量子点荧光探针的制备及其在细胞和活体成像中的应用
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摘要
量子点(quantum dots, QDs)是能够激发出荧光的纳米晶体,它们具有独特的光学特性：荧光波长可通过改变粒径大小和组成进行调节,荧光强,光稳定性好,激发光谱广和发射荧光光谱窄等。自从1998年量子点在生物学上的应用首次报道以来,量子点已在生物分子、细胞及活体荧光成像,生物传感,肿瘤标志物检测等生物医学研究领域广泛应用。
本文研究了Au掺杂可见-近红外量子点的光学性质及细胞毒性,量子点荧光探针在细胞及活体成像中的应用。利用量子点荧光探针进行肿瘤靶向多光谱成像,成功地进行了多光谱信号分离,实现了多重肿瘤标志物同时进行活体检测。本文主要研究内容如下：
首次将Au掺杂到半导体荧光量子点。采用谷胱甘肽和半胱氨酸作稳定剂,水相法合成Au:CdTe量子点。用所合成的量子点作探针对活细胞进行荧光成像,它们被细胞摄取进入细胞质,并长期非损伤性标记活细胞。所合成的Au:CdTe量子点具有较好的光稳定性、细胞亲和性,细胞毒性较小,适合活细胞长期无损伤标记和荧光成像。
制备了生物发光能量共振转移量子点偶联物,成功应用于小动物活体成像。在大肠杆菌表达和纯化了海肾荧光素酶(RLuc8),获得较好的酶活性。将纯化的RLuc8与RGD肽修饰的水溶性CdTe量子点连接,制备了自发光RGD-QD-RLuc8偶联物,并成功地应用于小鼠活体肿瘤靶向成像。证明所制备的生物发光RGD-QD-RLuc8偶联物适合用作肿瘤标记与活体成像的生物探针。
建立了“一步合成”量子点-生物靶向分子偶联物的方法,并将制备的偶联物成功地应用于活体肿瘤靶向成像。采用Cys-RGD肽和半胱氨酸做稳定剂,在水相中直接合成具有RGD肽连接的近红外水溶性CdHgTe量子点(RGD-CdHgTe),用PEG修饰RGD-CdHgTe量子点,并将近红外PEG-QD-RGD (770nm)成功地应用于活体肿瘤靶向成像。表明这种“一步合成”的量子点-RGD肽偶联物具有较好的肿瘤靶向性,并且在活体内具有较好的信号强度,适合活体成像,在活体成像及肿瘤检测方面具有应用潜力。
首次合成近红外Au:CdHgTe量子点,并成功地用于活体肿瘤靶向多光谱荧光成像。对所合成的量子点进行了表征,并测定了光稳定性、组织穿透度及细胞毒性等,结果表明所合成的量子点具有增强的荧光,较好的光漂白抗性,较低的细胞毒性,生物组织穿透能力强等优点。用所合成的量子点分别与RGD肽、抗CEACAM1抗体、抗EGFR抗体连接,制备了Au:CdHgTe量子点-生物分子偶联物荧光探针,进一步,同时利用三种荧光探针(QD800-RGD, QD820-anti-CEACAM1, QD840-anti-EGFR)进行活体肿瘤靶向多光谱荧光成像,对标记肿瘤标志物的荧光信号进行了光谱分离,实现了多重肿瘤标志物同时检测。结果表明所合成的Au:CdHgTe量子点适合作为活体多光谱荧光成像的荧光探针,有望成为肿瘤标志物的联合检测的有力工具,促进肿瘤早期诊断的准确性。
Quantum dots (QDs) are fluorescent nanocrystals which characterized unique optical properties such as size-and composition-dependent tunable emission spectra, high levels of brightness and photostability, broad excitation spectra and narrow emission bands. Since the first reports on the biological application of quantum dots in1998, they have been extensively applied to molecular, cellular, in vivo imaging, bio-analytical assays, and biosensing.
In this paper, the optical properties and cytotoxicity of Au-doped visible-near-infrared quantum dots, noninvasive living cell imaging and in vivo imaging using QDs fluorescent probes, were researched. The tumor targeting multispectral fluorescence imaging was performed using QDs fluorescent probes, the fluorescent signals were successfully unmixed, and the in vivo simultinously detection of multiple tumor markers was achieved. The detail content is as follows:
Gold was firstly doped into semiconductor fluorescent quantum dots. The gold-doped cadmium telluride (Au:CdTe) quantum dots were synthesized by aqueous solution route using L-glutathione and L-cysteine as stabilizers. As-prepared Au:CdTe NCs were used as probes for living cells fluorescence imaging. They were endocytic uptaken by cells and long-term noninvasive labeled the cells. The Au:CdTe quantum dots exhibited improved photostability, higher cellular affinity, and lower cytotoxicity, and were suitable for long-term noninvasive labeling and fluorescence imaging living cells.
Bioluminescence resonance energy transfer quantum dot conjugates were synthesized and used for small animal in vivo imaging. The Renilla reniformis luciferase (RLuc8) protein was expressed in BL21bacterial cytoplasm. The purified RLuc8was congjugated to RGD (arginine-glycine-aspartic acid containing) peptides capped CdTe quantum dots, and the RGD-QD-Rluc8self-illuminating quantum dots successfully used for mice in vivo tumor-targeted imaging, indicating that as prepared RGD-QD-Rluc8self-illuminating quantum dots could used as bioprobes for in vivo tumor labeling and fluorescence imaging.
The "one step synthesis" of quantum dots bioconjugates was developed. The RGD peptides capped, water-soluble near infrared gold-doped CdHgTe quantum dots were directly synthesized by aqueous solution route using Cys-RGD peptides and L-cysteine as stabilizers. The polyethylene glycol (PEG) was congjugated to the RGD-CdHgTe QDs. The tumor-targeting fluorescence imaging using PEG-QD-RGD congjugates as probes obtained idea efficiency, indicating that as prepared near infrared PEG-QD-RGD congjugates, which with good fluorescent signal in vivo, had great potential in tumor detection an in vivo imaging applications.
Near infrared gold-doped CdHgTe quantum dots were firstly synthesized and used for in vivo tumor-targeted multispectral fluorescence imagin. As prepared Au:CdHgTe QDs were characterized, and their photostability, cytotoxicity, and tissue penetration were tested. The results indicated that Au:CdHgTe QDs have improved photoluminescence, enhanced photostability againsting photobleaching, lower cytotoxicity, and deeper tissue penetration. Three Au:CdHgTe quantum dots were conjugated to RGD peptides, anti-epidermal growth factor receptor (EGFR) monoclonal antibody (MAb), and anti-carcinoembryonic antigen-related cell adhesion molecule1(CEACAM1) MAb respectively. Three bioconjugates (QD800-RGD, QD820-anti-CEACAM1and QD840-anti-EGFR) were successfully used as probes for in vivo tumor targeted multispectral fluorescence imaging; the fluorescent signals labeling tumor markers were spectrally unmixed, and the in vivo simultinously detection of multiple tumor markers was achieved. The results indicated that Au:CdHgTe QDs bioconjugates could be used as fluorescence probes for tumor targeted multispectral fluorescence imaging, and could potentially serve as a powerful tool for in vivo conjoined detection of multiple tumor markers, leading to improved accuracy of diagnosis of early stage cancer.
本文研究了Au掺杂可见-近红外量子点的光学性质及细胞毒性,量子点荧光探针在细胞及活体成像中的应用。利用量子点荧光探针进行肿瘤靶向多光谱成像,成功地进行了多光谱信号分离,实现了多重肿瘤标志物同时进行活体检测。本文主要研究内容如下：
首次将Au掺杂到半导体荧光量子点。采用谷胱甘肽和半胱氨酸作稳定剂,水相法合成Au:CdTe量子点。用所合成的量子点作探针对活细胞进行荧光成像,它们被细胞摄取进入细胞质,并长期非损伤性标记活细胞。所合成的Au:CdTe量子点具有较好的光稳定性、细胞亲和性,细胞毒性较小,适合活细胞长期无损伤标记和荧光成像。
制备了生物发光能量共振转移量子点偶联物,成功应用于小动物活体成像。在大肠杆菌表达和纯化了海肾荧光素酶(RLuc8),获得较好的酶活性。将纯化的RLuc8与RGD肽修饰的水溶性CdTe量子点连接,制备了自发光RGD-QD-RLuc8偶联物,并成功地应用于小鼠活体肿瘤靶向成像。证明所制备的生物发光RGD-QD-RLuc8偶联物适合用作肿瘤标记与活体成像的生物探针。
建立了“一步合成”量子点-生物靶向分子偶联物的方法,并将制备的偶联物成功地应用于活体肿瘤靶向成像。采用Cys-RGD肽和半胱氨酸做稳定剂,在水相中直接合成具有RGD肽连接的近红外水溶性CdHgTe量子点(RGD-CdHgTe),用PEG修饰RGD-CdHgTe量子点,并将近红外PEG-QD-RGD (770nm)成功地应用于活体肿瘤靶向成像。表明这种“一步合成”的量子点-RGD肽偶联物具有较好的肿瘤靶向性,并且在活体内具有较好的信号强度,适合活体成像,在活体成像及肿瘤检测方面具有应用潜力。
首次合成近红外Au:CdHgTe量子点,并成功地用于活体肿瘤靶向多光谱荧光成像。对所合成的量子点进行了表征,并测定了光稳定性、组织穿透度及细胞毒性等,结果表明所合成的量子点具有增强的荧光,较好的光漂白抗性,较低的细胞毒性,生物组织穿透能力强等优点。用所合成的量子点分别与RGD肽、抗CEACAM1抗体、抗EGFR抗体连接,制备了Au:CdHgTe量子点-生物分子偶联物荧光探针,进一步,同时利用三种荧光探针(QD800-RGD, QD820-anti-CEACAM1, QD840-anti-EGFR)进行活体肿瘤靶向多光谱荧光成像,对标记肿瘤标志物的荧光信号进行了光谱分离,实现了多重肿瘤标志物同时检测。结果表明所合成的Au:CdHgTe量子点适合作为活体多光谱荧光成像的荧光探针,有望成为肿瘤标志物的联合检测的有力工具,促进肿瘤早期诊断的准确性。
Quantum dots (QDs) are fluorescent nanocrystals which characterized unique optical properties such as size-and composition-dependent tunable emission spectra, high levels of brightness and photostability, broad excitation spectra and narrow emission bands. Since the first reports on the biological application of quantum dots in1998, they have been extensively applied to molecular, cellular, in vivo imaging, bio-analytical assays, and biosensing.
In this paper, the optical properties and cytotoxicity of Au-doped visible-near-infrared quantum dots, noninvasive living cell imaging and in vivo imaging using QDs fluorescent probes, were researched. The tumor targeting multispectral fluorescence imaging was performed using QDs fluorescent probes, the fluorescent signals were successfully unmixed, and the in vivo simultinously detection of multiple tumor markers was achieved. The detail content is as follows:
Gold was firstly doped into semiconductor fluorescent quantum dots. The gold-doped cadmium telluride (Au:CdTe) quantum dots were synthesized by aqueous solution route using L-glutathione and L-cysteine as stabilizers. As-prepared Au:CdTe NCs were used as probes for living cells fluorescence imaging. They were endocytic uptaken by cells and long-term noninvasive labeled the cells. The Au:CdTe quantum dots exhibited improved photostability, higher cellular affinity, and lower cytotoxicity, and were suitable for long-term noninvasive labeling and fluorescence imaging living cells.
Bioluminescence resonance energy transfer quantum dot conjugates were synthesized and used for small animal in vivo imaging. The Renilla reniformis luciferase (RLuc8) protein was expressed in BL21bacterial cytoplasm. The purified RLuc8was congjugated to RGD (arginine-glycine-aspartic acid containing) peptides capped CdTe quantum dots, and the RGD-QD-Rluc8self-illuminating quantum dots successfully used for mice in vivo tumor-targeted imaging, indicating that as prepared RGD-QD-Rluc8self-illuminating quantum dots could used as bioprobes for in vivo tumor labeling and fluorescence imaging.
The "one step synthesis" of quantum dots bioconjugates was developed. The RGD peptides capped, water-soluble near infrared gold-doped CdHgTe quantum dots were directly synthesized by aqueous solution route using Cys-RGD peptides and L-cysteine as stabilizers. The polyethylene glycol (PEG) was congjugated to the RGD-CdHgTe QDs. The tumor-targeting fluorescence imaging using PEG-QD-RGD congjugates as probes obtained idea efficiency, indicating that as prepared near infrared PEG-QD-RGD congjugates, which with good fluorescent signal in vivo, had great potential in tumor detection an in vivo imaging applications.
Near infrared gold-doped CdHgTe quantum dots were firstly synthesized and used for in vivo tumor-targeted multispectral fluorescence imagin. As prepared Au:CdHgTe QDs were characterized, and their photostability, cytotoxicity, and tissue penetration were tested. The results indicated that Au:CdHgTe QDs have improved photoluminescence, enhanced photostability againsting photobleaching, lower cytotoxicity, and deeper tissue penetration. Three Au:CdHgTe quantum dots were conjugated to RGD peptides, anti-epidermal growth factor receptor (EGFR) monoclonal antibody (MAb), and anti-carcinoembryonic antigen-related cell adhesion molecule1(CEACAM1) MAb respectively. Three bioconjugates (QD800-RGD, QD820-anti-CEACAM1and QD840-anti-EGFR) were successfully used as probes for in vivo tumor targeted multispectral fluorescence imaging; the fluorescent signals labeling tumor markers were spectrally unmixed, and the in vivo simultinously detection of multiple tumor markers was achieved. The results indicated that Au:CdHgTe QDs bioconjugates could be used as fluorescence probes for tumor targeted multispectral fluorescence imaging, and could potentially serve as a powerful tool for in vivo conjoined detection of multiple tumor markers, leading to improved accuracy of diagnosis of early stage cancer.
引文
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