WO2022089672A1 - 碳氮荧光量子点肿瘤细胞检测试剂盒及其使用方法 - Google Patents

碳氮荧光量子点肿瘤细胞检测试剂盒及其使用方法 Download PDF

Info

Publication number
WO2022089672A1
WO2022089672A1 PCT/CN2021/141681 CN2021141681W WO2022089672A1 WO 2022089672 A1 WO2022089672 A1 WO 2022089672A1 CN 2021141681 W CN2021141681 W CN 2021141681W WO 2022089672 A1 WO2022089672 A1 WO 2022089672A1
Authority
WO
WIPO (PCT)
Prior art keywords
solution
quantum dots
carbon
fluorescent quantum
nitrogen
Prior art date
Application number
PCT/CN2021/141681
Other languages
English (en)
French (fr)
Inventor
范先群
丁古巧
周慧芳
杨思维
李吉鹏
Original Assignee
上海交通大学医学院附属第九人民医院
中国科学院上海微系统与信息技术研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN202111227034.0A external-priority patent/CN115184313A/zh
Application filed by 上海交通大学医学院附属第九人民医院, 中国科学院上海微系统与信息技术研究所 filed Critical 上海交通大学医学院附属第九人民医院
Publication of WO2022089672A1 publication Critical patent/WO2022089672A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y20/00—Nanooptics, e.g. quantum optics or photonic crystals
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B21/00—Nitrogen; Compounds thereof
    • C01B21/082—Compounds containing nitrogen and non-metals and optionally metals
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
    • C09K11/65—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing carbon

Definitions

  • the invention relates to the technical field of tumor cell detection, in particular to a carbon-nitrogen fluorescent quantum dot tumor cell detection kit and a method for using the same.
  • tumor cells will fall off and release themselves into tissue fluids such as pleural effusion or urine, becoming a real-time tumor lesion detection marker, which has been widely used in clinical tumor diagnosis.
  • tissue fluids such as pleural effusion or urine
  • a real-time tumor lesion detection marker which has been widely used in clinical tumor diagnosis.
  • HE staining microscopy has problems such as high missed diagnosis rate, low precision and sensitivity, poor stability, and inability to sort tumor cells and analyze further tumor cells.
  • Flow cytometric DNA analysis and chromosome examination are complicated and costly, which limit their large-scale clinical applications.
  • the technical problem to be solved by the present invention is how to improve the specificity and sensitivity of tumor cell detection while reducing the complexity and cost of its operation.
  • the present invention provides a new use of carbon-nitrogen fluorescent quantum dots in the preparation of tumor cell detection products.
  • the tumor cell detection product should at least contain carbon-nitrogen fluorescent quantum dots.
  • the tumor cell detection product may be a carbon-nitrogen fluorescent quantum dot-based tumor cell detection kit, including a solution A and a solution B, wherein the solution A is a solution of carbon-nitrogen fluorescent quantum dots, and the solution B is a buffer. liquid; the carbon-nitrogen fluorescent quantum dot solution is composed of carbon-nitrogen fluorescent quantum dots and a solvent.
  • the B solution is phosphate buffered saline PBS.
  • the solvent is dimethyl sulfoxide DMSO.
  • concentration of carbon-nitrogen fluorescent quantum dots in the carbon-nitrogen fluorescent quantum dot solution is 0.01-50 ⁇ g/mL.
  • the carbon-nitrogen fluorescent quantum dots are N-doped graphene quantum dots, C 3 N 4 quantum dots, C 2 N quantum dots or C 3 N quantum dots, and the size of the carbon-nitrogen fluorescent quantum dots is 1-100 nm.
  • the carbon-nitrogen fluorescent quantum dots have tumor targeting properties.
  • the types of tumors that the kit can detect with high sensitivity include, but are not limited to, intraocular malignant tumors, liver cancer, lung cancer, gastric cancer, bladder cancer, breast cancer, ovarian cancer, thyroid cancer, melanoma, and colon cancer.
  • the present invention also provides a method for using the described carbon-nitrogen fluorescent quantum dot-based tumor cell detection kit, comprising the following steps:
  • Step 1 Collect the patient's pleural effusion or urine sample with a puncture needle and store it in a sterile centrifuge tube;
  • Step 2 centrifuge the sample obtained in the step 1, discard the supernatant, and obtain the sediment of the sample;
  • Step 3 adding the B solution, resuspending the precipitate obtained in the step 2, to obtain the resuspended liquid of the precipitate;
  • Step 4 adding the A solution, mixing with the resuspended solution obtained in the step 3, and incubating;
  • Step 5 centrifuge the resuspended liquid after incubation in step 4, discard the supernatant, and obtain the precipitate after incubation of the sample;
  • Step 6 adding the B solution, resuspending the precipitate after the incubation in the step 5, to obtain a uniformly mixed resuspension;
  • Step 7 use a fluorescence spectrophotometer to detect, and detect the fluorescence intensity of the resuspension obtained in the step 6;
  • Step 8 Perform semi-quantitative analysis on the sample according to the fluorescence intensity obtained in Step 7.
  • the volume of the pleural effusion or urine sample in the first step is 1-10 mL, and the temperature of the sample is 0°C-25°C.
  • the rotation speed of the centrifugation is 1000-2000 rpm, and the centrifugation time is 1-60 minutes.
  • the amount of liquid A added is 0.01-10 mL.
  • the amount of liquid B added in the third step and the sixth step is 0.1-10 mL.
  • the temperature of the incubation in the fourth step is 4°C to 40°C, and the incubation time is 10 to 480 minutes.
  • the excitation wavelength of the resuspended liquid of the sample to be tested is 400-800 nm.
  • the background fluorescence intensity of normal pleural effusion or urine is less than 5000.
  • the present invention provides a high-sensitivity detection kit for tumor cells based on tumor-targeted carbon-nitrogen fluorescent quantum dots. Compared with traditional methods, the advantages are:
  • the carbon-nitrogen fluorescent quantum dots in the present invention have tumor targeting properties, can respond to the microenvironment of tumor cells, identify tumor cells hidden in pleural effusion and urine, improve the accuracy of tumor cell detection, and have good Light stability to ensure repeatability of detection;
  • the present invention overcomes the problems of low detection accuracy, low sensitivity, poor stability, complex operation and high cost in the prior art, improves the diagnosis level of doctors for clinical tumor patients, and also provides a basis for tumor prognosis evaluation and drug use. Screening and individualized treatment help.
  • Fig. 1 is the schematic diagram of the use flow of the tumor cell detection kit based on carbon-nitrogen fluorescent quantum dots provided by the present invention
  • Fig. 2 is the contrast diagram of bright light and fluorescence of tumor cells in pleural effusion samples captured by carbon-nitrogen fluorescent quantum dots in Example 1 of the present invention
  • Fig. 3 is the correspondence between the tumor cell reference concentration and the fluorescence reference value in Example 1 of the present invention.
  • Example 4 is a comparison diagram of the bright light and fluorescence of the carbon-nitrogen fluorescent quantum dots capturing tumor cells in the urine sample in Example 4 of the present invention
  • FIG. 1 is a schematic diagram of the use of the carbon-nitrogen fluorescent quantum dot-based tumor cell detection kit disclosed in the present invention, and its operation steps at least include:
  • Step S10 providing kit A liquid, B liquid, patient's pleural effusion, urine samples
  • Step S20 high-speed centrifugation, after liquid B is resuspended, add liquid A for staining;
  • Step S30 high-speed centrifugation, and resuspending liquid B;
  • Step S40 the spectrophotometer detects the fluorescence intensity.
  • step S10 includes step S11: providing reagent kits A and B, and step S12: pleural effusion and urine samples of the patient.
  • the kit A solution is a solution of 0.01-50 ⁇ g of carbon-nitrogen fluorescent quantum dots dissolved in 1 mL of DMSO, wherein the carbon-nitrogen fluorescent quantum dots are N - doped graphene quantum dots, C3N4 quantum dots, C2 N quantum dots or C 3 N quantum dots; the size of carbon-nitrogen fluorescent quantum dots is 1-100 nm; kit B solution is PBS buffer.
  • step S12 a puncture needle is used to collect the patient's pleural effusion or urine sample, and the collected liquid volume is 1-10 mL, which is stored in a 50-mL centrifuge tube at 0°C to 25°C.
  • Step S20 includes step S21: high-speed centrifugation, step S22: resuspension with liquid B, and step S23: adding liquid A for dyeing.
  • step S21 the sample is centrifuged at high speed, the centrifugation speed is 1000-2000 rpm, and the centrifugation time is 1-60 minutes.
  • step S22 discard the supernatant in the centrifuge tube, add solution B of the kit, and add the solution in an amount of 0.1-10 mL, and resuspend the sediment at the bottom of the centrifuge tube.
  • step 23 the kit A solution is added in an amount of 0.01-10 mL, mixed with the resuspension solution, and incubated at a temperature of 4°C to 40°C and an incubation time of 10 to 480 minutes. Cell staining.
  • Step S30 includes step S31: high-speed centrifugation and step S32: resuspension of liquid B.
  • step S31 the suspension after the incubation of the liquid A is centrifuged at a high speed, the centrifugation speed is 1000-2000 rpm, and the centrifugation time is 1-60 minutes.
  • step S32 discard the supernatant in the centrifuge tube, add solution B of the kit, and resuspend the sediment at the bottom of the centrifuge tube.
  • the amount of solution B added is 0.1-10 mL.
  • Step S40 includes step S41: detecting the fluorescence intensity of the sample and step S42: semi-quantitative analysis.
  • step S41 a fluorescence spectrophotometer is used, and the excitation wavelength and the emission wavelength are set according to the particle size of the carbon-nitrogen fluorescent quantum dots, and the fluorescence intensity of the sample is read.
  • step S42 semi-quantitative analysis is performed on the fluorescence intensity of the pleural effusion or urine sample according to the corresponding relationship between the tumor cell reference concentration and the fluorescence reference value.
  • liquid A is N-doped graphene quantum dot solution
  • the solvent is DMSO
  • the concentration of carbon-nitrogen fluorescent quantum dots is 1 ⁇ g/mL
  • the size of carbon-nitrogen fluorescent quantum dots is 50 nm
  • liquid B is PBS buffer solution for detecting eye Pleural fluid samples from patients with choroidal melanoma liver metastases.
  • the patient's pleural effusion samples were collected with a puncture needle, and the volume of fluid collected was 10 mL, which was stored in a 50 mL centrifuge tube at room temperature of 25°C.
  • the pleural effusion samples were centrifuged at high speed at 1000 rpm for 20 minutes, and the supernatant was discarded to obtain the sediment of the pleural effusion samples.
  • the centrifugation speed was 1000 rpm, and the centrifugation time was 20 minutes, the supernatant was discarded, and 1 mL of B solution was added to obtain a re-suspension.
  • the detection conditions of the fluorescence spectrophotometer were set, the excitation wavelength was 400 nm, and the emission wavelength was 530 nm, and the pleural effusion samples of the patients were detected.
  • Figure 2 is a comparison of bright light and fluorescence of tumor cells in a patient's pleural effusion sample captured by carbon-nitrogen fluorescent quantum dots.
  • the cells in the dashed red circle are tumor cells in the patient's pleural effusion, and the cells outside the dashed red circle are the shed epithelial cells in the patient's pleural effusion.
  • the green fluorescent protein GFP channel Under the green fluorescent protein GFP channel, it can be clearly and clearly seen that the tumor cells show bright green fluorescence (the brighter part in the right part in Figure 2), while the epithelial cells are not fluorescent.
  • the fluorescence intensity of the patient's pleural effusion sample was 6300. Referring to the corresponding relationship between the reference concentration of tumor cells and the reference value of fluorescence in FIG. 3 , the count of tumor cells in the patient's pleural effusion is 10-100 cells/mL.
  • Example 1 Based on Example 1, under the condition that the concentration and size of the carbon-nitrogen fluorescent quantum dots and the sample volumes of liquid A and liquid B remain unchanged, the effects of incubation temperature and time on the detection are compared, and the detection results are as follows:
  • Incubation temperature Incubation time
  • the fluorescence intensity 4 degrees 480 minutes 6724 25 degree 60 minutes 6300 40 degree 10 minutes 5658
  • Embodiment 3 compares the concentration of carbon-nitrogen fluorescent quantum dots in the detection, the addition amount of liquid A and liquid B
  • Example 1 Based on Example 1, under the conditions that the size of the carbon-nitrogen fluorescent quantum dots is 50 nm, the incubation temperature is 25 degrees, and the incubation time is 60 minutes, the concentrations of the carbon-nitrogen fluorescent quantum dots are compared. The impact of detection
  • the concentrations of carbon-nitrogen fluorescent quantum dots are compared: 50 ⁇ g/mL, 1 ⁇ g/mL, 0.02 ⁇ g/mL, and the test results are as follows:
  • the reaction volume had no obvious effect on the fluorescence results, and the fluorescence intensity was distributed between 6000-6500.
  • solution A is C 3 N quantum dot solution
  • the solvent is DMSO
  • the concentration of C 3 N quantum dots is 1 ⁇ g/mL
  • the size of C 3 N quantum dots is 50 nm
  • solution B is PBS buffer solution for the detection of ureteral epithelial cancer Patient's urine sample.
  • the liquid collection volume is 10mL, and store it in a 50mL centrifuge tube at room temperature of 25 °C.
  • the urine samples were centrifuged at high speed at 1500 rpm for 20 minutes, and the supernatant was discarded to obtain the sediment of the urine samples.
  • the centrifugation speed was 1500 rpm
  • the centrifugation time was 20 minutes
  • the supernatant was discarded
  • 1 mL of B solution was added to obtain a resuspension.
  • the detection conditions of the fluorescence spectrophotometer were set, the excitation wavelength was 400 nm, and the emission wavelength was 530 nm, and the urine samples of the patients were detected.
  • Figure 4 is a comparison of bright light and fluorescence of C 3 N quantum dots capturing tumor cells in a patient's urine sample.
  • the cells in the dotted red circle (the dotted circle in the left part of Figure 4) in the figure are tumor cells in the patient's urine, and the cells outside the dotted red circle are epithelial cells exfoliated from the patient's urinary tract. Under the green fluorescent protein GFP channel, it can be clearly and clearly seen that the tumor cells show bright green fluorescence (the brighter part in the right part in Figure 4), while the epithelial cells are not fluorescent.
  • the fluorescence intensity of the patient's urine sample was 8400. Referring to the corresponding relationship between the reference concentration of tumor cells and the reference value of fluorescence in FIG. 3 , the count of tumor cells in the patient's urine is 100-1000 cells/mL.
  • Example 1 the sensitivity of using carbon-nitrogen fluorescent quantum dots to capture tumor cells in pleural effusion samples and urine samples is shown in FIG. 5 .
  • the sensitivity of using C 3 N quantum dots to capture tumor cells in patient urine samples exceeded 72%; the sensitivity of using carbon nitrogen fluorescent quantum dots to capture tumor cells in patient pleural fluid samples reached 100%.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Nanotechnology (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Inorganic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Condensed Matter Physics & Semiconductors (AREA)
  • Manufacturing & Machinery (AREA)
  • General Physics & Mathematics (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Materials Engineering (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biophysics (AREA)
  • Optics & Photonics (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

一种碳氮荧光量子点肿瘤细胞检测试剂盒及其使用方法，该试剂盒包括A液和B液；其中，该A液是碳氮荧光量子点溶液，该B液是缓冲液；该碳氮荧光量子点溶液由碳氮荧光量子点和溶剂组成。该使用方法至少包括以下步骤：步骤S10、提供试剂盒A液、B液，患者胸水、尿液样品；步骤S20、高速离心，B液重悬后，加入A液染色；步骤S30、高速离心，B液重悬；步骤S40、分光光度计检测荧光强度。该试剂盒和及方法对胸水和尿液组织液中肿瘤细胞的检测具有高特异性和高敏感性，且操作简单、成本小，不但能够提高医生对临床肿瘤患者的诊断水平，还能为肿瘤的预后评估、用药筛选、个体化治疗提供帮助。

Description

[根据细则37.2由ISA制定的发明名称]　碳氮荧光量子点肿瘤细胞检测试剂盒及其使用方法 技术领域
本发明涉及肿瘤细胞检测技术领域，尤其涉及一种碳氮荧光量子点肿瘤细胞检测试剂盒及其使用方法。
背景技术
在胸部、泌尿系统原发肿瘤病灶和转移瘤病灶中，肿瘤细胞会自行脱落释放进入胸水或尿液等组织液中，成为一种实时的肿瘤病灶检测标记物，一直广泛应用在临床的肿瘤诊断上。准确、灵敏的捕捉到胸水及尿液中的脱落肿瘤细胞，对于肿瘤的发生机制、早期诊断、病理分型、转移、治疗、判断患者预后、评估抗肿瘤药物的肿瘤耐药及制定个体化治疗等方面有着巨大的潜在临床应用价值。
随着生化检测技术的发展，胸水及尿液中脱落细胞检测确诊肿瘤病灶的准确率不断提高，但诊断常依据医生主观经验，容易出现误诊和遗漏等问题。目前常用的胸水、尿液实验室检查包括肿瘤细胞HE(苏木素-伊红)染色镜检，流式细胞DNA分析和染色体检查。其中，HE染色镜检存在漏诊率高，精度和敏感度低，稳定性差，无法分选肿瘤细胞及对进一步肿瘤细胞进行分析等问题。流式细胞DNA分析和染色体检查操作复杂，且成本高，局限了临床的大规模应用。
因此，本领域的技术人员致力于开发一种对胸水和尿液组织液中的肿瘤细胞具有高特异性和高敏感性，而且操作简单、成本低的肿瘤细胞检测试剂盒。
发明内容
有鉴于现有技术的上述缺陷，本发明所要解决的技术问题是如何提高肿瘤细胞检测的特异性和敏感性，同时减少其操作的复杂及其成本。
为实现上述目的，本发明提供了碳氮荧光量子点在肿瘤细胞检测产品制备中的新用途。所述肿瘤细胞检测产品中至少应包含碳氮荧光量子点。
所述肿瘤细胞检测产品可以是一种基于碳氮荧光量子点的肿瘤细胞检测试剂盒，包括A液和B液，其中，所述A液是碳氮荧光量子点溶液，所述B液是缓冲液；所述碳氮荧光量子点溶液由碳氮荧光量子点和溶剂组成。
进一步地，所述B液是磷酸盐缓冲液PBS。
进一步地，所述溶剂是二甲基亚砜DMSO。
进一步地，所述碳氮荧光量子点溶液中碳氮荧光量子点的浓度为0.01～50μg/mL。
进一步地，所述碳氮荧光量子点为N掺杂石墨烯量子点、C 3N 4量子点、C 2N量子 点或C 3N量子点，碳氮荧光量子点的尺寸为1～100nm。
进一步地，所述碳氮荧光量子点具有肿瘤靶向性。
进一步地，所述试剂盒可高灵敏检测的肿瘤种类包含但不仅限于眼内恶性肿瘤、肝癌、肺癌、胃癌、膀胱癌、乳腺癌、卵巢癌、甲状腺癌、黑色素瘤、结肠癌。
同时，本发明还提供了所述的基于碳氮荧光量子点的肿瘤细胞检测试剂盒的使用方法，包括以下步骤：
步骤一、用穿刺针采集患者的胸水或尿液样本，保存在无菌的离心管中；
步骤二、离心所述步骤一中得到的样本，弃上清液，得到所述样本的沉淀物；
步骤三、加入所述B液，重悬所述步骤二中得到的沉淀物，得到所述沉淀物的重悬液；
步骤四、加入所述A液，与所述步骤三中得到的重悬液混合均匀，孵育；
步骤五、离心所述步骤四中孵育后的重悬液，弃上清液，得到所述样本孵育后的沉淀物；
步骤六、加入所述B液，重悬所述步骤五中孵育后的沉淀物，得到混合均匀的重悬液；
步骤七、使用荧光分光光度计检测，检测所述步骤六中得到的重悬液的荧光强度；
步骤八、根据所述步骤七中得到的荧光强度，对所述样本进行半定量分析。
进一步地，所述步骤一中的胸水或尿液样本的体积为1～10mL，所述样本保存的温度为0℃～25℃。
进一步地，所述步骤二和所述步骤五中离心的转速为1000～2000转/分钟，所述离心的时间为1～60分钟。
进一步地，所述步骤四中A液的加液量是0.01～10mL。
进一步地，所述步骤三和所述步骤六中B液的加液量是0.1～10mL。
进一步地，所述步骤四中孵育的温度是4℃～40℃，所述孵育的时间为10～480分钟。
进一步地，所述步骤七中待测样本重悬液的激发波长为400～800nm。
进一步地，所述步骤八中，正常胸水或尿液的荧光强度本底小于5000。
技术效果
本发明提供了一种基于肿瘤靶向碳氮荧光量子点的肿瘤细胞高灵敏检测试剂盒。与传统方式相比，优点在于：
(1)本发明中的碳氮荧光量子点具有肿瘤靶向性，能够响应肿瘤细胞的微环境，识别藏匿在胸水、尿液中的肿瘤细胞，提高肿瘤细胞检测的精确度，同时有良好的光稳定性，保证检测具有可重复性；
(2)本发明克服了现有技术中检测精度低、敏感度低、稳定度差，且操作复杂、成本高等问题，提高了医生对临床肿瘤患者的诊断水平，还为肿瘤的预后评估、用药 筛选、个体化治疗提供了帮助。
以下将结合附图对本发明的构思、具体结构及产生的技术效果作进一步说明，以充分地了解本发明的目的、特征和效果。
附图说明
图1是本发明提供的基于碳氮荧光量子点的肿瘤细胞检测试剂盒的使用流程示意图；
图2是本发明实施例1中碳氮荧光量子点捕捉胸水样本中肿瘤细胞的明光和荧光对照图；
图3是本发明实施例1中肿瘤细胞参考浓度与荧光参考值之间的对应关系；
图4是本发明实施例4中碳氮荧光量子点捕捉尿液样本中肿瘤细胞的明光和荧光对照图；
图5是本发明实施例1和4中，碳氮荧光量子点捕捉胸水样本和尿液样本中肿瘤细胞的灵敏度。
具体实施方式
以下参考说明书附图介绍本发明的多个优选实施例，使其技术内容更加清楚和便于理解。本发明可以通过许多不同形式的实施例来得以体现，本发明的保护范围并非仅限于文中提到的实施例。
图1是本发明公开的基于碳氮荧光量子点的肿瘤细胞检测试剂盒的使用流程示意图，其操作步骤至少包括：
步骤S10、提供试剂盒A液、B液，患者胸水、尿液样品；
步骤S20、高速离心，B液重悬后，加入A液染色；
步骤S30、高速离心，B液重悬；
步骤S40、分光光度计检测荧光强度。
具体地，步骤S10包括步骤S11：提供试剂盒A液、B液和步骤S12：患者胸水、尿液样品。步骤S11中，试剂盒A液是0.01～50μg的碳氮荧光量子点溶于1mL DMSO的溶液，其中，碳氮荧光量子点为N掺杂石墨烯量子点、C 3N 4量子点、C 2N量子点或C 3N量子点；碳氮荧光量子点的尺寸为1～100nm；试剂盒B液是PBS缓冲液。步骤S12中，用穿刺针采集患者的胸水或尿液样本，采液量为1～10mL，保存在50mL离心管中，置于0℃～25℃。
步骤S20包括步骤S21：高速离心、步骤S22：B液重悬和步骤S23：加入A液染色。步骤S21中，高速离心样本，离心转速为1000～2000转/分钟，离心时间为1～60分钟。步骤S22中，弃离心管中的上清液，加入试剂盒B液，加液量为0.1～10mL，重悬离心管管底的沉淀物。步骤23中，加入试剂盒A液，加液量为0.01～10mL，与重悬液混合均匀，进行孵育，孵育温度为4℃～40℃，孵育时间为10～480分钟，对 样本中的肿瘤细胞染色。
步骤S30包括步骤S31：高速离心和步骤S32：B液重悬。步骤S31中，高速离心A液孵育后的悬浮液，离心转速为1000～2000转/分钟，离心时间为1～60分钟。步骤S32中，弃离心管中的上清液，加入试剂盒B液，重悬离心管管底的沉淀物，B液的加液量为0.1～10mL。
步骤S40包括步骤S41：检测样品的荧光强度和步骤S42：半定量分析。步骤S41中使用荧光分光光度计，根据碳氮荧光量子点的粒径大小，设置激发波长和发射波长，读取样本的荧光强度。步骤S42中根据肿瘤细胞参考浓度与荧光参考值的对应关系，对胸水或尿液样本的荧光强度进行半定量分析。
实施例1眼脉络膜黑色素瘤肝转移患者的胸水样本检测
使用基于碳氮荧光量子点的肿瘤细胞检测试剂盒，包括A液和B液。其中，A液是N掺杂石墨烯量子点溶液，溶剂是DMSO，碳氮荧光量子点浓度为1μg/mL，碳氮荧光量子点的尺寸为50nm，B液是PBS缓冲液，用于检测眼脉络膜黑色素瘤肝转移患者的胸水样品。
用穿刺针采集患者的胸水样本，采液量为10mL，保存在50mL离心管中，室温25℃。高速离心胸水样本，离心转速为1000转/分钟，离心时间为20分钟，弃上清，得到胸水样本的沉淀物。向离心管中加入10mL B液，重悬离心管管底胸水样本的沉淀物，再加入1mL A液，混合均匀，在25℃下孵育60分钟。60分钟后高速离心，离心转速为1000转/分钟，离心时间为20分钟，弃上清，加入1mL B液得到重悬液。设置荧光分光光度计的检测条件，激发波长为400nm，发射波长为530nm，对患者胸水样本进行检测。
图2是碳氮荧光量子点捕捉患者胸水样本中肿瘤细胞的明光和荧光对照图。图中虚线红圈(图2中左边部分的虚线圆圈)中的细胞是患者胸水中的肿瘤细胞，虚线红圈外的细胞是患者胸水中脱落的上皮细胞。在绿色荧光蛋白GFP通道下可以明显、清楚地看到，肿瘤细胞显示亮绿色荧光(图2中右边部分亮度较高的部分)，而上皮细胞无荧光。经检测，患者胸水样本的荧光强度为6300。参照图3中肿瘤细胞参考浓度与荧光参考值的对应关系，患者胸水中肿瘤细胞的计数为10～100个/mL。
实施例2比较检测中的孵育温度和时间
基于实施例1，在碳氮荧光量子点的浓度、尺寸及A液和B液加样量不变的情况下，比较孵育温度和时间对检测的影响，检测结果如下：
孵育温度 孵育时间 荧光强度
4度 480分钟 6724
25度 60分钟 6300
40度 10分钟 5658
实施例3比较检测中的碳氮荧光量子点的浓度，A液和B液的加液量
基于实施例1，在碳氮荧光量子点的尺寸为50nm，孵育温度为25度，孵育时间为60分钟的条件下，比较碳氮荧光量子点的浓度，A液和B液的加液量对检测的影响
(1)比较碳氮荧光量子点的浓度
在A液1mL，B液10mL，碳氮荧光量子点尺寸为50nm的条件下，比较碳氮荧光量子点的浓度：50μg/mL、1μg/mL、0.02μg/mL，检测结果如下：
碳氮荧光量子点的浓度 荧光强度
50μg/mL 17300
1μg/mL 6300
0.02μg/mL 4210
(2)比较A液和B液加液量的体积
在碳氮荧光量子点的浓度为1μg/mL，A液:B液(v/v)＝1:10，碳氮荧光量子点尺寸为50nm的条件下，比较反应体积：0.1mL、1mL、10mL，检测结果如下：
反应体积对荧光结果无明显影响，荧光强度在6000-6500之间分布。
实施例4输尿管上皮癌患者的尿液样本检测
使用基于碳氮荧光量子点的肿瘤细胞检测试剂盒，包括A液和B液。其中，A液是C 3N量子点溶液，溶剂是DMSO，C 3N量子点浓度为1μg/mL，C 3N量子点的尺寸为50nm，B液是PBS缓冲液，用于检测输尿管上皮癌患者的尿液样本。
取中段尿，采液量为10mL，保存在50mL离心管中，室温25℃。高速离心尿液样本，离心转速为1500转/分钟，离心时间为20分钟，弃上清，得到尿液样本的沉淀物。向离心管中加入10mL B液，重悬离心管管底尿液样本的沉淀物，再加入1mL A液，混合均匀，在25℃下孵育60分钟。60分钟后高速离心，离心转速为1500转/分钟，离心时间为20分钟，弃上清，加入1mL B液得到重悬液。设置荧光分光光度计的检测条件，激发波长为400nm，发射波长为530nm，对患者尿液样本进行检测。
图4是C 3N量子点捕捉患者尿液样本中肿瘤细胞的明光和荧光对照图。图中虚 线红圈(图4中左边部分的虚线圆圈)中的细胞是患者尿液中的肿瘤细胞，虚线红圈外的细胞是患者尿路脱落的上皮细胞。在绿色荧光蛋白GFP通道下可以明显、清楚地看到，肿瘤细胞显示亮绿色荧光(图4中右边部分亮度较高的部分)，而上皮细胞无荧光。经检测，患者尿液样本的荧光强度为8400。参照图3中肿瘤细胞参考浓度与荧光参考值的对应关系，患者尿液中肿瘤细胞的计数为100～1000个/mL。
在实施例1和4中，采用碳氮荧光量子点捕捉胸水样本和尿液样本中肿瘤细胞的灵敏度如图5所示。从图中可见，采用C 3N量子点捕捉患者尿液样本中肿瘤细胞的灵敏度超过了72％；采用碳氮荧光量子点捕捉患者胸水样本中的肿瘤细胞的灵敏度达到了100％。
以上详细描述了本发明的较佳具体实施例。应当理解，本领域的普通技术无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此，凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案，皆应在由权利要求书所确定的保护范围内。

Claims (10)

  1. 一种基于碳氮荧光量子点的肿瘤细胞检测试剂盒，其特征在于，包括A液和B液；其中，所述A液是碳氮荧光量子点溶液，所述B液是缓冲液；所述碳氮荧光量子点溶液由碳氮荧光量子点和溶剂组成。
  2. 如权利要求1所述的基于碳氮荧光量子点的肿瘤细胞检测试剂盒，其特征在于，所述碳氮荧光量子点溶液中碳氮荧光量子点的浓度为0.01～50μg/mL。
  3. 如权利要求2所述的基于碳氮荧光量子点的肿瘤细胞检测试剂盒，其特征在于，所述碳氮荧光量子点为N掺杂石墨烯量子点、C 3N 4量子点、C 2N量子点或C 3N量子点，所述碳氮荧光量子点的尺寸为1～100nm。
  4. 一种如权利要求1所述的基于碳氮荧光量子点的肿瘤细胞检测试剂盒的使用方法，其特征在于，包括以下步骤：
    步骤一、用穿刺针采集患者的胸水或收集患者尿液样本，保存在无菌的离心管中；
    步骤二、离心所述步骤一中得到的样本，弃上清液，得到所述样本的沉淀物；
    步骤三、加入所述B液，重悬所述步骤二中得到的沉淀物，得到所述沉淀物的重悬液；
    步骤四、加入所述A液，与所述步骤三中得到的重悬液混合均匀，孵育；
    步骤五、离心所述步骤四中孵育后的重悬液，弃上清液，得到所述样本孵育后的沉淀物；
    步骤六、加入所述B液，重悬所述步骤五中孵育后的沉淀物，得混合均匀的重悬液；
    步骤七、使用荧光分光光度计检测，检测所述步骤六中得到的重悬液的荧光强度；
    步骤八、根据所述步骤七中得到的荧光强度，对所述样本进行半定量分析。
  5. 如权利要求4所述的使用方法，其特征在于，所述步骤一中的胸水或尿液样本的体积为1～10mL，所述样本保存的温度为0℃～25℃。
  6. 如权利要求4所述的使用方法，其特征在于，所述步骤二和所述步骤五中离心的转速为1000～2000转/分钟，所述离心的时间为1～60分钟。
  7. 如权利要求4所述的使用方法，其特征在于，所述步骤四中A液的加液量是0.01～1mL。
  8. 如权利要求4所述的使用方法，其特征在于，所述步骤三和所述步骤六中B液的加液量是0.1～10mL。
  9. 如权利要求4所述的使用方法，其特征在于，所述步骤四中孵育的温度是4℃～40℃，所述孵育的时间为10～480分钟。
  10. 如权利要求4所述的使用方法，其特征在于，所述步骤七中待测样本重悬液的激发波长为400～800nm。
PCT/CN2021/141681 2020-10-27 2021-12-27 碳氮荧光量子点肿瘤细胞检测试剂盒及其使用方法 WO2022089672A1 (zh)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN202011164540.5 2020-10-27
CN202011164540 2020-10-27
CN202111227034.0 2021-10-21
CN202111227034.0A CN115184313A (zh) 2020-10-27 2021-10-21 一种碳氮荧光量子点肿瘤细胞检测试剂盒及其使用方法

Publications (1)

Publication Number Publication Date
WO2022089672A1 true WO2022089672A1 (zh) 2022-05-05

Family

ID=81381965

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/141681 WO2022089672A1 (zh) 2020-10-27 2021-12-27 碳氮荧光量子点肿瘤细胞检测试剂盒及其使用方法

Country Status (1)

Country Link
WO (1) WO2022089672A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117110256A (zh) * 2023-05-29 2023-11-24 兰州大学第一医院 一种基于N-GQDs荧光淬灭原理的尿液酪氨酸检测试剂及检测方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103911149A (zh) * 2013-01-07 2014-07-09 中国药科大学 基于离子液体的碳氮量子点的制备方法及其作为荧光探针在药物检测中的应用
US20150361334A1 (en) * 2014-06-16 2015-12-17 Postech Academy-Industry Foundation Process for preparing carbon quantum dots using emulsion
KR20170046063A (ko) * 2015-10-20 2017-04-28 한국과학기술연구원 질소가 도핑된 나노 카본 소재 및 이의 제조 방법
CN108310003A (zh) * 2018-01-22 2018-07-24 中国科学院上海微系统与信息技术研究所 一种用于高效肿瘤靶向治疗的表面修饰c3n量子点的制备方法
CN108557804A (zh) * 2018-05-23 2018-09-21 南京师范大学 一种Gd，N共掺杂碳量子点的制备方法及其产品和应用
CN111012908A (zh) * 2019-12-05 2020-04-17 南京大学深圳研究院 一种卫星状ucgm纳米复合材料及制备方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103911149A (zh) * 2013-01-07 2014-07-09 中国药科大学 基于离子液体的碳氮量子点的制备方法及其作为荧光探针在药物检测中的应用
US20150361334A1 (en) * 2014-06-16 2015-12-17 Postech Academy-Industry Foundation Process for preparing carbon quantum dots using emulsion
KR20170046063A (ko) * 2015-10-20 2017-04-28 한국과학기술연구원 질소가 도핑된 나노 카본 소재 및 이의 제조 방법
CN108310003A (zh) * 2018-01-22 2018-07-24 中国科学院上海微系统与信息技术研究所 一种用于高效肿瘤靶向治疗的表面修饰c3n量子点的制备方法
CN108557804A (zh) * 2018-05-23 2018-09-21 南京师范大学 一种Gd，N共掺杂碳量子点的制备方法及其产品和应用
CN111012908A (zh) * 2019-12-05 2020-04-17 南京大学深圳研究院 一种卫星状ucgm纳米复合材料及制备方法

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DU FENGYI, YUAN JING, ZHANG MIAOMIAO, LI JIANAN, ZHOU ZHOU, LI ZHANG, CAO MULAN, CHEN JUNHUI, ZHANG LIRONG, LIU XIA, GONG AIHUA, X: "Nitrogen-doped carbon dots with heterogeneous multi-layered structures", RSC ADVANCES, vol. 4, no. 71, 1 January 2014 (2014-01-01), pages 37536, XP055927521, DOI: 10.1039/C4RA06818A *
DU, FENGYI ET AL.: "Nitrogen-doped carbon dots as multifunctional fluorescent probes", J NANOPART RES, vol. 16, no. 11, 5 November 2014 (2014-11-05), XP035382688, ISSN: 1388-0764, DOI: 10.1007/s11051-014-2720-8 *
LI JIPENG, YANG SIWEI, DENG YUAN, CHAI PEIWEI, YANG YUCHENG, HE XIAOYU, XIE XIAOMING, KANG ZHENHUI, DING GUQIAO, ZHOU HUIFANG, FAN: "Emancipating Target-Functionalized Carbon Dots from Autophagy Vesicles for a Novel Visualized Tumor Therapy", ADVANCED FUNCTIONAL MATERIALS, WILEY - V C H VERLAG GMBH & CO. KGAA, DE, vol. 28, no. 30, 1 July 2018 (2018-07-01), DE , pages 1800881, XP055927518, ISSN: 1616-301X, DOI: 10.1002/adfm.201800881 *
LI JIPENG, YANG SIWEI, LIU ZEYANG, WANG GANG, HE PENG, WEI WEI, YANG MUYUE, DENG YUAN, GU PING, XIE XIAOMING, KANG ZHENHUI, DING G: "Imaging Cellular Aerobic Glycolysis using Carbon Dots for Early Warning of Tumorigenesis", ADVANCED MATERIALS, VCH PUBLISHERS, DE, vol. 33, no. 1, 1 January 2021 (2021-01-01), DE , pages 2005096, XP055927524, ISSN: 0935-9648, DOI: 10.1002/adma.202005096 *
LI JIPENG: "Experimental Studies on the Application of Graphene Quantum Dots in Retinoblastoma Imaging", JOURNAL OF CLINICAL OPHTHALMOLOGY, vol. 26, no. 4, 31 December 2018 (2018-12-31), pages 368 - 372, XP055927516 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117110256A (zh) * 2023-05-29 2023-11-24 兰州大学第一医院 一种基于N-GQDs荧光淬灭原理的尿液酪氨酸检测试剂及检测方法
CN117110256B (zh) * 2023-05-29 2024-04-19 兰州大学第一医院 一种基于N-GQDs荧光淬灭原理的尿液酪氨酸检测试剂及检测方法

Similar Documents

Publication Publication Date Title
CA2516795C (en) Circulating tumor cells (ctc's): early assessment of time to progression,_survival and response to therapy in metastatic cancer patients
CA2600225C (en) A method for predicting progression free and overall survival at each follow-up time point during therapy of metastatic breast cancer patients using circulating tumor cells
US20090061456A1 (en) Method for predicting progression free and overall survival at each follow-up time point during therapy of metastatic breast cancer patients using circulating tumor cells
JP6485759B2 (ja) 末梢循環腫瘍細胞単位の悪性度の検出方法及びそのキット
CN112666062B (zh) 流式细胞术在体液细胞免疫分析中联合检测的方法
WO2022089672A1 (zh) 碳氮荧光量子点肿瘤细胞检测试剂盒及其使用方法
CN110702589A (zh) 一种流式细胞仪检测外泌体的方法
Chudasama et al. Inertia based microfluidic capture and characterisation of circulating tumour cells for the diagnosis of lung cancer
Zhai et al. Diagnostic accuracy of tumor markers for malignant pleural effusion: a derivation and validation study
Kang et al. A handheld device for potential point-of-care screening of cancer
Wu et al. The diagnostic ability of high‐fluorescent cells combined with carcinoembryonic antigen for malignant pleural effusion
Giovanella et al. Thyroglobulin measurement on fine‐needle washout fluids: influence of sample collection methods
Muto et al. Isomorphic red blood cells using automated urine flow cytometry is a reliable method in diagnosis of bladder cancer
CN109975548B (zh) IgG4检测试剂在制备结直肠癌诊断剂方面的应用
CN112798567B (zh) 一种基于吖啶橙和碳点的比率荧光用于miRNA体外检测的方法
CN115561468A (zh) 一种评估患有肿瘤或特定肿瘤风险的方法
CN115261476A (zh) 筛选血清外泌体LncRNA HULC作为肝癌早期标记物的方法及其制备试剂盒的用途
CN110527726A (zh) 用于非小细胞肺癌检测及分期判断的外泌体检测装置及应用
CN115184313A (zh) 一种碳氮荧光量子点肿瘤细胞检测试剂盒及其使用方法
Romero et al. Diagnosis of intra-amniotic infection: the acridine orange stain
CN112462065A (zh) 一种用于检测实体肿瘤的抗体组合物、试剂盒及检测方法
Liu et al. Evaluation of acridine orange florescence in exfoliative urinary cytology for diagnosing bladder carcinoma
CN111518915A (zh) 用于局限性前列腺癌临床风险预测的标志物和试剂盒
Tian et al. Construction of high sensitivity electrochemiluminescence sensor and its application in nt-probnp detection
CN109777409B (zh) 一种长余辉近红外碳基荧光纳米材料及其制备方法和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21885398

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21885398

Country of ref document: EP

Kind code of ref document: A1