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CN-121975941-A - Bubble self-layering digital detection method and system for quantifying extracellular vesicle miRNA

CN121975941ACN 121975941 ACN121975941 ACN 121975941ACN-121975941-A

Abstract

The invention discloses a bubble self-layering digital detection method and system for quantifying extracellular vesicle miRNA, belonging to the technical fields of biomedical engineering and molecular diagnosis. Solves the technical problem of providing a simple and sensitive method for detecting tumor extracellular vesicle miRNA, which can detect a plurality of miRNAs simultaneously. The method comprises the steps of incubating a sample to be detected and immunocapture bubbles coupled with an anti-EpCAM antibody to enrich tumor extracellular vesicles, performing in-situ ultrasonic cleavage to obtain a miRNA sample to be detected, constructing an enzyme digestion system by the miRNA sample and fluorescent/DNA double-coded magnetic bead conjugates and the like, adding a multifunctional click glass microbubble to capture unreacted magnetic beads, standing for self-layering to collect bottom magnetic beads, and inputting an image into an AI automatic fluorescence counting decoding module for counting after fluorescence imaging to obtain a result. The method is used for detecting miR-21 and miR-155 in tumor extracellular vesicles, and is suitable for noninvasive early diagnosis of tumors.

Inventors

  • YANG FAN
  • XU XIAOHUAN
  • QIN XIAOJIE
  • LU HAO
  • Wei Binqi
  • ZHANG YUYUAN
  • LI XINCHUN

Assignees

  • 广西医科大学

Dates

Publication Date
20260505
Application Date
20260401

Claims (10)

  1. 1. The bubble self-layering digital detection method for quantifying extracellular vesicle miRNA is characterized by comprising the following steps of: s1, providing a sample to be tested, adding immunocapture bubbles, incubating for 10-60 min, centrifuging for 0.5-1.5 and min by 500-2000 rpm to remove supernatant, and washing with phosphate buffer solution for 3-5 times to obtain immunocapture bubbles enriched with tumor extracellular vesicles, wherein the immunocapture bubbles are hollow glass micro-bubble surfaces coupled with anti-EpCAM antibodies; S2, performing in-situ ultrasonic cleavage on the immunocapture bubbles enriched with tumor extracellular vesicles to release miRNA in the vesicles, centrifuging by 500-2000 rpm for 3-10 min, and taking a supernatant to obtain a miRNA sample to be detected; S3, mixing a miRNA sample to be detected with a fluorescent/DNA double-coded magnetic bead conjugate, double-strand specific nuclease, a double-strand specific nuclease reaction buffer, an RNase inhibitor and DEPC treatment water to form an enzyme digestion system, incubating enzyme digestion, adding a double-strand specific nuclease stopping solution to terminate reaction after incubation is finished, magnetically separating and collecting magnetic beads, and washing 3-5 times with PBS, wherein the fluorescent/DNA double-coded magnetic bead conjugate comprises a FITC-MB-DNA21-N 3 conjugate and a Cy3-MB-DNA155-N 3 conjugate, and is used for detecting miR-21 and miR-155 respectively; S4, re-suspending the magnetic beads collected in the step S3 in a multifunctional click glass microbubble solution, and incubating to enable the unreacted fluorescent/DNA double-coding magnetic bead conjugate to be combined to the surface of the multifunctional click glass microbubble through the click chemical reaction of azide and dibenzocyclooctyne; S5, standing to enable the multifunctional click glass microbubbles combined with unreacted magnetic beads to float up to the liquid level, and collecting the magnetic beads with settled bottoms; and S6, performing fluorescence imaging on the collected magnetic beads, respectively collecting fluorescence images of a FITC channel and a Cy3 channel, inputting the images into an AI automatic fluorescence counting and decoding module for counting, and obtaining detection results of miR-21 and miR-155.
  2. 2. The bubble self-layering digital detection method for quantification of extracellular vesicle mirnas according to claim 1, wherein the preparation of immunocapture bubbles comprises the steps of: Weighing 0.1-1 g of hollow glass microbubbles subjected to hydroxylation activation treatment, adding 5-15 of absolute ethyl alcohol solution mL containing 0.1-1 mL of (3- (2, 3-glycidoxy) propyl) triethoxysilane for silanization modification reaction 10-20 h, centrifuging 0.5-1.5 min by 3000-5000 rpm, discarding lower layer solution, washing 3-5 times by ultrapure water, and vacuum drying at 35-40 ℃ to obtain epoxy hollow glass microbubbles; Weighing 0.1-1 g epoxy hollow glass microbubbles, adding 1-10 mL phosphoric acid buffer, adding 80-120 mug/mL anti-EpCAM antibody solution, mixing and reacting 0.5-1.5 h, centrifuging 0.5-1.5 min by 1000-5000 rpm, discarding the lower solution, washing 3-5 times by phosphoric acid buffer solution, sealing with bovine serum albumin, washing and drying again, and obtaining the immunocapture bubbles of the surface coupling anti-EpCAM antibody.
  3. 3. The bubble self-layering digital detection method for extracellular vesicle miRNA quantification according to claim 1, wherein the preparation of the fluorescent/DNA double-encoded magnetic bead conjugate comprises the steps of: mixing streptavidin conjugated magnetic beads with fluorescein isothiocyanate-succinimidyl ester and Cy 3-succinimidyl ester respectively, performing fluorescent labeling by rotating reaction at room temperature and in the dark for 30-90 min times, removing supernatant by magnetic separation, and washing with 0.01g/100mL Tween 20 phosphate buffer solution for 3-5 times to obtain FITC-labeled magnetic beads and Cy 3-labeled magnetic beads respectively; Mixing Biotin-modified stippled DNA probe Biotin-DNA21-N 3 with FITC-labeled magnetic beads, and fixing by the affinity of Biotin and streptavidin to obtain FITC-MB-DNA21-N 3 conjugate; The Biotin-modified stippled DNA probe Biotin-DNA155-N 3 is mixed with Cy 3-labeled magnetic beads, and immobilized through the affinity of Biotin and streptavidin to obtain a Cy3-MB-DNA155-N 3 conjugate.
  4. 4. The method for detecting the number of the self-layering gas bubbles for quantifying the extracellular vesicle miRNA according to claim 1, wherein in the step S2, the parameters of in-situ ultrasonic cleavage are that the frequency is 10-30 kHz, the ultrasonic waves are 5-10S and 5-20S are suspended each time, and the cycle is 4-6 times.
  5. 5. The method for detecting the number of the self-contained air bubble layer for quantifying the extracellular vesicle miRNA according to claim 1, wherein in the step S3, the volume of the enzyme digestion system is 20. Mu.L, and the method comprises 6. Mu.L of an equimolar mixed fluorescent/DNA double-coded magnetic bead conjugate, 10. Mu.L of a miRNA sample to be detected, 1. Mu.L of 0.5U DSN enzyme, 1. Mu.L of 10 xDSN reaction buffer, 0.5. Mu.L of 20U RNase inhibitor and 1.5. Mu.L DEPC treatment water, and the incubation temperature is 40-60 ℃ and the incubation time is 0.5-1.5 h.
  6. 6. The bubble self-layering digital detection method for quantification of extracellular vesicle miRNA according to claim 1, wherein the preparation of the multifunctional click glass microbubbles comprises the following steps: Carrying out hydroxylation activation treatment on the hollow glass microbubbles to obtain activated hollow glass microbubbles; adding 100-1000 mg activated hollow glass microbubbles into 10-50 mL polyethyleneimine water solution with the mass-volume ratio of 1-5 mg/mL, performing rotary mixing reaction at room temperature for 10-20 min to form an amination coating on the surface through electrostatic adsorption, performing centrifugal treatment for 0.5-2 min through 3000-5000 rpm, washing for 3-5 times with ultrapure water, and performing vacuum drying at 30-40 ℃ to obtain the amination hollow glass microbubbles; 10-1000 mg of amination hollow glass microbubbles are added with 0.5-20 mL of phosphate buffer, 100-300 mu L of dibenzocyclooctyne-N-hydroxysuccinimide ester solution with the concentration of 10-30 mM is added, 0.5-1.5 h of dibenzocyclooctyne-N-hydroxysuccinimide ester solution is subjected to room temperature rotary mixing reaction to graft dibenzocyclooctyne groups through covalent reaction of surface amino groups and active ester, 0.5-2 min of the dibenzocyclooctyne-N-hydroxysuccinimide ester solution is subjected to 3000-5000 rpm centrifugation treatment, ultrapure water is used for washing for 3-5 times, and vacuum drying is carried out at 30-40 ℃ to obtain the multifunctional click glass microbubbles with bioorthogonal functions.
  7. 7. The method for detecting the number of the self-layering bubbles for quantifying extracellular vesicle miRNA according to claim 1, wherein the particle size of the multifunctional click glass microbubbles is 10-30 μm, the density is 0.5-0.7 g/cm 3 , and the floating time in a phosphate buffer solution is not more than 5min.
  8. 8. The bubble self-layering digital detection method for quantifying extracellular vesicle miRNA according to claim 1, wherein an AI automatic fluorescence counting decoding module is internally provided with an image classification algorithm, a graying algorithm, an adaptive binarization algorithm, an image segmentation algorithm based on an aspect ratio threshold value and an HSV parameter adaptive adjustment algorithm, and is used for automatically classifying, preprocessing, dividing and counting input FITC channel and Cy3 channel fluorescence images and outputting a double-target counting result.
  9. 9. The bubble self-layering digital detection method for extracellular vesicle miRNA quantification according to claim 1, wherein the AI automated fluorescence count decoding module performs the following process flow: 1) Calculating sub-pixel translation offset between two images of acquired FITC channel and Cy3 channel fluorescent images by adopting a phase correlation algorithm based on fast Fourier transform, correcting the images according to the offset, and accurately aligning the two channel images to ensure that the coordinates of magnetic beads in the same visual field are consistent in the double channels; 2) Converting registered FITC and Cy3 images from RGB color space to HSV color space respectively, extracting hue H, saturation S and brightness V components, performing hue matching on each pixel according to the characteristic hue range of pre-calibrated FITC and Cy3 fluorescent dyes, generating a binary mask, and primarily screening out pixel areas which are possibly magnetic beads so as to eliminate noise interference of non-target colors in the background; 3) In the mask region after tone screening, carrying out local self-adaptive threshold segmentation on the brightness component V by adopting a Bernsen method based on a pixel neighborhood mean value and a standard deviation to obtain a binary image of a magnetic bead candidate region so as to adapt to uneven illumination and background fluorescence change; 4) Sequentially carrying out morphological open operation on the binary images to remove tiny noise, closing operation to connect adjacent areas, and filling holes generated by uneven fluorescence in the magnetic beads by using a morphological reconstruction algorithm to obtain a complete magnetic bead connected domain; 5) Searching local maxima on the distance map as seed points, and dividing adhered magnetic beads by adopting a watershed algorithm to obtain independent areas of single magnetic beads, so as to solve counting deviation caused by magnetic bead aggregation; 6) Calculating the area, perimeter, circularity and length-width ratio of each independent region, setting a threshold according to the actual particle size and imaging magnification of the magnetic beads, removing the regions which do not accord with the circular characteristics or abnormal sizes of the magnetic beads, and filtering out the strip-shaped regions which still have adhesion due to incomplete segmentation; 7) And meanwhile, if one magnetic bead is detected in both channels, judging the magnetic beads as the same target according to the coordinates of the magnetic beads, and eliminating cross color interference to ensure the specificity and accuracy of double-target counting.
  10. 10. A bubble self-layering detection system for quantification of extracellular vesicle miRNA, comprising: Immunocapture bubbles, which are hollow glass microbubbles with anti-EpCAM antibodies coupled to the surface thereof, are used for specifically enriching tumor extracellular vesicles; fluorescent/DNA double-coded magnetic bead conjugates, including FITC-MB-DNA21-N 3 conjugate and Cy3-MB-DNA155-N 3 conjugate, for detecting miR-21 and miR-155, respectively; The multifunctional click glass micro-bubble is hollow glass micro-bubble, the surface of which is grafted with dibenzocyclooctyne groups, and the dibenzocyclooctyne groups are used for capturing unreacted fluorescent/DNA double-coded magnetic bead conjugates through azide-dibenzocyclooctyne click chemical reaction and realizing self-layering separation by means of self-buoyancy floating; Double-strand specific nuclease and a reaction buffer thereof are used for circularly cutting a DNA probe on a fluorescent/DNA double-coding magnetic bead conjugate in the presence of a target miRNA; an AI automated fluorescence count decoding module configured to perform the following image processing flow: 1) Performing phase correlation registration based on fast Fourier transform on acquired FITC channel and Cy3 channel fluorescence images, and eliminating sub-pixel offset among channels; 2) Converting the registered image into HSV color space, generating a binary mask according to the characteristic tone range of the pre-calibrated FITC and Cy3 fluorescent dyes, and eliminating background noise; 3) The brightness component V is segmented by adopting a local self-adaptive threshold value in the mask area, so as to obtain a magnetic bead candidate area; 4) Carrying out morphological reconstruction and hole filling on the candidate region to obtain a complete magnetic bead communication region; 5) Carrying out distance transformation on the connected domain, and dividing and adhering magnetic beads by adopting a watershed algorithm to obtain a single magnetic bead independent area; 6) Filtering the shape and the size based on the particle size, the circularity and the length-width ratio of the magnetic beads, and eliminating false positive targets; 7) Counting the number of the filtered magnetic beads in the double channels, eliminating crosstalk interference according to coordinate correlation, and outputting counting results of miR-21 and miR-155.

Description

Bubble self-layering digital detection method and system for quantifying extracellular vesicle miRNA Technical Field The invention belongs to the technical field of biomedical engineering and molecular diagnosis, and particularly relates to a bubble self-layering digital detection method and system for quantifying extracellular vesicle miRNA. Background Micrornas (mirnas) in Extracellular Vesicles (EVs) have been shown to be closely related to the development and progression of tumors, a promising class of liquid biopsy markers. However, specific enrichment of tumor-derived EVs from complex biological samples (e.g., plasma) and accurate quantification of miRNA within them remains a technical challenge. Existing EV separation methods based on ultracentrifugation or kits are low in efficiency and difficult to distinguish EV subgroup sources, and subsequent miRNA detection often depends on quantitative reverse transcription polymerase chain reaction (RT-qPCR) or second generation sequencing, and the methods generally require large sample size and total RNA extraction steps, and are easy to interfere with free miRNA with high abundance in a sample in the detection process, so that result deviation is caused. To circumvent such interference, researchers have attempted to introduce detection probes directly into EVs for in situ detection, but such methods are limited by membrane penetration efficiency and sensitivity to meet clinical needs. In recent years, the digital detection technology based on microwells or microdroplets realizes the sensitivity improvement of a single molecule level through physical compartmentalization, but the method often needs precise microfluidic control or multi-step surface capture operation, has the problems of large empty area chamber interference and low sampling efficiency, and leads to the limitation of the counting quantity of target molecules. Meanwhile, most of the prior art still needs to finish signal separation by means of external force fields such as centrifugation or magnetic separation, the operation flow is complicated, the final result depends on manual counting or commercial software analysis, and subjective deviation and missing meter and false meter of adhesion particles are difficult to avoid. Therefore, how to realize high-specificity enrichment, high-efficiency signal conversion and automatic unbiased quantification of specific miRNA in tumor source EV on the premise of not depending on complex equipment and complicated operation is still a difficult point to be solved in the field. Disclosure of Invention It is an object of the present invention to solve at least the above problems and to provide at least the advantages to be described later. The invention also aims to provide a bubble self-layering digital detection method for quantifying extracellular vesicle miRNA, which can realize efficient and specific enrichment of tumor extracellular vesicles by using immunocapture bubbles, release target miRNA by combining in-situ ultrasonic cleavage, realize synchronous detection of miR-21 and miR-155 double targets by double-strand specific nuclease-mediated circulating signal amplification and fluorescence/DNA double-coding magnetic bead conjugates, further effectively eliminate background signal interference by means of click chemistry capture of unreacted magnetic beads by multifunctional click glass microbubbles and self-buoyancy-driven self-layering separation, and finally finish unbiased counting and signal output of double-target magnetic beads by an AI automatic fluorescence counting decoding module, thereby providing an ultra-sensitive digital detection method for the tumor extracellular vesicle miRNA, which is simple to operate, does not need complex equipment, has high sensitivity and is suitable for clinical plasma samples. To achieve these objects and other advantages and in accordance with the purpose of the invention, there is provided a bubble self-layering digital detection method for quantification of extracellular vesicle miRNA, comprising the steps of: S1, providing a sample to be tested, adding immunocapture bubbles, incubating for 10-60 min, centrifuging for 0.5-1.5min by 500-2000 rpm to remove supernatant, and washing with a phosphate buffer solution for 3-5 times to obtain immunocapture bubbles enriched with tumor extracellular vesicles, wherein the immunocapture bubbles are hollow glass micro-bubble surfaces coupled with anti-epithelial cell adhesion molecule (EpCAM) antibodies; S2, performing in-situ ultrasonic cleavage on the immunocapture bubbles enriched with tumor extracellular vesicles to release miRNA in the vesicles, centrifuging by 500-2000 rpm for 3-10 min, and taking a supernatant to obtain a miRNA sample to be detected; S3, mixing a miRNA sample to be detected with a fluorescent/DNA double-coded magnetic bead conjugate, double-strand specific nuclease (DSN), a double-strand specific nuclease reaction buffer solution, a ribonuclea