RF biosensing system using RF sensor including microneedle

Abstract

Disclosed is an RF biosensing system using an RF sensor including a glucose-responsive hydrogel-based microneedle. The RF biosensing system measures a sensing signal with a VNA by detecting a sensing signal by using a reader antenna and a reader antenna sensing circuit through a readout coil on the basis of RF biosensing and wireless transmission, via EM coupling between a glucose-responsive hydrogel-based microneedle, which is provided below a flexible substrate so as to be minimally invasively applied in vivo to the skin surface (on-site) of epidermis / dermis / hypodermis of the skin or to skin / fat / muscle tissue, and an RF sensor, which is positioned over the flexible substrate and uses the difference in resonant frequencies within frequencies of 400 to 3,000 MHz, and performs biosensing such as detection of proteins, glucose, and fat in the skin, on the basis of changes in the capacitance of a sensing antenna circuit (LC resonator) of the RF sensor, shifts in resonant frequencies of the LC resonator, and changes in S-parameters.

Description

RF biosensing system using RF sensors equipped with microneedles

[0001] The present invention provides a biosensing system equipped with a glucose-responsive hydrogel-based microneedle, which detects a sensing signal using a reader antenna and a reader antenna sensing circuit through a readout coil via RF biosensing and wireless transmission using EM coupling of an RF sensor that utilizes a difference in resonant frequency within a frequency range of 400 to 3,000 MHz in a minimally invasive manner on the skin surface (on-site) of the epidermis / dermis / hypodermis or skin / fat / muscle in a living body, and measures the sensing signal using a VNA (Vector Network Analyzer), and provides biosensing such as detection of proteins, glucose, and fat in the skin according to a change in the capacitance of the sensing antenna circuit (LC resonator) of the RF biosensor / a shift in the resonant frequency of the LC resonator / a change in the S-parameter.

[0002] Carbohydrates consumed by humans are metabolized and transported into the bloodstream in the form of glucose, which is then supplied to cells through insulin secretion from the pancreas, acting as a source of energy. Diabetes mellitus can be caused by genetic factors, obesity, irregular eating habits (excessive sugar intake, fat intake, alcohol consumption), lack of exercise, stress, and existing diseases (hypothyroidism, pneumonia, pancreatitis, etc.). Diabetes is a disease characterized by hyperglycemia, in which blood glucose levels exceed the normal range and increase when the pancreas secretes insufficient insulin or its normal function is impaired. Diabetes is a complex disease that affects every tissue of the body due to complications such as hypertension (stroke), diabetic retinopathy, myocardial infarction (angina pectoris), chronic renal failure, and neuropathy. According to the World Health Organization (WHO), there were approximately 347 million people with diabetes in 2013, and the number of people with diabetes is increasing.

[0003] Diabetes is diagnosed by measuring blood sugar and glycated hemoglobin levels. Rather than a whole blood glucose test using a fingertip, blood is drawn from a vein, clots are allowed to settle, and only the clear plasma (serum) is separated to measure glucose concentration.

[0004] * Normal blood sugar level

[0005] 1) Plasma glucose less than 100 mg / dL after fasting for at least 8 hours

[0006] 2) Plasma glucose less than 140 mg / dL 2 hours after a 75 g oral glucose tolerance test

[0007] * Diabetes diagnosis criteria

[0008] 1) Symptoms of diabetes (polyuria, thirst and weight loss) Blood sugar level measured regardless of meal time is 200 mg / dL or higher

[0009] 2) Glycated hemoglobin (HbA1c) ≥ 6.5% or higher

[0010] 3) Fasting blood sugar level measured after 8 hours on an empty stomach is 126 mg / dL or higher

[0011] 4) Blood sugar level of 200 mg / dL or higher after 2 hours in a 75 mg oral glucose tolerance test

[0012] Blood sugar level standard (mg / dL) Fasting blood sugar Postprandial blood sugar Normal 70-100 mg / dL Less than 140 mg / dL Borderline diabetes 100-125 mg / dL 140-199 mg / dL Diabetes 126 mg / dL or higher 200 mg / dL or higher

[0013] Diabetes requires measuring blood sugar levels using biosensors and managing them through prescriptions such as diet, exercise, insulin injections, and oral medication. Various biosensors exist, utilizing conventional electrochemical, optical, and electromagnetic spectroscopy techniques. Additionally, numerous glucose biosensors relying on transduction techniques have been reported.

[0014] Blood sampling has the advantage of high accuracy because it directly measures the glucose concentration in the blood, but real-time blood sugar check is impossible because blood is collected intermittently, and there is a risk of secondary infection due to needle use during blood sampling, so research on non-invasive and continuous blood sugar measurement methods is needed.

[0015] Accurate measurement of blood sugar levels is crucial for diabetes treatment. Invasive diabetes detection methods exist, using electrochemical, metamaterial, and enzymatic oxidation methods.

[0016] Glucose biosensors are used to measure blood sugar levels. By measuring blood sugar levels, diabetic patients can manage their glucose levels through effective interventions such as diet, exercise, insulin injections, and oral medications, ultimately preventing various diabetic complications such as blindness, chronic renal failure, heart failure, and nerve damage.

[0017] As a related prior art, patent registration number 10-1887602 is registered for “a biosensor having an RF bandpass structure, a method for sensing biological data using the same, and a method for manufacturing the biosensor.”

[0018] Figure 1 shows a biosensor having an RF bandpass structure for measuring glucose concentration used in conventional diabetes diagnosis.

[0019] A biosensor with an RF bandpass structure

[0020] GaAs substrate used for high frequency in the 9 GHz band; and

[0021] A biosensor having an RF bandpass structure including a metal line formed on the substrate;

[0022] The metal line includes a first line and a second line spaced apart by a certain distance; at least two diagonal lines connecting the first line and the second line using an air-bridge structure; and a third line forming a closed loop including the first line and the second line and the at least two diagonal lines.

[0023] A passivation film formed between the two or more diagonal lines and the first metal layer of the first line and the second line,

[0024] The surface of the above metal line is coated with gold (Au), and a certain level of surface roughness is implemented using etching.

[0025] An RF biosensor having an RF bandpass structure is provided, which detects a change in resonant frequency due to capacitance of the biosensor that changes according to the dielectric constant of the measured substance (serum, D-glucose) located in the air-bridge region, and measures the concentration of the measured substance based on the change in resonant frequency.

[0026] Over the past several decades, the number of people with diabetes has increased dramatically worldwide, increasing the need for blood glucose monitoring (Saeedi et al., 2019; World Health Organization, 2023). Fortunately, glucose detection using biosensing technologies has provided advancements and innovations in the field of biosensing (Corrie et al., 2015; Fiedorova et al., 2022). Real-time invasive glucose level testing is now widely available to diabetic patients (Blicharz et al., 2018; Wang, 2008). Blood glucose detection aims to provide minimally invasive or even non-invasive testing to reduce pain in patients with diabetes, and a painless, reliable, and user-friendly glucose sensing system is urgently needed (Kim et al., 2018; Zhang et al., 2021; Zou et al., 2023).

[0027] Unlike attempts to non-invasively measure and extract blood glucose levels, minimally invasive microneedle-based detection systems are currently more reliable (Omar et al., 2023). Microneedles can extract and measure glucose levels in the interstitial fluid (ISF). The detection is painless, and the extraction marks disappear within minutes (Ma and Wu, 2017). Here, the glucose concentration in the interstitial fluid (ISF) of the subcutaneous tissue has been shown to closely match the dynamically changing plasma levels, and is expected to be a reliable alternative to the blood puncture of conventional glucose meters (Johnston et al., 2021). Various microneedle-based biosensing systems, primarily consisting of optical and electrochemical platforms, have been developed (Mohan et al., 2017; Wang et al., 2023; Wu et al., 2022). Ju et al. proposed a surface-enhanced Raman spectroscopy (SERS) glucose sensor based on a poly(methyl methacrylate) microneedle (PMMA MN) array.The tip of the microneedle is modified with Ag particles and the glucose capture agent 1-Decanethiol (1-DT), which displays different Raman spectra for different glucose concentrations under laser excitation, enabling fast and painless blood glucose testing (Ju et al., 2020). Liu et al. proposed an integrated microneedle electrochemical device through microfabrication, electroplating, and enzyme immobilization. This device generated an enzymatic reaction at the working electrode. Current signal ( The current signal produced by the enzymatic reaction on the working electrode is detected, which is highly correlated with the signal obtained from a commercial blood glucose meter (Liu et al., 2021). Recently, microneedles as minimally invasive platforms offer the possibility of replacing existing invasive detection methods (Tehrani et al., 2022; Xie et al., 2022). In particular, RF technology has emerged as a promising approach due to its passive detection, miniaturization, and remote sensing capabilities (Dautta et al., 2020; Kim et al., 2015; Qiang et al., 2017). RF biosensing technology offers the potential for non-stimulation, ultra-compact biosensing platforms that require no peripheral devices (Mannoor et al., 2012).

[0028] Hydrogel-based microneedles can interact strongly with microwave signals while undergoing significant morphological changes before and after incorporation with target molecules, leveraging their distinct swelling properties. This provides high sensitivity to microwave sensing (Manzanos et al., 2023; Sridhar and Takahata, 2009; Sun et al., 2023; Turner et al., 2021).

[0029] However, the cost of glucose testing for early diagnosis of diabetes is increasing, and a non-invasive, in vivo, intradermal glucose-responsive RF biosensing system has not been provided to replace existing invasive glucose biosensors.

[0030] [Prior art literature]

[0031] (Patent Document 1) Patent Registration No. 10-1887602 (Registration Date: August 6, 2018), “Biosensor having RF bandpass structure, biological data sensing method using the same, and method for manufacturing the biosensor”, Kwangwoon University Industry-Academic Cooperation Foundation

[0032] The purpose of the present invention to solve the above problems is to use a glucose-responsive hydrogel-based microneedle equipped on the lower part of a flexible substrate of an RF biosensor using a frequency of 400 to 3,000 MHz to detect a sensing signal using a microneedle with minimally invasive glucose-responsive hydrogels attached thereto on-site of the epidermis / dermis / hypodermis or skin / fat / muscle of the skin in vivo and a sensing antenna circuit and a reader antenna and a reader antenna sensing circuit through RF biosensing and wireless transmission through EM coupling from the sensing antenna, and a VNA (Vector Network Analyzer) measures the sensing signal, and the change in capacitance / LC of the sensing antenna circuit (LC resonator) of the RF biosensor An RF biosensing system using an RF biosensor having microneedles is provided, which provides biosensing such as detection of proteins, glucose, and fat in the skin according to the resonance frequency of the resonator and the change in S-parameter.

[0033] In order to achieve the object of the present invention, an RF biosensing system using an RF wireless biosensor having microneedles comprises: a substrate; microneedles provided on a lower portion of the substrate and configured to minimally invasively contact the skin in a living body; a sensing antenna circuit and a sensing antenna connected to the microneedles provided on the lower portion of the substrate and comprising an LC resonator of an RF biosensor provided on an upper portion of the substrate; and a reader antenna and a reader antenna sensing circuit connected through a circular readout coil that is EM coupled within an effective range and receives a sensing signal using RF biosensing and wireless transmission technology, and the RF biosensor is connected to a VNA; and a vector network analyzer (VNA) that measures a capacitance change of the sensing signal / a center frequency shift of the LC resonator / a change in the S parameter.

[0034] The RF biosensing system using the RF biosensor having the microneedle of the present invention uses a glucose-responsive hydrogel-based microneedle provided on the lower part of the flexible substrate of the RF biosensor to detect a sensor signal using a microneedle with minimally invasive glucose-responsive hydrogels attached thereto on the surface of the skin (on-site) of the epidermis / dermis / hypodermis or skin / fat / muscle in vivo through RF biosensing and wireless transmission from the sensing antenna circuit and the sensing antenna, and measures the sensing signal using a VNA (Vector Network Analyzer), and the sensing antenna circuit (LC resonator) of the RF biosensor. It has the effect of providing biosensing such as detection of proteins, glucose, and fat in the skin according to changes in capacitance / changes in the resonant frequency of the LC resonator / changes in the S-parameter.

[0035] An RF biosensing system having an RF biosensor with microneedles can measure protein in the skin, glucose concentration in the blood, and fat in vivo. An RF biosensing system using an RF biosensor with microneedles is connected to a VNA, and the VNA measures glucose concentration according to changes in the resonance frequency of the LC resonator of the RF biosensor (hypoglycemia frequency f1, normal frequency f2, hyperglycemia frequency f3) / changes in S-parameters (S1, S2, S3), thereby indicating hypoglycemia / normal / hyperglycemia and diagnosing diabetes.

[0036] Figure 1 shows a biosensor having an RF bandpass structure for measuring glucose concentration used in conventional diabetes diagnosis.

[0037] Figure 2 illustrates an RF biosensor using a glucose-responsive hydrogel-based microneedle according to the present invention.

[0038] FIG. 3 is a diagram showing the operation of the glucose-responsive hydrogel-based microneedle and sensing antenna circuit of FIG. 2, the sensing antenna and the leader antenna remotely wirelessly EM coupled, and the leader antenna sensing circuit.

[0039] Figure 4 is a drawing showing the microneedle fabrication process.

[0040] Figure 5 illustrates a biosensing system using an RF biosensor using a glucose-responsive hydrogel-based microneedle according to the present invention.

[0041] Figure 6. Preparation of CMC-pHEA GelMA-ConA hydrogel and fabrication of microneedle embedded system: (a) synthesis of CMC-pHEA pH-responsive particles and their chemical structural changes, (b) fabrication of microneedles from CMC-pHEA GelMA-ConA glucose-responsive hydrogel, (c) preparation of embedded system.

[0042] Figure 7. Material characterization: (A) FTIR spectra; (B) Characterization of CMC-pHEA and CMC-pHEA GelMA-ConA: (i) CMC-pHEA after freeze-drying and (ii) 1600X magnification SEM images; (iii) Temperature strain of CMC-pHEA GelMA-ConA and (iv) 1600X magnification SEM images after freeze-drying; (C) Swelling ability of CMC-pHEA GelMA-ConA at various glucose concentrations; (D) Comparison of morphology and SEM images of hydrogels after swelling. (i) Comparison of morphology; (ii) SEM images of hydrogels after immersion in 0 mM glucose solution; (iii) 3 mM glucose; (iv) 20 mM glucose.

[0043] Figure 8. Characterization of the microneedles: (A) Optical results of the microneedle patch: (i) overall shape; (ii) row of microneedles; (iii) single microneedle. (B) Extraction ability of the microneedle patch: (i) weight change; (ii) color change; (iii) mechanical properties of the displacement-force curve; (C) Response of dyed microneedles to insertion - worsened at different glucose concentrations. (D) Fluorescence intensity changes: (i) 0 mM; (ii) 3 mM; (iii) 20 mM.

[0044] Figure 9 shows the wireless sensing system simulation results: (A) size coupling effect; (B) different simulation results; (C) summarized results; (D) center shift and vertical distance effects on results; (E) center relative position effect on results; (F) vertical distance effect on results; (H) swelling condition effect on results; (I) effect of degree of swelling on results; (J) frequency shift toward swelling level.

[0045] Figure 10 shows wireless system detection results: (A) detection system configuration; (B) comparison of detection sizes of microneedle patches; (C) detection results comparing unloaded and loaded microneedle patches; (D) detection results for agarose with various glucose concentrations; (E) change in resonant frequency and (F) change in size.

[0046] Figure 11 shows the actual CMC-pHEA experimental process (Figure S1).

[0047] Figures 12 and 13 show the sensing antenna manufacturing process and HFSS simulation (Figure S2).

[0048] Fig. 14 is a photograph of the readout coil (circular reading coil).

[0049] Figure 15 shows a screen for measuring glucose concentration (hypoglycemia / normal / hyperglycemia) of a capacitance change of an RF biosensor (LC circuit of an LC resonator) equipped with a glucose-responsive microneedle -> change in the center frequency of the LC resonator.

[0050] Hereinafter, the configuration and operation of a preferred embodiment of the present invention will be described in detail with reference to the attached drawings.

[0051] The present invention is not limited to the disclosed embodiments, and can be implemented in various forms, as will be apparent to those skilled in the art. In the description of the present invention, detailed descriptions of known technologies or configurations may be omitted if they are deemed to unnecessarily obscure the gist of the present invention. Furthermore, the attached drawing numbers are assigned the same number in different drawings when indicating identical components.

[0052] This study is not limited to a specific embodiment, and should be understood to include all transformations, equivalents, or substitutes included in the spirit and technical scope of the present invention.

[0053] We propose a CMC-pHEA GelMA-ConA hydrogel microneedle-based RF detection platform (Wu et al., 2019). By leveraging the dramatic conformational changes of hydrogel microneedles in a selective response to glucose, we investigated the interaction of microneedles with RF signals and developed both in-situ and wireless readout platforms equipped with RF biosensors, resulting in a minimally invasive, passive, and wireless glucose detection system.

[0054] The RF biosensing system using an RF biosensor having microneedles uses a glucose-responsive hydrogel-based microneedle equipped on the lower part of a flexible substrate of an RF biosensor to minimally invasively attach glucose-responsive hydrogels to the epidermis / dermis / hypodermis of the skin or on-site of the skin / fat / muscle in vivo using a frequency of 400 to 3,000 MHz, and a sensing antenna circuit and a reader antenna and a reader antenna sensing circuit detect a sensing signal through RF biosensing and wireless transmission from the sensing antenna, and the sensing signal is measured by a VNA (Vector Network Analyzer), and the sensing antenna circuit (LC resonator) of the RF biosensor Biosensing such as detection of proteins, glucose, and fat in the skin is performed based on changes in capacitance, resonant frequency shift of the LC resonator, and changes in S-parameters.

[0055] Microneedles are used to obtain samples from a living body, and microneedles having a glucose-responsive hydrogel in a 15x15 array matrix structure on the bottom of a polyimide (PI) flexible substrate are used, and microneedles having a diameter of millimeters (mm) are used to measure the concentration of glucose by reacting with glucose contained in body fluids within the human skin.

[0056] The substrate uses a polyimide (PI) flexible substrate.

[0057] The lower part of the flexible substrate is equipped with microneedles in an nxn array matrix structure, and the RF biosensor equipped on the upper part of the flexible substrate uses a frequency of 400 to 3,000 MHz.

[0058] Microneedles are attached with glucose-responsive hydrogels.

[0059] The glucose-responsive hydrogel uses CMC-pHEA GelMA-ConA hydrogel.

[0060] The skin penetration depth of microneedles is 10 to 100 μm.

[0061] A biosensing system equipped with an RF biosensor with microneedles can measure protein, glucose, and fat in the skin in vivo.

[0062] 1. The antibody uses glucose oxidase as a specific identifier. Since glucose reacts with glucose oxidase to produce acid, the CMC-pHEA synthesized in this study is highly pH-sensitive and reacts only to glucose in the swelling state of the hydrogel.

[0063] 2. To create a swelling hydrogel microneedle, a type of composite material such as CMC, KPS, 2-HEA, PEGDA, gelMA, glucose oxidise, and a photoinitiator (photoreactive compound) is used.

[0064] 3. The antenna substrate of the RF biosensor uses either an FR4 substrate for wideband antennas or a polyimide (PI) flexible substrate. In addition to the FR4 wideband antenna substrate, a ceramic substrate using ceramic materials can also be used.

[0065] In the embodiment, the RF biosensor used a polyimide (PI) flexible substrate, and glucose-responsive hydrogel-based microneedles with a 15x15 matrix structure were used on the lower portion of the flexible substrate of the RF biosensor.

[0066] 4. Because it uses glucose oxidase, only glucose oxidase can react with glucose to produce acid and cause a pH change, and it has selectivity to react only to glucose.

[0067] Change in capacitance of RF biosensor (LC circuit of LC resonator) equipped with glucose-responsive microneedles -> Change / shift in center frequency of LC resonator -> Measurement of glucose concentration (hypoglycemia / normal / hyperglycemia)

[0068] Frequency of use of RF biosensor: 400~3,000MHz frequency

[0069] Measurements with a vector network analyzer (VNA): S-parameters for frequencies from 400 to 3,000 MHz ( ) Experimental results

[0070] Figure 2 illustrates an RF biosensor using a glucose-responsive hydrogel-based microneedle according to the present invention.

[0071] FIG. 3 is a diagram showing the operation of the glucose-responsive hydrogel-based microneedle and sensing antenna circuit of FIG. 2, the sensing antenna and the reader antenna remotely wirelessly EM-coupled and the reader antenna sensing circuit.

[0072] Figure 4 is a drawing showing the microneedle fabrication process.

[0073] An RF biosensor mounted on a flexible substrate is mounted on the upper side of the flexible substrate, and glucose-responsive hydrogel-based microneedles are mounted on the lower side of the flexible substrate. An antibody attached to the tip of the glucose-responsive hydrogel-based microneedles detects the glucose concentration of the body fluid (blood) minimally invasively within the skin of a living body, and measures the change in capacitance of the RFID wireless biosensor (LC circuit of the RF resonator) / the shift in the center frequency of the LC resonator / the change in the S-parameter.

[0074] An RF biosensing system having an RF biosensor wirelessly transmits a sensing signal to a readout coil, a reader antenna, and a reader antenna sensing circuit through EM coupling between a microneedle at the bottom of a substrate and a sensing antenna of an RF biosensor on the substrate, and the sensing antenna and the reader antenna, and measures the sensing signal in a VNA (Vector Network Analyzer) connected thereto.

[0075] Fabrication of microneedles

[0076] 1) DI water & CMC stir

[0077] CMC (carboxymethylcellulose): a polymer that dissolves in water (75°C, 400 rpm)

[0078] 2) Inject N2 gas into the solution

[0079] 3) Add KPS (26℃)

[0080] KPS: potassium peroxodisulfate

[0081] 4) Add 2-HEA

[0082] 2-hydroxyethyl acrylate (2-HEA) has a hydroxyl group (-OH), which has the advantage of improving adhesiveness due to hydrophilicity.

[0083] 5) Add PEGDA (biodegradable material)

[0084] PEGDA: poly ethylene glycol di acrylate (26℃, 400 rpm)

[0085] 6) Cool down solution

[0086] 7) Dialyzed for 3 days (Dialysis, 26℃)

[0087] 8) Dried using freeze dryer (Freeze dry, -85℃, 500 mm Torr)

[0088] 9) Powder was swollen in PBS and broken down (1000 rpm, 2h)

[0089] PBS: Phosphate buffered saline

[0090] 10) GelMA and LAP were dissolved in water

[0091] GelMA: gelatin methacryloyl

[0092] LAP: lithium acylphosphinate salt

[0093] Gelatin is obtained by hydrolyzing collagen at high temperatures. GelMA can be obtained by dissolving gelatin in phosphate-buffered saline (PBS) (pH = 7.4) at 50°C, then adding methacrylic anhydride (MA) and reacting.

[0094] 11) GelMA-LAP solution and CMC-pHEA mix

[0095] 12) Add GOx

[0096] To measure glucose concentration, glucose oxidase (GOx) is mainly applied to the electrode surface.

[0097] 13) Photocrosslinked under 405 nm

[0098] The fabrication of glucose-responsive hydrogel-based microneedles involves mixing the solution at 75°C to produce a hydrogel.

[0099] The fabrication of glucose-responsive hydrogel-based microneedles is as follows: solution mix 75℃ -> hydrogel drop in microneedle fabrication vessel -> centrifuge (3500 rpm, 5 min) -> scrape excess -> UV crosslink (40s) -> peel off -> UV crosslink (200s) -> resin drop

[0100] The microneedle material uses GelMA (gelatin methacryloyl) hydrogel. Hydrogels are hydrophilic polymer networks that form a matrix with a high water content. GelMA (gelatin methacryloyl) hydrogels, made from gelatin, a type of hydrogel, exhibit excellent biocompatibility and biodegradability. The swelling ratio of the hydrogel was measured in PBS buffer solutions at pH 7.4 and pH 2.5 at room temperature (23°C).

[0101] Swelling ratio(%) =

[0102] Here, Ws is the weight of the swollen hydrogel (g), and Wd is the weight of the dried hydrogel (g).

[0103] Hydrogels are hydrophilic polymeric materials with a three-dimensional network structure that are insoluble in water but swell and can hold large amounts of water. Hydrogels are thermodynamically stable after swelling in aqueous solutions, and their unique hydrophilicity and flexibility make them widely used in the pharmaceutical field.

[0104] [Ingredients]

[0105] Carboxymethyl cellulose,

[0106] 2-Hydroxyethyl acrylate

[0107] Potassium persulfate

[0108] (Example)

[0109] Figure 5 illustrates a biosensing system using an RF biosensor using a glucose-responsive hydrogel-based microneedle according to the present invention.

[0110] RF biosensing system using RF biosensor with glucose-responsive hydrogel-based microneedles

[0111] substrate;

[0112] A glucose-responsive hydrogel-based microneedles having a plurality of microneedles formed in an nxn matrix structure (n≥1 natural number) on the lower portion of the substrate, and which minimally invasively contacts the biological phase of the skin composed of epidermis / dermis / hypodermis or skin / fat / muscle in vivo;

[0113] A sensing antenna circuit and a sensing antenna connected to a glucose-responsive hydrogel-based microneedle provided on the lower portion of the substrate and composed of an LC resonator of an RF biosensor provided on the upper portion of the substrate; and a reader antenna and a reader antenna sensing circuit connected through a circular readout coil that receives a sensing signal using RF biosensing and wireless transmission technology by being EM coupled within an effective distance of 5 mm, and an RF biosensor (wireless detection) connected to a VNA; and

[0114] A biosensing system comprising a vector network analyzer (VNA) that measures capacitance changes / center frequency shifts of LC resonators / changes in S-parameters of the sensing signal of the RF biosensor,

[0115] It has a plurality of microneedles formed in a matrix structure on the lower part of the above substrate, and is used in a minimally invasive manner in contact with the skin in vivo, which is composed of the epidermis / dermis / hypodermis or skin / fat / muscle.

[0116] A vector network analyzer (VNA) has a plurality of microneedles formed in an nxn array matrix structure on the lower portion of the substrate, and is used in a minimally invasive manner in contact with the skin in vivo, which is composed of the epidermis / dermis / hypodermis or skin / fat / muscle.

[0117] A vector network analyzer (VNA) is used for RF and microwave measurements, and when a sample of the skin (blood) is measured by minimally invasively contacting the skin in vivo, the sensing signal from the sensing circuit of the sensing antenna circuit (LC resonator circuit) of the RF biosensor is wirelessly detected by the vector network analyzer (VNA) connected to the readout coil, and the glucose concentration of the body fluid is measured according to the change in capacitance of the LC resonator of the sensing antenna circuit / the center frequency shift of the LC resonator / the change in S-parameter.

[0118] The above substrate may be any one of an FR4 substrate, a flexible substrate, or a ceramic substrate for a wideband antenna for an antenna system of an RF biosensor.

[0119] In the embodiment, the substrate uses a flexible substrate, and the flexible substrate uses a PET substrate or a polyimide (PI) substrate.

[0120] An RF biosensor having a 15x15 array matrix structure of glucose-responsive hydrogel-based microneedles on the lower side of a flexible substrate and a sensing antenna and a sensing antenna circuit attached to the upper side of the flexible substrate, and connected to a VNA via a readout coil thereon, and when attached to the skin and measured, the readout coil senses the patch antenna from above within an effective distance of 1 to 5 mm to measure the resonant frequency.

[0121] In an embodiment, the sensing antenna of the RF biosensor is a patch antenna having a radiator at the center and a metal line having a square spiral structure with an N-turn structure, and the metal line uses gold (Au), silver (Ag), or copper (Cu), and the metal line has a line width W=0.1 to 1 mm, a line spacing S=0.1 to 1 mm, and is spaced apart by a certain distance. In an embodiment, the metal line used a line width W=0.5 mm, a line spacing S=0.5 mm.

[0122] A biosensing system having an RF biosensor uses an RF frequency in the range of 400 to 3,000 MHz for the sensing antenna and the reader antenna of the RF biosensor provided on the upper portion of the substrate.

[0123] A biosensing system using an RF biosensor having microneedles comprises a glucose-responsive hydrogel-based microneedle, a sensing antenna circuit, and a reader antenna and reader antenna sensing circuit that receive sensing signals wirelessly and remotely through a circular readout coil within an effective distance of 5 mm using RF biosensing and wireless transmission / wireless detection technology through EM coupling with the sensing antenna, and the readout coil, the reader antenna, and the reader antenna sensing circuit are connected to a vector network analyzer (VNA).

[0124] The RF biosensor having the above microneedles detects glucose and interstitial fluid (ISF) of subcutaneous fat in the skin or in microvessels in a minimally invasive manner during in vivo skin attachment measurement, and the complex permittivity and capacitance (C) for different glucose concentrations change, the resonant frequency of the LC resonator of the sensing antenna circuit of the RF biosensor changes, and the resonant frequency of the LC resonator of the sensing antenna of the RF biosensor shifts. By measuring this change and its resonant frequency, the glucose concentration can be accurately measured.

[0125] The complex permittivity of glucose changes depending on the concentration of glucose, which is expressed as a difference in the capacitance (C) of the RF biosensor. The complex permittivity and capacitance (C) change for different glucose concentrations, and the resonant frequency of the sensing antenna circuit of the RF biosensor is measured to measure the glucose concentration.

[0126] The RF biosensor, which utilizes microneedles made of glucose-responsive hydrogels embedded in a flexible substrate, accurately extracts interstitial fluid (ISF) from subcutaneous fat, minimally invasively, and painlessly. ISF is the fluid that forms between cells outside the microvasculature within the skin. Therefore, glucose levels are measured slightly later than blood glucose levels.

[0127] In an embodiment, when the RF biosensing system is in vivo to minimally invasively contact the skin consisting of epithelium / endothelium / hypodermis or skin / muscle / fat to measure a body fluid sample (blood), a vector network analyzer (VNA) receives a sensing signal through EM coupling and wireless detection in a swelling state through a sensing antenna circuit (LC resonator) of an RF biosensor (LC resonator) and a sensing antenna to a circular readout coil, a dither antenna, and a reader antenna sensing circuit, and the VNA connected to the readout coil measures the glucose concentration according to a change in capacitance / a shift in the center frequency of the LC resonator / a change in the S-parameter.

[0128] An RF biosensing system having an RF sensor having microneedles comprises a glucose-responsive hydrogel-based microneedle on a flexible substrate, for detecting a body fluid sample (blood, saliva) by minimally invasively contacting the skin in vivo, and a sensing antenna circuit (LC resonator) of an RF biosensor on the flexible substrate; a sensing antenna; and a sensor unit having a reader antenna and a reader antenna sensing circuit through a circular readout coil that is EM coupled to the RF biosensing and wireless transmission technology over an effective distance of 1 to 5 mm to wirelessly transmit a sensing signal; and the RF biosensor is connected to a vector network analyzer (VNA).

[0129] When making minimally invasive contact with the skin in a living body and measuring the skin, a sensing signal is detected through a readout coil above an effective distance of 1 to 5 mm of a sensing antenna of an RF biosensor having glucose-responsive hydrogel-based microneedles, and a VNA connected to the readout coil, the reader antenna, and the reader sensing circuit (wireless detection) measures the glucose concentration in the skin and microvessels according to the change in the capacitance of the LC resonator of the RF biosensor / the shift in the center frequency of the LC resonator circuit of the sensing antenna / the change in the S-parameter.

[0130] RF biosensors utilizing glucose-responsive hydrogel-based microneedles can measure glucose concentrations within the skin or microvasculature by utilizing the capacitance and permittivity of the RF biosensor, which vary with the glucose concentration. The overall impedance of the RF biosensor varies depending on the electrical properties (permittivity and capacitance) of the target substance (e.g., glucose).

[0131] When measuring within the skin, different glucose concentrations have different permittivities, so the capacitance of the LC resonator of the sensing antenna circuit of the RF biosensor changes and its resonant frequency ( ) are moved differently. That is, the electrical characteristics of the LC resonator of the RF biosensor during skin measurement ( ) measures the change.

[0132] Since the complex permittivity of glucose depends on its concentration, different electrical resonance phenomena appear in the RF biosensor (LC resonator) depending on the glucose concentration within the skin or microvasculature. Changes in permittivity for different glucose concentrations are reflected in differences in the capacitance (C) of the RF biosensor (the LC circuit of the RF biosensor).

[0133] In addition, in another embodiment, an RF biosensing system having an RF biosensor having glucose-responsive hydrogel-based microneedles can be manufactured as an RF diabetes meter, in which case the RF diabetes meter (IoT device) has a sensor unit connected to a control unit (MCU), and is provided with the control unit (MCU), a storage unit, and a display unit, and may further include a communication unit connected to the control unit (MCU) and providing Bluetooth or Wi-Fi communication. The RF diabetes meter can transmit and store glucose concentration to a diabetes measurement management server through a wired / wireless communication network, display the glucose concentration of the measured body fluid (blood, saliva) on a diabetes measurement App of a computer or a smartphone / tablet PC, and display hypoglycemia / normal / hyperglycemia on the screen of the computer.

[0134] An RF biosensing system having the RF biosensor may further include, for example, a control unit (MCU) that receives a sensing signal from a sensing antenna circuit of the RF biosensor and a readout coil, a reader antenna, and a reader antenna sensing circuit by EM coupling and wireless detection when the RF diabetes measuring device is used minimally invasively in the skin of a living body using a microneedle provided on the lower portion of a flexible substrate of the RF biosensor, and measures the glucose concentration according to a capacitance change of the sensing signal / center frequency shift / change (fc) of an LC resonator of the RF biosensor, a storage unit connected to the control unit (MCU), and a communication unit that provides Bluetooth and Wi-Fi communication connected to the control unit (MCU).

[0135] 2. Materials and Methods

[0136] 2.1 Reagents and Instruments

[0137] i) Materials for the synthesis of CMC-pHEA pH-responsive particles: sodium carboxymethyl cellulose (CMC, Mw~90,000), potassium persulfate (KPS), 2-hydroxyethyl acrylate (2-HEA)

[0138] ii) Materials for the further synthesis of glucose-responsive hydrogels: GelMA (DS~90) and LAP

[0139] iii) Glucose oxidase (GOx) and concanavalin A (ConA)

[0140] iv) Materials for artificial agarose skin model: agarose

[0141] * Materials and equipment for CMC-pHEA GelMA-ConA synthesis

[0142] Microneedles: A freeze dryer (ilShinBioBase, FD8512), cell disruption equipment (SONICS & MATERIALS, VC505), UV equipment (Kugou), and centrifuge (LABOGENE, 2236R) were used. Dialysis bags and UV resin were purchased from Beijing Yikang Prosperous Biotechnology Co., Ltd. and September Optoelectronics Technology Co., Ltd., respectively.

[0143] Characterization equipment: Fourier-transform infrared spectroscopy (FTIR) equipment (SS), scanning electron microscope (SEM) equipment (JOEL JSM-7001F), universal testing machine (MTS, CMT6103), and optical microscope (OLYMPUS, SZX16) were used.

[0144] 2.2 Preparation of CMC-pHEA pH-responsive particles

[0145] The preparation process of CMC-pHEA pH-responsive particles is illustrated in Figure 6. First, 1.2 g of CMC was added to DI water (CMC ratio: DI = 1.2 g:100 mL), placed on a hot plate (75 °C, 400 rpm), and completely dissolved. Then, it was placed in a vacuum atmosphere for 20 min to prevent oxidation. Next, 0.2 g of KPS was added to the mixture and reacted at room temperature for 20 min. Then, 18 mL of 2-HEA and 1 mL of PEGDA were each added to the CMC and KPS mixture and reacted at room temperature at 400 rpm for 3 h. The temperature should not be too high to prevent thermal cross-linking. Upon completion of the reaction, the mixture was poured into a dialysis bag, sealed, and kept in DI water for 3 days, changing the DI water once. After that, the mixture was refrigerated at -80℃ and freeze-dried in a freeze dryer (-85℃, 5 mTorr) for 3 days. The weight of the freeze-dried CMC-pHEA sample was measured, mixed with 6 times the weight of PBS (phosphate buffered saline) solution, and crushed into a viscous gel-like liquid in a cell crusher for 10 minutes. The unused freeze-dried sample was stored at -20℃, and the gelatinous liquid was stored at 4℃. It can be seen that after adding KPS to CMC, the H of the -OH group in the chemical structure of CMC is replaced in the mixture of CMC and KPS. When 2-HEA is added to the mixture of CMC and KPS, the 2-HEA molecules are replaced with the remaining 2-HEA molecules. After adding PEGDA, CMC-2-HEA molecules are linked to other CMC-2-HEA molecules via PEGDA molecules, forming a pH-responsive network.

[0146] Figure 6. Preparation of CMC-pHEA GelMA-ConA hydrogel and fabrication of microneedle embedded system: (a) synthesis of CMC-pHEA pH-responsive particles and their chemical structural changes, (b) fabrication of microneedles from CMC-pHEA GelMA-ConA glucose-responsive hydrogel, (c) preparation of embedded system.

[0147] 2.3 Fabrication of microneedles from CMC-pHEA GelMA-ConA glucose-responsive hydrogel

[0148] To fabricate glucose-responsive microneedles, the prepared CMC-pHEA pH-responsive gel was first mixed with GelMA (10% of the total mixture), LAP (5% of the total mixture), GOx (32 mg / mL), and ConA. The CMC-pHEA gelMA-ConA hydrogel solution was synthesized and stored at 4°C as shown in the figure. Although the liquid has low-temperature coagulation characteristics, excessive heat can inactivate the enzyme, so it should be stored in a water bath heated to approximately 50°C before use.

[0149] The microneedle sensing patch manufacturing process involved dropping a mixed hydrogel solution (250 μl) onto a microneedle mold and centrifuging for 5 minutes (3500 rpm, 25°C). After the gel had slightly hardened, the hydrogel was scraped from the microneedle pore surface using a spatula and exposed to UV light for 40 seconds. The patch was then left to dry for 2 hours in a well-ventilated area at room temperature. Subsequently, an appropriate resin was applied to the sensing antenna surface and the microneedle mold surface using a brush and bonded. To ensure a secure connection between the microneedle and the antenna, the patch was exposed to UV light for 40 seconds. After drying for 1 hour in a well-ventilated area at room temperature, the microneedles are peeled off vertically with tweezers using maximum force (microneedles are peeled off with tweezers), stored in a cool, dry place, and used within 1 week. Furthermore, the actual material synthesis and flexible antenna fabrication processes are illustrated in Figures 11 (Figure S1) and 13 (Figure S2).

[0150] Figure 11 shows the actual CMC-pHEA experimental process (Figure S1).

[0151] Figure S1 shows the actual process steps from the synthesis of CMC-pHEA microparticles to the freeze-dried sample, and the process of breaking and mixing materials such as GelMA, and finally uniformly filling each microneedle hole by centrifugation.

[0152] Figures 12 and 13 illustrate the sensing antenna manufacturing process and HFSS simulation (Figure S2). To manufacture a flexible antenna used as a sensing antenna of an RF biosensor, the surface of a polyimide (PI) substrate is first cleaned to make it smooth and clean, then copper is applied to the surface of the PI substrate and photoresist (PR) is applied thereon. A printed mask with a specific circuit pattern is then attached to the surface of the PI substrate and exposed to 405 nm UV light for 40 seconds. After UV light exposure, the mask is removed and the substrate is immersed in a solution. After the substrate is completely reacted, it is treated with acetone to manufacture a flexible antenna.

[0153] The sensing antenna of the actual RF biosensor is a square-shaped spiral antenna with an N-turn structure having a metal line formed of gold (Au) or copper (Cu). The metal line was manufactured with a size of line width W=0.5 mm and line spacing S=0.5 mm.

[0154] Referring to Fig. 14, the readout coil is a circular reading coil, and experiments were conducted with the circular diameters increasing by 18.5 mm, 19.5 mm, 20.5 mm, and 27 mm, respectively.

[0155] 2.4 Characteristics of CMC-pHEA GelMA-ConA glucose-responsive hydrogel

[0156] 2.4.1 Fourier transform infrared (FT-IR) analysis

[0157] The structures of CMC-pHEA pH-responsive gel and CMC-pHEA GelMA-ConA hydrogel were characterized by FT-IR absorption spectroscopy using a resolution in the range of 4000–400 cm-1.

[0158] 2.4.2 Morphological characterization

[0159] To observe the structure of CMC-pHEA pH-responsive lyophilized samples, the internal structure was observed under lyophilization conditions using SEM. In addition, to observe the internal structure of UV-cross-linked glucose-responsive hydrogels, gelled samples were first treated with liquid nitrogen for 5 min, then lyophilized for 2 days, and the internal porous structure was observed using SEM.

[0160] 2.4.3 Analysis of the swelling ability of hydrogels

[0161] Equal amounts of CMC-pHEA GelMA-ConA glucose-responsive hydrogels were dropped into multiple 3D-printed molds of the same design, and the weights were measured and recorded after UV treatment for the same duration. The UV-crosslinked samples were then immersed in glucose solutions of various concentrations for 10 minutes, and the surfaces were wiped with water. The weights were then measured and recorded. In addition, the soaked hydrogel samples were freeze-dried in liquid nitrogen, and differences in their internal structures were observed using SEM.

[0162] 2.5 Characterization of hydrogel microneedles

[0163] 2.5.1 Optical results of microneedles

[0164] To observe the surface condition of the microneedles, we used an optical microscope, a smartphone camera, and a scanning electron microscope (SEM) to fabricate the entire microneedles and obtain detailed height measurements. To enhance the visual appearance of the microneedles, a small amount of Rhodamine B was added to the hydrogel.

[0165] 2.5.2 Swelling and extraction ability

[0166] To investigate the swelling properties and in vitro extraction capacity of the microneedles, an agarose model of simulated artificial skin containing glucose was created (Supporting Information). Conventional quality characterization was used to compare quality changes before and after microneedle insertion, and optical microscopy was used to record height changes before and after microneedle insertion. To visualize extraction capacity, transparent microneedles were inserted into an agarose model containing Rhodamine B, and staining of the microneedles was observed.

[0167] 2.5.3 Mechanical property tests

[0168] Mechanical properties tests were conducted using a universal testing machine. The deformation of the microneedles was observed and recorded by applying various levels of vertical force.

[0169] The insertion ability of microneedles was initially tested by inserting them into an agarose model and observing the resulting holes. In the experiment, microneedles were inserted into pig skin and the resulting holes were observed.

[0170] A number of microneedles were placed in room-temperature unsealed, room-temperature relatively sealed, low-temperature unsealed, and low-temperature sealed environments, and morphological changes of each microneedle were observed and recorded daily under a microscope to confirm the storage stability of the microneedles.

[0171] 3. Results

[0172] 3.1 Characterization of CMC-pHEA and CMC-pHEA GelMA-ConA hydrogel particles

[0173] The FT-IR spectral results, as shown in Figure 7A, demonstrated that the cross-linking of CMC-pHEA by freeze-drying was successful. The peak at 1651 cm-1 in the spectrum, corresponding to the C=O double bond, demonstrated that the CMC molecules were highly crosslinked during the synthesis. In addition, the peak at 2853 cm-1 represented the symmetric stretching vibration of methylene CH, and the peak at 1325 cm-1 was caused by the symmetric stretching vibration band of carboxylate in CMC (-COO-). The lower frequency peak at 1121 cm-1 in the hydrogel spectra was assigned to the ester CO. The peak at 2362 cm-1 is attributed to carbon dioxide dissolving in solution from air and is not related to the sample synthesis. Based on the above results, the crosslinking of the CMC-pHEA particles was successfully confirmed (Park et al., 2018) (Schultz et al., 1996). Figure 7B shows the morphology. Characterization of the freeze-dried CMC-pHEA nanoparticles at 300x SEM magnification, revealing an internal filamentary mesh structure.The synthesized uncrosslinked CMC-pHEA GelMA-ConA hydrogel exhibits strong temperature rheology (Figure 7C), i.e., low-temperature solidification and high-temperature melting. A 300x SEM magnification of the dried crosslinked sample reveals its internal structure of small pores. In addition, Figures 7D and 7E verify the glucose responsiveness of the UV-crosslinked CMC-pHEA GelMA-ConA hydrogel. The morphologies obtained by a mobile phone camera are significantly different, as shown in Figure 7D. The swelling growth rates of hydrogels with almost identical initial weights were significantly different in different concentrations of glucose solutions, and the swelling ratios of hydrogels at 0 mM, 3 mM, and 20 mM at 60 min were 3.81, 2.98, and 2.674, respectively. The results show that the swelling rate decreases as the glucose concentration increases, indicating that CMC-pHEA GelMA-ConA is highly responsive to glucose. In addition, the bio Biocompatibility was verified in Figure 7F, which demonstrated that the hydrogel was non-toxic when attached to the human body.Furthermore, SEM observation of the internal microstructure revealed that all the soaked hydrogels had dense porous structures inside compared to the unswollen hydrogels. The porous size of the hydrogels immersed in water (Figure 7H) was much larger than that of the 3 mM glucose solution (Figure 3H), which was larger than that of the 20 mM glucose solution (Figure 7J). This indicates that the hydrogels can bind to glucose molecules through the internal network structure of the CMC-pHEA GelMA-ConA hydrogels, which can induce different swelling of the internal network. Additionally, the UV exposure time effect on reactivity requires an appropriate cross-linking time.

[0174] Figure 7. Material characterization: (A) FTIR spectra; (B) Characterization of CMC-pHEA and CMC-pHEA GelMA-ConA: (i) CMC-pHEA after freeze-drying and (ii) 1600X magnification SEM images; (iii) Temperature strain of CMC-pHEA GelMA-ConA and (iv) 1600X magnification SEM images after freeze-drying; (C) Swelling ability of CMC-pHEA GelMA-ConA at various glucose concentrations; (D) Comparison of morphology and SEM images of hydrogels after swelling. (i) Comparison of morphology; (ii) SEM images of hydrogels after immersion in 0 mM glucose solution; (iii) 3 mM glucose; (iv) 20 mM glucose.

[0175] 3.2 Characterization of CMC-pHEA GelMA-ConA microneedles

[0176] To verify the fabrication of microneedles, color-dye particles were added to the microneedle hydrogel, and then photographs of the microneedle patches were taken using an optical microscope and a camera.

[0177] The microneedle patch is fully formed as a 15x15 array. As shown in Figures 8A(ii) and 8A(iii), measuring the microneedles in a specific row reveals that both the height and the needle shape of the microneedles are very uniform. The extraction ability of the microneedles was determined by inserting them into rhodamine-containing agarose and observing the microneedles before and after insertion to determine the degree of microneedle expression. Further quantification of the weight change revealed that the microneedle patch can rapidly extract ISF, with an external ISF of 5 mg per 10 seconds when the microneedle patch itself weighs only 10 mg.

[0178] As shown in Figure 8B(i). 8B(ii), after insertion, the microneedles were stained and slightly swollen. The mechanical properties and insertion ability of the microneedles are important for their ability to insert into the skin and extract ISF. Universal testing machines were used to obtain accurate force-displacement curves. As the force increased, the displacement also increased. The relationship between force and compression was obtained and shown in Figure 8B(iii). MN could effectively penetrate the skin surface, withstanding a 10 N force at a displacement of 300 μm. To further investigate the swelling response of the microneedles to different glucose concentrations, rhodamine was mixed into the hydrogel, and after fabricating the microneedles, they were inserted into agarose containing different glucose concentrations and the fluorescence intensity was measured. As can be seen in Figure 8C, the transmission rates of microneedle inserting in different concentration agarose are different and regular, and the fluorescence intensity increases from 93.193, 172.460, and 290.256 as the glucose concentration increases, indicating a decrease in the swelling ratio (8D to 8F).

[0179] Figure 8. Characterization of the microneedles: (A) Optical results of the microneedle patch: (i) overall shape; (ii) row of microneedles; (iii) single microneedle. (B) Extraction ability of the microneedle patch: (i) weight change; (ii) color change; (iii) mechanical properties of the displacement-force curve; (C) Response of dyed microneedles to insertion - worsened at different glucose concentrations. (D) Fluorescence intensity changes: (i) 0 mM; (ii) 3 mM; (iii) 20 mM.

[0180] 3.3 Wireless detection system simulation and construction

[0181] Figure 9 shows the wireless sensing system simulation results: (A) size coupling effect; (B) different simulation results; (C) summarized results; (D) center shift and vertical distance effects on results; (E) center relative position effect on results; (F) vertical distance effect on results; (H) swelling condition effect on results; (I) effect of degree of swelling on results; (J) frequency shift toward swelling level.

[0182] To determine the optimal detection system and verify the detection effect, we first performed HFSS simulation (frequency-dependent A biosensing system equipped with an RF biosensor was simulated using HFSS. Considering the overall size of the microneedle (12 mm x 12 mm), the overall size of the sensing antenna was designed by ADS (Advanced Design Software), an RF design software, as shown in Fig. 9A, and set to 15 x 15 mm, and the number of turns, line width, and line spacing were optimized and adjusted. The HFSS simulation results are shown in Fig. 10B and Fig. 10C. This shows the sharpest response of -22 dB at 580 MHz. The antenna of structure 3-1 is shown showing the peak. The antenna of structure 3-1 has the sharpest peak of -22 dB at 580 MHz. It has a peak.

[0183] To build a highly sensitive and effective wireless detection system, we conducted exploratory experiments on several factors that significantly affect the results of the wireless detection system, as shown in Figure 9. Considering the size of the microneedle patch, the overall size of the sensing antenna was designed to be 12 mm × 12 mm. The wire width, number of turns, and coil of the helical coil of the sensing antenna were determined by different design factors. Furthermore, considering the reading coil sizes, all experiments were conducted at a fixed distance of 3 mm, with the dimensions shown in Figure 9A. Figure 9B shows the results of Coil-A, Coil-B, Coil-C, and Coil-D in response to the spiral-sensing antennas, along with insets of the magnitude variation summary. The analysis revealed that the size of the reading coil had a significant impact on the detection results. Factors such as the number of turns, wire width, and antenna size and morphology, distance, and location had irregular effects on the results. This highlights the importance of preliminary experiments to optimize the wireless sensing system. In a side-by-side comparison, the amplitude response of Coil-C consistently outperformed the other three groups. Furthermore, Figure 9c shows the vertical distance effect on the sensing results of the optimally selected group. Due to the presence of the sensing antenna, the peak generated by inductive coupling appears to be distributed between 650 and 700 MHz.As the distance between the two antennas in Fig. 9D increases (from 0 mm to 9 mm), the peak indensity gradually increases from -7 dB to -45 dB from 0 to 4 mm, and then decreases from -6 dB to 9 mm. The results indicate that the optimum distance for detection is approximately 2.5 mm. The measured original frequency values ​​can be used for calibration before sensing.

[0184] 3.4 Wireless detection results

[0185] Based on the above optimal detection conditions including antenna size and morphology, distance, and location (fixed at 2.5 mm vertically), the glucose detection capability of the wireless system was measured / investigated. The biosensing system that measures glucose concentration according to the capacitance change / resonance frequency change of the LC resonator of the sensing antenna circuit of the RF biosensor using a Vector Network Analyzer (VNA) is illustrated in Fig. 10(A). Fig. 10(B) presents a flexible sensing patch incorporating an integrated microneedle and antenna. The size of the flexible sensing patch is 15 mm x 15 mm, showing its adaptability to bending and microneedle size compared to wire size.

[0186] Figure 10 shows wireless system detection results: (A) detection system configuration; (B) comparison of detection sizes of microneedle patches; (C) detection results comparing unloaded and loaded microneedle patches; (D) detection results for agarose with various glucose concentrations; (E) change in resonant frequency and (F) change in size.

[0187] First, the results of the system equipped with microneedles and the basic antenna patch are shown in Fig. 10(C). Compared to the unloaded condition, the resonance appears with the addition of the sensing antenna, but is slightly reduced due to the adhesion of the microneedle. Fig. 10(D) shows the results for various glucose concentrations in agar over 10-second intervals. The spectral response is studied and displayed, and other results are shown in Figure S10. As the glucose concentration increases, the resonance shifts to higher frequencies and increases in magnitude. Figure 10(E) summarizes the resonance frequency change from 600 MHz at 0 mM to 748 MHz at 18 mM, and the curve follows a quadratic function y=597.6+16.89x-0.5x² with a COD of 0.998. Similarly, Figure 10(F) shows the magnitude change moving from -2.1 dB to -7.5 dB according to a quadratic function y=2.11+0.015x-0.018x² with a COD of 0.988. These results indicate that the glucose-sensitive microneedles are closely related to glucose concentration and have a significant impact on the antenna parameters. The glucose detection platform using various glucose microneedles is summarized in Table 1. Various types of microneedles have been developed and applied to detection methods such as electrochemistry and colorimetry based on various detection principles. We first proposed a combined hydrogel microneedle swelling and high-frequency detection method, achieving highly sensitive glucose detection.

[0188] Table 2 shows a comparison of different microneedle sensors.

[0189] ReferenceAnalytesMicroneedle typesRecognition elementsDetection approachPrincipleSensitivity(Liu et al., 2021)GlucoseAg / AgCl deposited Prussian blue / SolidGOxelectrochemicalDifferent current produced when GOx reacts with glucose0.0416 uA / mM(He et al., 2021)GlucoseHyaluronic acid (HA) / HydrogelGOxcolorimetricGOx reacts with glucose produces gluconic acid generates pH changes with color variation0.0125 Hue / mM(Lu et al.,2023)Glucosephotonic crystals / Hydrogelspectrum analyzerHydrogel swelling when PBA combines with glucose and generates color variation30 nm shift / mg / mL(Zheng et al.,2022)GlucoseSilk fibers / Hydrogel GOxelectrochemicalDifferent current produced by H2O2 when GOx reacts with glucose75 nA / mM(ZHANG et al.,2022)Glucosephotonic crystals PBA / HydrogelPBAcolorimetricHydrogel volume changes when exposure to glucose and generates optical wavelength vary25 nm / mM(Parrillaet al.,2022)GlucosePrussian blue (PB) / nickel hexacyanoferrateGOxelectrochemicalDifferent current produced by H2O2 when GOx reacts with glucose109 nA / mM(You et al., 2023)GlucosePEGDAGOxcolorimetricGOx reacts with glucose produces gluconic acid generates pH changes with color variationColor density / mMThisworkGlucoseCMC-pHEA GelMA-ConA hydrogel microneedleGOxRadio frequencyHydrogel swelling volume variation when reacting with glucose17 MHz / mM.

[0190]

[0191] Figure 15 shows a screen for measuring glucose concentration (hypoglycemia / normal / hyperglycemia) of a capacitance change of an RF biosensor (LC circuit of an LC resonator) equipped with a glucose-responsive microneedle -> change in the center frequency of the LC resonator.

[0192] A biosensing system having an RF biosensor with microneedles can measure protein in the skin, glucose concentration in the blood in microvessels, and fat in vivo. The biosensing system having microneedles and an RF biosensor is connected to a VNA, and the VNA measures glucose concentration according to changes in the resonance frequency of the LC resonator of the RF biosensor (hypoglycemia frequency f1, normal frequency f2, hyperglycemia frequency f3) / changes in S-parameters (S1, S2, S3), thereby indicating hypoglycemia / normal / hyperglycemia and diagnosing diabetes.

[0193] 4. Conclusion

[0194] We developed an integrated platform for glucose sensing that effectively combines extraction and detection processes by utilizing swelling hydrogel microneedles and an advanced wireless sensing system. A freeze-dried CMC-pHEA hydrogel network becomes glucose-specific after cross-linking with glucose oxidase and compounds like GelMA-ConA. When a compound like GelMA-ConA is combined with a flexible antenna, this sensing component can tap into the interstitial fluid, binding to its glucose molecules. The degree of swelling corresponds to glucose concentration, which results in a noticeable change in the signal from a wireless reading coil. Employing swelling-based hydrogel microneedles, we enable a minimally invasive and pain-free glucose monitoring system by using RF high-frequency signals to ensure rapid and precise glucose identification.

[0195] Microneedles made of glucose-responsive hydrogels can extract interstitial fluid from subcutaneous fat with minimal pain.

[0196] Glucose detection by hydrogel swelling is measured using RF biosensing technology.

[0197] The on-site measurement platform in vivo enables on-site sensing and detection.

[0198] The wireless detection platform enables passive sensing and remote reading without complex circuitry.

[0199] Glucose-responsive hydrogel-based microneedles are minimally invasively attached to the skin in vivo, and then glucose concentration is measured using a VNA based on the capacitance change of the sensing signal of the sensing circuit (wireless detection) of the RF biosensor / the center frequency of the LC resonator of the sensing antenna circuit / the change in S-parameter, thereby indicating hypoglycemia / normal / hyperglycemia and diagnosing diabetes.

[0200] A minimally invasive, wireless RF biosensor using hydrogel-based microneedle for glucose detection uses a glucose-responsive hydrogel-based microneedle under a flexible substrate to minimally invasively contact the epithelium / endothelium / hypodermis or skin / fat / muscle of the skin in a living body, and the VNA measures the glucose concentration according to the capacitance change of the sensing signal of the RF biosensor / the center frequency shift of the LC resonator of the sensing antenna circuit / the change in the S-parameter, thereby indicating hypoglycemia / normal / hyperglycemia to diagnose diabetes.

[0201] An RF biosensor having glucose-responsive hydrogel-based microneedles detects glucose in the skin or microvessels, interstitial fluid (ISF) of subcutaneous fat in a minimally invasive manner during in vivo skin-attached measurement, and the complex permittivity and capacitance (C) for different glucose concentrations change, and the resonant frequency of the LC resonator of the sensing antenna of the RF biosensor shifts. By measuring this change and its resonant frequency, the glucose concentration can be accurately measured.

[0202] Although the present invention has been described with reference to specific embodiments, the present invention is not limited to the same configuration and operation as the specific embodiments in order to illustrate the technical idea as described above, and may be implemented by various modifications within the scope that does not depart from the technical idea and scope of the present invention, and the scope of the present invention should be determined by the claims set forth below.

[0203] A biosensing system using an RF biosensor having microneedles uses a glucose-responsive hydrogel-based microneedle equipped on the lower part of a flexible substrate of an RF biosensor to detect a sensor in vivo on the epidermis / dermis / hypodermis or skin / fat / muscle using a frequency of 400 to 3,000 MHz, and detects a sensing signal from a sensing antenna circuit and a reader antenna and a reader antenna sensing circuit through a readout coil by RF biosensing and wireless transmission, and measures the sensing signal with a VNA (Vector Network Analyzer), and detects the sensing signal using a sensing antenna circuit (LC resonator) of the RF biosensor. It has the effect of providing biosensing such as detection of proteins, glucose, and fat in the skin according to changes in capacitance / changes in the resonant frequency of the LC resonator / changes in the S-parameter.

[0204] A biosensing system having an RF biosensor with microneedles can measure protein in the skin, glucose concentration in the blood, and fat in vivo. The biosensing system having microneedles and an RF biosensor is connected to a VNA, and the VNA measures glucose concentration according to changes in the resonance frequency of the LC resonator of the RF biosensor (hypoglycemia frequency f1, normal frequency f2, hyperglycemia frequency f3) / S-parameter changes (S1, S2, S3), thereby indicating hypoglycemia / normal / hyperglycemia and diagnosing diabetes.

Claims

1. Substrate; Microneedles provided on the lower part of the above substrate and that minimally invasively contact the skin within a living body; A sensing antenna circuit and a sensing antenna, which are connected to microneedles provided on the lower part of the substrate and are composed of an LC resonator of an RF biosensor provided on the upper part of the substrate; and an RF biosensor having a reader antenna and a reader antenna sensing circuit through a circular readout coil that is EM coupled within an effective distance and receives a sensing signal using RF biosensing and wireless transmission technology, and is connected to a VNA; and An RF biosensing system using an RF sensor having microneedles, comprising a vector network analyzer (VNA) that measures capacitance change / center frequency shift of the LC resonator / change in S parameter of the sensing signal.

2. In paragraph 1, An RF biosensing system using an RF sensor having microneedles, wherein the microneedles have a plurality of microneedles formed in an nxn matrix structure (n≥ 1 natural number).

3. In paragraph 1, An RF biosensing system using an RF sensor having microneedles, wherein the microneedles are glucose-responsive hydrogel-based microneedles.

4. In paragraph 3, The above glucose-responsive hydrogel is an RF biosensing system using an RF sensor having microneedles using CMC-pHEA GelMA-ConA hydrogel.

5. In paragraph 1, The above substrate uses any one of an FR4 substrate, a flexible substrate, or a ceramic substrate for a wideband antenna of the RF biosensor, The above flexible substrate is an RF biosensing system using an RF sensor having microneedles, wherein the RF biosensing system uses a PET substrate or a polyimide (PI) substrate.

6. In paragraph 1, An RF biosensing system using an RF sensor having microneedles, wherein the sensing antenna and the reader antenna of the RF biosensor provided on the upper part of the substrate use an RF frequency in the range of 400 to 3,000 MHz.

7. In paragraph 3, An RF biosensing system using an RF sensor equipped with microneedles, wherein the antibody equipped on the glucose-responsive hydrogel-based microneedles equipped on the lower portion of the above substrate uses glucose oxidise. Glucose reacts with glucose oxidise to generate an acid, so synthesized CMC-pHEA is used, and the synthesized CMC-pHEA is very sensitive to pH and selectively reacts only to glucose in the swelling state of the hydrogel.

8. In paragraph 7, The above glucose-responsive hydrogel-based microneedles are a kind of composite material, CMC, KPS, 2-HEA, PEGDA, gelMA, glucose oxidise, photoinitiator (photoinitiator, photoreactive compound), and an RF biosensing system using an RF sensor having microneedles.

9. In paragraph 3, An RF biosensing system using an RF sensor equipped with microneedles, wherein the material of the above glucose-responsive hydrogel-based microneedles is GelMA (gelatin methacryloyl) hydrogel with excellent biocompatibility.

10. In paragraph 1, The sensing antenna of the above RF biosensor is An RF biosensing system using an RF sensor having a patch antenna having a radiator at the center and a metal line having a square spiral structure with an N-turn structure, wherein the metal line uses gold (Au), silver (Ag) or copper (Cu), and the metal line has a line width W = 0.1 to 1 mm, a line spacing S = 0.1 to 1 mm, and microneedles spaced apart at a certain distance.

11. In paragraph 10, The above microneedles use a 15 x 15 array, and the skin penetration depth of the microneedles is 10 to 100 μm. An RF biosensing system using an RF sensor having microneedles, wherein the size of the sensing antenna is 12 mm x 12 mm, and the optimal distance for wireless biosensing between the sensing antenna of the RF biosensor and the readout coil is 1 to 5 mm.

12. In paragraph 1, The RF biosensor detects glucose and interstitial fluid (ISF) of subcutaneous fat in the skin or in microvessels in a minimally invasive manner during in vivo skin attachment measurement, and the complex permittivity and capacitance (C) for different glucose concentrations are changed, thereby shifting the resonance frequency of the LC resonator of the sensing antenna of the RF biosensor. An RF biosensing system using an RF sensor having microneedles, which changes and measures the resonant frequency thereof to measure glucose concentration.

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