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CN-115433767-B - Sensing chip based on DNA tetrahedron, preparation method and device thereof

CN115433767BCN 115433767 BCN115433767 BCN 115433767BCN-115433767-B

Abstract

The invention provides a sensing chip based on a DNA tetrahedron, and also provides a preparation method and a device thereof. The sensing chip based on the DNA tetrahedron is prepared by carrying out antibody functional modification on the surface of the sensing chip modified by the DNA tetrahedron. The sensing chip based on the DNA tetrahedron prepared by the invention can detect the Alzheimer's disease through a surface plasmon resonance imaging technology, has the advantages of high flux, high sensitivity and reusability of the chip, and the method shows good disease diagnosis performance. Under the condition of lacking an early detection means of the Alzheimer's disease at present, the invention selects a blood-based detection mode, and provides a miniaturized device for early detection and noninvasive detection of the Alzheimer's disease.

Inventors

  • YANG YANLIAN
  • SONG SHIJIE
  • ZHU LING
  • GAO HOUQIAN
  • WANG CHEN

Assignees

  • 国家纳米科学中心

Dates

Publication Date
20260508
Application Date
20220429

Claims (20)

  1. 1. The use of a DNA tetrahedron-based sensor chip for the preparation of a detection device for alzheimer's disease, characterized in that the DNA tetrahedron-based sensor chip is prepared by binding the DNA tetrahedron to the surface of the sensor chip and performing antibody functionalization modification, wherein: the DNA tetrahedron is a tetrahedron structure formed by DNA chains, and the base pair number of each side length of the DNA tetrahedron is 26bp; The planar dimensions of the triangles forming the DNA tetrahedron are the same and are uniformly dispersed on the substrate, wherein three vertexes of the triangle at the bottom of the DNA tetrahedron are respectively provided with one sulfhydryl group, the DNA chain extends out from the top to form a DNA double chain with carboxyl modification, the base pair number of the DNA chain extending out from the top is 15bp, and the number of the DNA chain extending out from the top is 1; The sequences of the DNA chains forming the DNA tetrahedron comprise SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5; the sensing chip is an SPR imaging sensing chip, and the antibody is one or more selected from the group consisting of Anti-Abeta 42, anti-NFL and Anti-Tau.
  2. 2. The Alzheimer's disease detection device is characterized by comprising a DNA tetrahedron-based sensing chip, wherein the DNA tetrahedron-based sensing chip is prepared by combining the DNA tetrahedron with the surface of the sensing chip and performing antibody functionalization modification, and the Alzheimer's disease detection device comprises: the DNA tetrahedron is a tetrahedron structure formed by DNA chains, and the base pair number of each side length of the DNA tetrahedron is 26bp; The planar dimensions of the triangles forming the DNA tetrahedron are the same and are uniformly dispersed on the substrate, wherein three vertexes of the triangle at the bottom of the DNA tetrahedron are respectively provided with one sulfhydryl group, the DNA chain extends out from the top to form a DNA double chain with carboxyl modification, the base pair number of the DNA chain extending out from the top is 15bp, and the number of the DNA chain extending out from the top is 1; The sequences of the DNA chains forming the DNA tetrahedron comprise SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5; the sensing chip is an SPR imaging sensing chip, and the antibody is one or more selected from the group consisting of Anti-Abeta 42, anti-NFL and Anti-Tau.
  3. 3. The use according to claim 1 or the detection device according to claim 2, wherein the method for manufacturing a DNA tetrahedron-based sensor chip comprises: The DNA tetrahedron is combined with the surface of the sensing chip, and antibody functionalization modification is carried out, wherein the combination is sulfhydryl bonding, and the antibody functionalization modification is amidation reaction modification.
  4. 4. A use or detection device according to claim 3, wherein the preparation method comprises the steps of: (1) Preparing a DNA single-chain mixed solution, and preparing a DNA tetrahedron solution after annealing treatment; (2) Bonding the DNA tetrahedral solution prepared in the step (1) with a sensing chip to obtain a DNA tetrahedral modified sensing chip, and (3) And (3) activating the DNA tetrahedron modified sensing chip prepared in the step (2), adding an antibody for incubation, and washing and drying to obtain the antibody functionalized DNA tetrahedron-based sensing chip.
  5. 5. The device for application or detection according to claim 4, wherein the step (1) further comprises preparing a single-stranded DNA mother solution from the single-stranded DNA, and then mixing the single-stranded DNA mother solution with a buffer solution to prepare a single-stranded DNA mixed solution.
  6. 6. The application or detection device according to claim 5, wherein: The sequence of the DNA single strand comprises SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5; The buffer solution is TM buffer solution, the pH value of the buffer solution is 7.5-8.5, and/or The concentration of the DNA single strand in the DNA single strand mixed solution is 0.4-5.0 mu mol/L.
  7. 7. The application or detection device according to claim 6, wherein: the pH of the buffer is 8, and/or The concentration of the DNA single strand in the DNA single strand mixed solution is 0.4-2.0 mu mol/L.
  8. 8. The use or detection apparatus according to claim 7, wherein the concentration of the single strand of DNA in the single strand mixed solution of DNA is 1.0. Mu. Mol/L.
  9. 9. The application or detection device according to claim 4, wherein in the step (1), the annealing treatment includes a temperature increasing reaction and a temperature decreasing reaction.
  10. 10. The application or detection device according to claim 9, wherein in step (1): The reaction temperature of the heating reaction is 95 ℃; The temperature rising reaction time is 2-10 min; the cooling reaction time is more than 30s, and/or The reaction temperature of the cooling reaction is 4 ℃.
  11. 11. The application or detection device according to claim 10, wherein in step (1): The temperature-rising reaction time is 4-9 min, and/or The cooling reaction time is more than 3min.
  12. 12. The application or detection device according to claim 11, wherein in step (1): the temperature-rising reaction time is 8min, and/or The cooling reaction time is 8min.
  13. 13. The application or detection device according to claim 4, wherein the step (2) further comprises spreading the DNA tetrahedron solution prepared in the step (1) on the surface of a sensing chip, incubating, bonding the DNA tetrahedron with the sensing chip, and cleaning to obtain the DNA tetrahedron-modified sensing chip.
  14. 14. The application or detection device according to claim 13, wherein in the step (2), the incubation time is 10-25 hours.
  15. 15. The application or detection device according to claim 14, wherein in the step (2), the incubation time is 10-20 hours.
  16. 16. The use or detection device according to claim 15, wherein in step (2), the incubation time is 12 hours.
  17. 17. The device for application or detection according to claim 4, wherein the step (3) further comprises immersing the DNA tetrahedrally modified sensor chip prepared in the step (2) in an activator for hydroxyl activation, washing and drying, and adding an antibody for incubation.
  18. 18. The application or detection apparatus according to claim 17, wherein in step (3): The activator is a mixed solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and nitrogen-hydroxysuccinimide (NHS); The volume of each sample drop added with the antibody is 0.4-1.0 mu L, and/or The incubation time is 10-25 h.
  19. 19. The application or detection apparatus according to claim 18, wherein in step (3): the volume of each sample drop added with the antibody is 0.4-0.5 mu L, and/or The incubation time is 10-20 h.
  20. 20. The application or detection apparatus according to claim 19, wherein in step (3): the antibody was added in a volume of 0.4. Mu.L per sample spot, and/or The incubation time was 12h.

Description

Sensing chip based on DNA tetrahedron, preparation method and device thereof Technical Field The invention belongs to the field of bioengineering and medical instruments, and particularly relates to a sensing chip based on a DNA tetrahedron, and a preparation method and a device thereof. Background Alzheimer's Disease (AD) is one of the most common neurodegenerative diseases, and is primarily manifested by progressive and irreversible decline in cognition. Alzheimer's disease and other dementias have led to 160 thousands of people losing life in 2019 as the seventh global fatal disease. The number of lost life per year due to the disease increases by 100 tens of thousands compared to 2000. With the development of aging population, the number of AD patients is increasing. However, accurate diagnosis and effective treatment of AD remains a difficult challenge to overcome, and such severe situations present a significant economic burden to society. As clinical symptoms become apparent, the progression of AD is divided into three stages, preclinical, mild Cognitive Impairment (MCI), and dementia. At present, clinical diagnostic methods mainly comprise investigation of patient and family medical history, neurophysiologic test, cognitive function evaluation, magnetic resonance imaging, positron emission tomography imaging and the like. Most of these methods can only be used for diagnosis in the dementia stage and the MCI stage with obvious clinical symptoms, and imaging examination often requires high cost, and in addition, the sampling mode of lumbar puncture brings great pain to patients. In order to obtain early detection equipment with convenient sampling, miniaturization and low cost, detection technology based on blood analysis is attracting attention. The sensitivity and specificity of the device and the parameters such as diagnostic sensitivity, specificity, accuracy and the like in the clinical application process are all important indexes for evaluating the detection technology based on blood analysis. Disclosure of Invention It is therefore an object of the present invention to overcome the drawbacks of the prior art and to provide a DNA tetrahedron based sensing chip, a method and a device for its preparation. The SPRi sensing chip based on DNA tetrahedron is coupled with a plurality of antibodies through amidation reaction, so that a plurality of protein biomarkers related to Alzheimer's disease can be detected simultaneously, and the effective distinction of plasma samples of dementia patients and mild cognitive impairment patients and normal people can be realized. Before setting forth the summary, the terms used herein are defined as follows: the term "AD" refers to Alzheimer's disease. The term "A.beta.40" refers to beta-amyloid 40. The term "A.beta.42" refers to beta-amyloid 42. The term "Anti-Abeta 42" refers to an antibody to beta-amyloid 40. The term "Anti-Abeta 42" refers to an antibody to beta-amyloid 42. The term "Anti-GFAP" refers to glial fibrillary acidic protein antibody The term "Anti-NFL" refers to neurofilament light chain protein antibodies. The term "Anti-Tau" refers to a Tau protein antibody. The term "Anti-p-Tau" refers to phosphorylated Tau protein antibodies. The term "Anti-p-Tau181" refers to a Tau protein antibody phosphorylated at position 181. The term "Anti-p-Tau217" refers to a Tau protein antibody phosphorylated at position 217. The term "AUC" refers to the area under the curve. The term "bp" refers to base pairs. The term "BSA" refers to bovine serum albumin. The term "EDC" refers to 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride. The term "HAS" refers to human serum albumin. The term "IgG" refers to immunoglobulin G. The term "IgM" refers to immunoglobulin M. The term "Ladder" refers to a trapezoidal strip. The term "MCI" refers to mild cognitive impairment. The term "NC" refers to a healthy control. The term "NFL" refers to neurofilament light chain proteins. The term "NHS" refers to a nitrogen-hydroxysuccinimide. The term "PBS" refers to phosphate buffer. The term "ROC" refers to the subject's operating characteristics. The term "SPR" refers to surface plasmon resonance. The term "SPRi" refers to SPR imaging. The term "TM" refers to a tris-magnesium buffer. The term "TRF" refers to transferrin. To achieve the above object, a first aspect of the present invention provides a DNA tetrahedron-based sensor chip prepared by binding the DNA tetrahedron to a surface of the sensor chip and performing antibody functionalization modification, wherein: the DNA tetrahedron is of a tetrahedron structure formed by DNA chains, and the base pair number of each side length of the DNA tetrahedron is 15-30 bp, preferably 20-30 bp, and most preferably 26 bp. The DNA tetrahedron-based sensor chip according to the first aspect of the present invention, wherein, The sequences of the DNA chains forming the DNA tetrahedron comprise SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID