技术领域technical field
本发明属于微纳操作技术领域，具体涉及一种基于近红外光镊激发上转换发光的定量检测装置及检测方法。The invention belongs to the technical field of micro-nano operation, and in particular relates to a quantitative detection device and detection method based on near-infrared optical tweezers excitation upconversion luminescence.
背景技术Background technique
光镊即单光束梯度力光阱，是基于散射力和辐射压梯度力相互作用而形成的能够捕获整个米氏和瑞利散射范围粒子的势阱。对粒子起捕获作用的是梯度力，将粒子稳定地束缚在光场势阱中的前提是轴向梯度力能够克服散射力。所以通常需要使用较大数值孔径的显微物镜将激光束高度会聚，从而产生足够强的梯度力来实现粒子的捕获。在生物医学领域，光镊技术往往和光学显微镜技术相结合，实现单个微粒的观察、捕获和操纵。光镊对微粒最常见的捕获是当散射力小于梯度力时，微粒被梯度力稳定在溶液之中，形成悬浮的状态，称为三维捕获。Optical tweezers, that is, single-beam gradient force optical traps, are potential wells formed based on the interaction of scattering force and radiation pressure gradient force, which can capture particles in the entire Mie and Rayleigh scattering range. It is the gradient force that traps the particles, and the prerequisite for stably confining the particles in the optical field potential well is that the axial gradient force can overcome the scattering force. Therefore, it is usually necessary to use a microscope objective lens with a large numerical aperture to highly converge the laser beam, so as to generate a strong enough gradient force to achieve particle capture. In the field of biomedicine, optical tweezers technology is often combined with optical microscopy technology to realize the observation, capture and manipulation of individual particles. The most common capture of particles by optical tweezers is that when the scattering force is smaller than the gradient force, the particles are stabilized in the solution by the gradient force and form a suspended state, which is called three-dimensional capture.
上转换发光纳米材料是一种特殊的稀土发光材料，它可以通过双光子或多光子机制将低频率的激发光转化成高频率的发射光。Up-conversion luminescent nanomaterials are special rare-earth luminescent materials, which can convert low-frequency excitation light into high-frequency emission light through a two-photon or multi-photon mechanism.
发明内容Contents of the invention
本发明所要解决的技术问题是提供一种基于近红外光镊定量检测装置及检测方法。The technical problem to be solved by the present invention is to provide a quantitative detection device and detection method based on near-infrared optical tweezers.
本发明将光镊技术和上转换发光技术相结合，提出一种通过采用近红外激发光以避免激发试样中自发荧光（提高信背比），从而提高检测灵敏度的定量检测装置及检测方法，用于各类复杂样品中的金属离子、生物分子和病毒粒子等的检测。The present invention combines optical tweezers technology with up-conversion luminescence technology, and proposes a quantitative detection device and detection method that improves detection sensitivity by using near-infrared excitation light to avoid excitation of autofluorescence in samples (increasing signal-to-background ratio), It is used for the detection of metal ions, biomolecules and virus particles in various complex samples.
一种基于近红外光镊定量检测装置，包括近红外半导体激光器，所述近红外半导体激光器发出激光束经单模光纤、光纤准直器后，由望远镜透镜组扩束，再经过二向色镜反射进入物镜后瞳，经物镜聚集在样品池中形成光镊，光阱中微球的前向散射光经过聚光镜，再由二向色镜反射，透镜汇聚后，照射到散射光检测器上；A quantitative detection device based on near-infrared optical tweezers, including a near-infrared semiconductor laser, the laser beam emitted by the near-infrared semiconductor laser passes through a single-mode optical fiber and an optical fiber collimator, is expanded by a telescope lens group, and then passes through a dichroic mirror Reflected into the rear pupil of the objective lens, it is collected in the sample cell by the objective lens to form optical tweezers. The forward scattered light of the microspheres in the optical trap passes through the condenser lens, and then reflected by the dichroic mirror. After the lens is converged, it is irradiated on the scattered light detector;
光阱中微球的上转换发光经阻挡滤光片和透镜后，通过针孔照射到上转换发光检测器上。The up-conversion luminescence of the microspheres in the light trap passes through the blocking filter and lens, and irradiates the up-conversion luminescence detector through the pinhole.
还包括支撑样品池的电动平移台和照明LED。Also includes a motorized translation stage to support the sample cell and lighting LEDs.
所述近红外半导体激光器为功率可调的多种类型的近红外半导体激光器，波长975nm-989nm。The near-infrared semiconductor lasers are various types of near-infrared semiconductor lasers with adjustable power, and the wavelength is 975nm-989nm.
所述近红外半导体激光器发出激光束经过单模光纤，在其出口处的能量分布为高斯分布。The laser beam emitted by the near-infrared semiconductor laser passes through the single-mode optical fiber, and the energy distribution at its exit is Gaussian distribution.
所述散射光检测器为四象限光电检测器(QPD)。The scattered light detector is a quadrant photodetector (QPD).
所述上转换发光信号检测器可以是点式光电检测器（如PMT和APD），也可以采用成像器件（如CCD或CMOS等）。本发明中所有光电检测器均具有将光信号转化成电信号输出的能力。The up-conversion luminescence signal detector can be a point photodetector (such as PMT and APD), or an imaging device (such as CCD or CMOS, etc.). All photodetectors in the present invention have the ability to convert optical signals into electrical signals for output.
所述样品池为覆盖盖玻片的载玻片，或无机高分子材料的透明容器，其体积为微升级。The sample pool is a glass slide covered with a cover glass, or a transparent container of inorganic polymer material, and its volume is microliter.
使用上述装置的定量检测方法，包括步骤：The quantitative detection method using the above-mentioned device, comprising the steps of:
步骤一，利用微球特异性捕获待测物，并用上转换发光探针对富集于微球表面的待测物进行标记，形成微球-待测物-上转换发光探针复合物，即富集待测物的微球；In step 1, the microsphere is used to specifically capture the analyte, and the analyte enriched on the surface of the microsphere is labeled with an upconversion luminescence probe to form a microsphere-analyte-upconversion luminescence probe complex, that is Microspheres enriched with analytes;
步骤二，将富集待测物的微球置于样品池中，利用物镜聚焦近红外半导体激光器产生的满足上转换发光探针激发条件的激光束并于样品池中形成光镊；Step 2, placing the microspheres enriched with the analyte in the sample cell, using the objective lens to focus the laser beam generated by the near-infrared semiconductor laser that meets the excitation conditions of the up-conversion luminescent probe, and forming optical tweezers in the sample cell;
步骤三，利用电动平移台移动样品池，捕获富集待测物的微球，散射光检测器同步检测来自样品池的散射光；当检测到的散射光强度达到预设阈值时，电动平移台停止移动，开始进行上转换发光信号的检测；Step 3: Use the motorized translation stage to move the sample cell to capture the microspheres enriched with the analyte, and the scattered light detector detects the scattered light from the sample cell synchronously; when the detected scattered light intensity reaches the preset threshold, the motorized translation stage Stop moving and start to detect the up-conversion luminescent signal;
步骤四，根据上转换发光检测器检测到的上转换发光强度对待测物进行定量分析。Step 4, performing quantitative analysis on the analyte according to the up-conversion luminescence intensity detected by the up-conversion luminescence detector.
所述待测物为金属离子、生物分子或病毒粒子等多种可以通过特异性生物亲和或化学偶联方式富集到微球表面的待测成分。The analytes are various analyte components that can be enriched on the surface of the microspheres through specific bioaffinity or chemical coupling, such as metal ions, biomolecules, or virus particles.
所述微球为透明的无机微球或透明的高分子微球，无机微球可以是二氧化硅等材料制备的微球，高分子微球可以是聚苯乙烯等材料制备的微球。The microspheres are transparent inorganic microspheres or transparent polymer microspheres. The inorganic microspheres can be microspheres made of materials such as silicon dioxide, and the polymer microspheres can be microspheres made of materials such as polystyrene.
所述的上转换发光探针为稀土掺杂的上转换发光纳米粒子。The up-conversion luminescence probes are rare earth-doped up-conversion luminescence nanoparticles.
本发明检测方法可以采用多种方法特异性富集待测物。如通过双抗免疫夹心法，利用微球和上转换发光探针特异性识别并捕获待测物，形成微球－待测物－上转换发光探针复合物；然后通过聚焦近红外激光的电控样品池的二维自动精密移动和采用QPD检测器定量监测微球表面散射光以逐一捕获多个复合物微球，同时利用上转换发光检测器对复合物微球进行上转换发光探测以实现待测物的定量分析。The detection method of the present invention can use various methods to specifically enrich the analyte. For example, through the double-antibody immune sandwich method, the microsphere and the up-conversion luminescence probe are used to specifically recognize and capture the analyte, forming a microsphere-analyte-up-conversion luminescence probe complex; Control the two-dimensional automatic and precise movement of the sample cell and use the QPD detector to quantitatively monitor the scattered light on the surface of the microspheres to capture multiple complex microspheres one by one. Quantitative analysis of analytes.
本发明装置采用的光镊捕获和上转换发光平台为显微镜，采用二维电动平移台达到移动样品的需求。同时，本发明检测方法用不同上转换发光探针来标记用微球富集的不同待测物，采用高灵敏度点式光电检测器检测单个微球的上转换发光信号的强度，也可采用成像器件获取单个微球的上转换发光图像。The optical tweezers capture and up-conversion luminescent platform used in the device of the present invention is a microscope, and a two-dimensional electric translation platform is used to meet the requirement of moving samples. At the same time, the detection method of the present invention uses different up-conversion luminescent probes to mark different analytes enriched with microspheres, and uses a high-sensitivity point-type photodetector to detect the intensity of the up-conversion luminescence signal of a single microsphere. The device acquires upconverted luminescence images of individual microspheres.
与现有技术相比，本发明具有以下特点和有益效果：Compared with the prior art, the present invention has the following characteristics and beneficial effects:
1、本发明装置将光镊技术和上转换发光技术结合，提出一种新型的定量检测装置，该装置可对多种待测成分进行定量检测。1. The device of the present invention combines optical tweezers technology and up-conversion luminescence technology to propose a new type of quantitative detection device, which can perform quantitative detection of various components to be measured.
2、本发明检测方法具有灵敏度高、选择性好、速度快、样品用量少且无需预处理等优势。2. The detection method of the present invention has the advantages of high sensitivity, good selectivity, fast speed, less sample consumption and no need for pretreatment.
3、本发明装置采用的980nm近红外激光不会激发生物体系中的自发荧光，避免了自发荧光的干扰，显著提高了检测的信背比，从而提高检测灵敏度，因此可适用于复杂样品中的多种成分的检测，无需分离待测微球，有效简化了检测流程。3. The 980nm near-infrared laser used in the device of the present invention will not excite the autofluorescence in the biological system, avoid the interference of autofluorescence, and significantly improve the signal-to-background ratio of the detection, thereby improving the detection sensitivity, so it can be applied to complex samples. For the detection of multiple components, there is no need to separate the microspheres to be tested, which effectively simplifies the detection process.
附图说明Description of drawings
图1为本发明装置光路及结构示意图；Fig. 1 is a schematic view of the light path and structure of the device of the present invention;
图2为工作曲线示意图。Figure 2 is a schematic diagram of the working curve.
具体实施方式Detailed ways
如图1所示，一种基于近红外光镊定量检测装置，包括近红外半导体激光器，所述近红外半导体激光器发出激光束经单模光纤、光纤准直器后，由望远镜透镜组扩束，再经过二向色镜反射进入物镜后瞳，经物镜聚集在样品池中形成光镊，光阱中微球的前向散射光经过聚光镜，再由二向色镜反射，透镜汇聚后，照射到散射光检测器上；As shown in Figure 1, a quantitative detection device based on near-infrared optical tweezers includes a near-infrared semiconductor laser. The near-infrared semiconductor laser emits a laser beam that passes through a single-mode optical fiber and an optical fiber collimator, and is expanded by a telescope lens group. After being reflected by the dichroic mirror, it enters the rear pupil of the objective lens, and is gathered in the sample cell by the objective lens to form optical tweezers. The forward scattered light of the microspheres in the optical trap passes through the condenser mirror, and then is reflected by the dichroic mirror. After the lens converges, it irradiates the on the scattered light detector;
光阱中微球的上转换发光经阻挡滤光片和透镜后，通过针孔照射到上转换发光检测器上。The up-conversion luminescence of the microspheres in the light trap passes through the blocking filter and lens, and irradiates the up-conversion luminescence detector through the pinhole.
本发明的近红外半导体激光器产生的激光束经准直镜和望远镜透镜组（L1，L2）调整光束汇聚程度，使激光束充满物镜的后瞳，经望远镜透镜组调整后的激光束射入二向色镜(DM)。The laser beam generated by the near-infrared semiconductor laser of the present invention is adjusted by the collimator and the telescope lens group (L1, L2) to converge the beam, so that the laser beam fills the rear pupil of the objective lens, and the laser beam adjusted by the telescope lens group enters the two dichroic mirror (DM).
二向色镜反射激光进入合适数值孔径的物镜，经物镜聚焦在样品池底部并形成光阱即光镊。来自微球的散射光信号（波长和激光相同）经过聚光镜汇聚为平行光，再由DM反射，经过透镜L3汇聚，落射到散射光检测器上。The dichroic mirror reflects the laser light into the objective lens with suitable numerical aperture, and is focused on the bottom of the sample cell by the objective lens to form an optical trap, that is, optical tweezers. The scattered light signal (with the same wavelength as the laser) from the microsphere is converged into parallel light by the condenser, reflected by the DM, converged by the lens L3, and falls onto the scattered light detector.
本发明中采用高数值孔径物镜。本发明中的样品池为盖玻片或材质为无机高分子透明材料的微小容器。A high numerical aperture objective lens is used in the present invention. The sample pool in the present invention is a cover glass or a tiny container whose material is an inorganic polymer transparent material.
二向色镜DM为短通滤光片，可以透过可见光，反射近红外光。因此激光束被二向色镜DM反射至物镜聚焦形成光镊；由样品池中微球散射的近红外前向散射光信号经过聚光镜的汇聚，由一个同样的二向色镜DM反射，经过透镜L3聚焦落射到散射光检测器。同时，由微球表面返回的位于可见光区的上转换发光信号则透过DM，通过阻挡滤光片（BF）和聚焦镜L4聚焦后，通过针孔并由上转换发光检测器进行检测。The dichroic mirror DM is a short-pass filter that can pass through visible light and reflect near-infrared light. Therefore, the laser beam is reflected by the dichroic mirror DM to the objective lens to focus to form optical tweezers; the near-infrared forward scattered light signal scattered by the microspheres in the sample cell is converged by the condenser mirror, reflected by the same dichroic mirror DM, and passed through the lens L3 focuses epi-incident onto the scattered light detector. At the same time, the up-conversion luminescence signal returned from the surface of the microsphere in the visible region passes through the DM, is focused by the blocking filter (BF) and the focusing lens L4, passes through the pinhole and is detected by the up-conversion luminescence detector.
本装置采用二维电动平移台来实现多微球的逐一快速捕获和上转换发光测定，且捕获和上转换发光检测可以同时进行。The device adopts a two-dimensional electric translation stage to realize rapid capture and up-conversion luminescence measurement of multiple microspheres one by one, and the capture and up-conversion luminescence detection can be performed simultaneously.
当样品池内溶液中存在一定浓度的微球－待测物－上转换发光探针复合物微球，且光阱力足够时，移动中的某一微球靠近光阱被会被光阱捕获。在这个过程中，散射光检测器全程检测来自微球的近红外光散射光，当其强度达到预先设定的阈值时，电动平移台系统停止移动，上转换发光检测器检测来自微球的上转换发光强度。当完成一个微球的测定后，电动平移台继续移动，捕获并探测下一个微球，直至完成一定数量微球的上转换发光检测。When there is a certain concentration of microspheres-analyte-upconversion luminescent probe complex microspheres in the solution in the sample cell, and the optical trap force is sufficient, a moving microsphere close to the optical trap will be captured by the optical trap. In this process, the scattered light detector detects the near-infrared scattered light from the microspheres throughout the whole process. When the intensity reaches the preset threshold, the motorized translation stage system stops moving, and the up-conversion luminescence detector detects the up-conversion light from the microspheres. Converts luminous intensity. After the determination of a microsphere is completed, the motorized translation stage continues to move to capture and detect the next microsphere until the up-conversion luminescence detection of a certain number of microspheres is completed.
本发明可通过测定的来自微球的上转换发光强度对微球表面的富集的待测物进行定量分析，可采用工作曲线法，具体如下：The present invention can quantitatively analyze the enriched analyte on the surface of the microsphere by measuring the upconversion luminous intensity from the microsphere, and can adopt the working curve method, specifically as follows:
首先，制备某待测物的免疫微球。然后，按照设定的浓度梯度配制至少6份待测物标准溶液（包括空白溶液），分别采用相同的微球免疫夹心法（所用上转换发光探针也相同）对该系列待测物标准溶液进行分析，对每份标样均测定至少50个微球的上转换发光强度，取其平均值，绘制工作曲线，即待测物浓度和上转换发光强度曲线，该曲线在一定范围内为直线。最后，按照相同方法测定未知样所含相同待测物的上转换发光强度，根据以上工作曲线计算待测物含量。First, immune microspheres of a certain substance to be tested are prepared. Then, prepare at least 6 standard solutions of the analyte (including the blank solution) according to the set concentration gradient, and use the same microsphere immune sandwich method (the same up-converting luminescence probe is used) to test the series of standard solutions of the analyte For analysis, measure the up-conversion luminous intensity of at least 50 microspheres for each standard sample, take the average value, and draw the working curve, that is, the concentration of the analyte and the up-conversion luminous intensity curve, which is a straight line within a certain range . Finally, the up-conversion luminescence intensity of the same analyte contained in the unknown sample was measured according to the same method, and the content of the analyte was calculated according to the above working curve.
图2为工作曲线法定量的示意图，共配制7份待测物标准溶液（含空白溶液），每个标准样品均至少平行测定50个微球的上转换发光，取平均值，同时计算其标准偏差并将其在图中每点上标识出来。该曲线的横坐标为待测物标准溶液浓度，纵坐标为上转换发光强度，若样品中待测物的上转换发光强度在工作曲线的线性范围内，则可实现对待测物的定量分析。Figure 2 is a schematic diagram of the quantification of the working curve method. A total of 7 standard solutions of the analyte (including the blank solution) were prepared. For each standard sample, the upconversion luminescence of at least 50 microspheres was measured in parallel, and the average value was taken, and its standard was calculated at the same time. Deviations are identified at each point on the graph. The abscissa of the curve is the concentration of the standard solution of the analyte, and the ordinate is the up-conversion luminescence intensity. If the up-conversion luminescence intensity of the analyte in the sample is within the linear range of the working curve, the quantitative analysis of the analyte can be realized.
下面将实施例1～2对本发明检测方法做进一步说明。The detection method of the present invention will be further described in Examples 1-2 below.
实施例1Example 1
甲型流感病毒H9N2的定量分析Quantitative Analysis of Influenza A Virus H9N2
以聚苯乙烯微球作为载体，采用双抗免疫夹心法对甲型流感病毒进行富集，具体是以单克隆抗体修饰微球表面，以UCNPs标记单克隆抗体。Polystyrene microspheres were used as carriers, and influenza A virus was enriched by the double-antibody immune sandwich method, specifically, the surface of the microspheres was modified with monoclonal antibodies, and the monoclonal antibodies were labeled with UCNPs.
首先，制备免疫微球。取表面羧基修饰的聚苯乙烯微球，采用化学偶联法在其表面偶联H9N2病毒的抗-HA 单克隆抗体得到免疫微球，备用。First, immunomicrospheres are prepared. Take the surface carboxyl-modified polystyrene microspheres, and use the chemical coupling method to couple the anti-HA monoclonal antibody of H9N2 virus on its surface to obtain immune microspheres, which are ready for use.
对H9N2禽流感病毒进行定量检测Quantitative detection of H9N2 avian influenza virus
在免疫微球上富集H9N2禽流感病毒：在含H9N2禽流感病毒的样品溶液中加入一定浓度（104-106个/mL）的免疫微球中，并加入标记有UCNPs的单克隆抗体于37 ℃下孵育1小时左右得到富集待测物的微球，所采用的UCNPs的发射波长范围为547±25nm。Enrichment of H9N2 avian influenza virus on immune microspheres: add a certain concentration (104 -106 /mL) of immune microspheres to the sample solution containing H9N2 avian influenza virus, and add monoclonal antibodies labeled with UCNPs Incubate at 37°C for about 1 hour to obtain microspheres enriched with the analyte, and the emission wavelength range of the UCNPs used is 547±25nm.
采用本装置测定50-100个微球的上转换发光信号强度，并用平均值定量；依据所测定的上转换发光信号强度，采用工作曲线法获取待测物的浓度，从而实现定量检测。检测结果精密度（RSD）可以达到5%，检测限为在100μL的反应体系中可检测出约50个病毒颗粒，和目前现在的病毒检测中最灵敏的荧光定量PCR方法的检测限在同一数量级上。The device is used to measure the up-conversion luminescence signal intensity of 50-100 microspheres, and the average value is used for quantification; according to the measured up-conversion luminescence signal intensity, the working curve method is used to obtain the concentration of the analyte, so as to realize quantitative detection. The precision (RSD) of the detection results can reach 5%, and the detection limit is about 50 virus particles can be detected in a 100 μL reaction system, which is in the same order of magnitude as the detection limit of the most sensitive fluorescent quantitative PCR method in current virus detection superior.
实施例2Example 2
肺癌标志物CEA的定量分析Quantitative Analysis of Lung Cancer Marker CEA
以聚苯乙烯微球作为载体，采用适配体偶联方法对癌胚抗原CEA进行富集，具体是以CEA的适配体修饰微球表面，以UCNPs标记适配体。两条适配体，适配体1序列为：3′-ATACCAGCTTATTCAATT-5′，适配体2序列为：3′-AGGGGGTGAAGGGATACCC-5′，两条适配体分别对应CEA抗原上的不同位点，适配体1用于制备捕获微球，适配体2用于制备上转换探针。Using polystyrene microspheres as a carrier, carcinoembryonic antigen CEA was enriched by the aptamer coupling method, specifically, the surface of the microspheres was modified with CEA aptamers, and the aptamers were labeled with UCNPs. Two aptamers, the sequence of aptamer 1 is: 3′-ATACCAGCTTATTCAATT-5′, the sequence of aptamer 2 is: 3′-AGGGGGTGAAGGGATACCC-5′, and the two aptamers correspond to different sites on the CEA antigen respectively , aptamer 1 was used to prepare capture microspheres, and aptamer 2 was used to prepare upconversion probes.
首先，制备捕获微球。取表面羧基修饰的聚苯乙烯微球，采用化学偶联法在其表面偶联CEA的适配体得到捕获微球，备用。First, capture microspheres are prepared. Take the polystyrene microspheres modified with carboxyl groups on the surface, and couple the CEA aptamer on its surface by a chemical coupling method to obtain capture microspheres for future use.
然后，在含CEA的样品溶液中加入一定浓度（104-106个/mL）的捕获微球，并加入标记有UCNPs的适配体于室温下孵育1小时左右，所采用的UCNPs的发射波长为547±25nm。Then, a certain concentration (104 -106 /mL) of capture microspheres was added to the sample solution containing CEA, and aptamers labeled with UCNPs were added and incubated at room temperature for about 1 hour. The emission of the UCNPs used The wavelength is 547±25nm.
采用本装置测定50-100个富集了CEA的微球的上转换发光信号强度，并用平均值定量；依据所测定的上转换发光信号强度，采用工作曲线法对CEA进行定量检测。检测结果精密度（RSD）可以达到5%，检测限在pg／mL的数量级。比常规的临床检测方法提升了约两个数量级，具有很好的应用前景。The device is used to measure the up-conversion luminescence signal intensity of 50-100 CEA-enriched microspheres, and the average value is used for quantification; according to the measured up-conversion luminescence signal intensity, the working curve method is used for quantitative detection of CEA. The precision (RSD) of the detection results can reach 5%, and the detection limit is in the order of pg/mL. Compared with conventional clinical detection methods, it is improved by about two orders of magnitude, and has a good application prospect.
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| CN201510190040.1ACN104749105A (en) | 2015-04-21 | 2015-04-21 | Quantitative detection device and detection method based on near-infrared optical tweezers excited up-conversion luminescence |
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