A radial multilayer microneedle sensing system and method for single hair follicle unit microenvironment analysis

Abstract

The application discloses a radial multilayer microneedle sensing system and method for single follicular unit microenvironment analysis, and belongs to the field of skin appendage detection and biological micro-electro-mechanical systems. The system comprises a positioning hole integrated with an optical collimation unit, concentric ring microneedles with a length ratio within a certain range, ring compression protruding structures between the microneedles, a differential negative pressure sampling module and a detection electrode modified with an aptamer probe, which can accurately position the follicle, perform layered puncture, construct a hydraulic barrier to prevent cross-flow, isolate and extract interstitial fluid between deep and shallow layers and target detection of disease markers such as androgen alopecia. The method comprises the steps of optical targeting positioning, compression sealing puncture, differential sampling, in-situ detection and gradient index calculation. The application can improve the spatial detection resolution of the markers, solve the problems of signal dilution and subcutaneous cross-flow, and provide a quantitative tool for follicular pathological research and drug non-diagnostic screening.

Description

Technical Field

[0001] This invention relates to the fields of skin appendage detection, wearable microfluidic devices and bio-microelectromechanical systems, and particularly to a radial multilayer microneedle sensing system and method for microenvironmental analysis of a single hair follicle unit. Background Technology

[0002] Hair follicles are highly heterogeneous microorganisms with complex self-renewal cycles. Under normal physiological conditions, terminal hairs on the human scalp are deeply embedded in the subcutaneous tissue. Different longitudinal anatomical layers (such as the superficial follicular infundibulum and the deep dermal papilla) exhibit significant spatial depth differences in biochemical metabolism, paracrine signal regulation, and cell receptor expression (Shim J, Park J, Abudureyimu G, et al. Comparative Spatial Transcriptomic and Single-Cell Analyses of Human Nail Units and Hair Follicles Show Transcriptional Similarities between the Onychodermis and Follicular Dermal Papilla[J]. Journal of Investigative Dermatology, 2022, 142(12): 3146-3157.e12.). In the pathological evolution of many hair degenerative diseases, especially androgenetic alopecia, the homeostasis of this spatial biochemical microenvironment is severely disrupted (Cuevas-Diaz Duran R, Martinez-Ledesma E, Garcia-Garcia M, et al. The Biology and Genomics of Human Hair Follicles: A Focus on Androgenetic Alopecia[J]. International Journal of Molecular Sciences, 2024,25(5): 2542.).

[0003] (a) The necessity of individual hair follicle testing Most existing scalp clinical biopsy and interstitial fluid extraction techniques rely on "group sampling" of macroscopic areas, such as using a uniform-length array of planar microneedles to perform overall aspiration of a scalp area of ​​several square centimeters (Sinclair R, Jolley D, Mallari R, et al. Morphological Approach to Hair Disorders[J]. Journal of Investigative Dermatology Symposium Proceedings, 2003, 8(1): 56-64.). However, one of the core histological features of AGA is the alteration and progressive miniaturization of the hair follicle cycle, and this process does not occur uniformly and synchronously throughout the scalp, but rather exhibits high spatial and temporal heterogeneity. In the baldness areas of patients in the early and middle stages of AGA, three different states of hair follicles coexisted on the scalp (Khunkhet S, Chanprapaph K, Rutnin S, et al. Histopathological Evidence of Occipital Involvement in Male Androgenetic Alopecia[J / OL]. Frontiers in Medicine, 2021, 8[2026-03-24].): 1. Healthy terminal hairs: diameter greater than 60 μm, dermal papillae depth up to 3.0 to 4.5 mm.

[0004] 2. Critical hair in the pathological transition period: with a diameter between 40 and 60 μm, and the dermal papilla begins to recede upwards.

[0005] 3. Completely degenerated lanugo: diameter less than 40 μm, depth less than 1.0 mm.

[0006] Diseased hair follicles and their adjacent healthy primary hair follicles may coexist in the same follicular unit (Sinclair R. Androgenetic alopecia. Modelling progression and regrowth[J]. Experimental Dermatology, 2016, 25(6): 424-425.). If traditional regional macroscopic interstitial fluid extraction is used, only a "mixed average concentration" at a certain depth in the subcutaneous tissue is obtained. This severe spatial averaging effect nonspecifically mixes normal signals from healthy hair follicles with pathological signals from diseased hair follicles, thereby diluting and masking the weak pathogenic signals emitted by individual hair follicles in the early stages of disease. Therefore, isolating the signal interference from surrounding normal tissue and focusing the detection field from "macroscopic scalp area" to "single hair follicle microenvironment" is a key technical approach to accurately capture pathogenic molecules in the very early stages of hair loss and evaluate the microscopic response of single targets to drugs (such as 5α-reductase inhibitors, minoxidil, etc.). Furthermore, existing technologies using two spatially separated microneedle units for stratified sampling also fail to achieve highly specific and accurate capture of pathological signals from a single hair follicle unit. The sampling ranges of the two sets of units typically belong to different hair follicle units, easily leading to spatial misalignment sampling: shallow samples are taken from hair follicle units containing secondary hair follicles with miniaturized lesions, while deep samples are taken from adjacent hair follicle units containing healthy primary hair follicles. This makes it difficult to guarantee that the shallow and deep samples originate from the same target hair follicle unit, differing only in sampling depth across different longitudinal anatomical levels. Consequently, the obtained detection data cannot objectively reflect the true pathophysiological state of a single target hair follicle unit, and the core technical problem of easily masking weak pathogenic signals in the early stages of disease and distorted detection results remains unresolved.

[0007] (II) Spatial heterogeneity and specific types of biomarkers for androgenic alopecia Numerous molecular biology and transcriptomics studies have shown that the core pathological mechanism of AGA (Awriggs Aging) is androgen-driven dermal papillary cell dysfunction and follicle miniaturization. During this process, key biomarkers in the follicular microenvironment not only exhibit abnormal concentrations but also show significant spatial concentration gradients. This system primarily targets and detects the following key biomarkers: Dihydrotestosterone (DHT): DHT is the initiating factor for alopecia-associated gyrus (AGA). The dermal papilla and outer root sheath of deep hair follicles are rich in type II 5α-reductase, which can convert testosterone in the circulation system into DHT, which has a very strong affinity for androgen receptors (AR) (5 times that of testosterone). The high local concentration of DHT in the deep dermal papilla is the root cause of hair follicle miniaturization (Anonymous. Molecular mechanisms of androgenetic alopecia[J]. Experimental Gerontology,2002, 37(8-9): 981-990.).

[0008] Prostaglandin D2 (PGD2): After DHT binds to AR, it triggers a downstream cascade reaction, significantly upregulating the expression of prostaglandin D2 synthase (PTGDS) in deep cells, leading to an explosive increase in PGD2 concentration in the deep region of hair follicles (i.e., the hair bulb and the area around the dermal papilla) (PGD2 levels in the lesion area of ​​AGA patients are more than 3 times higher than in the normal area). High concentrations of PGD2 inhibit the Wnt / β-catenin signaling pathway by binding to G protein-coupled receptor 44 (GPR44 / DP2), blocking hair growth and forcing hair follicles to enter the regression phase prematurely (Garza LA, Liu Y, Yang Z, et al. Prostaglandin D2 Inhibits HairGrowth and Is Elevated in Bald Scalp of Men with Androgenetic Alopecia[J].Science Translational Medicine, 2012, 4(126): 126ra34-126ra34.).

[0009] Transforming growth factor β2 (TGF-β2): Under the stimulation of DHT, the deep dermal papilla secretes TGF-β2, which acts as a paracrine signal to induce apoptosis of hair matrix cells and accelerate the shortening of the hair follicle cycle (Hibino T, Nishiyama T. Role of TGF-β2 in the human hair cycle[J]. Journal of Dermatological Science,2004, 35(1): 9-18.).

[0010] Vascular endothelial growth factor (VEGF): During the hair growth phase, the hair growth depends heavily on the dense capillary bed around the hair bulb for nutrition. In the course of AGA, the expression of deep angiogenesis-promoting VEGF is significantly reduced, leading to the degeneration of the hair papilla microvascular network (Yano K, Brown LF, Detmar M. Control of hair growth and follicle size by VEGF-mediated angiogenesis[J]. Journal of Clinical Investigation, 2001, 107(4): 409-417.).

[0011] Superficial microinflammatory markers (IL-1, IL-6, TNF-α, MCP-1, etc.): Although AGA has traditionally been considered a non-inflammatory alopecia, a large amount of histological and spatial transcriptomic evidence suggests that there is chronic low-grade microinflammation and immune cell infiltration in the superficial layer of the hair follicle (especially the area around the infundibulum and isthmus) (Charoensuksira S, Tantiwong S, Pongklaokam J, et al. Disturbance of Immune Microenvironment in Androgenetic Alopecia through Spatial Transcriptomics[J]. International Journal of Molecular Sciences, 2024, 25(16): 9031.;Plante J, Valdebran M, Forcucci J, et al. Perifollicular inflammation and follicular spongiosis in androgeneticalopecia[J]. Journal of the American Academy of Dermatology, 2022, 86(2):437-438.). Within this specific depth region, local immune responses and abnormal sebaceous gland metabolism release large amounts of pro-inflammatory cytokines, such as interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and monocyte chemoattractant protein-1 (MCP-1). These superficial inflammatory factors constitute important microenvironmental features of early AGA pathogenesis and play a key role in inducing perifolliculitis fibrosis and superficial stem cell damage.

[0012] In summary, the key pathogenic biochemical events that trigger hair loss (such as elevated DHT and PGD2 levels) and superficial immune response events (such as elevated IL-6 and TNF-α levels) exhibit distinctly different spatial distributions. Existing uniform-length microneedle technology cannot capture this "superficial-deep" gradient difference, making it difficult to comprehensively analyze the spatial dynamics of hair loss pathogenesis.

[0013] (III) Defects of existing stepped microneedle arrays at the hydrodynamic level To overcome the limitations of single-length microneedles, some studies have begun to explore non-uniform length microneedle arrays composed of needles of different lengths, attempting to achieve layered targeting through differentiation of physical depth (Barnum L, Quint J, Derakhshandeh H, et al. 3D-Printed Hydrogel-Filled Microneedle Arrays[J].Advanced Healthcare Materials, 2021, 10(13): 2001-922.). However, when performing deep gradient interstitial fluid aspiration in a real physiological environment, this design suffers from hydrodynamic defects that are difficult to overcome using conventional techniques: The dermis is not a free-flowing, homogeneous fluid pool, but rather a porous, elastic medium filled with collagen fibers, glycosaminoglycans, and containing a large amount of non-covalently bound water. When microneedles of varying lengths share the same system substrate to aspirate interstitial fluid, tissues at different depths exhibit drastically different fluid permeability resistances. The superficial epidermis and papillary dermis have relatively loose structures and lower flow resistance; while the deep reticular dermis has an extremely dense collagen network and extremely high flow resistance.

[0014] According to the physical laws of fluid flow in porous media, under negative pressure, fluids always tend to move along the path of least resistance. If a uniform negative pressure is applied to microneedles of varying lengths, the strong vacuum suction will inevitably cause a large amount of shallow fluid (low flow resistance) to rush into the deep needle pores, or cause lateral flow and cross-mixing of shallow and deep fluids in the subcutaneous matrix. This cross-contamination at the fluid dynamics level disrupts the original spatial concentration gradient of biomarkers, making it impossible to achieve the original design intent of "stratified extraction," and ultimately detecting contaminated and distorted data after averaging. Summary of the Invention

[0015] To address the problems of existing technologies, this invention provides a radial multilayer microneedle sensing system and non-diagnostic analysis method that performs optical positioning of a single hair follicle unit, performs in-situ layered sampling without crossflow, and generates spatial concentration gradient differential signals of androgenetic alopecia (AGA) related biomarkers through differential negative pressure. By biomimetic adaptation design of the three-dimensional anatomical morphology of the hair follicle (the inner longer and outer shorter design resembles an inverted cone shape in space, matching the funnel-shaped structure of the hair follicle), defining the microneedle length ratio (L1 / L2), and introducing a physical compression anti-crossflow sealing mechanism based on Darcy's law, the problem of subcutaneous lateral crossflow is solved, enabling in-situ layered sampling and biomarker gradient differential analysis of a single targeted hair follicle.

[0016] To achieve the objectives of this invention, the present invention provides a radial multilayer microneedle sensing system for microenvironment analysis of a single hair follicle unit, comprising: The hair follicle unit positioning module is used to determine the spatial location of the target hair follicle opening; The radial annular multilayer microneedle array module includes a first annular microneedle group and a second annular microneedle group that are concentrically and coaxially distributed around a central axis, and the microneedle length L1 of the first annular microneedle group is greater than the microneedle length L2 of the second annular microneedle group. The anti-flow sealing module includes an annular compression protrusion structure disposed on the bottom surface of the system substrate between the first annular microneedle group and the second annular microneedle group, which is used to block the lateral flow of interstitial fluid between deep and superficial tissues under negative pressure aspiration. The multi-layer independent sampling channel module includes a first annular passage connected to a first annular microneedle group and a second annular passage connected to a second annular microneedle group. The first annular passage and the second annular passage are physically isolated from each other in the fluid path. The detection module includes a first detection unit and a second detection unit, which are fluidly connected to the first annular pathway and the second annular pathway, respectively, and are used to specifically identify biomarkers collected from interstitial fluid at different depths and generate detection signals. The negative pressure sampling power module is connected to the drain end of the first annular passage and the second annular passage respectively, and is configured to provide differentiated negative pressure driving force according to the different tissue fluid resistances corresponding to the first annular passage and the second annular passage. The signal processing unit, which is connected in communication with the detection module, is used to calculate and output data characterizing the spatial gradient features of the microenvironment of a single hair follicle unit based on the received detection signals at different depths.

[0017] Furthermore, the detection module is configured to specifically identify at least two of the following biomarkers: dihydrotestosterone (DHT), prostaglandin D2 (PGD2), transforming growth factor β2 (TGF-β2), vascular endothelial growth factor (VEGF), interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and monocyte chemoattractant protein-1 (MCP-1).

[0018] Furthermore, the hair follicle unit positioning module includes a positioning hole disposed on the system substrate and an optical auxiliary collimation unit disposed in the positioning hole; The positioning hole is used to define the location of the target hair follicle opening and to form a limiting area that fits the skin surface around the central positioning hole, so that the central axis of the positioning hole substantially coincides with the extension axis of the target hair follicle opening. The optical collimation unit includes a light-emitting diode for obliquely illuminating the area around the target hair follicle opening, and a lens for capturing high-contrast stereoscopic artifact enhancement images of the local skin surface. This unit locks the opening position and surrounding surface structure features of the pre-selected target hair follicle through real-time dynamic optical feedback, assisting in achieving precise micron-level alignment between the central axis and the target hair follicle opening axis, thereby avoiding the signal dilution effect caused by group sampling.

[0019] Furthermore, the first annular microneedle group and the second annular microneedle group are concentrically and coaxially distributed around the central axis of the positioning hole.

[0020] Furthermore, a physical isolation wall is provided between the first annular passage and the second annular passage to physically isolate them from each other in the fluid path.

[0021] Furthermore, the annular compression protrusion structure is a continuous concentric circular protrusion integrally formed on the system base, made of rigid or semi-rigid biocompatible material, with a radial width W of 0.1 to 0.12 mm and a downward protrusion height of 50 to 150 μm; the annular compression protrusion structure can significantly reduce the hydraulic conductivity of the pressure area by squeezing the skin tissue, forming a high flow resistance hydraulic sealing isolation zone in situ.

[0022] Furthermore, the microneedle length L1 of the first annular microneedle group and the microneedle length L2 of the second annular microneedle group satisfy 1.3≤L1 / L2≤2.5, and the value range of L1 is 500 to 900 μm, and the value range of L2 is 300 to 500 μm.

[0023] Furthermore, the radius of the needle placement circle of the first annular microneedle group is R1, and the radius of the needle placement circle of the second annular microneedle group is R2, where R1 is 0.32 to 0.34 mm, R2 is 0.74 to 0.78 mm, and the difference between the two, R2-R1, is set to 0.40 to 0.46 mm, so as to accurately cover the spatial anatomical depth difference between the superficial infundibulum and the deep dermal papilla of a single hair follicle at different miniaturized pathological stages, while avoiding destructive stress concentration on the epidermis; the bottom radius of the first annular microneedle is r1, and the bottom radius of the second annular microneedle is r2; the radial width of the annular compression protrusion structure is W; the radius of the positioning hole is R0; where r1 is 120 to 150 μm, r2 is 70 to 90 μm, W is 0.10 to 0.12 mm, and R0 is 0.05 to 0.15 mm; and the system satisfies the geometric constraint: R2-R1>r1+r2+W.

[0024] Furthermore, the first annular microneedle group includes 3 to 6 microneedles arranged at equal intervals, and the second annular microneedle group includes 6 to 12 microneedles arranged at equal intervals; and the physical distance between the tips of adjacent microneedles within the same annular microneedle group is limited to the range of 400 μm to 1000 μm.

[0025] Furthermore, both the first detection unit and the second detection unit include microfluidic detection chambers respectively disposed in the first annular pathway and the second annular pathway. Each microfluidic detection chamber is provided with a reagent-free electrochemical sensor. The reagent-free electrochemical sensor includes a detection electrode. The surface of the detection electrode is modified with a biochemical blocking layer for resisting nonspecific adsorption of proteins and covalently coupled with a nucleic acid aptamer probe for specific binding to biomarkers.

[0026] Preferably, the detection electrode is located at the bottom of the microfluidic detection chamber; the detection module is a reagent-free electrochemical sensor, including a detection electrode located at the bottom of the microfluidic detection chamber; the surface of the detection electrode is self-assembled with a biochemical blocking layer of 6-mercapto-1-hexanol (MCH) or bovine serum albumin (BSA) to prevent non-specific adsorption.

[0027] Preferably, the microfluidic detection chamber is connected in series between the microneedle and the negative pressure sampling power module, so that the upward unidirectional flow of interstitial fluid completely covers the detection electrode.

[0028] Furthermore, the negative pressure sampling power module includes a passive flow resistance adjustment structure, at least one pre-vacuum negative pressure source, and an active dual-path feedback gas path connected to the pre-vacuum negative pressure source. The passive flow resistance regulation structure includes a miniature check valve to prevent fluid backflow and a static flow resistance regulation micro-column array, and an active dual-path feedback gas path with a first negative pressure regulation branch and a second negative pressure regulation branch; wherein, The second annular passage is equipped with the static flow resistance regulating micropillar array to weaken the effective negative pressure in the passage of the shallow branch through physical throttling, thereby applying differentiated suction forces to the shallow and deep tissues under the same source drive. The first and second negative pressure control branches are each equipped with the aforementioned miniature check valve, miniature proportional solenoid valve, and pressure sensor connected in series. They are controlled in a closed loop by a signal processing unit to apply a negative pressure with an absolute value greater than that of the second annular channel to the first annular channel, thereby compensating for the extremely high hydrodynamic resistance of the deep reticular dermis layer caused by the dense extracellular matrix.

[0029] Preferably, the absolute value of the opening threshold pressure of the miniature check valve located in the first annular passage is smaller than that of the miniature check valve located in the second annular passage.

[0030] Furthermore, a physical isolation wall is provided between the first annular channel and the second annular channel to physically isolate them from each other in the fluid path. Preferably, the surface of the physical isolation wall and the inner wall of the annular channel are both provided with a superhydrophobic coating to ensure that the shallow sample and the deep sample do not cross-mix or diffuse contamination.

[0031] Furthermore, the follicular depth feature index (FDCI) built into the signal processing unit is calculated using the following formula: S1 and S2 are respectively the deep concentration signal calculated and converted by the signal processing unit based on the electrical signal output by the first detection unit and the shallow concentration signal calculated and converted based on the electrical signal output by the second detection unit for the same specific biomarker; the signal processing unit is configured to: extract the deep concentration signal S1 and the shallow concentration signal S2 synchronously acquired from the same hair follicle unit for each type of biomarker, and substitute this set of data into the formula for independent differential calculation.

[0032] The present invention provides a non-diagnostic analysis method comprising the following steps: The central axis of the radial sensing system is aligned with the opening of the target single hair follicle by using the hair follicle unit positioning module. Pressing the sensing system downwards on the skin surface causes it to adhere tightly to and compress the epidermal surface. The annular compression protrusion structure applies directional mechanical stress to the porous elastic medium under the skin, forming a high flow resistance hydraulic sealing isolation zone with low hydraulic conductivity in situ in the pressure area. At the same time, the first and second annular microneedle groups are inserted into the deep dermal papilla area and the superficial infundibulum of the target single hair follicle, respectively. The negative pressure sampling power module is activated to apply differentiated negative pressure to the first and second annular channels to aspirate interstitial fluid from the deep and shallow layers, respectively. The same target biomarker was detected in the deep and shallow interstitial fluids flowing into the first and second annular pathways, respectively, by the detection module, and the deep concentration signal S1 and the shallow concentration signal S2 were obtained respectively. The signal processing unit calculates and outputs the hair follicle depth characteristic index, which characterizes the longitudinal biochemical gradient of the microenvironment, based on the deep concentration signal S1 and the shallow concentration signal S2.

[0033] Compared with the prior art, the present invention can achieve at least the following beneficial effects: (1) It helps reduce blind spots in group sampling and facilitates precise analysis at the micro-organ level: This invention integrates optical visual positioning and alignment structure for the first time, which can achieve tubular three-dimensional encircling minimally invasive puncture for pre-selected single hair follicle units, effectively avoiding the signal aliasing and dilution problems of adjacent healthy hair follicles that are difficult to avoid in large-area array sampling.

[0034] (2) Based on porous media mechanics, fluid crossflow suppression is achieved to ensure the fidelity of spatial signal detection: This invention innovatively utilizes an annular compression protrusion structure to exponentially reduce the permeability of local tissues. With the help of Darcy flow blocking effect, a high-resistance hydraulic sealing barrier is constructed in situ at the purely physical level, which is conducive to improving the collection specificity of deep and shallow samples.

[0035] (3) Precisely adapted to tissue resistance and anatomical morphology, enabling one-time deep and superficial layered sampling: The design of the L1 / L2 value range [1.3, 2.5] can match the spatial depth difference characteristics between different anatomical layers of hair follicles, and can effectively capture deep DHT / PGD2 lesion areas and superficial sebaceous gland peri-IL. 6. Related micro-inflammatory areas; combined with differentiated negative pressure compensation technology of active closed loop or passive throttling, it can improve problems such as poor suction or flow imbalance caused by the dense deep reticular dermal structure. Attached Figure Description

[0036] Figure 1 This is a cross-sectional view of the integrated structure of the radial multilayer microneedle sensing system and the microfluidic optical path in an embodiment of the present invention.

[0037] Figure 2 This is a top view of the radial multilayer microneedle sensing system structure in an embodiment of the present invention.

[0038] Figure 3 This is an enlarged schematic diagram illustrating the mechanism by which the annular compression protrusion structure blocks subcutaneous lateral flow of fluid dynamics in an embodiment of the present invention.

[0039] Figure 4 This is a schematic diagram showing the structural connection and flow path between the detection module, the microfluidic detection chamber, and the negative pressure sampling power module.

[0040] Figure 5 A flowchart illustrating the workflow for calculating the Follicle Depth Characteristic Index (FDCI) and performing closed-loop negative pressure regulation for the signal processing module.

[0041] Among them, 1. First annular microneedle; 2. Second annular microneedle; 3. Annular compression protrusion structure; 4. First annular passage; 5. Second annular passage; 6. Positioning hole; 7. Lens; 8. Light-emitting diode; 9. Miniature vacuum pump; 10. Detection electrode; 11. Check valve; 12. Waste liquid storage sponge; 13. System substrate; 14. Pressure sensor; 15. Miniature proportional solenoid valve; 16. Static flow resistance adjustment micro-pillar array. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0043] This invention provides a radial multilayer microneedle sensing system for spatial gradient difference detection of the microenvironment of a single hair follicle unit, the core hardware topology and microfluidic mechanism of which are as follows: The hair follicle unit positioning module includes a miniature optical collimation unit and a positioning hole 6 integrated at the center of the system base 13. The positioning hole 6 defines the position of the target hair follicle opening, and a limiting area that conforms to the skin surface is formed around the positioning hole 6 on the skin-facing side of the system base 13, so that the central axis of the positioning hole 6 substantially coincides with the extension axis of the target hair follicle opening. The miniature optical collimation unit includes a miniature light-emitting diode 8 for obliquely illuminating the periphery of the target hair follicle opening and a miniature gradient refractive index lens 7 for capturing a high-contrast stereoscopic artifact-enhanced image of the local skin surface. Both the light-emitting diode 8 and the lens 7 are located within the positioning hole 6, with the lens 7 located at the bottom of the positioning hole 6. The asymmetric oblique illumination of the light-emitting diode 8 can avoid strong reflections from the stratum corneum from obscuring the hair shaft details, and the configured lens 7 can utilize near-field optical characteristics to capture a high-contrast image of the hair follicle opening with three-dimensional artifact enhancement. With real-time visual feedback, the operator can accurately distinguish clusters of healthy hair follicles and precisely align the central axis of the microneedle with the opening of a single target hair follicle that is in a critical miniaturization state, thus solving the signal dilution problem of non-specific sampling of groups from the root.

[0044] The radial annular multilayer microneedle array module includes a first annular microneedle group and a second annular microneedle group, which are concentrically and coaxially distributed around a central axis. The first annular microneedle group includes multiple spaced-apart first annular microneedles 1, and the second annular microneedle group includes multiple spaced-apart second annular microneedles 2. The microneedle length of the first annular microneedle 1 is L1, and the microneedle length of the second annular microneedle 2 is L2, with L1 being greater than L2. This allows the first and second annular microneedle groups to penetrate the deep dermal papilla and superficial infundibulum of a single hair follicle at the same spatial center and time point. To simultaneously access the "superficial micro-inflammatory environment" and "deep core lesion" of a single hair follicle, this system uses first annular microneedles 1 (long needles, length L1) and second annular microneedles 2 (short needles, length L2) concentrically and coaxially distributed around the central axis of the system base 13 (the central axis of the positioning hole 6). In one embodiment, based on follicular morphology and subcutaneous fluid dynamics analysis, the L1 / L2 ratio is limited to the critical scientific range of [1.3, 2.5], with L1 set to 500-900 μm and L2 to 300-500 μm. The rationale for this design is as follows: (a) Biological Anatomical Depth Match: The dermal papilla of normal terminal hair on the human scalp is typically located 3.0 to 4.5 mm deep subcutaneously. However, when AGA occurs and the hair follicle becomes progressively miniaturized, the hair bulb and dermal papilla retract and shift upwards towards the epidermis to a depth of 1.0 to 1.5 mm or even shallower. The depth of the superficial pathological activity area (follicular infundibulum and sebaceous isthmus) that produces pro-inflammatory factors is almost fixed, usually maintained between 0.3 and 0.8 mm. Therefore, the first circular microneedle 1 is used to precisely penetrate the matrix layer rich in markers such as DHT and PGD2 around the deep dermal papilla during the miniaturization process; while the second circular microneedle 2 is used to stably extract pro-inflammatory cytokines (such as IL-1 and IL-6) from the superficial infundibulum.

[0045] (b) Spatial Distribution of the Microneedle Array: This embodiment of the invention employs an arrangement of "long inner ring needles and short outer ring needles." This "long center, short periphery" structure anatomically suits the characteristics of narrow deep lesions and extensive superficial inflammation in hair follicles. More importantly, it has the advantage of "central anchoring first, followed by peripheral insertion" in terms of puncture mechanics. During puncture, the long inner ring needles penetrate the skin first, fixing the center point of the target hair follicle. As the probe is pressed down, the short outer needles, with the central scalp already tightened and fixed, then smoothly penetrate the superficial tissue. This design avoids the mechanical defect of the short outer needles being pulled outwards by the force, causing the central area to sink downwards and preventing the long inner needles from penetrating. This design ensures that the microneedles and compression rings fit tightly against the skin, providing a reliable physical seal for preventing cross-flow sampling.

[0046] (c) Microneedle structure and material adaptation: If the L1 / L2 ratio is less than 1.3, the longitudinal spatial difference between the long and short needles is too small, and the puncture depth difference is insufficient to effectively cross the different biochemical metabolic zones of the hair follicle. The obtained interstitial fluid is highly homogeneous and cannot form an effective differential signal. If the ratio is greater than 2.5, when performing skin puncture, in order to ensure that the inner ring long needle is completely inserted into the target deep tissue, the bottom surface of the system base 13 needs to be pressed down more significantly. This will cause the outer ring short needle to excessively penetrate the skin and penetrate the target superficial area. At the same time, the system base excessively compresses the epidermis, causing pain to the patient, or causing uneven force on the microneedles, resulting in bending and breakage. To avoid the 'bed of nails effect' during puncture and ensure independent force and effective puncture depth for each microneedle, the system in this embodiment of the invention implements coordinated constraints on the number and spacing of microneedles based on the radial circumference of the ring and the safety spacing: the first annular microneedle group includes 3 to 6 long needles arranged at equal intervals, and the second annular microneedle group includes 6 to 12 short needles arranged at equal intervals, with the physical distance between the tips of adjacent microneedles within the same ring limited to the range of 400 μm to 1000 μm. Further, in one embodiment, to balance the mechanical strength of the needle body and the skin penetration capability under the condition of the liquid retrieval channel setting, the bottom radius r1 of the first annular microneedle 1 is preferably 120 to 150 μm, and the bottom radius r2 of the second annular microneedle 2 is preferably 70 to 90 μm. Based on the bottom diameter, the aspect ratio of the first annular microneedle 1 is controlled at approximately 1.67 to 3.75, and the aspect ratio of the second annular microneedle 2 is controlled at approximately 1.67 to 3.57. By limiting the aspect ratio within the aforementioned range, the risk of bending, kinking, or breakage due to excessively thin needles can be reduced while ensuring the tip's penetration capability. Furthermore, the first annular microneedle 1 and the second annular microneedle 2 are made of biocompatible materials with high mechanical strength and chemical inertness. Preferred materials include, but are not limited to, medical-grade polycarbonate, monocrystalline silicon, stainless steel alloys, SU-8 photoresist, or high-strength biodegradable polymers. These preferred materials ensure that, even with hollow or porous extraction channels internally, the microneedles still possess sufficient mechanical strength to safely penetrate the stratum corneum of the skin, and do not cause non-specific interference with the biochemical activity of the extracted interstitial fluid and internal biomarkers. In this embodiment of the invention, the lengths of all microneedles within the same annular microneedle group are configured to be equal. Due to limitations in the precision of existing micro-nano fabrication processes, normal manufacturing tolerances objectively exist for microneedles. These differences in the length of individual microneedles caused by manufacturing tolerances do not affect their technical effect of penetrating the target depth and are all within the scope of protection of this invention.

[0047] (d) Anatomical boundary definition of radial distance: According to clinical anatomical statistics of the scalp, in the healthy scalp area, the physical distance between adjacent hair follicle units is approximately 0.5 to 1.0 mm; while in the area where AGA miniaturization lesions occur, due to the retraction and dormancy of some hair follicles, the distance between adjacent diseased hair follicle units is increased to 1.5 mm or even wider. In one embodiment, to ensure that the system of this embodiment achieves targeted three-dimensional encapsulation of only a single target hair follicle unit and avoids cross-contamination of interstitial fluid between adjacent hair follicle units during sampling, the radial distance between the annular microneedles and the central axis of the positioning hole 6 located in the center is defined as follows: the needle radius R1 of the first annular microneedle group (long needle) is preferably 0.32 to 0.34 mm, which can accurately reach the peripheral matrix area of ​​the deep dermal papilla of the target hair follicle unit; the needle radius R2 of the second annular microneedle group (short needle) is preferably 0.74 to 0.78 mm, which can tightly surround and fit the peripheral tissue of the superficial infundibulum of the target hair follicle unit. Meanwhile, this system limits the maximum diameter of the needle circle of the second annular microneedle group (i.e., 2R2) to within 1.56 mm. Further considering the physical structure of the bottom surface of a single microneedle, the maximum radius of the outermost physical edge of the entire microneedle array from the central axis is R2+r2, and its corresponding maximum physical outer diameter (i.e., 2(R2+r2)) is limited to within 1.74 mm. In addition, the radius R0 of the positioning hole 6 located at the center of the system is limited to the range of 0.05 to 0.15 mm. This lower limit ensures that the positioning hole can accommodate hair shafts and follicle openings at different stages; this upper limit is constrained by the geometric inequality R1-r1>R0, ensuring sufficient physical support spacing between the edge of the positioning hole and the first annular microneedle group, thereby guaranteeing the overall mechanical structural integrity of the system.

[0048] The multi-layer independent sampling channel module includes a first annular channel 4 connected to a first annular microneedle group and a second annular channel 5 connected to a second annular microneedle group. A physical isolation wall is provided between the first annular channel 4 and the second annular channel 5 to physically isolate them from each other in the fluid path. In one embodiment, the physical isolation wall is disposed between the first annular channel 4 and the second annular channel 5, and the surface of the physical isolation wall and the inner wall of the annular channel are coated with polytetrafluoroethylene or functionalized superhydrophobic coating. The fluid network of the system uses a physical isolation wall to ensure insulation and non-communication between the two layers, and to ensure that shallow samples and deep samples do not cross-mix or diffuse contamination.

[0049] The detection module includes a first detection unit and a second detection unit. The first detection unit includes a microfluidic detection chamber embedded in a first annular pathway 4 and a reagent-free microelectrochemical sensor. The second detection unit includes a corresponding microfluidic detection chamber embedded in a second annular pathway 5 and a reagent-free microelectrochemical sensor. The reagent-free microelectrochemical sensor includes a detection electrode 10 located at the bottom of the corresponding microfluidic detection chamber. The detection electrode 10 is capable of contacting the fluid within the corresponding annular pathway, and its surface is modified with a dense biochemical blocking layer to resist non-specific protein adsorption. It is also covalently coupled with a nucleic acid aptamer probe that specifically binds to the biomarker. The detection module is used to specifically identify the biomarker and obtain an electrical signal positively correlated with the biomarker concentration. In one embodiment, the detection module is capable of specifically recognizing at least two biomarkers from the following group: dihydrotestosterone (DHT), prostaglandin D2 (PGD2), transforming growth factor β2 (TGF-β2), vascular endothelial growth factor (VEGF), interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and monocyte chemoattractant protein-1 (MCP-1). In one embodiment, the detection electrode surface is self-assembled with a dense biochemical blocking layer of 6-mercapto-1-hexanol (MCH) or bovine serum albumin (BSA) to prevent non-specific adsorption. The detection electrode 10 is covalently coupled with nucleic acid aptamer probes that specifically target DHT, PGD2, IL-6, and TNF-α, etc. To ensure the biochemical stability of this system as an in vitro analytical device during long-term storage and transportation, the nucleic acid aptamer probes are chemically modified to resist nuclease degradation. Furthermore, in the factory-packaged state, the microfluidic detection chamber and the detection electrode 10 with its surface modified with an aptamer are in a dehumidified freeze-dried state and packaged with vacuum or inert gas. To mitigate interference caused by unequal interstitial fluid extraction volumes due to differences in length, number, and channel flow resistance between the first annular microneedle 1 and the second annular microneedle 2, the microfluidic detection chambers in the first annular passage 4 and the second annular passage 5 are configured to have equal internal fixed volumes. The system uses negative pressure to drive the interstitial fluid into and fill the microfluidic detection chamber, thereby covering the detection electrode 10. Excess interstitial fluid is discharged into the waste fluid storage sponge 12 at the rear end.

[0050] The negative pressure sampling power module is located downstream of the flow path of the detection module and is connected to the drain ends of the first annular passage 4 and the second annular passage 5 respectively. It is configured to provide differentiated negative pressure driving force according to the different tissue fluid resistances corresponding to the first annular passage 4 and the second annular passage 5. The microfluidic detection chamber is connected in series between the microneedle and the negative pressure sampling power module, so that the upward unidirectional flow of interstitial fluid completely covers the detection electrode 10. In one embodiment, the negative pressure sampling power module includes at least one micro vacuum pump 9 (pre-vacuum negative pressure source) and a passive flow resistance adjustment structure for achieving differentiated negative pressure driving force, as well as an active dual-path feedback gas path connected to the pre-vacuum negative pressure source. The passive flow resistance adjustment structure includes a micro check valve 11 and a static flow resistance adjustment micro-column array 16. The active dual-path feedback gas path includes a first negative pressure control branch and a second negative pressure control branch. Each branch is equipped with a micro check valve 11, a waste liquid storage sponge 12, a high-precision micro proportional solenoid valve 15, and a pressure sensor 14 connected in series. The signal processing unit performs closed-loop control to continuously apply a negative pressure with an absolute value greater than that of the second annular path 5 (shallow layer) to the first annular passage 4 (deep layer) to compensate for the extremely high fluid permeability resistance of the deep reticular dermis layer due to the dense extracellular matrix.

[0051] The anti-flow sealing module features a coaxial annular pressure protrusion structure 3 extending downwards on the bottom surface of the system base 13 between the first and second annular microneedle groups. This annular pressure protrusion structure 3 significantly reduces the hydraulic conductivity of the pressure area by compressing the skin tissue, forming a high-resistance hydraulic seal isolation zone in situ to block the lateral flow of interstitial fluid between deep and superficial tissues under negative pressure suction. The hydrodynamic anti-flow mechanism of the physical pressure protrusion structure between the long and short needles is as follows: To block the lateral flow of subcutaneous interstitial fluid driven by negative pressure suction, this embodiment of the invention innovatively features a coaxial annular pressure protrusion structure 3 extending downwards on the surface of the system base 13 between the first and second annular microneedles 1 and 2. Its specific mechanical mechanism is that the dermis is essentially a porous, elastic biological medium filled with fluid. According to the fluid dynamics of porous media, the permeation of interstitial fluid follows Darcy's law: ,in Volumetric flow rate, To organize hydraulic conductivity or permeability, For seepage cross-sectional area, For fluid dynamic viscosity, For the applied pressure difference, This represents the effective seepage path length. In this formula, the permeability of skin tissue is... It is not a constant static constant, but rather exhibits an exponentially negative correlation with the compressive volumetric strain of the extracellular matrix (ECM). For example... Figure 3As shown, when the system adheres to the skin and applies downward pressure, the rigid annular compression protrusion 3 applies a concentrated normal compressive stress F to the skin surface of a specific annular area below. c The deep dermal collagen network in the compressed area undergoes elastic volume compression, local pores are instantly flattened, the microcapillary bed closes, and the free water filling the pores is forcibly squeezed out. This in-situ mechanical deformation can significantly reduce the hydraulic conductivity within the annular isolation zone. At this time, even if the first annular pathway 4 applies a high negative pressure to overcome the resistance of deep tissues (such as...), Figure 3 The upward arrow P inside the microneedle vac As shown, this is the applied pressure difference. (Increase), due to the increased permeability of the intermediate buffer zone. Approaching zero, according to Darcy's law, the volumetric flow rate of transverse fluid flow... (like Figure 3 The curved arrow Q at the tip of the microneedle isf The pressure (as shown) was also effectively suppressed to near zero. This annular compression protrusion structure 3, without introducing any exogenous chemical sealants, utilizes the skin's own elastoplastic mechanical response to dynamically construct an insurmountable "hydraulic sealing ring" in situ under the skin, fundamentally blocking the lateral short circuit between the deep and superficial fluids and ensuring the spatial purity of the sample. In the above porous media permeation model, the deep and superficial paths face different tissue permeation resistances and boundary conditions. To prevent flow rate imbalance caused by resistance difference, this system employs active asymmetric negative pressure compensation. With the micro vacuum pump 9 providing basic suction force, a closed-loop control network is formed by combining a high-precision micro proportional solenoid valve 15 and a pressure sensor 14, thereby applying high negative pressure to the high-resistance deep layer and low negative pressure to the low-resistance superficial layer. This differentiated pressure regulation mechanism can offset the subcutaneous resistance imbalance, allowing the two fluids to fill the detection chamber at dynamically matched flow rates, thus achieving high-fidelity equal-volume sampling. In one embodiment, the annular compression protrusion structure 3 is a continuous concentric circular protrusion integrally formed from the system base 13, made of rigid or semi-rigid biocompatible material, with a radial width W of 0.1 to 0.12 mm and a downward protrusion height of 50 to 150 μm.

[0052] The signal processing unit, communicatively connected to the detection module, is used to calculate and output data characterizing the spatial gradient features of the microenvironment of a single hair follicle unit based on detection signals at different depths. The signal processing unit, also communicatively connected to the detection module, is used to store, process, and compute the raw electrical signals output by the detection module according to different application scenarios of the system, in order to obtain follicle depth feature index (FDCI) data characterizing the spatial gradient features of a single hair follicle unit.

[0053] In one embodiment, the signal processing unit employs a local active computing architecture. The signal processing unit relies on a microcontroller and memory integrated within the system. The memory is preferably non-volatile, including but not limited to Flash or EEPROM. Standard operating curves, signal determination thresholds, and related calculation parameters corresponding to each target biomarker are pre-stored in the memory. The microcontroller sequentially performs error filtering, concentration conversion, and FDCI calculation on the raw biochemical electrical signals acquired by the detection module, and outputs the corresponding results.

[0054] In another embodiment, the signal processing unit employs a distributed edge computing architecture. At least some functional modules of the signal processing unit are located in an external smart terminal that is communicatively connected to the system. The external smart terminal includes, but is not limited to, smartphones, tablets, or personal computers. The detection module is used to acquire raw electrical signals and transmit the raw electrical signals to the external smart terminal via near-field communication, Bluetooth, or a wired interface. The standard working curves, signal determination thresholds, and preset calculation models corresponding to each target marker are stored in the local storage medium of the external smart terminal or in a server connected to it. The external smart terminal performs concentration conversion, FDCI calculation, and result output on the received raw electrical signals.

[0055] After the superficial and deep interstitial fluids enter their respective microfluidic detection chambers and cover the detection electrode 10, the aptamers bind to the target, causing a conformational change that results in a change in electron transfer resistance. The signal processing unit reads the deep concentration signal S1 and the superficial concentration signal S2 in real time and substitutes them into the system's preset mathematical kernel algorithm to calculate the hair follicle depth characteristic index (FDCI). The calculation formula is as follows: The Follicular Depth Characteristic Index (FDCI) calculates the rate of change of the concentration gradient of a specific biomarker along the longitudinal axis of the hair follicle per micrometer, quantifying the spatial pathological deflection of the microenvironment. FDCI characterizes the degree of longitudinal spatial gradient change of a specific biomarker between the superficial and deep layers within the same target hair follicle unit. FDCI values ​​obtained from different hair follicle units, at different sampling times, or under different experimental conditions may vary. This parameter can be used for recording the spatial characteristics of a single hair follicle microenvironment, inter-group comparative analysis, mechanism of action studies, and non-diagnostic evaluation of experimental effects. It should be noted that FDCI reflects the relative gradient characteristics of a specific biomarker under a given sampling structure and does not directly correspond to any disease diagnosis or individual disease grading result.

[0056] The system also includes a micro power supply, a microcontroller, and peripheral drive circuits. The micro power supply powers each active module within the system. The microcontroller is connected to a micro optical collimation unit, a negative pressure sampling power module, an electrochemical sensor array, and a signal processing unit, respectively, to achieve closed-loop control of the sampling process and acquisition and preprocessing of detection signals. The peripheral drive circuits specifically refer to auxiliary electronic circuits connected between the microcontroller and each physical actuator or sensing element, used for power amplification, electrical isolation, and signal conversion. They mainly consist of three parts: first, an LED constant current drive circuit for illuminating the light-emitting diode 8 in the hair follicle unit positioning module; second, a motor and valve power amplification circuit for providing sufficient operating voltage and current to the micro vacuum pump 9 and the micro proportional solenoid valve 15; and third, a micro potentiostat (analog front end) and a weak biochemical electrical signal amplification and filtering circuit for the electrochemical sensor array.

[0057] It is important to note that in the above FDCI calculation, S1 and S2 represent the concentration signal values ​​of the same analyte molecule (e.g., both PGD2 or both IL-6) in the superficial and deep layers. The system does not perform cross-subtraction between different types of biomarkers; instead, it independently derives the longitudinal spatial gradient rate of change for each target class. The rationality and innovation of this differential processing mechanism lies in the fact that, due to the significant differences in hormone and inflammatory basal metabolic levels among individual patients, obtaining absolute concentrations at a single depth often fails to accurately determine the severity of the lesion. This system simultaneously extracts interstitial fluid from different depths of the same hair follicle, using the layers where no specific pathological response occurs as "homogeneous natural internal controls." Through this differential calculation based on homologous physical distance, the system can filter out interference from individual systemic background fluctuations at the algorithmic level, achieving high-fidelity quantification of the degree of local pathological deterioration in microorganisms without needing to know the absolute standard values ​​of healthy individuals. Furthermore, the positive or negative sign of the FDCI index calculation result can intuitively characterize the spatial distribution of the biomarker. In one embodiment, to avoid interference from minor concentration fluctuations, the signal processing unit incorporates a signal-to-noise ratio (SNR) determination module and an error filtering algorithm based on variance analysis. The specific execution logic is as follows: Before sample contact, the signal processing unit performs blank baseline acquisition on the detection channels corresponding to the first annular path 4 and the second annular path 5, respectively, to obtain the background mean and standard deviation of each channel. During the detection phase, the two channels are continuously sampled at a preset frequency, and after low-pass filtering, the mean and relative standard deviation (RSD) are calculated using a sliding time window. When the RSD of multiple consecutive time windows is lower than a preset stability threshold (determined based on in vitro calibration experiments using standard buffer solution before the sensor leaves the factory), the corresponding signal is initially identified as a stable plateau signal, and outliers are removed. Further, the stable plateau region is divided into multiple consecutive sub-time windows, and variance analysis is performed on the mean of each sub-time window. When there is no significant difference between the sub-time windows, the mean of the stable plateau region is determined as the final valid detection signal. The signal-to-noise ratio (SNR) is determined by the ratio of the mean of the stable platform signal to the standard deviation of the corresponding channel's blank baseline. When this ratio exceeds a preset threshold, the channel signal is considered valid. Subsequently, based on pre-stored standard operating curves of the corresponding targets, the system converts the stable platform electrical signal into deep-layer concentration signal value S1 and shallow-layer concentration signal value S2. The system then uses the standard deviation of the blank baselines of the two channels... and Calculate the differential noise threshold ,in , and only if |S1 S2|greater than n When a recognizable effective spatial gradient is identified, n is the confidence level coefficient. In one embodiment, n is 3, which is set based on the 3σ confidence limit criterion, representing that the pathological deflection gradient has statistical validity of more than 99.7%, effectively eliminating the interference of random background noise of the instrument. The detection electrode 10 captures target molecules through a high-affinity nucleic acid aptamer probe and reduces background noise in conjunction with the physical isolation wall, thereby achieving a low limit of detection (LOD) at the nanomolar (nM) or even picomolar (pM) level. Further, the follicle depth feature index (FDCI) output by the signal processing unit can be used as a multidimensional input feature of the preset model for non-diagnostic calibration comparison with the previous histological assessment results. In one embodiment, when the output label is a biochemical distribution feature type, the preset model used is a multivariate logistic regression model or a support vector machine classification model obtained through training. In another embodiment, when the output label is a continuous variable such as the proportion of follicle miniaturization, the preset model used is a support vector regression model, a linear regression model, a ridge regression model, or a partial least squares regression model. The preset model uses biochemical distribution patterns, hair follicle morphological proportions, or other quantitative parameters obtained from previous in vitro tissue analysis as reference labels, and the hair follicle depth feature index (FDCI) values ​​corresponding to various biomarkers as input features for training and fitting. During detection, the feature matrix obtained from a single detection is input into the preset model. The preset model is configured to output the gradient feature parameters, relative change amplitude, or data grouping results of the hair follicle microenvironment at different sampling time points, under different experimental conditions, or between different sample groups, thereby providing a quantitative reference for clustering of biochemical gradient features of the hair follicle microenvironment, classification of spatial distribution patterns of biomarkers, or screening of non-diagnostic drugs.

[0058] This invention also discloses a working method (non-diagnostic analysis method) based on the above-mentioned system hardware. This method is not intended for the direct diagnosis or treatment of diseases, but primarily serves non-diagnostic screening and evaluation for in vitro analysis, basic research on pathogenic mechanisms, or drug metabolism pharmacokinetics. It includes the following steps: Optical positioning steps: Activate the miniature optical-assisted collimation unit to capture a high-contrast image of the hair follicle opening through oblique asymmetric illumination and a miniature gradient refractive index lens. The operator aligns the central axis of the positioning hole with the extension axis of the pre-selected single target hair follicle opening based on the image feedback. Compression sealing and puncture steps: Press down on the sensing system to closely adhere to and squeeze the epidermal surface, apply directional mechanical stress to the subcutaneous porous elastic medium, and construct a high flow resistance hydraulic sealing isolation zone with low hydraulic conductivity in situ in the pressure area; at the same time, the first ring microneedle group and the second ring microneedle group puncture the deep dermal papilla area and the superficial funnel part of a single hair follicle. Differential flow resistance matching sampling steps: Start the negative pressure sampling power module, apply high negative pressure to the first annular channel to overcome the high hydraulic resistance of the dense reticular dermis, and apply low negative pressure to the second annular channel to extract the superficial interstitial fluid. Under the isolation of the high flow resistance hydraulic sealing isolation zone, obtain the superficial and deep interstitial fluid without lateral flow. In-situ detection and signal extraction steps: Two interstitial fluids flow unidirectionally through physically isolated microfluidic detection chambers along the fluid path. Nucleic acid aptamer probes on the surface of the electrochemical sensor capture shallow and deep target markers respectively, and the working electrode converts them into two independent concentration electrical signals. Spatial gradient feature calculation steps: The signal processing unit reads two detection signals, calculates and outputs the hair follicle depth feature index that characterizes the longitudinal biochemical gradient of the microenvironment based on the preset mathematical model.

[0059] Based on the two extreme boundary points (1.3 and 2.5) of the microneedle length ratio (L1 / L2) interval and the typical median value (1.87), three core implementation cases were configured for the retraction depth of the dermal papilla of the hair follicle in different stages of AGA development. It should be clarified that the method steps included in this implementation only involve the acquisition and calculation of biochemical information and are not intended for the direct diagnosis of diseases.

[0060] Example 1: Minimum Length-to-Short Ratio Design (L1 / L2=1.3) – A very shallow probe for acquiring spatial gradient parameters of superficial and mid-deep hair follicles. This embodiment demonstrates the system configuration for the lower limit endpoint values ​​of the interval: the length of the first annular microneedle 1 is L1 = 650 μm, the length of the second annular microneedle 2 is L2 = 500 μm, and the L1 / L2 ratio is 1.3. The needle circle radius R1 of the first annular microneedle group can be 0.32 mm, and the needle circle radius R2 of the second annular microneedle group can be 0.78 mm, with a difference of R2-R1 of 0.46 mm; the bottom radius r1 of the first annular microneedle can be 120 μm, the bottom radius r2 of the second annular microneedle can be 90 μm, and the radial width W of the annular compression protrusion structure can be 0.10 mm. The above parameters satisfy the geometric requirements for the complete arrangement of the continuous annular compression protrusion structure between the inner and outer annular microneedles, while also taking into account both shallow surrounding coverage and localized encapsulation of the target hair follicle unit.

[0061] 1. Analysis of application scenarios and the rationality of design length: When follicular lesions enter the mid-to-late stage of AGA (Advanced Alopecia Areata), the terminal hairs that were originally several millimeters deep have degenerated into very shallow, fine, soft vellus hairs. The volume of the dermal papilla tissue shrinks, and irreversible retraction and upward migration occur in the subcutaneous tissue, typically reaching a depth of about 0.8 to 1.0 mm below the epidermis. At this point, the physical distance between the deep lesion target point and the superficial infundibulum is extremely compressed. If microneedles with a large length difference are still used at this stage, the deep long needle will directly penetrate the atrophied dermal papilla and enter the top of the subcutaneous fat layer, which has no detection value. Therefore, the 650 μm first circular microneedle 1 is just right to anchor the late-stage upward migration of the dermal papilla and hair bulb remnants, while the 500 μm second circular microneedle 2 is used to detect the accompanying high concentration of inflammatory lipid secretions and immune microenvironment areas.

[0062] 2. Specific work methods and steps: Optical positioning and locking: The micro-optical-assisted collimation unit is activated, and the light-emitting diode 8 located in the positioning hole 6 provides oblique illumination. The lens 7 breaks through the scattering of the stratum corneum and captures a micron-level image of the opening of the subcutaneous atrophic vellus hair follicle. Under optical feedback, the positioning hole 6 is moved to be coaxial with the opening of the target hair follicle.

[0063] Compression sealing and shallow / deep puncture: The probe base is pressed downwards evenly, causing the rigid annular compression protrusion 3, located between the two annular microneedle groups, to press tightly against the epidermal surface. Under physical surface pressure, the extracellular matrix (ECM) directly beneath this annular region experiences significant volumetric compressive strain, displacing pore water and constructing a dense, high-flow-resistance hydraulic seal. Simultaneously, the 650 μm first annular microneedle 1 and the 500 μm second annular microneedle 2 penetrate without interference.

[0064] Differential active aspiration extraction: The miniature vacuum pump 9 within the negative pressure sampling power module is activated under the control of the signal processing unit, applying a relatively high negative pressure of -35 kPa to the first annular passage 4 of the inner ring to overcome the residual resistance of the dermis, while simultaneously applying a mild negative pressure of -10 kPa to the second annular passage 5 of the outer ring to extract the superficial interstitial fluid. Under this pressure difference, the deep and superficial interstitial fluids isolated by the annular compression protrusion structure 3 cannot cross-flow and instead ascend along the porous channels within the needle.

[0065] In-situ biomarker detection and signal calculation: Interstitial fluid flows unidirectionally into two unconnected microfluidic detection chambers. Two detection electrodes 10, each covered with nucleic acid aptamers, are equipped with the same combination of nucleic acid aptamer probes to detect the same set of targets (including PGD2, IL-6, and MCP-1, etc.). For each specific biomarker, the deeper detection electrode captures its local concentration to obtain a deep signal S1, while the shallower detection electrode captures its homologous local concentration to obtain a shallow signal S2. Excess waste fluid enters a waste fluid storage sponge 12. The signal processing unit independently calculates the final FDCI value corresponding to each biomarker, and then uses this as input features into a preset model to output an objective quantitative index feature for the microenvironment of this miniaturized hair follicle.

[0066] Example 2: Design of the median length-to-length ratio (L1 / L2=1.87) – A handheld high-precision analyzer for hair follicles in the intermediate transitional stage. This embodiment uses a representative intermediate value system configuration within the range: the length of the first annular microneedle 1 is L1 = 750 μm, the length of the second annular microneedle 2 is L2 = 400 μm, and the L1 / L2 ratio is 1.87. The needle circle radius R1 of the first annular microneedle group can be 0.33 mm, and the needle circle radius R2 of the second annular microneedle group can be 0.76 mm, with a difference of R2-R1 of 0.43 mm; the bottom radius r1 of the first annular microneedle can be 140 μm, the bottom radius r2 of the second annular microneedle can be 80 μm, and the radial width W of the annular pressure protrusion structure can be 0.12 mm. The above parameters meet the geometric requirements for the complete arrangement of the continuous annular pressure protrusion structure between the inner and outer annular microneedles, while taking into account the dispersion of surface pressure stress and the localized encapsulation of a single hair follicle unit.

[0067] 1. Analysis of application scenarios and the rationality of design length: This ratio design targets the group with the greatest clinical need for hair loss testing—patients in the early or intermediate stages of hair loss. At this critical stage, the deep lesions of the hair follicle (dermal papilla area) are just beginning to detach from the subcutaneous fat layer, sinking and stagnating within the middle reticular dermis at a depth of approximately 1.0 to 1.5 mm. Using a 750 μm first circular microneedle 1, the tip precisely pierces and enters the microenvironment surrounding the middle hair follicle matrix, where DHT conversion is most active and PGD2 synthase expression is most intense. Meanwhile, a 400 μm second circular microneedle 2 stably extracts immune inflammatory markers such as interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) from the periphery of the infundibulum. This configuration effectively avoids over-puncture while capturing the gradient of the AGA pathogenesis core area.

[0068] 2. Specific working methods and details of the anti-channeling procedures: After targeting and locking using the same optical collimation method, a compression sealing step is performed. Because the reticular dermal tissue around the mid-stage hair follicle is extremely dense and tough, when mechanical puncture force is applied downwards, the annular compression protrusion structure 3 acts like a "hydraulic sealing ring" to press against the skin surface area between the deep and shallow needle tips, suppressing the potential flow velocity that might otherwise occur to zero.

[0069] The system then proceeds to a differentiated flow resistance matching sampling step. The dense dermal structure in the middle stage requires a greater suction force; the system applies a high vacuum of -45 kPa to the first annular passage 4 (750 μm deep). Meanwhile, under the insulating protection of a physical sealing barrier, a low negative pressure of -15 kPa is applied to the second annular passage 5 (400 μm shallow). Check valves 11 inside each flow path effectively prevent fluid backflow under different pressure drops.

[0070] In the in-situ detection step, the deep extract and the shallow liquid enter two microfluidic detection chambers in parallel. A biochemical blocking layer is densely arranged on the surface of the detection electrode 10 using self-assembled monolayer technology. The specific binding reaction instantaneously triggers a change in the electron transfer rate, outputting a significantly amplified biochemical gradient electrical signal. The waste liquid is discharged into the waste liquid storage sponge 12 via capillary action. The system ultimately calculates the FDCI value and inputs it into the aforementioned preset model, outputting the gradient characteristic parameters, relative change amplitude, or data grouping results of the hair follicle microenvironment at different sampling times, under different experimental conditions, or between different sample groups.

[0071] Example 3: Maximum Length-to-Short Ratio Design (L1 / L2=2.5) – Passive Micro-Patch for Low-Power Non-Medical Hair Follicle Microenvironment Parameter Acquisition in Non-Medical Scenarios This embodiment uses the upper limit of the ratio range: the length of the first annular microneedle 1 is L1 = 900 μm, the length of the second annular microneedle 2 is L2 = 360 μm, and the L1 / L2 ratio is 2.5. The needle radius R1 of the first annular microneedle group can be 0.34 mm, and the needle radius R2 of the second annular microneedle group can be 0.74 mm, with a difference of R2-R1 of 0.40 mm; the bottom radius r1 of the first annular microneedle can be 150 μm, the bottom radius r2 of the second annular microneedle can be 70 μm, and the radial width W of the annular compression protrusion structure can be 0.10 mm. The above parameters meet the geometric requirements for the complete arrangement of the continuous annular compression protrusion structure between the inner and outer annular microneedles, while also taking into account patch outer diameter control, shallow surrounding coverage, and localized encapsulation of the target hair follicle unit.

[0072] 1. Analysis of application scenarios and the rationality of design length: This embodiment is suitable for users who need continuous observation of hair follicle microenvironment parameters, and is used for the collection and recording of physicochemical parameters of the hair follicle microenvironment for non-medical purposes in a home setting. Since the dermal papilla of the hair follicle is still 3.0 to 4.5 mm deep under the skin, the first annular microneedle 1 is designed to be 900 μm in order to capture as many early chemotactic factors as possible that diffuse from the lesion area of ​​the deep dermal papilla; at the same time, in order to eliminate noise interference caused by the upward diffusion of potent metabolites from deep layers, the second annular microneedle 2 is compressed to 360 μm to remain completely in the superficial infundibulitis high-incidence area.

[0073] 2. Specific working methods (purely physical and passive wireless control mechanisms): In this embodiment, the sensing system is specifically configured as a disposable passive micro-patch. Its "passive" characteristic is reflected in the fact that no battery or active electromechanical components need to be integrated inside the patch during the entire sampling and detection process. This is achieved through the following mechanism: Passive positioning and locking: Since the patch is designed for home use, the internal LED 8, which requires power, is removed. A high-transmittance micro-magnifying lens is integrated at the bottom of the positioning hole 6. Under ambient light, the user can mechanically position the center hole with the target hair follicle by aligning it with the lens 7 using the naked eye or a smartphone camera.

[0074] Mechanical passive negative pressure drive: Eliminating the power-consuming miniature vacuum pump 9. A mechanically pre-vacuumed silicone capsule made of elastic material is integrated on the back of the patch as a shared initial negative pressure source. When the operator applies the patch to the scalp, pressing down expels the air from the silicone capsule, simultaneously creating a high-flow-resistance, water-sealed isolation zone on the skin surface due to the annular pressure protrusion 3. Upon release, the silicone capsule rebounds physically due to the material's elasticity, generating a passive mechanical vacuum suction force of approximately -50 kPa within the pathway. In one embodiment, the mechanically pre-vacuumed silicone capsule is integrally molded from highly elastic biocompatible materials such as medical-grade silicone or polydimethylsiloxane (PDMS) through micro-molding or injection molding processes, often in the form of a hemispherical or micro-corrugated tubular cavity. The passive suction mechanism works as follows: the operator presses the silicone capsule with their finger to cause elastic deformation, squeezing out the air inside the cavity; once the patch adheres tightly to the skin, forming a hydraulically sealed flow path, the operator releases their finger, and the silicone capsule attempts to return to its original structure under the elastic deformation recovery force of the material itself. Because the system is in a closed state, the forced rebound expansion of the cavity passively generates a continuous mechanical negative pressure inside the microfluidic pipeline, thereby achieving passive suction.

[0075] Passive physical throttling impedance matching: Under pure mechanical negative pressure, the fluid enters the dual channels. The second annular passage 5 uses a static flow resistance regulating micro-pillar array 16 (preferably staggered micro-pillars in this embodiment) molded or injection-molded on the inner wall to perform physical throttling. Through fluid turbulence, losses are generated, and the effective suction force reaching the superficial tissue is passively weakened to a lower -10 kPa; while the check valve 11 at the end of the first annular passage 4 is opened by high pressure, so that the dense deep tissue obtains a suction force of -45 kPa.

[0076] Passive electrode detection based on near-field communication (NFC): Extracted superficial and deep tissue fluids flow through separate microfluidic detection chambers and cover the detection electrode 10. Since the patch has no built-in battery, the detection electrode 10 is physically connected to a miniature NFC radio frequency antenna printed on the patch surface. When a user brings an NFC-enabled smartphone close to the patch, the electromagnetic field emitted by the phone provides instantaneous induced power to the radio frequency antenna. This power drives an electrochemical sensor to perform impedance measurement, and the concentration signals from the superficial and deep layers are wirelessly transmitted back to the smartphone via NFC. Finally, the mobile app acts as a signal processing unit to calculate the FDCI value and calls upon the aforementioned preset model built into the app to intuitively output qualitative or semi-quantitative indicators of the deviation of local scalp biochemical metabolism, providing non-medical data references for daily monitoring of the scalp's physiochemical state.

[0077] 3. Performance degradation analysis and application rationality explanation of passive design: The passive mechanical negative pressure and NFC inductive power supply detection architecture used in this embodiment involves reasonable compromises in performance compared to the active electronic vacuum pump and independent power supply architectures in Embodiments 1 and 2. Regarding fluid control, the initial negative pressure generated by the mechanical silicone capsule gradually decreases with fluid aspiration and deformation recovery, failing to maintain an absolutely constant high pressure differential like an active micro-pump, resulting in a slightly longer time to reach the desired extraction volume. In terms of signal detection, the micro-electrochemical sensor driven by NFC instantaneous radio frequency induction power has lower signal gain, signal-to-noise ratio, and extremely low detection limit (LOD) than the active potentiostat circuit powered by an independent stable power supply. However, this embodiment is specifically designed for monitoring the hair follicle microenvironment in a non-medical setting at home, with the core application requirement being the qualitative or semi-quantitative tracking of concentration gradient fluctuations in targeted biochemical markers. Under this basic physiological parameter daily monitoring scenario, the moderate decrease in detection accuracy and aspiration stability is within an acceptable range.

[0078] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined in this invention may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A radial multilayer microneedle sensing system for microenvironment analysis of a single hair follicle unit, characterized in that, include: The hair follicle unit positioning module is used to determine the spatial location of the target hair follicle opening; The radial annular multilayer microneedle array module includes a first annular microneedle group and a second annular microneedle group that are concentrically and coaxially distributed around a central axis, and the microneedle length L1 of the first annular microneedle group is greater than the microneedle length L2 of the second annular microneedle group. The anti-flow sealing module includes an annular compression protrusion structure disposed on the bottom surface of the system substrate between the first annular microneedle group and the second annular microneedle group, which is used to block the lateral flow of interstitial fluid between deep and superficial tissues under negative pressure aspiration. The multi-layer independent sampling channel module includes a first annular passage connected to a first annular microneedle group and a second annular passage connected to a second annular microneedle group. The first annular passage and the second annular passage are physically isolated from each other in the fluid path. The detection module includes a first detection unit and a second detection unit, which are fluidly connected to the first annular pathway and the second annular pathway, respectively, and are used to specifically identify biomarkers collected from interstitial fluid at different depths and generate detection signals. The negative pressure sampling power module is connected to the drain end of the first annular passage and the second annular passage respectively, and is configured to provide differentiated negative pressure driving force according to the different tissue fluid resistances corresponding to the first annular passage and the second annular passage. The signal processing unit, which is connected in communication with the detection module, is used to calculate and output data characterizing the spatial gradient features of the microenvironment of a single hair follicle unit based on the received detection signals at different depths.

2. The system according to claim 1, characterized in that, The hair follicle unit positioning module includes a positioning hole disposed on the system base and an optical auxiliary collimation unit disposed in the positioning hole; The positioning hole is used to define the location of the target hair follicle opening and to form a limiting area that fits the skin surface around the central positioning hole, so that the central axis of the positioning hole substantially coincides with the extension axis of the target hair follicle opening. The optically assisted collimation unit includes a light-emitting diode for illuminating the periphery of the target hair follicle opening, and / or a lens for capturing a high-contrast stereoscopic artifact enhancement image of the local skin surface.

3. The system according to claim 1, characterized in that, The annular compression protrusion structure is a continuous concentric circular protrusion integrally formed on the system base. It can reduce the hydraulic conductivity of the pressure area by squeezing the skin tissue, and form a high flow resistance hydraulic sealing isolation zone in situ.

4. The system according to claim 1, characterized in that, The microneedle lengths L1 of the first annular microneedle group and L2 of the second annular microneedle group satisfy 1.3≤L1 / L2≤2.5, and the value range of L1 is 500 to 900 μm, and the value range of L2 is 300 to 500 μm.

5. The system according to claim 1, characterized in that, The first annular microneedle group has a needle circle radius of R1, the second annular microneedle group has a needle circle radius of R2, the first annular microneedle has a bottom radius of r1, the second annular microneedle has a bottom radius of r2, the radial width of the annular compression protrusion structure is W, and the system satisfies the geometric constraints: R2-R1>r1+r2+W; and / or, R1 is 0.32 to 0.34 mm, R2 is 0.74 to 0.78 mm, the difference between the two R2-R1 is set to 0.40 to 0.46 mm, r1 is 120 to 150 μm, r2 is 70 to 90 μm, and W is 0.10 to 0.12 mm.

6. The system according to claim 1, characterized in that, Both the first detection unit and the second detection unit include microfluidic detection chambers respectively disposed in the first annular pathway and the second annular pathway. Each microfluidic detection chamber is provided with a reagent-free electrochemical sensor. The reagent-free electrochemical sensor includes a detection electrode. The surface of the detection electrode is modified with a biochemical blocking layer to resist non-specific protein adsorption and is covalently coupled with a nucleic acid aptamer probe that specifically binds to the biomarker.

7. The system according to claim 1, characterized in that, The negative pressure sampling power module includes a passive flow resistance adjustment structure, at least one pre-vacuum negative pressure source, and an active dual-path feedback gas path connected to the pre-vacuum negative pressure source. The passive flow resistance regulation structure includes a miniature check valve and a static flow resistance regulation micro-column array, and an active dual-path feedback gas path with a first negative pressure regulation branch and a second negative pressure regulation branch; among which... The static flow resistance regulating micropillar array is disposed within the second annular path; The miniature check valve, miniature proportional solenoid valve, and pressure sensor are connected in series in both the first and second negative pressure control branches, and closed-loop control is performed through a signal processing unit to apply a negative pressure to the first annular passage with an absolute value greater than that to the second annular passage.

8. The system according to claim 1, characterized in that, A physical isolation wall is provided between the first annular passage and the second annular passage to physically isolate them from each other in the fluid path.

9. The system according to any one of claims 1-8, characterized in that, The signal processing unit is configured to: acquire the deep concentration signal S1 and the superficial concentration signal S2 based on the detection module, and calculate the hair follicle depth feature index using the following formula: FDCI stands for Follicle Depth Characteristic Index.

10. A non-diagnostic analytical method, characterized in that, The system and method described in any one of claims 1-9, wherein the method is not for the direct purpose of diagnosing or treating a disease, but only for in vitro tissue analysis, basic biochemical research on the pathogenesis of androgenetic alopecia, or measurement of physiological parameters for non-medical purposes, comprises the following steps: The central axis of the radial sensing system is aligned with the opening of the target single hair follicle by using the hair follicle unit positioning module. Pressing the sensing system downwards on the skin surface causes it to adhere tightly to and compress the epidermal surface. The annular compression protrusion structure applies directional mechanical stress to the porous elastic medium under the skin, forming a high flow resistance hydraulic sealing isolation zone with low hydraulic conductivity in situ in the pressure area. At the same time, the first and second annular microneedle groups are inserted into the deep dermal papilla area and the superficial infundibulum of the target single hair follicle, respectively. The negative pressure sampling power module is activated to apply differentiated negative pressure to the first and second annular channels to aspirate interstitial fluid from the deep and shallow layers, respectively. The same target biomarker was detected in the deep and shallow interstitial fluids flowing into the first and second annular pathways, respectively, by the detection module, and the deep concentration signal S1 and the shallow concentration signal S2 were obtained respectively. The signal processing unit calculates and outputs the hair follicle depth characteristic index, which characterizes the longitudinal biochemical gradient of the microenvironment, based on the deep concentration signal S1 and the shallow concentration signal S2.

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