長周期光柵生物傳感器研究進展

李秋順，蔡 雷，馬耀宏，楊俊慧，楊 艷，孟慶軍，史建國

(齊魯工業(yè)大學(山東省科學院)，山東省科學院生物研究所 山東省生物傳感器重點實驗室，山東 濟南 250103)

1 Introduction

引 言

Photon biosensors have developed increasingly in recent years. Especially LPFG(long period fiber grating) has attracted much attention in the field of biosensing because it has many general characteristics of optical fibers such as immunity to electromagnetic interference, non-corrosiveness, small weight, miniature size, easy compatibility with optoelectronic systems,etc. as well as unique advantages including no retroreflection, low insertion loss, wide transmitted spectrum bandwidth, high sensitivity,etc.

近年來，光子生物傳感器快速發(fā)展。特別是長周期光纖光柵(long period fiber grating，LPFG)，由于其不僅具有抗電磁干擾、耐腐蝕、重量輕、體積小、易與光電系統(tǒng)兼容等諸多的光纖通有特點，還具有無后向反射、插入損耗低、透射光譜帶寬寬、靈敏度高等獨特的優(yōu)點，在生物傳感領域受到了廣泛的關注。

LPFG is a passive optical device[1-3]which is made from periodic refractive index modulation generated by carbon dioxide laser, ultraviolet laser or femtosecond laser source axially on the fiber using the tools and technologies such as photo mask, arc discharge, distortion,etc.

長周期光纖光柵是利用二氧化碳激光、紫外激光或飛秒激光光源，并結合掩模板、電弧放電、扭曲變形等工具和技術對光纖軸向上產生周期性的折射率調制制作而成、周期一般在幾百微米的一種無源光學器件[1-3]。

The transmission spectrum of LPFG is affected by cladding and surrounding environment extremely easily, so it is sensitive to the refractive index variation of environmental media[4-5]. In comparison with the extensively used SPR(surface plasma resonance) sensor, the LPFG sensor doesn't need huge prisms, rotary devices and deposited metal films, so that the LPFG sensor has very compact structure and low cost and can achieve more accurate measurement. As reported by some references[6-9], in comparison with other optical sensing devices such as Bragg grating[10-12], Fabry-Perot fiber[13], microring resonator[14-15], surface plasmon resonance[16-19], nanowire array[20-21], micro-nanofiber coupler[22],etc., LPFG has unique advantages such as higher sensitivityetc. Therefore, in recent years, many domestic and foreign researchers attempt to combine LPFG with bioactive materials for use in the detection of biological substances. Since Ashish M Vengsarkar et al. made the world's first LPFG in 1996, through over 20 years of development, LPFG has made great progress in the detection of biological substances. LPFG has been utilized extensively in the detection of many biological substances such as antigen-antibody, viruses, proteins, DNA, enzymes, nucleic acids,etc., and has become an advanced biological detection tool in the biosensing technique field. The following chapters and sections mainly describe the progress of application and study of LPFG in biological detection and look into its future development trend and prospect.

由于長周期光纖光柵的透射譜極易受包層和周圍環(huán)境的影響，因而其具有對環(huán)境介質的折射率變化比較敏感的性能[4-5]。與廣泛使用的表面等離子體共振傳感器相比，長周期光柵傳感器不需要龐大的棱鏡、無需旋轉部分器件及無需沉積金屬薄膜，這使得長周期光柵傳感器結構非常緊湊且成本低廉，測量更加精確。根據一些文獻報道[6-9]，與布拉格光柵[10-12]、光纖法珀諧振器[13]、微環(huán)諧振器[14-15]、表面等離子體共振[16-19]、納米線陣列[20,21]、微納光纖耦合器[22]等其他光學傳感器件相比，長周期光柵具有靈敏度更高的獨特優(yōu)勢。因而，近年來，國內外許多研究人員嘗試將長周期光柵與生物活性材料進行結合，用于生物物質的檢測。自從1996年Ashish M Vengsarkar等人制作出世界上第一根長周期光纖光柵以來，經過20余年的發(fā)展，長周期光柵在生物物質的檢測方面取得了很大的進展，已經被廣泛應用于抗原抗體、病毒、蛋白質、DNA、酶及核酸等多種生物物質的檢測，長周期光柵在生物傳感技術領域已經成為一種先進的生物技術檢測工具。本文主要介紹近年來長周期光柵在生物檢測方面的應用及研究進展，并對其未來發(fā)展趨勢及前景做了展望。

2 Fundamental principle of LPFG in the detection of biological substances

長周期光纖光柵檢測生物物質的基本原理

LPFG achieves the detection of biological substances through the refractive index variation caused by inductively adsorbed or immobilized on the grating surface. The fundamental principle of LPFG in the detection of biological substances is as follows :through periodic refractive index modulation in LPFG, lights are coupled from the forward-propagating fiber core basic mode to the equidirectional-propagating cladding mode, and energy is transferred between modes; after a certain propagation distance of the lights coupled into the cladding, due to the factors such as scattering between the cladding and air interface, fiber bending,etc., the cladding mode is converted into radiation mode and quickly attenuates so as to result in part energy loss; thus, a series of resonance peaks of specific wavelength can be observed in the transmission spectrum of LPFG. The matching conditions of coupling between LPFG core basic guide mode and equidirectional-propagating cladding mode conform to the formula[23]:

長周期光纖光柵是通過感應吸附或固定在光柵表面的生物物質所引起的折射率變化來實現(xiàn)對生物物質的檢測。其對生物物質檢測的基本原理是：長周期光纖光柵中周期性的折射率調制使光線從前向傳輸的纖芯基模耦合到同向傳輸的包層模，能量在模式之間發(fā)生相互轉移，耦合到包層中的光在經過一段傳輸距離后，由于包層與空氣界面的散射以及光纖彎曲等原因，包層模轉化成輻射模而迅速衰減掉，造成部分能量的損失，于是可以觀察到在長周期光纖光柵的透射譜中有一系列特定波長的諧振峰。長周期光纖光柵纖芯的基導模與同向傳輸的包層模之間耦合的相匹配條件滿

足公式[23]：

(1)

3 Application of LPFG in biological detection

長周期光纖光柵在生物檢測方面的應用

3.1 Application in the detection of antigen-antibody molecules

在抗原抗體分子檢測方面的應用

Bentley research group[24]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研究組[24]第一次將長周期光纖光柵用作生物傳感器，他們首先在長周期光柵表面進行單分子層氨基化修飾，然后用戊二醛偶聯(lián)固定抗體，進行抗原Human IgG的檢測。檢測范圍為2～100 μg/mL，在連續(xù)使用5次后，仍能達到初始信號的85%。但由于檢測限為700 μg/L，存在檢測靈敏度低的問題。

Kimetal.[25]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[25]在長周期光柵端面濺射鍍銀反射膜，用于測定抗原抗體的相互作用。當光遇到銀金屬膜后被反射，在長周期光纖光柵內產生干涉條紋。固定于光柵表面的抗體與抗原發(fā)生作用時，膜層折射率會發(fā)生變化，引起干涉條紋位置的移動，以此檢測抗原。在組裝了PAH/PSS薄膜后，用于抗兔IgG的檢測。隨著抗兔IgG的固定，諧振波長移動了約0.55 nm。

Through coupling of LPFG written in PCF with optical collimating flow cell, Heetal.[26]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.

He等人[26]將寫入光子晶體光纖中的長周期光柵與光學準直的流動池耦合，開發(fā)了一種用于無標記生物傳感的光學流體折射率傳導平臺，用于抗小鼠IgG的檢測。這種長周期光柵-光子晶體光纖體系對不同溶液介質中的局部結合所引起的折射率變化具有極高的靈敏度。通過一系列表面改性步驟，包括吸附聚烯丙胺鹽酸鹽單層、固定抗大鼠骨唾液蛋白單克隆一抗以及與非特異性山羊抗兔IgG(H+L)和特異性二抗山羊抗小鼠IgG(H+L)抗體結合的相互作用，在PCF的通道內進行模型免疫測定實驗。通過測量長周期光柵-光子晶體光纖諧振波長的位移變化，原位監(jiān)測了這些生化材料在長周期光柵表面的吸附和結合過程，生化試劑在光子晶體光纖-長周期光柵表面作用時所引起的諧振波長移動情況如圖1所示。從圖1可以看到，在長周期光柵表面單層材料的吸附和結合產生了顯著的共振波長變化，每納米厚度吸附/結合的生物分子約為0.75 nm，這表明該長周期光柵-光子晶體光纖生物傳感器可以檢測單層敏感的表面結合，在生物傳感與評估方面有很大的潛力。

Fig.1 Shift of resonance wavelength of the LPFG-PCF at various surface events 圖1 生化試劑在光子晶體光纖-長周期光柵表面作用時所引起的諧振波長移動情況

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. Chiavaiolietal. 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[27]. 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)[28].

將前向傳輸的纖芯基模耦合到靠近其轉折點的高階包層模式是實現(xiàn)無需額外的涂層(沉積在光纖上的感測層和選擇性生物層除外)而使長周期光柵具有良好生物傳感性能的一種策略。轉折點是指在長周期光纖光柵的特定包層模式下的相匹配曲線中的點，在該模式下，相同包層模式的兩個諧振峰合并成一個更寬的諧振峰。而諧振峰在轉折點附近的長周期光纖光柵具有最高的靈敏度，可以實現(xiàn)對生物物質的高靈敏檢測。Chiavaioli等人對轉折點長周期光柵的建模和制造進行了討論，構建了基于轉折點長周期光柵的新型無標簽光學生物傳感器。該研究組將Eudragit L100共聚物沉積在長周期光柵表面，隨后共價固定免疫球蛋白G(IgG)使長周期光柵表面功能化，然后實施沿光柵區(qū)域的IgG/抗IgG生物測定及抗體/抗原相互作用的動力學分析[27]。為了突出所提出的改進的免疫傳感器的優(yōu)點，對轉折點長周期光柵和非轉折點長周期光柵進行了定量比較。并用檢測復雜的人血清證明了轉折點長周期光柵生物傳感器的實際可行性和有效性及檢測的特異性，在人血清中的檢測限達到70 μg/L(460 pM)[28]。

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(10-11M)[29].

Chiavaioli研究組還提出了一種基于二氧化鈦-二氧化硅涂覆長周期光柵的倏逝波光纖生物傳感器，如圖2所示。他們采用浸涂技術在長周期光柵表面沉積了一層二氧化鈦-二氧化硅薄膜。在浸涂過程中通過改變溶膠粘度和提取速度調節(jié)薄膜覆蓋層的厚度，增加長周期光柵的折射率靈敏度。并進一步用甲基丙烯酸/甲基丙烯酸酯共聚物使長周期光柵表面功能化，之后將鼠IgG固定在長周期光柵表面，對鼠抗IgG進行檢測。通過結合過程結束時的諧振波長偏移和初始結合速率來確定分析物的濃度。這種結構化的長周期光柵對鼠抗IgG的檢測限達到8 μg/L(10-11M)[29]。

Fig.2 Schematic illustration of the optical fiber biosensor based on titania-silica coated LPFG 圖2 涂覆二氧化鈦-二氧化硅的長周期光柵生物傳感結構示意圖

Pillaetal. 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[30].

Pilla等人利用浸涂法在長周期光柵表面制備無規(guī)立構聚苯乙烯薄膜，并通過控制合適的薄膜厚度優(yōu)化長周期光柵對周圍介質折射率變化的敏感度，將長周期光柵調節(jié)至過度模式的高靈敏工作點?；谂Ｑ灏椎鞍讓郾揭蚁┍砻娴膹娬掣侥芰?，選擇牛血清白蛋白作為固定人IgG的媒介層，將牛血清白蛋白吸附于聚苯乙烯表面。然后使用戊二醛將BSA和作為原型生物感受器的人IgG偶聯(lián)，將人IgG共價固定到BSA上。并通過ELISA和表面等離子體共振分析來確定人IgG在戊二醛的暴露時間和最佳濃度。最后，在長周期光柵的過度模式區(qū)間實時監(jiān)測了人IgG與抗人IgG分子的相互作用，對人IgG分子進行檢測。結果顯示，這樣設計的長周期光柵傳感器對抗人IgG分子檢測限約為5 pg/mm2[30]。

Biswasetal. 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-SiO2thin 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等人也使用簡單和通用的溶膠-凝膠浸涂技術，將二氧化硅-二氧化鈦薄膜沉積在光柵部分，制備了超耦合長周期光纖光柵傳感器。并通過仔細調整覆蓋層的厚度和折射率，使長周期光柵處于過渡模式工作區(qū)域，從而使長周期光柵對折射率的傳感性能最優(yōu)化和最大化。并且在二氧化硅-二氧化鈦薄膜表面進一步生物功能化后，制備了一個經典的IgG/抗IgG生物測定的生物傳感器。在1.333～1.334環(huán)境折射率范圍內，涂覆159 nm厚度TiO2-SiO2薄膜時的長周期光柵的靈敏度達到7 000 nm/RIU，對抗IgG的檢測限達到0.025 mg/L[31-32]。

Queroetal. 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長周期光柵實時監(jiān)測了一種分化型甲狀腺癌的蛋白標志物—人甲狀腺球蛋白(Tg)。該研究組首先在長周期光柵表面覆蓋了一層無規(guī)立構聚苯乙烯涂層。這種無規(guī)立構聚苯乙烯涂層不僅提高了長周期光纖光柵對環(huán)境介質的折射率靈敏性，同時對抗Tg單克隆抗體具有特定的、高親和力，為固定抗Tg單克隆抗體提供了一種功能化表面。然后在聚苯乙烯涂層上面固定抗甲狀腺球蛋白，用于甲狀腺球蛋白的檢測。這種功能化的長周期光柵生物傳感器清晰地展示了提高生物傳感的有效性和靈敏性，可以以細針穿刺的形式進行無標記活體Tg檢測，用于術前和術后評估生物標志物水平的濃度。在先導測試中，使用生物素化蛋白質與HRP標記的鏈霉親和素進行光纖類ELISA法平行測定分析，證實可捕獲和識別分析物。劑量依賴性實驗表明，在0～4 ng/mL的濃度范圍內，信號與濃度呈現(xiàn)良好的線性關系；而在較高濃度時，發(fā)生抗體飽和。該傳感器的特征在于具有非常高的靈敏度和特異性，允許從不同患者的甲狀腺結節(jié)進行細針穿刺活檢，進行ng/mL級人Tg濃度的體外檢測[33-34]。

Tangetal. assembled colloidal gold nanoparticles on the LPFG surface. After optimizing assembling conditions, the sensitivity was remarkably improved and reached 10-4nm/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[35]. After further immobilization of dinitrobenzene antigen on gold surfaced, the detection limit reached 1.4×10-7g/mL or 9.5×10-10M[36]during the use in specific detection of dinitrobenzene antibody anti-DNP.

Tang等人在長周期光纖光柵表面組裝了金納米溶膠，在優(yōu)化了組裝的條件后，靈敏度顯著提高，對1.34～1.39范圍內的靈敏度達到10-4nm/URI的分辨率，說明這種傳感器非常適合于水溶液中生化物質的傳感[35]。在金表面進一步固定了二硝基苯抗原后，用于專一性檢測二硝基苯抗體anti-DNP時，檢測線達到1.4×10-7g/mL或9.5×10-10M[36]。

3.2 Application in the detection of nucleotide/DNA molecules

在核苷酸/DNA分子檢測方面的應用

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.

DNA是人們研究生物分子之間的相互作用時的一個重要代表。由于DNA序列的識別和鑒定在環(huán)境健康保護、疾病診斷、疾病治療、病毒的控制、藥物研發(fā)等領域有著重要的用途，DNA分子對的雜交檢測成為光纖生物傳感器應用的一個熱點。

Sozzietal. 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[37].

Sozzi等人通過在長周期光柵表面固定與DNA靶鏈相匹配的肽核酸探針，對光纖表面進行功能化，證明了長周期光柵用作DNA生物傳感器的可行性。在120 nM的DNA溶液中，諧振波長移動了1.2 nm[37]。

Chenetal. 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[38], 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等人利用雙諧振峰長周期光纖光柵實現(xiàn)了DNA序列雜交的檢測。他們首先使光柵表面硅烷化，然后用辛二亞氨二甲基醚把探針DNA共價固定于長周期光纖光柵表面，隨后在線監(jiān)測其與互補靶DNA序列雜交過程。結果顯示，在60 min內雜交反應過程有兩個階段：在前3 min，顯示出86 pm的移動速率；從3 min至60 min，以9 pm/min的速率進行更慢的反應過程。雜交反應在60 min內引起的總波長移動大約是715 pm。與先前報道的基于腐蝕的布拉格光柵DNA生物傳感器[38]相比，這種雙諧振峰長周期光纖光柵DNA生物傳感器實現(xiàn)了高達3倍的反應速率。由于采用了共價固定，在多次加熱/冷卻解離DNA后，該傳感器仍然能很好地識別目標DNA，顯示了良好的重現(xiàn)性[39-40]。

Delgado-Pinaretal. 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[41].

Delgado-Pinar等人將納米窄帶長周期光柵用于DNA的檢測。為了提高長周期光柵的生物傳感性能，該研究組通過選擇適當的制造參數(高數值孔徑、相對高階模式和較大的長度)，制造了只有1.5 nm的3-dB帶寬的長周期光柵。相對于在1 500 nm處的標準長周期光柵，這種長周期光柵的線寬減少一個數量級以上，從而使傳感器的分辨率顯著提高。在將長周期光柵表面功能化后，進行了DNA的雜交檢測。結果顯示，當該生物傳感器浸沒在2 μM的互補DNA鏈溶液中時，傳感器的靈敏度為-10%/μM，檢測限約為10 nM[41]。

Jangetal. 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[42].

Jang等人通過光刻技術在側面拋光的光纖表面上制作了長周期光纖光柵，用于實時檢測DNA的雜交。首先用聚-L-賴氨酸修飾長周期光纖光柵的二氧化硅表面，然后在聚-L-賴氨酸表面固定ssDNA探針，隨后用于與靶ssDNA的雜交。結果顯示：由于DNA雜交的快速反應，大部分的波長移動發(fā)生在前9 min，即0.94 nm的波長偏移；由DNA雜交引起的總體波長偏移是1.82 nm。這種側面拋光的長周期光柵DNA傳感器比先前Chen Xianfeng研究小組報道的雙諧振峰長周期光柵DNA傳感器提高了約2.5倍[42]。

Rindorfetal. 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[43].

Rindorf等人將光子晶體長周期光柵用于DNA的檢測。結果顯示，在光子晶體光纖的孔內壁固定一層聚-L-賴氨酸后，長周期光柵的諧振波長移動了6.7 nm；當進一步結合一單層雙鏈DNA時，諧振波長移動了2.3 nm。就每納米厚度的生物分子層所引起的移動諧振波長而言，這種光子晶體長周期光纖光柵DNA傳感器具有大約1.4 nm/nm的靈敏度[43]。

Chenetal. 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[44].

Chen等人利用雙諧振峰長周期光柵設計了寡核苷酸光纖生物傳感器。第一步直接用1-乙基-3-(3-二甲氨基丙基)碳二亞胺將未修飾的5′-磷酸化寡核苷酸探針共價固定到氨基酸衍生化的光纖光柵表面。由于是通過5′磷酸特異性連接，留下了可用于結合反應的寡核苷酸的其余部分。這種長周期光柵的折射率靈敏度是先前長周期光柵的50倍，功能化后的長周期光柵傳感器用于監(jiān)測互補的寡核苷酸，結果顯示，可以檢測到4 nM的雜交寡核苷酸的濃度[44]。

Fig.3 Schematic representation of DNA detection process using LPFG 圖3 長周期光柵檢測DNA過程示意圖

Based on LPFG and a component optical detection device, Goncalvesetal. 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[45].

Gon?alves等人基于長周期光柵和一種組件光學檢測裝置，提出了一種測定長周期光柵共振光譜的新方法，用于原位實時檢測DNA雜交。圖3給出了該傳感器系統(tǒng)對DNA檢測的過程示意圖。該傳感器對DNA的檢測限為(62±2) nM，定量限為(209±7) nM。通過與探針不同的堿基錯配的DNA靶點作用，證明了該傳感器系統(tǒng)對檢測樣本具有良好的特異性[45]。

3.3 Application in the detection of biotin-streptavidin system

在生物素-鏈霉親和素體系檢測方面的應用

There is a high specific binding affinity between biotin and streptavidin(affinity constant up to 1015mol/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.

生物素與鏈霉親和素之間具有高度的特異性結合能力(親和常數高達1015mol/L)，二者一旦結合就極難拆解，這兩種物質之間的作用是目前已知的最強的非共價鍵相互作用。而且它們的結合特性基本不受試劑濃度、酸堿pH、變性劑、緩沖液的離子強度、蛋白溶解酶、有機溶劑等環(huán)境的影響，不受反應試劑的非特異性作用的干擾。因而，在驗證傳感器件的生物傳感性能時，生物素與鏈霉親和素成為研究者最常用的一種生物傳感主客體配對體系。

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研究組在光柵表面浸涂了不同厚度的聚苯乙烯，對長周期光纖光柵的靈敏度進行優(yōu)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，隨后用于鏈霉親和素的檢測，動態(tài)地監(jiān)測了生物分子隨時間的吸附反應過程[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[48]. See Fig.4 for the modification process of coated LPFG interface with different biological agents.

由于直接吸附的方式僅僅依靠親疏水性結合，很容易受溶液的pH值、離子濃度的影響，該研究組對檢測方式做了進一步的改進，采用浸涂多層薄膜及共價耦合的方法設計了長周期光纖光柵生物傳感器，用于生物素-鏈霉親和素體系的檢測。首先，浸涂透明的聚合物無規(guī)立構聚苯乙烯提高長周期光纖光柵的靈敏度，同時調整光譜儀工作點使其處于長周期光纖光柵過渡區(qū)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價交聯(lián)的羧基官能團，這樣的處理大大降低了光柵表面功能化所帶來的包層模式過渡區(qū)域的調諧難度；第三步，使用EDC/NHS將羧基與鏈霉親和素偶聯(lián)，使其能高靈敏實時監(jiān)測鏈霉親和素與生物素化的牛血清白蛋白的相互作用過程[48]。不同生物試劑在長周期光柵表面修飾的過程如圖4所示。

Fig.4 Sketch of coated LPFG interface with different biological agents 圖4 長周期光柵表面不同生物試劑修飾示意圖

Wangetal. 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[49].

Wang等人用靜電自組裝技術在長周期光纖光柵的表面制備PAH/PCBS薄膜，然后吸附生物素，在用牛血清白蛋白封閉后，用于抗生物素蛋白的檢測。這種傳感器對0～0.075 mg/mL之間五種不同濃度的鏈霉親和素有很好的區(qū)分能力，檢測的最低濃度小于0.012 5 mg/mL[49]。

Using the LBL self-assembly method, Marquesetal. 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/mm2and 19 pg/mm2respectively. 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等人采用層層自組裝方法在長周期光柵表面沉積PAH/SiO2∶Au核殼納米粒子，制備了高靈敏性的長周期光柵生物傳感器，用于鏈霉親和素的檢測。在SiO2∶Au核殼納米粒子表面進一步修飾生物素，作為檢測鏈霉親和素的配體。SiO2∶Au核殼納米粒子的厚度和直徑有效地增加了固定生物素的面積，進而增加了長周期光柵對鏈霉親和素的靈敏度。組裝了3層PAH/SiO2∶Au薄膜的長周期光柵對鏈霉親和素的最低檢測濃度達到2.5 nM，靈敏度為6.9 nm/ng/mm2，檢測限為19 pg/mm2。該傳感器的靈敏度比先前報道的其它光纖生物傳感器[24,30,49-50]至少提高了兩個數量級。并可以通過改變配體來調整該傳感器的傳感性質[51]。

3.4 Application in the detection of E.coli/phage

在大腸桿菌/噬菌體檢測方面的應用

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.

大腸桿菌是與人們日常生活關系非常密切的一類細菌，學名稱作“大腸埃希菌”，屬于腸道桿菌大類中的一種。它是寄生在人體大腸和小腸里對人體無害的一種單細胞生物，結構簡單，繁殖迅速，培養(yǎng)容易，它是生物學上重要的實驗材料。正常棲居條件下大腸桿菌并不致病，但若進入膽囊、膀胱等處可引起炎癥。在基因工程中，大腸桿菌就被用于表達基因重組后的蛋白(例如胰島素，某些疫苗等)。大腸桿菌還常常作為模型生物參與細胞學實驗。

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.

噬菌體是通過表面特異性受體分子識別宿主的有機體，對細菌具有高度的敏感性和特異性，熱穩(wěn)定性好，而且噬菌體對人類無毒，以及廉價和生產快速，在生物傳感領域已經成為抗體的一個替代物。許多研究人員將長周期光纖光柵與噬菌體結合應用于大腸桿菌/噬菌體的檢測。

Smietanaetal. 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[52]after identification and immobilization of K12 cell of E.coil.

Smietana等人利用T4噬菌體作為檢測大腸桿菌K12細胞的識別元件，采用物理吸附的方法將T4噬菌體直接固定于長周期光柵表面，原位實時監(jiān)測了噬菌體與大腸桿菌K12細胞的相互作用過程。結果表明，大腸桿菌K12細胞被識別固定后，長周期光柵的諧振波長移動了～1.3 nm[52]。

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. Tripathietal. 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 103CFU/mL, and the experiment accuracy more than 99%[53].

Fig.5 SEM micrograph of the E.coli immobilized on the surface of the LPFG:109 CFU/mL of E.coli concentration in PBS 圖5 磷酸緩沖液中濃度為109 CFU/mL的大腸桿菌在長周期光柵表面固定后的掃描電鏡圖

由于物理吸附的方法僅僅是親疏水性吸附，T4噬菌體在光柵表面的覆蓋率很小，而且容易被緩沖溶液洗脫，影響后續(xù)的檢測。Tripathi等人對上述方法進行了改進，采用共價方法固定噬菌體，即首先用硅烷偶聯(lián)劑使光柵表面氨基化，然后用雙官能團交聯(lián)劑戊二醛共價固定噬菌體，使T4噬菌體在測量過程中保持穩(wěn)定，如圖5所示。他們同時改用高精度光纖解調儀、超敏感長周期光纖光柵等來檢測大腸桿菌，實現(xiàn)了穩(wěn)定的、無標記、以噬箘體為基礎的大腸桿菌檢測，對大腸桿菌檢測的最低濃度可以達到103CFU/mL，實驗的準確度在99%以上[53]。

Brzozowskaetal. 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[54].

Brzozowska等人提出了一種基于涂覆有T4噬菌體粘附素的新型高靈敏度長周期光柵傳感器。T4噬菌體粘附素(gp37)通過識別大腸桿菌脂多糖(LPS)而以其天然或變性形式結合大腸桿菌B，這種結合具有高度的特異性和不可逆性。T4噬菌體粘附素對LPS陽性的反應幾乎是陰性LPS的3倍。長周期光柵生物功能化的方法是用能夠與gp37組氨酸標簽可逆結合的鎳離子涂覆長周期光柵表面。重組粘附噬菌體蛋白被用作生物傳感體系中的受體分子。用長周期光柵裝置對粘附素與LPS結合的特異性進行了測試，并通過蛋白質印跡、酶聯(lián)免疫吸附試驗(ELISA)和BIACORE方法做了證實。這種傳感器能夠實時、準確地測量細菌污染[54]。

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[55].

該研究小組還在長周期光柵表面涂覆了T4噬箘體黏附素用于革蘭氏陰性菌的檢測。結果顯示，與ELISA和BIAcore方法相比，這種傳感器能更明顯地識別大腸桿菌K-12 (PCM2560)。大腸桿菌K-12所引起的諧振波長移動大約是大腸桿菌B(陽性對照)的一半。而在BIAcore表面等離子體共振傳感器中，大腸桿菌K-12所引起的檢測信號(RU)是大腸桿菌B的10%。這表明長周期光柵傳感器的靈敏度明顯高于BIAcore表面等離子體共振傳感器。這種涂覆有粘附素的長周期光柵傳感器不僅可選擇性檢測大腸桿菌B，而且可用于檢測在脂多糖結構中包含OmpC或在末端糖苷鍵周圍的特殊間構型的更廣泛的革蘭氏陰性細菌組，如大腸桿菌O111(PCM418), 肺炎克雷伯菌O1(PCM1)和結腸炎耶爾森菌O1(PCM1879)。還可以檢測含有OmpC的革蘭氏陰性菌和脂多糖結構中圍繞在末端糖苷鍵的特殊空間結構。此外，實驗結果還證明了使用粘附素替代噬菌體以獲得特異性細菌菌株識別的可能性。與噬菌體相比，粘附素更穩(wěn)定且可以儲存更長的時間，因而在商業(yè)應用中有很好的前景[55]。

Dandapatetal. 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 102CFU/mL. The biosensor has showed its capacity of reliable and quantitative detection of E.coil in water at 24-40 ℃ temperature[56].

Dandapat等人提出了一種溫度不敏感型級聯(lián)長周期光柵生物傳感器，用于水中大腸桿菌的精確定量檢測。這種傳感器對溫度的靈敏度僅為1.25 pm/℃，而其折射率靈敏度為1 929 nm/RIU，柵區(qū)長度大約為3.6 cm，非常適合于生物傳感。當改變大腸桿菌的濃度時，諧振波長移動的范圍在1.3～2.5 nm之間，檢測限達到102CFU/mL。在24～40 ℃的溫度范圍內下，該傳感器展示了可靠、定量檢測水中大腸桿菌的能力[56]。

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.

由于用長周期光柵直接檢測時，對生物物質檢測的靈敏度比較低。一些研究者嘗試用高折射率的納米薄膜對長周期光柵表面修飾，提高長周期光柵對環(huán)境介質的折射率靈敏度，然后再進行對生物物質的檢測。

Smietanaetal. prepared TiO2films on the LPFG surface using the atomic layer deposition technology. According to the result, when the thickness of the TiO2overlay 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 TiO2film surface functionalization by the LBP(adhesin) of T4 phage, the sensitivity of the LPFG enhanced by TiO2to bacterial endotoxin(E.coil B LPS) was improved obviously. After E.coil LPS incubation, the resonance wavelength shift of the LPFG without TiO2film was no more than 20 nm, while that of the LPFG enhanced by deposited TiO2film was about 40 nm[57].

Smietana等人用原子層沉積技術在長周期光柵表面制備了二氧化鈦薄膜，發(fā)現(xiàn)當二氧化鈦覆蓋層厚度達到70 nm時，對于折射率為1.34的環(huán)境介質，長周期光柵的折射率靈敏度達到6 200 nm/RIU。與未涂覆薄膜的長周期光柵相比，靈敏度提高了2.8倍。此外，當二氧化鈦薄膜表面被T4噬菌體的內毒素結合蛋白(粘附素)功能化后，二氧化鈦增敏長周期光柵對細菌內毒素(大腸桿菌B脂多糖)的敏感性明顯增加：在大腸桿菌脂多糖孵化后，沒有二氧化鈦薄膜的長周期光柵的諧振波長移動不超過20 nm，沉積二氧化鈦薄膜后的長周期光柵的諧振波長移動了大約40 nm[57]。

Kobaetal. 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[58].

Koba等人提出了一種在高階包層模式的色散轉折點附近工作的長周期光柵生物傳感器。將這種噬菌體粘附素功能化的長周期光柵傳感器用特定和非特異性細菌干重進行測試，結果顯示，這種生物傳感器能夠選擇性地結合，從而識別不同的細菌。他們用大腸桿菌B的細菌干重作為陽性測試，用大腸桿菌K12和腸道沙門氏菌作為陰性測試。大腸桿菌B引起的諧振波長漂移超過90 nm，而大腸桿菌K12和腸道沙門氏菌引起的諧振波長漂移分別約為40 nm和20 nm[58]。

Phage T 7 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.

噬菌體T 7是一種感染大腸桿菌的、烈性的、具有不能收縮的短尾的多面體噬菌體，其病毒顆粒有20面體的頭部和非常小的尾，結構較復雜，頭部由5個不同的蛋白質組成，對腸桿菌和相關革蘭氏陰性菌特異。

Janczuk-Richteretal. 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 5×103PFU/mL[59].

Janczuk-Richter等將長周期光柵用于T7病毒噬箘體的檢測。他們首先將長周期光柵調整到最佳的折射率靈敏度狀態(tài)，然后用3-(三乙氧基甲硅烷基)丙基琥珀酸酐使長周期光柵表面功能化以共價結合抗T7抗體。通過追蹤由生物覆蓋物的厚度和密度增加所引起的長周期光柵透射光譜中的諧振波長移動情況來判斷T7噬菌體的存在和濃度。長周期光柵表面從修飾步驟及其對噬菌體T7檢測的示意圖如圖6所示。這種傳感器對T7噬箘體有良好的選擇性和再生性，檢測限低于5×103PFU/mL[59]。

Fig.6 Schematic representation of the LPFG surface modification steps and phage T7 detection圖6 長周期光柵表面修飾步驟及噬菌體T7檢測示意圖

3.5 Application in the detection of Francisella tularensis

在土拉弗朗西斯菌檢測方面的應用

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. Cooperetal. 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[60].

土拉弗朗西斯菌(Francisella tularensis)是土拉菌病的致病菌，是一種具有較強傳染性的胞內寄生菌，能夠引起人和動物土拉熱，主要通過媒介昆蟲在哺乳動物和人之間傳染。在自然界中已發(fā)現(xiàn)一百種以上的動物感染此菌。因其傳播途徑多樣、易擴散、毒性強而被美國疾病控制預防中心列入A類生物恐怖制劑。土拉菌病是一種人畜共患病，致死率高，及時、準確的檢測土拉弗朗西斯菌對于土拉菌病患者及時治療和防止擴散具有重要的意義。Cooper等人通過層層靜電自組裝技術在長周期光柵表面制備納米薄膜，并進一步將抗土拉弗朗西斯菌的血清免疫球蛋白(IgG)沉積到納米薄膜上，然后對土拉弗朗西斯菌進行檢測。從特異性抗原的結合所引起的峰波長的減少檢測到了土拉弗朗西斯菌的存在。該研究為快速、無培養(yǎng)和野外診斷土拉弗朗西斯菌填補了空白[60]。

3.6 Application in the detection of methicillin-resistant staphylococci

在耐甲氧西林葡萄球菌檢測方面的應用

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.

耐甲氧西林金黃色葡萄球菌是臨床上常見的毒性較強的細菌，已成為醫(yī)院和社區(qū)感染的重要病原菌之一，是醫(yī)院、社區(qū)和動物的主要健康問題。為了保護人們的健康，對耐甲氧西林金黃色葡萄球菌進行無培養(yǎng)的、高靈敏性、特異性的快速診斷分析至關重要。

In order to meet this need, Bandaraetal. 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 102CFU/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 106CFU/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 104CFU/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[61].

為了滿足這一需要，Bandara等人用納米薄膜修飾的長周期光柵生物傳感器檢測了耐甲氧西林金黃色葡萄球菌。該研究組利用自組裝技術，在長周期光纖光柵表面沉積了PAH/PCBs多層薄膜，使長周期光柵多層膜的末端具有大量的羧基官能團。這些末端羧基官能團被進一步共價偶聯(lián)到對耐甲氧西林金黃色葡萄球菌的青霉素結合蛋白2a有特異性識別的單克隆抗體上。將該組裝多層薄膜的長周期光柵暴露到102CFU/mL的耐甲氧西林金黃色葡萄球菌(MRSA)中，50 min后透光率降低了19.7%。相反，當暴露到106CFU/mL的甲氧西林敏感的金黃色葡萄球菌(MSSA)中后，光傳輸衰減小于1.8%。將該多層膜長周期光柵暴露于感染MRSA的小鼠的肝、肺、脾的提取物中，光傳輸衰減了11.7%～73.5%。與此相反，將該多層膜長周期光柵暴露到感染MSSA的小鼠的提取液中，傳感器的光傳輸衰減等于或小于5.6%。當將該傳感器用于檢驗36株耐甲氧西林金黃色葡萄球菌、15株甲氧西林敏感的金黃色葡萄球菌、10株異種屬(所有菌株均為104CFU/mL)、感染MRSA的小鼠組織樣品時，當光傳輸衰減的臨界值為6.3%時，抗生素敏感性試驗和該生物傳感器測定的MR細菌和非MR細菌完全一致。因此，這種納米薄膜修飾的長周期光柵生物傳感器具有高靈敏性和特異性地檢測臨床樣品中的耐甲氧西林金黃色葡萄球菌的潛力[61]。

3.7 Application in the detection of fungal pathogens

在真菌病原體檢測方面的應用

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.

長周期光柵生物傳感器不僅在有關人及動物的生物成分檢測應用方面進展迅速，而且在植物病毒檢測方面的應用也有了一定的進展。

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. Gambhiretal. 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[62]

真菌病原體能導致嚴重的真菌病害，對作物產量造成巨大損失，而且，還會對人類造成致命的影響。木霉真菌種類的檢測和鑒定對于保護植物的根系免遭木霉病原菌的侵蝕具有重要的意義。Gambhir等人將銅蒸氣激光寫入的長周期光柵用于植物中真菌的檢測。在T.Harzianum、T.Viride和T.longibacterium等3種木霉菌溶液中，長周期光柵的諧振波長分別在1 524、1 520和1 522 nm，相應的光損耗分別從水中的63.75 dB變到54.85、57.34和59.76 dB，這說明長周期光柵能很好地區(qū)分不同種類的木霉菌[62]。

3.8 Application in the detection of aptamers

在適配體輔助體檢測方面的應用

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.

適配體是短的單鏈DNA或RNA寡核苷酸，它可以通過采用穩(wěn)定的三維序列依賴性結構與不同的靶標結合。它們的大小一般可以從25到90個堿基，允許選擇地控制與特定分子的反應。

Coelhoetal. 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 TiO2film on the LPFG and then a layer of polylysine on the TiO2film 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[63].

Coelho等人將凝血酶適配體固定在涂覆二氧化鈦的長周期光柵表面，用于檢測凝血酶。為了增加長周期光柵對環(huán)境介質的靈敏性，該研究組首先在長周期光柵覆蓋了30 nm厚的二氧化鈦薄膜，再在二氧化鈦薄膜表面覆蓋一層聚賴氨酸，隨后通過靜電作用將末端氨基化的凝血酶適配體固定到聚賴氨酸薄膜上，進而測定緩沖溶液中不同濃度的凝血酶，測定示意圖如圖7所示。該傳感器的檢測范圍為0.01～0.1 μM，檢測限為0.01 μM。該傳感器的功能表面可以通過5%次氯酸鈉溶液再生，有良好的再生性[63]。

Fig.7 Schematic of the sensing configurations for LPFG coated with 30 nm of TiO2 圖7 覆蓋30 nm二氧化鈦薄膜的長周期光柵傳感結構示意圖

Carrasquillaetal. 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[64]。

Carrasquilla等人首先將金納米顆粒摻雜的二氧化硅溶膠-凝膠薄膜修飾在邁克爾遜干涉式長周期光柵表面，然后把三磷酸腺苷(ATP)特異性適配體固定在金納米粒子摻雜的大孔溶膠-凝膠衍生薄膜上，使之與含有猝滅劑部分的互補序列雜交，進行無標記地探測ATP。由于大孔二氧化硅溶膠-凝膠薄膜材料能夠提供大的比表面積，可以高密度親和性地負載大量的DNA適配體，而二氧化硅薄膜內包含的金納米顆?？梢蕴峁└哒凵渎矢采w層，增強長周期光柵的折射率靈敏度。目標分子ATP的存在將誘導適配體的構象變化，使之與ATP結合，ATP的結合將導致寡核苷酸的釋放，折射率的有效變化被增強和反轉(即ATP的結合將導致分子量和折射率的凈減少)。結構轉換的適配體阻止了來自非特異性結合的信號。這種金納米粒子/二氧化硅薄膜具有改善ATP適配體結合密度和長周期光柵傳感器包層折射率的雙重功能，顯著提高了傳感器對小分子檢測的靈敏度，對ATP的檢測限達到400 μM[64]。

Queirósetal. 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[65].

Queirós等人用靜電自組裝和共價鍵耦合這兩種方法來在長周期光柵表面固定大腸桿菌適配體，用于大腸桿菌外膜蛋白(EcOMPs)的檢測。這種消失波光纖傳感器能無需標記地特異性識別水中的EcOMPs，在0.1 nM至10 nM的范圍內，諧振波長與EcOMPs的濃度呈現(xiàn)良好的線性關系。對于靜電自組裝和共價鍵固定這兩種方法，檢測EcOMPs的靈敏度分別為(-0.156 3±0.005) nm/decade和(-0.159 7± 0.004) nm/decade。而且，該傳感器在低pH條件下可以再生，連續(xù)檢測3次后偏差小于0.1%。這種傳感器已被應用于加標的環(huán)境水樣中大腸桿菌的檢測。經過對比發(fā)現(xiàn)，涂覆有一層聚賴氨酸的具有更好的再現(xiàn)性和表面均勻性，相反，通過共價結合適配體的傳感器獲得了更好的加標水樣品分析性能[65]。

3.9 Application in the detection of hemoglobin/BSA

在血紅蛋白/牛血清白蛋白檢測方面的應用

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. Chenetal. 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[66].

血紅蛋白又稱血色素，是紅細胞的主要組成部分，能與氧結合，運輸氧和二氧化碳。血紅蛋白是紅細胞內運輸氧的特殊蛋白質，是使血液呈紅色的蛋白，由珠蛋白和血紅素組成，其珠蛋白部分是由兩對不同的珠蛋白鏈(α鏈和β鏈)組成的四聚體。血紅蛋白的含量能很好地反映貧血程度。血紅蛋白的檢測對鑒別和診斷某些疾病具有重要的臨床意義。Chen等人用氫氟酸腐蝕光纖包層的方法，精細地調制了雙峰長周期光柵的色散模式來提高長周期光柵的靈敏度，并用于檢測蔗糖溶液中的血紅蛋白。 結果顯示，當血紅蛋白濃度從0變化到1.0%時，諧振峰值紅移19.8 nm，靈敏度高達20 nm/1%。如果使用0.1 nm的光學分辨率，這種傳感器可以檢測到濃度為0.005%血紅蛋白，這對于生物化學、醫(yī)學和環(huán)境傳感應用來說具有極大的吸引力[66]。

BSA is a globulin in bovine serum and has been widely applied in biochemical experiments. Ficeketal. 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[67].

牛血清白蛋白，是牛血清中的一種球蛋白，在生化實驗中有廣泛的應用。Ficek等人利用微波化學氣相沉積技術在長周期光柵表面制備了金剛石薄膜，并用牛血清白蛋白作為外部生物介質，研究了覆蓋金剛石薄膜的長周期光柵在生物傳感方面的應用。隨著牛血清白蛋白生物薄膜層在金剛石薄膜表面的固定和形成，長周期光柵的諧振峰出現(xiàn)輕微的分裂，并向長波方向移動了0.8 nm，對牛血清白蛋白的檢測限達到0.15 nM。證實了覆蓋金剛石薄膜的長周期光柵作為生物物質傳感的可行性。由于金剛石薄膜具有良好的透明性、高度的化學穩(wěn)定性和耐磨性、無毒和生物相容性等，在實現(xiàn)特定的生物傳感器方面有獨到的優(yōu)勢[67]。

3.10 Application in the detection of β-lactamase

在β-內酰胺酶檢測方面的應用

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內酰胺酶(AmpC BLs)，又稱作為頭孢菌素酶，是由腸桿菌科細菌或和綠膿假單胞菌的染色體或質粒介導產生的一類β內酰胺酶，屬β內酰胺酶Ambler分子結構分類法中的C類和Bush Jacoby Medeiros 功能分類法中第一群，即作用于頭孢菌素、且不被克拉維酸所抑制的β內酰胺酶。近年來，隨著β內酰胺類抗生素、尤其是頭孢菌素的廣泛應用，產AmpC酶的革蘭陰性桿菌越來越多見，尤其是出現(xiàn)了(去阻遏)持續(xù)高產AmpC酶和質粒介導的AmpC酶，導致耐藥菌株廣泛傳播和臨床對該類細菌感染的治療困難，已引起臨床的高度重視，故檢測AmpC酶具有非常重要的臨床意義。

Queroetal. 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[68].

Quero等人報道了一種快速檢測AmpC β內酰胺酶的反射型長周期光柵生物傳感器。為了增加長周期光柵對周圍折射率的靈敏度，在其表面涂覆了一層高折射率的無規(guī)聚苯乙烯薄膜。隨后在聚苯乙烯薄膜表面涂一層聚(甲基丙烯酸甲酯)-二甲基丙烯酸共聚物，以提供功能化表面，促進穩(wěn)定的生物受體的共價固定?；?-氨基苯硼酸對AmpC β內酰胺酶具有優(yōu)良的識別性能和特異性，因而采用3-氨基苯[68]。β內酰胺酶的生物識別元件。結果顯示，這種傳感器可以檢測磷酸緩沖液中純化的AmpC β內酰胺酶，檢測線達到幾十納摩nM。所提出的生物傳感器的實際效果也在含有過量表達AmpC β內酰胺酶的大腸桿菌的裂解物樣品中進一步得到了證實[68]。

3.11 Application in the detection of triglyceride

在甘油三酯檢測方面的應用

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. Baliyanetal. 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[69].

甘油三酯是脂質的組成成分，其主要功能是供給與儲存能源、固定和保護內臟。正常情況下，血漿中的甘油三酯保持著動態(tài)平衡；如果甘油三酯含量增高，可誘發(fā)糖尿病、甲狀腺功能減退、腎病綜合征和胰腺炎、動脈粥樣硬化、糖原貯積病等疾??；如果甘油三酯含量降低，可誘發(fā)甲狀腺功能亢進癥、腎上腺皮質功能降低、肝功能嚴重低下、慢性阻塞性肺疾患、腦梗塞、營養(yǎng)不良、先天性α-β脂蛋白血癥等疾病。血清甘油三酯測定成為血脂分析的常規(guī)項目。Baliyan等人將脂肪酶共價固定于長周期光纖光柵表面，并用于甘油三酯的檢測。其檢測原理是基于甘油三酯與酶的相互作用會引起長周期光柵透射譜中諧振波長的漂移。與廣泛使用的需要多種酶的安培型生物傳感器相比，該傳感器中僅僅只需要脂肪酶這一種酶。該傳感器在37 ℃和pH 7.4的條件下，在1 min內顯示出了最佳的響應。而且，該傳感器對甘油三酯具有高度的特異性，不受血清中其它多種干擾物質的影響。該傳感器對人血液中的甘油三酯顯示出了0.5 nm/mM的高靈敏度和17.71 mg/dL的檢測限[69]。

3.12 Application in glucose detection

在葡萄糖檢測方面的應用

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廣且最為重要的一種單糖，在生物學領域具有重要地位，是生物的主要供能物質。在人體中，葡萄糖能快速補充能量、促進肝臟解毒、加強記憶等。葡萄糖的濃度對生理活動有很大影響。如果濃度過低，可能造成中風或其他的血管疾病；濃度過高會導致肥胖、糖尿病、腎臟病、心臟病及神經損傷等。在發(fā)酵生產中，葡萄糖是菌體生長和產物合成的主要碳源，其含量直接決定著生產菌增殖、代謝的數量。此外，葡萄糖是食品加工、造酒、制藥、制鏡、印染制革等生產的重要原料。因此，準確測定葡萄糖含量對于糖尿病等疾病的治療決策和工業(yè)生產產量的控制具有重要意義。

Yangetal. 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 10-7M. But this paper doesn't involve the selectivity of glucose detection[70].

Yang等人利用靜電自組裝技術，在長周期光纖光柵表面組裝了TiO2/PSS雜交薄膜，通過組裝層數調整了長周期光纖光柵的折射率靈敏度。并利用這種多孔結構的薄膜檢測了葡萄糖，檢測限達到10-7M，但該文沒有涉及到對葡萄糖檢測的選擇性[70]。

Deepetal. 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[71].

Deep等人通過在長周期光柵表面修飾葡萄糖氧化酶，實現(xiàn)了對葡萄糖的專一性檢測。該研究組所使用的長周期光柵的折射率靈敏度為50～100 nm/RIU，該傳感器對葡萄糖濃度的檢測范圍是0.1～3.0 mg/mL，對葡萄糖檢測的靈敏度為0.806 mg/mL[71]。

4 Summary and prospect

總結和展望

This paper describes the working principle of LPFG in the detection of biological substances. LPFG has made significant progress in the detection of biological substances, but up to now, the research on biological substance detection with LPFG is only in the laboratory innovation research stage, and no commercial LPFG biosensor has appeared. It is nice to see that LPFG itself has already been commercialized. Looking around the world today, science and technology are developing rapidly and changing with each passing day. It can be expected that in the near future, with the promotion of multidisciplinary development in biological science, information science, materials science, etc., the development and application of LPFG in biosensing technology will be further quickened, and significant breakthroughs will be made in several aspects below:(1)the species and scope of biological substances to be detected by LPFG biosensors will be increased; (2)the sensitivity of LPFG biosensors to the detection of biological substances will be further enhanced, and LPFG biosensors are expected to be one of the most sensitive biosensors; (3)LPFG will be combined with other devices to achieve highly automated and integrated detection of biological substances, and LPFG biosensors are expected to develop into fast and portable integrated biosensors with high sensitivity, high specificity and low cost; (4) LPFG will achieve online continuous monitoring of biological substances in complex systems; (5)commercial LPFG biosensors will appear with further maturation of LPFG fabrication technology, optimization of LPFG surface bio-functional film modification technology and maturation of LPFG application technology. LPFG biosensors will become an important part of the emerging high-tech industry in the 21st century and will play an important role in various sectors of the national economy. LPFG biosensors have a wide range of application prospects in many fields such as food safety inspection, biological pharmacy, biochemical engineering, clinical examination, treatment monitoring, medical care, biomedicine, fermentation industry process monitoring and environmental monitoring and treatment,etc.

本文介紹了長周期光柵對生物物質檢測的工作原理，并對近年來長周期光柵在生物檢測方面的應用做了簡要的總結。雖然長周期光柵在生物物質檢測方面有了很大的進展，但是，到目前為止，長周期光柵在生物物質檢測方面的研究還僅僅集中于實驗室創(chuàng)新研究階段，尚沒有商品化的長周期光柵生物傳感器出現(xiàn)。值得喜賀的是，長周期光柵本身已實現(xiàn)了商品化?？v觀當今世界，科學技術發(fā)展迅猛，日新月異，可以預計，在不久的將來，隨著生物科學、信息科學和材料科學等多學科發(fā)展成果的推動，長周期光柵在生物傳感技術方面的開發(fā)和應用將會得到進一步的飛速發(fā)展，在以下幾個方面實現(xiàn)重大突破：(1)長周期光柵生物傳感器檢測的生物物質種類和范圍將會繼續(xù)地增多和擴大；(2)長周期光柵生物傳感器對生物物質檢測的靈敏度將會進一步被提升，有望成為最靈敏的生物傳感器之一；(3)長周期光柵將會與其它器件結合，實現(xiàn)對生物物質檢測的高度自動化與集成化，有望發(fā)展成為高靈敏度、高特異性、廉價、快速、便攜的集成化生物傳感器；(4)還將會實現(xiàn)長周期光柵在復雜的體系中進行對生物物質的在線連續(xù)監(jiān)測；(5)隨著長周期光柵制作技術的進一步成熟及其表面生物功能膜修飾技術工藝的優(yōu)化和應用技術的成熟，將會出現(xiàn)商用產品化的長周期光柵生物傳感器。長周期光柵生物傳感器將成為21世紀新興的高技術產業(yè)的重要組成部分，在國民經濟的各個部門發(fā)揮重要作用，在食品安全檢測、生物制藥、生物化工、臨床檢驗、治療時實施監(jiān)控、醫(yī)療保健、生物醫(yī)學、發(fā)酵工業(yè)過程監(jiān)測和環(huán)境監(jiān)測治理等諸多領域有廣泛的應用前景。

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