CN106198496B - A high-throughput electrochemiluminescence detection method - Google Patents

Abstract

The invention discloses a kind of electrochemical luminous detection methods of high throughput, electrochemical luminescence reaction is carried out by the porous micropore disk of conductive disc type, then after the micropore disk for completing electrochemical luminescence reaction being sent to image capturing system, using charge-coupled device or complementary metal oxide semiconductor as imaging sensor, in the case where gauge without light source, the sample image that shines is acquired；Through data analysis system by micropore disk image comparison, micropore disc-type is judged automatically；Gray value will be converted to after image digitazation, judges luminous point, to luminous point automatic ferrule, with true fixed position；By being continuously shot to the micropore disk after reaction, composite calulation average gray, it is last according to linear relationship, gray value is converted into concentration, realizes the analysis of qualitative or quantitative result；The present invention can detect multiple samples simultaneously, have high-throughput advantage；Each micropore can accommodate multiple albumen indexs, realize multiple determination；It can be carried out digitized a large amount of interpretations, detection speed is fast.

Description

A kind of electrochemical luminous detection method of high throughput

Technical field

The present invention relates to field of biosensors, the electrochemical luminous detection method of specifically a kind of high throughput.

Background technique

Electrochemiluminescent immunoassay (Electrochemiluminescence immunoassay, ECLI) is after radiationLabel immunoassay technology of new generation immune, enzyme is immune, fluorescence immunoassay, chemiluminescence immunoassay are later, electrochemical luminescenceSources of law are in electrochemical process and chemoluminescence method, and ECLI is electrochemical luminescence and the technology that immunoassays combine, and is that one kind existsThe specific chemical luminescence-producing reaction that electrode surface is caused by electrochemistry includes two processes of electrochemistry and chemiluminescence, electrificationLearning luminescence-producing reaction principle is that anode adds under certain voltage energy effect on the working electrode (s, the trichlorine bipyridyl ruthenium of divalent[Rubpy3] 2+ discharges trichlorine bipyridyl ruthenium [Rubpy3] 3+ that electronics occurs oxidation reaction and becomes trivalent, meanwhile, electrode tableThe TPA in face also discharges electronics and oxidation reaction occurs and becomes radical cation TPA+, and rapidly it is spontaneous slough a proton andTripropyl amine (TPA) free radical TPA is formed, in this way, there is the trichlorine bipyridyl ruthenium of the trivalent with strong oxidizing property in reaction system[Rubpy3] 3+ and tripropyl amine (TPA) free radical TPA with strong reducing property；At this moment the trichlorine bipyridyl of the trivalent with strong oxidizing propertyRedox reaction occurs for ruthenium [Rubpy3] 3+ and tripropyl amine (TPA) free radical TPA with strong reducing property, as a result makes the trichlorine of trivalentBipyridyl ruthenium [Rubpy3] 3+ is reduced into trichlorine bipyridyl ruthenium [Rubpy3] 2+ of the divalent of excitation state, and energy derives from trivalentTrichlorine bipyridyl ruthenium [Rubpy3] 3+ and tripropyl amine (TPA) free radical TPA between potential difference, excitation state [Rubpy3] 2+ is with fluorescenceMechanism decay is simultaneously released energy in a manner of releasing a wavelength and being 620nm photon, and becomes [Rubpy3] 2+ of ground state；OnAfter stating chemiluminescent process progresses, there are still trichlorine bipyridyl ruthenium [Rubpy3] 2+ and tripropyl amine (TPA) TPA of divalent in reaction system, so thatThe electrochemical reaction and chemiluminescent process progresses of electrode surface can recycle progress, by above-mentioned cyclic process, measure signal notDisconnected amplification, to make detection sensitivity greatly improve, so ECL measurement has the characteristics that highly sensitive, above-mentioned electrochemical luminescenceThe concentration of trichlorine bipyridyl ruthenium [Rubpy3] 2+ of the intensity and divalent for the optical signal that process generates is linear, by divalentIn conjunction with one of trichlorine bipyridyl ruthenium [Rubpy3] 2+ and immune response system substance, after being immunoreacted, detect immune anti-Light of trichlorine bipyridyl ruthenium [Rubpy3] 2+ through being issued for answering remaining divalent in system, it can be learnt that the concentration of object to be checked；SieveFamily name's Elecsys and Cobase series is current most popular electrochemical luminescence system, marks the antibody with high specificity of RutheniumAfter in conjunction with measured object in conjunction with paramagnetic beads, after adsorbing magnetic micro-beads using magnetic field, apply voltage starting electrochemical luminescenceReaction, last Photoelectric multiplier tube detection transmitting optical signal, emits optical signal and measured object concentration is linear, but at presentElectrochemiluminescince, can only once detect a sample, i.e., a kind of protein content, carry out multiple protein that can not be high-throughput containsThe detection of amount needs to improve the prior art, to solve the above problems.

Summary of the invention

The purpose of the present invention is to provide one kind to realize high throughput by microwell plate and Image Acquisition and data analysis systemElectrochemical luminous detection method, to solve the problems mentioned in the above background technology.

To achieve the above object, the invention provides the following technical scheme:

A kind of electrochemical luminous detection method of high throughput is used for the micropore disk and one of electrochemiluminescence analysis using oneA image capturing system, a data analysis system, the micropore disk are combined up and down by substrate boards (1) and mesh grid frame (4)Form, the length of substrate boards (1) is 16cm, width 9cm, with a thickness of 0.5cm, substrate boards (1) upper surface utilizes magnetron sputteringMethod be coated with indium oxide layer tin film (2), the setting of indium oxide tin film (2) so that substrate boards (1) have conducting function, thusIt is used as an electrode, substrate boards (1) surface for being coated with indium oxide tin film (2), which is also adhered to, to be had conductivity excellent and have eggThe graphene film decorative layer (3) of white matter Molecular Adsorption function；The length of the mesh grid frame (4) is 16cm, width 9cm,With a thickness of 1cm, mesh grid frame (4) and substrate boards (1) are all made of polyethylene terephthalate, polyvinyl chloride or polyethylenePlastic cement material is made；After mesh grid frame (4) and substrate boards (1) composition micropore disk, so that each micropore (5) on micropore disk is equalA sample can be measured, and is not interfere with each other between each micropore (5), multiple samples is realized while measuring；The detection method packetInclude following steps:

1) micropore disk electrochemical luminescence reacts: when carrying out electrochemiluminescence analysis measurement, connecting and is marked with electrochemical luminescenceThe substance of object, is powered to substrate boards, and electrochemical luminescence reaction will occur for electrochemical luminescence object；

2) then the micropore disk for completing electrochemical luminescence reaction is sent to after image capturing system and acquires, browses sample graphPicture utilizes charge-coupled device (Charge Coupled Device, CCD) or complementary metal oxide semiconductor(Complementary Metal-Oxide-Semiconductor, CMOS) is used as imaging sensor, the gauge without light source the case whereUnder, the luminous sample image after acquisition electrochemical luminescence reaction, the sample image of acquisition is carried out qualitative by data analysis systemOr quantitative analysis and interpretation judges automatically micropore disc-type first by micropore disk image comparison；Data analysis system hasAutomatic ferrule function will be converted to gray value after image digitazation, judge luminous point, to luminous point automatic ferrule, passes through ashAngle value difference delimit effective image area, grid dividing, check point spacing, battle array spacing, with true fixed position, by reactionMicropore disk afterwards is continuously shot, synthesizes, calculates, and obtains average gray, last according to linear relationship, gray value is converted into denseDegree is analyzed to qualitative or quantitative result.

Preferably, the graphene film decorative layer (3) is that graphene oxide is reduced into graphite by chemical reduction reactionAlkene is made, and the making step of the graphene film decorative layer (3) includes:

1) molten for the graphene oxide of 0.1~0.4g/L in substrate boards (1) surface 5~15 microlitres of concentration of dropwise addition cleanedLiquid spontaneously dries under room temperature；

2) 5~15 microlitres of water, hydrazine and ammonium hydroxide volume ratio is added dropwise in substrate boards (1) surface handled in step 1100:(0.4~1): the hydrazine solution of (0.8~4) or 1 × 10^-3~1 × 10^-2The hydrogen bromide solution of mol/L as reducing agent,It is placed at 25~30 DEG C 12~24 hours until dry；

3) it washes with water step 2 and handles obtained substrate boards (1), obtain the substrate boards with graphene film decorative layer (3).

Preferably, the specification of the mesh grid frame includes: 96 grids, grid area 8mm*8mm, each grid receiving4 albumen Indexs measures；Or 24 grids, grid cotton knot are 1.6cm*1.6cm, each grid accommodates 16 albumen index inspectionsIt surveys.

Compared with prior art, the beneficial effects of the present invention are: the present invention is compared to tubular type detection method, by using disc typeThe mode of Electrochemical Detection at most may be implemented disposable while detect 96 samples, has high-throughput advantage；By in microporePlating sets indium oxide tin film and graphene film decorative layer in the substrate boards of disk, so that micropore disk of the invention is conductive, makes itIt can be directly used for electrochemical luminescence reaction, have both the characteristic of conductive agent adsorbed proteins；Each micropore can accommodate 4 or 16A albumen index realizes multiple determination, greatly improves sensitivity and specificity, provide more kinds of disease detection meanings；WithCCD or COMS interception image, realization disposably carry out digitized a large amount of interpretations, amplify interpretation far faster than photoelectricity times pipe single,Detection is quick.

Detailed description of the invention

Fig. 1 is the structural schematic diagram of substrate boards part in the present invention.

Fig. 2 is the structural schematic diagram for the mesh grid frame that specification is 8mm*8mm in the present invention.

Fig. 3 is the structural schematic diagram for the mesh grid frame that specification is 1.6cm*1.6cm in the present invention.

Fig. 4 is workflow schematic block diagram of the invention.

Specific embodiment

Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, completeSite preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based onEmbodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every otherEmbodiment shall fall within the protection scope of the present invention.

Please refer to Fig. 1~4, in the embodiment of the present invention, a kind of electrochemical luminous detection method of high throughput, using a useDetection, the micropore disk are realized jointly in the micropore disk of chemical analysis and an image capturing system, a data analysis systemBe composed of substrate boards 1 and about 4 mesh grid frame, the length of substrate boards 1 is 16cm, width 9cm, with a thickness of 0.5cm,1 upper surface of substrate boards is coated with indium oxide layer tin film 2 using the method for magnetron sputtering, and the setting of indium oxide tin film 2 is so that substratePlate 1 has conducting function, to use as an electrode, 1 surface of substrate boards for being coated with indium oxide tin film 2 is also attached with conductionFunction admirable and the graphene film decorative layer 3 with protein adsorption function, the graphene film decorative layer 3 is passing throughGraphene oxide is reduced into graphene and is made by reduction reaction, and 3 making step of graphene film decorative layer includes:

1) molten for the graphene oxide of 0.1~0.4g/L in 1 surface of substrate boards, 5~15 microlitres of concentration of dropwise addition cleanedLiquid spontaneously dries under room temperature；

2) it is 100 that 5~15 microlitres of water, hydrazine and ammonium hydroxide volume ratio, which is added dropwise, in 1 surface of substrate boards handled in step 1:(0.4~1): the hydrazine solution of (0.8~4) or 1 × 10^-3~1 × 10^-2The hydrogen bromide solution of mol/L as reducing agent, 25~It is placed at 30 DEG C 12~24 hours until dry；

3) it washes with water step 2 and handles obtained substrate boards 1, obtain the substrate boards 1 with graphene film decorative layer 3.

The length of the mesh grid frame 4 is 16cm, width 9cm, with a thickness of 1cm, the specification of mesh grid frame includes:(1) 96 grid, grid area 8mm*8mm, each grid accommodate 4 albumen Indexs measures；(2) 24 grids, grid cotton knotFor 1.6cm*1.6cm, each grid accommodates 16 albumen Indexs measures；Mesh grid frame 4 and substrate boards 1 are all made of poly- to benzene twoFormic acid glycol ester, polyvinyl chloride or polyethylene plastic cement material are made；After mesh grid frame 4 and substrate boards 1 form micropore disk, makeThe each micropore 5 obtained on micropore disk can measure a sample, and not interfere with each other between each micropore 5, realize that multiple samples are sameWhen measure.

It is described that detection method includes the following steps:

1) micropore disk electrochemical luminescence reacts: when carrying out electrochemiluminescence analysis measurement, connecting and is marked with electrochemical luminescenceThe substance of object, is powered to substrate boards, and electrochemical luminescence reaction will occur for electrochemical luminescence object；

2) then the micropore disk for completing electrochemical luminescence reaction is sent to after image capturing system and acquires, browses sample graphPicture utilizes charge-coupled device (Charge Coupled Device, CCD) or complementary metal oxide semiconductor(Complementary Metal-Oxide-Semiconductor, CMOS) is used as imaging sensor, the gauge without light source the case whereUnder, the luminous sample image after acquisition electrochemical luminescence reaction, the sample image of acquisition is carried out qualitative by data analysis systemOr quantitative analysis and interpretation judges automatically micropore disc-type first by micropore disk image comparison；Data analysis system hasAutomatic ferrule function will be converted to gray value after image digitazation, judge luminous point, to luminous point automatic ferrule, passes through ashAngle value difference delimit effective image area, grid dividing, check point spacing, battle array spacing etc., with true fixed position, by anti-Micropore disk after answering is continuously shot, synthesizes, calculates, and obtains average gray, last according to linear relationship, and gray value is converted intoConcentration is analyzed to qualitative or quantitative result.

It is obvious to a person skilled in the art that invention is not limited to the details of the above exemplary embodiments, Er QieIn the case where without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matterFrom the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended powerBenefit requires rather than above description limits, it is intended that all by what is fallen within the meaning and scope of the equivalent elements of the claimsVariation is included within the present invention, and any reference signs in the claims should not be construed as limiting the involved claims.

In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only wrappedContaining an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art shouldIt considers the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the artThe other embodiments being understood that.

Claims (3)

Translated fromChinese

1.一种高通量的电化学发光检测方法，采用一个用于电化学发光分析的微孔盘和一个图像采集系统、一个数据分析系统，其特征在于：所述微孔盘由基片板(1)及网状格子架(4)上下组合而成，基片板(1)的长度为16cm、宽度为9cm、厚度为0.5cm，基片板(1)上表面利用磁控溅射的方法镀有一层氧化铟锡膜(2)，氧化铟锡膜(2)的设置使得基片板(1)具有导电功能，从而作为一个电极使用，镀有氧化铟锡膜(2)的基片板(1)表面还附着有导电性能优良且具有蛋白质分子吸附功能的石墨烯膜修饰层(3)；所述网状格子架(4)的长度为16cm、宽度为9cm、厚度为1cm，网状格子架(4)与基片板(1)均采用聚对苯二甲酸乙二醇酯、聚氯乙烯或聚乙烯塑胶材质制成；网状格子架(4)与基片板(1)组成微孔盘后，使得微孔盘上的每个微孔(5)均能测定一个样本，且每个微孔(5)之间互不干扰，实现多个样本同时测定；所述检测方法包括以下步骤：1. a high-throughput electrochemiluminescence detection method, adopts a microporous disk for electrochemiluminescence analysis and an image acquisition system, a data analysis system, it is characterized in that: the microporous disk is composed of a substrate plate. (1) and the mesh lattice frame (4) are assembled up and down, the length of the substrate plate (1) is 16cm, the width is 9cm, and the thickness is 0.5cm, and the upper surface of the substrate plate (1) is formed by magnetron sputtering. The method is coated with a layer of indium tin oxide film (2), the arrangement of the indium tin oxide film (2) enables the substrate plate (1) to have a conductive function, so as to be used as an electrode, the substrate coated with the indium tin oxide film (2) The surface of the plate (1) is also attached with a graphene film modification layer (3) with excellent electrical conductivity and protein molecule adsorption function; the length of the mesh lattice (4) is 16cm, the width is 9cm, the thickness is 1cm, and the mesh The grid (4) and the substrate plate (1) are both made of polyethylene terephthalate, polyvinyl chloride or polyethylene plastic material; the mesh grid (4) and the substrate plate (1) After forming the microwell plate, each microwell (5) on the microwell disc can measure one sample, and each microwell (5) does not interfere with each other, so as to realize the simultaneous measurement of multiple samples; the detection method Include the following steps:1)微孔盘电化学发光反应：进行电化学发光分析测定时，接上标记有电化学发光物的物质，对基片板进行通电，电化学发光物就会发生电化学发光反应；1) Electrochemiluminescence reaction of microwell plate: when performing electrochemiluminescence analysis and measurement, connect a substance marked with electrochemiluminescence, and energize the substrate plate, and the electrochemiluminescence reaction will occur;2)接着将完成电化学发光反应的微孔盘送到图像采集系统后采集、浏览样本图像，利用电荷耦合器(Charge Coupled Device,CCD)或是互补金属氧化物半导体(ComplementaryMetal-Oxide-Semiconductor,CMOS)作为图像传感器，在无光源的情况下，采集电化学发光反应后的发光样本图像，采集的样本图像通过数据分析系统进行定性或定量的分析与判读，首先通过微孔盘图像对比，自动判断微孔盘类型；数据分析系统具有自动套圈功能，将图像数字化后转换为灰度值，判断出发光点，对发光点自动套圈，通过灰度值差异，划定图像有效区域、网格划分、校正点间距、阵间距，以确定点位置，通过对反应后的微孔盘连续拍摄、合成、计算，获取灰度平均值，最后依据线性关系，将灰度值换算成浓度，用以定性或是定量结果分析。2) Next, send the microwell plate that has completed the electrochemiluminescence reaction to the image acquisition system to collect and browse the sample images, and use a Charge Coupled Device (CCD) or a Complementary Metal-Oxide-Semiconductor (Complementary Metal-Oxide-Semiconductor, CMOS) as an image sensor, in the absence of a light source, the luminescent sample image after the electrochemiluminescence reaction is collected, and the collected sample image is analyzed and interpreted qualitatively or quantitatively through the data analysis system. Determine the type of micro-hole disk; the data analysis system has the function of automatic circling, converts the image into gray value after digitization, determines the light-emitting point, automatically circles the light-emitting point, and demarcates the effective area and mesh of the image through the difference of gray value. Grid division, correction of point spacing, and matrix spacing to determine the point position, through continuous shooting, synthesis, and calculation of the microplate after the reaction, the gray average value is obtained, and finally the gray value is converted into concentration according to the linear relationship, using Analysis with qualitative or quantitative results.2.根据权利要求1所述的高通量的电化学发光检测方法，其特征在于，所述石墨烯膜修饰层(3)是通过化学还原反应将氧化石墨烯还原成石墨烯制成，所述石墨烯膜修饰层(3)的制作步骤包括：2. high-throughput electrochemiluminescence detection method according to claim 1, is characterized in that, described graphene film modification layer (3) is made by reducing graphene oxide into graphene by chemical reduction reaction, so The manufacturing steps of the graphene film modification layer (3) include:1)在清洗过的基片板(1)表面滴加5～15微升浓度为0.1～0.4g/L的氧化石墨烯溶液，常温下自然干燥；1) dripping 5-15 microliters of graphene oxide solution with a concentration of 0.1-0.4 g/L on the surface of the cleaned substrate plate (1), and naturally drying at normal temperature;2)在步骤1中处理得到的基片板(1)表面滴加5～15微升水、肼和氨水体积比为100：(0.4～1)：(0.8～4)的肼溶液，或1×10^-3～1×10^-2mol/L的溴化氢溶液作为还原剂，在25～30℃下放置12～24小时直至干燥；2) Drop 5-15 microliters of water, hydrazine and ammonia solution with a volume ratio of 100:(0.4-1):(0.8-4) on the surface of the substrate plate (1) obtained in step 1, or 1× 10^-3 to 1×10^-2 mol/L of hydrogen bromide solution as a reducing agent, placed at 25 to 30 ° C for 12 to 24 hours until dry;3)用水清洗步骤2处理得到的基片板(1)，得到具有石墨烯膜修饰层(3)的基片板。3) Washing the obtained substrate plate (1) in step 2 with water to obtain a substrate plate having a graphene film modification layer (3).3.根据权利要求1所述的高通量的电化学发光检测方法，其特征在于，所述网状格子架的规格包括：96个方格，方格面积8mm*8mm，每个方格容纳4个蛋白指标检测；或者24个方格，方格棉结为1.6cm*1.6cm，每个方格容纳16个蛋白指标检测。3. The high-throughput electrochemiluminescence detection method according to claim 1, wherein the specification of the mesh grid frame comprises: 96 squares, the square area is 8mm*8mm, and each square accommodates 4 protein index detection; or 24 squares, the square neps is 1.6cm*1.6cm, and each square can accommodate 16 protein index detections.