An epidermal sweat patch for non-invasive monitoring of bilirubin level and a preparation method and application thereof

Abstract

The present application belongs to the technical field of electrochemical detection, and more particularly relates to a non-invasive skin sweat patch for monitoring bilirubin level, and a preparation method and application thereof. The patch is composed of a microfluidic sweat collection and detection chip and a flexible portable electrochemical workstation. The chip is provided with a three-electrode system, and the surface of the working electrode is modified with p-toluenesulfonic acid functionalized gold aerogel. The porous network of the gold aerogel enhances electron transmission, and the sulfonic acid group and aromatic ring provide specific recognition sites for bilirubin, realizing efficient enrichment and electrochemical oxidation. The patch collects sweat through capillary action, and the signal is detected by differential pulse voltammetry, amplified, filtered, and wirelessly transmitted to a mobile terminal to realize monitoring. The preparation method includes microfluidic chip reverse molding, one-pot synthesis of aerogel, and electrode modification and packaging. The product is suitable for liver disease detection, drug-liver monitoring, postoperative evaluation, cholestasis, and neonatal jaundice screening, and has the advantages of non-invasiveness, portability, high sensitivity, and strong anti-interference.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical detection technology, and more specifically, relates to a non-invasive epidermal sweat patch for monitoring bilirubin levels, its preparation method, and its application. Background Technology

[0002] Bilirubin is a major byproduct of hemoglobin metabolism in the body. The metabolism and excretion of bilirubin largely depend on liver function; therefore, bilirubin levels are frequently used as an indicator for diagnosing and monitoring liver diseases. Clinically, it is commonly seen in viral / alcoholic hepatitis, cholestatic liver disease, cirrhosis / decompensated liver disease, and drug-induced liver injury, and is closely related to jaundice. Changes in bilirubin concentration are not only a warning sign of liver disease but also crucial for assessing treatment effectiveness and disease progression.

[0003] Current clinical monitoring mainly relies on blood analysis. However, routine blood analysis has the following limitations: (1) invasive sampling leads to discomfort and infection risks, limiting high-frequency retesting; (2) the tests are mostly intermittent and offline, and can only obtain the instantaneous level of bilirubin, making it difficult to reflect rapid fluctuations in bilirubin; (3) compliance with home and long-term management outside of hospital / outpatient settings is low and the cost is high.

[0004] With the rapid development of electronic information and sensing technologies, wearable electrochemical sensing technology has brought hope for non-invasive monitoring of biochemical indicators. Among these, sweat-based wearable electrochemical sensing technology has been widely used to monitor biochemical indicators such as glucose, lactate, and inorganic ions, primarily targeting patients with chronic diseases or those undergoing exercise and health management. However, its application in liver disease scenarios such as outpatient follow-up, adverse drug reaction monitoring, postoperative recovery assessment, cholestasis progression monitoring, and neonatal / infant jaundice risk screening remains unexplored. Therefore, developing a non-invasive and continuous bilirubin level monitoring product is of great significance for the early diagnosis, treatment efficacy evaluation, and long-term management of liver diseases. Summary of the Invention

[0005] The purpose of this invention is to provide a non-invasive epidermal sweat patch for monitoring bilirubin levels, its preparation method, and its application.

[0006] This invention provides a non-invasive epidermal sweat patch for monitoring bilirubin levels, comprising: A microfluidic sweat collection and detection chip includes a microfluidic chip layer and a sensing electrode layer. The sensing electrode layer includes an electrode substrate and a working electrode, a counter electrode, and a reference electrode disposed on the surface of the electrode substrate. The surface of the working electrode is modified with p-toluenesulfonic acid-functionalized gold aerogel for recognizing bilirubin. The microfluidic chip layer includes a chip body with channels disposed thereon. The side of the microfluidic chip layer with channels is irreversibly bonded to the side of the sensing electrode layer with electrodes. A flexible portable electrochemical workstation, which is connected to the electrodes of the sensing electrode layer; The p-toluenesulfonic acid functionalized gold aerogel is prepared by the following steps: a chloroauric acid solution with a mass fraction of 5%~15%, β-cyclodextrin, and a sodium borohydride solution with a concentration of 0.5~1.5 mg / mL are mixed and a gold nanoparticle suspension is synthesized by a one-pot wet chemical method. A toluenesulfonic acid aqueous solution with a mass fraction of 0.1%~1% is added dropwise to the gold nanoparticle suspension. After standing for 30~40 hours, the mixture is freeze-dried to obtain the p-toluenesulfonic acid functionalized gold aerogel. The mass-to-volume ratio of the chloroauric acid solution, β-cyclodextrin, sodium borohydride solution, and toluenesulfonic acid aqueous solution is 20~30µL:300~400mg:400~600µL:100µL.

[0007] This invention marks the first time wearable electrochemical sensing technology has been extended to liver disease management, constructing a fully integrated solution encompassing "sweat collection – microfluidic transport – specific recognition – electrochemical quantification – wireless transmission." By modifying the working electrode surface with p-toluenesulfonic acid-functionalized gold aerogel, a three-dimensional hierarchical porous conductive framework is formed. This significantly improves the specific surface area and charge transfer rate, and through the electrostatic and π-π synergistic effects between the negatively charged sulfonate interface and the bilirubin conjugated structure, it endows the electrode with excellent selective enrichment capabilities. Compared to unmodified aerogels or those modified with other aromatic / phenolic / carboxyl groups, this material exhibits stronger response, a wider linear range (0.01~1000 μmol / L), and a lower detection limit in cyclic voltammetry, differential pulse voltammetry, and open-circuit potential tests, while showing almost no response to common interfering substances such as glucose and lactic acid. By employing a PDMS microfluidic chip to achieve passive sweat drainage and storage, combined with signal conditioning, Bluetooth transmission, and mobile app display from a flexible electrochemical workstation, bilirubin monitoring is upgraded from single-point blood sampling to continuous, dynamic, non-invasive monitoring. This concept provides a novel wearable solution for home management of liver disease, postoperative recovery tracking, and early screening for neonatal jaundice.

[0008] Furthermore, the microfluidic chip layer is made of PDMS, and the flow channel includes an inlet, a reservoir, and an outlet connected in sequence. The number of inlets is 2 to 4, and the diameter is 0.5 to 1.5 mm. Sweat is driven through the inlets into the reservoir by capillary force. The diameter of the reservoir is 5 to 15 mm, and excess sweat is discharged from the outlet, which has a diameter of 1 to 3 mm. The depth of the inlet, reservoir, and outlet of the microfluidic chip layer is 0.1 to 0.2 mm.

[0009] Furthermore, the electrode substrate is PDMS, the working electrode and the counter electrode are carbon electrodes, and the reference electrode is an Ag / AgCl electrode.

[0010] Furthermore, the surface of the working electrode is also modified with a chitosan layer, which covers the outer layer of the p-toluenesulfonic acid functionalized gold aerogel.

[0011] Furthermore, the irreversible bonding encapsulation includes hydrophilic treatment of the side of the microfluidic chip layer with flow channels and the side of the sensing electrode layer with electrodes.

[0012] Furthermore, the hydrophilic treatment employs oxygen plasma treatment under the following conditions: oxygen flow rate 10~20 sccm, radio frequency power 30~50W, cavity vacuum degree 0.3~0.5 Torr, and treatment time 40~60s.

[0013] The present invention also provides a method for preparing the aforementioned epidermal sweat patch, comprising the following steps: S1. Mix PDMS and curing agent evenly at a mass ratio of 5~15:1, degas under vacuum, pour into a mold, cure at 50~70℃, and obtain a microfluidic chip layer after demolding. S2. The p-toluenesulfonic acid functionalized gold aerogel is drop-coated onto the surface of the working electrode in the form of a dispersion of 0.5~1.5 mg / mL, with a drop volume of 4~6 µL. Then, a chitosan layer is modified on the outer layer of the p-toluenesulfonic acid functionalized gold aerogel to obtain the sensing electrode layer. S3. After irreversibly bonding and encapsulating the microfluidic chip layer with the flow channel and the sensing electrode layer with the electrode, a microfluidic sweat collection and detection chip is obtained. The sweat introduced from the inlet gathers in the storage cavity and can completely cover the working electrode, the counter electrode and the reference electrode. The sensing electrode uses differential pulse voltammetry to perform electrochemical quantitative detection of bilirubin in the sweat and outputs an electrical signal. S4. Connect the electrodes of the sensing electrode layer in the microfluidic sweat collection and detection chip to the flexible portable electrochemical workstation through the electrode interface to obtain an epidermal sweat patch.

[0014] The present invention also provides the application of the aforementioned epidermal sweat patch in the preparation of devices for monitoring liver disease, adverse drug reactions, postoperative recovery assessment, cholestasis monitoring, or neonatal / infant jaundice risk screening.

[0015] The present invention has the following beneficial effects: 1. This invention uses p-toluenesulfonic acid-functionalized gold aerogel as a bilirubin recognition element. The three-dimensional hierarchical porous network of the gold aerogel is used to improve the effective specific surface area and electron transport efficiency of the electrode. At the same time, the aromatic ring and sulfonic acid group recognition sites provided by the p-toluenesulfonic acid molecule are used to enhance the bilirubin interface enrichment ability.

[0016] 2. Compared with unmodified gold aerogel, p-toluenesulfonic acid functionalized gold aerogel exhibits stronger cyclic voltammetric and differential pulse voltammetric responses, a wider linear detection range, a lower detection limit, a faster open-circuit potential response, and better repeatability in bilirubin detection, indicating that p-toluenesulfonic acid modification brings about a clear performance improvement.

[0017] 3. Compared with other modified molecules such as p-cresol, dopamine, and p-toluenesulfonic acid, p-toluenesulfonic acid functionalized gold aerogel exhibits a stronger response to bilirubin and better interfacial adsorption capacity, indicating that its technical effect cannot be obtained by arbitrary molecular modification, but is the result of the synergistic effect of p-toluenesulfonic acid and gold aerogel.

[0018] 4. In the presence of common sweat interfering substances such as glucose, lactic acid, urea, ascorbic acid and uric acid, the response of the toluenesulfonic acid-functionalized gold aerogel electrode showed little change, while it showed a significant response to bilirubin, indicating that it has good selectivity and anti-interference performance in complex sweat matrices.

[0019] 5. This invention integrates microfluidic sweat collection, flexible electrochemical sensing, signal processing, and wireless transmission, enabling the transformation of bilirubin level detection from traditional offline point detection to continuous dynamic monitoring. It is suitable for scenarios such as outpatient follow-up for liver diseases, monitoring of adverse drug reactions, postoperative recovery assessment, and jaundice risk screening for newborns / infants. Attached Figure Description

[0020] Figure 1 This is a layer diagram of a microfluidic chip, where A is a schematic diagram of the microfluidic chip structure (unit: mm), and B is a physical image of the microfluidic chip.

[0021] Figure 2 This is a diagram of the sensing electrode layer, where A is a schematic diagram of the electrode structure and B is a physical image of the electrode.

[0022] Figure 3 Images of the epidermal sweat patch are shown, where A is a physical image of the portable flexible electrochemical workstation and B is a physical image of the epidermal sweat patch.

[0023] Figure 4 Cyclic voltammetry for the oxidation of bilirubin using a p-toluenesulfonic acid-functionalized gold aerogel electrode.

[0024] Figure 5 This is a cyclic voltammogram of bilirubin oxidation catalyzed by a gold aerogel electrode.

[0025] Figure 6 The graphs show the differential pulse voltammetry (CPV) curves of the p-toluenesulfonic acid-functionalized gold aerogel electrode in bilirubin solutions of different concentrations. In the graphs, A is the CPV curve of the p-toluenesulfonic acid-functionalized gold aerogel sensing electrode in bilirubin solutions of concentrations of 0.01, 0.1, 1, 10, 100, and 1000 µmol / L, and B is the calibration curve of the p-toluenesulfonic acid-functionalized gold aerogel sensing electrode at the above bilirubin concentrations.

[0026] Figure 7 Cyclic voltammetry diagrams for the oxidation of bilirubin using p-cresol, dopamine, and p-toluic acid-functionalized gold aerogel electrodes are shown. In these diagrams, A represents the cyclic voltammetry diagram for the oxidation of bilirubin using p-cresol-functionalized gold aerogel electrodes, B represents the cyclic voltammetry diagram for the oxidation of bilirubin using dopamine-functionalized gold aerogel electrodes, and C represents the cyclic voltammetry diagram for the oxidation of bilirubin using p-toluic acid-functionalized gold aerogel electrodes.

[0027] Figure 8 Open-circuit potential diagram of bilirubin adsorption on a gold aerogel electrode functionalized with p-toluenesulfonic acid, p-cresol, dopamine, and p-methylbenzoic acid.

[0028] Figure 9 The diagram shows the open-circuit potentials of the p-toluenesulfonic acid-functionalized gold aerogel electrode adsorbing other interfering substances. Specifically, A represents the open-circuit potential of the p-toluenesulfonic acid-functionalized gold aerogel electrode adsorbing glucose; B represents the open-circuit potential of the p-toluenesulfonic acid-functionalized gold aerogel electrode adsorbing lactic acid; C represents the open-circuit potential of the p-toluenesulfonic acid-functionalized gold aerogel electrode adsorbing urea; D represents the open-circuit potential of the p-toluenesulfonic acid-functionalized gold aerogel electrode adsorbing ascorbic acid; E represents the open-circuit potential of the p-toluenesulfonic acid-functionalized gold aerogel electrode adsorbing uric acid; and F is a bar chart comparing the changes in the open-circuit potential response in the above A, B, and C diagrams.

[0029] Figure 10 The image shows the IT response curve of a p-toluenesulfonic acid-functionalized gold aerogel electrode in the presence of bilirubin and interfering substances. Detailed Implementation

[0030] The present invention will now be described in detail with reference to specific embodiments, but these should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained commercially unless otherwise specified.

[0031] Example 1 I. Fabrication of microfluidic chip layers.

[0032] This microfluidic structure consists of three 1mm diameter inlets, a 10mm diameter reservoir, and a 2mm diameter outlet. All regions have a depth of 0.2mm. The microfluidic chip was fabricated using a casting technique. First, PDMS and curing agent were mixed uniformly at a mass ratio of 10:1. The mixture was then allowed to stand under vacuum for 20 minutes to eliminate air bubbles. The mixture was then poured onto a corresponding mold (Anhui Chixin Biotechnology Co., Ltd.), heated to 60℃ for curing, and finally peeled off to obtain the corresponding microfluidic chip layer, as shown below. Figure 1 As shown. The curing agent was purchased from Hangzhou Weisicheng Technology Co., Ltd.

[0033] II. Sensing Electrode Layer.

[0034] The sensing electrodes were purchased from Sanjun Biotechnology Co., Ltd., such as Figure 2 As shown, the sensor has a size of 2cm×3cm and uses a complete three-electrode system (working electrode, counter electrode and reference electrode). The sensing electrodes are fabricated using screen printing technology with PDMS as the printing substrate and polyimide printing insulation layer. The working electrode and counter electrode are carbon electrodes and the reference electrode is Ag / AgCl.

[0035] III. Preparation of p-Toluenesulfonic acid functionalized gold aerogel.

[0036] Gold nanoparticles were synthesized using a one-pot wet chemical method with chloroauric acid solution (10 wt%, 25.6 µL) as a precursor, β-cyclodextrin (5 wt%, 343 mg) as a ligand, and sodium borohydride solution (1 mg / mL, 0.5 mL) as a reducing agent. A 0.5% (w / w) TsOH aqueous solution was prepared by dissolving p-toluenesulfonic acid (TsOH) in ultrapure water. Under continuous gentle magnetic stirring (150 rpm), the above TsOH aqueous solution (100 µL) was added dropwise to the β-cyclodextrin-stabilized gold nanoparticle suspension prepared in the first step using a microsyringe. The dropping rate was controlled at 2 µL / s to ensure uniform dispersion of TsOH in the system. After the addition was complete, stirring was stopped, and the mixed solution was transferred to a sealed container and allowed to stand at 25°C for 36 hours. During the incubation process, the color of the system gradually changed from wine red (characteristic plasma resonance color of dispersed gold nanoparticles) to grayish brown hydrogel. After freeze-drying, p-toluenesulfonic acid functionalized gold aerogel was obtained, and then it was prepared into a 2 mg / mL dispersion for later use.

[0037] During gel formation, the sulfonic acid groups (−SO3H) of the TsOH molecules are stably anchored to the gold surface of the aerogel framework through sulfur-gold interactions (S−Au), forming a monolayer sulfonic acid-modified interface. Under physiological pH conditions (sweat pH is 4.5~7.5), the −SO3H groups completely dissociate into sulfonate groups (−SO3⁻), endowing the aerogel framework with stable hydrophilicity and negatively charged surface properties. On the one hand, this improves the dispersibility and resistance to non-specific adsorption of the aerogel in aqueous detection media (sweat). On the other hand, the negatively charged functional group surface provides an activation interface for the subsequent efficient fixation of bilirubin molecules through electrostatic interactions, thus achieving simultaneous destabilization gel formation and aerogel surface functionalization modification in a single step.

[0038] IV. Preparation of p-Toluenesulfonic acid functionalized gold aerogel electrode.

[0039] p-Toluenesulfonic acid-functionalized gold aerogel was thoroughly dispersed in ultrapure water (1 mg / mL). Using a physical adsorption method, 5 µL of the p-toluenesulfonic acid-functionalized gold aerogel dispersion was coated onto the surface of the working electrode. Subsequently, a layer of chitosan (1 wt%, 3 µL) was added to prevent material detachment. Neither the electrode nor the reference electrode underwent functionalization, thus obtaining the p-toluenesulfonic acid-functionalized gold aerogel-modified electrode. The PDMS microfluidic chip layer was irreversibly bonded and encapsulated with the p-toluenesulfonic acid-functionalized gold aerogel-modified electrode to form a microfluidic sweat collection and detection chip. The specific steps are as follows:

[0040] (a) Surface activation pretreatment: The PDMS microfluidic chip layer and the sensing electrode layer were simultaneously placed in the oxygen plasma cleaner chamber, with the sensing electrode layer containing the electrode functional surface facing upwards, and the PDMS microfluidic chip layer with the microchannel structure surface facing upwards. The treatment parameters were set as follows: oxygen flow rate 15 sccm, RF power 40 W, chamber vacuum degree 0.4 Torr, and treatment time 50 s. Under the bombardment of oxygen plasma, the Si−CH3 (methyl) groups on the PDMS surface were oxidized and broken, generating a high density of silanol (Si−OH) active groups in situ, changing the PDMS surface from hydrophobic (water contact angle > 100°) to hydrophilic (contact angle < 10°), providing an activation interface for subsequent irreversible covalent bonding. At the same time, plasma treatment can also remove trace amounts of organic contaminants from the microchannel and electrode surfaces.

[0041] (b) Alignment and Bonding: Immediately after oxygen plasma treatment, the chip is removed and, in a clean bench, precisely aligned with the PDMS microfluidic chip layer (microchannel side down) and the sensing electrode layer (electrode side up) using an optical microscope. The alignment criteria are: the liquid reservoir (6mm diameter) of the microfluidic chip is located directly above the three-electrode sensing area, ensuring that the sweat introduced from the inlet, after converging through the microchannels, completely covers the sensing areas of the working electrode, counter electrode, and reference electrode; the inlet and outlet positions should avoid the conductive lead areas of the electrodes to prevent short circuits or signal crosstalk caused by sweat contacting the leads. After alignment, apply uniform, light pressure (0.5MPa) using clean tweezers to ensure tight adhesion. Allow it to stand at room temperature for 30 seconds to complete the bonding, thus obtaining the microfluidic sweat collection and detection chip.

[0042] Example 2 This invention provides a non-invasive, real-time epidermal sweat patch for monitoring bilirubin levels, such as... Figure 3 As shown, this patch integrates a microfluidic sweat collection and detection chip and a portable flexible electrochemical workstation (Shenzhen Haoyang Technology Co., Ltd., model: HY-EC Cube), enabling continuous monitoring and remote display of bilirubin concentration. The microfluidic sweat collection and detection chip and the flexible portable electrochemical workstation are stably connected and mechanically fixed via a pluggable electrode interface, which is integrated into the flexible portable electrochemical workstation. (The working principle of the flexible portable electrochemical workstation is described in sections: III. Analog signal amplification and filtering; IV. Analog-to-digital conversion and digital signal processing; V. Bluetooth wireless data transmission; and VI. Mobile terminal data display and auxiliary analysis.)

[0043] I. Sweat collection and microfluidic transport.

[0044] The patch is conformally attached to the skin surface (areas with moderate sweat gland distribution, such as the flexor surface of the forearm and wrist) using medical-grade acrylic pressure-sensitive adhesive. Sweat continuously secreted by the skin's sweat glands is automatically drawn into the microchannels at the three inlets of the microfluidic chip by capillary action. This capillary force originates from the high surface energy wetting effect of the microchannel walls (PDMS, after oxygen plasma treatment, has a surface rich in silanol groups, resulting in a water contact angle <10°) on the aqueous sweat. Capillary pressure drives the sweat to passively collect along the microchannels into the reservoir chamber, which can hold 5.7 µL of sweat, meeting the sample volume (3–5 µL) required for a single electrochemical detection. Excess sweat is discharged through the outlet (2 mm in diameter) to prevent pressure buildup in the reservoir chamber from affecting the stability of the sensing signal.

[0045] II. Electrochemical oxidation detection of bilirubin.

[0046] After sweat enters the reservoir, it completely covers the three-electrode sensing area located directly below the reservoir. The sensing electrodes use differential pulse voltammetry to perform electrochemical quantitative detection of bilirubin in sweat.

[0047] With a stable potential reference provided by the Ag / AgCl reference electrode, the working electrode potential is linearly scanned in a pulsed step manner within the range of 0V to +0.6V (vs. Ag / AgCl). Typical parameters are set as follows: pulse amplitude 50mV, pulse width 50ms, scan rate 10~20mV / s, and sampling period 0.5s. Bilirubin molecules undergo an irreversible electrochemical oxidation reaction with two electrons and two protons on the surface of the p-toluenesulfonic acid-functionalized gold aerogel working electrode.

[0048] III. Analog signal amplification and filtering.

[0049] The weak current signal (typically in the nA to µA range) output by the microfluidic sweat collection and detection chip first enters the preamplifier circuit in the signal processing electronic components. The circuit employs a transimpedance amplifier topology, with a core component being a low-noise operational amplifier (input bias current <1pA, gain-bandwidth product >1MHz), and the transimpedance gain is set to 10. 5 -10 7 The current signal is linearly converted to a voltage signal (mV to V level) at a V / A ratio, with a signal-to-noise ratio >60dB. The voltage signal, after transimpedance amplification, passes through a second-order active low-pass filter, effectively filtering out interference components from power frequency interference (50Hz), motion artifacts (1~5Hz), and high-frequency circuit noise (>100Hz), while retaining the effective low-frequency signal component (0.1~1Hz) of the bilirubin electrochemical response. The filtered signal then enters a subsequent programmable gain amplifier, whose gain is adaptively adjusted (1~128 times) according to the dynamic range of bilirubin concentration, ensuring that the output signal amplitude is always within the optimal input range of the analog-to-digital converter (0~3.3V).

[0050] IV. Analog-to-digital conversion and digital signal processing.

[0051] The conditioned analog voltage signal is input to the ADC input of the microcontroller (MCU). The ADC discretizes the signal at a sampling rate of 100~500Hz, with a resolution of 12 bits (corresponding to a voltage resolution of approximately 0.8mV). The quantized digital signal sequence is then processed by the MCU in the following real-time digital signal processing: Digital filtering: Using a moving average filter (window width 10~50 points) or median filter algorithm, residual random noise interference in the signal is further smoothed; Baseline correction and drift compensation: By performing real-time subtraction on the reference current signal of blank sweat (without bilirubin), baseline drift caused by factors such as temperature drift and electrode aging is eliminated; Concentration conversion: Based on a preset standard working curve (y [current] =0.42x[concentration] +1.13), the filtered and corrected current / voltage signal y [current] Directly convert to bilirubin molar concentration (µmol / L) x [concentration] .

[0052] V. Bluetooth wireless data transmission.

[0053] Real-time bilirubin concentration data, processed by the MCU, is transmitted in data packet format to the paired mobile terminal via the Bluetooth Low Energy (BLE) wireless module integrated on the portable flexible electrochemical workstation. The data packet format includes fields such as timestamp, bilirubin concentration value (µmol / L), sensor ID, battery level, and signal quality identifier. BLE transmission utilizes the 2.4GHz ISM band, with a transmit power of 0dBm, a transmission distance >10m, an adjustable data update frequency of 1~10Hz, and an average operating current <5mA, meeting the low-power, long-endurance requirements of portable battery-powered devices.

[0054] VI. Mobile terminal data display and auxiliary analysis.

[0055] After receiving the BLE data packet, the HY-FlexiSens app running on the paired smartphone displays the real-time bilirubin concentration (µmol / L) on a digital dial on the user interface. The app also supports local storage and cloud synchronization of historical data, allowing users or clinicians to access historical records at any time to view diurnal fluctuation patterns of bilirubin concentration, dynamic changes before and after dietary / exercise events, and to continuously assess liver function and provide risk warnings in conjunction with clinical symptoms.

[0056] Example 3 I. Experimental Methods.

[0057] 1. Preparation of unmodified gold aerogel electrode (control group) A β-cyclodextrin-stabilized gold nanoparticle suspension was synthesized according to the method described in Example 1. An unmodified gold aerogel was obtained by a one-step gelation method using sodium borohydride (i.e., adding 20 mg / mL and 1 mL of sodium borohydride to the above β-cyclodextrin-stabilized gold nanoparticle suspension). After freeze-drying, the unmodified gold aerogel was ultrasonically dispersed in ultrapure water to form a 1 mg / mL suspension. 5 µL of the suspension was dropped onto the surface of the working electrode and dried at 25 °C for 60 min. Then, 3 µL of 1 wt% chitosan solution was added, and the electrode was dried at 25 °C for 2 h to obtain the unmodified gold aerogel working electrode, which was stored at 4 °C for later use.

[0058] 2. p-Toluenesulfonic acid functionalized gold aerogel electrode (experimental group) The p-toluenesulfonic acid-functionalized gold aerogel electrode prepared in Example 1. Except for the different modifying materials, the two sets of electrodes maintained strict consistency in electrode substrate type, modifying solution concentration and volume, drying conditions, and chitosan encapsulation process to eliminate interference from differences in preparation processes on the detection results.

[0059] 3. Preparation of bilirubin standard solution Weigh 10 mg of bilirubin standard and dissolve it in 200 µL of 0.1 mol / L NaOH solution in the dark. After complete dissolution, quickly bring the volume to 10 mL with 0.1 mol / L PBS solution (pH=7.4) to prepare a bilirubin stock solution. Dilute the stock solution serially with PBS solution to prepare the following bilirubin standard concentration series: 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 500, and 1000 µmol / L. All bilirubin solutions should be prepared fresh and used within 30 minutes of preparation. The containers should be wrapped in aluminum foil to protect them from light throughout the preparation and testing process.

[0060] 4. Electrochemical testing conditions All electrochemical tests were performed at 25°C in the dark using an electrochemical workstation (CHI 660E, Shanghai Chenhua Instrument Co., Ltd.), with the control and experimental group electrodes as the testing platform. 50 µL of bilirubin standard solution was added dropwise to cover the three-electrode sensing area, and after standing for 30 seconds to reach diffusion equilibrium, cyclic voltammetry and differential pulse voltammetry tests were started.

[0061] II. Experimental Results.

[0062] like Figure 4 and Figure 5 Cyclic voltammetry results showed that the unmodified gold aerogel electrode produced only a weak oxidation response to bilirubin, while the oxidation peak current of the p-toluenesulfonic acid-functionalized gold aerogel electrode was significantly enhanced, indicating that p-toluenesulfonic acid modification improved the interfacial electron transfer efficiency during the electrochemical oxidation of bilirubin. Figure 6 As shown, the differential pulse voltammetry results further demonstrate that the p-toluenesulfonic acid-functionalized gold aerogel electrode exhibits a significant, stable, and quantifiable peak current change with increasing bilirubin concentration. Its linear range covers 0.01–1000 μmol / L, with a detection limit at the nanomolar level and a sensitivity of 5.99 μA·μM⁻¹·cm⁻².

[0063] Example 4 I. Experimental Methods.

[0064] To demonstrate that p-toluenesulfonic acid modification cannot be replaced by ordinary molecular modification, p-cresol-functionalized gold aerogel, dopamine-functionalized gold aerogel, and p-methylbenzoic acid-functionalized gold aerogel working electrodes were prepared, and cyclic voltammetry and open circuit potential were tested using an electrochemical workstation at the same bilirubin concentration (2 mmol).

[0065] 1. Preparation of gold aerogels modified with different functional molecules Four portions of the same batch of β-CD stable gold nanoparticle suspension (5 mL each, prepared using the same method as in Example 1) were subjected to the following treatments: TsOH group (experimental group): Add TsOH aqueous solution (0.5wt%, 100µL), incubate at 25℃ for 48h to form a gel, wash, freeze dry, and disperse into a 1mg / mL suspension.

[0066] p-Cresol group (control group 1): p-Cresol aqueous solution (0.5wt%, 100µL) was used instead of TsOH, and the same conditions were applied. The phenolic hydroxyl group of p-Cresol has a pKa≈10.2 and hardly dissociates under near-neutral conditions. It mainly relies on the hydrophobic interaction of the aromatic ring to bind to the surface of gold nanoparticles.

[0067] Dopamine group (control group 2): TsOH was replaced with dopamine hydrochloride aqueous solution (0.5wt%, 100µL, prepared with Tris-HCl buffer 10mmol / L, pH=8.5 to promote the self-polymerization and anchoring of dopamine under alkaline conditions). In an alkaline oxygen-containing environment, dopamine can undergo self-polymerization by oxidizing catechol groups to ortho-quinones, forming a polydopamine film that coats the surface of gold nanoparticles, rather than forming a monolayer through physical adsorption. Treatment conditions were adjusted: static incubation at 25℃ for 48h, with complete protection from light to reduce non-specific oxidation of dopamine.

[0068] p-Toluene benzoic acid group (control group 3): TsOH was replaced with sodium p-toluene aqueous solution (0.5 wt%, 100 µL), and the treatment was carried out under the same conditions. The carboxyl group of p-toluene benzoic acid has a pKa of ≈4.36, and it can partially dissociate into carboxylate ions (−COO-) under the pH conditions of the system. ‒ Interface modification is achieved through the coordination or electrostatic interaction between carboxylate groups and the gold surface.

[0069] (2) The subsequent washing (immersion in ultrapure water 3 times, 1h each time), freeze drying (pre-freezing at -80℃ for 2h, vacuum freeze drying for 72h), dispersion (1mg / mL) and electrode modification (dropping 5µL onto the working electrode, fixing 3µL with chitosan, drying conditions as in Example 2) of the four groups of samples were completely consistent, eliminating the interference of preparation process variables.

[0070] II. Experimental Results.

[0071] Experimental results are as follows Figure 7 and Figure 8 As shown, p-toluenesulfonic acid-functionalized gold aerogel exhibits the strongest oxidative response to bilirubin. While p-cresol contains an aromatic ring and phenolic hydroxyl groups, it lacks a strong acidic sulfonic acid group, resulting in lower interfacial recognition ability compared to p-toluenesulfonic acid. Although p-methylbenzoic acid contains carboxyl and aromatic rings, the carboxylic acid group's interfacial interaction and electron transfer-promoting effect on bilirubin are lower than those of the sulfonic acid group. Dopamine contains catechol and amino groups, but it is easily affected by oxidation in complex electrochemical environments, leading to lower detection stability and selectivity compared to p-toluenesulfonic acid.

[0072] The above results indicate that the superior performance of p-toluenesulfonic acid functionalized gold aerogels is due to the synergistic effect of the p-toluenesulfonic acid molecular structure and the porous conductive network of the gold aerogel, and cannot be achieved by any modification with aromatic molecules, phenolic hydroxyl molecules, carboxylic acid molecules or amino molecules.

[0073] Example 5 I. Experimental Methods.

[0074] To verify the selectivity of p-toluenesulfonic acid-functionalized gold aerogel for bilirubin detection, common sweat interfering agents, including 2 mM glucose, 5 mM lactic acid, 5 mM urea, 0.5 mM ascorbic acid, and 0.5 mM uric acid, were added to the detection system, and the open-circuit potential response of the p-toluenesulfonic acid-functionalized gold aerogel electrode was recorded.

[0075] Take a p-toluenesulfonic acid-functionalized gold aerogel electrode, add 50 µL of PBS buffer (0.1 mol / L, pH=7.4) to cover the three-electrode sensing area, connect it to an electrochemical workstation, select the open circuit potential-time (OCP-t) test mode, with a sampling interval of 1 s, and record the OCP baseline until it stabilizes (drift <1 mV / min). After the OCP baseline stabilizes, add the interfering substances and bilirubin to the detection system in the following order: glucose (2 mmol / L, final concentration) – wait for the OCP signal to stabilize after sample addition; lactate (5 mmol / L, final concentration) – wait for stabilization; urea (5 mmol / L, final concentration) – wait for stabilization; ascorbic acid (0.5 mmol / L, final concentration) – wait for stabilization; uric acid (0.5 mmol / L, final concentration) – wait for stabilization; bilirubin (500 µmol / L, final concentration) – wait for stabilization. To further verify the sensor's anti-interference capability in actual electrochemical detection mode (with applied potential), the potentiostatic amperometric method (it) was used for supplementary verification.

[0076] A p-toluenesulfonic acid-functionalized gold aerogel electrode was used, and 50 µL of PBS buffer was added. A constant working potential of +0.30 V (vs. Ag / AgCl) was applied, and the baseline current (it) was recorded until it stabilized. After baseline stabilization, each interfering substance and bilirubin were added sequentially under the same sample addition order and concentration conditions as the OCP test, and the it current-time response curve was continuously recorded. Each addition was spaced 5 min apart to ensure sufficient current response stabilization. The step change in current ΔI (µA) after each addition was used as the response index to compare the differences in current response between the interfering substances and bilirubin. Each test was performed in triplicate, and the average value was taken.

[0077] II. Experimental Results.

[0078] The results are as follows Figure 9 and 10 As shown, the potential signal only changed slightly after the addition of the aforementioned interfering substance, without producing a significant step response similar to that observed with the addition of bilirubin. This result indicates that the p-toluenesulfonic acid-functionalized gold aerogel has a significantly higher adsorption effect on bilirubin than common sweat interfering substances.

[0079] This selectivity stems from the stronger non-covalent interactions and interfacial enrichment capacity between bilirubin molecules and the p-toluenesulfonic acid functionalized interface. Bilirubin contains a large conjugated structure and multiple polar groups, enabling it to form stronger interfacial interactions with the p-toluenesulfonic acid modified interface; while interfering substances such as glucose, lactic acid, urea, ascorbic acid, and uric acid exhibit weak adsorption and electrochemical responses at this interface, thus not significantly affecting bilirubin detection.

[0080] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0081] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0082] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A non-invasive epidermal sweat patch for monitoring bilirubin levels, characterized in that, include: A microfluidic sweat collection and detection chip includes a microfluidic chip layer and a sensing electrode layer. The sensing electrode layer includes an electrode substrate and a working electrode, a counter electrode, and a reference electrode disposed on the surface of the electrode substrate. The surface of the working electrode is modified with p-toluenesulfonic acid-functionalized gold aerogel for recognizing bilirubin. The microfluidic chip layer includes a chip body with channels disposed thereon. The side of the microfluidic chip layer with channels is irreversibly bonded to the side of the sensing electrode layer with electrodes. A flexible portable electrochemical workstation, which is connected to the electrodes of the sensing electrode layer; The p-toluenesulfonic acid functionalized gold aerogel is prepared by the following steps: a chloroauric acid solution with a mass fraction of 5%~15%, β-cyclodextrin, and a sodium borohydride solution with a concentration of 0.5~1.5 mg / mL are mixed and a gold nanoparticle suspension is synthesized by a one-pot wet chemical method. A toluenesulfonic acid aqueous solution with a mass fraction of 0.1%~1% is added dropwise to the gold nanoparticle suspension. After standing for 30~40 hours, the mixture is freeze-dried to obtain the p-toluenesulfonic acid functionalized gold aerogel. The mass-to-volume ratio of the chloroauric acid solution, β-cyclodextrin, sodium borohydride solution, and toluenesulfonic acid aqueous solution is 20~30µL:300~400mg:400~600µL:100µL.

2. The epidermal sweat patch according to claim 1, characterized in that, The microfluidic chip layer is made of PDMS. The flow channel includes an inlet, a reservoir, and an outlet connected in sequence. There are 2 to 4 inlets with a diameter of 0.5 to 1.5 mm. Sweat is driven through the inlets into the reservoir by capillary force. The reservoir has a diameter of 5 to 15 mm. Excess sweat is discharged through the outlet with a diameter of 1 to 3 mm. The depth of the inlet, reservoir, and outlet of the microfluidic chip layer is 0.1 to 0.2 mm.

3. The epidermal sweat patch according to claim 2, characterized in that, The electrode substrate is PDMS, the working electrode and the counter electrode are carbon electrodes, and the reference electrode is an Ag / AgCl electrode.

4. The epidermal sweat patch according to claim 3, characterized in that, The working electrode surface is also modified with a chitosan layer, which covers the outer layer of the p-toluenesulfonic acid functionalized gold aerogel.

5. The epidermal sweat patch according to claim 4, characterized in that, The irreversible bonding encapsulation includes hydrophilic treatment of the side of the microfluidic chip layer with flow channels and the side of the sensing electrode layer with electrodes.

6. The epidermal sweat patch according to claim 5, characterized in that, The hydrophilic treatment employs oxygen plasma treatment under the following conditions: oxygen flow rate 10~20 sccm, radio frequency power 30~50W, cavity vacuum degree 0.3~0.5 Torr, and treatment time 40~60s.

7. The method for preparing the epidermal sweat patch according to claim 6, characterized in that, Includes the following steps: S1. Mix PDMS and curing agent evenly at a mass ratio of 5~15:1, degas under vacuum, pour into a mold, cure at 50~70℃, and obtain a microfluidic chip layer after demolding. S2. The p-toluenesulfonic acid functionalized gold aerogel is drop-coated onto the surface of the working electrode in the form of a dispersion of 0.5~1.5 mg / mL, with a drop volume of 4~6 µL. Then, a chitosan layer is modified on the outer layer of the p-toluenesulfonic acid functionalized gold aerogel to obtain the sensing electrode layer. S3. After irreversibly bonding and encapsulating the microfluidic chip layer with the flow channel and the sensing electrode layer with the electrode, a microfluidic sweat collection and detection chip is obtained. The sweat introduced from the inlet gathers in the storage cavity and can completely cover the working electrode, the counter electrode and the reference electrode. The sensing electrode uses differential pulse voltammetry to perform electrochemical quantitative detection of bilirubin in the sweat and outputs an electrical signal. S4. Connect the electrodes of the sensing electrode layer in the microfluidic sweat collection and detection chip to the flexible portable electrochemical workstation through the electrode interface to obtain an epidermal sweat patch.

8. The use of the epidermal sweat patch according to any one of claims 1 to 6 in the preparation of a device for monitoring liver disease, adverse drug reactions, postoperative recovery assessment, cholestasis monitoring, or neonatal / infant jaundice risk screening.

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