Protein monitoring device and application thereof

Abstract

The invention discloses a protein monitoring device and application thereof.The monitoring device is provided with a microneedle with a hollow channel, a working electrode, a reference electrode and a counter electrode are arranged in the microneedle and connected with the hollow channel in the microneedle, a P1 probe is fixed to the working electrode, and a P2 probe is fixed to the counter electrode. The P2 aptamer and the P1 probe are paired through a hydrogen bond to form double strands, the P2 aptamer has a free end combined with a target protein, and the working electrode is connected with an alternating voltage. According to the monitoring device disclosed by the invention, non-covalent binding such as hydrogen bonds and Van der Waals force between the P2 aptamer and the target protein is broken by applying oscillation potential, so that the dissociation time is remarkably shortened, the sensor can be quickly recovered to an unbound state, dynamic and continuous monitoring is supported, and the requirement of real-time diagnosis is met.

Description

Technical Field

[0001] The present invention relates to the field of medical sensors, and more specifically to a protein monitoring device and its application, in particular to an implantable biosensor technology for continuous monitoring of protein biomarkers such as inflammation-related proteins in vivo. These biomarkers are of great significance in a variety of diseases, and can reflect the dynamic changes in the pathological process of chronic diseases and acute inflammatory states, providing key data for early diagnosis, real-time management, and therapeutic intervention of diseases. Background Art

[0002] In the prior art, there are a variety of sensors and devices for the detection and monitoring of biomarkers, especially in the fields of electrochemical sensors, wearable devices and microfluidics. However, these technologies are mainly widely used in the in vitro or non-invasive monitoring of small molecules such as glucose, lactate, drug metabolites, and electrolytes, and it is difficult to directly extend them to the detection and monitoring of proteins. For example, flexible pressure sensors achieve high-sensitivity detection of small pressure changes through carbon nanotubes or polymer composites such as PDMS microspheres, which are suitable for continuous monitoring of tactile or mechanical signals. Wearable strain sensors and skin interface microfluidic systems achieve real-time tracking of sweat loss, electrolyte composition and flow rate through hydrogels, strain sensing fabrics or wireless electronic modules, solving the limitation of traditional methods that cannot be continuously monitored. The skin interface system based on microfluidics achieves real-time monitoring of sweat by optimizing channel design. Some studies have proposed wearable antibacterial patches or antifouling coating monitoring devices, which respectively transport sweat in a directional manner through Janus structures or reduce nonspecific adsorption to improve stability. Although these technologies show high sensitivity and stability in small molecule monitoring, they are difficult to be used directly for protein detection and monitoring due to the large molecular weight, complex structure and high affinity binding of proteins to receptors, especially in real-time in vivo scenarios.

[0003] At present, the detection of protein biomarkers mainly relies on electrochemical sensor technology, especially electrochemical methods based on aptamers or antibodies. For example, aptamer-based sensors achieve in vitro detection of low-concentration biomarkers through nanostructures such as tetrahedral DNA or stem-loop structures, covering thrombin (1 pM to 10 nM), Tau381 (detection limit 0.70 pM) and AβO (detection limit 0.002 pM), etc. These technologies significantly improve the sensitivity and selectivity by optimizing detection methods such as time-series amperometry and alternating current voltammetry, and are suitable for in vitro analysis. However, these technologies are mainly limited to the in vitro detection of a single target molecule, and do not solve the problem of long dissociation time after high-affinity receptors bind to proteins, which limits their application in dynamic and continuous monitoring. Therefore, it is difficult for existing technologies to achieve real-time and continuous monitoring of proteins in vivo, especially when low-concentration proteins need to be dynamically tracked in inflammatory-related diseases, which faces significant technical challenges. Summary of the Invention

[0004] For the above reasons, the present invention provides a protein monitoring device and its application. Specifically, to achieve the objectives of the present invention, the following technical solutions are proposed: On the one hand, the present invention relates to a protein monitoring device, which has a microneedle with a hollow channel. Inside the microneedle, there are a working electrode, a reference electrode, and a counter electrode. The working electrode, the reference electrode, and the counter electrode are connected to the hollow channel in the microneedle. A P1 probe is fixed on the working electrode. The P2 aptamer forms a double strand with the P1 probe through hydrogen bonding. The P2 aptamer has a free end that binds to the target protein, and an alternating voltage is connected to the working electrode.

[0005] In a preferred embodiment of the present invention, the diameter of the hollow channel of the microneedle is 200 - 500 μm, and the hollow channel has a hydrophilic surface.

[0006] In a preferred embodiment of the present invention, the working electrode is a gold electrode, and its surface is modified with gold nanoparticles.

[0007] In a preferred embodiment of the present invention, the alternating voltage generates a square wave oscillating potential.

[0008] In a preferred embodiment of the present invention, the P1 probe is fixed on the working electrode through a thiol group.

[0009] In a preferred embodiment of the present invention, the P1 probe is a single-stranded DNA with the sequence 5'-SH-MC6-TACCAGCTATGTATCTAATAAGA-Fc-3'. Its 5' end forms an Au-S covalent bond with the surface of the gold electrode through a thiol group (SH-MC6), and its 3' end is connected to a ferricyanide redox reporter molecule for signal detection.

[0010] In a preferred embodiment of the present invention, the P2 aptamer contains the sequence: 5′-TCTTATTAGATACAATAGCTGGTA-3′.

[0011] On the other hand, the present invention also relates to the application of the above protein monitoring device in quantitatively detecting the protein concentration in vivo.

[0012] In a preferred embodiment of the present invention, the change in current is measured by chronoamperometry, and the current difference between the detected current and the baseline current in the unbound state is detected. This current difference is proportional to the protein concentration, thereby realizing quantitative monitoring of the protein concentration.

[0013] The beneficial effects of the present invention include but are not limited to: 1. Active reset mechanism achieved through oscillating potential: By applying an oscillating potential, the non-covalent bonds such as hydrogen bonds and van der Waals forces between the P2 aptamer and the target protein are broken, significantly shortening the dissociation time, enabling the sensor to quickly return to the unbound state, supporting dynamic and continuous monitoring, and meeting the requirements of real-time diagnosis.

[0014] 2. Monitoring of multiple inflammations achieved using flexible P2 aptamers: The free ends of the P2 aptamers are sequences specific to different inflammation-related proteins screened from a random nucleic acid library through SELEX technology. These sequences can form unique secondary structures such as stem-loops or G-quadruplexes, and specifically bind to specific epitopes of the target protein through non-covalent interactions. For each protein, the free-end sequence of P2 is optimized in terms of base composition and conformation to match its unique binding site, thus enabling the detection of multiple inflammation markers and meeting the requirements of multi-marker monitoring in complex diseases.

[0015] 3. Microneedles integrated with miniaturized oscillating electrodes support in-vivo continuous protein monitoring: The present invention embeds an oscillating potential system into an implantable microneedle device, supports in-vivo real-time protein monitoring through a rapid dissociation mechanism, and combines the electrochemical signal detection of the probe to track the change in protein concentration in real time, thereby expanding from in-vitro detection to in-vivo real-time monitoring.

[0016] 4. Utilizing the perturbation effect of the electric field to reduce non-specific adsorption and accumulation of proteins on the probe surface and enhance stability: The active reset technology applies a square-wave oscillating potential, and the perturbation effect of the electric field reduces non-specific adsorption and accumulation of proteins on the probe surface, preventing the microneedle channels from failing due to blockage by proteins or biomolecules. The capillary action of the hydrophilic IP-Q material combined with the main channels of the microneedles enables the interstitial fluid to fill the channels within 5 ms and reach protein concentration equilibrium within 20 minutes, significantly improving the sampling efficiency.

[0017] 5. Using a reagentless electrochemical oscillation method without additional reagents: The active reset technology directly acts on the P1-P2 probe system, breaks the non-covalent bond between the high-affinity aptamer and the protein, and can achieve protein dissociation and sensor reset without any external reagents. It simplifies the manufacturing process, reduces costs, and reduces the potential biocompatibility risks introduced by complex structures in in-vivo applications, achieving efficient and simple protein detection. Description of the Drawings

[0018] Figure 1 : Schematic diagram of the microneedle inserted into the skin; Figure 2 : Three views of the microneedle, where I is the perspective view; II is the front view; III is the top view; Figure 3 : Structure of the aptamer probe; Figure 4 : Current change of the ferrocene oxidation peak before and after hybridization of the P1 probe and the P2 aptamer; Figure 5 : Test results of current signals in solutions without the target protein (Unbound) and with 100 pg / mL of the target protein (Bound); Figure 6 : Oscillation-based active reset; Figure 7 : Continuous protein monitoring based on active reset; Figure 8 : Change in the number of hydrogen bonds between the aptamer and the protein before and after oscillation; Figure 9 : Change in the solvent-accessible area before and after oscillation; Figure 10 : Control experiments for passive reset and active reset. Detailed implementation manners

[0019] To further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0020] Unless otherwise specified, the reagents involved in the embodiments of the present invention are all commercially available products and can be obtained through commercial channels.

[0021] Example 1: The present invention uses a microneedle device to collect interstitial fluid for protein monitoring, as Figure 1 shown. The main channel diameter of the microneedle structure is about 300 μm, the inlet nozzle size is 300 μm × 400 μm, and the height is 1000 μm. The interstitial fluid is filled within 5 ms by capillary action through the hydrophilic surface and the protein concentration between the inside of the microneedle and the interstitial fluid in the body reaches equilibrium within 20 minutes by diffusion. A three-electrode system is integrated inside the microneedle: the working electrode is made of a gold rod (diameter 100 μm), the surface is modified with gold nanoneedles to enhance the surface area, the reference electrode is made of a silver rod (diameter 100 μm), and the counter electrode is made of a platinum rod (diameter 250 μm). These electrodes are connected to the main channel through side channels (width 10 μm) to ensure contact with the interstitial fluid.

[0022] The probe for monitoring protein concentration is fixed on the surface of the gold working electrode, and the presence of the protein is detected by an electrochemical method. As Figure 3As shown, the probe consists of a P1 probe and a P2 aptamer. The P1 probe is a single-stranded DNA (5'-SH-MC6-TACCAGCTATGTATCTAATAAGA-Fc-3'), whose 5' end forms an Au-S covalent bond with the gold electrode surface through a thiol group (SH-MC6). MC6 is a 6-mercaptohexyl modification group (-C6H12-), serving as a linker. The 3' end is connected to the redox reporter molecule ferrocene Fe(C5H5)2 (ferrocene, Fc), which is responsible for generating an electrochemical signal for signal detection. The P2 aptamer contains a general "P2-base" structure (5′-TCTTATTAGATACAATAGCTGGTA-3′) and a free end that forms a specific secondary structure. The free end is a variant sequence customized for different proteins through SELEX technology and is responsible for specifically binding to a specific target. It forms a secondary structure through folding and specifically binds to the target protein. Different target proteins require unique free end sequences to ensure high affinity and specificity. In the present invention, the free end specifically binding to the inflammatory marker myeloperoxidase (MPO) is 5'-AGCAGCACAGAGGTCAGATGGTCTGGAAACGACGAGGGCCACTGATTAACGTAGTTAATTGGTCTTGTCGAAGTGTAGTGTCTCCGT-3'. The "P2-base" is used to form a double strand with the middle sequence of the P1 probe through hydrogen bonding, while the free end binds to the target protein through hydrogen bonding, van der Waals forces, and electrostatic interactions. To verify the successful hybridization of the P1 probe and the P2 aptamer, the current change of the ferrocene oxidation peak before and after the hybridization of the P1 probe and the P2 aptamer was measured by square wave voltammetry, and 5 independent repeated experiments were carried out. The results are as Figure 4As shown. After hybridization, the peak current decreases because the formation of double strands increases the rigidity of the probe, hindering the diffusion of the ferrocene reporter molecule to the electrode, blocking electron transfer, and significantly reducing the current, which reflects the success of hydrogen bond pairing. After the protein binds to the free end of P2, the conformation of P1-P2 changes, moving the reporter molecule away from the gold electrode surface and reducing the electron transfer efficiency. The current change is measured by amperometry. The working electrode records the oxidation current of ferrocene, the reference electrode provides a stable potential, and the auxiliary electrode balances the circuit. A constant potential is applied by a potentiostat to measure the change in current over time. The current difference between the detected current and the baseline current in the unbound state is proportional to the protein concentration, thus enabling quantitative monitoring of protein concentration. To prove that the aptamer can bind to the protein successfully, a control experiment was designed to compare the current signals in the solution without the target protein (Unbound) and with 100 pg / mL target protein (Bound). The experiment used a three-electrode system to detect the current: the working electrode records the oxidation current of ferrocene, the reference electrode provides a stable potential, and the auxiliary electrode balances the circuit. A constant potential is applied by a potentiostat to measure the change in current over time. The results are as Figure 5 shown. Amperometry records the change trend of the normalized current over time in the Unbound and Bound states. In the Unbound state, the P1-P2 double strand maintains its initial conformation, the ferrocene is close to the electrode, and the electron transfer efficiency is high, so the current signal decays rapidly. In the Bound state, the target protein binds to the free end of P2, resulting in the adjustment of the double-strand conformation, pulling the ferrocene away from the electrode surface, blocking the electron transfer path, and the current signal decays slowly, directly reflecting the protein binding and the resulting conformational changes. ΔI is defined as the difference in the normalized current between the Unbound and Bound states and is used to quantify the protein concentration.

[0023] As Figure 6 shown, to solve the problem that the slow natural dissociation rate of the protein-probe complex after binding affects real-time monitoring, an alternating voltage is applied to the working electrode to accelerate the dissociation of the probe from the protein through oscillation. The oscillating electric field changes the conformation of the free end of the P2 aptamer and the protein, breaks the non-covalent interactions, promotes protein dissociation, shortens the reset time, and enables the probe to quickly return to its initial state, supporting continuous monitoring. Figure 7 The complete monitoring process is demonstrated. The microneedles are inserted into the skin to collect interstitial fluid, and the protein binds to the probe through diffusion, causing a change in current. After the binding is completed, an alternating voltage is applied to actively reset the probe, dissociating the protein and resetting the electrical signal. When the protein concentrations in the interstitial fluid and inside the microneedles reach equilibrium again and a new round of monitoring is completed, active reset is performed again. This operation is repeated continuously to achieve continuous and dynamic protein monitoring.

[0024] The preparation of the protein sensor based on active reset proposed by the present invention is as follows: 1. Preparation of microdevices: It is completed by two-dimensional two-photon polymerization (2PP) technology using IP-Q photoresist. First, a single-crystalline silicon (100) wafer (25 × 25 mm) substrate is cleaned with Piranha solution (a mixture of sulfuric acid and hydrogen peroxide) to remove organic matter, and then rinsed successively with acetone and isopropyl alcohol (IPA). Subsequently, IP-Q photoresist is drop-coated on the substrate on a Nanoscribe Photonic Professional GT system, and a micro-needle structure (main channel diameter 300 μm, inlet nozzle 300 μm × 400 μm, height 1000 μm, side channel width 10 μm) is written using a 10× objective lens with parameters of a speed of 40000 μm / s and a filling pitch of 0.75 μm for seamless splicing. After completion, it is immersed in propylene glycol monomethyl ether acetate (PGMEA) for 30 minutes to dissolve the uncured photoresist, washed twice with IPA (10 minutes each time), and after peeling, washed again with PGMEA (1 hour) and IPA (30 minutes). Finally, UV curing is performed for 3 minutes (360 nm, 17.0 mW / cm 2 ) to complete the micro-needle structure.

[0025] 2. Preparation of electrodes: First, a gold working electrode is cleaned by cyclic voltammetry (scan rate 100 mV / s, 30 cycles) with 0.5 M H2SO4, and then gold nanoneedles are deposited by chronoamperometry in a solution of 0.1 mM L-cysteine, 0.1 M H2SO4, and 5 mM HAuCl4. The potential for the first step is 0 mV (100 seconds), and the potential for the second step is -800 mV (5 seconds) to form gold nanoneedles to enhance the surface area. Finally, the modified gold working electrode, silver reference electrode, and platinum counter electrode are embedded in the microdevice and fixed with nuts, exposing the electrode ends to the side channel to contact the interstitial fluid in the main channel. After rinsing with 1× PBS, it is ready for probe fixation.

[0026] 3. Preparation of probes: The P1 probe is formulated into a 1 μM solution and 1 mM TCEP is added. After incubating for 1 hour in the dark to break disulfide bonds, it is heated to 55°C (5 minutes) and then cooled to room temperature. The P2 aptamer is heated at 90°C for 3 minutes to disrupt the secondary structure and then cooled, and incubated with the P1 solution for 15 minutes to form a double-stranded structure. Subsequently, the gold working electrode is immersed in the mixture (containing 9 μM MCH), and fixed overnight (16 hours) in a dark and humid environment through Au-S bonds. Then, it is incubated with 1 mM MCH for 1 hour to fill the gaps, incubated with 1% BSA for 30 minutes to form an anti-fouling layer, and finally washed with 1× PBS and 0.1× PBS to complete probe assembly.

[0027] To reveal the microscopic mechanism of the active reset mechanism, molecular dynamics simulations were performed to investigate the effect of an oscillating electric field on the dissociation of the P2 aptamer from the MPO protein. First, a complex system of the P2 aptamer (predicted secondary structure by RNAfold, modeled by AMBER tleap) and the MPO protein (obtained from the PDB database) was constructed and placed in a TIP3P water box with a side length of approximately 12 nm (about 180,000 atoms, 150 mM NaCl). The parameters were set using the AMBER ff14SB, OL3, and GAFF force fields, and the electrostatic interaction was treated with the PME method. Subsequently, energy minimization was carried out (5000 steps each for the steepest descent and conjugate gradient methods, where the steepest descent refers to rapidly adjusting the atomic coordinates to descend along the steepest energy gradient to eliminate the initial unreasonable geometric conformations and atomic collisions in the molecular system), heating to 300 K in the NVT ensemble (100 ps), equilibrating for 100 ps in the NPT ensemble (1 bar), followed by 50 ns of equilibration simulation and 20 ns of oscillating electric field simulation (0.0125 - 0.03 V / Å). Each group was repeated three times, and the trajectories were analyzed using VMD and GROMACS to calculate the number of hydrogen bonds and SASA. The results showed that the number of hydrogen bonds decreased after oscillation ( Figure 8 ), and the SASA increased ( Figure 9 ). The oscillating electric field changed the conformation of P2 through perturbation, enhanced local flexibility, disrupted the hydrogen bond network at the binding interface, and reduced non-covalent interactions. The increase in SASA made the interface more exposed to the solvent, and the penetration of water molecules weakened the van der Waals force and electrostatic interaction, reducing the binding affinity and accelerating the dissociation process.

[0028] Figure 10 The comparative experiments in Figure 10The ΔI vs. time curves of the passive reset and active reset groups are shown, reflecting the differences in dissociation rates. The passive reset group did not fully recover to the baseline even after 60 minutes, indicating low natural dissociation efficiency and more residual MPO protein; after applying an oscillating electric field, the signal of the active reset group quickly recovered to the baseline, indicating rapid dissociation of MPO protein and the sensor returning to its initial state. The results show that active reset significantly accelerates the dissociation process through an oscillating electric field. Compared with passive reset, active reset achieves complete dissociation within 1 minute, demonstrating its high efficiency and providing an effective strategy for the rapid reset of aptasensors.

[0029] The preferred embodiments of the present invention have been described above, but they are not intended to limit the present invention. Those skilled in the art can make improvements and changes to the embodiments disclosed herein without departing from the scope and spirit of the present invention.

Claims

1. A protein monitoring device, which has a microneedle with a hollow channel. Inside the microneedle, there are a working electrode, a reference electrode and a counter electrode. The working electrode, the reference electrode and the counter electrode are connected to the hollow channel in the microneedle. A P1 probe is fixed on the working electrode. The P2 aptamer forms a double strand with the P1 probe through hydrogen bonding. The P2 aptamer has a free end that binds to the target protein. An alternating voltage is connected to the working electrode.

2. The monitoring device according to claim 1, wherein the alternating voltage generates a square wave oscillating potential.

3. The monitoring device according to claim 1, wherein the diameter of the hollow channel of the microneedle is 200 - 500 μm, and the hollow channel has a hydrophilic surface.

4. The monitoring device according to claim 1, wherein the working electrode is a gold electrode, and its surface is modified with gold nanoparticles.

5. The monitoring device according to claim 4, wherein the P1 probe is fixed on the working electrode through a thiol group.

6. The monitoring device according to claim 5, wherein the P1 probe is a single-stranded DNA with the sequence 5'-SH-MC6-TACCAGCTATGTATCTAATAAGA-Fc-3'.

7. The monitoring device according to claim 6, wherein the P2 aptamer contains the sequence: 5'-TCTTATTAGATACAATAGCTGGTA-3′.

8. Use of the protein monitoring device according to any one of claims 1 - 7 in the preparation of a detection device for quantitatively detecting the concentration of proteins in vivo.

9. The use according to claim 8, wherein the change in current is measured by chronoamperometry, and the current difference between the detected current and the baseline current in the unbound state is detected.

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