Application in the detection of antigen-antibody molecules
Bentley research group first used LPFG as biosensors, who firstly carried out monomolecular layer amination modification on the LPFG surface and then immobilized antibody through glutaric dialdehyde coupling to detect antigen Human IgG. The detection range was 2-100 μg/mL. After continuous 5 times of use, 85% of initial signal could still be reached. However, the detection limit was 700 μg/L, so the detection sensitivity was low.
Bentley研究组第一次将长周期光纤光栅用作生物传感器, 他们首先在长周期光栅表面进行单分子层氨基化修饰, 然后用戊二醛偶联固定抗体, 进行抗原Human IgG的检测。检测范围为2～100 μg/mL, 在连续使用5次后, 仍能达到初始信号的85%。但由于检测限为700 μg/L, 存在检测灵敏度低的问题。
Kim et al. carried out sputtering of silver-plated reflective film on the LPFG end face to detect antigen-antibody interaction. After encountering a silver-plated film, lights are reflected, and thus interference fringes were generated in the LPFG. When the antibody immobilized on the grating surface was of interaction with antigen, the refractive index of the film will change so as to result in interference fringe shifting. Based on this, antigen was detected. The assembled PAH/PSS film was used in anti-rabbit IgG detection. With anti-rabbit IgG immobilization, resonance wavelength was shifted by 0.55 nm.
Kim等人在长周期光栅端面溅射镀银反射膜, 用于测定抗原抗体的相互作用。当光遇到银金属膜后被反射, 在长周期光纤光栅内产生干涉条纹。固定于光栅表面的抗体与抗原发生作用时, 膜层折射率会发生变化, 引起干涉条纹位置的移动, 以此检测抗原。在组装了PAH/PSS薄膜后, 用于抗兔IgG的检测。随着抗兔IgG的固定, 谐振波长移动了约0.55 nm。
Through coupling of LPFG written in PCF with optical collimating flow cell, He et al. developed an optical fluid refractive index transmission platform used in label-free biosensing and detection of anti-mouse IgG. Such LPFG-PCF system has ultra-high sensitivity to the refractive index variation caused by partial binding in different solution media. Model immunoassay experiment is conducted in the PCF channel through a series of surface modification steps including PPAam hydrochloride monolayer adsorption, immobilization of anti-rat BSP primary MCAB, and interaction of non-specific goat anti-rabbit IgG(H+L) and specific secondary goat anti-mouse IgG(H+L) antibodies. By measuring the shift of LPFG-PCF resonance wavelength, the process of adsorption and binding of these biochemical materials in the LPFG surface has been monitored in site. See Figure 1 for the shift of resonance wavelength of the LPFG-PCF at various surface events. As shown in Fig. 1, remarkable resonance wavelength variation is caused by the adsorption and binding of monolayer materials on the LPFG surface, and the wavelength of biomolecules adsorbed/bonded per nm thickness is about 0.75nm. This indicates that the LPFG-PCF biosensor can be used to detect monolayer sensitive surface binding and has a very large potential in biosensing and evaluation.
Shift of resonance wavelength of the LPFG-PCF at various surface events
He等人将写入光子晶体光纤中的长周期光栅与光学准直的流动池耦合, 开发了一种用于无标记生物传感的光学流体折射率传导平台, 用于抗小鼠IgG的检测。这种长周期光栅-光子晶体光纤体系对不同溶液介质中的局部结合所引起的折射率变化具有极高的灵敏度。通过一系列表面改性步骤, 包括吸附聚烯丙胺盐酸盐单层、固定抗大鼠骨唾液蛋白单克隆一抗以及与非特异性山羊抗兔IgG(H+L)和特异性二抗山羊抗小鼠IgG(H+L)抗体结合的相互作用, 在PCF的通道内进行模型免疫测定实验。通过测量长周期光栅-光子晶体光纤谐振波长的位移变化, 原位监测了这些生化材料在长周期光栅表面的吸附和结合过程, 生化试剂在光子晶体光纤-长周期光栅表面作用时所引起的谐振波长移动情况如图 1所示。从图 1可以看到, 在长周期光栅表面单层材料的吸附和结合产生了显著的共振波长变化, 每纳米厚度吸附/结合的生物分子约为0.75 nm, 这表明该长周期光栅-光子晶体光纤生物传感器可以检测单层敏感的表面结合, 在生物传感与评估方面有很大的潜力。
Coupling of the forward-propagating fiber core basic mode to the high-order cladding mode near the turn-around point is a strategy for enabling LPFG to have good biosensing performance without need for additional coating (except the sensing layer and selective biological layer deposited on the fiber). The turn-around point refers to the point in the matching curve under the specific cladding mode of LPFG, where two resonance peaks of the same cladding mode are merged into a wider resonance peak. The LPFG with resonance peaks near the turn-around point has the highest sensitivity and can achieve high sensitivity detection of biological substances. Chiavaioli et al. discussed the modeling and fabrication of turn-around point LPFG and built a new label-free biosensor based on turn-around point LPFG. The research group deposited Eudragit L100 copolymer onto the LPFG surface, carried out covalent immobilization of immune globulin G(IgG) for LPFG surface functionalization, and then conducted IgG/anti-IgG biological assay along the grating area and dynamic analysis of antibody/antigen interaction. In order to highlight the proposed improved immunosensor, quantitative comparison was conducted between turn-around point LPFG and non-turn-around point LPFG. In addition, the actual feasibility, effectiveness and detection specificity of turn-around point LPFG were verified by detecting complex human serum, and the detection limit in human serum reached 70 μg/L(460 pM).
将前向传输的纤芯基模耦合到靠近其转折点的高阶包层模式是实现无需额外的涂层(沉积在光纤上的感测层和选择性生物层除外)而使长周期光栅具有良好生物传感性能的一种策略。转折点是指在长周期光纤光栅的特定包层模式下的相匹配曲线中的点, 在该模式下, 相同包层模式的两个谐振峰合并成一个更宽的谐振峰。而谐振峰在转折点附近的长周期光纤光栅具有最高的灵敏度, 可以实现对生物物质的高灵敏检测。Chiavaioli等人对转折点长周期光栅的建模和制造进行了讨论, 构建了基于转折点长周期光栅的新型无标签光学生物传感器。该研究组将Eudragit L100共聚物沉积在长周期光栅表面, 随后共价固定免疫球蛋白G(IgG)使长周期光栅表面功能化, 然后实施沿光栅区域的IgG/抗IgG生物测定及抗体/抗原相互作用的动力学分析。为了突出所提出的改进的免疫传感器的优点, 对转折点长周期光栅和非转折点长周期光栅进行了定量比较。并用检测复杂的人血清证明了转折点长周期光栅生物传感器的实际可行性和有效性及检测的特异性, 在人血清中的检测限达到70 μg/L(460 pM)。
Chiavaioli research group also proposed an evanescent wave optical fiber biosensor based on titania-silica coated LPFG, as shown in Fig. 2. The research group deposited a layer of titania-silica film on the LPFG surface using the dip-coating technology. The thickness of the film cladding was adjusted and the refractive index sensitivity of LPFG was improved by changing sol viscosity and extraction rate during dip-coating. The research group further used methacrylic acid/methacrylate copolymer for LPFG surface functionalization; afterwards, mouse IgG was immobilized on the LPFG surface to detect anti-mouse IgG. The analyte concentration was determined according to the resonance wavelength shift at the end of the binding process and initial binding rate. The detection limit of this structural LPFG to anti-mouse IgG is up to 8 μg/L(10-11 M).
Schematic illustration of the optical fiber biosensor based on titania-silica coated LPFG
Chiavaioli研究组还提出了一种基于二氧化钛-二氧化硅涂覆长周期光栅的倏逝波光纤生物传感器, 如图 2所示。他们采用浸涂技术在长周期光栅表面沉积了一层二氧化钛-二氧化硅薄膜。在浸涂过程中通过改变溶胶粘度和提取速度调节薄膜覆盖层的厚度, 增加长周期光栅的折射率灵敏度。并进一步用甲基丙烯酸/甲基丙烯酸酯共聚物使长周期光栅表面功能化, 之后将鼠IgG固定在长周期光栅表面, 对鼠抗IgG进行检测。通过结合过程结束时的谐振波长偏移和初始结合速率来确定分析物的浓度。这种结构化的长周期光栅对鼠抗IgG的检测限达到8 μg/L(10-11 M)。
Pilla et al. prepared an atactic polypropylene film on LPFG surface using the dip-coating method. They optimized the sensitivity of LPFG to the refractive index variation of the surrounding medium by controlling an appropriate film thickness, and adjusted LPFG to the high sensitivity working point in transition mode. They selected BSA (Bovine Serum Albumin) as the medium layer for immobilizing human IgG based on the strong adhesion of BSA to polystyrene surface, and adsorbed BSA onto polystyrene surface. Then they used glutaric dialdehyde to couple BSA with human IgG as the prototype biosensor, and carried out covalent immobilization of human IgG onto BSA. In addition, they determined the exposure time and optimum concentration of human IgG in glutaric dialdehyde through the resonance analysis of ELISA and surface plasmons. Finally they monitored the interaction of IgG with anti-human IgG molecules in the transition mode range of LPFG in real time and detected human IgG molecules. According to the result, the detection limit of such designed LPFG sensor to anti-human IgG molecules is about 5 pg/mm2.
Pilla等人利用浸涂法在长周期光栅表面制备无规立构聚苯乙烯薄膜, 并通过控制合适的薄膜厚度优化长周期光栅对周围介质折射率变化的敏感度, 将长周期光栅调节至过度模式的高灵敏工作点。基于牛血清白蛋白对聚苯乙烯表面的强粘附能力, 选择牛血清白蛋白作为固定人IgG的媒介层, 将牛血清白蛋白吸附于聚苯乙烯表面。然后使用戊二醛将BSA和作为原型生物感受器的人IgG偶联, 将人IgG共价固定到BSA上。并通过ELISA和表面等离子体共振分析来确定人IgG在戊二醛的暴露时间和最佳浓度。最后, 在长周期光栅的过度模式区间实时监测了人IgG与抗人IgG分子的相互作用, 对人IgG分子进行检测。结果显示, 这样设计的长周期光栅传感器对抗人IgG分子检测限约为5 pg/mm2。
Biswas et al. also deposited silica-titania thin film on the grating using the simple and general sol-gel dip-coating technology and fabricated an over-coupled LPFG sensor. By carefully adjusting the thickness and refractive index of the cladding, the LPFG was in the working area of transition mode and thus the sensing performance of the LPFG to refractive index was optimized and maximized. In addition, after further bio-functionalization of silica-titania thin film surface, they fabricated a classical biosensor for IgG/anti-IgG biological assay. The sensitivity of the LPFG coated with 159 nm thick TiO2-SiO2 thin films with the environmental refractive index range of 1.333-1.334 is up to 7000 nm/RIU, and its detection limit to anti-IgG is up to 0.025 mg/L[31-32].
Biswas等人也使用简单和通用的溶胶-凝胶浸涂技术, 将二氧化硅-二氧化钛薄膜沉积在光栅部分, 制备了超耦合长周期光纤光栅传感器。并通过仔细调整覆盖层的厚度和折射率, 使长周期光栅处于过渡模式工作区域, 从而使长周期光栅对折射率的传感性能最优化和最大化。并且在二氧化硅-二氧化钛薄膜表面进一步生物功能化后, 制备了一个经典的IgG/抗IgG生物测定的生物传感器。在1.333～1.334环境折射率范围内, 涂覆159 nm厚度TiO2-SiO2薄膜时的长周期光栅的灵敏度达到7 000 nm/RIU, 对抗IgG的检测限达到0.025 mg/L[31-32]。
Quero et al. monitored a protein marker of differentiated thyroid carcinoma—human thyroglobulin(Tg) in real time using the LPFG in reflection mode. The research group firstly covered a layer of atactic polystyrene coating on the LPFG surface. This atactic polystyrene coating can improve the sensitivity of LPFG to the refractive index of environmental media, has specific and high affinity to anti-Tg MCAB, and provides a functionalized surface for immobilizing anti-Tg MCAB. Then the research group immobilized antithyroglobulin on the polystyrene coating for use in thyroglobulin detection. This functionalized LPFG biosensor has clearly indicated the improvement of the effectiveness and sensitivity of biosensing and can be used to carry out label-free living body Tg detection in the form of fine needle aspiration(FNA) and to evaluate the biomarker concentration before and after operation. The research group conducted optical fiber ELISA parallel determination and analysis using biotinylated protein and HRP labeled streptavidin and verified the identification and capture of analytes in the pilot test. According to the dose-dependent experiment result, signals are of good linear relation with concentration within the concentration range of 0-4 ng/mL, while antibody saturation occurs at a high concentration. The sensor is characterized in that it has very high sensitivity and specificity and allows fine needle aspiration biopsy from thyroid nodules of different patients and IVD of ng/mL grade human Tg concentration[33-34].
Quero等人利用反射模式的长周期光栅实时监测了一种分化型甲状腺癌的蛋白标志物—人甲状腺球蛋白(Tg)。该研究组首先在长周期光栅表面覆盖了一层无规立构聚苯乙烯涂层。这种无规立构聚苯乙烯涂层不仅提高了长周期光纤光栅对环境介质的折射率灵敏性, 同时对抗Tg单克隆抗体具有特定的、高亲和力, 为固定抗Tg单克隆抗体提供了一种功能化表面。然后在聚苯乙烯涂层上面固定抗甲状腺球蛋白, 用于甲状腺球蛋白的检测。这种功能化的长周期光栅生物传感器清晰地展示了提高生物传感的有效性和灵敏性, 可以以细针穿刺的形式进行无标记活体Tg检测, 用于术前和术后评估生物标志物水平的浓度。在先导测试中, 使用生物素化蛋白质与HRP标记的链霉亲和素进行光纤类ELISA法平行测定分析, 证实可捕获和识别分析物。剂量依赖性实验表明, 在0～4 ng/mL的浓度范围内, 信号与浓度呈现良好的线性关系; 而在较高浓度时, 发生抗体饱和。该传感器的特征在于具有非常高的灵敏度和特异性, 允许从不同患者的甲状腺结节进行细针穿刺活检, 进行ng/mL级人Tg浓度的体外检测[33-34]。
Tang et al. assembled colloidal gold nanoparticles on the LPFG surface. After optimizing assembling conditions, the sensitivity was remarkably improved and reached 10-4 nm/URI resolution within the range of 1.34-1.39, indicating that this sensor is very suitable for sensing of biochemical substances in water solutions. After further immobilization of dinitrobenzene antigen on gold surfaced, the detection limit reached 1.4×10-7 g/mL or 9.5×10-10 M during the use in specific detection of dinitrobenzene antibody anti-DNP.
Tang等人在长周期光纤光栅表面组装了金纳米溶胶, 在优化了组装的条件后, 灵敏度显著提高, 对1.34～1.39范围内的灵敏度达到10-4 nm/URI的分辨率, 说明这种传感器非常适合于水溶液中生化物质的传感。在金表面进一步固定了二硝基苯抗原后, 用于专一性检测二硝基苯抗体anti-DNP时, 检测线达到1.4×10-7 g/mL或9.5×10-10 M。
Application in the detection of nucleotide/DNA molecules
DNA is an important representative in the study of interaction among biomolecules. Identification and appraisal of DNA sequence play an important role in environmental health protection, disease diagnosis, treatment of diseases, virus control, drug R&D, etc., so hybridization detection of DNA molecular pairs has become a hot spot of application of optical fiber biosensors.
Sozzi et al. carried out optical fiber surface functionalization by immobilizing the peptide nucleic acid probe matching with DNA target chain on the LPFG surface, and verified the feasibility of using LPFG as a DNA biosensor. The resonance wavelength was shifted by 1.2 nm in the 120nM DNA solution.
Sozzi等人通过在长周期光栅表面固定与DNA靶链相匹配的肽核酸探针, 对光纤表面进行功能化, 证明了长周期光栅用作DNA生物传感器的可行性。在120 nM的DNA溶液中, 谐振波长移动了1.2 nm。
Chen et al. achieved the detection of DNA sequence hybridization using dual-resonance peak LPFG. They firstly carried out silanization of the grating surface and covalent immobilization of DNA probe on the LPFG surface using DME-suberimidate and then monitored the process of hybridization with complementary target DNA sequence on line. According to the result, the hybridization reaction process included two stages within 60 min. The shift rate was 86 pm within the first 3 minutes and slower reaction occurred at the rate of 9 pm/min from 3 min to 60 min. The total wavelength shift caused by hybridization reaction was about 715 pm within 60 min. In comparison with the previously reported etched core fiber Bragg grating DNA biosensor, this dual-resonance peak LPFG DNA biosensor has achieve the reaction rate three times higher. Because of covalent immobilization, the sensor could still identify the target DNA very well after multiple times of heating/cooling dissociation of DNA, showing good repeatability[39-40].
Chen等人利用双谐振峰长周期光纤光栅实现了DNA序列杂交的检测。他们首先使光栅表面硅烷化, 然后用辛二亚氨二甲基醚把探针DNA共价固定于长周期光纤光栅表面, 随后在线监测其与互补靶DNA序列杂交过程。结果显示, 在60 min内杂交反应过程有两个阶段:在前3 min, 显示出86 pm的移动速率; 从3 min至60 min, 以9 pm/min的速率进行更慢的反应过程。杂交反应在60 min内引起的总波长移动大约是715 pm。与先前报道的基于腐蚀的布拉格光栅DNA生物传感器相比, 这种双谐振峰长周期光纤光栅DNA生物传感器实现了高达3倍的反应速率。由于采用了共价固定, 在多次加热/冷却解离DNA后, 该传感器仍然能很好地识别目标DNA, 显示了良好的重现性[39-40]。
Delgado-Pinar et al. used a nano narrow bandwidth LPFG in DNA detection. In order to improve the biosensing performance of LPFG, the research group fabricated the LPFG of only 1.5 nm 3-dB bandwidth by choosing appropriate fabrication parameters(high numerical aperture, relatively high order mode and large length). In comparison with the standard LPFG at 1 500 nm, the line width of such LPFG is reduced by more than one order of magnitude, thereby remarkably increasing sensor resolution. After functionalization of LPFG surface, the research group conducted DNA hybridization detection. According to the result, when the biosensor was immersed in 2 μM complementary DNA chain solution, the sensitivity and detection limit of the biosensor were respectively ～10%/μM and about 10 nM.
Delgado-Pinar等人将纳米窄带长周期光栅用于DNA的检测。为了提高长周期光栅的生物传感性能, 该研究组通过选择适当的制造参数(高数值孔径、相对高阶模式和较大的长度), 制造了只有1.5 nm的3-dB带宽的长周期光栅。相对于在1 500 nm处的标准长周期光栅, 这种长周期光栅的线宽减少一个数量级以上, 从而使传感器的分辨率显著提高。在将长周期光栅表面功能化后, 进行了DNA的杂交检测。结果显示, 当该生物传感器浸没在2 μM的互补DNA链溶液中时, 传感器的灵敏度为-10%/μM, 检测限约为10 nM。
Jang et al. fabricated a LPFG on the side-polished fiber surface with the photolithography for use in the detection of DNA hybridization. They firstly modified the silica surface of the LPFG using poly-L-lysine and then immobilized ssDNA probe on the poly-L-lysine surface for use in hybridization with target ssDNA. According to the result, due to the quick reaction of DNA hybridization, the majority of wavelength shift occurred within the first 9 minutes, i.e. 0.94 nm wavelength shift. The total wavelength shift caused by DNA hybridization was 1.82 nm. The reaction rate of this side-polished LPFG DNA biosensor is about 2.5 times higher than that of the dual-resonance peak LPFG DNA biosensor previously reported by Xianfeng Chen research group.
Jang等人通过光刻技术在侧面抛光的光纤表面上制作了长周期光纤光栅, 用于实时检测DNA的杂交。首先用聚-L-赖氨酸修饰长周期光纤光栅的二氧化硅表面, 然后在聚-L-赖氨酸表面固定ssDNA探针, 随后用于与靶ssDNA的杂交。结果显示:由于DNA杂交的快速反应, 大部分的波长移动发生在前9 min, 即0.94 nm的波长偏移; 由DNA杂交引起的总体波长偏移是1.82 nm。这种侧面抛光的长周期光栅DNA传感器比先前Chen Xianfeng研究小组报道的双谐振峰长周期光栅DNA传感器提高了约2.5倍。
Rindorf et al. used a photonic crystal fiber(PCF) long period grating in DNA detection. According to the result, after immobilizing a layer of poly-L-lysine on the inner wall of PCF holes, the resonance wavelength of the long period grating was shifted by 6.7 nm. In case of binding a monolayer dual-chain DNA, the resonance wavelength was shifted by 2.3 nm. With respect to resonance wavelength shift caused by biomolecule layer per nm thickness, this PCF long period grating DNA biosensor has about 1.4 nm/nm sensitivity.
Rindorf等人将光子晶体长周期光栅用于DNA的检测。结果显示, 在光子晶体光纤的孔内壁固定一层聚-L-赖氨酸后, 长周期光栅的谐振波长移动了6.7 nm; 当进一步结合一单层双链DNA时, 谐振波长移动了2.3 nm。就每纳米厚度的生物分子层所引起的移动谐振波长而言, 这种光子晶体长周期光纤光栅DNA传感器具有大约1.4 nm/nm的灵敏度。
Chen et al. designed an oligonucleotide optical fiber biosensor using dual-resonance peak long period grating. Firstly, they carried out covalent immobilization of the non-modified 5′-phosphorylated oligonucleotide probe onto the aminoacyl derivatization fiber grating surface directly using 1-ethyl-3-(3-dimethylamino propyl)carbodiimide. Because of binding via the specificity of 5′-phosphoric acid, other parts of oligonucleotide which can be used for conjugation reaction were left. The refractive index sensitivity of this LPFG is 50 times that of pervious LPFGs. The LPFG biosensor after functionalization is used to monitor complementary oligonucleotide. According to the result, the concentration of 4 nM hybridization oligonucleotide can be detected.
Chen等人利用双谐振峰长周期光栅设计了寡核苷酸光纤生物传感器。第一步直接用1-乙基-3-(3-二甲氨基丙基)碳二亚胺将未修饰的5′-磷酸化寡核苷酸探针共价固定到氨基酸衍生化的光纤光栅表面。由于是通过5′磷酸特异性连接, 留下了可用于结合反应的寡核苷酸的其余部分。这种长周期光栅的折射率灵敏度是先前长周期光栅的50倍, 功能化后的长周期光栅传感器用于监测互补的寡核苷酸, 结果显示, 可以检测到4 nM的杂交寡核苷酸的浓度。
Based on LPFG and a component optical detection device, Goncalves et al. proposed a new method for determining the resonance spectrum of LPFG and for in-situ detection of DNA hybridization in real time. Fig. 3 shows the schematic representation of the DNA detection process using the biosensor system. The detection limit and quantification limit of this biosensor to DNA are respectively 62±2 nM and 209±7 nM. It has been proved that the biosensor system has good specificity to detection samples according to the effect of DNA target mismatching with different base pairs of the probe.
Schematic representation of DNA detection process using LPFG
Gonçalves等人基于长周期光栅和一种组件光学检测装置, 提出了一种测定长周期光栅共振光谱的新方法, 用于原位实时检测DNA杂交。图 3给出了该传感器系统对DNA检测的过程示意图。该传感器对DNA的检测限为(62±2) nM, 定量限为(209±7) nM。通过与探针不同的碱基错配的DNA靶点作用, 证明了该传感器系统对检测样本具有良好的特异性。
Application in the detection of biotin-streptavidin system
There is a high specific binding affinity between biotin and streptavidin(affinity constant up to 1015 mol/L). Once bound, they are very difficult to disassemble. The action between the two substances is the currently known as the strongest non-covalent interaction. In addition, their binding property is not basically affected by reagent concentration, pH value, denaturant, ionic strength of buffer solution, proteolytic enzyme, organic solvent, etc. and doesn′t suffer from the interference from the non-specific effect of reaction reagents. Therefore, the biotin-streptavidin system is the most common biosensing subject-object pairing system for researchers to verify the biosensing performance of sensing devices.
生物素与链霉亲和素之间具有高度的特异性结合能力(亲和常数高达1015 mol/L), 二者一旦结合就极难拆解, 这两种物质之间的作用是目前已知的最强的非共价键相互作用。而且它们的结合特性基本不受试剂浓度、酸碱pH、变性剂、缓冲液的离子强度、蛋白溶解酶、有机溶剂等环境的影响, 不受反应试剂的非特异性作用的干扰。因而, 在验证传感器件的生物传感性能时, 生物素与链霉亲和素成为研究者最常用的一种生物传感主客体配对体系。
Pilla research group dip-coated different thickness of polystyrene on the grating surface and optimized the sensitivity of the LPFG; then the research group directly adsorbed biotinylated BSA on the polystyrene surface for use in the detection of streptavidin. The research group dynamically monitored the adsorption reaction process of biomolecules with time[46-47].
Pilla研究组在光栅表面浸涂了不同厚度的聚苯乙烯, 对长周期光纤光栅的灵敏度进行优化, 然后在聚苯乙烯表面直接吸附生物素化的牛血清白蛋白, 随后用于链霉亲和素的检测, 动态地监测了生物分子随时间的吸附反应过程[46-47]。
The direct adsorption mode relies on only hydrophily-hydrophobicity combination and is affected by solution pH value and ionic concentration very easily. Therefore, the research group further improved this mode, and designed a LPFG biosensor using the method of coating multilayer films and covalent coupling for use in the detection of biotin-streptavidin system. Firstly the sensitivity of LPFG is improved through dip-coating of transparent atactic polystyrene. Meanwhile, the working point of a spectrograph is adjusted so that it is in the transition area of the LPFG. Secondly, through dip-coating of transparent methyl methacrylate-methacrylic acid block copolymer, the film surface is rich in a large quantity of carboxyl function groups which can be covalent-crosslinked. Such treatment can greatly reduce the resonating problem with the cladding mode transition area caused by grating surface functionalization. Thirdly, carboxyl is coupled with streptavidin using EDC/NHS, so that the biosensor can monitor the process of interaction between streptavidin and biotinylated BSA highly sensitively in real time. See Fig. 4 for the modification process of coated LPFG interface with different biological agents.
Sketch of coated LPFG interface with different biological agents
由于直接吸附的方式仅仅依靠亲疏水性结合, 很容易受溶液的pH值、离子浓度的影响, 该研究组对检测方式做了进一步的改进, 采用浸涂多层薄膜及共价耦合的方法设计了长周期光纤光栅生物传感器, 用于生物素-链霉亲和素体系的检测。首先, 浸涂透明的聚合物无规立构聚苯乙烯提高长周期光纤光栅的灵敏度, 同时调整光谱仪工作点使其处于长周期光纤光栅过渡区域; 然后, 浸涂透明的甲基丙烯酸甲酯-甲基丙烯酸嵌段共聚物, 使薄膜表面富含大量可以共价交联的羧基官能团, 这样的处理大大降低了光栅表面功能化所带来的包层模式过渡区域的调谐难度; 第三步, 使用EDC/NHS将羧基与链霉亲和素偶联, 使其能高灵敏实时监测链霉亲和素与生物素化的牛血清白蛋白的相互作用过程。不同生物试剂在长周期光栅表面修饰的过程如图 4所示。
Wang et al. prepared PAH/PCBS films on the LPFG surface using the electrostatic self-assembly technology and then adsorbed biotins. After enclosure by BSA, they used the biosensor to detect avidin. Such biosensor can make a distinction between streptavidins of five different concentrations ranging from 0 to 0.075 mg/mL very well, and the lowest detection concentration is less than 0.012 5 mg/mL.
Wang等人用静电自组装技术在长周期光纤光栅的表面制备PAH/PCBS薄膜, 然后吸附生物素, 在用牛血清白蛋白封闭后, 用于抗生物素蛋白的检测。这种传感器对0～0.075 mg/mL之间五种不同浓度的链霉亲和素有很好的区分能力, 检测的最低浓度小于0.012 5 mg/mL。
Using the LBL self-assembly method, Marques et al. deposited PAH/SiO2:Au core shell nanoparticles on the LPFG surface and prepared a highly sensitive LPFG biosensor for use in streptavidin detection. The biotin further modified on the surface of PAH/SiO2:Au core shell nanoparticles was used as the detection ligand of streptavidin. The thickness and diameter of PAH/SiO2:Au core shell nanoparticles effectively increased the area of immobilized biotin and then the sensitivity of the LPFG to streptavidin. The lowest detection concentration of the LPFG assembled with 3 layers of PAH/SiO2:Au films to streptavidin is up to 2.5 nM, and its sensitivity and detection limit 6.9 nm/ng/mm2 and 19 pg/mm2 respectively. The sensitivity of this biosensor is at least two orders of magnitude higher than that of the previously reported other optical fiber biosensors[24, 30, 49-50]. The sensing property of the biosensor can be adjusted by changing the ligand.
Marques等人采用层层自组装方法在长周期光栅表面沉积PAH/SiO2:Au核壳纳米粒子, 制备了高灵敏性的长周期光栅生物传感器, 用于链霉亲和素的检测。在SiO2:Au核壳纳米粒子表面进一步修饰生物素, 作为检测链霉亲和素的配体。SiO2:Au核壳纳米粒子的厚度和直径有效地增加了固定生物素的面积, 进而增加了长周期光栅对链霉亲和素的灵敏度。组装了3层PAH/SiO2:Au薄膜的长周期光栅对链霉亲和素的最低检测浓度达到2.5 nM, 灵敏度为6.9 nm/ng/mm2, 检测限为19 pg/mm2。该传感器的灵敏度比先前报道的其它光纤生物传感器[24, 30, 49-50]至少提高了两个数量级。并可以通过改变配体来调整该传感器的传感性质。
Application in the detection of E.coli/phage
E.coil is a kind of bacterium closely related to people′s daily lives. The scientific name of E.coil is "escherichia coli", which belongs to a kind of enteric bacilli. E.coil is a kind of unicellular organism which is parasitic in human large intestines and small intestines and harmless to human bodies. E.coil has features such as simple structure, quick reproduction and easy culture and is an important experiment material biologically. E.coli is not pathogenic under normal habitat conditions. However, in case of entering gall bladder, bladder, etc., E.coil can cause inflammation. E.coil is used to express proteins(e.g. insulin, some vaccines, etc.) after genetic recombination in genetic engineering. E.coil is often used as model organisms for cytological experiments.
大肠杆菌是与人们日常生活关系非常密切的一类细菌, 学名称作"大肠埃希菌", 属于肠道杆菌大类中的一种。它是寄生在人体大肠和小肠里对人体无害的一种单细胞生物, 结构简单, 繁殖迅速, 培养容易, 它是生物学上重要的实验材料。正常栖居条件下大肠杆菌并不致病, 但若进入胆囊、膀胱等处可引起炎症。在基因工程中, 大肠杆菌就被用于表达基因重组后的蛋白(例如胰岛素, 某些疫苗等)。大肠杆菌还常常作为模型生物参与细胞学实验。
Phage is an organism which recognizes a host through surface-specific receptor molecules. Phase has features such as high sensitivity and specificity to bacteria, good thermal stability, low cost and quick production, is non-toxic to people, and has become an alternative to antibody in the field of biosensing. Many researchers combine LPFG with phage for use in the detection of E.coil/phage.
噬菌体是通过表面特异性受体分子识别宿主的有机体, 对细菌具有高度的敏感性和特异性, 热稳定性好, 而且噬菌体对人类无毒, 以及廉价和生产快速, 在生物传感领域已经成为抗体的一个替代物。许多研究人员将长周期光纤光栅与噬菌体结合应用于大肠杆菌/噬菌体的检测。
Smietana et al. used T4 phage as the recognition element for detecting K12 cell of E.coil, directly immobilized T4 phage onto the LPFG surface using the physical adsorption method, and monitored the process of interaction between phage and K12 cell of E.coil in situ in real time. According to the result, the resonance wavelength of the LPFG was shifted by ～1.3 nm after identification and immobilization of K12 cell of E.coil.
Smietana等人利用T4噬菌体作为检测大肠杆菌K12细胞的识别元件, 采用物理吸附的方法将T4噬菌体直接固定于长周期光栅表面, 原位实时监测了噬菌体与大肠杆菌K12细胞的相互作用过程。结果表明, 大肠杆菌K12细胞被识别固定后, 长周期光栅的谐振波长移动了～1.3 nm。
The physical adsorption method is only hydrophilic-hydrophobic combination. The coverage rate of T4 phage on the grating surface is very small and the phage is easily eluted by PBS, thus affecting subsequent detection. Tripathi et al. improved the above method and immobilized the phage using the covalent method. For instance, silane coupling agent was firstly used for amination of the grating surface, and then dual-function group crosslinking agent-glutaric dialdehyde was used for covalent immobilization of the phage so as to keep T4 phage stable in the measurement process, as shown in Fig. 5. In addition, they used high precision optical fiber demodulator, ultra-high sensitivity LPFG, etc. in the detection of E.coil so as to achieve stable and label-free phage-based E. coil detection. The lowest detection concentration of E.coil can reach 103 CFU/mL, and the experiment accuracy more than 99%.
SEM micrograph of the E.coli immobilized on the surface of the LPFG:109 CFU/mL of E.coli concentration in PBS
由于物理吸附的方法仅仅是亲疏水性吸附, T4噬菌体在光栅表面的覆盖率很小, 而且容易被缓冲溶液洗脱, 影响后续的检测。Tripathi等人对上述方法进行了改进, 采用共价方法固定噬菌体, 即首先用硅烷偶联剂使光栅表面氨基化, 然后用双官能团交联剂戊二醛共价固定噬菌体, 使T4噬菌体在测量过程中保持稳定, 如图 5所示。他们同时改用高精度光纤解调仪、超敏感长周期光纤光栅等来检测大肠杆菌, 实现了稳定的、无标记、以噬箘体为基础的大肠杆菌检测, 对大肠杆菌检测的最低浓度可以达到103 CFU/mL, 实验的准确度在99%以上。
Brzozowska et al. proposed a new high-sensitivity T4 phage-adhesin-coated LPFG biosensor. T4 phage adhesin(gp37) is bound with E.coil B in natural form or modified form by recognizing LPS. This kind of binding has high specificity and irreversibility. The reaction of T4 phage adhesin to positive LPS is almost three times that to negative LPS. The method of LPFG bio-functionalization is to coat LPFG surface with nickel ions which can be reversibly bound with gp37 His-tag. Recombinant adhesin protein phage proteins were used as the receptor molecules in the biosensing system. A test was conducted on the specificity of binding of adhesin with LPS using LPFG, and the specificity was verified using the methods such as Western blotting, ELISA and BIACORE. This biosensor can be used to measure bacterial contamination accurately in real time.
Brzozowska等人提出了一种基于涂覆有T4噬菌体粘附素的新型高灵敏度长周期光栅传感器。T4噬菌体粘附素(gp37)通过识别大肠杆菌脂多糖(LPS)而以其天然或变性形式结合大肠杆菌B, 这种结合具有高度的特异性和不可逆性。T4噬菌体粘附素对LPS阳性的反应几乎是阴性LPS的3倍。长周期光栅生物功能化的方法是用能够与gp37组氨酸标签可逆结合的镍离子涂覆长周期光栅表面。重组粘附噬菌体蛋白被用作生物传感体系中的受体分子。用长周期光栅装置对粘附素与LPS结合的特异性进行了测试, 并通过蛋白质印迹、酶联免疫吸附试验(ELISA)和BIACORE方法做了证实。这种传感器能够实时、准确地测量细菌污染。
The research group also coated T4 phage adhesin on the LPFG surface for use in Gram-negative bacteria detection. According to the result, in comparison with ELISA and BIAcore, this biosensor can be used to recognize E.coil K-12 (PCM2560) more obviously. The resonance wavelength shift caused by E.coil K-12 is about half that caused by E.coil B(positive control). The detection signal(RU) caused by E. coil K-12 in BIAcore SPR sensor is 10% of that caused by E.coil B. This indicates that the sensitivity of the LPFG sensor is obviously higher than that of BIAcore SPR sensor. This adhesin-coated long-period grating sensor can not only be used to selectively detect E. coil B but also to detect more extensive Gram-negative bacteria groups containing OmpC in LPS structure or having special configuration around tail end glycosidic bond, e.g. E.coil O111(PCM418), klebsiella pneumoniae O1(PCM1) and Yersinia enterocolitica O1(PCM1879). In addition, this sensor can be used to detect Gram-negative bacteria containing OmpC and the special spatial configuration around tail end glycosidic bond in LPS structure. Moreover, the experiment result has proved the possibility of using adhesin to replace phage so as to recognize specific bacterial strains. In comparison with phage, adhesin is more stable and can be stored for a longer time, so it has a very good prospect in commercial application.
该研究小组还在长周期光栅表面涂覆了T4噬箘体黏附素用于革兰氏阴性菌的检测。结果显示, 与ELISA和BIAcore方法相比, 这种传感器能更明显地识别大肠杆菌K-12 (PCM2560)。大肠杆菌K-12所引起的谐振波长移动大约是大肠杆菌B(阳性对照)的一半。而在BIAcore表面等离子体共振传感器中, 大肠杆菌K-12所引起的检测信号(RU)是大肠杆菌B的10%。这表明长周期光栅传感器的灵敏度明显高于BIAcore表面等离子体共振传感器。这种涂覆有粘附素的长周期光栅传感器不仅可选择性检测大肠杆菌B, 而且可用于检测在脂多糖结构中包含OmpC或在末端糖苷键周围的特殊间构型的更广泛的革兰氏阴性细菌组, 如大肠杆菌O111(PCM418), 肺炎克雷伯菌O1(PCM1)和结肠炎耶尔森菌O1(PCM1879)。还可以检测含有OmpC的革兰氏阴性菌和脂多糖结构中围绕在末端糖苷键的特殊空间结构。此外, 实验结果还证明了使用粘附素替代噬菌体以获得特异性细菌菌株识别的可能性。与噬菌体相比, 粘附素更稳定且可以储存更长的时间, 因而在商业应用中有很好的前景。
Dandapat et al. proposed a temperature-insensitive cascade LPFG biosensor used in accurate and quantitative detection of E.coil in water. The temperature sensitivity of this biosensor is only 1.25 pm/℃, and its refractive index sensitivity is 1 929 nm/RIU. The grating region length of the biosensor is about 3.6 cm and it is very suitable for biosensing. In case of changing E.coil concentration, the resonance wavelength shift range is about 1.3-2.5 nm and the detection limit is up to 102 CFU/mL. The biosensor has showed its capacity of reliable and quantitative detection of E.coil in water at 24-40 ℃ temperature.
Dandapat等人提出了一种温度不敏感型级联长周期光栅生物传感器, 用于水中大肠杆菌的精确定量检测。这种传感器对温度的灵敏度仅为1.25 pm/℃, 而其折射率灵敏度为1 929 nm/RIU, 栅区长度大约为3.6 cm, 非常适合于生物传感。当改变大肠杆菌的浓度时, 谐振波长移动的范围在1.3～2.5 nm之间, 检测限达到102 CFU/mL。在24～40 ℃的温度范围内下, 该传感器展示了可靠、定量检测水中大肠杆菌的能力。
In case of using LPFG in direct detection, the sensitivity to detection of biological substances is low. Therefore, some researchers attempt to modify LPFG surface using nano-films of high refractive index so as to improve the refractive index sensitivity of LPFG to environmental media, and then detect biological substances.
由于用长周期光栅直接检测时, 对生物物质检测的灵敏度比较低。一些研究者尝试用高折射率的纳米薄膜对长周期光栅表面修饰, 提高长周期光栅对环境介质的折射率灵敏度, 然后再进行对生物物质的检测。
Smietana et al. prepared TiO2 films on the LPFG surface using the atomic layer deposition technology. According to the result, when the thickness of the TiO2 overlay is up to 70 nm, the refractive index sensitivity of LPFG to the environmental medium of 1.34 in refractive index reaches 6 200 nm/RIU. The sensitivity of the LPFG is 2.8 times higher than that of the non-film-coated LPFG. In addition, after TiO2 film surface functionalization by the LBP(adhesin) of T4 phage, the sensitivity of the LPFG enhanced by TiO2 to bacterial endotoxin(E.coil B LPS) was improved obviously. After E.coil LPS incubation, the resonance wavelength shift of the LPFG without TiO2 film was no more than 20 nm, while that of the LPFG enhanced by deposited TiO2 film was about 40 nm.
Smietana等人用原子层沉积技术在长周期光栅表面制备了二氧化钛薄膜, 发现当二氧化钛覆盖层厚度达到70 nm时, 对于折射率为1.34的环境介质, 长周期光栅的折射率灵敏度达到6 200 nm/RIU。与未涂覆薄膜的长周期光栅相比, 灵敏度提高了2.8倍。此外, 当二氧化钛薄膜表面被T4噬菌体的内毒素结合蛋白(粘附素)功能化后, 二氧化钛增敏长周期光栅对细菌内毒素(大肠杆菌B脂多糖)的敏感性明显增加:在大肠杆菌脂多糖孵化后, 没有二氧化钛薄膜的长周期光栅的谐振波长移动不超过20 nm, 沉积二氧化钛薄膜后的长周期光栅的谐振波长移动了大约40 nm。
Koba et al. proposed a high-order layer-coated LPFG biosensor working near the dispersion turn-around point. A test was conducted on the phage adhesin-functionalized LPFG biosensor using the dry weight of specific and non-specific bacteria. According to the test result, this biosensor can recognize bacteria through selective binding. They used the dry weight of E.coil B for positive test and E.coil K12 and salmonella enterica for negative test. The resonance wavelength shift caused by E.coil B was over 90nm, while the resonance wavelength shift caused by E.coil K12 and salmonella enterica was about 40nm and 20nm respectively.
Koba等人提出了一种在高阶包层模式的色散转折点附近工作的长周期光栅生物传感器。将这种噬菌体粘附素功能化的长周期光栅传感器用特定和非特异性细菌干重进行测试, 结果显示, 这种生物传感器能够选择性地结合, 从而识别不同的细菌。他们用大肠杆菌B的细菌干重作为阳性测试, 用大肠杆菌K12和肠道沙门氏菌作为阴性测试。大肠杆菌B引起的谐振波长漂移超过90 nm, 而大肠杆菌K12和肠道沙门氏菌引起的谐振波长漂移分别约为40 nm和20 nm。
Phage T7 is a virulent polyhedral phage which is infected with E.coli and has irretractive short tail. The virus particle of phage T7 has icosahedron head, with very small tail and complex structure. The head consists of 5 different proteins. Phage T7 is specific to enteric bacilli and relevant Gram-negative bacteria.
噬菌体T7是一种感染大肠杆菌的、烈性的、具有不能收缩的短尾的多面体噬菌体, 其病毒颗粒有20面体的头部和非常小的尾, 结构较复杂, 头部由5个不同的蛋白质组成, 对肠杆菌和相关革兰氏阴性菌特异。
Janczuk-Richter et al. used LPFG in the detection of T7 virus phage. They firstly adjusted the LPFG to the best refractive index sensitivity state and then functionalized the LPFG surface using 3-(triethoxy silyl) propyl succinic anhydride for covalent binding of anti-T7 antibody. The existence and concentration of T7 phage were judged by tracking the shift of the resonance wavelength in the transmission spectrum of the LPFG caused by the increase in the thickness and density of biological coverings. See Fig. 6 for the schematic representation of the LPFG surface modification steps and phage T7 detection. This sensor has good selectivity and reproducibility to T7 phage and the detection limit is lower than 5×103 PFU/mL.
Schematic representation of the LPFG surface modification steps and phage T7 detection
Janczuk-Richter等将长周期光栅用于T7病毒噬箘体的检测。他们首先将长周期光栅调整到最佳的折射率灵敏度状态, 然后用3-(三乙氧基甲硅烷基)丙基琥珀酸酐使长周期光栅表面功能化以共价结合抗T7抗体。通过追踪由生物覆盖物的厚度和密度增加所引起的长周期光栅透射光谱中的谐振波长移动情况来判断T7噬菌体的存在和浓度。长周期光栅表面从修饰步骤及其对噬菌体T7检测的示意图如图 6所示。这种传感器对T7噬箘体有良好的选择性和再生性, 检测限低于5×103 PFU/mL。
Application in the detection of Francisella tularensis
Francisella tularensis is the pathogen of tularemia and a highly infectious intracellular bacterial parasite and can cause human and animal tularemia. More than one hundred species of animals have been found in nature to infect Francisella tularensis. Due to a wide variety of transmission routes, easy diffusion, and high toxicity, Francisella tularensis has been listed by CDC as class A bioterrorism agent. Tularemia is a zoonosis and has a high mortality rate. Timely and accurate detection of Francisella tularensis is of much significance to timely treatment of tularemia patients and prevention of tularemia diffusion. Cooper et al. prepared nano-films on the LPFG surface using the layer-by-layer electrostatic self-assembly technology, further deposited anti-Francisella tularensis IgG onto nano-films, and then detected Francisella tularensis. The existence of Francisella tularensis was detected according to the reduction of peak wavelength caused by specific antigen binding. The research has filled up the gap of quick and culture-free field diagnosis of Francisella tularensis.
土拉弗朗西斯菌(Francisella tularensis)是土拉菌病的致病菌, 是一种具有较强传染性的胞内寄生菌, 能够引起人和动物土拉热, 主要通过媒介昆虫在哺乳动物和人之间传染。在自然界中已发现一百种以上的动物感染此菌。因其传播途径多样、易扩散、毒性强而被美国疾病控制预防中心列入A类生物恐怖制剂。土拉菌病是一种人畜共患病, 致死率高, 及时、准确的检测土拉弗朗西斯菌对于土拉菌病患者及时治疗和防止扩散具有重要的意义。Cooper等人通过层层静电自组装技术在长周期光栅表面制备纳米薄膜, 并进一步将抗土拉弗朗西斯菌的血清免疫球蛋白(IgG)沉积到纳米薄膜上, 然后对土拉弗朗西斯菌进行检测。从特异性抗原的结合所引起的峰波长的减少检测到了土拉弗朗西斯菌的存在。该研究为快速、无培养和野外诊断土拉弗朗西斯菌填补了空白。
Application in the detection of methicillin-resistant staphylococci
Methicillin-resistant staphylococci is a common clinical bacterium with high toxicity, which has become one of important pathogenic bacteria for hospital and community infection, and mainly affects the health of hospitals, communities and animals. In order to protect the health of people, it is crucially important to carry out culture-free, highly sensitive and specific quick diagnosis and analysis of methicillin-resistant staphylococci.
耐甲氧西林金黄色葡萄球菌是临床上常见的毒性较强的细菌, 已成为医院和社区感染的重要病原菌之一, 是医院、社区和动物的主要健康问题。为了保护人们的健康, 对耐甲氧西林金黄色葡萄球菌进行无培养的、高灵敏性、特异性的快速诊断分析至关重要。
In order to meet this need, Bandara et al. detected methicillin-resistant staphylococci using the nano-film modified LPFG. The research group deposited PAH/PCBs multilayer films on the LPFG surface, so that the terminal of the multilayer films of the LPFG had a large quantity of carboxyl function groups. These terminal carboxyl function groups were further covalently coupled onto the monoclonal antibodies which have specificity to recognize methicillin-resistant staphylococci penicillin-binding protein 2a. The LPFG assembled with multilayer films was exposed into 102 CFU/mL MRSA. After 50 min, the light transmittance was reduced by 19.7%. On the contrary, light transmission was attenuated by less than 1.8% after exposure into 106 CFU/mL MSSA. Light transmission was attenuated by 11.7%-73.5% after exposure of the LPFG with multilayer films into the extracts of the liver, lung and spleen of the mouse infected with MRSA. On the contrary, light transmission of the sensor was attenuated by ≤5.6% after exposure of the LPFG with multilayer films into the extracts of the mouse infected with MSSA. When the sensor was used to detect 36 strains of methicillin-resistant staphylococci, 15 strains of Methicillin-sensitive staphylococci, 10 strains of heterogenetic species(all strains 104 CFU/mL) and mouse tissue samples infected with MRSA and the critical value of light transmission attenuation was 6.3%, the antibiotics sensitivity test result was completely consistent with MR bacteria and non-MR bacteria measured by the biosensor. Therefore, this nano-film modified LPFG biosensor has the potential of detecting methicillin-resistant staphylococci in clinical samples highly sensitively and specifically.
为了满足这一需要, Bandara等人用纳米薄膜修饰的长周期光栅生物传感器检测了耐甲氧西林金黄色葡萄球菌。该研究组利用自组装技术, 在长周期光纤光栅表面沉积了PAH/PCBs多层薄膜, 使长周期光栅多层膜的末端具有大量的羧基官能团。这些末端羧基官能团被进一步共价偶联到对耐甲氧西林金黄色葡萄球菌的青霉素结合蛋白2a有特异性识别的单克隆抗体上。将该组装多层薄膜的长周期光栅暴露到102 CFU/mL的耐甲氧西林金黄色葡萄球菌(MRSA)中, 50 min后透光率降低了19.7%。相反, 当暴露到106 CFU/mL的甲氧西林敏感的金黄色葡萄球菌(MSSA)中后, 光传输衰减小于1.8%。将该多层膜长周期光栅暴露于感染MRSA的小鼠的肝、肺、脾的提取物中, 光传输衰减了11.7%～73.5%。与此相反, 将该多层膜长周期光栅暴露到感染MSSA的小鼠的提取液中, 传感器的光传输衰减等于或小于5.6%。当将该传感器用于检验36株耐甲氧西林金黄色葡萄球菌、15株甲氧西林敏感的金黄色葡萄球菌、10株异种属(所有菌株均为104 CFU/mL)、感染MRSA的小鼠组织样品时, 当光传输衰减的临界值为6.3%时, 抗生素敏感性试验和该生物传感器测定的MR细菌和非MR细菌完全一致。因此, 这种纳米薄膜修饰的长周期光栅生物传感器具有高灵敏性和特异性地检测临床样品中的耐甲氧西林金黄色葡萄球菌的潜力。
Application in the detection of fungal pathogens
LPFG biosensor not only has developed quickly in the detection of biotic components of humans and animals but also has made some progress in the detection of plant viruses.
Fungal pathogens can cause serious fungal diseases and huge losses to crop yield, and will also have a fatal impact on humans. Detection and appraisal of trichoderma Fungi species are of much significance to protecting plant root systems from being eroded by trichoderma pathogens. Gambhir et al. used the CVL-written LPFG in the detection of fungi in plants. The resonance wavelength of LPFG was respectively 1 524 nm, 1 520 nm and 1 522 nm in the three trichoderma solutions such as T. Harzianum, T. Viride and T. longibacterium, and the corresponding optical loss was changed from 63.75 dB in water to 54.8 5, 57.34 and 59.6 dB respectively. This indicates that LPFG can be used to distinguish different species of trichoderma very well
真菌病原体能导致严重的真菌病害, 对作物产量造成巨大损失, 而且, 还会对人类造成致命的影响。木霉真菌种类的检测和鉴定对于保护植物的根系免遭木霉病原菌的侵蚀具有重要的意义。Gambhir等人将铜蒸气激光写入的长周期光栅用于植物中真菌的检测。在T.Harzianum、T.Viride和T.longibacterium等3种木霉菌溶液中, 长周期光栅的谐振波长分别在1 524、1 520和1 522 nm, 相应的光损耗分别从水中的63.75 dB变到54.85、57.34和59.76 dB, 这说明长周期光栅能很好地区分不同种类的木霉菌。
Application in the detection of aptamers
Aptamer is short single-stranded DNA or RNA oligonucleotides and can be bound with different targets by using stable sequence-dependent structure. The size of aptamers can range from 25 to 90 basic groups in general, allowing selective control of reaction with specific molecules.
适配体是短的单链DNA或RNA寡核苷酸, 它可以通过采用稳定的三维序列依赖性结构与不同的靶标结合。它们的大小一般可以从25到90个碱基, 允许选择地控制与特定分子的反应。
Coelho et al. immobilized thrombin aptamer onto the TiO2-coated LPFG surface for thrombin detection. In order to enhance the sensitivity of the LPFG to environmental media, the research group firstly covered 30 nm thick TiO2 film on the LPFG and then a layer of polylysine on the TiO2 film surface. Afterwards, the research group immobilized the terminal aminated thrombin aptamer onto the polylysine film through electrostatic interaction and measured different concentrations of thrombin in the buffer solution. The measurement schematic, see Fig. 7. The detection range and detection limit of the sensor are respectively 0.01-0.1 μM and 0.01 μM. The function surface of the sensor can be regenerated by means of 5% sodium hypochlorite solution and has good reproducibility.
Schematic of the sensing configurations for LPFG coated with 30 nm of TiO2
Coelho等人将凝血酶适配体固定在涂覆二氧化钛的长周期光栅表面, 用于检测凝血酶。为了增加长周期光栅对环境介质的灵敏性, 该研究组首先在长周期光栅覆盖了30 nm厚的二氧化钛薄膜, 再在二氧化钛薄膜表面覆盖一层聚赖氨酸, 随后通过静电作用将末端氨基化的凝血酶适配体固定到聚赖氨酸薄膜上, 进而测定缓冲溶液中不同浓度的凝血酶, 测定示意图如图 7所示。该传感器的检测范围为0.01～0.1 μM, 检测限为0.01 μM。该传感器的功能表面可以通过5%次氯酸钠溶液再生, 有良好的再生性。
Carrasquilla et al. firstly modified the surface of Michelson interferometric LPFG using gold nanoparticle-doped silica sol-gel film and then immobilized ATP specific aptamer onto the gold nanoparticle-doped macroporous sol-gel derivative film for hybridization with the complementary sequence containing quencher and label-free detection of ATP. The macroporous silica sol-gel derivative film material can provide a large specific surface area and bear a large quantity of DNA aptamers at high density and affinity, and the gold nanoparticles contained in the silica film can provide a cladding of high refractive index so as to enhance the refractive index sensitivity of LPFG. The existence of the target molecule ATP will induce aptamer conformation variation for binding with ATP, which will result in oligonucleotides release. Moreover, effective variation of refractive index is enhanced and reversed(i.e. ATP binding will lead to net reduction of molecular weight and refractive index). The use of structure-switching aptamers prevented the signal from non-specific binding. This gold nanoparticle/silica film has dual functions such as improvement of ATP aptamer binding density and LPFG sensor cladding refractive index and can remarkably enhance the sensor's sensitivity to micromolecule detection, and its detection limit to ATP is up to 400 μM。
Carrasquilla等人首先将金纳米颗粒掺杂的二氧化硅溶胶-凝胶薄膜修饰在迈克尔逊干涉式长周期光栅表面, 然后把三磷酸腺苷(ATP)特异性适配体固定在金纳米粒子掺杂的大孔溶胶-凝胶衍生薄膜上, 使之与含有猝灭剂部分的互补序列杂交, 进行无标记地探测ATP。由于大孔二氧化硅溶胶-凝胶薄膜材料能够提供大的比表面积, 可以高密度亲和性地负载大量的DNA适配体, 而二氧化硅薄膜内包含的金纳米颗粒可以提供高折射率覆盖层, 增强长周期光栅的折射率灵敏度。目标分子ATP的存在将诱导适配体的构象变化, 使之与ATP结合, ATP的结合将导致寡核苷酸的释放, 折射率的有效变化被增强和反转(即ATP的结合将导致分子量和折射率的净减少)。结构转换的适配体阻止了来自非特异性结合的信号。这种金纳米粒子/二氧化硅薄膜具有改善ATP适配体结合密度和长周期光栅传感器包层折射率的双重功能, 显着提高了传感器对小分子检测的灵敏度, 对ATP的检测限达到400 μM。
Queirós et al. immobilized E.coil aptamer on the LPFG surface using the two methods such as electrostatic self-assembly and covalent bond coupling for detection of EcOMPs. This evanescent wave optical fiber sensor can achieve label-free specific recognition of EcOMPs in water. Within the range of 0.1～10 nM, resonance wavelength has a good linear relation with EcOMPs concentration. Using the two methods such as electrostatic self-assembly and covalent bond immobilization, the EcOMPs detection sensitivity is respectively -0.1563±0.005 nm/decade and -0.1597±0.004 nm/decade. In addition, the sensor can be regenerated at low pH value. After continuous 3 times of detection, the deviation is less than 0.1%. This sensor has been applied in the detection of E. coil in labeled environmental water samples. According to the comparison result, the sensor coated with a layer of polylysine has better reproducibility and surface uniformity. On the contrary, the sensor covalently bound with aptamer has better performance of analyzing labeled water samples.
Queirós等人用静电自组装和共价键耦合这两种方法来在长周期光栅表面固定大肠杆菌适配体, 用于大肠杆菌外膜蛋白(EcOMPs)的检测。这种消失波光纤传感器能无需标记地特异性识别水中的EcOMPs, 在0.1 nM至10 nM的范围内, 谐振波长与EcOMPs的浓度呈现良好的线性关系。对于静电自组装和共价键固定这两种方法, 检测EcOMPs的灵敏度分别为(-0.156 3±0.005) nm/decade和(-0.159 7± 0.004) nm/decade。而且, 该传感器在低pH条件下可以再生, 连续检测3次后偏差小于0.1%。这种传感器已被应用于加标的环境水样中大肠杆菌的检测。经过对比发现, 涂覆有一层聚赖氨酸的具有更好的再现性和表面均匀性, 相反, 通过共价结合适配体的传感器获得了更好的加标水样品分析性能。
Application in the detection of hemoglobin/BSA
Hemoglobin is also called hemachrome, which is the main component of RBC and can combine with oxygen to transport oxygen and carbon dioxide. Hemoglobin is a special protein for transporting oxygen in RBC and the protein which makes blood red. Hemoglobin is composed of globin and heme. The globin part is the tetramer composed of two pairs of different globin chains(α chain and β chain). The content of hemoglobin can reflect the degree of anemia very well. Hemoglobin detection is of much clinical significance to recognition and diagnosis of some diseases. Chen et al. finely modulated the dispersion mode of dual-peak LPG using the method of hydrofluoric acid etched fiber cladding to enhance the sensitivity of LPG and detect the hemoglobin in sucrose solution. According to the result, when the concentration of hemoglobin changes from 0 to 1.0%, the red shift of resonance peak is 19.8 nm and the sensitivity is up to 20 nm/1%. In case of using 0.1 nm optical resolution, this sensor can detect 0.005% hemoglobin, which is a great attraction for biochemical, medical and environmental sensing applications.
血红蛋白又称血色素, 是红细胞的主要组成部分, 能与氧结合, 运输氧和二氧化碳。血红蛋白是红细胞内运输氧的特殊蛋白质, 是使血液呈红色的蛋白, 由珠蛋白和血红素组成, 其珠蛋白部分是由两对不同的珠蛋白链(α链和β链)组成的四聚体。血红蛋白的含量能很好地反映贫血程度。血红蛋白的检测对鉴别和诊断某些疾病具有重要的临床意义。Chen等人用氢氟酸腐蚀光纤包层的方法, 精细地调制了双峰长周期光栅的色散模式来提高长周期光栅的灵敏度, 并用于检测蔗糖溶液中的血红蛋白。结果显示, 当血红蛋白浓度从0变化到1.0%时, 谐振峰值红移19.8 nm, 灵敏度高达20 nm/1%。如果使用0.1 nm的光学分辨率, 这种传感器可以检测到浓度为0.005%血红蛋白, 这对于生物化学、医学和环境传感应用来说具有极大的吸引力。
BSA is a globulin in bovine serum and has been widely applied in biochemical experiments. Ficek et al. prepared diamond films on LPFG surface using the microwave chemical vapor deposition technology and studied the application of the LPFG coated with diamond films in biosensing using BSA as the external biological medium. With immobilization and forming of the BSA biological film layer on the diamond film surface, the resonance peak of the LPFG was split slightly and shifted by 0.8 nm towards the long wave direction. The detection limit of the LPFG to BSA reached 0.15 nM. This has verified the feasibility of using the LPFG coated with diamond films as a biosensor. Diamond films have good transparency, high chemical stability and abrasive resistance, nontoxicity, biocompatibility, etc., so they have unique advantages in achieving specific biosensors.
牛血清白蛋白, 是牛血清中的一种球蛋白, 在生化实验中有广泛的应用。Ficek等人利用微波化学气相沉积技术在长周期光栅表面制备了金刚石薄膜, 并用牛血清白蛋白作为外部生物介质, 研究了覆盖金刚石薄膜的长周期光栅在生物传感方面的应用。随着牛血清白蛋白生物薄膜层在金刚石薄膜表面的固定和形成, 长周期光栅的谐振峰出现轻微的分裂, 并向长波方向移动了0.8 nm, 对牛血清白蛋白的检测限达到0.15 nM。证实了覆盖金刚石薄膜的长周期光栅作为生物物质传感的可行性。由于金刚石薄膜具有良好的透明性、高度的化学稳定性和耐磨性、无毒和生物相容性等, 在实现特定的生物传感器方面有独到的优势。
Application in the detection of β-lactamase
AmpCβ lactamase(AmpC BLs) is also called AmpC, which is a kind of β-lactamase generated from mediation of chromosomes or plasmids of enterobacteriaceae or bacillus pyocyaneus and belongs to class A in β-lactamase Ambler molecular structure classification system and group Ⅰ in Bush Jacoby Medeiros functional classification system, i.e. β-lactamase which acts on cephalosporin and is not inhibited by clavulanic acid. In recent years, with extensive application of β-lactam antibiotics and especially cephalosporin, more and more gram-negative bacilli producing AmpC enzyme have appeared, and especially (derepressed) continuously high-producing AmpC enzyme and plasmid-mediated AmpC enzyme have appeared, thus leading to extensive spreading of drug-resistant strains and making it to treat the infection by this kind of bacteria. This has attracted much clinical attention. Therefore, the detection of AmpC enzyme is of much clinical significance.
AmpCβ内酰胺酶(AmpC BLs), 又称作为头孢菌素酶, 是由肠杆菌科细菌或和绿脓假单胞菌的染色体或质粒介导产生的一类β内酰胺酶, 属β内酰胺酶Ambler分子结构分类法中的C类和Bush Jacoby Medeiros功能分类法中第一群, 即作用于头孢菌素、且不被克拉维酸所抑制的β内酰胺酶。近年来, 随着β内酰胺类抗生素、尤其是头孢菌素的广泛应用, 产AmpC酶的革兰阴性杆菌越来越多见, 尤其是出现了(去阻遏)持续高产AmpC酶和质粒介导的AmpC酶, 导致耐药菌株广泛传播和临床对该类细菌感染的治疗困难, 已引起临床的高度重视, 故检测AmpC酶具有非常重要的临床意义。
Quero et al. reported a LPFG biosensor working in reflection mode for quick detection of AmpC β-lactamase. In order to enhance of the sensitivity of the LPFG to the ambient refractive index, they coated a layer of atactic polystyrene film with high refractive index on the LPFG surface. Afterwards, they coated a layer of poly(MMA)-dimethylacrylic acid copolymer on the polystyrene film surface so as to provide a functionalized surface and promote covalent immobilization of stable bioreceptors. They used 3-aminophenylboronic acid as the biological recognition element for AmpC β-lactamase in view of the fact that 3-aminophenylboronic acid has excellent recognition performance and specificity to AmpC β-lactamase. According to the result, this biosensor can detect the purified AmpC β-lactamase in PBS and its detection limit is dozens of nM. The actual effect of the proposed biosensor has also been further verified in the lysate sample of E.coil containing overexpressed AmpC β-lactamase.
Quero等人报道了一种快速检测AmpC β内酰胺酶的反射型长周期光栅生物传感器。为了增加长周期光栅对周围折射率的灵敏度, 在其表面涂覆了一层高折射率的无规聚苯乙烯薄膜。随后在聚苯乙烯薄膜表面涂一层聚(甲基丙烯酸甲酯)-二甲基丙烯酸共聚物, 以提供功能化表面, 促进稳定的生物受体的共价固定。基于3-氨基苯硼酸对AmpC β内酰胺酶具有优良的识别性能和特异性, 因而采用3-氨基苯。β内酰胺酶的生物识别元件。结果显示, 这种传感器可以检测磷酸缓冲液中纯化的AmpC β内酰胺酶, 检测线达到几十纳摩nM。所提出的生物传感器的实际效果也在含有过量表达AmpC β内酰胺酶的大肠杆菌的裂解物样品中进一步得到了证实。
Application in the detection of triglyceride
Triglyceride is the constituent of lipids, and its main function is to supply and store energy, fix and protect internal organs. Normally, triglycerides in plasma maintain a homeostasis. If the triglyceride content increases, this can induce the diseases such as diabetes, hypothyroidism, nephrotic syndrome and pancreatitis, atherosclerosis, glycogenosis, etc. If the triglyceride content is reduced, this can induce the diseases such as hyperthyroidism, decreased adrenocortical function, severe liver dysfunction, chronic obstructive pulmonary disease, cerebral infarction, malnutrition, congenital α-β lipoproteinemia, etc. Serum triglyceride assay is a conventional item of blood lipid analysis. Baliyan et al. carried out covalent immobilization of lipase on LPFG surface for triglyceride detection. The detection principle is that the interaction of triglyceride with enzyme will cause the shift of resonance wavelength in the transmission spectrum of LPFG. In comparison with the amperometric biosensor which is widely used and needs multiple enzymes, this sensor needs only a kind of enzyme, i.e. lipase. The sensor showed the best response at 37 ℃ and pH=7.4 within 1min. In addition, the sensor has high specificity to triglyceride and is not affected by other multiple interfering substances in serum. The sensor showed 0.5 nm/mM high sensitivity and 17.71 mg/dL detection limit to triglyceride in human blood.
甘油三酯是脂质的组成成分, 其主要功能是供给与储存能源、固定和保护内脏。正常情况下, 血浆中的甘油三酯保持着动态平衡; 如果甘油三酯含量增高, 可诱发糖尿病、甲状腺功能减退、肾病综合征和胰腺炎、动脉粥样硬化、糖原贮积病等疾病; 如果甘油三酯含量降低, 可诱发甲状腺功能亢进症、肾上腺皮质功能降低、肝功能严重低下、慢性阻塞性肺疾患、脑梗塞、营养不良、先天性α-β脂蛋白血症等疾病。血清甘油三酯测定成为血脂分析的常规项目。Baliyan等人将脂肪酶共价固定于长周期光纤光栅表面, 并用于甘油三酯的检测。其检测原理是基于甘油三酯与酶的相互作用会引起长周期光栅透射谱中谐振波长的漂移。与广泛使用的需要多种酶的安培型生物传感器相比, 该传感器中仅仅只需要脂肪酶这一种酶。该传感器在37 ℃和pH 7.4的条件下, 在1 min内显示出了最佳的响应。而且, 该传感器对甘油三酯具有高度的特异性, 不受血清中其它多种干扰物质的影响。该传感器对人血液中的甘油三酯显示出了0.5 nm/mM的高灵敏度和17.71 mg/dL的检测限。
Application in glucose detection
Glucose is the most widely distributed and most important monosaccharide in nature. It has an important position in the biological field and is the main energy source for organisms. In the human body, glucose can quickly replenish energy, promote liver detoxification, strengthen memory, etc. The concentration of glucose has a great influence on physiological activities. Too low glucose concentration may cause stroke or other vascular diseases. Too high glucose concentration will lead to obesity, diabetes, kidney disease, heart disease, nerve damage, etc. Glucose is the main source of carbon for cell growth and product synthesis in fermentation production, and the content of glucose directly determines the amount of proliferation and metabolism of producing strains. In addition, glucose is an important raw material for food processing, winemaking, pharmacy, mirror making, printing, dyeing and leather making, etc. Therefore, accurate determination of glucose content is of much significance to the decisions on the treatment of diseases such as diabetes etc. and the control of industrial production.
葡萄糖是自然界分布最广且最为重要的一种单糖, 在生物学领域具有重要地位, 是生物的主要供能物质。在人体中, 葡萄糖能快速补充能量、促进肝脏解毒、加强记忆等。葡萄糖的浓度对生理活动有很大影响。如果浓度过低, 可能造成中风或其他的血管疾病; 浓度过高会导致肥胖、糖尿病、肾脏病、心脏病及神经损伤等。在发酵生产中, 葡萄糖是菌体生长和产物合成的主要碳源, 其含量直接决定着生产菌增殖、代谢的数量。此外, 葡萄糖是食品加工、造酒、制药、制镜、印染制革等生产的重要原料。因此, 准确测定葡萄糖含量对于糖尿病等疾病的治疗决策和工业生产产量的控制具有重要意义。
Yang et al. assembled TiO2/PSS hybrid films on the LPFG surface using the electrostatic self-assembly technology and adjusted the refractive index sensitivity of the LPFG by changing the assembly layer number. They used this kind of porous films to detect glucose, with the detection limit up to 10-7 M. But this paper doesn't involve the selectivity of glucose detection.
Yang等人利用静电自组装技术, 在长周期光纤光栅表面组装了TiO2/PSS杂交薄膜, 通过组装层数调整了长周期光纤光栅的折射率灵敏度。并利用这种多孔结构的薄膜检测了葡萄糖, 检测限达到10-7 M, 但该文没有涉及到对葡萄糖检测的选择性。
Deep et al. modified the LPFG surface with glucose oxidase so as to achieve specific detection of glucose. The refractive index sensitivity of the LPFG used by the research group is 50-100 nm/RIU. The detection range of the sensor to glucose concentration is 0.1-3.0 mg/mL, and its sensitivity to glucose detection is 0.806 mg/mL.
Deep等人通过在长周期光栅表面修饰葡萄糖氧化酶, 实现了对葡萄糖的专一性检测。该研究组所使用的长周期光栅的折射率灵敏度为50～100 nm/RIU, 该传感器对葡萄糖浓度的检测范围是0.1～3.0 mg/mL, 对葡萄糖检测的灵敏度为0.806 mg/mL。