硒化物纳米材料的制备及其在生物传感器中的应用
摘要
本文使用水热合成法,制备了Ag_2Se、PbSe与ZnSe三种硒化物纳米粒子。在制备过程中对纳米粒子的表面进行修饰,使其在水体系中具有很好的分散性和生物相容性。使用TEM、SEM、XRD、EDS、XPS、UV-Vis、FT-IR等对产物进行了表征,探讨了它们的形成机理,并对制备的硒化物纳米粒子在生物传感器方面的应用进行了初步探究。
(1)以AgNO_3,KI,PVP形成的配合物[PVP(Ag_mI_n)]～((n-m)-)(n≧m)为前驱体与Na_2SeSO_3在180℃条件下水热反应20小时,制备了单分散Ag_2Se纳米粒子。透射电镜和扫描电镜测试显示制备的Ag_2Se为大米粒状,长约为60-80nm,宽约为30-40nm。配合物[PVP(Ag_mI_n)]～((n-m)-)对控制Ag_2Se晶核的生成速度和生长方向起到了至关重要的作用,使得在高浓度PVP条件下可以制备出尺寸均一、大米粒状的Ag_2Se纳米粒子。通过XPS和FT-IR的检测证实PVP与Ag_2Se纳米粒子间以配位键形式的结合,并表明在Ag_2Se纳米粒子表面有大量PVP存在。利用PVP中C=O与DNA分子中-NH2易于形成氢键,将制备的PVP修饰的Ag_2Se纳米粒子作为DNA探针的标记物用于电化学溶出检测DNA。用该方法对对互补序列、三碱基错配及非互补序列DNA的控制表明,PVP修饰的Ag_2Se对互补的20碱基PEP基因片段具有很好的选择性,其检测的线性范围为1.0×10~(-12)～1.0×10~(-8) mol·L~(-1),检测限为2.3×10~(-13) mol·L~(-1)。
(2)在表面活性剂CTAB存在的条件下,Pb(CH3COO)2与NaHSe在去离子水中于150℃条件下水热反应24小时制备了PbSe纳米粒子。XRD显示制备的PbSe为立方晶型,TEM显示PbSe纳米粒子粒径大约40nm。FT-IR检测证实了CTAB在PbSe纳米粒子表面的存在。PbSe-CTA~+使得PbSe纳米粒子表面带正电性,通过与DNA中的-COO-的负电性的静电作用,将制备的PbSe纳米粒子标记寡核苷酸制成了新型的DNA探针。用该方法对对互补序列、三碱基错配及非互补序列DNA的控制表明,CTAB修饰的PbSe对互补的18碱基CaMV35S基因片段具有很好的选择性,其检测的线性范围为5.0×10-12 mol·L~(-1)到5.0×10~(-7) mol·L~(-1),检测限为6.1×10~(-13) mol·L~(-1)。
(3)以生物分子L-谷氨酸辅助水热法合成了ZnSe纳米粒子。XRD检测显示制备的ZnSe为立方晶型,TEM图像表明制备的ZnSe为尺寸在100纳米左右的球形粒子。制备过程中,生物分子L-谷氨酸,起到了配位剂和生物分子模板的作用。FT-IR检测表明,ZnSe纳米粒子表面吸附有L-谷氨酸,谷氨酸作为ZnSe纳米粒子与DNA分子的连接物,使ZnSe纳米粒子标记于DNA分子上。将ZnSe纳米粒子标记于合成的5'端氨基修饰的寡聚核苷酸片段上,制成具有电化学活性的ZnSe纳米粒子标记DNA探针。测定目标DNA的线性范围为1.0×10~(-11)至1.0×10~(-7) mol/L,检测限为4.7×10~(-12) mol·L~(-1)。
Three kinds of selenide nanoparticals have been synthesized through hydrothermal route. The synthesized nanoparticles are well dispersed and have biology consistent ability as their surfaces are modified in the process of synthesis. The morphology and characteristic were characterized by TEM、SEM、XRD、EDS、XPS、UV-Vis、FT-IR respectively, and the possible growth mechanisms were elucidated based on our experimental results. The application of nanoparticles-based novel electrochemical DNA biosensor was studied and discussed.
(1) Monodispersed silver selenide (Ag_2Se) nanoparticles have been prepared successfully by a hydrothermal reaction of Na2SeSO3 with [PVP(AgmIn)](n-m)- which is formed by AgNO3,KI and PVP. TEM revealed that the nanoparticles are much like husked rice with length of about 60～80 nm and width of about 30～40nm. [PVP(AgmIn)](n-m)- acted an important role in the reaction. The formation rate of Ag_2Se crystal cores and the growth direction could be well controlled as the protection effect of PVP. Husked rice-like Ag_2Se nanoparticles with uniform size were obtained in the condition of high concentration of PVP. The XPS and FT-IR test indicted that there was a great number of PVP modified on the surface of Ag_2Se nanoparticles. The modification effect was not only a physical adsorption but also a chemical coordination. Ag_2Se was bound to the probe DNA via the formation of the hydrogen bond between–C=O of PVP in the modified Ag_2Se and–NH2 in the probe DNA. The proposed method showed a good and distinguishable ability to the three-base mismatch or non-complementary sequences with the complementary sequences. DNA specific-sequence related to PEP promoter gene in the transgenic plants was determined with a detection range from 1.0×10~(-12) to 1.0×10~(-8) mol L~(-1) and a detection limit of 2.3×10~(-13) mol L~(-1)(3σ).
(2) Lead selenide (PbSe) nanoparticles were synthesized by the reaction between Pb(CH3COO)2 and NaHSe under hydrothermal conditions in the presence of CTAB at 150℃for 24h. The X-ray diffraction (XRD) pattern indicated that the product was cubic PbSe. Transmission electron microscopy (TEM) revealed that the nanoparticales are about 40 nm in diameter. The FT-IR test indicted the adsorption of CTAB on the surface of PbSe nanoparticles. The ionization of CTAB produced cation CTA+ on the surface of PbSe nanoparticles and made the nanoparticles with positive charge. As the -COO- of DNA made the DNA with negative charge, the PbSe nanoparticles can adsorbed the negative charged oligonucleotides by electrostatic interaction to form a nanoparticle labeled oligonucleotides probe. The PbSe nanoparticles showed a good distinguishable ability to the two-base mismatch or non-complementary sequences with the complementary sequences. DNA specific-sequence related to CaMV35S promoter gene in the transgenic plants was determined with a detection range from 5.0×10~(-12) to 5.0×10~(-7) mol/L and a detection limit of 6.1×10~(-13) mol/L (3σ).
(3) A simple biomolecule-assisted hydrothermal approach was developed to synthesize zinc selenide (ZnSe) nanoparticles. The X-ray diffraction (XRD) pattern indicated that the as-prepared product was cubic ZnSe. The TEM images showed the size of the ZnSe nanosphere was 100nm in diameter. The L-glutamic acid acted as complexing agent and biomolecule templet to combine with Zn2+ in the process of the reaction. The FT-IR studies of the as-prepared ZnSe provide preliminary proof for confirming the adsorption between ZnSe nanoparticles and glutamic acid. Glutamic acid was the bridging agent between DNA molecule and zinc selenide nanospheres. So the ZnSe nanoshperes could be used for the detection of the DNA hybridization. DNA specific-sequence related to PAT gene in the transgenic plants was determined with a detection range from 1.0×10-11 to 1.0×10~(-7) mol L~(-1) and a detection limit of 4.7×10~(-12) mol L~(-1) (3σ).
(1)以AgNO_3,KI,PVP形成的配合物[PVP(Ag_mI_n)]～((n-m)-)(n≧m)为前驱体与Na_2SeSO_3在180℃条件下水热反应20小时,制备了单分散Ag_2Se纳米粒子。透射电镜和扫描电镜测试显示制备的Ag_2Se为大米粒状,长约为60-80nm,宽约为30-40nm。配合物[PVP(Ag_mI_n)]～((n-m)-)对控制Ag_2Se晶核的生成速度和生长方向起到了至关重要的作用,使得在高浓度PVP条件下可以制备出尺寸均一、大米粒状的Ag_2Se纳米粒子。通过XPS和FT-IR的检测证实PVP与Ag_2Se纳米粒子间以配位键形式的结合,并表明在Ag_2Se纳米粒子表面有大量PVP存在。利用PVP中C=O与DNA分子中-NH2易于形成氢键,将制备的PVP修饰的Ag_2Se纳米粒子作为DNA探针的标记物用于电化学溶出检测DNA。用该方法对对互补序列、三碱基错配及非互补序列DNA的控制表明,PVP修饰的Ag_2Se对互补的20碱基PEP基因片段具有很好的选择性,其检测的线性范围为1.0×10~(-12)～1.0×10~(-8) mol·L~(-1),检测限为2.3×10~(-13) mol·L~(-1)。
(2)在表面活性剂CTAB存在的条件下,Pb(CH3COO)2与NaHSe在去离子水中于150℃条件下水热反应24小时制备了PbSe纳米粒子。XRD显示制备的PbSe为立方晶型,TEM显示PbSe纳米粒子粒径大约40nm。FT-IR检测证实了CTAB在PbSe纳米粒子表面的存在。PbSe-CTA~+使得PbSe纳米粒子表面带正电性,通过与DNA中的-COO-的负电性的静电作用,将制备的PbSe纳米粒子标记寡核苷酸制成了新型的DNA探针。用该方法对对互补序列、三碱基错配及非互补序列DNA的控制表明,CTAB修饰的PbSe对互补的18碱基CaMV35S基因片段具有很好的选择性,其检测的线性范围为5.0×10-12 mol·L~(-1)到5.0×10~(-7) mol·L~(-1),检测限为6.1×10~(-13) mol·L~(-1)。
(3)以生物分子L-谷氨酸辅助水热法合成了ZnSe纳米粒子。XRD检测显示制备的ZnSe为立方晶型,TEM图像表明制备的ZnSe为尺寸在100纳米左右的球形粒子。制备过程中,生物分子L-谷氨酸,起到了配位剂和生物分子模板的作用。FT-IR检测表明,ZnSe纳米粒子表面吸附有L-谷氨酸,谷氨酸作为ZnSe纳米粒子与DNA分子的连接物,使ZnSe纳米粒子标记于DNA分子上。将ZnSe纳米粒子标记于合成的5'端氨基修饰的寡聚核苷酸片段上,制成具有电化学活性的ZnSe纳米粒子标记DNA探针。测定目标DNA的线性范围为1.0×10~(-11)至1.0×10~(-7) mol/L,检测限为4.7×10~(-12) mol·L~(-1)。
Three kinds of selenide nanoparticals have been synthesized through hydrothermal route. The synthesized nanoparticles are well dispersed and have biology consistent ability as their surfaces are modified in the process of synthesis. The morphology and characteristic were characterized by TEM、SEM、XRD、EDS、XPS、UV-Vis、FT-IR respectively, and the possible growth mechanisms were elucidated based on our experimental results. The application of nanoparticles-based novel electrochemical DNA biosensor was studied and discussed.
(1) Monodispersed silver selenide (Ag_2Se) nanoparticles have been prepared successfully by a hydrothermal reaction of Na2SeSO3 with [PVP(AgmIn)](n-m)- which is formed by AgNO3,KI and PVP. TEM revealed that the nanoparticles are much like husked rice with length of about 60～80 nm and width of about 30～40nm. [PVP(AgmIn)](n-m)- acted an important role in the reaction. The formation rate of Ag_2Se crystal cores and the growth direction could be well controlled as the protection effect of PVP. Husked rice-like Ag_2Se nanoparticles with uniform size were obtained in the condition of high concentration of PVP. The XPS and FT-IR test indicted that there was a great number of PVP modified on the surface of Ag_2Se nanoparticles. The modification effect was not only a physical adsorption but also a chemical coordination. Ag_2Se was bound to the probe DNA via the formation of the hydrogen bond between–C=O of PVP in the modified Ag_2Se and–NH2 in the probe DNA. The proposed method showed a good and distinguishable ability to the three-base mismatch or non-complementary sequences with the complementary sequences. DNA specific-sequence related to PEP promoter gene in the transgenic plants was determined with a detection range from 1.0×10~(-12) to 1.0×10~(-8) mol L~(-1) and a detection limit of 2.3×10~(-13) mol L~(-1)(3σ).
(2) Lead selenide (PbSe) nanoparticles were synthesized by the reaction between Pb(CH3COO)2 and NaHSe under hydrothermal conditions in the presence of CTAB at 150℃for 24h. The X-ray diffraction (XRD) pattern indicated that the product was cubic PbSe. Transmission electron microscopy (TEM) revealed that the nanoparticales are about 40 nm in diameter. The FT-IR test indicted the adsorption of CTAB on the surface of PbSe nanoparticles. The ionization of CTAB produced cation CTA+ on the surface of PbSe nanoparticles and made the nanoparticles with positive charge. As the -COO- of DNA made the DNA with negative charge, the PbSe nanoparticles can adsorbed the negative charged oligonucleotides by electrostatic interaction to form a nanoparticle labeled oligonucleotides probe. The PbSe nanoparticles showed a good distinguishable ability to the two-base mismatch or non-complementary sequences with the complementary sequences. DNA specific-sequence related to CaMV35S promoter gene in the transgenic plants was determined with a detection range from 5.0×10~(-12) to 5.0×10~(-7) mol/L and a detection limit of 6.1×10~(-13) mol/L (3σ).
(3) A simple biomolecule-assisted hydrothermal approach was developed to synthesize zinc selenide (ZnSe) nanoparticles. The X-ray diffraction (XRD) pattern indicated that the as-prepared product was cubic ZnSe. The TEM images showed the size of the ZnSe nanosphere was 100nm in diameter. The L-glutamic acid acted as complexing agent and biomolecule templet to combine with Zn2+ in the process of the reaction. The FT-IR studies of the as-prepared ZnSe provide preliminary proof for confirming the adsorption between ZnSe nanoparticles and glutamic acid. Glutamic acid was the bridging agent between DNA molecule and zinc selenide nanospheres. So the ZnSe nanoshperes could be used for the detection of the DNA hybridization. DNA specific-sequence related to PAT gene in the transgenic plants was determined with a detection range from 1.0×10-11 to 1.0×10~(-7) mol L~(-1) and a detection limit of 4.7×10~(-12) mol L~(-1) (3σ).
引文
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