A protein monitoring device and its application

Abstract

The present invention discloses a protein monitoring device and its application. The monitoring device comprises a microneedle with a hollow channel. The microneedle contains 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 to the working electrode. A P2 aptamer and the P1 probe are paired through hydrogen bonds to form a double chain. The P2 aptamer has a free end that binds to a target protein. An alternating voltage is connected to the working electrode. The monitoring device of the present invention significantly shortens the dissociation time by applying an oscillating potential to break the non-covalent binding between the P2 aptamer and the target protein, such as hydrogen bonds and van der Waals forces, so that the sensor can quickly return to an unbound state, supporting dynamic and continuous monitoring and meeting the needs of real-time diagnosis.

Description

Technical Field

[0001] The present invention relates to the field of medical sensors, specifically to a protein monitoring device and its applications, particularly implantable biosensor technology for continuous in vivo monitoring of protein biomarkers, such as inflammation-related proteins. These biomarkers are of significant importance in a variety of diseases, reflecting the dynamic changes in the pathological processes of chronic diseases and acute inflammatory conditions, providing key data for early disease diagnosis, real-time management, and therapeutic intervention. Background Art

[0002] In the existing technology, 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 for in vitro or non-invasive monitoring of small molecules such as glucose, lactate, drug metabolites, and electrolytes, and are difficult to directly extend 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. Microfluidics-based skin interface systems achieve real-time monitoring of sweat by optimizing channel design. Some studies have proposed wearable antibacterial patches or anti-fouling coating monitoring devices, which respectively use Janus structures to transport sweat in a directional manner 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] Currently, the detection of protein biomarkers primarily relies on electrochemical sensor technologies, particularly aptamer- or antibody-based electrochemical methods. For example, aptamer-based sensors utilize nanostructures such as tetrahedral DNA or stem-loop structures to enable in vitro detection of low-concentration biomarkers, including thrombin (1 pM to 10 nM), Tau381 (detection limit 0.70 pM), and AβO (detection limit 0.002 pM). These technologies significantly improve sensitivity and selectivity through optimized detection methods such as time-series amperometry and alternating current voltammetry, making them suitable for in vitro analysis. However, these technologies are primarily limited to the in vitro detection of single target molecules and fail to address the long dissociation time of high-affinity receptors after binding to proteins, limiting their application in dynamic, continuous monitoring. Consequently, existing technologies struggle to achieve real-time, continuous monitoring of proteins in vivo, particularly when dynamic tracking of low-concentration proteins is required in inflammatory diseases, which presents significant technical challenges. Summary of the Invention

[0004] Based on the above reasons, the present invention proposes a protein monitoring device and its application. Specifically, in order to achieve the purpose of the present invention, the present invention intends to adopt the following technical solutions:

[0005] One aspect of the present invention relates to a protein monitoring device, comprising a microneedle with a hollow channel. The microneedle contains a working electrode, a reference electrode, and a counter electrode, which are connected to the hollow channel in the microneedle. A P1 probe is fixed to the working electrode, and a P2 aptamer and the P1 probe are paired through hydrogen bonds to form a double chain. The P2 aptamer has a free end that binds to the target protein. An alternating voltage is connected to the working electrode.

[0006] 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.

[0007] In a preferred embodiment of the present invention, the working electrode is a gold electrode, the surface of which is modified with gold nanoparticles.

[0008] In a preferred embodiment of the present invention, the alternating voltage generates a square wave oscillating potential.

[0009] In a preferred embodiment of the present invention, the P1 probe is immobilized on the working electrode via a thiol group.

[0010] 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 gold electrode surface via a thiol group (SH-MC6), and its 3' end is linked to an iron redox reporter molecule for signal detection.

[0011] In a preferred embodiment of the present invention, the P2 aptamer comprises the sequence: 5′-TCTTATTAGATACAATAGCTGGTA-3′.

[0012] Another aspect of the present invention also relates to the use of the above protein monitoring device in quantitatively detecting protein concentration in the body.

[0013] In a preferred embodiment of the present invention, the current change is measured by chronoamperometry, and the current difference between the detection current and the baseline current of the unbound state is proportional to the protein concentration, thereby achieving quantitative monitoring of protein concentration.

[0014] The beneficial effects of the present invention include but are not limited to:

[0015] 1. Active reset mechanism via oscillating potential: Applying an oscillating potential breaks the non-covalent binding between the P2 aptamer and the target protein, such as hydrogen bonds and van der Waals forces, significantly shortening the dissociation time. This allows the sensor to quickly return to the unbound state, supporting dynamic and continuous monitoring and meeting the needs of real-time diagnosis.

[0016] 2. Using the flexible P2 aptamer to monitor multiple inflammations: The free end of the P2 aptamer uses SELEX technology to screen specific sequences for different inflammation-related proteins from a random nucleic acid library. These sequences can form unique secondary structures, such as stem-loops or G-quadruplexes, and bind specifically to specific epitopes of the target protein through non-covalent interactions. For each protein, the free end sequence of P2 is optimized in base composition and conformation to match its unique binding site, enabling the detection of multiple inflammatory markers and meeting the needs of multi-marker monitoring in complex diseases.

[0017] 3. Microneedles with integrated miniaturized oscillating electrodes support continuous in vivo protein monitoring: This invention embeds an oscillating potential system into an implantable microneedle device, supporting real-time protein monitoring in vivo through a rapid dissociation mechanism. Combined with the electrochemical signal detection of the probe, it can track changes in protein concentration in real time, thus expanding from in vitro detection to real-time in vivo monitoring.

[0018] 4. Utilizing the perturbative effect of the electric field to reduce nonspecific adsorption and accumulation of proteins on the probe surface, enhancing stability: Active reset technology applies a square wave oscillating potential, allowing the perturbative effect of the electric field to reduce nonspecific adsorption and accumulation of proteins on the probe surface, preventing microneedle channel failure due to protein or biomolecule clogging. The main microneedle channel, combined with the capillary action of the hydrophilic IP-Q material, allows interstitial fluid to fill the channel within 5 ms and reach protein concentration equilibrium within 20 minutes, significantly improving sampling efficiency.

[0019] 5. A reagent-free electrochemical oscillation method eliminates the need for additional reagents: Active resetting technology directly acts on the P1-P2 probe system, breaking the non-covalent binding of the high-affinity aptamer to the protein, enabling protein dissociation and sensor resetting without any external reagents. This simplifies the manufacturing process, reduces costs, and mitigates potential biocompatibility risks introduced by complex structures in in vivo applications, enabling efficient and simple protein detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : Schematic diagram of microneedle insertion into the skin;

[0021] Figure 2 : Three views of the microneedle, where I is a perspective view; II is a front view; III is a top view;

[0022] Figure 3: Structure of aptamer probe;

[0023] Figure 4 : Current changes of ferrocene oxidation peak before and after hybridization of P1 probe and P2 aptamer;

[0024] Figure 5 : Current signal test results of solutions without target protein (Unbound) and with 100 pg / mL target protein (Bound);

[0025] Figure 6 : Active reset based on oscillation;

[0026] Figure 7 : Continuous protein monitoring based on active resetting;

[0027] Figure 8 : Changes in the number of hydrogen bonds between the aptamer and the protein before and after oscillation;

[0028] Figure 9 : Changes in solvent accessible area before and after shaking;

[0029] Figure 10 : A controlled experiment of passive and active reset. DETAILED DESCRIPTION

[0030] To further understand the present invention, the following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0031] Unless otherwise specified, all reagents involved in the embodiments of the present invention are commercially available products and can be purchased through commercial channels.

[0032] Example 1:

[0033] The present invention uses a microneedle device to collect interstitial fluid for protein monitoring, such as Figure 1As shown in Figure 2, the microneedle structure has a main channel diameter of approximately 300 μm, an inlet nozzle measuring 300 μm × 400 μm, and a height of 1000 μm. The hydrophilic surface allows capillary action to fill the microneedle with interstitial fluid within 5 ms, and diffusion allows the protein concentration within the microneedle to equilibrate with that in vivo within 20 minutes. The microneedle incorporates a three-electrode system: a working electrode made of a gold rod (100 μm diameter) modified with gold nanoneedles to increase surface area, a reference electrode made of a silver rod (100 μm diameter), and a counter electrode made of a platinum rod (250 μm diameter). These electrodes are connected to the main channel via a side channel (10 μm wide), ensuring contact with the interstitial fluid.

[0034] The probe for monitoring protein concentration is fixed on the surface of the gold working electrode and the presence of protein is detected by electrochemical method. Figure 3 As 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'). Its 5' end forms a covalent bond between the Au and S atoms of the gold electrode surface via a thiol group (SH-MC6). MC6, a 6-mercaptohexyl modification group (-C6H12-), serves as a linker. The 3' end is linked to the redox reporter molecule ferrocene (Fe(C5H5)2) (Fc), responsible for generating an electrochemical signal for signal detection. The P2 aptamer consists of a universal "P2-base" structure (5'-TCTTATTAGATACAATAGCTGGTA-3') and a free end that forms a specific secondary structure. The free end is a protein-specific variant sequence customized using SELEX technology. It is responsible for specific binding to a specific target and forms a secondary structure by folding to the target protein. Different target proteins require unique free end sequences to ensure high affinity and specificity. The free end specifically binding to the inflammatory marker myeloperoxidase (MPO) used in the present invention is 5'-AGCAGCACAGAGGTCAGATGGTCTGGAAACGACGAGGGCCACTGATTAACGTAGTTAATTGGTCTTGTCGAAGTGTAGTGTCTCCGT-3'. "P2-base" is used to form a double chain 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 force and electrostatic interaction. In order 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 performed. The results are as follows: Figure 4As shown. The peak current decreases after hybridization. This is because the formation of double strands increases the rigidity of the probe, hindering the diffusion of the ferrocene reporter molecule to the electrode, hindering electron transfer, and significantly reducing the current, reflecting the success of hydrogen bond pairing. After the protein binds to the free end of P2, the conformation of P1-P2 changes, causing the reporter molecule to move away from the gold electrode surface, reducing the efficiency of electron transfer. The current change is measured by chronoamperometry. The working electrode records the ferrocene oxidation current, the reference electrode provides a stable potential, the auxiliary electrode balances the circuit, and a constant potential is applied by a constant potentiostat to measure the current change over time. The current difference between the detection current and the baseline current of the unbound state is proportional to the protein concentration, thereby achieving quantitative monitoring of protein concentration. In order to prove that the aptamer can successfully bind to the protein, a control experiment was designed to compare the current signals of the solution without target protein (Unbound) and containing 100 pg / mL target protein (Bound). The experiment uses a three-electrode system to detect the current: the working electrode records the ferrocene oxidation current, the reference electrode provides a stable potential, the auxiliary electrode balances the circuit, and a constant potential is applied by a constant potentiostat to measure the current change over time. The results are shown in Figure 2. Figure 5 As shown in Figure 2 , chronoamperometry records the time-dependent trend of the normalized current in the unbound and bound states. In the unbound state, the P1-P2 duplex maintains its initial conformation, ferrocene is close to the electrode, electron transfer efficiency is high, and the current signal decays rapidly. In the bound state, the target protein binds to the free end of P2, causing the duplex to adjust its conformation. Ferrocene is pulled away from the electrode surface, electron transfer pathways are blocked, and the current signal decays slowly, directly reflecting protein binding and the conformational changes it triggers. ΔI, defined as the normalized current difference between the unbound and bound states, is used to quantify protein concentration.

[0035] like Figure 6 As shown in the figure, to address the issue of slow natural dissociation of proteins after binding to the probe, which affects real-time monitoring, an alternating voltage is applied to the working electrode, causing the probe to accelerate its dissociation from the protein through oscillation. The oscillating electric field changes the conformation of the free end of the P2 aptamer and the protein, breaking the non-covalent interaction, promoting protein dissociation, shortening the reset time, and quickly returning the probe to its initial state, supporting continuous monitoring. Figure 7 The complete monitoring process is demonstrated. A microneedle is inserted into the skin to collect interstitial fluid. Proteins bind to the probe through diffusion, causing a change in current. Once binding is complete, an alternating voltage is applied to actively reset the probe, dissociating the proteins and resetting the electrical signal. Once the interstitial fluid and protein concentrations within the microneedle reach equilibrium again, completing a new round of monitoring, another active reset is performed. This cycle repeats, enabling continuous, dynamic protein monitoring.

[0036] The preparation of the protein sensor based on active resetting proposed in the present invention is as follows:

[0037] 1. Preparation of microdevices: IP-Q photoresist was used by two-dimensional photopolymerization (2PP) technology. First, the single crystal silicon (100) wafer (25 × 25 mm) substrate was cleaned with Piranha solution (a mixture of sulfuric acid and hydrogen peroxide). After removing organic matter, it was rinsed with acetone and isopropyl alcohol (IPA) in sequence. Then, IP-Q photoresist was drop-coated on the substrate on a Nanoscribe Photonic Professional GT system. The microneedle structure (main channel diameter 300 μm, inlet nozzle 300 μm × 400 μm, height 1000 μm) was written using a 10× objective lens at a speed of 40,000 μm / s and a filling spacing of 0.75 μm. μm, with a side channel width of 10 μm) for seamless splicing. After completion, the uncured photoresist was dissolved in propylene glycol methyl ether acetate (PGMEA) for 30 minutes, and then washed twice with IPA (10 minutes each). After stripping, the film was cleaned again with PGMEA (1 hour) and IPA (30 minutes), and finally UV cured for 3 minutes (360 nm, 17.0 mW / cm 2 ) to complete the microneedle structure.

[0038] 2. Electrode Preparation: First, the gold working electrode was cleaned using 0.5 M H₂SO₄ using cyclic voltammetry (scan rate 100 mV / s, 30 cycles). Then, gold nanoneedles were deposited using chronoamperometry in a solution of 0.1 mM L-cysteine, 0.1 M H₂SO₄, and 5 mM HAuCl₄. The first step was at a potential of 0 mV for 100 seconds, followed by a second step at a potential of -800 mV for 5 seconds. This formed the gold nanoneedles to enhance the surface area. Finally, the modified gold working electrode, silver reference electrode, and platinum counter electrode were embedded in the microdevice and secured with a nut, exposing the electrode tip to the side channel and contacting the interstitial fluid in the main channel. After rinsing with 1× PBS, the electrodes were prepared for probe fixation.

[0039] 3. Probe Preparation: The P1 probe was prepared as a 1 μM solution and 1 mM TCEP was added. The solution was incubated in the dark for 1 hour to break disulfide bonds, then heated to 55°C for 5 minutes and cooled to room temperature. The P2 aptamer was heated at 90°C for 3 minutes to disrupt its secondary structure, cooled, and incubated with the P1 solution for 15 minutes to form a duplex structure. The gold working electrode was then immersed in the solution (containing 9 μM MCH) and incubated in a dark, humid environment overnight (16 hours) to immobilize the Au-S bond. The gaps were then filled with 1 mM MCH and incubated with 1% BSA for 30 minutes to form an antifouling layer. The probe assembly was then completed by washing with 1× PBS and 0.1× PBS.

[0040] In order to reveal the microscopic mechanism of the active reset mechanism, molecular dynamics simulation was used to explore the effect of oscillating electric field on the dissociation of P2 aptamer and MPO protein. The simulation first constructed a complex system of the P2 aptamer (secondary structure predicted by RNAfold, AMBER tleap modeling) and the MPO protein (obtained from the PDB database) and placed it in a TIP3P water box with a side length of approximately 12 nm (approximately 180,000 atoms, 150 mM NaCl). The parameters were set using AMBER ff14SB, OL3, and GAFF force fields, and the PME method was used to treat electrostatic interactions. Energy minimization was then performed (5000 steps each of the steepest descent and conjugate gradient method, where the steepest descent refers to the elimination of initial unreasonable geometric conformations and atomic collisions in the molecular system by rapidly adjusting the atomic coordinates to descend along the steepest energy gradient). The NVT ensemble was heated to 300 K (100 ps), the NPT ensemble was equilibrated for 100 ps (1 bar), and then a 50 ns equilibrium simulation and a 20 ns oscillating electric field simulation (0.0125-0.03 V / Å) were performed. Each group was repeated three times. 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 ), SASA increases ( Figure 9 The oscillating electric field perturbs the P2 conformation, enhancing local flexibility, disrupting the hydrogen bond network at the binding interface, and reducing non-covalent interactions. The increased SASA exposes the interface to the solvent, and water molecule penetration weakens van der Waals and electrostatic interactions, reducing binding affinity and accelerating the dissociation process.

[0041] Figure 10 Comparative experiments in the literature verified the accelerated effect of active resetting on the dissociation rate between the aptamer and the MPO protein. The experiment used a P1-P2-based gold nanostructured microelectrode sensor. The sensor was first incubated in a PBS buffer containing 100 pg / ml of the target protein for 30 minutes at a pH of 7.4, allowing the MPO protein to specifically bind to the aptamer. The passive resetting group did not apply any external electric field and was rinsed with protein-free PBS, relying solely on natural dissociation to complete protein dissociation. The active resetting group applied an oscillating electric field with a potential of 0.25 V, a frequency of 90 Hz, and an application time of 1 minute. Both experiments recorded current signals every 1 minute for a period of 0 to 60 minutes. The experiments were conducted at room temperature (25°C), with five replicates per group and the average value taken. Figure 10The ΔI curves for the passive and active reset groups are shown over time, reflecting the difference in dissociation rates. The passive reset group had not fully recovered to baseline after 60 minutes, indicating low natural dissociation efficiency and a high level of residual MPO protein. In the active reset group, the signal quickly recovered to baseline after application of an oscillating electric field, indicating rapid dissociation of the MPO protein and restoration of the sensor to its initial state. The results demonstrate that active reset significantly accelerates the dissociation process through the oscillating electric field. Compared to passive reset, active reset achieved complete dissociation within 1 minute, demonstrating its high efficiency and providing an effective strategy for rapidly resetting aptamer sensors.

[0042] The above describes the preferred embodiments of the present invention, which is not intended to limit the present invention. Those skilled in the art may 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 comprising a microneedle with a hollow channel. The microneedle contains a working electrode, a reference electrode, and a counter electrode, which are connected to the hollow channel in the microneedle. A P1 probe is fixed to the working electrode. A P2 aptamer and the P1 probe form a double strand through hydrogen bonding. The P2 aptamer has a free end that binds to a target protein. An alternating voltage is connected to the working electrode; the alternating voltage generates a square wave oscillating potential. The P1 probe is a single-stranded DNA with the sequence 5'-SH-MC6-TACCAGCTATGTATCTAATAAGA-Fc-3'; the P2 aptamer comprises the sequence 5'-TCTTATTAGATACAATAGCTGGTA-3'. 2 . 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.

3. The monitoring device according to claim 1, wherein the working electrode is a gold electrode, and the surface of the electrode is modified with gold nanoparticles. The monitoring device according to claim 1 , wherein the P1 probe is fixed on the working electrode via a thiol group.

5. Use of the protein monitoring device according to any one of claims 1 to 4 in preparing a detection device for quantitatively detecting protein concentration in vivo.

6. The use according to claim 5, wherein the current change is measured by chronoamperometry, and the current difference between the detection current and the baseline current of the unbound state is detected.

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