WO2021203567A1 - 高稳定性强荧光发射的铁离子配位黑磷量子点的制备方法 - Google Patents

高稳定性强荧光发射的铁离子配位黑磷量子点的制备方法 Download PDF

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WO2021203567A1
WO2021203567A1 PCT/CN2020/100533 CN2020100533W WO2021203567A1 WO 2021203567 A1 WO2021203567 A1 WO 2021203567A1 CN 2020100533 W CN2020100533 W CN 2020100533W WO 2021203567 A1 WO2021203567 A1 WO 2021203567A1
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bpqds
water
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金辉
姜晓文
桂日军
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青岛大学
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    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
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    • 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
    • C09K11/70—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing phosphorus
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    • 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
    • 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
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells

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  • the invention belongs to the technical field of preparation of novel fluorescent nanomaterials, and specifically relates to a method for preparing black phosphorus quantum dots based on transition metal iron ion coordination.
  • the prepared iron ion coordination black phosphorus quantum dots have high stability and strong fluorescence emission , Can be used for fluorescence imaging of living cells and small animals in vitro.
  • BP black phosphorus
  • the armchair direction is a stacking layer structure
  • the zigzag direction is a double-layer conformation.
  • BP has an anisotropic layered structure, and its electronic properties can be adjusted by the band gap structure, type and width.
  • BP nanostructures especially two-dimensional BP nanosheets, one-dimensional phosphorene or BP nanoribbons, have been widely reported.
  • researchers used liquid-phase ultrasound to peel off bulk BP crystals and prepared zero-dimensional black phosphorous quantum dots (BPQDs) for the first time.
  • BPQDs have more excellent structural features, such as higher energy band gap, smaller size, larger specific surface area and more edge active sites, giving BPQDs unique and excellent properties and applications.
  • BPQDs have potential applications in important fields such as fluorescence (FL) detection, biological imaging, and nonlinear optics.
  • FL fluorescence
  • BPQDs it is very important to passivate the surface and edges of BP, which can effectively control the luminescence properties of BPQDs.
  • Small-layer BP nanosheets can be prepared by ultrasonic-assisted liquid phase exfoliation, and this method is suitable for preparing BPQDs. As the number of BP stacking layers decreases, BP is more unstable and easily oxidized and degraded in the presence of water and oxygen.
  • Lian Peichao et al. disclosed a method for preparing BPQDs, using BP powder as a negative electrode active material to assemble a battery, and performing constant current discharge. The supernatant was collected to obtain BPQDs (Lian Peichao; Zhang Qian; Mei Yi; Yang Ying; Liu Honghong. A method for preparing black phosphorous quantum dots. National Invention Patent. Publication No. CN109573970A).
  • the present invention discloses a method for preparing a new type of iron ion coordinated black phosphorous quantum dots (Fe@BPQDs) with high stability and strong fluorescence emission.
  • Fe@BPQDs iron ion coordinated black phosphorous quantum dots
  • the novel FL nanomaterials and FL imaging agents have important applications in the field of FL imaging of living cells and small animals in vitro.
  • no domestic and foreign literature and patent reports on the preparation and application of transition metal iron ion coordinated black phosphorus quantum dots with high stability and strong fluorescence emission have been retrieved.
  • the purpose of the present invention is to overcome the shortcomings of the above-mentioned prior art, and design a method for preparing transition metal iron ion coordinated black phosphorous quantum dots with high stability and strong fluorescence emission.
  • the present invention relates to a transition metal iron ion coordinated black phosphorous quantum dot with high stability and strong fluorescence emission, and its preparation method includes the following steps:
  • step (4) the Fe@BPQDs are dispersed in phosphate water (PBS) buffer and left for 1 to 14 days.
  • PBS phosphate water
  • the UV-Vis absorption spectrum and FL emission spectrum of the mixed solution are measured, and the absorbance and FL intensity change with incubation time are calculated.
  • Excite with 365nm ultraviolet light in a dark place record the color change of Fe@BPQDs PBS dispersion FL with a smart phone, and evaluate the colloidal and photochemical stability of Fe@BPQDs.
  • Fe@BPQDs were dispersed in PBS buffer, and the cultured living cells, such as mouse breast cancer 4T1 cells, human cervical adenocarcinoma HeLa cells, and human liver normal L02 cells, were combined with Fe@BPQDs in PBS. Incubate together at 37°C for 0-72h, and perform FL imaging of live cells using a confocal laser fluorescence microscope (CLFM).
  • CLFM confocal laser fluorescence microscope
  • step (4) Fe@BPQDs were dispersed in PBS water buffer, and small aquatic animals such as zebrafish, glass fish, green prawns and star-moon jellyfish were cultured in tap water, and Fe@BPQDs PBS dispersion was added. Carry out artificial breeding. After cultivating for 1 to 14 days, the small animals were taken out of the water, placed on a clean glass slide, excited with 365nm ultraviolet light in a dark place, and recorded FL imaging of live small animals after different breeding time with a smart phone.
  • small aquatic animals such as zebrafish, glass fish, green prawns and star-moon jellyfish were cultured in tap water, and Fe@BPQDs PBS dispersion was added. Carry out artificial breeding. After cultivating for 1 to 14 days, the small animals were taken out of the water, placed on a clean glass slide, excited with 365nm ultraviolet light in a dark place, and recorded FL imaging of live small animals after different breeding time with a smart phone.
  • the effect of the present invention is: a novel black phosphorous quantum dot based on the coordination of transition metal iron ions, namely a preparation method of Fe@BPQDs, is disclosed.
  • the Fe@BPQDs has high stability and strong fluorescence emission, and can be used in living cells in vitro And FL imaging of live small animals.
  • the empty orbitals of transition metal atoms such as Ti, Mn, Fe, Co, Ni, Cu, Ag, etc. can coordinate with the lone pair of BP electrons. After the coordination, BP is occupied by the lone pair of electrons and no longer generates phosphorus-oxygen with oxygen. reaction.
  • a protective layer of metal phosphide M x P such as Co 2 P on the surface of BP effectively improves the stability and optical properties of Fe@BPQDs.
  • the water-soluble thiol ligand binds to Fe x P through Fe-S bond, which is helpful for the functional modification and potential application of Fe@BPQDs.
  • Figure 1 is a schematic diagram of the preparation process and theoretical calculation of iron ion-coordinated black phosphorus quantum dots (Fe@BPQDs);
  • Figure 2 shows the prepared Fe@BPQDs fluorescence excitation spectrum and fluorescence emission spectrum
  • Figure 3 shows the fluorescence emission spectra and ultraviolet-visible absorption spectra measured after the PBS dispersion of Fe@BPQDs is placed for 1-14 days, comparing the relative fluorescence emission peak intensity (a) and the change of the absorption spectrum characteristic peak (b).
  • This embodiment relates to a method for preparing black phosphorous quantum dots (Fe@BPQDs) based on the coordination of transition metal iron ions.
  • the preparation process and theoretical calculation of the Fe@BPQDs are shown in Figure 1.
  • the specific preparation steps are as follows:
  • the ultrasonically treated mixture was transferred to a miniature magnetic high-pressure reactor containing 100 mL of polytetrafluoroethylene lining, heated to 120° C. under the protection of N 2, and continuously stirred and reacted for 8 hours.
  • the reaction product was cooled to room temperature, centrifuged at 3500 rpm for 15 min, and the upper reaction solution was taken, and centrifuged at 12000 rpm for 10 min.
  • the precipitate after centrifugation was repeatedly washed three times with ethanol and double-distilled water, and dried in vacuum to obtain Fe@BPQDs functionalized with surface carboxyl groups.
  • Disperse Fe@BPQDs in PBS buffer for 1 to 14 days measure the ultraviolet-visible absorption spectrum and FL emission spectrum of the mixed solution (as shown in Figure 2), calculate the absorbance and FL intensity changes with incubation time ( As shown in Figure 3). Excite with 365nm ultraviolet light in a dark place, record the color change of the mixed dispersion FL with a smart phone, and evaluate the colloidal and photochemical stability of Fe@BPQDs.
  • the cultured mouse breast cancer 4T1 live cells were incubated with the PBS dispersion of Fe@BPQDs at 37°C for 36 hours, and CLFM was used to perform FL imaging of live cells. Zebrafish were cultured in tap water, and Fe@BPQDs PBS dispersion was added for artificial culture.
  • the zebrafish were taken out of the water and placed on a clean glass slide, excited with 365nm ultraviolet light in a dark place, and recorded FL imaging of live zebrafish after different culturing time with a smart phone.
  • This embodiment relates to a method for preparing black phosphorous quantum dots (Fe@BPQDs) based on the coordination of transition metal iron ions.
  • the preparation process and theoretical calculation of Fe@BPQDs are the same as in Example 1.
  • the specific preparation steps are as follows:
  • the treated homogeneous mixture was transferred to an ultrasonic cleaner, and ultrasonic treatment was performed in a water bath for 8 hours.
  • the ultrasonically treated mixture was transferred to a miniature magnetic high-pressure reactor containing 100 mL of polytetrafluoroethylene lining, heated to 140° C. under the protection of N 2, and continuously stirred and reacted for 10 hours.
  • the reaction product was cooled to room temperature, centrifuged at 3500 rpm for 20 min, and the upper reaction solution was taken and centrifuged at 12000 rpm for 12 min.
  • the precipitate after centrifugation was repeatedly washed 4 times with ethanol and double distilled water, and dried in vacuum to obtain Fe@BPQDs functionalized with surface carboxyl groups.
  • Disperse Fe@BPQDs in a PBS buffer solution for 1 to 14 days measure the ultraviolet-visible absorption spectrum and FL emission spectrum of the mixed solution, and calculate the absorbance and FL intensity changes with incubation time. Excite with 365nm ultraviolet light in a dark place, record the color change of the mixed dispersion FL with a smart phone, and evaluate the colloidal and photochemical stability of Fe@BPQDs.
  • the cultivated cells HeLa live cells of human cervical adenocarcinoma were incubated with Fe@BPQDs PBS dispersion at 37°C for 36 hours, and CLFM was used to perform FL imaging of live cells.
  • the green prawns were cultured in tap water, and the Fe@BPQDs PBS dispersion was added for artificial culture.
  • the green prawns are taken out of the water body, placed on a clean glass slide, excited with 365nm ultraviolet light in a dark place, and the FL imaging of the live green prawns after different cultivation time is recorded with a smart phone.
  • This embodiment relates to a method for preparing black phosphorous quantum dots (Fe@BPQDs) based on the coordination of transition metal iron ions.
  • the preparation process and theoretical calculation of Fe@BPQDs are the same as in Example 1.
  • the specific preparation steps are as follows:
  • the treated homogeneous mixture was transferred to an ultrasonic cleaner, and ultrasonic treatment was performed in a water bath for 10 hours.
  • the ultrasonically treated mixture was transferred into a miniature magnetic high pressure reactor containing 100 mL of polytetrafluoroethylene lining, heated to 150° C. under the protection of N 2, and continuously stirred and reacted for 10 hours.
  • the reaction product was cooled to room temperature, centrifuged at 3500 rpm for 25 min, and the upper reaction solution was taken, and centrifuged at 12000 rpm for 15 min.
  • the precipitate after centrifugation was repeatedly washed 5 times with ethanol and double-distilled water, and dried in vacuum to obtain Fe@BPQDs functionalized with surface carboxyl groups.
  • Disperse Fe@BPQDs in a PBS buffer solution for 1 to 14 days measure the ultraviolet-visible absorption spectrum and FL emission spectrum of the mixed solution, and calculate the absorbance and FL intensity changes with incubation time. Excite with 365nm ultraviolet light in a dark place, record the color change of the mixed dispersion FL with a smart phone, and evaluate the colloidal and photochemical stability of Fe@BPQDs.
  • the cultured human liver normal L02 live cells and the PBS dispersion of Fe@BPQDs were incubated at 37°C for 36 hours, and CLFM was used to perform FL imaging of live cells.
  • the Xingyue jellyfish were cultured in tap water, and the Fe@BPQDs PBS dispersion was added for artificial culture.

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Abstract

本发明公开了高稳定性强荧光发射的铁离子配位黑磷量子点Fe@BPQDs的制备方法，在Fe离子存在下，采用超声辅助溶剂热处理黑磷BP晶体，制备Fe配位的少层Fe@BP纳米片。加入水溶性短链硫醇配体，采用溶剂热处理Fe@BP纳米片,制备硫醇稳定的Fe@BPQDs。由于过渡金属M可通过M–π相互作用自发地吸附在BP表面，钝化BP的孤对电子，使BP在空气和水中更稳定。在BP表面形成金属磷化物M xP，有效提高了Fe@BPQDs的稳定性与光学性能。水溶性硫醇配体通过Fe–S键与Fe xP结合，有助于Fe@BPQDs的功能化改性和潜在应用，尤其是应用于活细胞和活体小动物的荧光成像。

Description

高稳定性强荧光发射的铁离子配位黑磷量子点的制备方法 技术领域：
本发明属于新型荧光纳米材料的制备技术领域，具体涉及一种基于过渡金属铁离子配位黑磷量子点的制备方法，其制备的铁离子配位黑磷量子点具有高稳定性和强荧光发射，可用于体外活细胞和活体小动物的荧光成像。
背景技术：
通过范德华力层间堆垛作用，黑磷(BP)呈现褶皱蜂窝状结构，扶手椅方向为堆垛层结构，锯齿型方向为双层构象。BP具有各向异性的层状结构，其电子性质可通过带隙结构、类型和宽度来调节。近年来，BP纳米结构尤其是二维BP纳米片、一维磷烯或BP纳米带的制备和应用已大量报道。2015年，科研人员采用液相超声法剥离块体BP晶体，首次制备了零维黑磷量子点(BPQDs)。相比其它类型BP结构，BPQDs具备更优异的结构特征，如能带隙更高，尺寸更小，比表面积更大和边缘活性位点更多，赋予了BPQDs独特且优异的性质和应用。
BPQDs在荧光(FL)检测、生物成像和非线性光学等重要领域具备潜在应用。在BPQDs制备过程中，对BP表面和边缘进行钝化至关重要，可有效调控BPQDs发光性质。少层BP纳米片可采用超声辅助液相剥离制备，该方法适用于制备BPQDs。随着BP堆垛层数减少，BP越不稳定，易在水和氧存在下氧化和降解。在光激发下，氧分子在BP表面产生超氧负离子O 2 –，而BP与O 2 –结合引起BP表面氧化成P xO y。P xO y快速与水中氧反应降解为PO 4 3–或PO 3 3–，新暴露BP继续氧化和降解，降解后BP结构与性能快速消失。基于此，在制备高稳定性和强荧光发射BPQDs的过程中，阻断BP氧化和降解是十分必要的。
在先前的文献资料报道中，Lee等以块体BP晶体为起始原料，采用超声辅助溶剂热反应制备了BPQDs，平均直径10nm，荧光量子产率(FLQY)为7.2％。该BPQDs具有激发波长依赖的FL发射，放置10天后其FL下降至初试强度的20％，显示出较低FL稳定性(H.U.Lee,S.Y.Park,S.C.Lee,S.Choi,S.Seo,H.Kim,J.Won,K.Choi,K.S.Kang,H.G.Part,H.S.Kim,H.R.An,K.H.Jeong,Y.C.Lee,J. Lee,Black phosphorus(BP)nanodots for potential biomedical applications.Small,2016,12,214–219.)。廉培超等公开了一种BPQDs的制备方法，将BP粉末作为负极活性材料组装电池，进行恒电流放电，放电过程结束后取出负极片，有机溶剂浸泡洗涤，放入去离子水中超声、离心、收集上清液得到BPQDs(廉培超；张倩；梅毅；杨颖；刘红红.一种黑磷量子点的制备方法.国家发明专利.公开号CN109573970A)。
尽管现有技术已经报道了BPQDs的制备，但BPQDs的胶体和光化学稳定性，以及FLQY普遍较低，限制了在生化分析和生物传感领域的应用。过渡金属原子如Ti,Mn,Fe,Co,Ni,Cu,Ag等的空轨道可与BP孤对电子形成配位，配位后的BP因其孤对电子被占据，不再与氧发生磷-氧反应。基于过渡金属离子(M)配位策略，本发明公开了一种高稳定性和强荧光发射的新型铁离子配位黑磷量子点(Fe@BPQDs)的制备方法，该Fe@BPQDs作为一种新型的FL纳米材料和FL成像剂，在体外活细胞和活体小动物的FL成像领域具有重要的应用。截止目前，尚未检索到具有高稳定性和强荧光发射的过渡金属铁离子配位黑磷量子点的制备及其应用的国内外文献和专利报道。
发明内容：
本发明的目的在于克服上述现有技术存在的不足，设计一种具有高稳定性和强荧光发射的过渡金属铁离子配位黑磷量子点的制备方法。
为实现上述目的，本发明涉及的一种具有高稳定性和强荧光发射的过渡金属铁离子配位黑磷量子点，其制备方法包括以下步骤：
(1)称取避光处密封保存的黑磷块体晶体5～30mg，加入20～60mL氮甲基吡咯烷酮中，磁力搅拌均匀。然后加入新鲜配制的0.5～2.0mol/L硝酸铁水溶液1～5mL，搅拌均匀后加入溶解了5～10mg巯基丙酸水溶液1～5mL，充分搅拌，直至形成均质混合液；
(2)将上述混合液转入超声波细胞粉碎仪中，超声处理3～10分钟，然后冷却10～30分钟，反复执行超声3～10分钟，然后间隔10～30分钟的操作，共计5～10次。将处理后的均质混合液转入超声波清洗器中，水浴超声处理6～12h；
(3)将上述超声处理的混合液转入含有100mL聚四氟乙烯内衬的微型磁力高压反应釜中，在N 2保护下加热至100～160℃，连续搅拌反应6～12h；
(4)冷却反应产物至室温，在3500rpm转速下离心10～30min，取上层反应液，在12000rpm转速下离心5～15min。离心后的沉淀物用乙醇和二次蒸馏水反复冲洗3～6次，真空干燥，得到表面羧基功能化Fe@BPQDs。
步骤(4)中Fe@BPQDs分散在磷酸盐水(PBS)缓冲液中，放置1～14天，测量混合液的紫外-可见吸收光谱和FL发射光谱，计算吸光值和FL强度随孵育时间的改变；在避光处用365nm紫外光激发，用智能手机记录Fe@BPQDs的PBS分散液FL颜色变化，评估Fe@BPQDs的胶体和光化学稳定性。
步骤(4)中Fe@BPQDs分散在PBS缓冲液中，将培育的活细胞如小鼠乳腺癌4T1细胞、人宫颈腺癌HeLa细胞和人肝脏正常L02细胞等与Fe@BPQDs的PBS分散液在37℃下共同孵育0～72h，采用共聚焦激光荧光显微镜(CLFM)执行活细胞的FL成像。
步骤(4)中Fe@BPQDs分散在PBS水缓冲液中，将斑马鱼、玻璃鱼、青虾和星月水母等水生活体小动物分别养殖在自来水中，加入Fe@BPQDs的PBS分散液后进行人工养殖。养殖1～14天后，将小动物从水体中取出，放置洁净玻璃片上，在避光处用365nm紫外光激发，用智能手机记录不同养殖时间后活体小动物的FL成像。
本发明的效果是：公开了一种基于过渡金属铁离子配位的新型黑磷量子点，即Fe@BPQDs的制备方法，该Fe@BPQDs具有高稳定性和强荧光发射，可用于体外活细胞和活体小动物的FL成像。过渡金属原子如Ti,Mn,Fe,Co,Ni,Cu,Ag等的空轨道可与BP孤对电子配位，配位后BP因其孤对电子被占据，不再与氧发生磷-氧反应。在制备高稳定性和强荧光发射BPQDs的过程中，阻断BP氧化和降解是十分必要的。在Fe离子存在下，采用超声辅助溶剂热处理BP晶体，制备Fe配位的少层Fe@BP纳米片。加入水溶性短链硫醇配体，采用溶剂热处理Fe@BP纳米片制备硫醇稳定的Fe@BPQDs。由于过渡金属(M)可通过M–π相互作用自发地吸附在BP表面，以钝化BP的孤对电子，使BP在空气和水中更加稳定。在BP表面形成金属磷化物M xP如Co 2P保护层，有效提高了Fe@BPQDs的稳定性及其光学性能。水溶性硫醇配体(HS-X-COOH)通过Fe–S键与Fe xP结合，这有助于Fe@BPQDs功能化改性和潜在应用。
附图说明：
图1为铁离子配位黑磷量子点(Fe@BPQDs)的制备过程与理论计算示意图；
图2为制备的Fe@BPQDs荧光激发光谱和荧光发射光谱；
图3为Fe@BPQDs的PBS分散液放置1-14天后分别测定的荧光发射光谱和紫外-可见吸收光谱，对比相对荧光发射峰强度(a)和吸收光谱特征峰(b)的改变。
具体实施方式：
下面结合附图并通过具体实施例对本发明进行详细说明。
实施例1：
本实施例涉及的一种基于过渡金属铁离子配位黑磷量子点(Fe@BPQDs)的制备方法，该Fe@BPQDs的制备过程与理论计算如图1所示，具体制备步骤如下：
称取避光处密封保存的黑磷块体晶体10mg，加入30mL氮甲基吡咯烷酮中，磁力搅拌均匀。然后加入新鲜配制的1.0mol/L硝酸铁水溶液2mL，搅拌均匀后加入溶解了5mg巯基丙酸水溶液2mL，充分搅拌，直至形成均质混合液。将此混合液转入超声波细胞粉碎仪中，超声处理4分钟，然后冷却10分钟，反复执行超声4分钟，然后间隔10分钟的操作，共计5次。将处理后的均质混合液转入超声波清洗器中，水浴超声处理6h。将超声处理的混合液转入含有100mL聚四氟乙烯内衬的微型磁力高压反应釜中，在N 2保护下加热至120℃，连续搅拌反应8h。冷却反应产物至室温，在3500rpm转速下离心15min，取上层反应液，在12000rpm转速下离心10min。离心后的沉淀物用乙醇和二次蒸馏水反复冲洗3次，真空干燥，得到表面羧基功能化Fe@BPQDs。
将Fe@BPQDs分散在PBS缓冲液中，放置1～14天，测量混合液的紫外-可见吸收光谱和FL发射光谱(如图2所示)，计算吸光值和FL强度随孵育时间的改变(如图3所示)。在避光处用365nm紫外光激发，用智能手机记录混合分散液FL颜色变化，评估Fe@BPQDs的胶体和光化学稳定性。将培育的小鼠乳腺癌4T1活细胞与Fe@BPQDs的PBS分散液在37℃下共同孵育36h，采用CLFM执行活细胞的FL成像。将斑马鱼养殖在自来水中，加入Fe@BPQDs的PBS分散液后进行人工养殖。养殖1～14天后，将斑马鱼从水体中取出放置洁净玻璃片上，在避光处用365nm紫外光激发，用智能手机记录不同养殖时间后活 斑马鱼的FL成像。
实施例2：
本实施例涉及的一种基于过渡金属铁离子配位黑磷量子点(Fe@BPQDs)的制备方法，该Fe@BPQDs的制备过程与理论计算同实施例1，具体制备步骤如下：
称取避光处密封保存的黑磷块体晶体20mg，加入40mL氮甲基吡咯烷酮中，磁力搅拌均匀。然后加入新鲜配制的1.5mol/L硝酸铁水溶液3mL，搅拌均匀后加入溶解了6mg巯基丙酸水溶液3mL，充分搅拌，直至形成均质混合液。将此混合液转入超声波细胞粉碎仪中，超声处理5分钟，然后冷却15分钟，反复执行超声5分钟，然后间隔15分钟的操作，共计6次。将处理后的均质混合液转入超声波清洗器中，水浴超声处理8h。将超声处理的混合液转入含有100mL聚四氟乙烯内衬的微型磁力高压反应釜中，在N 2保护下加热至140℃，连续搅拌反应10h。冷却反应产物至室温，在3500rpm转速下离心20min，取上层反应液，在12000rpm转速下离心12min。离心后的沉淀物用乙醇和二次蒸馏水反复冲洗4次，真空干燥，得到表面羧基功能化Fe@BPQDs。
将Fe@BPQDs分散在PBS缓冲液中，放置1～14天，测量混合液的紫外-可见吸收光谱和FL发射光谱，计算吸光值和FL强度随孵育时间的改变。在避光处用365nm紫外光激发，用智能手机记录混合分散液FL颜色变化，评估Fe@BPQDs的胶体和光化学稳定性。将培育的细胞人宫颈腺癌HeLa活细胞与Fe@BPQDs的PBS分散液在37℃下共同孵育36h，采用CLFM执行活细胞的FL成像。将青虾养殖在自来水中，加入Fe@BPQDs的PBS分散液后进行人工养殖。养殖1～14天后，将青虾从水体中取出，放置洁净玻璃片上，在避光处用365nm紫外光激发，用智能手机记录不同养殖时间后活青虾的FL成像。
实施例3：
本实施例涉及的一种基于过渡金属铁离子配位黑磷量子点(Fe@BPQDs)的制备方法，该Fe@BPQDs的制备过程与理论计算同实施例1，具体制备步骤如下：
称取避光处密封保存的黑磷块体晶体25mg，加入50mL氮甲基吡咯烷酮中，磁力搅拌均匀。然后加入新鲜配制的1.8mol/L硝酸铁水溶液4mL，搅拌均匀后加入溶解了8mg巯基丙酸水溶液4mL，充分搅拌，直至形成均质混合液。 将此混合液转入超声波细胞粉碎仪中，超声处理8分钟，然后冷却20分钟，反复执行超声8分钟，然后间隔20分钟的操作，共计8次。将处理后的均质混合液转入超声波清洗器中，水浴超声处理10h。将超声处理的混合液转入含有100mL聚四氟乙烯内衬的微型磁力高压反应釜中，在N 2保护下加热至150℃，连续搅拌反应10h。冷却反应产物至室温，在3500rpm转速下离心25min，取上层反应液，在12000rpm转速下离心15min。离心后的沉淀物用乙醇和二次蒸馏水反复冲洗5次，真空干燥，得到表面羧基功能化Fe@BPQDs。
将Fe@BPQDs分散在PBS缓冲液中，放置1～14天，测量混合液的紫外-可见吸收光谱和FL发射光谱，计算吸光值和FL强度随孵育时间的改变。在避光处用365nm紫外光激发，用智能手机记录混合分散液FL颜色变化，评估Fe@BPQDs的胶体和光化学稳定性。将培育的人肝脏正常L02活细胞与Fe@BPQDs的PBS分散液在37℃下共同孵育36h，采用CLFM执行活细胞的FL成像。将星月水母养殖在自来水中，加入Fe@BPQDs的PBS分散液后进行人工养殖。养殖1～14天后，将星月水母从水体中取出，放置洁净玻璃片上，在避光处用365nm紫外光激发，用智能手机记录不同养殖时间后活星月水母的FL成像。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (4)

  1. 高稳定性强荧光发射的铁离子配位黑磷量子点的制备方法，其特征在于，该方法具体包括以下步骤：
    (1)称取避光处密封保存的黑磷块体晶体5～30mg，加入20～60mL氮甲基吡咯烷酮中，磁力搅拌均匀；然后加入新鲜配制的0.5～2.0mol/L硝酸铁水溶液1～5mL，搅拌均匀后加入溶解了5～10mg巯基丙酸水溶液1～5mL，充分搅拌，直至形成均质混合液；
    (2)将上述混合液转入超声波细胞粉碎仪中，超声处理3～10分钟，然后冷却10～30分钟，反复执行超声3～10分钟，然后间隔10～30分钟的操作，共计5～10次；将处理后的均质混合液转入超声波清洗器中，水浴超声处理6～12h；
    (3)将上述超声处理的混合液转入含有100mL聚四氟乙烯内衬的微型磁力高压反应釜中，在N 2保护下加热至100～160℃，连续搅拌反应6～12h；
    (4)冷却反应产物至室温，在3500rpm转速下离心10～30min，取上层反应液，在12000rpm转速下离心5～15min；离心后的沉淀物用乙醇和二次蒸馏水反复冲洗3～6次，真空干燥，得到表面羧基功能化Fe@BPQDs。
  2. 一种如权利要求1所述的制备方法，其特征在于，步骤(4)中Fe@BPQDs分散在磷酸盐水(PBS)缓冲液中，放置1～14天，测量混合液的紫外-可见吸收光谱和FL发射光谱，计算吸光值和FL强度随孵育时间的改变；在避光处用365nm紫外光激发，用智能手机记录Fe@BPQDs的PBS分散液FL颜色变化，评估Fe@BPQDs的胶体和光化学稳定性。
  3. 一种如权利要求1所述的制备方法，其特征在于，步骤(4)中Fe@BPQDs分散在PBS缓冲液中，将培育的活细胞如小鼠乳腺癌4T1细胞、人宫颈腺癌HeLa细胞和人肝脏正常L02细胞等与Fe@BPQDs的PBS分散液在37℃下共同孵育0～72h，采用共聚焦激光荧光显微镜(CLFM)执行活细胞的FL成像。
  4. 一种如权利要求1所述的制备方法，其特征在于，步骤(4)中Fe@BPQDs分散在PBS水缓冲液中，将斑马鱼、玻璃鱼、青虾和星月水母等水生活体小动物分别养殖在自来水中，加入Fe@BPQDs的PBS分散液后进行人工养殖；养殖1～14天后，将小动物从水体中取出，放置洁净玻璃片上，在避光处用365nm紫外光激发，用智能手机记录不同养殖时间后活体小动物的FL成像。
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