An integrated microfluidic biosensor chip based on micro-column array D-shaped optical fiber

Abstract

The application discloses a kind of integrated microfluidic biological sensing chip based on micro column array D type optical fiber, belong to biological detection field.This integrated microfluidic biological sensing chip based on micro column array D type optical fiber includes fiber base micro column array D type sensing optical fiber;Periodic micro column array is arranged on the side edge polishing surface of fiber base micro column array D type sensing optical fiber;Micro column array surface is successively coated with sensitizing layer and specific modification layer;The raw material of sensitizing layer includes sensitizing material, and sensitizing material includes MXene material and / or metal nano material, and the raw material of specific modification layer includes biological aptamer material.Micro column array structure can be used as cell capture enhancement interface, forms a large number of capture sites, increases the contact area of cell and chip surface, improves cell capture efficiency.Sensitizing layer can greatly enhance the evanescent field intensity of optical fiber, and biological aptamer material realizes specific identification, and also has good sensitivity and detection accuracy to very low concentration circulating tumor cell.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and in particular to an integrated microfluidic biosensor chip based on a micropillar array of D-type optical fibers. Background Technology

[0002] Circulating tumor cells (CTCs) are tumor cells that detach from solid tumors or metastatic tumors and enter the peripheral blood circulation system. They are a key factor leading to tumor metastasis. Most CTCs undergo apoptosis or are phagocytosed after entering the peripheral blood, but a small number can escape and anchor to develop into metastatic lesions, increasing the risk of death for patients with malignant tumors. Detection of CTCs mainly involves capturing and detecting the presence of circulating tumor cells in peripheral blood, monitoring changes in the type and number of CTCs. This monitoring is of great significance for early cancer diagnosis, prognostic assessment, and treatment monitoring.

[0003] Currently, the detection technology for circulating tumor cells often requires complex sample preprocessing and multi-step operation procedures, which not only increases the detection time and cost, but also easily introduces errors. Furthermore, since the concentration of circulating tumor cells in peripheral blood is usually extremely low, existing detection methods often cannot achieve accurate detection, leading to missed detections.

[0004] To improve the detection technology of circulating tumor cells (CTCs), microfluidics has been applied due to its rapid detection and minimal impact on cell viability. However, while traditional microfluidic chips offer high throughput and rapid sample processing within tiny channels, they lack in-situ detection capabilities. Samples must be transferred to external detection equipment for pre-analysis before detection, increasing operational complexity and the risk of sample contamination. Furthermore, in the detection of low concentrations of CTCs, other components in the blood sample can interfere, affecting the accuracy and reliability of the detection. Using optical fibers as the light transmission medium and embedding them into the chip allows for improved sensitivity and precise detection of light signals using fiber optic sensors. However, this approach still struggles to capture low concentrations of cells, and the integration of both methods relies on external optical components, hindering miniaturization and integration, thus limiting its application in portable devices.

[0005] Therefore, it is of great significance to obtain an integrated detection chip that can efficiently capture low-concentration cells and detect them in situ with high sensitivity. Summary of the Invention

[0006] This invention provides an integrated microfluidic biosensor chip based on a micropillar array of D-type optical fibers, which can solve the problems of high difficulty in capturing low concentrations of circulating tumor cells, low sensitivity, and complex operation in the detection process of existing technologies.

[0007] An integrated microfluidic biosensor chip based on a micropillar array D-type optical fiber, comprising a fiber-based micropillar array D-type sensing optical fiber;

[0008] The side polished surface of the fiber-based micropillar array D-type sensing fiber is provided with a periodic micropillar array.

[0009] The surface of the micropillar array is sequentially coated with a sensitizing layer and a specific modification layer;

[0010] The raw materials for the sensitizing layer include sensitizing materials, such as MXene materials and / or metal nanomaterials; the raw materials for the specific modification layer include bioaptamer materials.

[0011] Preferably, the height of the micropillars in the periodic micropillar array is 35–45 μm, the diameter of the micropillars is 10–20 μm, and the spacing between the micropillars is 5–15 μm.

[0012] Preferably, the MXene material includes M n+1 X n T x MXene nanosheets; the M n+1 X n T x In MXene nanosheets, M includes any one of Ti, Nb, and V, and X includes C and T. x Includes any one of -OH, -F, -O and -Cl, 1≤n≤3;

[0013] Metal nanomaterials include one or a combination of two of gold nanorods and gold nanostars.

[0014] Preferably, the bioaptamer material includes one or more combinations of Anti-EpCAM antibody, folic acid, biotin, biotinylated streptavidin, and biotinylated Anti-EpCAM antibody.

[0015] By adopting the above technical solution, the fiber-based micropillar array D-type sensing fiber is used as the substrate of the microfluidic biosensor chip of the present invention. When the light signal is transmitted in the D-type sensing fiber, the evanescent field interacts with the cells captured by the fiber, thereby causing changes in the light signal. By detecting the changes in the light signal, quantitative detection of target biomolecules can be achieved.

[0016] Using D-type optical fiber as the sensing fiber substrate, compared with other fiber types, it can remove part of the cladding through side polishing, so that the evanescent field of the fiber core can be effectively exposed. Moreover, its flat polished surface also provides an ideal substrate for the fabrication and integration of micropillar arrays.

[0017] A periodic micropillar array is set on the side polished surface of the fiber-based micropillar array D-type sensing fiber. This micropillar array structure can serve as a cell capture enhancement interface. The vertical micropillars greatly increase the effective surface area of ​​the chip. When sample cells flow through the fluid, they will collide and contact with the sides, top surfaces, and gaps between multiple micropillars, transforming the original two-dimensional capture interface into a three-dimensional structure, forming a large number of potential capture sites. Moreover, the presence of the micropillar array will change the distribution of sample flow, increase the contact area between the target cells and the chip, prolong the residence time of the target cells in the capture area, improve the probability of specific binding, and increase the cell capture efficiency.

[0018] Furthermore, the surface of the micropillar array is sequentially coated with an enhancing layer and a specific modification layer. The enhancing layer is made of MXene and / or metal nanomaterials. The collective oscillation of free electrons in the metal nanomaterials resonates with photons, thereby generating a highly localized enhanced electromagnetic field around the metal nanomaterials with an intensity much higher than that of the incident light. When this field overlaps with the evanescent field region of the optical fiber, it significantly amplifies the intensity of the evanescent field. MXene itself has extremely high conductivity and dielectric constant, which can effectively increase the reflection of light waves at the interface, thereby confining and extending more light energy into the evanescent field. The coating of the enhancing layer can create a more powerful optical field environment than the original evanescent field at the micropillar array interface, i.e., the cell capture interface. When target cells enter the significantly enhanced evanescent field region, even if the target cell concentration is extremely low, their slight perturbation to the optical field can be synchronously amplified, outputting an optical signal. This helps the microfluidic biosensor chip to more easily detect subtle signal changes caused by single or a small number of target cells.

[0019] Moreover, MXene materials include M n+1 X n T x MXene nanosheets, with their graphene-like two-dimensional layered structure and large specific surface area, provide numerous binding sites for the immobilization of subsequent specific modification materials. They also enable efficient and stable coupling of bioaptamer materials using their natural functional groups, further improving cell capture efficiency. Moreover, MXene materials exhibit excellent chemical stability and biocompatibility in biosensing environments, without interfering with samples. They can also form uniform and dense films on the complex microstructure surface of micropillar arrays, enhancing evanescent field intensity and improving chip sensitivity.

[0020] Furthermore, after coating the sensitizing layer, a specific modification layer is coupled and coated on the surface of the micropillar array. This introduces a bio-recognition element with high affinity and high selectivity onto the surface of the optical fiber. The bio-aptamer material can pair with receptors on the surface of target cells, thereby achieving specific recognition of target biomolecules such as circulating tumor cells from the sample and improving detection accuracy.

[0021] The D-type sensing fiber obtained through the above modification process utilizes the structural characteristics of the micropillar array to increase the contact area between the target cells and the chip, significantly improving the capture efficiency of low-concentration cells. Its high specific surface area also increases the number of capture sites. By enhancing the evanescent field intensity of the fiber, it is easier to detect minute signal changes caused by single or small numbers of target cells. Furthermore, by leveraging the specific recognition function of bioaptamer materials, precise identification of target cells can be achieved. Integrating the D-type fiber with microfluidic channels enables a unified process for target cell capture and optical detection, greatly simplifying the operation and reducing the risk of sample loss and contamination.

[0022] Preferably, the fiber-based micropillar array D-type sensing fiber is prepared according to the following method:

[0023] S1. Polish the side of the single-mode fiber to form a side polished surface to obtain a D-type fiber, and clean the side polished surface.

[0024] S2. The side polished surface is etched to form a periodic micropillar array, resulting in a micropillar array D-type optical fiber;

[0025] S3. Immerse the micropillar array D-type optical fiber in an alkaline aqueous solution, control the pH value of the alkaline aqueous solution to 7-10, and soak for 1-2 hours to obtain hydroxylated micropillar array D-type optical fiber;

[0026] S4. The hydroxylated micropillar array D-type optical fiber is pretreated by immersing it in an aminosilane coupling agent alcohol solution, dried, and then immersed in an aqueous solution containing sensitizing material for 30-60 minutes. Then the temperature is raised until the solvent evaporates, and the immersion is repeated to form a sensitizing layer.

[0027] S5. Add a bifunctional crosslinking agent to an organic solvent and stir to obtain a crosslinking agent solution; immerse the optical fiber material obtained above in the crosslinking agent solution for 10-20 min, dry it and transfer it to a bioaptamer material solution, incubate it at 10-25℃ for 12-24 h to form a specific modification layer, and finally dry it to obtain a fiber-based micropillar array D-type sensing optical fiber.

[0028] Preferably, the polishing depth of the side polishing surface is 60-80 μm, the polishing length is 10-15 mm, and the transition zone length is 4-8 mm.

[0029] Preferably, the bifunctional crosslinking agent comprises 4-maleimide butyric acid-N-succinimide ester; the concentration of the crosslinking agent solution is 0.1-10 mg / L.

[0030] More preferably, the cladding diameter of the single-mode fiber is 125 μm.

[0031] More preferably, the alkaline aqueous solution includes an aqueous solution of sodium hydroxide.

[0032] More preferably, the aminosilane coupling agent alcohol solution is an aminosilane coupling agent ethanol solution with a concentration of 0.02 to 0.05 mg / mL; the aminosilane coupling agent includes one or more combinations of 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropylmethyldimethoxysilane and γ-aminopropylmethyldiethoxysilane.

[0033] More preferably, the concentration of the aqueous solution containing the sensitizing material is 0.1 to 1 mg / mL.

[0034] More preferably, the thickness of the sensitizing layer is 8–12 nm.

[0035] More preferably, the concentration of the bioaptamer material solution is 0.1 μmol to 1 mmol.

[0036] By adopting the above technical solution, single-mode optical fiber is polished to obtain D-type optical fiber, whose polished side surface is flat and smooth, which can be used as a substrate for micropillar array. Then, the micropillar array is formed by etching. Then, the surface of the micropillar array is hydroxylated to introduce active hydroxyl groups. Subsequently, it is immersed in an aminosilane coupling agent alcohol solution. The aminosilane coupling agent hydrolyzes to obtain silanol groups, which combine with the hydroxyl groups on the surface of the hydroxylated micropillar array D-type optical fiber to form Si-O-Si chemical bonds.

[0037] Then, the micropillar array is immersed in an aqueous solution containing the sensitizing material. Since the surface of the micropillar array modified by the aminosilane coupling agent is positively charged, while the surface of the sensitizing material is negatively charged, the electrostatic attraction is utilized, and the material is gradually heated until the solvent evaporates, so that the sensitizing material can be firmly adsorbed onto the surface of the micropillar array. Through repeated immersion, the appropriate thickness of the sensitizing layer is achieved.

[0038] The D-type optical fiber, after the above treatment, is then immersed in a crosslinking agent solution. Due to the combination between the aminosilane coupling agent and the optical fiber, amino groups are introduced onto the surface of the optical fiber. The amino groups will further react and crosslink with the bifunctional crosslinking agent. Then, it is immersed in a bioaptamer material solution, where the crosslinking agent combines with the bioaptamer, thereby modifying the surface of the micropillar array with the bioaptamer material, resulting in a fiber-based micropillar array D-type sensing optical fiber with high sensitivity and high precision, integrating cell capture and detection.

[0039] Preferably, step S2 specifically includes:

[0040] S201. Aluminum film is deposited on the surface of a D-type optical fiber by magnetron sputtering;

[0041] S202. A micropillar array pattern is formed on the side polished surface by photolithography, and UV adhesive is coated on the unpolished area and the transition area;

[0042] S203. The micropillar array is gradually formed by etching in a nitrogen-containing etching gas. Finally, it is cleaned with a piranha solution to obtain the micropillar array D-type optical fiber.

[0043] By adopting the above technical solution, an aluminum film is deposited on the entire surface of the D-shaped optical fiber using magnetron sputtering technology. The deposition thickness of the aluminum film can be adjusted according to the height of the micropillars in the required micropillar array. Then, through photolithography, that is, inkjet printing using negative photoresist, a mask is first made on the polished side surface of the D-shaped optical fiber with a printed pattern. After UV exposure and curing, the aluminum film in the unprinted areas on the polished side surface is removed using a phosphoric acid solution, thus forming the prototype of the micropillar array. Then, UV adhesive is coated on the unpolished area and the transition area to protect the aluminum film in other locations of the D-shaped optical fiber from being removed in subsequent processing.

[0044] The resulting D-shaped optical fiber was then placed in a fluorine-containing etching gas. The silicon dioxide in the fiber reacted with the fluorine in the etching gas to form silicon tetrafluoride, which etched the uncoated aluminum areas of the fiber, gradually forming a micropillar array. Finally, a piranha solution was used to remove the residual photoresist and aluminum film from the surface of the D-shaped optical fiber, yielding the micropillar array D-shaped optical fiber.

[0045] Preferably, the integrated microfluidic biosensor chip based on a micropillar array of D-type optical fibers is prepared according to the following method:

[0046] After mixing PDMS with a curing agent, the mixture is poured into a microfluidic mold and cured at 40–60°C to obtain a PDMS microfluidic substrate with a single microchannel structure. A fiber-based micropillar array D-type sensing fiber is embedded into the microchannel of the PDMS microfluidic substrate, and finally encapsulated to obtain the final product.

[0047] More preferably, the mass ratio of PDMS (polydimethylsiloxane) to the curing agent is 1:(0.1 to 0.15); the curing agent includes one or more combinations of dibutyl phthalate, benzoyl peroxide and dicumyl peroxide.

[0048] By adopting the above technical solution, the integrated microfluidic biosensor chip based on micropillar array D-type optical fiber of the present invention directly uses the sensing optical fiber as the flow channel, realizing "flow channel as sensor". This eliminates the complex process of separately processing the flow channel and embedding the sensor in the traditional design, greatly simplifying the chip structure and detection process. Moreover, it can further improve the detection sensitivity. Since the fiber-based micropillar array D-type sensing optical fiber is the only flow channel, the target cell will be directly located within the evanescent field range of the optical fiber, ensuring the signal strength, thereby improving the sensitivity of the microfluidic biosensor chip, meeting the detection accuracy of low concentration circulating tumor cells, and improving the capture efficiency of extremely low concentration target substances.

[0049] Preferably, the integrated microfluidic biosensor chip based on micropillar array D-type optical fiber is used for the detection of circulating tumor cells.

[0050] More preferably, the detection of circulating tumor cells using the integrated microfluidic biosensor chip based on a micropillar array D-type optical fiber obtained by the present invention mainly involves injecting a sample containing the target biomolecule into a microchannel through the inlet of the microfluidic chip. As the sample flows within the microchannel, the target biomolecule specifically binds to the specific modification layer on the chip surface. Simultaneously, by inputting light of a specific wavelength at one end of the optical fiber, the change in the light signal is detected at the other end of the optical fiber. Based on the relationship between the change in the light signal and the concentration of the target biomolecule, quantitative detection of the target biomolecule is achieved.

[0051] More preferably, the light input wavelength is 1200–1800 nm.

[0052] The beneficial effects of this invention are:

[0053] 1. The integrated microfluidic biosensor chip based on micropillar array D-type optical fiber of the present invention includes fiber-based micropillar array D-type sensing optical fiber, which realizes the detection of target biomolecules by utilizing changes in optical signals. Furthermore, a periodic micropillar array is provided on the side polished surface of the fiber-based micropillar array D-type sensing optical fiber. Its micropillar array structure can serve as a cell capture enhancement interface, which can form a large number of capture sites while increasing the contact area between the cell and the chip surface, thereby improving the cell capture efficiency.

[0054] 2. The surface of the micropillar array of the fiber-based micropillar array D-type sensing fiber of the present invention is further coated with a sensitizing layer and a specific modification layer in sequence. The sensitizing material in the sensitizing layer has excellent optical and electrical properties, which can significantly enhance the evanescent field intensity of the fiber, thereby making it easier to detect small signal changes caused by single or a small number of target cells. The bioaptamer material in the specific modification layer can coordinate with the target biomolecules to achieve specific recognition, improve detection accuracy, and also have good sensitivity and detection accuracy for extremely low concentrations of circulating tumor cells.

[0055] 3. In the integrated microfluidic biosensor chip based on micropillar array D-type optical fiber of the present invention, the fiber-based micropillar array D-type sensing optical fiber is used as the only liquid channel, realizing "flow channel as sensor", which simplifies the structure of the chip and the detection process, realizes the integration of target cell capture and optical detection process, greatly simplifies the operation steps, and reduces the risk of sample loss and contamination. Attached Figure Description

[0056] Figure 1 This is a schematic diagram of the structure of the fiber-based micropillar array D-type sensing fiber obtained in Embodiment 1 of the present invention, wherein 101 is a periodic micropillar array; 102 is a side polished surface; 103 is a transition region; 104 is a single-mode fiber core; 105 is a probe light input port; and 106 is a signal light output port.

[0057] Figure 2 This is a schematic diagram of the integrated microfluidic biosensor chip based on micropillar array D-type optical fiber obtained in Embodiment 1 of the present invention, wherein 201 is fiber-based micropillar array D-type sensing optical fiber; 202 is PDMS microfluidic substrate; 203 is cover plate; 204 is sample inlet; and 205 is sample outlet.

[0058] Figure 3 The spectrum obtained by the integrated microfluidic biosensor chip based on micropillar array D-type optical fiber obtained in Embodiment 1 of the present invention is the spectrum obtained for test samples of different concentrations and pure water during performance testing. Detailed Implementation

[0059] The specific embodiments of the present invention will be described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0060] Example

[0061] Example 1: An integrated microfluidic biosensor chip based on a micropillar array of D-type optical fibers was prepared according to the following method:

[0062] Fabrication of fiber-based micropillar array D-type sensing fiber:

[0063] S1. Polish the side of a single-mode fiber (cladding diameter of 125μm) to form a side polishing surface to obtain a D-type fiber, and clean the side polishing surface. The polishing depth of the side polishing surface is 70μm, the polishing length is 10mm, and the transition zone length is 5mm.

[0064] S2. The side-polished surface is etched to form a periodic micropillar array, resulting in a micropillar array D-type optical fiber. Specifically:

[0065] S201. Aluminum film is deposited on the surface of a D-type optical fiber by magnetron sputtering;

[0066] S202. A micropillar array pattern is formed on the side polished surface by photolithography, and UV adhesive is coated on the unpolished area and the transition area;

[0067] S203. The micropillar array is gradually formed by etching in a nitrogen-containing etching gas for 30 minutes. Finally, the micropillar array D-type optical fiber is obtained by cleaning with piranha solution.

[0068] The obtained periodic micropillar array has a micropillar height of 40 μm, a micropillar diameter of 15 μm, and a spacing of 10 μm between micropillars;

[0069] S3. Immerse the micropillar array D-type optical fiber in an aqueous sodium hydroxide solution, control the pH value to 8, and soak for 2 hours to obtain hydroxylated micropillar array D-type optical fiber;

[0070] S4. The hydroxylated micropillar array D-type optical fiber was pretreated by immersing it in a 0.03 mg / mL solution of 3-aminopropyltriethoxysilane-ethanol, and then dried before immersion in a 0.5 mg / mL solution containing the sensitizing material Ti3C2T. x The nanosheets (with a diameter of 2-10 μm) were immersed in an aqueous solution for 40 min, and then the temperature was raised until the solvent evaporated. The immersion was repeated to form a sensitizing layer, and the thickness of the sensitizing layer was controlled to be 10 nm.

[0071] S5. Add 4-maleimide butyric acid-N-succinimide ester to dimethyl sulfoxide and stir to obtain a crosslinking agent solution with a concentration of 4 mg / L; immerse the optical fiber material obtained above in the crosslinking agent solution for 10 min, dry it and then transfer it to a biotinylated streptavidin solution with a concentration of 30 μmol, incubate it at 15 °C for 12 h to form a specific modification layer, and finally dry it to obtain a fiber-based micropillar array D-type sensing optical fiber;

[0072] Fabrication of an integrated microfluidic biosensor chip based on a micropillar array of D-type optical fibers:

[0073] PDMS and curing agent dibutyl phthalate were mixed at a mass ratio of 1:0.1, then poured into a microfluidic mold and cured at 50°C to obtain a PDMS microfluidic substrate with a microchannel structure. The obtained fiber-based micropillar array D-type sensing fiber was embedded in the microchannel of the PDMS microfluidic substrate and finally encapsulated.

[0074] Example 2: An integrated microfluidic biosensor chip based on a micropillar array D-type optical fiber. The only difference from Example 1 is that, in the fabrication process of the fiber-based micropillar array D-type sensing optical fiber, an equal amount of gold nanorods (with an average particle size of 5 nm) are used to replace Ti3C2T. xNanosheets; biotinylated streptavidin was replaced with an equal amount of biotinylated Anti-EpCAM antibody.

[0075] Example 3 is an integrated microfluidic biosensor chip based on a micropillar array D-type optical fiber. The only difference from Example 1 is that, in the fabrication process of the fiber-based micropillar array D-type sensing optical fiber, the height of the micropillars in the obtained periodic micropillar array is 30 μm, the diameter of the micropillars is 10 μm, and the spacing between the micropillars is 20 μm.

[0076] Example 4: An integrated microfluidic biosensor chip based on a micropillar array D-type optical fiber. The only difference from Example 1 is that, in the fabrication process of the fiber-based micropillar array D-type sensing optical fiber, the height of the micropillars in the obtained periodic micropillar array is 50 μm, the diameter of the micropillars is 25 μm, and the spacing between the micropillars is 5 μm.

[0077] Comparative Example

[0078] Comparative Example 1, an integrated microfluidic biosensor chip based on a micropillar array D-type optical fiber, differs from Example 1 only in that the fiber-based micropillar array D-type sensing optical fiber is prepared according to the following method:

[0079] S1. Polish the side of a single-mode fiber (cladding diameter of 125μm) to form a side polishing surface to obtain a D-type fiber, and clean the side polishing surface. The polishing depth of the side polishing surface is 70μm, the polishing length is 10mm, and the transition zone length is 5mm.

[0080] S2. The side-polished surface is etched to form a periodic micropillar array, resulting in a micropillar array D-type optical fiber. Specifically:

[0081] S201. Aluminum film is deposited on the surface of a D-type optical fiber by magnetron sputtering;

[0082] S202. A micropillar array pattern is formed on the side polished surface by photolithography, and UV adhesive is coated on the unpolished area and the transition area;

[0083] S203. The micropillar array is gradually formed by etching in a nitrogen-containing etching gas for 30 minutes. Finally, the micropillar array D-type optical fiber is obtained by cleaning with piranha solution.

[0084] The obtained periodic micropillar array has a micropillar height of 40 μm, a micropillar diameter of 15 μm, and a spacing of 10 μm between micropillars;

[0085] S3. Immerse the micropillar array D-type optical fiber in an aqueous sodium hydroxide solution, control the pH value to 8, and soak for 2 hours to obtain hydroxylated micropillar array D-type optical fiber;

[0086] S4. The hydroxylated micropillar array D-type optical fiber was pretreated by immersing it in a 0.03 mg / mL 3-aminopropyltriethoxysilane ethanol solution and then dried.

[0087] S5. Add 4-maleimide butyric acid-N-succinimide ester to dimethyl sulfoxide and stir to obtain a crosslinking agent solution with a concentration of 4 mg / L; immerse the optical fiber material obtained above in the crosslinking agent solution for 10 min, dry it and then transfer it to a biotinylated streptavidin solution with a concentration of 30 μmol, incubate it at 15 °C for 12 h to form a specific modification layer, and finally dry it to obtain a fiber-based micropillar array D-type sensing optical fiber.

[0088] Comparative Example 2, an integrated microfluidic biosensor chip based on a micropillar array D-type optical fiber, differs from Example 1 only in that the fiber-based micropillar array D-type sensing optical fiber is prepared according to the following method:

[0089] Fabrication of fiber-based micropillar array D-type sensing fiber:

[0090] S1. Polish the side of a single-mode fiber (cladding diameter of 125μm) to form a side polishing surface to obtain a D-type fiber, and clean the side polishing surface. The polishing depth of the side polishing surface is 70μm, the polishing length is 10mm, and the transition zone length is 5mm.

[0091] S2. The side-polished surface is etched to form a periodic micropillar array, resulting in a micropillar array D-type optical fiber. Specifically:

[0092] S201. Aluminum film is deposited on the surface of a D-type optical fiber by magnetron sputtering;

[0093] S202. A micropillar array pattern is formed on the side polished surface by photolithography, and UV adhesive is coated on the unpolished area and the transition area;

[0094] S203. The micropillar array is gradually formed by etching in a nitrogen-containing etching gas for 30 minutes. Finally, the micropillar array D-type optical fiber is obtained by cleaning with piranha solution.

[0095] The obtained periodic micropillar array has a micropillar height of 40 μm, a micropillar diameter of 15 μm, and a spacing of 10 μm between micropillars;

[0096] S3. Immerse the micropillar array D-type optical fiber in an aqueous sodium hydroxide solution, control the pH value to 8, and soak for 2 hours to obtain hydroxylated micropillar array D-type optical fiber;

[0097] S4. The hydroxylated micropillar array D-type optical fiber was pretreated by immersing it in a 0.03 mg / mL solution of 3-aminopropyltriethoxysilane-ethanol, and then dried before immersion in a 0.5 mg / mL solution containing the sensitizing material Ti3C2T. x Nanosheets (with a diameter of 2–10 μm) were immersed in an aqueous solution for 40 min, and then the temperature was raised until the solvent evaporated. This process was repeated to form an enhancement layer. The thickness of the enhancement layer was controlled to be 10 nm, resulting in a fiber-based micropillar array D-type sensing fiber.

[0098] Comparative Example 3: An integrated microfluidic biosensor chip based on D-type optical fiber was prepared according to the following method:

[0099] Fabrication of fiber-based D-type sensing optical fibers:

[0100] S1. Polish the side of a single-mode fiber (cladding diameter of 125μm) to form a side polishing surface to obtain a D-type fiber, and clean the side polishing surface. The polishing depth of the side polishing surface is 70μm, the polishing length is 10mm, and the transition zone length is 5mm.

[0101] S2. Immerse D-type optical fiber in an aqueous sodium hydroxide solution, control the pH value to 8, and soak for 2 hours to obtain hydroxylated D-type optical fiber;

[0102] S3. The hydroxylated D-type optical fiber was pretreated by immersing it in a 0.03 mg / mL solution of 3-aminopropyltriethoxysilane-ethanol, and then dried before immersion in a 0.5 mg / mL solution containing the sensitizing material Ti3C2T. x The nanosheets (with a diameter of 2-10 μm) were immersed in an aqueous solution for 40 min, and then the temperature was raised until the solvent evaporated. The immersion was repeated to form a sensitizing layer, and the thickness of the sensitizing layer was controlled to be 10 nm.

[0103] S4. Add 4-maleimide butyric acid-N-succinimide ester to dimethyl sulfoxide and stir to obtain a crosslinking agent solution with a concentration of 4 mg / L; immerse the optical fiber material obtained above in the crosslinking agent solution for 10 min, dry it and then transfer it to a biotinylated streptavidin solution with a concentration of 30 μmol, incubate it at 15 °C for 12 h to form a specific modification layer, and finally dry it to obtain fiber-based D-type sensing optical fiber;

[0104] Fabrication of an integrated microfluidic biosensor chip based on D-type optical fiber:

[0105] PDMS and curing agent dibutyl phthalate were mixed at a mass ratio of 1:0.1, then poured into a microfluidic mold and cured at 50°C to obtain a PDMS microfluidic substrate with a microchannel structure. The fiber-based D-type sensing optical fiber obtained above was embedded in the microchannel of the PDMS microfluidic substrate and finally encapsulated to obtain the final product.

[0106] Performance testing

[0107] Sample preparation: Leukemia cancer cells K562 were selected as the target biomolecule. The target biomolecule was added to PBS buffer and the concentration of leukemia cancer cells K562 was controlled at 1 cell / mL and 10 cells / mL to obtain the experimental samples.

[0108] Performance testing: Based on the microfluidic biosensor chip obtained in the examples and comparative examples, light with a wavelength of 1500nm was input at one end. 2mL of test samples of different concentrations and 2mL of pure water (as a control group) were injected into the microchannel through the inlet of the microfluidic chip, respectively. Finally, the changes in the light signal of the test samples and control pure water were observed by a spectrometer at the other end of the optical fiber.

[0109] The experimental results are shown in Table 1:

[0110] Table 1 Performance test results

[0111]

[0112] According to Table 1, and in conjunction with Examples 1, 3, and 4, it can be seen that the light signals detected in Examples 3 and 4 do not change significantly compared to Example 1. The reason for this is that Examples 3 and 4 changed the structural parameters of the micropillar array. When the structural parameters of the micropillars are too large, the contact area between the chip and the sample decreases, the cell capture efficiency decreases, and the amplitude of the output light signal change is small. When the structural parameters of the micropillars are too small, it will affect the flow distribution of the sample, which will also lead to a decrease in capture efficiency.

[0113] Based on Example 1 and Comparative Examples 1 to 3, it can be seen that the output optical signals of Comparative Examples 1 to 3 are basically not significantly different from those of the control group. The reason is that no sensitizing layer was added in Comparative Example 1, so the evanescent field intensity of the optical fiber could not be enhanced, which led to a decrease in sensitivity when facing extremely low concentrations of target biomolecules, and no optical signal shift. No specific modification layer was added in Comparative Example 2, so the specific recognition ability of target biomolecules was greatly reduced, and it was impossible to accurately judge when facing target biomolecules of different concentrations. No micropillar array was set in Comparative Example 3, so the contact area between the sample and the chip was greatly reduced, and the cell capture ability was greatly reduced, resulting in almost no optical signal shift.

[0114] The above-disclosed embodiments are merely a few specific examples of the present invention. However, the embodiments of the present invention are not limited thereto, and any variations that can be conceived by those skilled in the art should fall within the protection scope of the present invention.

Claims

1. An integrated microfluidic biosensor chip based on a micropillar array of D-type optical fibers, characterized in that, Including fiber-based micropillar array D-type sensing fiber; The side polished surface of the fiber-based micropillar array D-type sensing fiber is provided with a periodic micropillar array. The surface of the periodic micropillar array is sequentially coated with a sensitizing layer and a specific modification layer; The raw material of the sensitizing layer includes sensitizing materials, which include MXene materials and / or metal nanomaterials; the raw material of the specific modification layer includes bioaptamer materials. The periodic micropillar array has a micropillar height of 35–45 μm, a micropillar diameter of 10–20 μm, and a spacing of 5–15 μm between micropillars.

2. The integrated microfluidic biosensor chip based on a micropillar array D-type optical fiber according to claim 1, characterized in that, The MXene material includes M n+1 X n T x MXene nanosheets; the M n+1 X n T x In MXene nanosheets, M includes any one of Ti, Nb, and V, and X includes C and T. x Includes any one of -OH, -F, -O and -Cl, 1≤n≤3; The metallic nanomaterials include one or a combination of two of gold nanorods and gold nanostars.

3. The integrated microfluidic biosensor chip based on a micropillar array D-type optical fiber according to claim 1, characterized in that, The bioaptamer material includes one or more combinations of Anti-EpCAM antibody, folic acid, biotin, biotinylated streptavidin, and biotinylated Anti-EpCAM antibody.

4. The integrated microfluidic biosensor chip based on a micropillar array D-type optical fiber according to claim 1, characterized in that, The fiber-based micropillar array D-type sensing fiber is prepared according to the following method: S1. Polish the side of the single-mode fiber to form a side polished surface to obtain a D-type fiber, and clean the side polished surface. S2. The side polished surface is etched to form a periodic micropillar array, resulting in a micropillar array D-type optical fiber; S3. Immerse the micropillar array D-type optical fiber in an alkaline aqueous solution, control the pH value of the alkaline aqueous solution to 7-10, and soak for 1-2 hours to obtain hydroxylated micropillar array D-type optical fiber; S4. The hydroxylated micropillar array D-type optical fiber is pretreated by immersing it in an aminosilane coupling agent alcohol solution, dried, and then immersed in an aqueous solution containing sensitizing material for 30-60 minutes. Then the temperature is raised until the solvent evaporates, and the immersion is repeated to form a sensitizing layer. S5. Add a bifunctional crosslinking agent to an organic solvent and stir to obtain a crosslinking agent solution; immerse the optical fiber material obtained above in the crosslinking agent solution for 10-20 min, dry it and transfer it to a bioaptamer material solution, incubate it at 10-25℃ for 12-24 h to form a specific modification layer, and finally dry it to obtain a fiber-based micropillar array D-type sensing optical fiber.

5. The integrated microfluidic biosensor chip based on a micropillar array D-type optical fiber according to claim 4, characterized in that, The polishing depth of the side polishing surface is 60-80 μm, the polishing length is 10-15 mm, and the transition zone length is 4-8 mm.

6. The integrated microfluidic biosensor chip based on a micropillar array D-type optical fiber according to claim 4, characterized in that, The S2 step specifically includes: S201. Aluminum film is deposited on the surface of a D-type optical fiber by magnetron sputtering; S202. A micropillar array pattern is formed on the side polished surface by photolithography, and UV adhesive is coated on the unpolished area and the transition area; S203. The micropillar array is gradually formed by etching in a nitrogen-containing etching gas. Finally, it is cleaned with a piranha solution to obtain the micropillar array D-type optical fiber.

7. The integrated microfluidic biosensor chip based on a micropillar array D-type optical fiber according to claim 4, characterized in that, The bifunctional crosslinking agent comprises 4-maleimide butyric acid-N-succinimide ester; the concentration of the crosslinking agent solution is 0.1–10 mg / L.

8. The integrated microfluidic biosensor chip based on a micropillar array D-type optical fiber according to claim 1, characterized in that, It was prepared according to the following method: After mixing PDMS with a curing agent, the mixture is poured into a microfluidic mold and cured at 40–60°C to obtain a PDMS microfluidic substrate with a single microchannel structure. A fiber-based micropillar array D-type sensing fiber is embedded into the microchannel of the PDMS microfluidic substrate, and finally encapsulated to obtain the final product.

9. The integrated microfluidic biosensor chip based on a micropillar array D-type optical fiber according to claim 1, characterized in that, It is used for the detection of circulating tumor cells.

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