CN115920984B - Integrated paper-based micro-fluidic chip for high-sensitivity detection of anemia markers - Google Patents

Abstract

The invention provides an integrated paper-based microfluidic chip for high-sensitivity detection of an anemia marker, which comprises a hemoglobin detection layer, a sample addition layer, ferritin, folic acid and vitamin B ₁₂ detection layer groups, wherein the ferritin, folic acid and vitamin B ₁₂ detection layer groups comprise a fluorescent layer and an enrichment layer, hydrophilic areas are arranged on the hemoglobin detection layer, the sample addition layer and the fluorescent layer, a plurality of fluorescence enhancement detection areas are arranged on the fluorescent layer, the fluorescence enhancement detection areas are connected with the hydrophilic areas of the fluorescent layer, a plurality of identification enrichment areas and a plurality of waste liquid areas are arranged on the enrichment layer, and the identification enrichment areas are connected with the corresponding waste liquid areas. The integrated paper-based microfluidic chip for high-sensitivity detection of anemia markers is used for high-sensitivity and rapid detection of various anemia markers (hemoglobin, ferritin, folic acid and vitamin B ₁₂) in whole blood.

Description

An integrated paper-based microfluidic chip for highly sensitive detection of anemia markers

Technical Field

The invention belongs to the field of disease detection, and in particular relates to an integrated paper-based microfluidic chip for high-sensitivity detection of anemia markers.

Background technique

Anemia, characterized by low hemoglobin (Hb) levels, especially nutritional anemia, is a high-risk disease in pregnant women, young children, and elderly patients. Due to the high prevalence and significant clinical impact of anemia, early identification and diagnosis of the cause of anemia is a top priority for developing prevention and treatment plans. Providing accurate anemia diagnostic information and simultaneous detection of clinically relevant anemia biomarkers are of great significance, but the main challenge is that the relevant biomarkers are distributed in serum or cells, and their concentrations range from pg/mL to mg/mL levels. For example, hemoglobin, ferritin, folic acid (FA), and vitamin B ₁₂ (VB ₁₂ ) are typical biomarkers of nutritional anemia. Simultaneous detection of these markers can significantly improve _the accuracy of diagnosis, shorten the time to test results, and improve diagnostic efficiency. Hb is a protein present in red blood cells, and the normal level in the human body is 11-16g/dL (male: 12-16g/dL, female: 11-15g/dL). Other biomarkers are present in serum, and the normal concentration in the human body is very low, ferritin is 12-200ng/mL (male: 15-200ng/mL, female: 12-150ng/mL), FA is 2-20ng/mL, and VB ₁₂ is 200-900pg/mL. The simultaneous detection of these related biomarkers requires the detection method to have high sensitivity and a wide detection range (from pg/mL to mg/mL). Although a lot of research has been done to build disease diagnostic tools, current technologies still face challenges such as lack of simultaneous detection of multiple targets, limited detection range, and time-consuming or expensive instrument dependence.

Paper-based detection technology has become a universal platform for chemical/biosensors due to its low cost and ease of mass production. Paper-based microfluidic diagnostic devices are becoming increasingly popular among researchers because they can improve current diagnostic capabilities in terms of simultaneous detection of multiple indicators and allow continuous chemical reactions in different areas of a device. Compared with traditional methods, paper-based microfluidic devices consume less reagents, are less time-consuming, and provide highly integrated and automated diagnostics, providing a broad platform for multiple biological detection. However, current integrated paper-based microfluidics still have limitations such as insufficient sensitivity and narrow detection range, which may be due to the inability to quickly capture and enrich the target and perform highly sensitive detection. Therefore, it is of great significance to develop a universal technology that can quickly capture and enrich trace targets.

Summary of the invention

In view of this, the present invention aims to provide an integrated paper-based microfluidic chip for highly sensitive detection of anemia markers.

To achieve the above object, the technical solution of the present invention is achieved as follows:

An integrated paper-based microfluidic chip for highly sensitive detection of anemia markers, characterized in that it comprises a hemoglobin detection layer, a sample loading layer, and a ferritin, folic acid, and vitamin _B12 detection layer group, wherein the ferritin, folic acid, and vitamin _B12 detection layer group comprises a fluorescent layer and an enrichment layer, the hemoglobin detection layer, the sample loading layer, and the fluorescent layer are all provided with hydrophilic areas, the fluorescent layer is provided with a plurality of fluorescence enhancement detection areas, and the fluorescence enhancement detection areas are all connected to the hydrophilic areas of the fluorescent layer, the enrichment layer is provided with a plurality of identification enrichment areas and a plurality of waste liquid areas, and the identification enrichment areas are connected to the corresponding waste liquid areas.

Furthermore, the number of the fluorescence enhancement detection area, the identification enrichment area and the waste liquid area are the same; and the positions of the fluorescence enhancement detection area and the identification enrichment area correspond to each other.

Furthermore, the size and structure of the hemoglobin detection layer, sample addition layer, fluorescent layer and enrichment layer are the same.

Furthermore, the material of the hydrophilic area of the sample loading layer is a microporous filter membrane; the materials of the hydrophilic area of the fluorescent layer and the hydrophilic area of the enrichment layer are both hydrophilic materials; preferably, the materials of the hydrophilic area of the fluorescent layer and the hydrophilic area of the enrichment layer are both glass fiber paper.

Furthermore, one end of the sample addition layer is connected to the hemoglobin detection layer, and the other end is connected to the fluorescent layer, and the enrichment layer is connected to the fluorescent layer.

The method for preparing the integrated paper-based microfluidic chip for highly sensitive detection of anemia markers comprises the following steps:

(1) Fixing quantum dot-aptamer fluorescent probe in the hydrophilic region of the hemoglobin detection layer;

(2) a microporous filter membrane is arranged in the hydrophilic area of the sample loading layer;

(3) Immobilizing antibody-modified ultrabright nanoclusters in the fluorescence-enhanced detection region of the fluorescent layer;

(4) Fixing the microneedle of the biorecognition element in the recognition enrichment area of the enrichment layer.

Furthermore, the nucleotide sequence of the aptamer in step (1) is shown in SEQ ID NO: 1.

Furthermore, the quantum dot-aptamer fluorescent probe in step (1) is prepared by a method comprising the following steps: using Agaricus bisporus as a raw material to synthesize quantum dots as fluorescent signals, using aptamers as recognition elements, and connecting the aptamers to the surface of the quantum dots, thereby constructing a quantum dot-aptamer fluorescent probe.

Furthermore, the antibody-modified ultra-bright nanoclusters in step (3) are prepared by a method comprising the following steps: mixing gold nanorods (AuNRs), _K2CO3 and _polyethylene glycol (PEG) solution for reaction, centrifuging, removing the lower layer of precipitate and resuspending it in pure water, adding ferritin, FA and _VB12 antibodies thereto, shaking for reaction, centrifuging, removing the lower layer of precipitate and resuspending it in pure water.

Furthermore, the microneedles of the biorecognition element in step (4) are prepared by a method comprising the following steps: 2-hydroxy-2-methylpropiophenone (HMPP) and ethoxylated trimethylolpropane triacrylate (ETPTA) are mixed and placed in a mold, and the microneedles are obtained after curing and demolding, and then the obtained microneedles are placed in a dopamine hydrochloride solution, reacted, and rinsed. Then, the microneedle stickers are immersed in ferritin antibody, folic acid antigen, and VB ₁₂ antigen solutions with the needle tip facing downward for coating, and then rinsed.

The method for using the integrated paper-based microfluidic chip for highly sensitive detection of anemia markers comprises the following steps:

(1) Whole human blood is dripped onto the hydrophilic area of the sample layer, and then red blood cell lysis solution is added to dissolve hemoglobin;

(2) Folding the sample layer to overlap with the hemoglobin detection layer to perform Hb detection;

(3) folding the fluorescent layer and the enrichment layer so that the identification enrichment area overlaps with the fluorescence enhancement detection area to form a ferritin, folic acid, and vitamin _B12 detection layer group;

(4) The ferritin, folic acid, and vitamin _B12 detection layer groups are folded and overlapped with the sample layer. The serum in the hydrophilic area of the sample layer can be filtered through the microporous filter membrane to penetrate into the hydrophilic area of the fluorescent layer, and migrate to each enrichment area and the fluorescence enhancement detection area through capillary action; after the reaction, the imager is used for measurement.

A nucleic acid aptamer, the nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID NO: 1.

Compared with the prior art, the present invention has the following advantages:

The integrated paper-based microfluidic chip for high-sensitivity detection of anemia markers of the present invention is used for high-sensitivity and rapid detection of multiple anemia markers (hemoglobin, ferritin, folic acid and vitamin B ₁₂ ) in whole blood.

The integrated paper-based microfluidic chip for highly sensitive detection of anemia markers described in the present invention integrates sample pretreatment and detection, and can achieve rapid (10min), sensitive (pg/mL), and accurate detection of Hb in a small volume (50μL) whole blood sample and three anemia markers in serum. The integrated paper-based microfluidic chip constructed by the present invention has the advantages of no need for complex pretreatment, high sensitivity, rapidity, portability, low cost, and easy batch production, and can provide a feasibility analysis method for accurate diagnosis of anemia and analysis of its causes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of an integrated paper-based microfluidic chip according to an embodiment of the present invention;

FIG2 is a schematic diagram of the integrated paper-based microfluidic chip after folding according to an embodiment of the present invention;

FIG3 is a graph showing a standard hemoglobin detection curve according to an embodiment of the present invention;

FIG4 is a representation diagram of the morphology of a three-dimensional microarray according to an embodiment of the present invention;

FIG5 is a graph showing the adsorption of FITC-Ab of biomacromolecules at different concentrations by the three-dimensional microarray according to an embodiment of the present invention;

FIG6 is an adsorption diagram of different concentrations of rhodamine by the three-dimensional microarray according to an embodiment of the present invention;

FIG7 is a transmission electron microscope characterization image of ultra-bright nanoclusters according to an embodiment of the present invention;

FIG8 is a graph showing the measurement of the ultra-bright nanocluster plasma resonance wavelength and the excitation and emission bands of the fluorophore according to an embodiment of the present invention;

FIG9 is a comparison diagram of the fluorescence intensity of the ultra-bright fluorescent nanoclusters and the pure dye BSA-ICG according to an embodiment of the present invention;

FIG10 is a standard curve diagram of serum anemia index detection according to an embodiment of the present invention: 10-A is a standard curve for ferritin detection, 10-B is a standard curve for folic acid detection, and 10-C is a standard curve for VB ₁₂ detection;

FIG11 is a diagram showing a conventional method detection method according to an embodiment of the present invention;

FIG. 12 is a detection diagram of the integrated paper-based microfluidic chip described in an embodiment of the present invention.

Description of reference numerals:

1. Hemoglobin detection layer; 2. Sample loading layer; 3. Fluorescence layer; 4. Enrichment layer; 5. Hydrophilic area; 6. Fluorescence enhancement detection area; 7. Identification and enrichment area; 8. Waste liquid area.

Detailed ways

Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods.

The folic acid and VB ₁₂ antigens, antibodies, and standard products described in the embodiments of the present invention are from Shandong Ludu Biotechnology Co., Ltd.

ETPTA, HMPP, NHS, dopamine hydrochloride, ascorbic acid described in the embodiments of the present invention: Aladdin Reagent (Shanghai) Co., Ltd.;

BSA, EDCHCl, PBS, CTAB, K ₂ CO ₃ described in the examples of the present invention: Beijing Solebow Biotechnology Co., Ltd.;

The fluorescent dyes ICG and PEG described in the embodiments of the present invention were obtained from Xi'an Ruixi Biotechnology Co., Ltd.

The glass fiber paper described in the embodiment of the present invention: Shanghai Jinbiao Biotechnology Co., Ltd.;

Whatman microporous membrane described in the embodiment of the present invention: Tianjin Puruisi Biotechnology Co., Ltd.;

The gel imager described in the embodiment of the present invention: ChemiDoc MP, Bio-Rad, USA;

The present invention will be described in detail below with reference to the embodiments.

Example 1 Construction of paper-based microfluidic chip

Hemoglobin detection layer: 1cm×1cm glass fiber paper containing quantum dot-aptamer fluorescent probes is glued to the hydrophilic area on the back of the filter paper; sample loading layer: the filter paper in the hydrophilic area is cut off, and the 1cm×1cm microporous filter membrane is glued to the hydrophilic area of the sample loading layer; fluorescent layer: 1cm×1cm ultra-bright nanocluster glass fiber paper modified with ferritin, FA and VB ₁₂ antibodies is glued to the corresponding hydrophilic area; enrichment layer: microneedles coupled with ferritin, FA and VB ₁₂ biorecognition elements are glued to the corresponding hydrophilic area of the filter paper, and then, the hemoglobin detection layer→sample loading layer, enrichment layer→fluorescence layer, fluorescence layer→sample loading layer are folded and assembled, and stored at 4°C for use. The chip structure and folding structure are shown in Figure 1-2.

Example 2 Construction of hemoglobin detection method based on fluorescence quenching

1. Synthesis of Agaricus bisporus quantum dots: 5 g of crushed Agaricus bisporus and 20 mL of distilled water were sealed in a 50 mL polytetrafluoroethylene-lined stainless steel autoclave for hydrothermal carbonization reaction at 200 °C for 5 h. The naturally cooled quantum dot solution was dialyzed through a 0.22 μm filter to remove small molecules and salts for further use.

2. Synthesis of quantum dot-aptamer fluorescent probe: 200 μL of nucleic acid aptamer, 50 μL of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCHCl, 10 mg/ml) and 50 μL of N-hydroxysuccinimide (NHS, 10 mg/ml) were added to 1 mL of quantum dot solution and oscillated for 1 hour. The nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID NO: 1.

3. Preparation of glass fiber paper containing quantum dots-aptamers: Take 1 mL of quantum dots-aptamer fluorescent probe solution and drop it onto 4 cm×10 cm glass fiber paper, soak it evenly, vacuum freeze-dry it for 12 hours, and cut it into 1 cm×1 cm for later use.

4. Construction of hemoglobin detection method based on fluorescence quenching

50 mL of hemoglobin solution of different concentrations (0, 0.07, 0.15, 0.31, 0.62, 1.25, 2.5, 5 g/dL) prepared according to the anemia diagnostic index was added dropwise to the glass fiber paper containing quantum dots-aptamers and reacted in the dark for 10 min. Hb specifically bound to the aptamer, resulting in the quenching of the fluorescence of the quantum dots. The fluorescence intensity of the glass fiber paper was measured at 368 nm, and grayscale analysis was performed using Image J software. The values were statistically analyzed and a standard curve of hemoglobin was drawn. As shown in FIG3 , the standard curve of hemoglobin detection is Y _Hb =7577X+10484, R ² =0.977. Finally, a hemoglobin detection method based on fluorescence quenching was constructed.

Example 3 Construction of a microneedle-based serum marker detection method

1. Microneedle patch production

(1) HMPP and ETPTA were mixed in a volume ratio of 1:100, 100 μL of the mixture was taken on the PDMS negative mold, and the bubbles and excess solution were removed by vacuum treatment. The mold was then irradiated under a UV lamp for 2 min and demolded after curing. The morphology of the microarray is shown in FIG4 .

The prepared microneedle patch was placed in a 2 mg/mL dopamine hydrochloride solution, irradiated under ultraviolet light for 10 minutes, and rinsed with clean water to obtain a polydopamine-modified microneedle patch.

The dopamine-modified microneedle patch was immersed in ferritin antibody, folic acid antigen and VB ₁₂ antigen solutions with the needle tip facing downward, coated at 4°C for 12 h, washed three times with phosphate-tween buffer (PBST, containing 0.5% Tween-20), blocked with 3% bovine serum albumin solution (BSA) for 30 min, washed, and stored at -20°C for later use.

(2) Adsorption of FITC-Ab by the microneedle patch: 50 μL of fluorescein isothiocyanate-conjugated antibody (FITC-Ab) solution of different concentrations (0, 1, 5, 10, 100, 200 ng/mL) was added dropwise to a plate, and the microneedle patch was immersed in the FITC-Ab solution with the needle tip facing downward for 10 min. The patch was rinsed with PBST five times, and the fluorescence value was measured under an imaging device. The results are shown in FIG5 , indicating that the microneedle patch has the ability to enrich low-concentration biomacromolecules.

(3) Adsorption of small molecule rhodamine by the microneedle patch: 50 μL of rhodamine solution of different concentrations (0, 50, 100, 1000, 5000, 10000 pg/mL) was added dropwise to a plate, and the microneedle patch was immersed in the rhodamine solution with the needle tip facing downward for 10 min. The patch was rinsed with PBST five times, and the fluorescence value was measured under an imaging device. The results are shown in FIG6 , indicating that the microneedle patch has the ability to enrich low-concentration biological small molecules.

2. Synthesis of ultra-bright fluorescent nanoclusters (AuNRs@BSA-ICG)

(1) Synthesis of AuNRs: HAuCl ₄ (10 mM, 2 mL), CTAB (0.1 M, 38 mL), AgNO ₃ (10 mM, 1 mL), HCl (1 M, 0.9 mL) and ascorbic acid solution (0.1 M, 0.22 mL) were mixed in order, and 5 μL of gold seed solution was added dropwise. After mixing, the mixture was grown in the dark for 24 h. The AuNRs were collected by centrifugation at 7000 rpm for 40 min and resuspended in pure water.

(2) Preparation of ultra-bright fluorescent nanoclusters: 1 mL of AuNRs solution was mixed with 10 μL of K ₂ CO ₃ (100 mM) and 20 μL of PEG (1 mM) solution for 10 min, centrifuged at 4000 rpm for 8 min, and the lower precipitate was resuspended in pure water; 500 μg of BSA-ICG and 50 μg of Ab (ferritin, FA and VB ₁₂ antibodies) were added and oscillated for 30 min, centrifuged at 4000 rpm for 8 min, and the remaining precipitate was resuspended in 500 μL of pure water.

(3) Preparation of ultra-bright fluorescent nanocluster glass fiber paper: 1 mL of each of the fluorescent nanocluster solutions modified with ferritin, FA, and _VB12 antibodies was dripped onto 4 cm × 10 cm glass fiber paper, soaked evenly, vacuum freeze-dried for 12 h, and cut into 1 cm × 1 cm pieces for later use.

(4) Characterization of fluorescence enhancement effect: The fabricated ultrabright nanoclusters were characterized by transmission electron microscopy, and the results are shown in Figure 7. The large overlap between the plasma resonance wavelength band of the plasma nanostructure and the excitation and emission bands of the fluorophore is crucial for maximizing fluorescence enhancement. As shown in Figure 8, the plasma resonance wavelength band of AuNRs overlaps with the excitation and emission bands of the fluorophore ICG over a large area, indicating that AuNRs can resonate with the fluorophore ICG, allowing the ultrabright fluorescent nanoclusters to enhance fluorescence. Figure 9 compares the fluorescence intensity of the ultrabright fluorescent nanoclusters with that of the pure dye BSA-ICG, confirming that the ultrabright fluorescent nanoclusters can significantly enhance fluorescence.

3. Construction of microneedle-based serum marker detection method

(1) The steps for sensitive detection of serum anemia markers are as follows:

a) According to the anemia diagnostic indicators, different concentrations of anemia markers ferritin, FA and VB ₁₂ solutions were prepared: ferritin was 0, 0.1, 0.5, 1, 10, 50, 100, 500 ng/mL; FA was 0, 0.1, 1, 5, 10, 50, 100 ng/mL; VB ₁₂ was 0, 0.05, 0.1, 1, 5, 10, 50 ng/mL.

b) Super bright fluorescent nano cluster glass fiber paper modified with ferritin, FA and VB ₁₂ was overlapped with the corresponding microneedle patch, 50 μL of the corresponding target of different concentrations was added to the super bright nano cluster glass fiber paper, reacted for 10 min, the microneedle patch was removed, and washed with PBST for 5 times; the fluorescence intensity of the microneedle was measured at 785 nm, grayscale analysis was performed using Image J software, the values were statistically analyzed and standard curves for detecting the three markers ferritin, FA and VB ₁₂ were drawn, as shown in FIG10 , FIG10-A is the standard curve for ferritin detection: Y _ferritin = 326201X + 554793, R ² = 0.982; FIG10-B is the standard curve for folic acid detection: Y _FA = 331212X + 604602, R ² = 0.980; FIG10-C is the standard curve for VB12 detection: Y _VB12 = 182720X + 571924, R ² =0.986; finally, a microneedle-based serum marker detection method was constructed.

The gold seed solution was prepared by adding 0.6 mL of 4 °C NaBH ₄ solution to 10 mL of a mixed solution of HAuCl ₄ (10 mM, 0.25 mL) and CTAB (0.1 M, 9.75 mL), stirring vigorously, and shaking for 2 h;

BSA-ICG is prepared by mixing BSA and fluorescent dye indocyanine green (ICG) at a molar ratio of 1:20 in phosphate buffer solution (PBS), shaking for 30 minutes, collecting by ultrafiltration, and storing at -20°C for future use.

(2) The same method was used to compare the detection sensitivity of pure dye BSA-ICG.

The concentration of ferritin in normal human blood is 13-400ng/mL. If it is lower than 12ng/mL, it is anemia. After calculation, the detection limit of pure dye BSA-ICG is 0.847ng/mL, and the detection limit of ultra-bright fluorescent nanoclusters is 35pg/mL. The concentration of folic acid in normal human blood is 1.896-13.98ng/mL. If it is lower than the lower limit, it is anemia. The detection limit of pure dye BSA-ICG is 0.318ng/mL, and the detection limit of ultra-bright fluorescent nanoclusters is 12pg/mL. The concentration of VB ₁₂ in normal human blood is 200-900pg/mL. If it is lower than 200pg/mL, it is anemia. The detection limit of pure dye BSA-ICG is 93pg/mL, and the detection limit of ultra-bright fluorescent nanoclusters is 7pg/mL. The ultra-bright fluorescent nanoclusters we used can fully detect the three indicators in the blood. The comparison of the detection limits of the ultra-bright fluorescent nanoclusters and the pure dye BSA-ICG shows that the ultra-bright fluorescent nanoclusters have an excellent fluorescence enhancement effect and proves the feasibility of using this method to detect anemia indicators.

Example 4 Multiplex detection of anemia markers in whole blood samples

1. Operation steps of integrated paper-based microfluidic chip to detect anemia indicators in whole blood

(1) 20 μL of whole blood sample and 60 μL of red blood cell lysate are dripped onto the microporous filter membrane of the hemoglobin detection layer. The filter paper of the hemoglobin detection layer is folded and overlapped with the filter paper of the sample loading layer. Hb reacts with the quantum dot-aptamer fluorescent probe of the hemoglobin detection layer, and the hemoglobin is detected.

(2) The fourth layer of filter paper is folded so that the microneedles on it overlap with the ultra-bright fluorescent nanocluster glass fiber paper of the fluorescent layer, and then the fluorescent layer filter paper is folded to overlap with the sample layer filter paper. The sample layer serum can be filtered through the microporous filter membrane and penetrate into the circular hydrophilic filter paper of the fluorescent layer, and migrate to the ultra-bright nanocluster glass fiber paper area modified with ferritin, FA and VB ₁₂ under capillary action. The microneedles quickly enrich the three trace markers in the serum, and then recognize and enhance the fluorescence through immune reaction with the ultra-bright fluorescent nanoclusters.

(3) The fluorescence signals of Hb and serum anemia markers were collected by gel imaging at excitation wavelengths of 365 nm and 785 nm. Grayscale numerical analysis was performed using Image J software, and the marker concentrations corresponding to the samples were calculated based on the standard curves of the four markers.

2. Use commercial ELISA kits according to the instructions to measure the concentration of anemia markers in 37 whole blood samples.

3. The detection results of the two methods are shown in Figures 11-12. The results of the two detection methods are basically consistent, indicating the feasibility of the integrated paper-based chip to simultaneously detect anemia markers in whole blood.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. An integrated paper-based microfluidic chip for high-sensitivity detection of an anemia marker, which is characterized in that: the device comprises a hemoglobin detection layer, a sample addition layer, a ferritin, folic acid and vitamin B ₁₂ detection layer group, wherein the ferritin, folic acid and vitamin B ₁₂ detection layer group comprises a fluorescent layer and an enrichment layer, hydrophilic areas are arranged on the hemoglobin detection layer, the sample addition layer and the fluorescent layer, a plurality of fluorescence enhancement detection areas are arranged on the fluorescent layer, the fluorescence enhancement detection areas are connected with the hydrophilic areas of the fluorescent layer, a plurality of identification enrichment areas and a plurality of waste liquid areas are arranged on the enrichment layer, and the identification enrichment areas are connected with the corresponding waste liquid areas; the hydrophilic area of the sample adding layer is made of a microporous filter membrane; one end of the sample adding layer is connected with the hemoglobin detection layer, the other end of the sample adding layer is connected with the fluorescent layer, and the enrichment layer is connected with the fluorescent layer;

The application method of the integrated paper-based microfluidic chip for high-sensitivity detection of the anemia marker comprises the following steps:

(1) Dripping human whole blood into a hydrophilic area of a sample adding layer, and then dripping erythrocyte lysate to dissolve out hemoglobin;

(2) Folding the sample adding layer and overlapping the hemoglobin detection layer to detect Hb;

(3) Folding the fluorescent layer and the enrichment layer to enable the identification enrichment region and the fluorescence enhancement detection region to coincide to form a ferritin, folic acid and vitamin B ₁₂ detection layer group;

(4) Folding a detection layer group of ferritin, folic acid and vitamin B ₁₂ to coincide with the sample adding layer, filtering and penetrating serum in a hydrophilic region of the sample adding layer to a hydrophilic region of a fluorescent layer through a microporous filter membrane, and migrating the serum to each enrichment region and a fluorescence enhancement detection region through capillary action; and measuring by using an imager after the reaction.

2. The integrated paper-based microfluidic chip for highly sensitive detection of anemia markers according to claim 1, wherein: the number of the fluorescence enhancement detection area, the number of the identification enrichment area and the number of the waste liquid area are all the same; the fluorescence enhancement detection zone corresponds to a location of the identification enrichment zone.

3. The integrated paper-based microfluidic chip for highly sensitive detection of anemia markers according to claim 1, wherein: the size and the structure of the hemoglobin detection layer, the sample adding layer, the fluorescent layer and the enrichment layer are the same.

4. An integrated paper-based microfluidic chip for highly sensitive detection of anemia markers according to claim 3, wherein: the hydrophilic areas of the fluorescent layer and the hydrophilic areas of the enrichment layer are made of hydrophilic materials.

5. The integrated paper-based microfluidic chip for highly sensitive detection of anemia markers of claim 4, wherein: and the hydrophilic area of the fluorescent layer and the hydrophilic area of the enrichment layer are made of glass fiber paper.

6. The method for preparing the integrated paper-based microfluidic chip for high-sensitivity detection of anemia markers according to any one of claims 1-5, which is characterized by comprising the following steps: the method comprises the following steps:

(1) Fixing a quantum dot-aptamer fluorescent probe in a hydrophilic region of the hemoglobin detection layer;

(2) A microporous filter membrane is arranged in a hydrophilic area of the sample adding layer;

(3) Fixing the ultra-bright nanoclusters modified by the antibody in a fluorescence enhancement detection area of the fluorescent layer;

(4) Microneedles for fixing biological recognition elements in a recognition and enrichment region of an enrichment layer.

7. The method for preparing the integrated paper-based microfluidic chip for high-sensitivity detection of anemia markers, which is disclosed in claim 6, is characterized in that: the nucleotide sequence of the aptamer in the step (1) is shown as SEQ ID NO. 1.

8. The method for preparing the integrated paper-based microfluidic chip for high-sensitivity detection of anemia markers, which is disclosed in claim 6, is characterized in that: the quantum dot-aptamer fluorescent probe in the step (1) is prepared by a method comprising the following steps: the method comprises the steps of synthesizing quantum dots by using agaricus bisporus as a raw material, using an aptamer as an identification element, and connecting the aptamer to the surface of the quantum dots, so as to construct a quantum dot-aptamer fluorescent probe.

9. The method for preparing the integrated paper-based microfluidic chip for high-sensitivity detection of anemia markers, which is disclosed in claim 6, is characterized in that: the ultra-bright nanoclusters modified by the antibody in the step (3) are prepared by a method comprising the following steps: mixing AuNRs solution, K ₂CO₃ and PEG solution, centrifuging, taking out the lower layer precipitate, re-suspending in pure water, adding ferritin, FA and VB ₁₂ antibodies, oscillating, centrifuging, and taking out the lower layer precipitate, re-suspending in pure water.

10. The method for preparing the integrated paper-based microfluidic chip for high-sensitivity detection of anemia markers, which is disclosed in claim 6, is characterized in that: the microneedle of the biological recognition element in the step (4) is manufactured by a method comprising the following steps: mixing HMPP and ETPTA, putting into a mould, solidifying and demoulding to obtain a microneedle, putting the obtained microneedle into a dopamine hydrochloride solution, reacting and washing.