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CN-118604344-B - Preparation method of HER2 ECD (human immunodeficiency Virus) detector for detecting gastric cancer serum

CN118604344BCN 118604344 BCN118604344 BCN 118604344BCN-118604344-B

Abstract

The invention relates to the technical field of fluorescence detection methods of nonlinear hybridization chain reactions, and discloses a preparation method of a HER2 ECD (human immunodeficiency virus) detector for detecting gastric cancer serum, which comprises the following steps of designing a trigger probe sequence and a hairpin chain of the nonlinear hybridization chain reaction according to sequences of an aptamer of the HER2 ECD of the gastric cancer serum, synthesizing and purifying the trigger probe sequence and the hairpin chain, modifying fluorescent groups and quenching groups at two ends of the hairpin chain, respectively dissolving the trigger probe sequence and the aptamer of the HER2 ECD in a buffer solution, carrying out mixed heating, cooling to room temperature to obtain a solution 1, dissolving the hairpin chain in the buffer solution to obtain a corresponding solution 2, a solution 3, a solution 4 and a solution 5, forming a mixed solution by the HER2 ECD protein solution and the solution, adjusting the pH value of the mixed solution, incubating, and obtaining the detector. The invention has higher sensitivity when being used for detecting gastric cancer serum HER 2.

Inventors

  • REN LI
  • LIU FEIFEI
  • SHI YUE
  • JIANG WENNA

Assignees

  • 天津市肿瘤医院（天津医科大学肿瘤医院）

Dates

Publication Date
20260508
Application Date
20240627

Claims (9)

  1. 1. The preparation method of the HER2 ECD detector for detecting gastric cancer serum is characterized by comprising the following steps of: Step S1, designing a trigger probe sequence and a hairpin chain of a nonlinear hybridization chain reaction according to the sequence of an aptamer of gastric cancer serum HER2 ECD, synthesizing and purifying, and modifying a fluorescent group and a quenching group at two ends of the hairpin chain; Step S2, respectively dissolving the trigger probe sequence and the HER2 ECD aptamer in a buffer solution, mixing and heating, and cooling to room temperature to obtain a solution 1; The hairpin chain includes a hairpin NLH1, a hairpin NLH2, a hairpin NLH3, a hairpin NLH4, Dissolving hairpin NLH1, hairpin NLH2, hairpin NLH3 and hairpin NLH4 in buffer solution, heating, and cooling to room temperature to obtain corresponding solution 2, solution 3, solution 4 and solution 5 respectively; Mixing HER2 ECD protein solution with solution 1 at room temperature, adding solution 2, solution 3, solution 4, solution 5, magnesium chloride solution and dipotassium hydrogen phosphate solution to form mixed solution, adjusting pH value of the mixed solution in the process of forming mixed solution, and incubating to form a detection object; the trigger probe sequence is shown as SEQ ID NO. 1; the sequence of the hairpin NLH1 is shown as SEQ ID NO.2, the sequence of the hairpin NLH2 is shown as SEQ ID NO.3, the sequence of the hairpin NLH3 is shown as SEQ ID NO.4, and the sequence of the hairpin NLH4 is shown as SEQ ID NO. 5.
  2. 2. The method of claim 1, wherein the buffer is a TE buffer.
  3. 3. The method according to claim 1, wherein in step S2, the trigger probe sequence and the aptamer of HER2 ECD are dissolved in a buffer solution, respectively, and the molar concentration ratio of the trigger probe sequence to the aptamer is 1:1 before the mixed heating is performed; In step S2, the trigger probe sequence and the HER2 ECD aptamer are dissolved in a buffer, and the molar concentration of the trigger probe sequence is 10 μm before mixing and heating.
  4. 4. The method of claim 1, wherein the buffer with trigger probe sequence is mixed with a buffer with HER2 ECD aptamer prior to the mixing and heating, the ratio of buffer with trigger probe sequence to buffer with HER2 ECD aptamer was 1:1 by volume.
  5. 5. The method according to claim 1, wherein the molar concentration ratio of hairpin NLH1 in solution 2, hairpin NLH2 in solution 3, hairpin NLH3 in solution 4, hairpin NLH4 in solution 5 is 1:2:2:4; the molar concentration of hairpin NLH1 in the solution 2 was 0.1. Mu.M.
  6. 6. The method of claim 1, wherein the HER2 ECD protein solution has a mass concentration of 20ng/mL.
  7. 7. The method of claim 1, wherein the HER2 ECD protein solution, solution 1, solution 2, solution 3, solution 4, solution 5 are present in a volume ratio of 1:1:1:1:1:1 by volume; the volume ratio of HER2 ECD protein solution, solution 1, solution 2, solution 3, solution 4 and solution 5 to magnesium chloride solution and dipotassium hydrogen phosphate solution is 20:2:1.
  8. 8. The method according to claim 1, wherein the molar concentration of the magnesium chloride solution is 45mM-55mM; the molar concentration ratio of the magnesium chloride solution to the dipotassium hydrogen phosphate solution is 2:1.
  9. 9. The method according to claim 1, wherein the pH of the mixed solution is 4.5-8.5; the temperature of the mixed heating is 94-96 ℃, and the heating time is 1.5-2.5 min; the heating temperature is 94-96 ℃, and the heating time is 2.5-3.5 min; the temperature of the incubation is 36-40 ℃ and the time of the incubation is 0.8-1.2 h.

Description

Preparation method of HER2 ECD (human immunodeficiency Virus) detector for detecting gastric cancer serum Technical Field The invention relates to the technical field of fluorescent detection methods based on nonlinear hybridization chain reaction, in particular to a preparation method of a HER2 ECD (human serum extracellular domain) detector for detecting gastric cancer. Background Gastric cancer is a malignant tumor originated from gastric mucosal epithelium, has obvious regional difference in incidence, and has obviously higher incidence rate in northwest and eastern coastal areas than in southern areas in China. Gastric cancer can occur in any part of the stomach, with more than half occurring in the antrum, the greater curvature, lesser curvature and anterior and posterior walls of the stomach all affected. Most stomach cancer belongs to adenocarcinoma, has no obvious symptoms in early stage, or has nonspecific symptoms such as upper abdomen discomfort, eructation and the like, is often similar to symptoms of chronic gastric diseases such as gastritis, gastric ulcer and the like, and is easy to ignore, so that the early diagnosis rate of stomach cancer in China is still lower. Based on the deep knowledge of the molecular mechanism of tumor development, gastric cancer patients should determine HER2 state as early as possible, and through accurate HER2 detection, the state and type of the disease can be comprehensively known, so that effective personalized treatment can be realized, and the survival period can be prolonged. In the prior art, the hybridization chain reaction has important significance in the field of medical detection analysis due to the advantages of no enzyme, constant temperature, high amplification efficiency and the like, however, the traditional hybridization chain reaction only provides a linear assembly process, and has poorer analysis sensitivity. Therefore, development of a more sensitive detection technique for dynamically monitoring changes in HER2 expression has become an urgent need. Disclosure of Invention The nonlinear hybridization chain reaction has a plurality of advantages in the aspect of developing the biosensor based on a plurality of detection methods, and has the advantages of simplicity, high sensitivity, low cost, good specificity and the like. But the design of trigger probe sequences in the nonlinear hybridization chain reaction process is a key technical point of whether the nonlinear hybridization chain reaction is successful or not. According to the design of the trigger probe sequence by the aptamer, the trigger probe can be obtained not only through the complementary base sequence of the aptamer, but also through the design of the number of the base sequences in the trigger probe sequence, the trigger probe and the aptamer can exist in a solution in a stable double-chain structure without reacting with a hairpin sequence under the condition that no detection target exists, and when the detection target exists, the target protein can be combined with the aptamer in high specificity and high affinity, so that the trigger probe is well exposed, and the exposed probe can further perform an assembly reaction with the hairpin NLH1-NLH4 to finally form a branched DNA cross-linking structure. In the process, the design and optimization of the trigger probe sequence and the optimization of the number of non-complementary bases in the hairpin sequence are key, determine whether the whole reaction process can be smoothly carried out, and screen non-specific fluorescent signals. If the design is not reasonable, the nonspecific detection signal is generated. Thus, the design of trigger probe sequences is a complex and critical step. In order to solve the technical problems, the invention provides a preparation method of a gastric cancer serum HER2 ECD detection object, and the gastric cancer serum HER2 detection object obtained by the method has higher sensitivity and specificity when being used for detecting gastric cancer serum HER 2. The invention provides a preparation method of a HER2 ECD (human immunodeficiency Virus) detector for detecting gastric cancer serum, which comprises the following steps: Step S1, designing a trigger probe sequence and a hairpin chain of a nonlinear hybridization chain reaction according to the sequence of an aptamer of gastric cancer serum HER2 ECD, synthesizing and purifying, and modifying a fluorescent group and a quenching group at two ends of the hairpin chain; Step S2, respectively dissolving the trigger probe sequence (TRIGGER DNA) and the HER2 ECD aptamer in a buffer solution, carrying out mixed heating, and cooling to room temperature to obtain a solution 1; The hairpin chain includes a hairpin NLH1, a hairpin NLH2, a hairpin NLH3, a hairpin NLH4, Dissolving hairpin NLH1, hairpin NLH2, hairpin NLH3 and hairpin NLH4 in buffer solution, heating, and cooling to room temperature to obtain corresponding solution 2, solution 3, solu