal fluid extraction module. The sensor can be used for diagnosis and treatment monitoring of necrotic soft tissue infection by virtue of the painless transdermal sampling capability of the microneedle, the high-specificity recognition characteristic of the aptamer and the high-sensitivity detection advantage of the electrochemical sensor, and has the advantages of minimally invasive property, portability, continuous monitoring, real-time data acquisition and the like; the problems that an existing detection method is long in consumed time, painful, complex in equipment and the like are solved.

Description

Technical Field

[0001] The invention belongs to the technical field of electrochemical sensors, and in particular relates to a method for preparing a microneedle-mediated dual-channel electrochemical aptamer sensor. Background Art

[0002] Necrotising soft-tissue infections (NSTIs) are severe, life-threatening bacterial infections characterized by necrosis of subcutaneous tissue, fascia, or muscle. NSTIs have a high mortality rate, with 20-50% of patients dying in the early stages. Therefore, rapid, accurate, inexpensive, and portable detection methods are essential to help early diagnosis and treatment of NSTIs patients and improve their survival rates.

[0003] At present, the traditional detection method for necrotizing soft tissue infection is mainly clinical diagnosis, such as advanced imaging technologies such as magnetic resonance imaging and CT; based on the traditional blood sample collection and analysis is the gold standard for the detection of disease-related biochemical indicators, researchers have also developed a necrotizing fasciitis laboratory risk index score (LRINEC) standard, by analyzing the content of C-reactive protein (CRP), white blood cell count (WBC), hemoglobin, sodium, creatinine and glucose, and scoring abnormal values; the existing sampling method for detection is mainly venous blood collection. Studies have found that the original LRINEC score of serum CRP is more predictive of NSTIs, and NSTIs should be highly suspected when CRP is elevated. Adults in good health usually have CRP values ​​as low as 10 mg / L, and CRP concentrations rise rapidly during inflammation, reaching 350-400 mg / L. At present, the treatment of NSTIs relies on antibiotics and early surgical debridement, and vancomycin (VA) is used to treat severe infections caused by sensitive strains of methicillin-resistant Staphylococcus, including skin and soft tissue infections, and patients with penicillin allergy. Therefore, infection can be quickly detected by monitoring the increase in CRP concentration, and the treatment effect of NSTIs can be improved by monitoring the concentration of VA. However, the current traditional detection methods for necrotizing soft tissue infection, such as advanced imaging technologies such as magnetic resonance imaging and CT, are time-consuming and not very practical in emergency situations; the necrotizing fasciitis LRINEC standard developed by researchers has low sensitivity and specificity, so it is not widely used; the existing sampling method for detection is mainly venous blood sampling, but it is usually invasive, causing tissue damage and patient discomfort, and there is a risk of secondary infection.

[0004] Therefore, there is an urgent need in the art for a method for diagnosing and monitoring necrotizing soft tissue infection to solve the problems of time-consuming, painful, and complex equipment in the prior art. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a microneedle-mediated dual-channel electrochemical aptamer sensor in response to the problems of the prior art detection methods for necrotizing soft tissue infection, such as long detection time, pain, and complex equipment. The prepared microneedle-mediated dual-channel electrochemical aptamer sensor can be used for the diagnosis and treatment monitoring of necrotizing soft tissue infection, and has the advantages of minimally invasive, miniaturization, portability, continuous monitoring, and real-time data acquisition.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A method for preparing a microneedle-mediated dual-channel electrochemical aptamer sensor comprises the following steps:

[0008] (1) Construct an interstitial fluid extraction module based on hollow microneedles;

[0009] The interstitial fluid extraction module includes a substrate and a hollow microneedle array disposed on the substrate, wherein a liquid storage chamber is disposed in the substrate, the hollow microneedle has a hollow cavity structure, and the bottom of the hollow microneedle is connected to the liquid storage chamber. The substrate and the hollow microneedle array are made of photosensitive resin and are integrally formed by light-curing 3D printing;

[0010] (2) Flower-like gold nanostructures were deposited on the surface of dual-channel screen-printed electrode (DSPCE) using a constant potential method to obtain DSPCE-Au electrode;

[0011] (3) The electrochemical signal molecule-labeled vancomycin aptamer (VA-Apt) and C-reactive protein aptamer (CRP-Apt) / auxiliary probe hybrid double strands were connected to the working electrode A and working electrode B of DSPCE-Au through Au-S bonds, respectively, to obtain the DSPCE-Au electrochemical detection module;

[0012] (4) The DSPCE-Au electrochemical detection module was integrated with the interstitial fluid extraction module to obtain a microneedle-mediated dual-channel electrochemical aptamer sensor.

[0013] Furthermore, in step (1), the microneedles of the hollow microneedle array are conical in shape, with a cone bottom diameter of 600 μm, a needle length of 1200 μm, a distance between adjacent needle tips of 1200 μm, and each microneedle is provided with a hole connecting its hollow cavity structure at a distance of 200 μm from the needle tip.

[0014] Furthermore, in step (1), the photosensitive resin is selected from one of polyurethane acrylate, epoxy acrylate or polyester acrylate.

[0015] Furthermore, in step (2), the method of depositing flower-like gold nanostructures on the surface of the dual-channel screen-printed electrode using a constant potential method is as follows: immersing the dual-channel screen-printed electrode in a polyacrylic acid (PAA) and polyethyleneimine (PEI) solution to form a PAA / PEI film on the electrode surface; after rinsing and drying, 20 μL of 50 mM HAuCl4 is dripped onto the three-electrode area of ​​the dual-channel screen-printed electrode; and then, the flower-like gold nanostructure is obtained by electrodeposition.

[0016] Further, in step (3), the nucleotide sequence of the vancomycin aptamer is:

[0017] 5'-CGAGGGTACCGCAATAGTACTTATTGTTCGCCTATTGTGGGTCGG-3' (SEQ ID NO: 1);

[0018] The nucleotide sequence of the auxiliary probe is:

[0019] 5'-TTTTTTTCACCAAAACACGCTTTCTCCCCTCTTTTCCCCTTCG-3' (SEQ ID NO: 2);

[0020] The nucleotide sequence of the C-reactive protein aptamer is:

[0021] 5'-CGAAGGGGATTCGAGGGGTGATTGCGTGCTCCATTTGGTG-MB-3' (SEQ ID NO: 3).

[0022] Furthermore, in step (3), the electrochemical signal molecule is methylene blue.

[0023] Furthermore, in step (3), the vancomycin aptamer and C-reactive protein aptamer / auxiliary probe hybrid double strands labeled with electrochemical signal molecules are respectively connected to the working electrode A and the working electrode B of DSPCE-Au through Au-S bonds: after activating the vancomycin aptamer with tri(2-carboxyethyl)phosphine (TCEP), it is dropped on the surface of the working electrode A of DSPCE-Au and incubated at room temperature; the C-reactive protein aptamer and the auxiliary probe are mixed with equal volumes at the same concentration, activated with TCEP for 0.5 to 3 hours, annealed at 80 to 100° C. for 5 to 60 minutes, and then dropped on the surface of the working electrode B of DSPCE-Au and incubated at room temperature.

[0024] The dual-channel electrochemical aptasensor prepared by the above method.

[0025] The dual-channel electrochemical aptamer sensor prepared by the above method is used to detect the markers of necrotizing soft tissue infection, vancomycin VA and CRP. Application method: The electrode substrate signal without VA and CRP is used as the starting signal, and then the microneedle-mediated dual-channel electrochemical aptamer sensor is used to collect interstitial fluid, and the electrochemical signal of the DSPCE-Au sensor is detected by square wave voltammetry and recorded as the end point signal;

[0026] If the difference between the endpoint signal and the starting signal is very small (less than 5%), the target VA and CRP are not present in the analyte;

[0027] If the endpoint signal increases or decreases compared with the start signal, the target VA or CRP is present in the analyte.

[0028] Beneficial effects of the present invention:

[0029] 1) The present invention constructs an electrochemical microneedle POCT sensor device that integrates sampling and testing, which has the advantages of minimally invasive, miniaturized, portable, continuous monitoring and real-time data acquisition, breaking through the bottleneck of traditional detection instruments that are large and heavy and cannot be sampled and tested immediately.

[0030] 2) The present invention constructs a dual-channel electrochemical aptamer sensing interface with a three-dimensional flower-like gold nanostructure, which has the function of coordinated detection of antibiotic drugs and infection markers, and is convenient for dynamic synchronous diagnosis and treatment monitoring of necrotizing soft tissue infections.

[0031] 3) The present invention designs a microneedle with a hollow microcavity structure as a fluid sample extraction module. Compared with a blood collection needle for collecting blood samples, it has the advantages of painless, efficient and continuous extraction of interstitial fluid, while reducing the risk of secondary infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a detection principle diagram of the microneedle-mediated dual-channel electrochemical aptamer sensor of the present invention;

[0033] Figure 2 This is a comparison diagram of electrochemical responses obtained when the DSPCE-Au-WE1 of the DSPCE-Au electrochemical detection module prepared in Example 1 detects artificial interstitial fluid containing 80 μM vancomycin;

[0034] Figure 3 The electrochemical signal response result diagram of the DSPCE-Au-WE1 of the DSPCE-Au electrochemical detection module prepared in Example 1 detecting vancomycin at different concentrations;

[0035] Figure 4This is a comparison diagram of electrochemical responses obtained when the DSPCE-Au-WE2 of the DSPCE-Au electrochemical detection module prepared in Example 1 detects artificial interstitial fluid containing 100 μg / mL C-reactive protein;

[0036] Figure 5 This is a graph showing the electrochemical signal response results of detecting C-reactive protein of different concentrations using the DSPCE-Au-WE2 of the DSPCE-Au electrochemical detection module prepared in Example 1;

[0037] Figure 6 The electrochemical response diagram of vancomycin detected by DSPCE-WE1 of the DSPCE electrochemical detection module prepared in Comparative Example 1;

[0038] Figure 7 This is the electrochemical response diagram of the DSPCE-WE2 of the DSPCE electrochemical detection module prepared in Comparative Example 1 for detecting C-reactive protein. DETAILED DESCRIPTION

[0039] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0040] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0041] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0042] Used in the embodiments of the present invention:

[0043] TCEP (tri(2-carboxyethyl)phosphine) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; VA-Apt, CRP-Apt, and auxiliary probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd.;

[0044] The specific material sources of the DSPCE electrode were purchased from Ningbo Yuangan Biotechnology Co., Ltd., type: CL20s, model: public dual channel.

[0045] Based on the characteristics of safe, effective and painless extraction of 3D printed hollow microneedles, the present invention uses stable and low-cost specific nucleic acid aptamers to construct a portable electrochemical aptamer sensor for efficient extraction and capture of biomarkers, which is used to monitor the expression level of C-reactive protein in necrotizing soft tissue infection and the therapeutic effect of vancomycin, and strives to improve the sensitivity and specificity of diagnosis, providing new theoretical and experimental basis for the identification, diagnosis and treatment of necrotizing soft tissue infection.

[0046] Figure 1 The detection principle diagram of the microneedle-mediated dual-channel electrochemical aptamer sensor of the present invention is shown in the figure. First, the dual-channel screen-printed electrode (DSPCE) after electroplating gold is inserted into the 3D printed microneedle module. After the C-reactive protein aptamer and the vancomycin aptamer are incubated on the two working electrodes respectively, the conformational changes produced by the binding of the aptamer to the target correspond to the changes in the electrical signal measured by the sensor, thereby realizing the diagnosis and treatment monitoring of necrotizing soft tissue infection. Specifically, the gold nanostructure is electroplated on the cleaned and pretreated DSPCE to obtain the DSPCE-Au electrode; the DNA chain VA-Apt and CRP-Apt / auxiliary probe hybrid double-stranded labeled with thiol and electrochemical signal molecules are dripped onto the two working electrodes, and the electrodes are assembled and connected through the Au-S bond to form an electrochemical detection platform. The electrochemical signal changes before and after the capture of CRP and VA are detected by square wave voltammetry and the signal gain value is calculated.

[0047] Example 1

[0048] A method for preparing a microneedle-mediated dual-channel electrochemical aptamer sensor comprises the following steps:

[0049] (1) Construct an interstitial fluid extraction module based on hollow microneedles;

[0050] The interstitial fluid extraction module includes a substrate and a hollow microneedle array arranged on the substrate, wherein a liquid storage chamber is arranged in the substrate, the hollow microneedle has a hollow cavity structure, and the bottom of the hollow microneedle is connected to the liquid storage chamber. The substrate and the hollow microneedle array are made of polyurethane acrylate and are integrally formed by light-curing 3D printing using digital light processing (DLP) technology;

[0051] In this embodiment, the microneedles of the hollow microneedle array are conical in shape, with a cone bottom diameter of 600 μm, a needle length of 1200 μm, and a distance between adjacent needle tips of 1200 μm. Each microneedle is provided with a hole (hole diameter of 200 μm) connecting to its hollow cavity structure at a distance of 200 μm from the needle tip, and the microneedles are arranged in a 6×6 array; the size of the entire module is 18 mm×18 mm×4.7 mm; the size of the liquid storage chamber in the substrate is 14 mm×14 mm×0.5 mm.

[0052] (2) flower-like gold nanostructures were deposited on the surface of the dual-channel screen-printed electrode using a constant potential method to obtain a DSPCE-Au electrode;

[0053] The method for depositing flower-like gold nanostructures on the surface of a dual-channel screen-printed electrode using a constant potential method is as follows: immersing the dual-channel screen-printed electrode in a polyacrylic acid (PAA) and polyethyleneimine (PEI) solution to form a PAA / PEI film on the electrode surface; after rinsing and drying, 20 μL of 50 mM HAuCl4 is dripped onto the three-electrode area of ​​the dual-channel screen-printed electrode; and then, the flower-like gold nanostructure is obtained by electrodeposition (deposition potential is -0.3 V, deposition time is 900 s).

[0054] (3) 1 μM vancomycin aptamer labeled with thiol and methylene blue and 10 mM tri(2-carboxyethyl)phosphine (TCEP) were mixed and reacted at room temperature in the dark for 1 h, and then 5 μL was added to the surface of DSPCE-Au working electrode A (DSPCE-Au-WE1) and incubated at room temperature overnight; then C-reactive protein aptamer and auxiliary probe were mixed with equal volume and concentration in a 1:1 ratio to 1 μM, mixed with 10 mM TCEP and reacted at room temperature in the dark for 1 h, annealed at 80°C for 10 min, and then 5 μL was added to the surface of DSPCE-Au working electrode B (DSPCE-Au-WE2), incubated at room temperature overnight, and finally rinsed and dried, and 5 μL 2 mM 6-mercaptohexanol (MCH) was added to each of the two working electrodes, incubated at room temperature for 1 h, rinsed and dried, and then 5 μL 10% bovine serum albumin was added to each to block nonspecific adsorption, and rinsed and dried to obtain the DSPCE-Au electrochemical detection module;

[0055] The sequence of the vancomycin aptamer labeled with thiol and methylene blue is:

[0056] 5'-SH-CGAGGGTACCGCAATAGTACTTATTGTTCGCCTATTGTGGGTCGG-MB-3';

[0057] The nucleotide sequence of the auxiliary probe is:

[0058] 5'-TTTTTTTCACCAAAACACGCTTTCTCCCCTCTTTTCCCCTTCG-3';

[0059] The nucleotide sequence of the C-reactive protein aptamer is:

[0060] 5'-CGAAGGGGATTCGAGGGGTGATTGCGTGCTCCATTTGGTG-MB-3'.

[0061] (4) The electrode chip of the DSPCE-Au electrochemical detection module is inserted into the substrate storage chamber of the interstitial fluid extraction module to obtain a microneedle-mediated dual-channel electrochemical aptamer sensor.

[0062] The detection performance of the DSPCE-Au electrochemical detection module prepared in Example 1 for VA and CRP was tested:

[0063] 1) preparing an artificial interstitial solution, wherein the formula of the artificial interstitial solution is: 3.5 mM KCl, 123 mM NaCl, 1.5 mM NaH2PO4, 2.2 mM CaCl2, 0.7 mM MgCl2, pH = 7.4; using a DSPCE-Au electrochemical detection module to detect electrochemical signals in the artificial interstitial solution by square wave voltammetry (SWV), the signal being used as a starting signal for detection; the SWV scanning potential window range is -0.6 to 0 V, the amplitude is 25 mV, and the frequency is 50 Hz.

[0064] 2) A series of artificial interstitial solutions containing different concentrations of VA (1 μM, 10 μM, 20 μM, 40 μM, and 80 μM) and a series of artificial interstitial solutions containing different concentrations of CRP (10 ng / mL, 100 ng / mL, 1 μg / mL, 10 μg / mL, and 100 μg / mL) were prepared, and the electrochemical signals in the artificial interstitial solutions were detected by square wave voltammetry (SWV) using a DSPCE-Au electrochemical detection module. The SWV scanning potential window range was -0.6 to 0 V, the amplitude was 25 mV, and the frequency was 50 Hz.

[0065] Figure 2 This is a comparison diagram of the electrochemical responses obtained when the DSPCE-Au-WE1 of the DSPCE-Au electrochemical detection module prepared in Example 1 detects artificial interstitial fluid containing 80 μM vancomycin. In the figure, the dotted line is the starting signal (VA concentration is 0), recorded as I0. The solid line is the end signal, recorded as I t .

[0066] Figure 3 The electrochemical signal response result diagram of the DSPCE-Au-WE1 of the DSPCE-Au electrochemical detection module prepared in Example 1 for detecting different concentrations of vancomycin shows the electrochemical signal response obtained when the WE1 channel electrode (working electrode A) is used to detect different concentrations of vancomycin in artificial interstitial fluid using the SWV determination method (scanning potential window range of -0.6 to 0V, amplitude of 25mV, frequency of 50Hz) at room temperature by an electrochemical workstation. Signal Change (%) = (I t -I0) / I0×100%.

[0067] It can be seen that as the concentration of VA increases, the electrochemical signal of methylene blue detected also gradually increases. This is reasonable because when the single chain is fixed on the electrode, the methylene blue is far away from the electrode surface and the electrochemical signal generated is small. After binding with VA (80μM), the single chain undergoes conformational changes, causing the methylene blue to approach the electrode surface, thereby generating a larger electrochemical signal. Figure 3 It can also be seen that the electrochemical signal change of methylene blue is related to the VA concentration. The greater the VA concentration, the greater the electrochemical signal change rate, and it tends to a stable value after a concentration of 40 μM. The VA concentration is linearly correlated with the electrochemical signal change rate in the range of 0-40 μM; the linear equation is Signal Change (%)

[0068] =1.99×Vancomycin concentration(μM)+8.31(R 2 =0.999), which proves that the detection platform has good detection performance for VA.

[0069] Likewise, Figure 4 The electrochemical response comparison diagram is obtained when the DSPCE-Au-WE2 of the DSPCE-Au electrochemical detection module prepared in Example 1 detects artificial interstitial fluid containing 100 μg / mL C-reactive protein. The dotted line is the starting signal (CRP concentration is 0), recorded as I0. The solid line is the end signal, recorded as I t .

[0070] Figure 5 The electrochemical signal response result diagram of the DSPCE-Au-WE2 of the DSPCE-Au electrochemical detection module prepared in Example 1 detecting different concentrations of C-reactive protein shows the electrochemical signal obtained when different concentrations of C-reactive protein are detected in artificial interstitial fluid using the WE2 channel electrode at room temperature using the SWV determination method (scanning potential window range of -0.6 to 0 V, amplitude of 25 mV, frequency of 50 Hz) of the electrochemical workstation. Signal Change (%) = (I t -I0) / I0×100%.

[0071] It can be seen that as the CRP concentration increases, the electrochemical signal of methylene blue detected gradually decreases. This is reasonable because when the CRP-Apt / Assisted probe hybrid double strand is fixed on the electrode, the methylene blue is closer to the electrode surface and the electrochemical signal generated is larger. After binding with CRP (100μg / mL), CRP-Apt will specifically bind to CRP and cause it to detach from the electrode surface, thereby reducing the electrochemical signal. Figure 5It can also be seen that the CRP concentration in the range of 10 ng / mL-100 μg / mL is linearly correlated with the electrochemical signal change rate, and the linear equation is Signal Change (%) = -13.46 × LgCRP (μg·mL-1) -39.81 (R 2 =0.997), which proves that the detection platform has good detection performance for CRP.

[0072] Comparative Example 1

[0073] In order to compare with the DSPCE-Au electrochemical detection module prepared by using the DSPCE-Au electrode modified with flower-like gold nanostructures, the DSPCE electrochemical detection module was prepared by using the DSPCE electrode without Au:

[0074] 1 μM vancomycin aptamer labeled with thiol and methylene blue and 10 mM tri(2-carboxyethyl)phosphine (TCEP) were mixed and reacted at room temperature in the dark for 1 hour, and then 5 μL was added to the surface of the working electrode A (DSPCE-WE1) of DSPCE and incubated at room temperature overnight; then the C-reactive protein aptamer and the auxiliary probe were mixed with equal volumes of the same concentration in a 1:1 ratio to 1 μM, mixed with 10 mM TCEP and reacted at room temperature in the dark for 1 hour, annealed at 80°C for 10 minutes, and then 5 μL was added to the surface of the working electrode B (DSPCE-WE2) of DSPCE and incubated at room temperature overnight, and finally rinsed and dried, and 5 μL of 2 mM 6-mercaptohexanol (MCH) was dropped on each of the two working electrodes, incubated at room temperature for 1 hour, rinsed and dried, and then 5 μL of 10% bovine serum albumin was dropped on each to block nonspecific adsorption, and after rinsing and drying, the DSPCE electrochemical detection module was obtained.

[0075] Square wave voltammetry (SWV) was used to detect electrochemical signals in artificial interstitial solution, and this signal was used as the starting signal for detection. The SWV scanning potential window range was -0.6 to 0 V, the amplitude was 25 mV, and the frequency was 50 Hz. The detection results are shown in Figure 6 , Figure 7 As shown,

[0076] Figure 6 The electrochemical response diagram of vancomycin detected by DSPCE-WE1 of the DSPCE electrochemical detection module prepared in Comparative Example 1; Figure 7The electrochemical response diagram of DSPCE-WE2 detecting C-reactive protein using the DSPCE electrochemical detection module prepared in Comparative Example 1. It can be seen that the curve is flat without obvious oxidation peaks, indicating that on the DSPCE without Au, the VA-Apt and CRP-Apt / Assisted probe hybrid double chains labeled with the electrochemical signal molecule (methylene blue) cannot be connected to the electrode interface through the Au-S bond, so that VA and CRP cannot be detected, proving the necessity of the flower-like gold nanostructure modified electrode.

[0077] The dual-channel electrochemical aptamer sensor based on microneedle mediation for diagnosis and treatment monitoring of necrotizing soft tissue infection of the present invention cleverly combines hollow microneedles with portable screen-printed electrodes to construct a microneedle-mediated dual-channel electrochemical biosensor by leveraging the painless transdermal sampling ability of microneedles, the high-specificity recognition characteristics of nucleic acid aptamers and the high-sensitivity detection advantages of electrochemical sensors.

[0078] The present invention proposes a microneedle collection method for painless, minimally invasive and efficient extraction of interstitial fluid. Through reasonable design, the hollow microneedle array is customized on demand, and the advantages of its special microcavity structure and minimally invasive and painless penetration of the epidermis to the dermis are utilized to collect dermal interstitial fluid by pressing or vacuum-assisted methods. Compared with the existing wick extraction method, suction bubble method, microdialysis method, electroosmosis method, etc., the efficiency of interstitial fluid sampling is significantly improved.

[0079] The present invention constructs an electrochemical nucleic acid aptamer sensing interface for simultaneous detection of drugs and markers, achieving disease diagnosis and treatment monitoring on a single platform, and improving monitoring efficiency and diagnostic accuracy. The density of aptamer probe fixation is enhanced by a three-dimensional flower-shaped gold nanosensing interface using a constant potential deposition method; two functional aptamers are fixed on the surface of the same sensor unit containing dual working electrodes, which not only achieves simultaneous detection of dual targets, but also avoids the use of high-cost antibodies and shortens the time for separate detection of single electrodes, thus playing a role in reducing costs and increasing efficiency.

[0080] The present invention integrates sample collection and detection in one portable sensor device. Clinical testing experiments often involve complex processes such as sample collection and pretreatment, marker extraction, instrument testing and data analysis. By miniaturizing and integrating the design of an electrochemical microneedle sensor that meets the above requirements, energy consumption and sample usage are significantly reduced; in terms of improving quality, by simplifying the operating procedures and real-time on-site testing, the risk of contamination or degradation that may occur during sample transportation is reduced, ensuring the accuracy and reliability of the test results.

[0081] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A method for preparing a microneedle-mediated dual-channel electrochemical aptamer sensor, characterized in that: The steps include: (1) constructing an interstitial fluid extraction module based on hollow microneedles, the interstitial fluid extraction module comprising a substrate and a hollow microneedle array disposed on the substrate, the substrate being provided with a liquid storage chamber, the hollow microneedles having a hollow cavity structure, the bottom of the hollow microneedles being connected to the liquid storage chamber, the substrate and the hollow microneedle array being made of photosensitive resin and integrally formed by light-curing 3D printing; (2) flower-like gold nanostructures were deposited on the surface of the dual-channel screen-printed electrode using a constant potential method to obtain a DSPCE-Au electrode; (3) connecting the electrochemical signal molecule-labeled vancomycin aptamer and C-reactive protein aptamer / auxiliary probe hybrid double strands to the working electrode A and working electrode B of DSPCE-Au respectively through Au-S bonds to obtain a DSPCE-Au electrochemical detection module; (4) The DSPCE-Au electrochemical detection module was integrated with the interstitial fluid extraction module to obtain a microneedle-mediated dual-channel electrochemical aptamer sensor.

2. The method for preparing the microneedle-mediated dual-channel electrochemical aptasensor according to claim 1, characterized in that: In step (1), the microneedles of the hollow microneedle array are conical in shape, with a cone bottom diameter of 600 μm, a needle length of 1200 μm, a distance between adjacent needle tips of 1200 μm, and each microneedle is provided with a hole connecting its hollow cavity structure at a distance of 200 μm from the needle tip.

3. The method for preparing the microneedle-mediated dual-channel electrochemical aptasensor according to claim 1, characterized in that: In step (1), the photosensitive resin is selected from one of polyurethane acrylate, epoxy acrylate or polyester acrylate.

4. The method for preparing the microneedle-mediated dual-channel electrochemical aptasensor according to claim 1, characterized in that: In step (2), the method of depositing flower-like gold nanostructures on the surface of the dual-channel screen-printed electrode using a constant potential method is as follows: immersing the dual-channel screen-printed electrode in a PAA and PEI solution to form a PAA / PEI film on the electrode surface; after rinsing and drying, 20 μL of 50 mM HAuCl4 is dripped onto the three-electrode area of ​​the dual-channel screen-printed electrode; and then, the flower-like gold nanostructure is obtained by electrodeposition.

5. The method for preparing the microneedle-mediated dual-channel electrochemical aptasensor according to claim 1, characterized in that: In step (3), the nucleotide sequence of the vancomycin aptamer is shown in SEQ ID NO: 1, the nucleotide sequence of the auxiliary probe is shown in SEQ ID NO: 2, and the nucleotide sequence of the C-reactive protein aptamer is shown in SEQ ID NO:

3.

6. The method for preparing the microneedle-mediated dual-channel electrochemical aptasensor according to claim 1, characterized in that: In step (3), the electrochemical signal molecule is methylene blue.

7. The method for preparing the microneedle-mediated dual-channel electrochemical aptasensor according to any one of claims 1 to 6, characterized in that: In step (3), the method of connecting the electrochemical signal molecule-labeled vancomycin aptamer and C-reactive protein aptamer / auxiliary probe hybrid double strands to the working electrode A and working electrode B of DSPCE-Au respectively through Au-S bonds is as follows: after activating the vancomycin aptamer with tri(2-carboxyethyl)phosphine, dripping it on the surface of the working electrode A of DSPCE-Au, incubating it at room temperature, mixing the C-reactive protein aptamer and the auxiliary probe with the same concentration and volume, activating them with tri(2-carboxyethyl)phosphine for 0.5 to 3 hours, annealing them at 80 to 100°C for 5 to 60 minutes, and then dripping them on the surface of the working electrode B of DSPCE-Au, and incubating them at room temperature.

8. A dual-channel electrochemical aptasensor prepared by the method according to any one of claims 1 to 7.

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