技术领域Technical Field
本发明涉及在线吸光度检测技术领域，具体涉及一种微流控芯片和在线吸光度检测系统。The invention relates to the technical field of online absorbance detection, and in particular to a microfluidic chip and an online absorbance detection system.
背景技术Background Art
许多细菌代谢物是低分子量化合物，气相色谱-质谱法(MS)、液相色谱-MS及其衍生物通常用于细菌代谢物检测。尽管这些传统方法具有准确和实用等优点，但它们复杂、耗时、昂贵，不适合快速和实时检测。部分研究者开发了比色和电化学方法，以实现精确的原位代谢物检测。这些较新的检测方法通常设计用于定向传感，这可能会阻碍其检测复杂代谢物混合物中未知化学物种的能力，因此开发检测细菌代谢物的新方法及其重要。于是有研究者将液滴微流控系统和吸光度检测应用于细菌代谢物检测。Many bacterial metabolites are low molecular weight compounds, and gas chromatography-mass spectrometry (MS), liquid chromatography-MS and their derivatives are often used for bacterial metabolite detection. Although these traditional methods have advantages such as accuracy and practicality, they are complex, time-consuming, expensive, and not suitable for rapid and real-time detection. Some researchers have developed colorimetric and electrochemical methods to achieve accurate in situ metabolite detection. These newer detection methods are often designed for directional sensing, which may hinder their ability to detect unknown chemical species in complex metabolite mixtures. Therefore, it is very important to develop new methods for detecting bacterial metabolites. Therefore, some researchers applied droplet microfluidics systems and absorbance detection to bacterial metabolite detection.
与连续流动的微流控系统相比，用于单细胞的液滴微流控系统的优势在于：液滴之间互不相容，每个液滴皆可作为独立的微反应器，可以将待检测的细胞样品单独包埋，进行单细胞及其胞外分泌代谢产物的分析。液滴微流控系统短时间内可以生成大量的液滴，速度达到数千个液滴每秒，适合高通量的生物和化学分析。液滴直径均一可控，液滴体积从皮升至纳升不等，降低了样品与试剂的消耗。液滴微流控系统提供了在动态环境中研究单个细菌的方法，液滴微流控系统可以准确控制灵敏度更高的细菌微环境，有助于在更小尺度上进一步探索细菌生命。液滴微流控系统实现了小型化，实现了皮升体积液滴的处理。Compared with the continuous flow microfluidic system, the advantage of the droplet microfluidic system for single cells is that the droplets are incompatible with each other, and each droplet can be used as an independent microreactor. The cell sample to be tested can be embedded separately to analyze the single cell and its extracellular secretory metabolites. The droplet microfluidic system can generate a large number of droplets in a short period of time, with a speed of thousands of droplets per second, which is suitable for high-throughput biological and chemical analysis. The droplet diameter is uniform and controllable, and the droplet volume ranges from picoliters to nanoliters, which reduces the consumption of samples and reagents. The droplet microfluidic system provides a method for studying single bacteria in a dynamic environment. The droplet microfluidic system can accurately control the bacterial microenvironment with higher sensitivity, which helps to further explore bacterial life on a smaller scale. The droplet microfluidic system has achieved miniaturization and realized the processing of picoliter droplets.
吸光度检测是一种基于物质对光的吸收特性进行分析的方法，是评估微流体分析中细胞动力学的有效和常用方法。由于分析物和产品会影响光学吸收光谱，可以通过吸收检测来估计各种细胞行为。当光束照射到样品溶液时，不同化合物具有不同的吸收光谱特征，这些特征可用于识别和区分不同化合物。通过测量样品溶液的吸收强度，可以对化合物的浓度进行定量分析。吸光度检测对微量的目标化合物及低浓度的样品具有较强的检测能力。利用吸光度检测分析细菌代谢物的吸收特性，可以对细菌的生长状态、代谢活性等进行监测和研究。吸光度检测的依据是朗伯比尔定律，朗伯定律表明吸收物介质的厚度会影响吸收响应，比尔定律表明吸收物的浓度也会影响吸收响应。两者相结合表达了物质吸收光的定量关系，吸光度检测是一种应用广泛的通用光学检测方法，具有可测定物质种类多、结构简单等优点，也是较早用于微流控分析系统的检测方法之一。Absorbance detection is a method based on the absorption characteristics of light by a substance. It is an effective and common method for evaluating cell dynamics in microfluidic analysis. Since analytes and products affect the optical absorption spectrum, various cell behaviors can be estimated by absorption detection. When the light beam is irradiated to the sample solution, different compounds have different absorption spectrum characteristics, which can be used to identify and distinguish different compounds. By measuring the absorption intensity of the sample solution, the concentration of the compound can be quantitatively analyzed. Absorbance detection has a strong detection ability for trace target compounds and low-concentration samples. Using absorbance detection to analyze the absorption characteristics of bacterial metabolites, the growth status and metabolic activity of bacteria can be monitored and studied. The basis of absorbance detection is Lambert-Beer's law. Lambert's law shows that the thickness of the absorbent medium will affect the absorption response, and Beer's law shows that the concentration of the absorbent will also affect the absorption response. The combination of the two expresses the quantitative relationship of the absorption of light by the substance. Absorbance detection is a widely used general optical detection method with the advantages of many types of substances that can be measured and simple structure. It is also one of the earlier detection methods used in microfluidic analysis systems.
将液滴微流控系统和吸光度检测应用于细菌代谢物检测的过程中面临以下技术问题：The following technical issues are faced in the process of applying droplet microfluidics system and absorbance detection to bacterial metabolite detection:
在液滴微流控系统中，尽管努力将流体处理系统小型化，但在检测和分析系统小型化方面的发展非常有限。传统的微流体检测和分析系统依赖于传统的自由空间光学器件，带有一系列透镜和二色镜以及荧光显微镜。这些自由空间光学器件的复杂集成和对齐使整个微流体检测和分析系统变得笨重和昂贵。刚性固定的光学器件在设计实验时几乎没有自由度，并且需要繁琐的处理和维护。In droplet microfluidics, despite efforts to miniaturize fluid handling systems, there has been very limited progress in miniaturizing detection and analysis systems. Traditional microfluidic detection and analysis systems rely on traditional free-space optics with a series of lenses and dichroic mirrors and fluorescence microscopes. The complex integration and alignment of these free-space optics make the entire microfluidic detection and analysis system bulky and expensive. Rigidly fixed optics provide little freedom in designing experiments and require cumbersome handling and maintenance.
在液滴微流控系统中，由于微流控通道检测区域的液滴检测体积小、吸收光程短，导致吸光度检测的灵敏度较低，对将吸光度检测和液滴微流控系统结合起来进行细菌代谢物检测形成很大的限制。In droplet microfluidic systems, the small droplet detection volume and short absorption optical path in the microfluidic channel detection area result in low sensitivity of absorbance detection, which greatly limits the combination of absorbance detection and droplet microfluidic systems for bacterial metabolite detection.
发明内容Summary of the invention
本发明的目的是提出一种微流控芯片和在线吸光度检测系统，以减轻或消除至少一个上述的技术问题。The purpose of the present invention is to provide a microfluidic chip and an online absorbance detection system to alleviate or eliminate at least one of the above-mentioned technical problems.
本发明所述的一种微流控芯片，所述微流控芯片应用于在线吸光度检测系统，所述微流控芯片内部设置有样品流道、入射光纤通道和收集光纤通道。The microfluidic chip described in the present invention is applied to an online absorbance detection system, and a sample flow channel, an incident optical fiber channel and a collecting optical fiber channel are arranged inside the microfluidic chip.
所述样品流道包括依次连通的第一流道段、第二流道段以及第三流道段，所述第一流道段用于向所述第二流道段输送样品液滴，所述第三流道段用于输出所述样品液滴，所述第二流道段的横截面面积小于所述第一流道段的横截面面积，所述第二流道段为直线流道段。The sample flow channel includes a first flow channel section, a second flow channel section and a third flow channel section which are connected in sequence. The first flow channel section is used to transport sample droplets to the second flow channel section, and the third flow channel section is used to output the sample droplets. The cross-sectional area of the second flow channel section is smaller than the cross-sectional area of the first flow channel section, and the second flow channel section is a straight flow channel section.
所述入射光纤通道用于安装能够向所述第二流道段的一端出射检测光束的入射光纤，所述收集光纤通道用于安装能够收集从所述第二流道段的另一端出射的检测光束的收集光纤。The incident optical fiber channel is used to install an incident optical fiber capable of emitting a detection beam to one end of the second flow channel segment, and the collecting optical fiber channel is used to install a collecting optical fiber capable of collecting the detection beam emitted from the other end of the second flow channel segment.
可选的，所述入射光纤通道和所述收集光纤通道分别间隔的设置在所述第二流道段的两侧，所述入射光纤通道、所述收集光纤通道和所述第二流道段在同一中心线上。Optionally, the incident optical fiber channel and the collecting optical fiber channel are respectively arranged at intervals on both sides of the second flow channel segment, and the incident optical fiber channel, the collecting optical fiber channel and the second flow channel segment are on the same center line.
可选的，所述第一流道段垂直于所述第二流道段，所述第三流道段垂直于所述第二流道段。Optionally, the first flow channel segment is perpendicular to the second flow channel segment, and the third flow channel segment is perpendicular to the second flow channel segment.
可选的，所述第三流道段的横截面面积大于所述第二流道段的横截面面积。Optionally, a cross-sectional area of the third flow channel segment is greater than a cross-sectional area of the second flow channel segment.
可选的，所述样品流道还包括油相入样口、水相入样口、出样口、一端与所述第一流道段连通的第四流道段以及与所述第四流道段垂直且相交的第五流道段，所述油相入样口与所述第五流道段的两端连通，所述水相入样口与所述第四流道段的另一端连通，所述出样口与所述第三流道段远离所述第二流道段的一端连通。Optionally, the sample flow channel also includes an oil phase sample inlet, a water phase sample inlet, a sample outlet, a fourth flow channel section whose one end is connected to the first flow channel section, and a fifth flow channel section that is perpendicular to and intersects with the fourth flow channel section, the oil phase sample inlet is connected to both ends of the fifth flow channel section, the water phase sample inlet is connected to the other end of the fourth flow channel section, and the sample outlet is connected to one end of the third flow channel section away from the second flow channel section.
可选的，所述微流控芯片包括键合在一起的玻璃层和PDMS层，所述第一流道段、所述第二流道段、所述第三流道段、所述入射光纤通道和所述收集光纤通道均设置在所述PDMS层中。Optionally, the microfluidic chip includes a glass layer and a PDMS layer bonded together, and the first flow channel segment, the second flow channel segment, the third flow channel segment, the incident optical fiber channel and the collecting optical fiber channel are all arranged in the PDMS layer.
本发明还提出了一种在线吸光度检测系统，包括入射单元、检测单元、入样单元和出样单元和上述任一项所述的微流控芯片，所述入射单元用于向所述微流控芯片入射检测光束，所述入射单元包括输出端嵌装在所述入射光纤通道中的入射光纤，所述检测单元用于检测分析所述微流控芯片输出的检测光束，所述检测单元包括输入端嵌装在所述收集光纤通道中的收集光纤，所述入样单元用于向所述样品流道输入样品，所述出样单元用于收集所述样品流道输出的样品。The present invention also proposes an online absorbance detection system, comprising an incident unit, a detection unit, a sample input unit and a sample output unit and a microfluidic chip as described in any of the above items, wherein the incident unit is used to incident a detection light beam onto the microfluidic chip, the incident unit comprises an incident optical fiber whose output end is embedded in the incident optical fiber channel, the detection unit is used to detect and analyze the detection light beam output by the microfluidic chip, the detection unit comprises a collection optical fiber whose input end is embedded in the collection optical fiber channel, the sample input unit is used to input samples into the sample flow channel, and the sample output unit is used to collect samples output by the sample flow channel.
可选的，所述入射单元还包括依次设置的LED光源、第一光纤准直器和第一滤波器，所述LED光源用于提供检测光束，所述第一光纤准直器用于准直所述LED光源提供的检测光束，所述第一滤波器用于滤除所述第一光纤准直器输出的检测光束中的杂散光。Optionally, the incident unit also includes an LED light source, a first fiber optic collimator and a first filter which are arranged in sequence, the LED light source is used to provide a detection beam, the first fiber optic collimator is used to collimate the detection beam provided by the LED light source, and the first filter is used to filter out stray light in the detection beam output by the first fiber optic collimator.
可选的，所述检测单元还包括依次设置的第二滤波器、第二光纤准直器、探测器和示波器，所述第二滤波器用于滤除所述收集光纤收集的检测光束中的杂散光，所述第二光纤准直器用于准直所述第二滤波器输出的检测光束，所述探测器用于将所述第二光纤准直器输出的光信号的转换成电信号，所述示波器用于显示所述电信号。Optionally, the detection unit also includes a second filter, a second fiber optic collimator, a detector and an oscilloscope which are arranged in sequence, the second filter is used to filter out stray light in the detection light beam collected by the collection optical fiber, the second fiber optic collimator is used to collimate the detection light beam output by the second filter, the detector is used to convert the optical signal output by the second fiber optic collimator into an electrical signal, and the oscilloscope is used to display the electrical signal.
可选的，所述入样单元包括微量进样泵、油相进样管和水相进样管，所述油相进样管和所述水相进样管分别连接在所述微量进样泵和所述样品流道之间。Optionally, the sample injection unit includes a micro-injection pump, an oil-phase injection tube and a water-phase injection tube, and the oil-phase injection tube and the water-phase injection tube are respectively connected between the micro-injection pump and the sample flow channel.
本发明优化了微流控芯片的样品流道，能够提高检测灵敏度，为将吸光度检测和液滴微流控系统结合起来进行细菌代谢物检测提供了便利；本发明将吸光度检测、液滴微流控系统以及光纤微纳技术结合起来形成在线吸光度检测系统，具有实现低成本、结构简单、自由度高、试样和试剂消耗少、流道微小以及分析速度快的特点。The present invention optimizes the sample flow channel of the microfluidic chip, can improve the detection sensitivity, and provides convenience for combining absorbance detection with a droplet microfluidic system to detect bacterial metabolites; the present invention combines absorbance detection, a droplet microfluidic system and optical fiber micro-nano technology to form an online absorbance detection system, which has the characteristics of low cost, simple structure, high degree of freedom, low sample and reagent consumption, tiny flow channel and fast analysis speed.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为部分实施例中所述的微流控芯片的俯视图；FIG1 is a top view of a microfluidic chip described in some embodiments;
图2为部分实施例中所述的微流控芯片的局部放大视图之一；FIG2 is one of the partial enlarged views of the microfluidic chip described in some embodiments;
图3为部分实施例中所述的微流控芯片的局部放大视图之二；FIG3 is a second partial enlarged view of the microfluidic chip described in some embodiments;
图4为部分实施例中所述的微流控芯片的侧视图；FIG4 is a side view of a microfluidic chip described in some embodiments;
图5为部分实施例中所述在线吸光度检测系统的结构示意图。FIG. 5 is a schematic diagram of the structure of the online absorbance detection system described in some embodiments.
其中，1-微流控芯片；2-LED光源；3-第一光纤准直器；4-第一滤波器；5-入射光纤；6-收集光纤；7-第二滤波器；8-第二光纤准直器；9-探测器；10-示波器；11-微量进样泵；12-油相进样管；13-水相进样管；14-出样管；15-样品容器；Among them, 1-microfluidic chip; 2-LED light source; 3-first fiber collimator; 4-first filter; 5-incident fiber; 6-collecting fiber; 7-second filter; 8-second fiber collimator; 9-detector; 10-oscilloscope; 11-micro injection pump; 12-oil phase injection tube; 13-water phase injection tube; 14-sample outlet tube; 15-sample container;
101-玻璃层；102-PDMS层；103-样品流道；104-油相入样口；105-水相入样口；106-出样口；107-入射光纤通道；108-收集光纤通道；101-glass layer; 102-PDMS layer; 103-sample flow channel; 104-oil phase sample inlet; 105-water phase sample inlet; 106-sample outlet; 107-incident optical fiber channel; 108-collecting optical fiber channel;
1031-第五流道段；1032-第四流道段；1033-第一流道段；1034-第二流道段；1035-第三流道段；1036-第六流道段；1037-第七流道段；1038-第八流道段；1039-第九流道段。1031 - fifth flow channel section; 1032 - fourth flow channel section; 1033 - first flow channel section; 1034 - second flow channel section; 1035 - third flow channel section; 1036 - sixth flow channel section; 1037 - seventh flow channel section; 1038 - eighth flow channel section; 1039 - ninth flow channel section.
具体实施方式DETAILED DESCRIPTION
以下将参照附图和优选实施例来说明本发明的实施方式，本领域技术人员可由本说明书中所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。应当理解，优选实施例仅为了说明本发明，而不是为了限制本发明的保护范围。The following will describe the embodiments of the present invention with reference to the accompanying drawings and preferred embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are only for illustrating the present invention, not for limiting the scope of protection of the present invention.
需要说明的是，以下实施例中所提供的图示仅以示意方式说明本发明的基本构想，遂图式中仅显示与本发明中有关的组件而非按照实际实施时的组件数目、形状及尺寸绘制，其实际实施时各组件的型态、数量及比例可为一种随意的改变，且其组件布局型态也可能更为复杂。It should be noted that the illustrations provided in the following embodiments are only schematic illustrations of the basic concept of the present invention, and thus the drawings only show components related to the present invention rather than being drawn according to the number, shape and size of components in actual implementation. In actual implementation, the type, quantity and proportion of each component may be changed arbitrarily, and the component layout may also be more complicated.
如图1和图2所示一种微流控芯片1，微流控芯片1应用于在线吸光度检测系统，微流控芯片1内部设置有样品流道103、入射光纤通道107和收集光纤通道108，样品流道103为微流道，样品流道103包括依次连通的第一流道段1033、第二流道段1034以及第三流道段1035，第一流道段1033用于向第二流道段1034输送样品液滴，第三流道段1035用于输出样品液滴，第二流道段1034的横截面面积小于第一流道段1033的横截面面积，第二流道段1034为直线流道段，入射光纤通道107用于安装能够向第二流道段1034的一端出射检测光束的入射光纤5，收集光纤通道108用于安装能够收集从第二流道段1034的另一端出射的检测光束的收集光纤6。As shown in Figures 1 and 2, a microfluidic chip 1 is applied to an online absorbance detection system. A sample flow channel 103, an incident optical fiber channel 107 and a collecting optical fiber channel 108 are arranged inside the microfluidic chip 1. The sample flow channel 103 is a microflow channel. The sample flow channel 103 includes a first flow channel section 1033, a second flow channel section 1034 and a third flow channel section 1035 which are connected in sequence. The first flow channel section 1033 is used to transport sample droplets to the second flow channel section 1034, and the third flow channel section 1035 is used to output sample droplets. The cross-sectional area of the second flow channel section 1034 is smaller than the cross-sectional area of the first flow channel section 1033. The second flow channel section 1034 is a straight flow channel section. The incident optical fiber channel 107 is used to install an incident optical fiber 5 that can emit a detection light beam to one end of the second flow channel section 1034, and the collecting optical fiber channel 108 is used to install a collecting optical fiber 6 that can collect the detection light beam emitted from the other end of the second flow channel section 1034.
采用上述的技术方案，利用入射光纤5出射的检测光束照射第二流道段1034内的样品液滴，利用收集光纤6收集从第二流道段1034出射的检测光束，能够对第二流道段1034内的样品液滴进行吸光度检测；通过设置横截面面积较小的第二流道段1034作为检测流道段，第二流道段1034能够将从第一流道段1033输送来的样品液滴压缩得更长，能够延长检测光束在样品液滴中的光程，进而提高在线吸光度检测系统的检测灵敏度，为将吸光度检测和液滴微流控系统结合起来进行细菌代谢物检测提供了便利。光程越长，信号越强，通过延长检测光束在样品液滴中的光程，能够提高信号强度，从而进一步提高了对样品液滴的检测能力。By adopting the above technical solution, the sample droplets in the second flow channel section 1034 are irradiated with the detection light beam emitted by the incident optical fiber 5, and the detection light beam emitted from the second flow channel section 1034 is collected by the collecting optical fiber 6, so that the sample droplets in the second flow channel section 1034 can be detected by absorbance; by setting the second flow channel section 1034 with a smaller cross-sectional area as the detection flow channel section, the second flow channel section 1034 can compress the sample droplets transported from the first flow channel section 1033 longer, and can extend the optical path of the detection light beam in the sample droplets, thereby improving the detection sensitivity of the online absorbance detection system, and providing convenience for combining absorbance detection with the droplet microfluidic system for bacterial metabolite detection. The longer the optical path, the stronger the signal. By extending the optical path of the detection light beam in the sample droplets, the signal intensity can be increased, thereby further improving the detection capability of the sample droplets.
作为一种优选示例，第一流道段1033和第二流道段1034截面均为方形，第一流道段1033的宽度为40nm-200nm，深度为50nm-100nm；第二流道段1034的宽度为30nm-190nm，深度为50nm-100nm，长度为100nm-600nm。通过对第二流道段1034做减窄处理，第二流道段1034能够将从第一流道段1033流过来的样品液滴挤压成长条形液滴，能够延长检测光束在样品液滴中的光程。经过试验验证，上述优选示例所采用的方案具有最佳的使用效果。As a preferred example, the cross-sections of the first flow channel section 1033 and the second flow channel section 1034 are both square, the width of the first flow channel section 1033 is 40nm-200nm, and the depth is 50nm-100nm; the width of the second flow channel section 1034 is 30nm-190nm, the depth is 50nm-100nm, and the length is 100nm-600nm. By narrowing the second flow channel section 1034, the second flow channel section 1034 can squeeze the sample droplets flowing from the first flow channel section 1033 into long strip-shaped droplets, which can extend the optical path of the detection light beam in the sample droplets. After experimental verification, the scheme adopted in the above preferred example has the best use effect.
在一些实施例中，入射光纤通道107和收集光纤通道108分别间隔的设置在第二流道段1034的两侧，入射光纤通道107、收集光纤通道108和第二流道段1034在同一中心线上。采用上述的技术方案，第二流道段1034和入射光纤通道107之间以及第二流道段1034和收集光纤通道108之间间隔设置，能够防止出现漏液的情况，降低了对入射光纤通道107和收集光纤通道108的密封要求，降低了入射光纤5和收集光纤6的安装难度，为减小第二流道段1034的横截面面积提供了便利，并且嵌入的光纤可以重复利用，更换芯片以后光纤还可以继续插入使用。入射光纤通道107、收集光纤通道108和第二流道段1034在同一中心线上，便于向第二流道段1034入射检测光束和便于收集从第二流道段1034出射的检测光束。In some embodiments, the incident optical fiber channel 107 and the collection optical fiber channel 108 are arranged at intervals on both sides of the second flow channel section 1034, and the incident optical fiber channel 107, the collection optical fiber channel 108 and the second flow channel section 1034 are on the same center line. With the above technical solution, the second flow channel section 1034 and the incident optical fiber channel 107 and the second flow channel section 1034 and the collection optical fiber channel 108 are arranged at intervals, which can prevent leakage, reduce the sealing requirements for the incident optical fiber channel 107 and the collection optical fiber channel 108, reduce the installation difficulty of the incident optical fiber 5 and the collection optical fiber 6, and provide convenience for reducing the cross-sectional area of the second flow channel section 1034. In addition, the embedded optical fiber can be reused, and the optical fiber can continue to be inserted and used after the chip is replaced. The incident optical fiber channel 107, the collection optical fiber channel 108 and the second flow channel section 1034 are on the same center line, which is convenient for incident detection beams to the second flow channel section 1034 and for collecting detection beams emitted from the second flow channel section 1034.
由于第二流道段1034和入射光纤通道107之间以及第二流道段1034和收集光纤通道108之间间隔设置，入射光纤采用嵌装的方式安装在入射光纤通道107中，收集光纤采用嵌装的方式安装在收集光纤通道108中，整体机械结构很稳定，为入射光纤何收集光纤提供了稳定的工作平台，为将微纳光纤应用于在线吸光度检测系统提供了便利。Since the second flow channel section 1034 and the incident optical fiber channel 107 are spaced apart as well as the second flow channel section 1034 and the collecting optical fiber channel 108, the incident optical fiber is installed in the incident optical fiber channel 107 by an embedded manner, and the collecting optical fiber is installed in the collecting optical fiber channel 108 by an embedded manner. The overall mechanical structure is very stable, which provides a stable working platform for the incident optical fiber and the collecting optical fiber, and facilitates the application of micro-nano optical fiber in online absorbance detection systems.
作为一种具体示例，第二流道段1034和入射光纤通道107之间以及第二流道段1034和收集光纤通道108之间的间隔为200um，在保证工作性能的同时能够防止出现因为光纤戳穿通道而导致漏液的情况。As a specific example, the interval between the second flow channel section 1034 and the incident optical fiber channel 107 and between the second flow channel section 1034 and the collecting optical fiber channel 108 is 200um, which can prevent leakage caused by the optical fiber piercing the channel while ensuring working performance.
在一些实施例中，第一流道段1033垂直于第二流道段1034，第三流道段1035垂直于第二流道段1034。采用上述的技术方案，能够减少杂散光的干扰，提高了检测光束与样品液滴对准的精度，增加了出射光的光通量，同时也提高了收集的光信号强度。In some embodiments, the first flow channel segment 1033 is perpendicular to the second flow channel segment 1034, and the third flow channel segment 1035 is perpendicular to the second flow channel segment 1034. The above technical solution can reduce the interference of stray light, improve the accuracy of alignment between the detection light beam and the sample droplet, increase the luminous flux of the emitted light, and also improve the intensity of the collected light signal.
在一些实施例中，第三流道段1035的横截面面积大于第二流道段1034的横截面面积。将第三流道段1035的流断面面积设置的较大，能够顺利的排出样品液滴。In some embodiments, the cross-sectional area of the third flow channel segment 1035 is larger than the cross-sectional area of the second flow channel segment 1034. The cross-sectional area of the third flow channel segment 1035 is set larger so that the sample droplets can be discharged smoothly.
在一些实施例中，如图1至图4所示，样品流道103还包括油相入样口104、水相入样口105、出样口106、一端与第一流道段1033连通的第四流道段1032以及与第四流道段1032垂直且相交的第五流道段1031，油相入样口104与第五流道段1031的两端连通，水相入样口105与第四流道段1032的另一端连通，出样口106与第三流道段1035远离第二流道段1034的一端连通。采用上述的技术方案，第四流道段1032和第五流道段1031构成了十字型流道，能够利用油相剪切水相来生成油包水型的样品液滴，并且可以通过控制油相和水相的流速来控制样品液滴的大小。In some embodiments, as shown in FIGS. 1 to 4 , the sample flow channel 103 further includes an oil phase sample inlet 104, a water phase sample inlet 105, a sample outlet 106, a fourth flow channel section 1032 whose one end is connected to the first flow channel section 1033, and a fifth flow channel section 1031 which is perpendicular to and intersects with the fourth flow channel section 1032. The oil phase sample inlet 104 is connected to both ends of the fifth flow channel section 1031, the water phase sample inlet 105 is connected to the other end of the fourth flow channel section 1032, and the sample outlet 106 is connected to one end of the third flow channel section 1035 away from the second flow channel section 1034. With the above technical solution, the fourth flow channel section 1032 and the fifth flow channel section 1031 form a cross-shaped flow channel, which can generate water-in-oil type sample droplets by shearing the water phase with the oil phase, and the size of the sample droplets can be controlled by controlling the flow rates of the oil phase and the water phase.
作为一种具体示例，样品流道103还包括第六流道段1036、连接在第六流道段1036的一端与第五流道段1031的一端之间的第七流道段1037、连接在第六流道段1036的另一端与第五流道段1031的另一端之间的第八流道段1038以及一端与第六流道段1036的中部连接的第九流道段1039，第五流道段1031、第六流道段1036、第七流道段1037和第八流道段1038构成矩形框状流道结构，油相入样口104与第九流道段1039的另外一端连通。采用上述的技术方案，采用矩形框架状流道结构结合第四流道段1032来构成能够生成油包水型的样品液滴的液滴生成流道，具有结构简单、易于实现的特点。As a specific example, the sample flow channel 103 also includes a sixth flow channel section 1036, a seventh flow channel section 1037 connected between one end of the sixth flow channel section 1036 and one end of the fifth flow channel section 1031, an eighth flow channel section 1038 connected between the other end of the sixth flow channel section 1036 and the other end of the fifth flow channel section 1031, and a ninth flow channel section 1039 connected at one end to the middle of the sixth flow channel section 1036. The fifth flow channel section 1031, the sixth flow channel section 1036, the seventh flow channel section 1037 and the eighth flow channel section 1038 constitute a rectangular frame-shaped flow channel structure, and the oil phase sample inlet 104 is connected to the other end of the ninth flow channel section 1039. The above technical solution uses a rectangular frame-shaped flow channel structure combined with the fourth flow channel section 1032 to form a droplet generation flow channel capable of generating oil-in-water type sample droplets, which has the characteristics of simple structure and easy implementation.
在一些实施例中，如图4所示，微流控芯片1包括键合在一起的玻璃层101和PDMS层102，第一流道段1033、第二流道段1034、第三流道段1035、第五流道段1031、第六流道段1036、第七流道段1037、第八流道段1038、第九流道段1039、入射光纤通道107和收集光纤通道108均设置在PDMS层102中，油相入样口104、水相入样口105和出样口106设置在玻璃层101中。PDMS层102即为聚二甲基硅氧烷层。In some embodiments, as shown in FIG4 , the microfluidic chip 1 includes a glass layer 101 and a PDMS layer 102 bonded together, a first flow channel segment 1033, a second flow channel segment 1034, a third flow channel segment 1035, a fifth flow channel segment 1031, a sixth flow channel segment 1036, a seventh flow channel segment 1037, an eighth flow channel segment 1038, a ninth flow channel segment 1039, an incident optical fiber channel 107, and a collecting optical fiber channel 108 are all arranged in the PDMS layer 102, and an oil phase sample inlet 104, a water phase sample inlet 105, and a sample outlet 106 are arranged in the glass layer 101. The PDMS layer 102 is a polydimethylsiloxane layer.
在具体实施时，可以使用CAD或L-Edit画图软件设计微流控芯片1的结构，微流控芯片1的制作材料选用玻璃及聚二甲基硅氧烷(PDMS)，可以通过软光刻技术制得PDMS层102并与玻璃层101键合。In specific implementation, CAD or L-Edit drawing software can be used to design the structure of the microfluidic chip 1. The microfluidic chip 1 is made of glass and polydimethylsiloxane (PDMS). The PDMS layer 102 can be made by soft lithography technology and bonded to the glass layer 101.
入射光纤5和收集光纤6可以选用纤芯为50um或100um的多模光纤，在PDMS层102倒膜时，使用与光纤同样粗细的钢丝粘于预留的光纤通道处，保证倒模后所得的PDMS层102能够顺利的嵌入光纤，光纤嵌入微流控芯片1使得检测时免于聚焦。The incident optical fiber 5 and the collecting optical fiber 6 can be multimode optical fibers with a core of 50um or 100um. When the PDMS layer 102 is molded, a steel wire of the same thickness as the optical fiber is used to stick to the reserved optical fiber channel to ensure that the PDMS layer 102 obtained after molding can be smoothly embedded in the optical fiber. The optical fiber is embedded in the microfluidic chip 1 to avoid focusing during detection.
如图5所示，本发明还提出了一种在线吸光度检测系统，包括入射单元、检测单元、入样单元和出样单元和上述任一项所述的微流控芯片1，入射单元用于向微流控芯片1入射检测光束，入射单元包括输出端嵌装在入射光纤通道107中的入射光纤5，检测单元用于检测分析微流控芯片1输出的检测光束，检测单元包括输入端嵌装在收集光纤通道108中的收集光纤6，入样单元用于向样品流道103输入样品，出样单元用于收集样品流道103输出的样品。As shown in Figure 5, the present invention also proposes an online absorbance detection system, including an incident unit, a detection unit, a sample input unit and a sample output unit and the microfluidic chip 1 described in any of the above items, the incident unit is used to incident the detection light beam onto the microfluidic chip 1, the incident unit includes an incident optical fiber 5 whose output end is embedded in an incident optical fiber channel 107, the detection unit is used to detect and analyze the detection light beam output by the microfluidic chip 1, the detection unit includes a collection optical fiber 6 whose input end is embedded in a collection optical fiber channel 108, the sample input unit is used to input samples into the sample flow channel 103, and the sample output unit is used to collect samples output by the sample flow channel 103.
在一些实施例中，入射单元还包括依次通过光纤连接的LED光源2、第一光纤准直器3和第一滤波器4，第一滤波器4的输出端与入射光纤5的输入端连接，LED光源2用于提供检测光束，第一光纤准直器3用于准直LED光源2提供的检测光束，第一滤波器4用于滤除第一光纤准直器3输出的检测光束中的杂散光，第一滤波器4输出的检测光束通过入射光纤5输送至微流控芯片1。采用上述的技术方案，能够提供优质的检测光束。In some embodiments, the incident unit further includes an LED light source 2, a first fiber collimator 3, and a first filter 4 connected in sequence through optical fibers, the output end of the first filter 4 is connected to the input end of the incident optical fiber 5, the LED light source 2 is used to provide a detection beam, the first fiber collimator 3 is used to collimate the detection beam provided by the LED light source 2, the first filter 4 is used to filter out stray light in the detection beam output by the first fiber collimator 3, and the detection beam output by the first filter 4 is delivered to the microfluidic chip 1 through the incident optical fiber 5. The above technical solution can provide a high-quality detection beam.
在一些实施例中，检测单元还包括依次通过光纤连接的第二滤波器7、第二光纤准直器8、探测器9和示波器10，第二滤波器7用于滤除收集光纤6收集的检测光束中的杂散光，第二光纤准直器8用于准直第二滤波器7输出的检测光束，探测器9用于将第二光纤准直器8输出的光信号的转换成电信号，示波器10用于显示电信号。采用上述的技术方案，可以获得更准确的检测结果，并且利用示波器10可以实时、直观的观察峰形的变化。In some embodiments, the detection unit further includes a second filter 7, a second fiber collimator 8, a detector 9 and an oscilloscope 10 connected in sequence through optical fibers, the second filter 7 is used to filter out stray light in the detection beam collected by the collection optical fiber 6, the second fiber collimator 8 is used to collimate the detection beam output by the second filter 7, the detector 9 is used to convert the optical signal output by the second fiber collimator 8 into an electrical signal, and the oscilloscope 10 is used to display the electrical signal. By adopting the above technical solution, more accurate detection results can be obtained, and the change of the peak shape can be observed in real time and intuitively using the oscilloscope 10.
在一些实施例中，入样单元包括微量进样泵11、油相进样管12和水相进样管13，油相进样管12和水相进样管13分别连接在微量进样泵11和样品流道103之间。采用上述的技术方案，水相和油相可以分开输送，可以对不同浓度的待测物(水相)进行测试，对其进行浓度定量。在具体实施时，油相进样管12的输出端与油相入样口104连接，水相进样管13的输出端与水相入样口105连接。In some embodiments, the sample injection unit includes a micro-injection pump 11, an oil phase injection tube 12 and a water phase injection tube 13, and the oil phase injection tube 12 and the water phase injection tube 13 are respectively connected between the micro-injection pump 11 and the sample flow channel 103. With the above technical solution, the water phase and the oil phase can be transported separately, and different concentrations of the analyte (water phase) can be tested and the concentration can be quantified. In the specific implementation, the output end of the oil phase injection tube 12 is connected to the oil phase injection port 104, and the output end of the water phase injection tube 13 is connected to the water phase injection port 105.
在一些实施例中，出样单元包括样品容器15以及连接在样品容器15和出样口106之间的出样管14，样品容器15用于收集从出样口106输出的样品液滴。在具体实施时，样品容器15可以选用锥形瓶。In some embodiments, the sample output unit includes a sample container 15 and a sample output tube 14 connected between the sample container 15 and the sample output port 106. The sample container 15 is used to collect sample droplets output from the sample output port 106. In a specific implementation, the sample container 15 can be a conical flask.
采用上述在线吸光度检测系统，搭建了单波长吸光度光学检测平台,具有整体结构实现成本低、简洁的特点，光源采用LED光源2，检测光束经过纤准和滤光后穿过微流控芯片1，检测光束透射过样品液滴后，经滤光后最终被探测器9接收，探测器9连接示波器10观察峰形的变化。A single-wavelength absorbance optical detection platform was built using the above-mentioned online absorbance detection system, which has the characteristics of low cost and simplicity in overall structure. The light source is an LED light source 2. The detection light beam passes through the microfluidic chip 1 after being aligned and filtered. After the detection light beam passes through the sample droplet, it is finally received by the detector 9 after filtering. The detector 9 is connected to the oscilloscope 10 to observe the change of the peak shape.
选用单波长是因为每种物质吸收光的波长不同，本检测系统采用单波长可对某种特定物质进行精准的测量，不同物质更换不同的窄带光源，相对比于宽光源光路，所涵盖的波长极大的减少了误差，极大的提高了检测的准确性，采用LED光源2为实现单波长吸光度光学检测平台提供了便利。A single wavelength is selected because each substance absorbs light at a different wavelength. This detection system uses a single wavelength to accurately measure a specific substance. Different narrow-band light sources are replaced for different substances. Compared with the wide light source optical path, the wavelengths covered greatly reduce errors and greatly improve the accuracy of detection. The use of LED light source 2 provides convenience for realizing a single-wavelength absorbance optical detection platform.
基于光纤的在线吸光度检测系统比使用传统的自由空间光检测系统具有显著优势，由于经过光纤的光占据体积更小，从而产生更大的光场强度。此外，在上述的在线吸光度检测系统中，光纤与微流控结合，由于光可以直接发射到光纤中，因此可以实现光与样品液滴精准聚焦。因此不需要为了实现最佳光场强度而在聚焦样品液滴之前扩大检测光束。这些优点可以达到更低的样品检测极限，此类检测系统适合分析大量样品。The fiber-based online absorbance detection system has significant advantages over the traditional free-space light detection system, because the light passing through the fiber optic occupies a smaller volume, thereby generating a greater light field intensity. In addition, in the above-mentioned online absorbance detection system, the fiber optic is combined with microfluidics, and since the light can be directly emitted into the fiber optic, precise focusing of the light and the sample droplets can be achieved. Therefore, there is no need to expand the detection beam before focusing on the sample droplets in order to achieve the optimal light field intensity. These advantages can achieve a lower sample detection limit, and this type of detection system is suitable for analyzing a large number of samples.
吸收率的有效光程会影响其整体检测的灵敏度，本申请通过改变截面通道、压缩通道来延长光程以提高检测灵敏度，吸光度与微流控技术及光纤微纳技术结合具有试样和试剂消耗少、流道微小及分析速度快等优点。The effective optical path of the absorbance rate will affect the sensitivity of its overall detection. The present application extends the optical path by changing the cross-sectional channel and compressing the channel to improve the detection sensitivity. The absorbance is combined with microfluidics technology and fiber optic micro-nano technology, which has the advantages of less sample and reagent consumption, tiny flow channel and fast analysis speed.
在具体实施时，可以根据待测物选择合适的光源，进行最后的光路系统整合优化；可以使用油包水型的微液滴进行检测，待测物为水相，油相为硅油或氟化油。可以通过对不同浓度的待测物进行测试，对其进行浓度定量。In specific implementation, a suitable light source can be selected according to the object to be tested, and the final optical path system integration optimization can be performed; oil-in-water type micro-droplets can be used for detection, the object to be tested is the water phase, and the oil phase is silicone oil or fluorinated oil. The concentration of the object to be tested can be quantified by testing different concentrations.
吸光度检测原理为：吸光度是物质对光的吸收程度。常见的液体都会对光有一定的吸收程度，吸收的大小和物质的固有属性和浓度等有关，朗伯-比尔定律中溶液对光的吸收程度与溶液浓度和液层厚度的乘积正比，通过对该定律的演算可以得出所检测物质的浓度等相关特性。朗伯-比尔定律的数学表达式：The principle of absorbance detection is: absorbance is the degree of absorption of light by a substance. Common liquids have a certain degree of absorption of light, and the magnitude of the absorption is related to the inherent properties and concentration of the substance. In the Lambert-Beer law, the degree of absorption of light by the solution is proportional to the product of the solution concentration and the thickness of the liquid layer. By calculating this law, the concentration and other related characteristics of the detected substance can be obtained. The mathematical expression of the Lambert-Beer law is:
式中各参数的意义为：A：吸光度：Io：入射光强度：It：为透射光强度；T：为透射比；ε：物质的吸收常数；b：光束通过液层的厚度(cm)；c：物质的浓度(mol/L)；The meanings of the parameters in the formula are: A: absorbance;Io : incident light intensity;It : transmitted light intensity; T: transmittance; ε: absorption constant of the substance; b: thickness of the liquid layer through which the light beam passes (cm); c: concentration of the substance (mol/L);
根据朗伯-比尔定律，在理想情况下，对于同一种物质，同一种波长光照射，可以认为其吸光系数固定，当通过液层的厚度确定后，通过测量透射光的强度和入射光的强度的比值，可以反演出物质的浓度。According to the Lambert-Beer law, under ideal conditions, for the same substance, when irradiated with light of the same wavelength, its absorption coefficient can be considered to be fixed. Once the thickness of the liquid layer is determined, the concentration of the substance can be inverted by measuring the ratio of the intensity of the transmitted light to the intensity of the incident light.
以上实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”或“一些示例”等的描述意指结合该实施例或示例的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。此外，本领域的技术人员可以将本说明书中描述的不同实施例或示例进行接合和组合。The above embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. In the description of this specification, the description of reference terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics of the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine the different embodiments or examples described in this specification.
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