An air-core coaxial dual-cavity differential quantum well-liquid crystal micro-laser biosensor and a detection method thereof

Abstract

This invention relates to a hollow coaxial dual-cavity differential quantum well-liquid crystal microlaser biosensor and its detection method, belonging to the technical field of biosensing and liquid crystal photonic devices. It includes a detection microcavity, a reference microcavity, a pump source, a spectral acquisition unit, and a signal processing unit. Both the detection and reference microcavities are hollow coaxial microcavity structures. Each hollow coaxial microcavity, from the inside out, includes a central fluid channel, a liquid crystal response layer, a quantum well gain layer, and a low-refractive-index protective layer. The inner wall of the central fluid channel of the detection microcavity is provided with a functional molecular layer for contacting the sample to be tested and generating a specific response to the target analyte. This invention combines hollow flow-through sample introduction, liquid crystal response transduction, quantum well active microlaser output, and dual-cavity differential reference, offering advantages such as high detection stability, strong anti-interference ability, and good quantitative accuracy. It is suitable for the detection of targets such as enzymes, proteins, nucleic acids, small molecules, ions, or pathogens.

Description

Technical Field

[0001] This invention relates to the fields of biosensing and liquid crystal photonic devices, and in particular to a hollow coaxial dual-cavity differential quantum well-liquid crystal micro-laser biosensor and its detection method. Background Technology

[0002] Biosensing technology is a technology that converts biometric processes into detectable physical signals. It is widely used in fields such as disease diagnosis, food safety, environmental monitoring, and biomanufacturing process control. Traditional biosensing methods include colorimetry, fluorescence, and enzyme-linked immunosorbent assay (ELISA), which usually require labeling, color development, and complex sample pretreatment. The detection process is lengthy and cannot meet the needs of real-time, continuous, and micro-volume sample detection.

[0003] Microcavity optical sensors are a type of high-sensitivity sensor that has seen rapid development in recent years. They utilize microscale optical resonant cavities to strongly confine the light field. When there are slight changes in the refractive index of the medium surrounding the microcavity, the surface adsorption state, or the cavity size, the resonant peak position, peak width, or output intensity will change accordingly. Whispering-gallery mode microcavities are widely used for the detection of biomarkers such as proteins, nucleic acids, and viruses due to their high quality factor and strong light field confinement capability. Introducing a gain medium into a passive microcavity can form an active microlaser sensor, which outputs a narrow linewidth and high signal-to-noise ratio laser spectrum, suitable for the amplified detection of weak biological reactions. Colloidal quantum wells are gradually becoming the preferred gain material due to their high luminous efficiency, narrow emission spectrum, and good photostability.

[0004] Liquid crystal materials are characterized by ordered orientation, significant optical anisotropy, and high sensitivity to interface disturbances, making them suitable for interface biosensing. When liquid crystals come into contact with functionalized interfaces, the biochemical reactions triggered by the target material change the orientation of the liquid crystals, thereby modulating the optical signals. Traditional liquid crystal sensors mostly rely on polarizing microscopes to observe changes in brightness, which has limited quantitative capabilities. In recent years, a technical approach has emerged that combines liquid crystal response interfaces with microcavity laser structures. By modulating the effective refractive index of the microcavity through changes in liquid crystal orientation, quantitative spectral readout can be achieved.

[0005] The prior art that is relatively similar to this invention includes the following two categories: The first type is a liquid crystal-coated silica microbubble WGM sensor (Peiyao Anatel., Optics Letters, 2024, 49:7230-7233). This scheme uses silica microbubbles as the main resonant cavity, covered with a liquid crystal layer. The WGM resonant spectrum is excited by coupling through a tapered optical fiber. Penicillin G is hydrolyzed under the action of penicillinase, causing a change in liquid crystal orientation, which in turn changes the effective refractive index around the microbubble, causing the resonant peak to shift. This scheme is similar to the present invention in that it uses liquid crystal response to modulate the WGM spectrum, but its shortcomings are: it adopts a single cavity structure, lacks a synchronous reference microcavity, and it is difficult to subtract background drift such as temperature, pump fluctuations and mechanical disturbances; it is a passive resonant structure, and no gain medium is introduced to form an active microlaser output; the liquid crystal layer is set on the outer surface, and the sample acts on the outer coating layer, without forming a coaxial layered structure of "central fluid channel-liquid crystal response layer-gain layer-protective layer".

[0006] The second type is the self-referenced optofluidic ring resonant cavity sensor (MingLietal.,Anal.Chem.,2013,85(19):9328-9332). This scheme optically couples the sensing cavity and the reference cavity, and realizes detection by monitoring the splitting of coupling modes or the change of mode spacing, so as to reduce common-mode noise. It is similar to the present invention in that both adopt the dual-cavity concept of the sensing cavity and the reference cavity, but its shortcomings are: this scheme is a labelless refractive index sensing, does not combine a liquid crystal response layer, and cannot convert the enzyme-catalyzed reaction or molecular recognition process into a change in liquid crystal orientation; it does not introduce a quantum well gain layer and does not generate micro-laser output; it does not disclose a hollow coaxial layered structure, and cannot realize the integrated control of sample flow path, reaction interface limitation and gain layer protection.

[0007] In summary, existing liquid crystal micro-laser biosensors typically employ a single microfiber, microsphere, microring, or other single-cavity resonant structure as the sensing unit. Detection is achieved through changes in liquid crystal orientation, interfacial refractive index, or resonant peak position caused by the analyte. While this type of structure can convert biochemical reactions into readable spectral signals, in practical applications, the output signal of a single resonant cavity is easily affected by both the target reaction and non-target factors, such as changes in ambient temperature, pump power fluctuations, mechanical disturbances, changes in the overall refractive index of the sample, liquid flow disturbances, and device background drift. Therefore, existing single-cavity structures struggle to effectively distinguish between the specific response signal caused by the target analyte and the non-specific background signal caused by the external environment or the system itself, thus limiting the stability, repeatability, and quantitative accuracy of the detection results.

[0008] Meanwhile, existing external surface contact microcavity structures typically place the liquid crystal response layer, functional molecular layer, and gain layer on the outer surface of the microcavity, directly exposing the sensing area to the test liquid environment. This structure has problems such as difficulty in defining the sample contact area, difficulty in accurately controlling the reaction interface, reliance on external immersion or drop addition for sample introduction, and difficulty in achieving stable and continuous flow detection. In the process of detecting complex samples, salt ions, enzymes, proteins, surfactants, pH fluctuations, or other impurities in the test liquid may also directly affect the liquid crystal layer and gain layer, thereby affecting the liquid crystal orientation stability, laser output intensity, and long-term device lifespan.

[0009] Furthermore, existing single-cavity liquid crystal microlaser sensors typically lack a synchronous reference structure, making it impossible to subtract background drift in real time under the same detection conditions. When the detection environment undergoes temperature changes, pump light intensity changes, or slight disturbances in the microcavity structure, the resonant peak position will also shift. This shift may be superimposed on the spectral drift caused by the target analyte, leading to false positives, error amplification, or quantitative deviation. This is especially true when detecting low-concentration target analytes, where the target response signal is weak and the impact of background drift on detection accuracy is more pronounced. To address this, a hollow coaxial dual-cavity differential quantum trap-liquid crystal microlaser biosensor and its detection method are proposed. Summary of the Invention

[0010] This invention provides a hollow coaxial dual-cavity differential quantum well-liquid crystal microlaser biosensor and its detection method, which solves the technical problems of existing single-cavity liquid crystal microlaser biosensors, such as difficulty in subtracting background drift, uncontrollable sample flow path, difficulty in defining the reaction interface, susceptibility of the gain layer to interference from complex samples, insufficient continuous detection capability, and insufficient quantitative accuracy at low concentrations. By designing the microcavity as a hollow coaxial structure, the sample flows in the central fluid channel, and the liquid crystal response layer, quantum well gain layer, and low refractive index protective layer are arranged radially in sequence, thereby defining the sample flow path and reaction interface, improving the controllability of the detection area, reducing the direct interference of the analyte on the gain layer, and adapting to the needs of continuous sample injection and flow-through detection.

[0011] The solution to the above-mentioned technical problems of the present invention is as follows: a hollow coaxial dual-cavity differential quantum well-liquid crystal micro-laser biosensor, comprising a detection microcavity, a reference microcavity, a hollow fluid channel, a pump light source, a spectral acquisition unit, and a signal processing unit.

[0012] The detection microcavity is used to contact the sample to be tested and generate a specific response to the target analyte. As the core sensing unit, the functional molecular layer inside the detection microcavity can specifically recognize or react with the target analyte, thereby converting the biochemical signal into a measurable optical signal. This configuration enables the sensor to specifically identify the target analyte, improving the selectivity and accuracy of detection.

[0013] The reference microcavity is used to synchronously record non-specific background signals such as temperature changes, pump fluctuations, mechanical disturbances, overall refractive index changes of the sample, and device background drift. The reference microcavity has a basically the same structure and material composition as the detection microcavity, but does not produce a specific response to the target analyte. This setting enables the reference microcavity to record common-mode interference signals such as environmental fluctuations and system drift in real time, providing a benchmark for subsequent differential processing, thereby effectively deducting background noise.

[0014] The pump source employs a pump optical coupler, which couples the pump light into the detection microcavity and the reference microcavity to excite the quantum well gain layer in the detection microcavity and the reference microcavity to generate a micro-laser signal. Through the configuration of the pump optical coupler, the pump light can be efficiently introduced into the detection microcavity and the reference microcavity, ensuring that the quantum well gain layer obtains sufficient excitation energy, thereby generating a stable active micro-laser output and improving the signal-to-noise ratio and stability of the detection signal.

[0015] The spectral acquisition unit employs a micro-laser output coupler, which acquires the micro-laser spectra output from the detection microcavity and the reference microcavity. The micro-laser output coupler can efficiently collect the micro-laser signals generated by the detection microcavity and the reference microcavity and transmit them to the spectral analysis equipment, ensuring complete acquisition of the spectral signals and improving the sensitivity and accuracy of the detection.

[0016] The signal processing unit is used to extract the spectral peak position, peak width, peak intensity, mode spacing, mode splitting, or Vernier envelope of the detection microcavity and the reference microcavity, and perform differential calculations. By performing synchronous processing and differential analysis on the dual-cavity spectral signals through the signal processing unit, common-mode background drift can be effectively deducted, and specific signals related to the concentration or activity of the target analyte can be extracted, thereby significantly improving the stability, repeatability, and quantitative accuracy of the detection.

[0017] Both the detection microcavity and the reference microcavity are hollow coaxial microcavity structures. Each hollow coaxial microcavity includes, from the inside out: a central fluid channel, a liquid crystal response layer, a quantum well gain layer, and a low refractive index protective layer.

[0018] The central fluid channel is located at the center of the microcavity and is used for the passage of the sample to be tested or the reference liquid. The central fluid channel serves as the path for sample flow, confining the sample to flow in the central region of the microcavity. This makes the contact between the sample and the inner wall functional layer more sufficient and controllable. This structure makes the sample flow path clear and the reaction area fixed, which is conducive to realizing continuous flow detection and improving the repeatability and controllability of the detection.

[0019] The liquid crystal response layer is arranged around the central fluid channel to convert interfacial reactions, enzymatic reactions, or molecular recognition processes caused by the target into changes in the orientation of liquid crystal molecules. The liquid crystal response layer utilizes the high sensitivity of liquid crystal materials to interfacial physicochemical changes to convert biochemical reactions into changes in the orientation of liquid crystal molecules, thereby changing the effective refractive index of the microcavity. This arrangement enables biochemical reactions to be efficiently converted into optical signals, achieving highly sensitive biological detection.

[0020] The quantum well gain layer is disposed outside the liquid crystal response layer or adjacent to the liquid crystal response layer, and is used to generate an active micro-laser signal under external pump light excitation. The quantum well gain layer adopts a semiconductor quantum well material with high photoluminescence efficiency and good photostability. Under pump light excitation, it can generate a micro-laser output with narrow linewidth and high signal-to-noise ratio. This configuration enables the sensor to output an active laser signal, which has higher spectral resolution and detection sensitivity compared to a passive resonant signal.

[0021] The low-refractive-index protective layer is disposed outside the quantum well gain layer to enhance optical field confinement, stabilize the resonant mode, and reduce the interference of the external environment on the quantum well gain layer. On the one hand, the low-refractive-index protective layer acts as an optical cladding to enhance the confinement of the optical field in the microcavity, and on the other hand, it acts as a physical isolation layer to protect the quantum well gain layer from direct damage from complex components such as salt ions, proteins, enzymes, surfactants, or pH fluctuations in the sample. This arrangement not only improves the quality factor and resonant stability of the microcavity, but also extends the lifespan of the device and enhances the reliability of the sensor in complex sample systems.

[0022] Furthermore, the inner wall of the central fluid channel is provided with an inner wall functional layer. At least within the detection microcavity region, the inner wall functional layer is a functional molecular layer capable of specifically interacting with the target analyte. This functional molecular layer is selected from any one of the following: enzyme substrate layer, antibody layer, antigen layer, nucleic acid aptamer layer, peptide chain layer, carbohydrate molecular layer, polysaccharide derivative layer, ion-responsive molecular layer, or molecularly imprinted layer. Within the reference microcavity region, the inner wall functional layer is a reaction layer, which includes an inert layer, blank layer, blocking layer, or inactivation layer, used to simultaneously reflect temperature, pump fluctuations, and other mechanical stresses. Non-specific factors such as mechanical disturbances, changes in the overall refractive index of the sample, and device background drift can mitigate the impact of enzyme activity detection. The functional molecular layer is a substrate molecular layer that can be hydrolyzed, cleaved, or transformed by the target enzyme. By setting a specific functional molecular layer on the inner wall of the detection microcavity, the sensor can achieve specific identification and detection of different targets. By setting an inert layer or sealing layer on the inner wall of the reference microcavity, the reference microcavity only responds to non-specific background signals and does not respond to the target analyte. This configuration allows the dual-cavity differential structure to work effectively, ensuring both detection specificity and background subtraction.

[0023] Furthermore, the central fluid channel is formed by a quartz capillary, a silica microtube, a borosilicate glass capillary, or a polymer hollow microtube, and the inner diameter of the central fluid channel is 20 μm to 200 μm; the thickness of the liquid crystal response layer is 1 μm to 20 μm; the thickness of the quantum well gain layer is 50 nm to 2 μm; the thickness of the low refractive index protective layer is 0.5 μm to 10 μm, and the quantum well gain layer can be made of colloidal quantum wells, colloidal semiconductor nanosheets, core-shell quantum wells, or other nanoluminescent materials with optical gain characteristics. Preferably, the quantum well gain layer is CdSe / CdS, CdSe / CdZnS, CdSe / ZnS, CdSe / CdS / ZnS, or other II-VI group semiconductor quantum well materials. The low refractive index protective layer is made of fluorinated polymer, silicon dioxide, PMMA, CYTOP, PDMS, epoxy resin, or transparent low refractive index material. The selection of the above parameter range is based on the following considerations: the inner diameter of the central fluid channel within the above range can ensure smooth flow of the sample liquid and meet the conditions for whispering-gallery mode resonance, achieving a high quality factor; the thickness of the liquid crystal response layer within the above range can ensure sufficient modulation of the effective refractive index of the microcavity by the change in liquid crystal molecule orientation, while avoiding response delay caused by excessive thickness; the thickness of the quantum well gain layer within the above range can provide sufficient optical gain, while avoiding increased scattering loss caused by excessive thickness; the thickness of the low refractive index protective layer within the above range can effectively constrain the light field and isolate external interference. The selection of the above materials is based on their comprehensive performance such as optical transparency, refractive index matching, and biocompatibility. This preferred parameter range ensures the best optical performance and detection sensitivity of the sensor.

[0024] Furthermore, the detection microcavity and the reference microcavity are arranged sequentially along the axial direction of the same hollow capillary. The inner wall of the detection microcavity region is provided with a target response functional layer, and the inner wall of the reference microcavity region is provided with an inert layer, a sealing layer, a deactivation functional layer, or a control layer that does not react with the target. An isolation zone is provided between the detection microcavity region and the reference microcavity region to reduce fluid cross-interference between the detection microcavity and the reference microcavity. This axial arrangement structure has the following advantages: the detection cavity and the reference cavity are located on the same microtube and have the same or similar temperature, mechanical state, and pumping environment, which facilitates synchronous reference and differential subtraction; the isolation zone effectively prevents cross-contamination between the detection liquid and the reference liquid, ensuring the purity of the reference signal; this structure simplifies the device fabrication process and improves the reliability of dual-cavity differential detection.

[0025] Furthermore, the detection microcavity and the reference microcavity are two parallel hollow coaxial microtubes. These two microtubes are connected to the sample channel and the reference channel, respectively, or can be split through the same injection channel, allowing the detection liquid and the reference liquid to enter the detection microcavity and the reference microcavity, respectively. A target response functional layer is provided within the detection microcavity, and an inert functional layer or a blank functional layer is provided within the reference microcavity. This parallel dual-tube structure has the following advantages: clearer sample flow path separation, avoiding any fluid cross-interference between the detection microcavity and the reference microcavity; flexible injection methods, allowing for both independent and split injection; and easy integration with microfluidic chips, suitable for multi-channel parallel detection and lab-on-a-chip applications. This structure improves the sensor's integration and application flexibility.

[0026] Furthermore, the detection microcavity and the reference microcavity are positioned close to each other and satisfy the evanescent field coupling condition. When the detection microcavity and the reference microcavity are optically coupled, the two microcavities form a coupled mode, mode splitting, or Vernier envelope. When the target object acts only on the detection microcavity, it causes a change in the effective refractive index of the detection microcavity, thereby changing the coupling mode spacing, mode splitting amount, or Vernier envelope position. This optical coupling structure has the following advantages: by converting the refractive index change of the detection microcavity into a change in the coupling mode parameters through evanescent field coupling, it has higher sensitivity compared to the detection method of single peak position drift; the change in coupling mode spacing or Vernier envelope can amplify small refractive index changes and achieve signal enhancement; this structure makes full use of the interaction between the two cavities, further improving the detection resolution and sensitivity.

[0027] On the other hand, the present invention also provides a hollow coaxial dual-cavity differential quantum well-liquid crystal microlaser biodetection method using the above-mentioned sensor, comprising the following steps: S1, Establishing a baseline: Buffer solution or blank sample is introduced into the detection microcavity and the reference microcavity, the pump light source is turned on, and the pump light is coupled into the detection microcavity and the reference microcavity respectively through the pump light coupler, so that the detection microcavity and the reference microcavity generate stable micro-laser output. The initial spectral parameters λs(0) of the detection microcavity and the initial spectral parameters λr(0) of the reference microcavity are recorded respectively through the micro-laser output coupler. This baseline establishment step ensures that the reference standard for all subsequent spectral changes is consistent, and eliminates the influence of individual device differences and initial state differences on the detection results.

[0028] S2, Introduce the sample to be tested: Introduce the sample to be tested into the central fluid channel of the detection microcavity, and simultaneously introduce the blank sample or reference liquid into the central fluid channel of the reference microcavity. This synchronous injection step ensures that the detection microcavity and the reference microcavity are under the same environmental conditions, so that the subsequent differential processing can effectively deduct the common mode background drift.

[0029] S3, Acquisition of micro-laser spectrum: During the detection process, the micro-laser spectrum parameters λs(t) of the detection microcavity and the micro-laser spectrum parameters λr(t) of the reference microcavity are simultaneously acquired through the micro-laser output coupler. The synchronous acquisition ensures the consistency of the detection signal and the reference signal in time and avoids differential error caused by time delay.

[0030] S4, Calculate spectral changes: Calculate the spectral changes of the detection microcavity and the reference microcavity relative to their initial states, respectively. Δλs(t)=λs(t)-λs(0) Δλr(t) = λr(t) - λr(0); Where λs(0) is the initial spectral peak position of the detection microcavity and λr(0) is the initial spectral peak position of the reference microcavity. By calculating the change relative to the initial state, the influence of the static background signal is eliminated, making the detection signal purer.

[0031] S5, Perform differential processing: Calculate the difference between the spectral change of the detected microcavity and the spectral change of the reference microcavity to obtain the differential detection signal. Δλdiff(t)=Δλs(t)-k·Δλr(t) Where k is a correction coefficient used to compensate for the sensitivity difference between the detection microcavity and the reference microcavity. This differential processing step is the core algorithm of this invention. By subtracting the common-mode background drift recorded by the reference microcavity, a specific signal related only to the target analyte is obtained, which significantly improves the detection accuracy and anti-interference ability.

[0032] S6, Determine the concentration or activity of the target analyte: Compare the differential detection signal Δλdiff(t) with a pre-established standard curve, kinetic model or calibration database to obtain the concentration, activity or reaction rate of the target analyte. By comparing with the standard curve or calibration database, quantitative analysis of the target analyte is achieved, improving the quantitative accuracy of the detection.

[0033] Further, the correction coefficient k in step S5 is obtained through temperature calibration, blank sample calibration, or standard solution calibration. The spectral parameters are selected from any one or more of the following: laser peak position, laser intensity, laser threshold, peak width, mode spacing, mode splitting amount, or Vernier envelope position. In the embodiment where the detection microcavity and the reference microcavity are optically coupled, a single peak position drift is not directly used as the detection signal. Instead, the coupling mode splitting amount, double peak spacing, mode crossover position, or Vernier envelope displacement is used as the detection signal. In this case, the differential signal represents the difference between the detection coupling mode parameter and the reference coupling mode parameter. The outer diameter of the hollow coaxial microcavity is 50 μm to 300 μm. Through the selection of various spectral parameters and the calibration of the correction coefficient, the method of the present invention has good adaptability and flexibility, and can select the optimal signal readout method for different detection systems and different target objects.

[0034] On the other hand, the present invention also provides a method for fabricating a hollow coaxial dual-cavity differential quantum well-liquid crystal micro-laser biosensor, comprising the following steps: S1, Preparation of hollow microcavity substrate: Quartz capillary, silica microtube, glass capillary or transparent polymer hollow microtube are selected as substrate, and its outer diameter, inner diameter and wall thickness are adjusted by drawing, hot melting, etching, laser processing or micro-nano processing methods to meet the whispering gallery mode resonance conditions. This step ensures the optical quality and geometric accuracy of the microcavity substrate.

[0035] S2. Surface treatment of the inner wall of the hollow microcavity: Plasma treatment, silanization treatment, dopamine coating, polymer grafting or self-assembly monolayer modification methods are used to enable the inner wall to fix functional molecules. The surface treatment makes the inner wall surface have suitable functional groups, which provides a basis for the subsequent fixation of functional molecules.

[0036] S3, Constructing the functional layer of the detection microcavity: Immobilizing functional molecules that can specifically interact with the target analyte within the detection microcavity region. The functional molecules are immobilized on the inner wall surface through covalent bonding, physical adsorption, electrostatic adsorption, affinity binding, or embedding. This step achieves the specific functionalization of the detection microcavity.

[0037] S4, Constructing a reference microcavity control layer: An inert layer, sealing layer, deactivation layer, or blank layer is set in the reference microcavity area so that it does not produce a specific response to the target analyte under the same detection environment. This step achieves the inertization treatment of the reference microcavity.

[0038] S5, Forming a liquid crystal response layer: Introducing liquid crystal material near the wall of the hollow microcavity to form a response layer between the central fluid channel and the microcavity wall. The liquid crystal material is 5CB, E7, nematic liquid crystal, cholesteric liquid crystal, reactive liquid crystal monomer or liquid crystal polymer. An orientation regulator, chiral agent, surface active molecule, crosslinking agent or target response molecule is added to the liquid crystal to improve the orientation response capability of the liquid crystal.

[0039] S6, Depositing a quantum well gain layer: The colloidal quantum well solution is formed on the outer surface of the liquid crystal response layer or the outer surface of the microcavity by dip coating, spin coating, drop coating, spray coating, capillary coating, layer-by-layer self-assembly or microfluidic deposition, so as to form a continuous or semi-continuous gain layer.

[0040] S7, forming a low refractive index protective layer: a low refractive index transparent material is coated on the outside of the quantum well gain layer to form a protective layer. The thickness of the protective layer is adjusted according to the light field distribution and protection requirements to simultaneously meet the requirements of optical resonance and environmental isolation.

[0041] S8, Encapsulate the microfluidic interface: Connect the detection microcavity and the reference microcavity to the inlet, outlet, buffer channel, cleaning channel or microfluidic chip respectively, and complete the overall encapsulation.

[0042] S9, Perform optical calibration: Use a standard solution, blank solution, or temperature scan to calibrate the detection microcavity and the reference microcavity to determine the initial peak position, sensitivity coefficient, differential correction coefficient k, and standard detection curve.

[0043] The steps of the above preparation method work together to achieve the complete construction of the sensor of the present invention. The method is controllable, repeatable, and suitable for mass production and standardized manufacturing.

[0044] Furthermore, the sensor is used to detect one or more of the following targets: enzyme targets selected from cellulase, penicillinase, lipase, protease, amylase, glucose oxidase, or nuclease; protein targets selected from antigens, antibodies, cytokines, tumor markers, or viral proteins; nucleic acid targets selected from DNA, RNA, miRNA, or aptamer recognition targets; small molecule targets selected from glucose, uric acid, lactic acid, antibiotics, toxins, or metabolites; and ion targets selected from Hg. 2+ Cu 2+ Pb 2+ Ca 2+ K + or H +The pathogens or particulate targets are selected from bacteria, viruses, exosomes, or cell vesicles. For different targets, only the functional molecular layer of the inner wall of the detection microcavity needs to be changed to construct a corresponding specific detection system. This broad coverage of detection targets gives the invention good versatility and application value.

[0045] The beneficial effects of this invention are as follows: This invention provides a hollow coaxial dual-cavity differential quantum well-liquid crystal micro-laser biosensor and its detection method, which has the following advantages: 1. A hollow coaxial microcavity structure is adopted, in which the sample flow path is set in the central fluid channel, and the liquid crystal response layer, quantum well gain layer and protective layer are arranged radially. As a result, the flow path and reaction area of ​​the sample under test are more clearly defined, which can reduce the problems of uneven sample contact and difficult-to-define reaction interface in traditional external surface contact structures. It is more suitable for continuous sample injection and flow-through detection. This structure integrates the sample flow path, reaction interface, liquid crystal response area and micro laser resonant area in the same coaxial microcavity, realizing a high degree of integration and controllability of the detection process. 2. A dual-cavity differential structure is constructed by setting up a detection microcavity and a reference microcavity. The detection microcavity is used to respond to the target analyte, while the reference microcavity is used to synchronously record non-specific factors such as temperature changes, pump fluctuations, mechanical disturbances, overall sample refractive index changes, and device background drift. Therefore, common-mode interference can be subtracted or weakened through differential calculation, resulting in better stability, repeatability, and quantitative accuracy of the detection results compared to the single-cavity liquid crystal WGM sensing structure. This dual-cavity differential structure is one of the core innovations of this invention, effectively solving the technical problem of difficult background drift subtraction in the single-cavity structure. 3. By introducing a quantum well gain layer into a hollow coaxial microcavity, the sensor can generate active micro-laser output under pump light excitation. Compared with sensing methods that rely solely on passive resonant peak changes, the micro-laser signal has the characteristics of narrow spectral lines, high signal-to-noise ratio, and easy resolution of spectral changes. Therefore, it is beneficial to improve the detection capability of weak signals and the reliability of low-concentration target detection. Compared with organic dyes, quantum well materials have better photostability and longer service life, further improving the long-term working capability of the sensor. 4. By utilizing a liquid crystal response layer to convert enzymatic reactions, molecular recognition reactions, or interface structure changes caused by the target analyte into liquid crystal orientation changes, and then modulating the effective refractive index and micro-laser spectrum of the microcavity by the liquid crystal orientation changes, this scheme retains the advantage of the liquid crystal interface being sensitive to biochemical reactions, and achieves quantitative readout through micro-laser spectroscopy. It overcomes the problem of traditional liquid crystal sensing mainly relying on image brightness changes and having insufficient quantitative capabilities, and realizes the leap from "image qualitative" to "spectral quantitative". 5. By using a protective layer and a coaxial layered structure, a certain degree of isolation is formed between the quantum well gain layer and the liquid to be tested, which can reduce the direct impact of salt ions, proteins, enzymes, surfactants or pH fluctuations in complex samples on the gain layer. This is beneficial to improving the stability of micro-laser output and the lifespan of the device. This protective layer design enables the sensor to be used in more complex sample systems, thus broadening the application range of the sensor.

[0046] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Specific embodiments of the present invention are given in detail below with reference to the accompanying drawings. Attached Figure Description

[0047] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings: Figure 1 This is a schematic diagram of the structure of a hollow coaxial dual-cavity differential quantum well-liquid crystal micro-laser biosensor and its detection method according to an embodiment of the present invention; Figure 2 This is a schematic diagram of the detection microcavity of a hollow coaxial dual-cavity differential quantum well-liquid crystal micro-laser biosensor and its detection method provided in an embodiment of the present invention. Figure 3 This is a schematic diagram of a reference microcavity cross-section of a hollow coaxial dual-cavity differential quantum well-liquid crystal micro-laser biosensor and its detection method provided in an embodiment of the present invention. Figure 4 A schematic diagram of the parallel coupling structure of a hollow coaxial dual-cavity differential quantum well-liquid crystal micro-laser biosensor and its detection method provided in an embodiment of the present invention; Figure 5 This is an actual microlaser spectrum of a protein detection embodiment in a hollow coaxial dual-cavity differential quantum trap-liquid crystal microlaser biosensor and its detection method provided in an embodiment of the present invention; Figure 6 This is a time-resolved microlaser spectrum of a protein detection embodiment in a hollow coaxial dual-cavity differential quantum trap-liquid crystal microlaser biosensor and its detection method provided in an embodiment of the present invention; Figure 7 Concentration-spectral distribution diagram of protein detection embodiment in a hollow coaxial dual-cavity differential quantum trap-liquid crystal microlaser biosensor and its detection method provided in an embodiment of the present invention; Figure 8This is an actual microlaser spectrum of a nucleic acid detection embodiment in a hollow coaxial dual-cavity differential quantum trap-liquid crystal microlaser biosensor and its detection method provided in an embodiment of the present invention; Figure 9 This is a time-spectral distribution diagram of a nucleic acid detection embodiment in a hollow coaxial dual-cavity differential quantum trap-liquid crystal microlaser biosensor and its detection method provided in an embodiment of the present invention; Figure 10 The selective microlaser spectrum of a nucleic acid detection embodiment of a hollow coaxial dual-cavity differential quantum trap-liquid crystal microlaser biosensor and its detection method provided in an embodiment of the present invention.

[0048] The attached diagram lists the components represented by each number as follows: 1. Central fluid channel; 2. Liquid crystal response layer; 3. Quantum well gain layer; 4. Protective layer; 5. Functionalized layer on the inner wall of the central channel; 6. Isolation region; 7. Reflection layer; 8. Pump optical coupler; 9. Micro laser output coupler. Detailed Implementation

[0049] The following is in conjunction with the appendix Figure 1-10 The principles and features of the present invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. The invention is described more specifically in the following paragraphs by way of example with reference to the accompanying drawings. The advantages and features of the invention will become clearer from the following description. It should be noted that the drawings are in a very simplified form and use non-precise proportions, and are only used to facilitate and clarify the illustration of the embodiments of the invention.

[0050] It should be noted that when a component is described as "fixed to" another component, it can be directly on the other component or may have a component in between. When a component is considered "connected to" another component, it can be directly connected to the other component or may have a component in between. When a component is considered "set on" another component, it can be directly set on the other component or may have a component in between. The terms "vertical," "horizontal," "left," "right," and similar expressions used in this document are for illustrative purposes only.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0052] Example 1: Parallel dual-tube hollow coaxial dual-cavity differential quantum trap-liquid crystal microlaser biosensor and its application in protein detection; This embodiment provides a parallel dual-tube hollow coaxial dual-cavity differential quantum trap-liquid crystal microlaser biosensor for the detection of protein biomolecules.

[0053] Sensor structure as follows Figure 1 and Figure 4 As shown, two identical hollow coaxial microtubes are used, one as a detection microcavity and the other as a reference microcavity. The two microtubes are fixed on the same substrate and simultaneously excited by the same pump light source.

[0054] like Figure 2 and Figure 3 As shown, both the detection microcavity and the reference microcavity are hollow coaxial microcavity structures. Each hollow coaxial microcavity includes, from the inside out: a central fluid channel 1, a liquid crystal response layer 2, a quantum well gain layer 3, and a low refractive index protective layer 4.

[0055] The central fluid channel 1 is formed by a quartz capillary with an inner diameter of 50 μm and an outer diameter of 150 μm. The central fluid channel 1 is located at the center of the microcavity and is used to allow the sample to be tested or the reference liquid to pass through.

[0056] A liquid crystal responsive layer 2 is disposed around the central fluid channel 1, and is made of nematic liquid crystal 5CB with a thickness of 5 μm. The liquid crystal responsive layer 2 is used to convert interfacial reactions, enzymatic reactions, or molecular recognition processes caused by the target substance into changes in the orientation of liquid crystal molecules.

[0057] The quantum well gain layer 3 is disposed outside the liquid crystal response layer 2, and is made of CdSe / CdS core-shell quantum well with a thickness of 100 nm. The quantum well gain layer 3 is used to generate an active microlaser signal under external pump light excitation.

[0058] A low-refractive-index protective layer 4 is disposed on the outside of the quantum well gain layer 3. The material is a fluorinated polymer CYTOP, and the thickness is 2 μm. The low-refractive-index protective layer 4 is used to enhance optical field confinement, stabilize resonant modes, and reduce interference from the external environment on the quantum well gain layer 3.

[0059] The inner wall of the central fluid channel 1 of the detection microcavity is provided with a functional molecular layer 5, which is an antibody molecular layer that specifically binds to the target protein and is used to specifically interact with the target analyte.

[0060] The inner wall of the central fluid channel 1 of the reference microcavity is provided with a reaction layer 7, which is a non-specific IgG inert layer used to simultaneously reflect non-specific factors such as temperature, pump fluctuations, mechanical disturbances, changes in the overall refractive index of the sample, and device background drift.

[0061] Two parallel hollow coaxial microtubes are connected to the sample channel and the reference channel, respectively, allowing the detection liquid and the reference liquid to enter the detection microcavity and the reference microcavity, respectively.

[0062] The sensor in this embodiment is prepared according to the following steps: The first step is to prepare a hollow microcavity substrate: two quartz capillaries are selected as the substrate, and their outer and inner diameters are adjusted by laser heating and drawing to meet the resonance conditions of the whispering-gallery mode.

[0063] The second step is to treat the surface of the inner wall of the hollow microcavity: the inner wall of the two quartz capillaries is treated with plasma to activate the inner wall surface and enable it to fix functional molecules.

[0064] The third step is to construct the functional layer 5 of the detection microcavity: In the detection microcavity area, the inner wall is first silanized, and then the antibody molecules that specifically bind to the target protein are covalently fixed to the inner wall surface by glutaraldehyde cross-linking method to form the functional molecular layer 5.

[0065] The fourth step is to construct a reference microcavity control layer 7: a non-specific IgG inert layer is set in the reference microcavity area as the reaction layer 7 so that it does not produce a specific response to the target analyte under the same detection environment.

[0066] Fifth step, forming liquid crystal response layer 2: 5CB liquid crystal material is introduced into the vicinity of the hollow microcavity wall through capillary action, so that a response layer with a thickness of 5μm is formed between the central fluid channel 1 and the microcavity wall.

[0067] Step 6, depositing quantum well gain layer 3: CdSe / CdS colloidal quantum well solution is formed on the outside of liquid crystal response layer 2 by dip coating, so that it constitutes a continuous gain layer with a thickness of 100nm.

[0068] Step 7: Forming a low-refractive-index protective layer 4: Coating CYTOP material on the outside of the quantum well gain layer 3 to form a protective layer with a thickness of 2μm. The thickness of the protective layer is adjusted according to the optical field distribution and protection requirements to simultaneously meet the requirements of optical resonance and environmental isolation.

[0069] Step 8, encapsulating the microfluidic interface: Connect the detection microcavity and the reference microcavity to the inlet and outlet respectively, fix the two microtubes on the same substrate, excite them simultaneously by the same pump light source, and complete the overall encapsulation.

[0070] Step 9: Perform optical calibration: Use blank phosphate buffer to calibrate the detection microcavity and the reference microcavity to determine the initial peak position, sensitivity coefficient, and differential correction coefficient k.

[0071] The method for detecting protein molecules using the above-mentioned sensor includes the following steps: S1, Establishing a baseline: PBS buffer solution is introduced into the detection microcavity and the reference microcavity, the pump light source is turned on, and the pump light is coupled into the detection microcavity and the reference microcavity respectively through the pump light coupler 8, so that the detection microcavity and the reference microcavity generate stable micro-laser output. The initial spectral peak position λs(0) of the detection microcavity and the initial spectral peak position λr(0) of the reference microcavity are recorded through the micro-laser output coupler 9 respectively.

[0072] S2, Introduce the test sample: Introduce the test sample containing the target protein into the central fluid channel 1 of the detection microcavity, and simultaneously introduce blank PBS buffer into the central fluid channel 1 of the reference microcavity.

[0073] S3, Acquisition of micro-laser spectrum: During the detection process, the micro-laser spectrum peak position λs(t) of the detection microcavity and the micro-laser spectrum peak position λr(t) of the reference microcavity are simultaneously acquired through the micro-laser output coupler 9.

[0074] S4, Calculate spectral changes: Calculate the spectral changes of the detection microcavity and the reference microcavity relative to their initial states, respectively. Δλs(t)=λs(t)-λs(0) Δλr(t) = λr(t) - λr(0); S5, Perform differential processing: Calculate the difference between the spectral change of the detected microcavity and the spectral change of the reference microcavity to obtain the differential detection signal. Δλdiff(t)=Δλs(t)-k·Δλr(t) Where k is the correction coefficient, used to compensate for the sensitivity difference between the detection microcavity and the reference microcavity. The correction coefficient k is obtained in advance through blank sample calibration.

[0075] S6, Determine the target protein concentration: Compare the differential detection signal Δλdiff(t) with the pre-established standard curve to obtain the concentration of the target protein.

[0076] Test results and analysis as follows Figure 5As shown, after immobilizing target protein recognition molecules on the inner wall of the detection microcavity, target protein solutions of different concentrations were introduced, and the microlaser spectra output from the detection microcavity were simultaneously acquired. As the target protein concentration increased from low to high, the microlaser spectral lines exhibited a regular shift. The detection mechanism is as follows: after the target protein specifically binds to the functional molecular layer 5 on the inner wall of the detection microcavity, it causes a change in the interface state of the inner wall of the central fluid channel 1, further inducing a change in the orientation of liquid crystal molecules in the liquid crystal response layer 2. Due to the optical anisotropy of the liquid crystal material, the change in liquid crystal orientation causes a change in the effective refractive index of the microcavity. The whispering galleries mode in the hollow coaxial microcavity satisfies the resonance condition; when the effective refractive index changes, a measurable peak shift occurs in the microlaser spectrum. This result indicates that the present invention can achieve concentration-related detection of protein molecules using microlaser spectral response.

[0077] like Figure 6 As shown, under the action of a target protein sample at a fixed concentration, the microlaser spectral lines gradually shift with increasing reaction time. This result indicates that the binding process between the target protein and the recognition layer on the inner wall of the detection microcavity is time-dependent. The binding reaction gradually changes the interfacial anchoring state of the liquid crystal response layer 2, leading to a continuous change in the effective refractive index of the microcavity. The spectral shift gradually stabilizes over time, indicating that the detection reaction has reached a relative equilibrium state. This result demonstrates that the present invention can not only perform endpoint detection but also monitor the binding process of protein molecules in real time.

[0078] like Figure 7 As shown, the microlaser spectral distribution corresponding to different concentrations of target protein exhibits a continuous changing trend. With increasing target protein concentration, the positions of the dominant laser modes in the spectral intensity distribution gradually shift, indicating a correlation between the detection signal and the target protein concentration. This concentration-spectral two-dimensional distribution map can more intuitively display the changing patterns of microlaser modes at different concentrations, which is beneficial for establishing a standard curve between target protein concentration and spectral response. These results demonstrate that the present invention is suitable for the quantitative detection of protein molecules.

[0079] In this embodiment, the reference microcavity synchronously records the background drift caused by temperature fluctuations, pump power variations, and system disturbances. Through differential processing, the aforementioned common-mode interference is effectively subtracted, resulting in a significantly improved signal-to-noise ratio for the detected signal compared to a single-cavity structure.

[0080] Example 2: Coaxial Axially Arranged Hollow Coaxial Dual-Cavity Differential Quantum Trap-Liquid Crystal Microlaser Biosensor and Its Application in Nucleic Acid Detection This embodiment provides a coaxially arranged hollow coaxial dual-cavity differential quantum trap-liquid crystal microlaser biosensor for the detection of nucleic acid molecules.

[0081] Sensor structure as follows Figure 1As shown, in this embodiment, the detection microcavity and the reference microcavity are arranged sequentially along the axial direction of the same hollow capillary. An isolation region 6 is provided between the detection microcavity region and the reference microcavity region to reduce fluid cross-interference between the detection microcavity and the reference microcavity.

[0082] The inner wall of the central fluid channel 1 in the detection microcavity region is immobilized with a single-stranded nucleic acid probe complementary to the target nucleic acid as a functional molecular layer 5. The inner wall of the central fluid channel 1 in the reference microcavity region is immobilized with a mismatched nucleic acid probe as a reaction layer 7 as a control.

[0083] Both the detection microcavity and the reference microcavity are hollow coaxial microcavity structures. Each hollow coaxial microcavity, from the inside out, includes: a central fluid channel 1, a liquid crystal response layer 2, a quantum well gain layer 3, and a low refractive index protective layer 4. Specific parameters are as follows: the central fluid channel 1 is formed by silicon dioxide microtubes with an inner diameter of 30 μm and an outer diameter of 120 μm; the liquid crystal response layer 2 is made of E7 liquid crystal material with a thickness of 8 μm; the quantum well gain layer 3 is a CdSe / ZnS core-shell quantum well with a thickness of 150 nm; and the low refractive index protective layer 4 is made of PMMA material with a thickness of 3 μm.

[0084] The method for nucleic acid molecular detection using the above-mentioned sensor is basically the same as that in Example 1, except that: When establishing the baseline in S1, hybridization buffer is introduced into the detection microcavity and the reference microcavity.

[0085] When the sample to be tested is introduced into S2, the sample containing the target nucleic acid is introduced into the central fluid channel 1 of the detection microcavity, and the blank hybridization buffer is introduced into the central fluid channel 1 of the reference microcavity.

[0086] Steps S3 to S6 are the same as in Example 1, and will not be repeated here.

[0087] Test results and analysis as follows Figure 8 As shown, after fixing a nucleic acid probe complementary to the target nucleic acid on the inner wall of the detection microcavity, different concentrations of target nucleic acid samples were introduced. The microlaser spectrum shifted regularly with increasing target nucleic acid concentration. This result indicates that after the target nucleic acid and the fixed probe hybridize, the interfacial charge, molecular conformation, and interfacial thickness near the inner wall of the central fluid channel 1 are altered, further affecting the orientation state of the liquid crystal response layer 2. The change in liquid crystal orientation leads to a change in the effective refractive index of the microcavity, ultimately manifested as a change in the quantum well microlaser peak position. This result demonstrates that the present invention can be used for the specific spectroscopic detection of nucleic acid molecules.

[0088] like Figure 9As shown, under a certain concentration of target nucleic acid, the microlaser spectrum changes continuously with reaction time. As the hybridization reaction proceeds, the dominant laser mode gradually shifts, indicating that the binding process between the target nucleic acid and the probe layer can be recorded in real time by the hollow coaxial microcavity structure of this invention. This time-spectral distribution map reflects the dynamic process of the nucleic acid hybridization reaction and can be used to analyze the reaction rate, binding process, and detection response time. This result demonstrates that the present invention has the ability to monitor the nucleic acid molecule recognition process in real time.

[0089] like Figure 10 As shown, after introducing fully complementary target nucleic acids, single-base mismatched nucleic acids, triple-base mismatched nucleic acids, random sequence nucleic acids, and blank samples into the detection system, the microlaser peak position change caused by the fully complementary target nucleic acid was the most significant, while the mismatched sequence, random sequence, and blank sample only produced small spectral changes. This result indicates that the nucleic acid probe fixed to the inner wall of the detection microcavity has good specific recognition ability for the target nucleic acid, and non-specific sequences are unlikely to produce the same degree of liquid crystal orientation change and microlaser spectral response. This result demonstrates that the present invention has good selectivity and anti-interference ability in nucleic acid molecule detection.

[0090] In this embodiment, the detection microcavity and the reference microcavity are sequentially arranged along the axial direction of the same hollow capillary tube. The two microcavities have the same or similar temperature, mechanical state, and pumping environment. The isolation zone 6 effectively prevents cross-contamination between the detection liquid and the reference liquid, ensuring the purity of the reference signal. Through differential processing, common-mode interference such as ambient temperature and pump fluctuations is effectively subtracted.

[0091] Example 3: Optically Coupled Hollow Coaxial Dual-Cavity Differential Quantum Trap-Liquid Crystal Microlaser Biosensor; This embodiment provides an optically coupled hollow coaxial dual-cavity differential quantum well-liquid crystal microlaser biosensor.

[0092] Sensor structure as follows Figure 4 As shown, in this embodiment, the detection microcavity and the reference microcavity are two hollow coaxial microtubes arranged side by side, close to each other, with a distance of 0.5 μm between them, satisfying the evanescent field coupling condition. When the detection microcavity and the reference microcavity undergo optical coupling, the two microcavities form a coupling mode.

[0093] Both the detection microcavity and the reference microcavity are hollow coaxial microcavity structures. Each hollow coaxial microcavity, from the inside out, includes: a central fluid channel 1, a liquid crystal response layer 2, a quantum well gain layer 3, and a low refractive index protective layer 4. Specific parameters are as follows: the central fluid channel 1 is formed by a borosilicate glass capillary with an inner diameter of 40 μm and an outer diameter of 160 μm; the liquid crystal response layer 2 is cholesteric liquid crystal with a thickness of 10 μm; the quantum well gain layer 3 is a CdSe / CdS / ZnS core-shell quantum well with a thickness of 200 nm; and the low refractive index protective layer 4 is PDMS material with a thickness of 5 μm.

[0094] The inner wall of the central fluid channel 1 of the detection microcavity is provided with an enzyme substrate functional molecule layer 5 for detecting the activity of the target enzyme. An inactivated substrate layer is provided on the inner wall of the central fluid channel 1 of the reference microcavity as a reaction layer 7 for control.

[0095] When the above-mentioned sensor is used for detection, since the detection microcavity and the reference microcavity are optically coupled, the single peak position drift is not directly used as the detection signal. Instead, the coupling mode splitting amount is used as the detection signal.

[0096] The specific testing steps are basically the same as in Example 1, with the following differences: When S1 establishes the baseline, it records the initial value of the splitting amount of the coupling mode between the detected microcavity and the reference microcavity.

[0097] When S3 acquires signals, it acquires the real-time value of the coupling mode splitting quantity.

[0098] In steps S4 to S6, the changes in the coupling mode splitting amount and the differential signal are calculated. The differential signal is represented as the difference between the detected coupling mode parameters and the reference coupling mode parameters.

[0099] When the target object acts solely on the detection microcavity, it causes a change in the effective refractive index of the microcavity, thereby altering the coupling mode spacing or mode splitting. Through evanescent field coupling, the minute refractive index change in the detection microcavity is amplified into a significant change in the coupling mode parameters. Non-specific background drift is simultaneously recorded in the reference microcavity. This method can further amplify the signal under test and improve detection sensitivity.

[0100] Example 4: Enzyme Activity Detection This embodiment provides an example of enzyme activity detection using an optically coupled hollow coaxial dual-cavity differential quantum trap-liquid crystal microlaser biosensor.

[0101] The sensor structure is the same as in Example 3. The inner wall of the central fluid channel 1 of the detection microcavity is provided with a substrate molecule layer of the target enzyme as a functional molecular layer 5. Taking penicillinase detection as an example, the functional molecular layer 5 is a penicillin G substrate layer. An inactivated penicillin G substrate layer or a blocking layer is provided on the inner wall of the central fluid channel 1 of the reference microcavity as a reaction layer 7.

[0102] The detection method includes the following steps: S1, Establish baseline: Introduce reaction buffer into the detection microcavity and the reference microcavity, turn on the pump light source, and record the initial value of the coupling mode splitting amount of the detection microcavity and the reference microcavity.

[0103] S2, Introduce the test sample: Introduce the test sample containing penicillinase into the central fluid channel 1 of the detection microcavity, and simultaneously introduce the blank reaction buffer into the central fluid channel 1 of the reference microcavity.

[0104] S3, Signal Acquisition: During the detection process, the coupling mode splitting amount of the detection microcavity and the reference microcavity is acquired simultaneously.

[0105] S4, Calculate spectral changes: Calculate the changes in coupling mode splitting of the detection microcavity and the reference microcavity relative to the initial state.

[0106] S5, perform differential processing: calculate the difference between the change in the detected microcavity and the change in the reference microcavity to obtain the differential detection signal.

[0107] S6, Determine enzyme activity: Compare the differential detection signal with a pre-established enzyme activity standard curve to obtain the activity of penicillinase.

[0108] The detection principle is as follows: When penicillinase in the sample enters the central fluid channel 1 of the detection microcavity, it undergoes a hydrolysis reaction with the penicillin G substrate layer on the inner wall, causing changes in molecular arrangement, hydrophilicity / hydrophobicity, or surface anchoring at the liquid crystal / aqueous phase interface. These interfacial changes further induce orientation changes in the liquid crystal molecules in the liquid crystal response layer 2. This change in liquid crystal orientation causes a change in the effective refractive index of the microcavity, which is amplified into a change in the coupling mode splitting amount through evanescent field coupling. Non-specific background drift is synchronously recorded in the reference microcavity, and common-mode interference is subtracted through differential processing to obtain a differential signal related to penicillinase activity.

[0109] Alternative solutions Alternative Option 1: Arrangement of the detection microcavity and the reference microcavity; In addition to the axial arrangement mentioned in the above embodiments ( Figure 1 ), parallel arrangement ( Figure 4 Besides the three arrangements of detection microcavities and optical coupling, the detection microcavities and reference microcavities can also be arranged in other ways. For example, they can be arranged at a certain angle and optical coupling can be achieved through the evanescent field near the intersection point; or they can be arranged in different fluid layers and formed into a three-dimensional integrated structure by vertical stacking. Regardless of the arrangement, as long as the detection microcavities and reference microcavities can work synchronously and the detection microcavities produce a specific response to the target object while the reference microcavities record background drift, they fall within the protection scope of this invention.

[0110] Alternative Option 2: Material for forming the central fluid channel; The central fluid channel 1 can be formed from quartz capillaries, silica microtubes, borosilicate glass capillaries, polymer hollow microtubes, or other transparent media materials. Different materials have different optical properties, surface chemical properties, and mechanical strengths, and can be selected according to specific detection requirements. For example, for applications requiring strong surface modification capabilities, silica microtubes with hydroxyl-rich surfaces can be selected; for applications requiring flexible devices, polymer hollow microtubes can be selected.

[0111] Alternative Option 3: Materials and Additives for the Liquid Crystal Response Layer; The material of the liquid crystal responsive layer 2 can be any one of 5CB, E7, nematic liquid crystal, cholesteric liquid crystal, reactive liquid crystal monomer, or liquid crystal polymer. If necessary, any one or more combinations of orientation modifiers, chiral agents, surfactants, crosslinking agents, or target responsive molecules can be added to the liquid crystal to improve the orientation responsiveness of the liquid crystal.

[0112] Alternative Option 4: Materials for the quantum well gain layer; The quantum well gain layer 3 can be any of the following nanoluminescent materials with optical gain characteristics: colloidal quantum well, colloidal semiconductor nanosheet, or core-shell quantum well, including but not limited to CdSe / CdS, CdSe / CdZnS, CdSe / ZnS, CdSe / CdS / ZnS, and other II-VI or III-V group semiconductor quantum well materials.

[0113] Alternative Option 5: Materials for a low-refractive-index protective layer; The low-refractive-index protective layer 4 can be made of any one of fluorinated polymers, silica, PMMA, CYTOP, PDMS, epoxy resin, or other transparent low-refractive-index materials. The thickness of the protective layer can be adjusted according to the optical field distribution and protection requirements to simultaneously meet the requirements of optical resonance and environmental isolation.

[0114] Alternative Option Six: Types and Fixation Methods of Functional Molecular Layers; The functional molecules in functional molecular layer 5 can be any of the following: enzyme substrates, antibodies, antigens, nucleic acid aptamers, peptide chains, carbohydrate molecules, polysaccharide derivatives, ion-responsive molecules, or molecularly imprinted layers. For different target analytes, only the functional molecular layer on the inner wall of the detection microcavity needs to be replaced to construct the corresponding specific detection system. Functional molecules can be immobilized on the inner wall surface of the central fluid channel 1 through any of the following methods: covalent bonding, physical adsorption, electrostatic adsorption, affinity binding, or embedding.

[0115] Alternative Option Seven: Type of Reflection Layer; The reaction layer 7 can be any one of the following: an inert molecular layer, a blank layer, a sealing layer, a deactivated functional layer, or a control layer that does not react with the target material. It is used to simultaneously reflect non-specific factors such as temperature, pump fluctuations, mechanical disturbances, changes in the overall refractive index of the sample, and device background drift in the reference microcavity.

[0116] Alternative Solution 8: Calculation method for differential signals; In the differential processing, besides the linear differential method Δλdiff(t) = Δλs(t) - k·Δλr(t), other differential algorithms can also be used. For example, weighted differential, nonlinear differential, multi-parameter joint differential, or machine learning-based data fusion methods can be employed. When optical coupling occurs between the detection microcavity and the reference microcavity, the coupling mode splitting amount, the bimodal spacing, the mode crossover position, or the Vernier envelope displacement can also be used as the detection signal. Regardless of the differential algorithm or detection signal type used, as long as the dual-cavity differential structure of the detection microcavity and the reference microcavity is used to subtract the background drift, it falls within the protection scope of this invention.

[0117] Alternative Option Nine: Detectable Objects; This invention can be used to detect a variety of biological or chemical targets, including but not limited to: enzyme targets (such as cellulase, penicillinase, lipase, protease, amylase, glucose oxidase, or nuclease), protein targets (such as antigens, antibodies, cytokines, tumor markers, or viral proteins), nucleic acid targets (such as DNA, RNA, miRNA, or aptamer recognition targets), small molecule targets (such as glucose, uric acid, lactic acid, antibiotics, toxins, or metabolites), and ion targets (such as Hg). 2+ Cu 2 + Pb 2+ Ca 2+ K + or H + This includes pathogens or particulate targets (such as bacteria, viruses, exosomes, or cell vesicles). For different targets, only the functional molecular layer 5 on the inner wall of the detection microcavity needs to be replaced to construct the corresponding specific detection system.

[0118] The hollow coaxial dual-cavity differential quantum trap-liquid crystal microlaser biosensor and its detection method provided by this invention can be widely used in disease diagnosis, food safety testing, environmental monitoring, enzyme activity analysis, and biomanufacturing process control. Due to its advantages such as high detection sensitivity, strong anti-interference ability, good quantitative accuracy, and real-time continuous detection capability, it is particularly suitable for the detection of low-concentration biomarkers and the analysis of target substances in complex sample systems.

[0119] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Content not described in detail in this specification is prior art known to those skilled in the art.

[0120] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Those skilled in the art can readily implement the present invention based on the accompanying drawings and the above description. However, any modifications, alterations, or variations made by those skilled in the art without departing from the scope of the present invention, utilizing the disclosed technical content, are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, or variations made to the above embodiments based on the essential technology of the present invention are still within the protection scope of the present invention.

Claims

1. A hollow coaxial dual-cavity differential quantum well-liquid crystal micro-laser biosensor, comprising a detection microcavity, a reference microcavity, a hollow fluid channel, a pump source, a spectral acquisition unit, and a signal processing unit, characterized in that: The detection microcavity is used to contact the sample to be tested and generate a specific response to the target analyte. The reference microcavity is used to synchronously record temperature changes, pump fluctuations, mechanical disturbances, overall refractive index changes of the sample, and non-specific background signals of device background drift. The pump light source uses a pump optical coupler (8), which couples the pump light into the detection microcavity and the reference microcavity to excite the quantum well gain layer in the detection microcavity and the reference microcavity to generate a micro laser signal; The spectral acquisition unit uses a micro-laser output coupler (9), which acquires the micro-laser spectra output by the detection microcavity and the reference microcavity; The signal processing unit is used to extract the spectral peak position, peak width, peak intensity, mode spacing, mode splitting or Vernier envelope of the detection microcavity and the reference microcavity, and to perform differential calculations. Both the detection microcavity and the reference microcavity are hollow coaxial microcavity structures. Each hollow coaxial microcavity includes, from the inside out: a central fluid channel (1), a liquid crystal response layer (2), a quantum well gain layer (3), and a low refractive index protection layer (4). The central fluid channel (1) is located at the center of the microcavity and is used to allow the sample to be tested or the reference liquid to pass through; The liquid crystal response layer (2) is arranged around the central fluid channel (1) to convert the interfacial reaction, enzymatic reaction or molecular recognition process caused by the target into a change in the orientation of liquid crystal molecules. The quantum well gain layer (3) is disposed outside the liquid crystal response layer (2) or adjacent to the liquid crystal response layer (2) and is used to generate an active micro-laser signal under external pump light excitation. The low refractive index protective layer (4) is disposed outside the quantum well gain layer (3) to enhance the optical field confinement, stabilize the resonance mode and reduce the interference of the external environment on the quantum well gain layer (3).

2. The hollow coaxial dual-cavity differential quantum well-liquid crystal microlaser biosensor according to claim 1, characterized in that, The inner wall of the central fluid channel (1) is provided with an inner wall functional layer. At least in the detection microcavity area, the inner wall functional layer is a functional molecular layer (5) that can specifically interact with the target analyte. The functional molecular layer (5) is selected from any one of the following: enzyme substrate layer, antibody layer, antigen layer, nucleic acid aptamer layer, peptide chain layer, carbohydrate molecular layer, polysaccharide derivative layer, ion-responsive molecular layer or molecular imprinted layer; Within the reference microcavity region, the inner wall functional layer is a reaction layer (7), which includes an inert layer, a blank layer, a sealing layer or an inactivation layer, used to synchronously reflect non-specific factors such as temperature, pump fluctuations, mechanical disturbances, changes in the overall refractive index of the sample and device background drift. For enzyme activity detection, the functional molecular layer (5) is a substrate molecular layer that can be hydrolyzed, sheared or transformed by the target enzyme.

3. The hollow coaxial dual-cavity differential quantum well-liquid crystal microlaser biosensor according to claim 1, characterized in that, The central fluid channel (1) is formed by a quartz capillary, a silica microtube, a borosilicate glass capillary, or a polymer hollow microtube. The inner diameter of the central fluid channel (1) is 20 μm to 200 μm; the thickness of the liquid crystal response layer (2) is 1 μm to 20 μm; the thickness of the quantum well gain layer (3) is 50 nm to 2 μm; and the thickness of the low refractive index protective layer (4) is 0.5 μm to 10 μm. The quantum well gain layer (3) is made of CdSe / CdS, CdSe / CdZnS, CdSe / ZnS, CdSe / CdS / ZnS or other II-VI group semiconductor quantum well materials; The low refractive index protective layer (4) is made of fluorinated polymer, silicon dioxide, PMMA, CYTOP, PDMS, epoxy resin or transparent low refractive index material.

4. The hollow coaxial dual-cavity differential quantum well-liquid crystal micro-laser biosensor according to claim 1, characterized in that, The detection microcavity and the reference microcavity are arranged sequentially along the axial direction of the same hollow capillary. The inner wall of the detection microcavity region is provided with a target response functional layer (5), and the inner wall of the reference microcavity region is provided with an inert layer, a sealing layer, an inactivation functional layer or a control layer that does not react with the target (7). An isolation zone (6) is provided between the detection microcavity region and the reference microcavity region to reduce fluid cross-interference between the detection microcavity and the reference microcavity.

5. The hollow coaxial dual-cavity differential quantum well-liquid crystal micro-laser biosensor according to claim 1, characterized in that, The detection microcavity and the reference microcavity are two hollow coaxial microtubes arranged in parallel. The two hollow coaxial microtubes are respectively connected to the sample channel and the reference channel, or are split through the same injection channel so that the detection liquid and the reference liquid enter the detection microcavity and the reference microcavity respectively. A target response functional layer (5) is provided in the detection microcavity, and an inert functional layer or a blank functional layer (7) is provided in the reference microcavity.

6. The hollow coaxial dual-cavity differential quantum well-liquid crystal micro-laser biosensor according to claim 1, characterized in that, The detection microcavity and the reference microcavity are positioned close to each other and satisfy the evanescent field coupling condition. When the detection microcavity and the reference microcavity are optically coupled, the two microcavities form a coupling mode, mode splitting, or Vernier envelope. When the target object acts only on the detection microcavity, it causes a change in the effective refractive index of the detection microcavity, thereby changing the coupling mode spacing, mode splitting amount, or Vernier envelope position.

7. A detection method using the hollow coaxial dual-cavity differential quantum well-liquid crystal microlaser biosensor according to any one of claims 1 to 6, characterized in that, Includes the following steps: S1, establish a baseline, introduce buffer solution or blank sample into the detection microcavity and the reference microcavity, turn on the pump light source, and couple the pump light into the detection microcavity and the reference microcavity respectively through the pump light coupler (8) so that the detection microcavity and the reference microcavity generate stable micro laser output. Record the initial spectral parameters λs(0) of the detection microcavity and the initial spectral parameters λr(0) of the reference microcavity respectively through the micro laser output coupler (9); S2, introduce the sample to be tested, and introduce the sample to be tested into the central fluid channel (1) of the detection microcavity, while introducing the blank sample or reference liquid into the central fluid channel (1) of the reference microcavity. S3, collect micro-laser spectrum. During the detection process, the micro-laser spectrum parameters λs(t) of the detection microcavity and the micro-laser spectrum parameters λr(t) of the reference microcavity are collected synchronously through the micro-laser output coupler (9). S4, Calculate the spectral changes, specifically the spectral changes of the detection microcavity and the reference microcavity relative to their initial states: Δλs(t)=λs(t)-λs(0) Δλr(t)=λr(t)-λr(0) Where λs(0) is the initial spectral peak position of the detection microcavity, and λr(0) is the initial spectral peak position of the reference microcavity; S5, perform differential processing, calculate the difference between the spectral change of the detected microcavity and the spectral change of the reference microcavity to obtain the differential detection signal: Δλdiff(t)=Δλs(t)-k·Δλr(t) Where k is the correction coefficient, used to compensate for the sensitivity difference between the detection microcavity and the reference microcavity; S6, determine the concentration or activity of the target analyte, and compare the differential detection signal Δλdiff(t) with a pre-established standard curve, kinetic model or calibration database to obtain the concentration, activity or reaction rate of the target analyte.

8. The detection method according to claim 7, characterized in that, The correction coefficient k mentioned in step S5 is obtained through temperature calibration, blank sample calibration, or standard solution calibration. The spectral parameters are selected from any one or more of the following: laser peak position, laser intensity, laser threshold, peak width, mode spacing, mode splitting amount, or Vernier envelope position. In the embodiment where optical coupling occurs between the detection microcavity and the reference microcavity, a single peak position shift is not directly used as the detection signal. Instead, the coupling mode splitting amount, the spacing between the two peaks, the mode crossover position, or the Vernier envelope shift is used as the detection signal. In this case, the differential signal represents the difference between the detection coupling mode parameters and the reference coupling mode parameters. The outer diameter of the hollow coaxial microcavity is 50 μm to 300 μm.

9. A method for fabricating a hollow coaxial dual-cavity differential quantum well-liquid crystal micro-laser biosensor, characterized in that, The sensor is the hollow coaxial dual-cavity differential quantum well-liquid crystal micro-laser biosensor according to any one of claims 1 to 6, and the preparation method includes the following steps: S1, Preparation of hollow microcavity substrate: Select quartz capillary, silica microtube, glass capillary or transparent polymer hollow microtube as substrate, and adjust its outer diameter, inner diameter and wall thickness by stretching, hot melting, etching, laser processing or micro-nano processing methods to make it meet the resonance conditions of the whispering gallery mode. S2, Surface treatment of the inner wall of the hollow microcavity: Plasma treatment, silanization treatment, dopamine coating, polymer grafting or self-assembly monolayer modification methods are used to enable the inner wall to fix functional molecules. S3, Construct a detection microcavity functional layer (5): Fix functional molecules that can specifically interact with the target analyte in the detection microcavity region. The functional molecules are fixed on the inner wall surface by covalent bonding, physical adsorption, electrostatic adsorption, affinity binding or embedding. S4, Construct a reference microcavity control layer (7): Set an inert layer, a sealing layer, an inactivation layer or a blank layer in the reference microcavity area so that it does not produce a specific response to the target analyte under the same detection environment; S5, forming a liquid crystal response layer (2): introducing liquid crystal material near the wall of the hollow microcavity to form a response layer between the central fluid channel (1) and the microcavity wall; The liquid crystal material is 5CB, E7, nematic liquid crystal, cholesteric liquid crystal, reactive liquid crystal monomer or liquid crystal polymer; an orientation modifier, chiral agent, surfactant molecule, crosslinking agent or target response molecule is added to the liquid crystal to improve the orientation response capability of the liquid crystal. S6, Deposit quantum well gain layer (3): The colloidal quantum well solution is formed on the outer side of the liquid crystal response layer (2) or the outer surface of the microcavity by dip coating, spin coating, drop coating, spray coating, capillary coating, layer-by-layer self-assembly or microfluidic deposition, so that it constitutes a continuous or semi-continuous gain layer. S7, forming a low refractive index protective layer (4): a low refractive index transparent material is coated on the outside of the quantum well gain layer (3) to form a protective layer. The thickness of the protective layer is adjusted according to the optical field distribution and protection requirements to simultaneously meet the requirements of optical resonance and environmental isolation. S8, Encapsulate the microfluidic interface: Connect the detection microcavity and the reference microcavity to the inlet, outlet, buffer channel, cleaning channel or microfluidic chip respectively, and complete the overall encapsulation; S9, Perform optical calibration: Use a standard solution, blank solution, or temperature scan to calibrate the detection microcavity and the reference microcavity to determine the initial peak position, sensitivity coefficient, differential correction coefficient k, and standard detection curve.

10. The hollow coaxial dual-cavity differential quantum well-liquid crystal micro-laser biosensor according to claim 1, characterized in that, The sensor is used to detect one or more of the following targets: The target enzyme is selected from cellulase, penicillinase, lipase, protease, amylase, glucose oxidase or nuclease; Protein targets are selected from antigens, antibodies, cytokines, tumor markers, or viral proteins; Nucleic acid targets are selected from DNA, RNA, miRNA, or aptamer recognition targets; Small molecule targets, selected from glucose, uric acid, lactic acid, antibiotics, toxins or metabolites; Ion target analytes, selected from Hg 2+ Cu 2+ Pb 2+ Ca 2+ K + or H + ; Pathogens or particulate targets are selected from bacteria, viruses, exosomes, or cellular vesicles.

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