CN112798377A - 一种荧光淬灭恢复剂及其应用 - Google Patents

一种荧光淬灭恢复剂及其应用 Download PDF

Info

Publication number
CN112798377A
CN112798377A CN202110125784.0A CN202110125784A CN112798377A CN 112798377 A CN112798377 A CN 112798377A CN 202110125784 A CN202110125784 A CN 202110125784A CN 112798377 A CN112798377 A CN 112798377A
Authority
CN
China
Prior art keywords
fluorescence
fluorescence quenching
parts
tissue slice
sample
Prior art date
Legal status (The legal status 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 status listed.)
Granted
Application number
CN202110125784.0A
Other languages
English (en)
Other versions
CN112798377B (zh
Inventor
姜云瀚
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
West China Hospital of Sichuan University
Original Assignee
West China Hospital of Sichuan University
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
Application filed by West China Hospital of Sichuan University filed Critical West China Hospital of Sichuan University
Priority to CN202110125784.0A priority Critical patent/CN112798377B/zh
Publication of CN112798377A publication Critical patent/CN112798377A/zh
Application granted granted Critical
Publication of CN112798377B publication Critical patent/CN112798377B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/34—Purifying; Cleaning
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N2001/302—Stain compositions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6432—Quenching
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P10/00—Technologies related to metal processing
    • Y02P10/20—Recycling

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Optics & Photonics (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

本发明公开了一种荧光淬灭恢复剂及其应用，涉及免疫分析和生物技术应用领域，按体积份数计，由下列组分组成：35‑45份无水乙醇，55‑65份PBS溶液，荧光淬灭恢复剂的应用，包括如下步骤：A1：使用PBS溶液清洗荧光淬灭的组织切片或细胞样本，清洗次数为1‑2次，每次5min；A2：在组织切片或细胞样本上滴加所述的荧光淬灭恢复剂，室温孵育10min；A3：用PBS溶液清洗组织切片或细胞样本3次，每次5min，得到荧光淬灭恢复后的组织切片或细胞样本。可在荧光淬灭的免疫荧光样本上重新染色，保证重新染色免疫荧光样本的荧光能很好的显示，能增加免疫荧光实验的容错率，节约样本数量，节省实验时间。

Description

一种荧光淬灭恢复剂及其应用
技术领域
本发明涉及免疫分析和生物技术应用领域，具体涉及一种荧光淬灭恢复剂及其应用。
背景技术
免疫荧光常常是一次性的，即一次实验后马上用荧光显微镜或者激光共聚焦观察。但免疫荧光孵育荧光二抗后如果操作失误，例如，照射白光或者其他原因引起荧光淬灭，现有技术只能通过重新实验来弥补失误。如果实验样品珍贵，例如，少见疾病的组织样本或者需要长时间造模得到的实验样本，常常没有多余样本用于犯错。同时，对于珍贵的样本，想用免疫荧光检测多个指标，而现有免疫荧光技术最多只能检测五种颜色，还要受限于检测仪器的检测通道个数，这就导致需要的样本量增加，延长实验时间。
发明内容
本发明针对现有技术存在的问题，提供了一种荧光淬灭恢复剂及其应用，可在荧光淬灭的免疫荧光样本上重新染色，保证重新染色免疫荧光样本的荧光能很好的显示，能增加免疫荧光实验的容错率，节约样本数量，节省实验时间。
本发明通过下述技术方案实现：一种荧光淬灭恢复剂，按体积份数计，由下列组分组成：35-45份无水乙醇，55-65份PBS溶液。
进一步，根据权利要求1所述的一种荧光淬灭恢复剂，按体积份数计，由下列组分组成：40份无水乙醇，60份PBS溶液。
一种荧光淬灭恢复剂的应用，包括如下步骤：
A1：使用PBS溶液清洗荧光淬灭的组织切片或细胞样本，清洗次数为1-2次，每次5min；
A2：在组织切片或细胞样本上滴加所述的荧光淬灭恢复剂，室温孵育10min；
A3：用PBS溶液清洗组织切片或细胞样本3次，每次5min，得到荧光淬灭恢复后的组织切片或细胞样本。
上述至少包括以下优点：
1、本发明一种荧光淬灭恢复剂及其应用，可在荧光淬灭的免疫荧光样本上重新染色，保证重新染色免疫荧光样本的荧光能很好的显示，能增加免疫荧光实验的容错率，节约样本数量，节省实验时间；
2、本发明一种荧光淬灭恢复剂及其应用，对于一些样本需要二次拍照和检测的样本，荧光较弱，可通过荧光淬灭恢复剂的应用获得荧光增强的效果。
附图说明
此处所说明的附图用来提供对本发明实施例的进一步理解，构成本申请的一部分，并不构成对本发明实施例的限定。在附图中：
图1为荧光淬灭恢复剂的使用效果图；
图2为AF488组荧光染料的样本使用荧光淬灭恢复剂后效果图；
图3为FITC组荧光染料的样本使用荧光淬灭恢复剂后效果图；
图4为AF555组荧光染料的样本使用荧光淬灭恢复剂后效果图；
图5为TRITC组荧光染料的样本使用荧光淬灭恢复剂后效果图；
图6为CY3组荧光染料的样本使用荧光淬灭恢复剂后效果图；
图7为AF647组荧光染料的样本使用荧光淬灭恢复剂后效果图；
图8为DAPI组荧光染料的样本使用荧光淬灭恢复剂后效果图；
图9为Hoechst组荧光染料的样本使用荧光淬灭恢复剂后效果图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白，下面结合实施例和附图，对本发明作进一步的详细说明，本发明的示意性实施方式及其说明仅用于解释本发明，并不作为对本发明的限定。
实施例
在一个实施例中，一种荧光淬灭恢复剂，按体积份数计，由下列组分组成：35-45份无水乙醇，55-65份PBS溶液。
在一个实施例中，一种荧光淬灭恢复剂，按体积份数计，由下列组分组成：40份无水乙醇，60份PBS溶液。
在一个实施例中，一种荧光淬灭恢复剂，按体积份数计，由下列组分组成：35份无水乙醇，55份PBS溶液。
在一个实施例中，一种荧光淬灭恢复剂，按体积份数计，由下列组分组成：45份无水乙醇，65份PBS溶液。
在一个实施例中，将无水乙醇和PBS溶液充分混合制得荧光淬灭恢复剂。
在一个实施例中，一种荧光淬灭恢复剂的应用，包括如下步骤：
A1：使用PBS溶液清洗荧光淬灭的组织切片或细胞样本，清洗次数为1-2次，每次5min；
A2：在组织切片或细胞样本上滴加所述的荧光淬灭恢复剂，室温孵育10min；
A3：用PBS溶液清洗组织切片或细胞样本3次，每次5min，得到荧光淬灭恢复后的组织切片或细胞样本。
实验例
荧光淬灭恢复效果实验：如图1所示，图1（a）为原始样本，原始样本为免疫荧光孵育荧光二抗后的样本，将图1（a）分成两组，分别用紫外线照射2小时进行处理，处理后并分别滴加PBS缓冲液和本发明荧光淬灭恢复剂，图1（b）即为滴加PBS缓冲液的对照组，图1（c）为滴加本发明荧光淬灭恢复剂的处理组，处理组应用荧光淬灭恢复剂的方法如实施例所述。由图可以看出，本发明荧光淬灭恢复剂能够对荧光淬灭的样本进行有效恢复。
不同组荧光染料的样本恢复效果实验：根据荧光染料的品牌不同，设立7个小组，分别为AF488组、FITC组、AF555组、TRITC组、CY3组、AF647组、DAPI组和Hoechst组，如图2-9，七个小组样本为免疫荧光孵育荧光二抗后的样本，淬灭后用本发明荧光淬灭恢复剂进行处理，处理方法如实施例所述，处理效果如图2-9所示。由图可知，AF488组、FITC组、AF555组、TRITC组、CY3组、AF647组淬灭恢复效果明显，DAPI组和Hoechst组淬灭恢复效果稍弱；恢复后的荧光强度达到最初的荧光强度的50%-80%左右；恢复后的荧光强度相比淬灭后的荧光强度能够提高2-5倍。
以上所述的具体实施方式，对本发明的目的、技术方案和有益效果进行了进一步详细说明，所应理解的是，以上所述仅为本发明的具体实施方式而已，并不用于限定本发明的保护范围，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (3)

1.一种荧光淬灭恢复剂，其特征在于，按体积份数计，由下列组分组成：35-45份无水乙醇，55-65份PBS溶液。
2.根据权利要求1所述的一种荧光淬灭恢复剂，其特征在于，按体积份数计，由下列组分组成：40份无水乙醇，60份PBS溶液。
3.一种荧光淬灭恢复剂的应用，其特征在于，包括如下步骤：
A1：使用PBS溶液清洗荧光淬灭的组织切片或细胞样本，清洗次数为1-2次，每次5min；
A2：在组织切片或细胞样本上滴加如权利要求1所述的荧光淬灭恢复剂，室温孵育10min；
A3：用PBS溶液清洗组织切片或细胞样本3次，每次5min，得到荧光淬灭恢复后的组织切片或细胞样本。
CN202110125784.0A 2021-01-29 2021-01-29 一种荧光淬灭恢复剂及其应用 Active CN112798377B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110125784.0A CN112798377B (zh) 2021-01-29 2021-01-29 一种荧光淬灭恢复剂及其应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110125784.0A CN112798377B (zh) 2021-01-29 2021-01-29 一种荧光淬灭恢复剂及其应用

Publications (2)

Publication Number Publication Date
CN112798377A true CN112798377A (zh) 2021-05-14
CN112798377B CN112798377B (zh) 2023-03-17

Family

ID=75812828

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110125784.0A Active CN112798377B (zh) 2021-01-29 2021-01-29 一种荧光淬灭恢复剂及其应用

Country Status (1)

Country Link
CN (1) CN112798377B (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115112656A (zh) * 2022-07-08 2022-09-27 四川大学华西医院 一种结合免疫荧光染色的黏膜淋巴管整体成像的方法
CN116990101A (zh) * 2023-09-27 2023-11-03 四川大学华西医院 一种易掉片组织的预处理方法及其多重免疫荧光染色方法

Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101639441A (zh) * 2008-08-01 2010-02-03 中国科学院海洋研究所 一种用于海水双壳类减数分裂器的免疫荧光显微观察方法
CN101646458A (zh) * 2007-02-01 2010-02-10 健泰科生物技术公司 使用血管发生抑制剂的联合疗法
CN101802013A (zh) * 2007-07-16 2010-08-11 健泰科生物技术公司 人源化抗cd79b抗体和免疫偶联物及使用方法
WO2011091086A1 (en) * 2010-01-19 2011-07-28 Sirigen Inc. Novel reagents for directed biomarker signal amplification
CN105062465A (zh) * 2015-07-31 2015-11-18 山东大学 一类环境敏感型的α1-肾上腺素能受体近红外荧光配体及其应用
CN107727844A (zh) * 2017-11-18 2018-02-23 安徽师范大学 一种荧光免疫定量检测mfap纳米药物载体的方法
CN109298174A (zh) * 2018-09-26 2019-02-01 姜云瀚 一种多色免疫荧光标记方法和成像方法
CN110179981A (zh) * 2019-06-18 2019-08-30 四川大学华西医院 一种线性-树状给药系统及其制备方法和用途
CN110632292A (zh) * 2019-06-12 2019-12-31 江苏莱尔生物医药科技有限公司 一种检测pd-l1和cd8抗原的免疫荧光试剂盒及应用方法
CN111811909A (zh) * 2020-07-01 2020-10-23 北京基谱生物科技有限公司 一种组织自发荧光淬灭剂的制备及应用
CN112179879A (zh) * 2019-07-02 2021-01-05 南京工业大学 一种左多巴纳米颗粒的制备方法及其生物传感应用

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101646458A (zh) * 2007-02-01 2010-02-10 健泰科生物技术公司 使用血管发生抑制剂的联合疗法
CN101802013A (zh) * 2007-07-16 2010-08-11 健泰科生物技术公司 人源化抗cd79b抗体和免疫偶联物及使用方法
CN101639441A (zh) * 2008-08-01 2010-02-03 中国科学院海洋研究所 一种用于海水双壳类减数分裂器的免疫荧光显微观察方法
WO2011091086A1 (en) * 2010-01-19 2011-07-28 Sirigen Inc. Novel reagents for directed biomarker signal amplification
CN105062465A (zh) * 2015-07-31 2015-11-18 山东大学 一类环境敏感型的α1-肾上腺素能受体近红外荧光配体及其应用
CN107727844A (zh) * 2017-11-18 2018-02-23 安徽师范大学 一种荧光免疫定量检测mfap纳米药物载体的方法
CN109298174A (zh) * 2018-09-26 2019-02-01 姜云瀚 一种多色免疫荧光标记方法和成像方法
CN110632292A (zh) * 2019-06-12 2019-12-31 江苏莱尔生物医药科技有限公司 一种检测pd-l1和cd8抗原的免疫荧光试剂盒及应用方法
CN110179981A (zh) * 2019-06-18 2019-08-30 四川大学华西医院 一种线性-树状给药系统及其制备方法和用途
CN112179879A (zh) * 2019-07-02 2021-01-05 南京工业大学 一种左多巴纳米颗粒的制备方法及其生物传感应用
CN111811909A (zh) * 2020-07-01 2020-10-23 北京基谱生物科技有限公司 一种组织自发荧光淬灭剂的制备及应用

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
KIM IJ ET AL: "Spectroscopic Analysis of Fe Ion-Induced Fluorescence Quenching of the Green Fluorescent Protein ZsGreen" *
ZHANG LD ET AL: "Selective and sensitive detection and detoxification of Pd2+ in living cells with a water-soluble fluorescent probe" *
全国卫生专业技术资格考试专家委员会: "《卫生专业技术资格考试指导 临床医学检验（上）》", 28 February 2004 *
杨启文等: "临床微生物实验室真菌检测能力建设基本要求专家共识" *
温书恒等: "小鼠骨骼肌挫伤修复过程中周细胞数量变化及其与损伤时间相关性" *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115112656A (zh) * 2022-07-08 2022-09-27 四川大学华西医院 一种结合免疫荧光染色的黏膜淋巴管整体成像的方法
CN116990101A (zh) * 2023-09-27 2023-11-03 四川大学华西医院 一种易掉片组织的预处理方法及其多重免疫荧光染色方法
CN116990101B (zh) * 2023-09-27 2023-12-15 四川大学华西医院 一种易掉片组织的预处理方法及其多重免疫荧光染色方法

Also Published As

Publication number Publication date
CN112798377B (zh) 2023-03-17

Similar Documents

Publication Publication Date Title
CN112798377B (zh) 一种荧光淬灭恢复剂及其应用
Sun et al. A whole-brain map of long-range inputs to GABAergic interneurons in the mouse medial prefrontal cortex
Fuzik et al. Integration of electrophysiological recordings with single-cell RNA-seq data identifies neuronal subtypes
Yuan et al. PLD3 affects axonal spheroids and network defects in Alzheimer’s disease
Rey et al. Mitochondrial RNA granules are fluid condensates positioned by membrane dynamics
Strettoi et al. The number of unidentified amacrine cells in the mammalian retina
Rocco et al. Markedly lower glutamic acid decarboxylase 67 protein levels in a subset of boutons in schizophrenia
Sung et al. Simplified three-dimensional tissue clearing and incorporation of colorimetric phenotyping
Silvestri et al. Universal autofocus for quantitative volumetric microscopy of whole mouse brains
WO2016108359A1 (ko) 조직 투명화 방법
Wang et al. Chemical sectioning fluorescence tomography: high-throughput, high-contrast, multicolor, whole-brain imaging at subcellular resolution
CN110308031A (zh) 一种稳定的真菌荧光染色液
CN104155160A (zh) 一种不含苯的组织脱蜡透明剂
Yu et al. Development of the mouse and human cochlea at single cell resolution
Zheng et al. Review of micro-optical sectioning tomography (MOST): technology and applications for whole-brain optical imaging
Fish et al. Laminar distribution of subsets of GABAergic axon terminals in human prefrontal cortex
Heiple et al. pH changes in pinosomes and phagosomes in the ameba, Chaos carolinensis.
Zhang et al. Use of TAI-FISH to visualize neural ensembles activated by multiple stimuli
Tehrani et al. Two-photon deep-tissue spatially resolved mitochondrial imaging using membrane potential fluorescence fluctuations
Gurdita et al. InVision: An optimized tissue clearing approach for three-dimensional imaging and analysis of intact rodent eyes
Currens et al. Evaluation and optimization of PolyJet 3D-printed materials for cell culture studies
Wu et al. Age related extracellular matrix and interstitial cell phenotype in pulmonary valves
Sicherman et al. Estimating and correcting for off-target cellular contamination in brain cell type specific RNA-seq data
Constantinople et al. Quantitative analysis of neurons with Kv3 potassium channel subunits, Kv3. 1b and Kv3. 2, in macaque primary visual cortex
Woo et al. A modified magnified analysis of proteome (MAP) method for super-resolution cell imaging that retains fluorescence

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant