Needle type flexible biosensor and preparation method thereof

Abstract

The invention relates to the technical field of biosensors, in particular to a needle type flexible biosensor and a preparation method thereof. The sensor includes a plurality of working electrode layers formed on a corresponding plurality of first flexible substrates, a reference electrode layer formed on a second flexible substrate, a counter electrode layer, a counter electrode conductive contact layer, and an encapsulation layer. A plurality of first flexible substrates and a plurality of second flexible substrates are laminated to form an integral electrode array needle body, the electrode array needle body is formed to form a flexible probe, and the flexible probe exposes a plurality of working electrode layers and the reference electrode layers in a fine needle working area of the electrode array needle body in a layered manner. A plurality of detection channels which are electrically isolated from one another are integrated in a single needle body, the structure is compact, multi-parameter detection can be carried out, the stability is high, and implantation wounds are reduced.

Description

Technical Field

[0001] The present application relates to the technical field of biosensors, and in particular to a needle-type flexible biosensor and a preparation method thereof. Background Art

[0002] With the rapid development of biosensor technology, implantable medical devices have shown tremendous potential in areas such as health management, disease monitoring, and personalized medicine. Biosensors that continuously monitor physiological indicators are gradually evolving from single-parameter detection to multi-parameter integration, providing more comprehensive and accurate data support for medical health monitoring. In particular, multi-parameter biosensors can provide real-time data on multiple metabolites in scenarios such as chronic disease management and exercise physiology assessment, enabling more efficient health management and disease prevention.

[0003] At present, in order to meet the needs of multi-parameter detection, the industry often adopts the following solutions: First, by implanting multiple independent probes under the skin, different objects to be tested are monitored in real time; second, using a multi-probe array structure, using multiple sensor probes to achieve multi-index simultaneous detection; third, trying to set multiple detection areas in the same needle body, and realizing a multi-channel layout through a separated cavity structure. In addition, the manufacture of existing multi-parameter sensors mainly relies on a full-process screen printing process, where the conductive layer and the insulating layer are both superimposed and formed by screen printing, and UV curable resin or other materials are used for interlayer isolation. For example, invention patent application publication number CN116448849A discloses an implantable multifunctional electrochemical flexible biosensor and its preparation method, the implantable multifunctional electrochemical flexible biosensor includes a sensor body, the sensor body is a sheet structure, the sensor body includes a polymer base layer, an electrode material layer, a biosensitive membrane, an insulating layer, a biorestriction membrane and electrode contacts, and the sensor body also includes a three-electrode system. Among them, multiple electrode contacts are integrated on the sheet structure sensor body to form a multi-probe array structure.

[0004] However, the relevant technology still has significant defects. The use of multiple independent probes will lead to the expansion of the wound surface, prolonged wound recovery time and increased risk of rejection; the multi-probe array structure is difficult to meet the needs of miniaturization due to its large size (the electrode spacing in the invention patent application publication number CN116448849A is ≥2mm); and the separated cavity structure cannot achieve a highly integrated single-needle multi-channel layout due to the large electrode spacing. In addition, the traditional screen printing process faces problems such as uneven insulation layer thickness and insufficient interface bonding force when multiple channels are densely arranged, resulting in shortened sensor life and reduced detection accuracy (interlayer resistance fluctuations of ±15%). Therefore, how to achieve a highly integrated layout of multi-channel electrodes in a single-needle structure with an extremely small volume while ensuring electrical isolation and long-term stability has become a technical problem that needs to be solved urgently. Summary of the Invention

[0005] In order to integrate multi-channel electrodes in a sensor with a single needle structure and ensure the electrical isolation and long-term stability of the sensor, the present application provides a needle-type flexible biosensor and a preparation method thereof.

[0006] On the one hand, a needle-type flexible biosensor is provided, which adopts the following technical solution.

[0007] A needle-type flexible biosensor, comprising: a plurality of working electrode layers formed on the corresponding plurality of first flexible substrates; A reference electrode layer is formed on a second flexible substrate; wherein the first flexible substrates and the second flexible substrates are heat-pressed together to form an integral electrode array needle body, and the working electrode layers and the reference electrode layer have different lengths in the fine needle working area of the electrode array needle body; a counter electrode layer formed on the back side of the electrode array needle body; An encapsulation layer is formed on the surface of the electrode array needle body and exposes the fine needle working area and contact area of the electrode array needle body; the electrode array needle body is formed to form a flexible probe needle, and the flexible probe needle exposes multiple working electrode layers and reference electrode layers in the fine needle working area of the electrode array needle body in a hierarchical manner.

[0008] By adopting the above technical solution, multiple working electrode layers and reference electrode layers are pressed together to form a single needle body structure, and are exposed in layers in the fine needle working area to form an integrated stacked soft needle structure. At the same time, a counter electrode layer and a packaging layer with vias on the back are provided, so that multiple detection channels electrically isolated from each other are integrated into a single needle body. The structure is compact, multi-parameter detection can be performed, the stability is strong, and the implantation trauma is reduced.

[0009] Optionally, the Young's modulus of the first flexible substrate (11) and the second flexible substrate (12) is between 0.5 and 2 GPa, and the pressing temperature of the hot pressing process is 120±15°C; an insulating structure is provided between the first flexible substrates and the second flexible substrates, comprising a polyimide buffer layer or a fluoroplastic isolation film; the first flexible substrate (11) has a nanoscale roughness (Ra) of 50 to 100 nm, a contact angle of the hydrophilic coating is less than 30°, and has a gradient pore structure.

[0010] By adopting the above technical solution, the Young's modulus of the first flexible substrate and the second flexible substrate is set at 0.5-2GPa, and the pressing temperature of the hot pressing process is 120±15℃, which is conducive to making multiple first flexible substrates and second flexible substrates form an integral electrode array needle body with good performance during the hot pressing process; an insulating structure containing a polyimide buffer layer or a fluoroplastic isolation film is provided between the multiple first flexible substrates and the second flexible substrate, which can further ensure the electrical isolation between different electrode layers in the stacked structure, prevent signal crosstalk, and improve the accuracy and stability of detection; the nanoscale roughness of the first flexible substrate is set to 50-100nm, the contact angle of the hydrophilic coating is <30°, and it has a gradient pore structure, which helps to improve the surface properties of the first flexible substrate and better adapt to the working electrode layer and related processes.

[0011] Optionally, the working electrode layer includes a working electrode, a working electrode conductive circuit and a working electrode contact, and the working electrode is connected to the working electrode contact through the working electrode conductive circuit; the reference electrode layer includes a reference electrode, a reference conductive circuit and a reference electrode contact, and the reference electrode is connected to the reference electrode contact through the reference conductive circuit.

[0012] The above technical solution clarifies the specific composition of the working electrode layer and the reference electrode layer, including electrodes for sensing detection, conductive lines for signal transmission, and contacts for external connection, ensuring the realization of the basic electrochemical detection function of the sensor and the effective extraction of signals.

[0013] Optionally, the plurality of working electrodes and the reference electrodes are exposed in layers in the fine needle working area of the electrode array needle body, and the plurality of working electrode contacts and the reference electrode contacts are arranged alternately in the contact area of the electrode array needle body, with a first dividing gap between the working electrode contacts.

[0014] By adopting the above technical solution, the hierarchical exposed structure of the fine needle working area is conducive to the simultaneous detection of different substances by multiple working electrodes in a limited space; the staggered arrangement and clear boundaries of the contact area facilitate the independent and reliable connection of each electrode to the external circuit, reducing the connection difficulty and short circuit risk.

[0015] Optionally, an electrode via is provided in the electrode array needle body, and an electrode conductive contact layer is formed on the front side of the electrode array needle body, the electrode conductive contact layer is connected to the electrode layer through the electrode via, and there is a second dividing gap between the electrode conductive contact layer and the reference electrode contact; the electrode layer includes an electrode and an electrode conductive circuit, and the electrode conductive contact layer is connected to the electrode conductive circuit through the electrode via.

[0016] By adopting the above technical solution, a counter electrode layer is set on the back and led out through vias and a conductive contact layer on the front, forming a complete three-electrode detection system, thereby ensuring the integrity of the electrochemical reaction circuit.

[0017] Optionally, the packaging layer includes a front packaging layer covering the front of the electrode array needle body and a back packaging layer covering the back of the electrode array needle body, wherein in the fine needle working area, the front packaging layer exposes a plurality of the working electrodes and the reference electrodes, and the back packaging layer exposes the counter electrode, and in the contact area, the front packaging layer exposes a plurality of the working electrode contacts, the reference electrode contacts and the counter electrode conductive contact layer, and the back packaging layer covers the counter electrode conductive circuit and the counter electrode via; the needle-type flexible biosensor has a microfluidic channel, which is arranged between the first flexible substrate and the front packaging layer, and has a trapezoidal cross-section.

[0018] Using this technical solution, the encapsulation layer precisely exposes the electrode sensing area required for operation and the contact area required for external connections, while protecting the remaining areas, ensuring the functionality, stability, and durability of the sensor in the operating environment. A microfluidic channel with a trapezoidal cross-section is provided between the first flexible substrate and the front encapsulation layer to facilitate fluid flow and distribution, improving sensor performance.

[0019] On the other hand, a method for preparing a needle-type flexible biosensor is also provided, which adopts the following technical solution.

[0020] A method for preparing a needle-type flexible biosensor comprises the following steps: S1. preparing corresponding working electrode layers on multiple first flexible substrates; S2. preparing a reference electrode layer on a second flexible substrate; S3. Thermally pressing the first flexible substrates and the second flexible substrates to form an integrated electrode array needle body, wherein the working electrode layers and the reference electrode layers have different lengths in the fine needle working area of the electrode array needle body; S4, preparing a counter electrode layer on the back side of the electrode array needle body; S6. Forming an encapsulation layer on the surface of the electrode array needle body, wherein the encapsulation layer covers the surface of the electrode array needle body and exposes the fine needle working area and contact area of the electrode array needle body; S7. Cutting the electrode array needle body into shapes to form a flexible probe needle, wherein the flexible probe needle exposes a plurality of working electrode layers and a reference electrode layer in a layered manner in the fine needle working area of the electrode array needle body.

[0021] By adopting the above technical solution, the electrode layers are prepared in layers and then pressed together, which avoids the deformation problem that may be caused by multiple high-temperature curing on the same substrate, improves the process stability and product yield of multi-layer flexible device manufacturing, and can accurately control the exposure length of each electrode layer in the fine needle working area. Finally, a flexible probe needle with a layered exposure structure is formed by cutting.

[0022] Optionally, before step S3 , an insulating structure is provided between the plurality of first flexible substrates and the second flexible substrates.

[0023] By adopting the above technical solution, an insulating structure is set before pressing, which ensures reliable electrical isolation between different electrode layers during the subsequent pressing process, providing a guarantee for the preparation of multi-channel sensors with low crosstalk and high stability.

[0024] Optionally, in step S1, the working electrode layer includes a working electrode, a working electrode conductive circuit and a working electrode contact, and the working electrode is connected to the working electrode contact through the working electrode conductive circuit; in step S2, the reference electrode layer includes a reference electrode, a reference conductive circuit and a reference electrode contact, and the reference electrode is connected to the reference electrode contact through the reference conductive circuit; in step S3, the plurality of working electrodes and the reference electrodes are exposed in layers in the fine needle working area of the electrode array needle body, and the plurality of working electrode contacts and the reference electrode contacts are staggered in the contact area of the electrode array needle body. , there is a first dividing gap between the working electrode contacts; the counter electrode layer in step S4 includes a counter electrode and a counter electrode conductive circuit, and a counter electrode via is also opened in the electrode array needle body; step S5 is also included between step S4 and step S6, step S5 includes preparing a counter electrode conductive contact layer on the front side of the electrode array needle body, the counter electrode conductive contact layer is connected to the counter electrode layer through the counter electrode via, there is a second dividing gap between the counter electrode conductive contact layer and the reference electrode contact, and the counter electrode conductive contact in step S5 is connected to the counter electrode conductive circuit through the counter electrode via.

[0025] By adopting the above technical solution, the correct formation and connection of the key functional parts of the sensor (working electrode, reference electrode, counter electrode and its wires and contacts) are ensured through specific preparation steps. The layered exposure of the fine needle working area and the staggered arrangement of the contact area are achieved through pressing, ensuring the integrity and functionality of the final sensor structure.

[0026] Optionally, in step S6, the packaging layer includes a front packaging layer covering the front of the electrode array needle body and a back packaging layer covering the back of the electrode array needle body, wherein in the fine needle working area, the front packaging layer exposes a plurality of the working electrodes and the reference electrodes, and the back packaging layer exposes the counter electrode, and in the contact area, the front packaging layer exposes a plurality of the working electrode contacts, the reference electrode contacts and the counter electrode conductive contact layer, and the back packaging layer covers the counter electrode conductive circuit and the counter electrode via.

[0027] By adopting the above technical solution, by precisely controlling the range of the encapsulation layer, the sensing function area of the fine needle working area and the external connection area of the contact area can be accurately exposed, while other areas are effectively protected, ensuring that the sensor can perform electrochemical detection normally and reliably connect with external equipment.

[0028] In summary, this application includes at least one of the following beneficial technical effects: 1. Multiple working electrode layers and reference electrode layers are pressed together to form a single needle body structure. These layers are exposed in layers in the fine needle working area, forming an integrated laminated soft needle structure. A counter electrode layer and an encapsulation layer with vias on the back are also provided. This allows for the integration of multiple electrically isolated detection channels within a single needle body. This results in a compact structure, enabling multi-parameter detection and reducing implant trauma. 2. The hierarchical exposed structure of the fine needle working area facilitates the simultaneous detection of different substances by multiple working electrodes within the limited space of the stacked soft needle structure. The staggered arrangement of the contact area facilitates the independent and reliable connection of each electrode to the external circuit, reducing connection difficulty and short circuit risk. 3. By preparing the electrode layers in layers and then pressing them together, the deformation problem that may be caused by multiple high-temperature curing on the same substrate is avoided. The soft needle structure is stacked in the layered exposed fine needle working area. Even if deformation occurs, the multiple working electrode layers, the reference electrode layers and the counter electrode will not touch each other, thereby improving the process stability and product yield of the multi-layer flexible device manufacturing, and being able to accurately control the exposure length of each electrode layer in the fine needle working area, and finally forming a flexible probe needle with a layered exposure structure by cutting. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a front view of a needle-type flexible biosensor according to an embodiment of the present application; Figure 2 is a rear view of the needle-type flexible biosensor according to an embodiment of the present application; Figure 3 It is along Figure 1 Cross-sectional view along line AA; Figure 41 is a schematic flow chart of the main steps of the method for preparing a needle-type flexible biosensor according to an embodiment of the present application; Figure 5 It is executed Figure 4 A front view of the structure after main step S1 in FIG. 1 ((A) is a blood glucose working electrode layer formed on a first flexible substrate, (B) is a uric acid working electrode layer formed on a first flexible substrate, and (C) is a lactic acid working electrode layer formed on a first flexible substrate); Figure 6 It is executed Figure 4 A main view of the structure after the main step S2 in FIG. Figure 7 It is executed Figure 4 A diagram showing the arrangement relationship between the plurality of first flexible substrates and the second flexible substrates in the main step S3; Figure 8 It is executed Figure 4 Schematic diagram of the hot pressing process of main step S3 in FIG. 1 ((A) printing hot pressing adhesive on the first flexible substrate and the second flexible substrate, (B) arranging the multiple first flexible substrates and the second flexible substrate after printing the hot pressing adhesive, (C) hot pressing the multiple first flexible substrates and the second flexible substrate so that the hot pressing adhesive penetrates the fiber structure of the first flexible substrates and the second flexible substrate, and the multiple first flexible substrates and the second flexible substrate form an integrated structure); Figure 9 It is executed Figure 4 A front perspective view of the structure after the main step S3 in FIG. Figure 10 It is executed Figure 4 A rear view of the structure after the main step S4 in FIG. Figure 11 It is executed Figure 4 A front perspective view of the structure after the main step S5; Figure 12 It is executed Figure 4 Schematic diagram of the structure after the main step S6 in FIG. 1 ((A) is a front perspective view, (B) is a rear view).

[0030] Explanation of the accompanying drawings: 11. first flexible substrate; 12. second flexible substrate; 13. hot pressing layer; 20. working electrode layer; 21. blood glucose working electrode layer; 21a. blood glucose working electrode; 21b. blood glucose working electrode conductive circuit; 21c. blood glucose working electrode contact; 22. uric acid working electrode layer; 22a. uric acid working electrode; 22b. uric acid working electrode conductive circuit; 22c. uric acid working electrode contact; 23. lactic acid working electrode layer; 23a. lactic acid working electrode; 23b , lactic acid working electrode conductive circuit; 23c, lactic acid working electrode contact; 24, hollow window; 30, reference electrode layer; 40, electrode array needle body; 41, fine needle working area; 42, contact area; 50, counter electrode layer; 51, counter electrode; 52, counter electrode conductive circuit; 53, counter electrode via; 54, counter electrode conductive contact layer; 61, front packaging layer; 62, back packaging layer; 63, dividing line; 70, flexible probe needle; 71, first dividing gap; 72, second dividing gap. DETAILED DESCRIPTION

[0031] The following is combined with the accompanying drawings Figures 1 to 12 This application is described in further detail.

[0032] The embodiment of the present application discloses a needle-type flexible sensor.

[0033] Figure 1 is a front view of the needle-type flexible biosensor according to an embodiment of the present application, Figure 2 : is a rear view of the needle-type flexible biosensor according to an embodiment of the present application, Figure 3 It is along Figure 1 Sectional view along line AA. Figure 1-Figure 3 The needle-type flexible biosensor includes a plurality of working electrode layers 20, a reference electrode layer 30, a counter electrode layer 50 and an encapsulation layer.

[0034] The plurality of working electrode layers 20 are formed on corresponding plurality of first flexible substrates 11. The thickness of the first flexible substrate 11 may be 0.01-0.1 mm, and the material of the first flexible substrate 11 may be one of polyethylene terephthalate (PET), polyimide (PI), and polyurethane (PU). The Young's modulus of the first flexible substrate 11 is between 0.5 and 2 GPa. The nanoscale roughness (Ra) of the first flexible substrate 11 is between 50 and 100 nm, the contact angle of the hydrophilic coating of the first flexible substrate 11 is less than 30°, and the first flexible substrate 11 has a gradient pore structure with a pore size gradient of 5-20 μm.

[0035] The working electrode layer 20 may be one of a blood glucose working electrode layer 21 , a uric acid working electrode layer 22 , and a lactic acid working electrode layer 23 .

[0036] The blood glucose working electrode layer 21 can be formed on the first flexible substrate 11 by screen printing. The blood glucose working electrode layer 21 includes a blood glucose working electrode 21a, a blood glucose working electrode conductive circuit 21b, and a blood glucose working electrode contact 21c. The blood glucose working electrode 21a has a sensing area, which can be made of a composite material such as carbon, platinum carbon, or carbon nanotubes, for electrochemical detection of blood glucose. The blood glucose working electrode conductive circuit 21b is formed by printing with a carbon paste material for electrical signal transmission. The blood glucose working electrode contact 21c is formed by printing with a carbon material for connection to an external circuit.

[0037] The uric acid working electrode layer 22 can be formed on the first flexible substrate 11 through a screen printing process. The uric acid working electrode layer 22 includes a uric acid working electrode 22a, a uric acid working electrode conductive circuit 22b, and a uric acid working electrode contact 22c. The uric acid working electrode 22a has a sensing region, which can be made of a mixture of carbon-based conductive materials and functional fillers (such as carbon, platinum carbon, carbon nanotubes, etc.) for electrochemical reaction detection of uric acid. The uric acid working electrode conductive circuit 22b is formed by printing using a carbon paste material for electrical signal transmission. The uric acid working electrode contact 22c is formed by printing using a carbon material for connection to an external circuit.

[0038] The lactic acid working electrode layer 23 can be formed on the first flexible substrate 11 by a screen printing process. The lactic acid working electrode layer 23 includes a lactic acid working electrode 23a, a lactic acid working electrode conductive circuit 23b, and a lactic acid working electrode contact 23c. The lactic acid working electrode 23a has a sensing area, and the sensing area can be made of a composite material such as carbon, platinum carbon, or carbon nanotubes. The lactic acid working electrode conductive circuit 23b is formed by printing with a carbon paste material for electrical signal transmission. The lactic acid working electrode contact 23c is formed by printing with a carbon material for connection to an external circuit.

[0039] The reference electrode layer 30 is formed on the second flexible substrate 12. The first flexible substrates 11 and the second flexible substrates 12 are thermally pressed together to form an integrated electrode array needle body 40. The working electrode layers 20 and the reference electrode layer 30 have different lengths within the fine needle working area 41 of the electrode array needle body 40. The thermal pressing process is performed at a temperature of 120±15°C.

[0040] The thickness of the second flexible substrate 12 can be 0.01-0.1 mm, and the material of the second flexible substrate 12 can be one of polyethylene terephthalate (PET), polyimide (PI), and polyurethane (PU). The Young's modulus of the second flexible substrate 12 is between 0.5 and 2 GPa. The reference electrode layer 30 can be formed on the second flexible substrate 12 by a screen printing process. The reference electrode layer 30 includes a reference electrode 31, a reference electrode conductive circuit 32, and a reference electrode contact 33. The reference electrode 31 can be made of silver, silver chloride, or the like, which can enhance the stability and selectivity of the reference electrode 31. The reference electrode conductive circuit 32 is formed by printing using a carbon material for electrical signal transmission. The reference electrode contact 33 is formed by printing using a carbon material for connection to an external circuit.

[0041] An insulating structure (not shown) is provided between the multiple first flexible substrates 11 and the second flexible substrates 12. The insulating structure can be a polyimide buffer layer or a fluoroplastic isolation film. The polyimide buffer layer can be 0.05 mm thick, and the fluoroplastic isolation film can be 0.02 mm thick. The blood glucose working electrode 21a, the uric acid working electrode 22a, the lactic acid working electrode 23a, and the reference electrode 31 are layered within the fine needle working area 41 of the electrode array needle body 40. The blood glucose working electrode contacts 21c, the uric acid working electrode contacts 22c, the lactic acid working electrode contacts 23c, and the reference electrode contacts 33 are arranged in an alternating pattern within the contact area 42 of the electrode array needle body 40. First demarcation notches 71 are defined between the blood glucose working electrode contacts 21c and the uric acid working electrode contacts 22c, and between the uric acid working electrode contacts 22c and the lactic acid working electrode contacts 23c, ensuring electrical isolation between the working areas.

[0042] The counter electrode layer 50 is formed on the back of the electrode array needle body 40, and the electrode array needle body 40 is provided with a counter electrode via 53. The counter electrode layer 50 includes a counter electrode 51 and a counter electrode conductive circuit 52. The counter electrode 51 and the counter electrode conductive circuit 52 are formed on the back of the electrode array needle body 40 by screen printing. The material of the counter electrode 51 and the counter electrode conductive circuit 52 can be pure carbon. The counter electrode via 53 is also provided in the electrode array needle body 40 by laser engraving, so that it can be connected to the front of the electrode array needle body 40 and provide an electrical connection for the counter electrode 51.

[0043] The counter electrode conductive contact layer 54 is formed on the front face of the electrode array needle body 40, and the counter electrode conductive contact layer 54 is connected to the counter electrode layer 50 through the counter electrode via 53. The counter electrode conductive contact layer 54 is formed on the front face of the electrode array needle body 40 by a screen printing process, and the material of the counter electrode conductive contact layer 54 is carbon. The counter electrode conductive contact layer 54 covers the counter electrode via 53, and during the printing process, the electroconductive carbon ink covers the surface of the counter electrode via 53, so that the counter electrode conductive contact layer 54 is located on the front face of the electrode array needle body 40. The counter electrode conductive contact layer 54 can be electrically connected to the counter electrode conductive line 52 located on the back face of the electrode array needle body 40, and is electrically connected to the counter electrode 51. There is a second dividing gap 72 between the counter electrode conductive contact layer 54 and the reference electrode contact 33 to ensure electrical isolation between the working areas. In addition, the insertion of the fine needle working area 41 will produce sliding linkage, and the first boundary notch 71 and the second boundary notch 72 are positioned on both sides of the contact area 42 to prevent the sliding of each electrode contact.

[0044] The encapsulation layer is formed on the surface of the electrode array needle body 40, exposing the fine needle working area 41 and contact area 42 of the electrode array needle body 40. The electrode array needle body 40 is formed into a flexible probe needle 70, which exposes multiple working electrode layers 20 and reference electrode layers 30 in a layered manner within the fine needle working area 41 of the electrode array needle body 40. The encapsulation layer includes a front encapsulation layer 61 covering the front of the electrode array needle body 40 and a back encapsulation layer 62 covering the back of the electrode array needle body 40. The front encapsulation layer 61 exposes the blood glucose working electrode 21a, the uric acid working electrode 22a, the lactic acid working electrode 23a, and the reference electrode 31 in the fine needle working area 41. The contact area 42 exposes the blood glucose working electrode contact 21c, the uric acid working electrode contact 22c, the lactic acid working electrode contact 23c, the reference electrode contact 33, the counter electrode conductive contact layer 54, and the area where the first and second dividing notches 71 and 72 are to be formed. The back encapsulation layer 62 exposes the counter electrode 51 in the fine needle working area 41. A microfluidic channel is provided between the first flexible substrate 11 and the front encapsulation layer 61. The cross-section of the microfluidic channel is trapezoidal, wherein the bottom length of the trapezoid can be 100 μm and the length of the trapezoidal opening can be 50 μm.

[0045] After experimental verification and comparison of the needle-type flexible biosensor of the embodiment of the present application with the flexible biosensor in the related art, it was found that the electrode impedance of the flexible biosensor in the related art is 5.8±1.2kΩ, the crosstalk rate is ≥12%, the bending fatigue life is less than 200 times, and the retention time in the body is less than 30 days. The electrode impedance of the needle-type flexible biosensor in the embodiment of the present application is 2.1±0.3kΩ, the crosstalk rate is ≤5%, the bending fatigue life is greater than 500 times, and the retention time in the body can exceed 90 days. It can be seen that compared with the related art, the needle-type flexible biosensor in the embodiment of the present application has obvious optimization and improvement in terms of electrode impedance, crosstalk rate, bending fatigue life and in vivo retention time.

[0046] The implementation principle of a needle-type flexible biosensor in an embodiment of the present application is: multiple working electrode layers 20 and reference electrode layers 30 are pressed together to form a single needle body structure, and are exposed in layers in the fine needle working area 41. At the same time, a counter electrode layer 50 with a counter electrode via 53 connected to the back and a back packaging layer 62 covering the counter electrode conductive circuit 52 are provided. This realizes the integration of multiple electrically isolated detection channels in a single needle body. The structure is compact, multi-parameter detection can be performed, the stability is strong, and the implantation trauma is reduced.

[0047] The embodiments of the present application also disclose a method for preparing a needle-type flexible biosensor.

[0048] Figure 4 This is a schematic flow chart of the main steps of a method for preparing a needle-type flexible biosensor according to an embodiment of the present application. Figure 4 , the method comprises the following main steps: S1. Prepare corresponding working electrode layers 20 on multiple first flexible substrates 11.

[0049] S2 , preparing a reference electrode layer 30 on the second flexible substrate 12 .

[0050] S3. Thermally press-bond the first flexible substrates 11 and the second flexible substrates 12 to form an integrated electrode array needle body 40 . The working electrode layers 20 and the reference electrode layers 30 have different lengths in the fine needle working area 41 of the electrode array needle body 40 .

[0051] S4 , preparing a counter electrode layer 50 on the back side of the electrode array needle body 40 , and opening a counter electrode via hole 53 in the electrode array needle body 40 .

[0052] S5 , preparing a counter electrode conductive contact layer 54 on the front surface of the electrode array needle body 40 , wherein the counter electrode conductive contact layer 54 is connected to the counter electrode layer 50 through the counter electrode via 53 .

[0053] S6. Forming a packaging layer on the surface of the electrode array needle body 40 , wherein the packaging layer covers the surface of the electrode array needle body 40 and exposes the fine needle working area 41 and the contact area 42 of the electrode array needle body 40 .

[0054] S7 , cutting the electrode array needle body 40 into shape to form a flexible probe needle 70 , wherein the flexible probe needle 70 exposes a plurality of working electrode layers 20 and a reference electrode layer 30 in a hierarchical manner in the fine needle working area 41 of the electrode array needle body 40 .

[0055] The main steps of this method are described in detail below.

[0056] In the main step S1 , corresponding working electrode layers 20 are prepared on a plurality of first flexible substrates 11 . Figure 5 It is executed Figure 4 A front view of the structure after the main step S1 in FIG. 1 ((A) is a blood glucose working electrode layer 21 formed on the first flexible substrate 11, (B) is a uric acid working electrode layer 22 formed on the first flexible substrate 11, and (C) is a lactic acid working electrode layer 23 formed on the first flexible substrate 11). Figure 5 A blood glucose working electrode layer 21 can be prepared on one of the first flexible substrates 11 , a uric acid working electrode layer 22 can be prepared on another of the first flexible substrates 11 , and a lactic acid working electrode layer 23 can be prepared on another of the first flexible substrates 11 .

[0057] The thickness of the first flexible substrate 11 can be 0.01-0.1 mm, and the material of the first flexible substrate 11 can be one of polyethylene terephthalate (PET), polyimide (PI), and polyurethane (PU). The blood glucose working electrode layer 21 can be formed on the first flexible substrate 11 using a screen printing process. The blood glucose working electrode layer 21 includes a blood glucose working electrode 21a, a blood glucose working electrode conductive circuit 21b, and a blood glucose working electrode contact 21c. The blood glucose working electrode 21a has a sensing area, which can be made of a composite material such as carbon, platinum carbon, or carbon nanotubes, for electrochemically detecting blood glucose. The blood glucose working electrode conductive circuit 21b is formed by printing using a carbon paste material for electrical signal transmission. The blood glucose working electrode contact 21c is formed by printing using a carbon material for connection to an external circuit.

[0058] The uric acid working electrode layer 22 can be formed on the other first flexible substrate 11 through a screen printing process. The uric acid working electrode layer 22 includes a uric acid working electrode 22a, a uric acid working electrode conductive circuit 22b, and a uric acid working electrode contact 22c. The uric acid working electrode 22a has a sensing area, which can be made of a mixture of carbon-based conductive materials and functional fillers (such as carbon, platinum carbon, carbon nanotubes, etc.) for electrochemical reaction detection of uric acid. The uric acid working electrode conductive circuit 22b is formed by printing using a carbon paste material for electrical signal transmission. The uric acid working electrode contact 22c is formed by printing using a carbon material for connection to an external circuit. The uric acid working electrode layer 22 has a hollow window 24 opened by laser engraving in the fine needle working area 41 to expose the blood glucose working electrode 21a during subsequent hot pressing and bonding, ensuring that the functional areas between channels do not interfere with each other.

[0059] The lactic acid working electrode layer 23 can be formed on another first flexible substrate 11 by a screen printing process. The lactic acid working electrode layer 23 includes a lactic acid working electrode 23a, a lactic acid working electrode conductive circuit 23b and a lactic acid working electrode contact 23c. The lactic acid working electrode 23a has a sensing area, and the sensing area can be made of composite materials such as carbon, platinum carbon, and carbon nanotubes. The lactic acid working electrode conductive circuit 23b is formed by printing with a carbon paste material for electrical signal transmission. The lactic acid working electrode contact 23c is formed by printing with a carbon material for connection to an external circuit. The uric acid working electrode layer 22 is provided with a hollow window 24 in the fine needle working area 41 by laser engraving, which is used to expose the blood glucose working electrode 21a and the uric acid working electrode 22a during subsequent hot pressing and laminating, thereby realizing single-needle multi-channel coordinated output.

[0060] Figure 6 It is executed Figure 4 The main view of the structure after the main step S2 in FIG. Figure 6In the main step S2, a reference electrode layer 30 is prepared on the second flexible substrate 12. The thickness of the second flexible substrate 12 can be 0.01-0.1 mm, and the material of the second flexible substrate 12 can be one of polyethylene terephthalate (PET), polyimide (PI), and polyurethane (PU). The reference electrode layer 30 can be formed on the second flexible substrate 12 by a screen printing process. The reference electrode layer 30 includes a reference electrode 31, a reference electrode conductive circuit 32, and a reference electrode contact 33. The material of the reference electrode 31 can be silver, silver chloride, etc. These materials can enhance the stability and selectivity of the reference electrode 31. The reference electrode conductive circuit 32 is formed by printing using a carbon material for electrical signal transmission. The reference electrode contact 33 is formed by printing using a carbon material for connection to an external circuit. The reference electrode layer 30 is provided with a hollow window 24 in the fine needle working area 41 by laser engraving, which is used to expose the blood glucose working electrode 21a, the uric acid working electrode 22a and the lactic acid working electrode 23a during subsequent hot pressing and bonding, thereby ensuring that the functional areas of each detection channel do not interfere with each other.

[0061] Figure 7 It is executed Figure 4 FIG. 4 is a diagram showing the arrangement relationship between the plurality of first flexible substrates and the second flexible substrates in the main step S3 of FIG. Figure 8 It is executed Figure 4 Schematic diagram of the hot pressing process of main step S3 in FIG (A) printing hot pressing adhesive on the first flexible substrate and the second flexible substrate, (B) stacking multiple first flexible substrates and second flexible substrates after printing the hot pressing adhesive, (C) hot pressing the multiple first flexible substrates and the second flexible substrates so that the hot pressing adhesive penetrates the fiber structure of the first flexible substrates and the second flexible substrates, and the multiple first flexible substrates and the second flexible substrates form an integrated structure. Figure 9 It is executed Figure 4 A front perspective view of the structure after the main step S3 in FIG. Figure 7-Figure 9 In the main step S3, multiple first flexible substrates 11 and second flexible substrates 12 are heat-pressed to form an integral electrode array needle body 40, and multiple working electrode layers 20 and reference electrode layers 30 have different lengths in the fine needle working area 41 of the electrode array needle body 40.

[0062] Prior to step S3, an insulating structure is provided between the first flexible substrates 11 and the second flexible substrates 12. The insulating structure may be a polyimide film or a fluoroplastic film, and its thickness can be adjusted as needed. This insulating structure effectively prevents short circuits between the first flexible substrates 11 and the second flexible substrates 12, improving the reliability of the overall structure. Furthermore, the insulating structure acts as a buffer, reducing the impact of the external environment on the electrode layer, further improving detection performance.

[0063] Then, the blood glucose working electrode layer 21, the uric acid working electrode layer 22, the lactic acid working electrode layer 23 and the reference electrode layer 30 prepared separately are stacked together and fused into an integral electrode array needle body 40 through a hot pressing process. During the hot pressing process, hot pressing adhesive is first printed on the back of the first flexible substrate 11 on which the uric acid working electrode layer 22 is formed, the first flexible substrate 11 on which the lactic acid working electrode layer 23 is formed, and the second flexible substrate 12 on which the reference electrode layer 30 is formed to form a hot pressing layer 13; after printing the hot pressing adhesive, multiple first flexible substrates 11 and second flexible substrates 12 are stacked, so that the blood glucose working electrode layer 21, the uric acid working electrode layer 22, the lactic acid working electrode layer 23 and the reference electrode layer 30 are stacked in a specific order to ensure that the fine needle working area 41 is accurately docked; then, the different layers of materials are combined by hot pressing technology, so that the hot pressing adhesive in the hot pressing layer 13 penetrates into the fiber structure of the first flexible substrate 11 and the second flexible substrate 12 under a certain temperature and pressure, forming a strong bond to form an integral electrode array needle body 40. The blood glucose working electrode 21a, the uric acid working electrode 22a, the lactic acid working electrode 23a and the reference electrode 31 are exposed in layers in the fine needle working area 41 of the electrode array needle body 40, and the blood glucose working electrode contact 21c, the uric acid working electrode contact 22c, the lactic acid working electrode contact 23c and the reference electrode contact 33 are arranged alternately in the contact area of the electrode array needle body 40.

[0064] The application temperature of the hot pressing process can be in the range of 100°C-160°C, which can ensure that the materials are fully bonded and avoid material degradation caused by overheating. The application time of the hot pressing process can be 30 seconds to 180 seconds, and the specific time is adjusted according to the thickness of the material and the required bonding strength. The application pressure of the hot pressing process can be 0.5MPa to 3MPa to ensure close bonding between the layers, reduce bubbles and gaps between the layers, and ensure the electrical performance and stability of the overall electrode. During the hot pressing process, it is necessary to ensure that the insulation structure between the layers is fully bonded to avoid bubbles, cracks or detachment between the layers to ensure electrical stability during long-term use.

[0065] Figure 10It is executed Figure 4 Rear view of the structure after the main step S4 in FIG. Figure 10 In the main step S4, a counter electrode layer 50 is prepared on the back side of the electrode array needle body 40, and a counter electrode via 53 is opened in the electrode array needle body 40. The counter electrode layer 50 includes a counter electrode 51 and a counter electrode conductive circuit 52. The counter electrode 51 and the counter electrode conductive circuit 52 are formed on the back side of the electrode array needle body 40 by screen printing. The material of the counter electrode 51 and the counter electrode conductive circuit 52 can be pure carbon. The counter electrode via 53 is opened in the electrode array needle body 40 by laser engraving so as to be subsequently connected to the front side of the electrode array needle body 40 and provide an electrical connection for the counter electrode 51.

[0066] Figure 11 It is executed Figure 4 A front perspective view of the structure after the main step S5 in FIG. Figure 11 In the main step S5, a counter electrode conductive contact layer 54 is prepared on the front surface of the electrode array needle body 40, and the counter electrode conductive contact layer 54 is connected to the counter electrode layer 50 through the counter electrode via 53. The counter electrode conductive contact layer 54 is formed on the front surface of the electrode array needle body 40 by a screen printing process, and the material of the counter electrode conductive contact layer 54 is carbon. The counter electrode conductive contact layer 54 covers the counter electrode via 53. During the printing process, the electroconductive carbon ink covers the surface of the counter electrode via 53, so that the counter electrode conductive contact layer 54 located on the front surface of the electrode array needle body 40 can be connected to the counter electrode conductive line 52 located on the back surface of the electrode array needle body 40 and electrically connected to the counter electrode 51.

[0067] Figure 12 It is executed Figure 4 Schematic diagram of the structure after the main step S6 in ((A) is a front perspective view, (B) is a rear view). Figure 12In main step S6, an encapsulation layer is formed on the surface of the electrode array needle body 40. The encapsulation layer covers the surface of the electrode array needle body 40 and exposes the fine needle working area 41 and contact area 42 of the electrode array needle body 40. The encapsulation layer includes a front encapsulation layer 61 covering the front surface of the electrode array needle body 40 and a back encapsulation layer 62 covering the back surface of the electrode array needle body 40. The front packaging layer 61 exposes the blood glucose working electrode 21a, the uric acid working electrode 22a, the lactic acid working electrode 23a, the reference electrode 31 and the area where the first dividing gap 71 and the second dividing gap 72 are predetermined to be formed in the fine needle working area 41, and exposes the blood glucose working electrode contact 21c, the uric acid working electrode contact 22c, the lactic acid working electrode contact 23c, the reference electrode contact 33 and the counter electrode conductive contact layer 54 in the contact area 42; the back packaging layer 62 exposes the counter electrode 51 in the fine needle working area 41.

[0068] The encapsulation layer can be made of a UV-insulating material, screen-printed onto the surface of the electrode array needle body 40. This material offers excellent insulation and stability, ensuring electrical isolation between the electrodes and enhancing the sensor's long-term stability, preventing environmental interference or damage. The encapsulation layer's dividing line 63 demarcates the needle's working area 41, which is inserted into the body, from the external connection area, ensuring a stable data transmission path.

[0069] Figure 1 is a front view of the needle-type flexible biosensor according to an embodiment of the present application, Figure 2 : is a rear view of the needle-type flexible biosensor according to an embodiment of the present application, Figure 3 It is along Figure 1 Sectional view along line AA. Figure 1-Figure 3 In step S7, the electrode array needle body 40 is cut into a shape to form a flexible probe needle 70, completing the preparation of the needle-type flexible biosensor. The flexible probe needle 70 exposes multiple working electrode layers 20 and reference electrode layers 30 in a layered manner within the fine needle working area 41 of the electrode array needle body 40.

[0070] Among them, the electrode array needle body 40 can be finely cut by a laser engraving process to ensure that the shape and functional area of the needle-type flexible biosensor are clearly divided. During the engraving process, the packaged electrode array needle body 40 is precisely cut using laser engraving technology to engrave the required shape and size to ensure the integrity of the contact area 42. Laser engraving not only cuts the sensor shape, but also forms a clear first dividing notch 71 between the blood glucose working electrode contact 21c and the uric acid working electrode contact 22c, and between the uric acid working electrode contact 22c and the lactic acid working electrode contact 23c, and forms a clear second dividing notch 72 between the reference electrode contact 33 and the counter electrode conductive contact layer 54, and divides the external electrode working area and the implanted human body area to ensure that the functions of each area are clear. After engraving is completed, it is ensured that the implanted human body area is connected to the external electrode working area, and at the same time, the electrical isolation of each functional area is complete, ensuring smooth signal transmission and avoiding cross interference.

[0071] The implementation principle of the preparation method of a needle-type flexible biosensor in the embodiment of the present application is: by preparing the electrode layer in layers and then pressing them together, the deformation problem that may be caused by multiple high-temperature curing on the same substrate is avoided, the process stability and product yield of the multi-layer flexible device manufacturing are improved, and the exposure length of each electrode layer in the fine needle working area 41 can be accurately controlled, and finally a flexible probe needle 70 with a layered exposure structure is formed by cutting.

[0072] The above are all preferred embodiments of the present application, and are not intended to limit the scope of protection of the present application. Therefore, any equivalent changes made based on the structure, shape, and principle of the present application should be included in the scope of protection of the present application.

Claims

1. A needle-type flexible biosensor, characterized in that: include: A plurality of working electrode layers (20) are formed on corresponding plurality of first flexible substrates (11); A reference electrode layer (30) is formed on a second flexible substrate (12); wherein a plurality of the first flexible substrates (11) and the second flexible substrates (12) are pressed together to form an integral electrode array needle body (40); and a plurality of the working electrode layers (20) and the reference electrode layer (30) have different lengths in a fine needle working area (41) of the electrode array needle body (40); a counter electrode layer (50) formed on the back side of the electrode array needle body (40); The encapsulation layer is formed on the surface of the electrode array needle body (40) and exposes the fine needle working area (41) and the contact area (42) of the electrode array needle body (40); the electrode array needle body (40) is formed to form a flexible probe needle (70), and the flexible probe needle (70) exposes a plurality of working electrode layers (20) and the reference electrode layer (30) in layers in the fine needle working area (41) of the electrode array needle body (40).

2. The needle-type flexible biosensor according to claim 1, characterized in that: The Young's modulus of the first flexible substrate (11) and the second flexible substrate (12) is between 0.5 and 2 GPa, and the pressing temperature of the hot pressing process is 120±15°C; an insulating structure is provided between the plurality of the first flexible substrates (11) and the second flexible substrates (12), comprising a polyimide buffer layer or a fluoroplastic isolation film; the nanoscale roughness (Ra) of the first flexible substrate (11) is 50 to 100 nm, the contact angle of the hydrophilic coating is less than 30°, and the substrate has a gradient pore structure.

3. The needle-type flexible biosensor according to claim 1, characterized in that: The working electrode layer (20) includes working electrodes (21a, 22a, 23a), working electrode conductive circuits (21b, 22b, 23b) and working electrode contacts (21c, 22c, 23c), wherein the working electrodes (21a, 22a, 23a) are connected to the working electrode contacts (21c, 22c, 23c) via the working electrode conductive circuits (21b, 22b, 23b); the reference electrode layer (30) includes a reference electrode (31), a reference conductive circuit (32) and a reference electrode contact (33), wherein the reference electrode (31) is connected to the reference electrode contact (33) via the reference conductive circuit (32).

4. The needle-type flexible biosensor according to claim 3, characterized in that: The plurality of working electrodes (21a, 22a, 23a) and the reference electrode (31) are exposed in layers in the fine needle working area (41) of the electrode array needle body (40), and the plurality of working electrode contacts (21c, 22c, 23c) and the reference electrode contacts (33) are arranged alternately in the contact area (42) of the electrode array needle body (40), and a first dividing gap (71) is provided between the working electrode contacts (21c, 22c, 23c).

5. The needle-type flexible biosensor according to claim 3, characterized in that: The electrode array needle body (40) is provided with an electrode via hole (53), and a counter electrode conductive contact layer (54) is formed on the front surface of the electrode array needle body (40). The counter electrode conductive contact layer (54) is connected to the counter electrode layer (50) through the counter electrode via hole (53), and a second boundary gap (72) is provided between the counter electrode conductive contact layer (54) and the reference electrode contact (33); the counter electrode layer (50) includes a counter electrode (51) and a counter electrode conductive circuit (52), and the counter electrode conductive contact layer (54) is connected to the counter electrode conductive circuit (52) through the counter electrode via hole (53).

6. The needle-type flexible biosensor according to claim 5, characterized in that: The packaging layer includes a front packaging layer (61) covering the front of the electrode array needle body (40) and a back packaging layer (62) covering the back of the electrode array needle body (40); in the fine needle working area (41), the front packaging layer (61) reveals a plurality of the working electrodes (21a, 22a, 23a) and the reference electrode (31), and the back packaging layer (62) reveals the counter electrode (51); in the contact area (42), the front packaging layer (61) reveals a plurality of the working electrode contacts (21c, 22c, 23c), the reference electrode contact (33) and the counter electrode conductive contact layer (54), and the back packaging layer (62) covers the counter electrode conductive circuit (52) and the counter electrode via (53); the needle-type flexible biosensor has a microfluidic channel, which is arranged between the first flexible substrate (11) and the front packaging layer (61) and has a trapezoidal cross-section.

7. A method for preparing a needle-type flexible biosensor, characterized in that: The following steps are involved: S1, preparing corresponding working electrode layers (20) on a plurality of first flexible substrates (11); S2, preparing a reference electrode layer (30) on a second flexible substrate (12); S3, thermally pressing a plurality of the first flexible substrates (11) and the second flexible substrates (12) to form an integral electrode array needle body (40), wherein the plurality of the working electrode layers (20) and the reference electrode layers (30) have different lengths in the fine needle working area (41) of the electrode array needle body (40); S4, preparing a counter electrode layer (50) on the back side of the electrode array needle body (40); S6, forming an encapsulation layer on the surface of the electrode array needle body (40), wherein the encapsulation layer covers the surface of the electrode array needle body (40) and exposes the fine needle working area (41) and the contact area (42) of the electrode array needle body (40); S7. Cutting the electrode array needle body (40) into shape to form a flexible probe needle (70), wherein the flexible probe needle (70) exposes a plurality of working electrode layers (20) and reference electrode layers (30) in a hierarchical manner in the fine needle working area (41) of the electrode array needle body (40).

8. The method for preparing a needle-type flexible biosensor according to claim 7, wherein: Before step S3, an insulating structure is provided between the plurality of first flexible substrates (11) and the second flexible substrates (12).

9. The method for preparing a needle-type flexible biosensor according to claim 7, wherein: In step S1, the working electrode layer (20) includes working electrodes (21a, 22a, 23a), working electrode conductive lines (21b, 22b, 23b) and working electrode contacts (21c, 22c, 23c), and the working electrodes (21a, 22a, 23a) are connected to the working electrode contacts (21c, 22c, 23c) through the working electrode conductive lines (21b, 22b, 23b); in step S2, the reference electrode layer (30) includes a reference electrode (31), a reference conductive line (32) and a reference electrode contact (33), and the reference electrode (31) is connected to the reference electrode contact (33) through the reference conductive line (32); in step S3, a plurality of the working electrodes (21a, 22a, 23a) and the reference electrode (31) are exposed in layers in the fine needle working area (41) of the electrode array needle body (40), and a plurality of the working electrode contacts (21c, 22c, 23c) and the reference electrode contacts (33) are arranged alternately in the contact area (42) of the electrode array needle body (40). 23c) have a first dividing gap (71) between them; in step S4, the counter electrode layer (50) includes a counter electrode (51) and a counter electrode conductive circuit (52), and a counter electrode via (53) is also provided in the electrode array needle body (40); between step S4 and step S6, step S5 is also included, step S5 includes preparing a counter electrode conductive contact layer (54) on the front surface of the electrode array needle body (40), the counter electrode conductive contact layer (54) is connected to the counter electrode layer (50) through the counter electrode via (53), a second dividing gap (72) is provided between the counter electrode conductive contact layer (54) and the reference electrode contact (33), and in step S5, the counter electrode conductive contact layer (54) is connected to the counter electrode conductive circuit (52) through the counter electrode via (53).

10. The method for preparing a needle-type flexible biosensor according to claim 9, wherein: In step S6, the packaging layer includes a front packaging layer (61) covering the front of the electrode array needle body (40) and a back packaging layer (62) covering the back of the electrode array needle body (40); in the fine needle working area (41), the front packaging layer (61) reveals the plurality of working electrodes (21a, 22a, 23a) and the reference electrode (31), and the back packaging layer (62) reveals the counter electrode (51); in the contact area (42), the front packaging layer (61) reveals the plurality of working electrode contacts (21c, 22c, 23c), the reference electrode contact (33) and the counter electrode conductive contact layer (54), and the back packaging layer (62) covers the counter electrode conductive circuit (52) and the counter electrode via (53).

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