CN111487403A - Portable enzyme-linked immunosorbent assay (ELISA) sample liquid absorbance detection instrument and detection method - Google Patents
Portable enzyme-linked immunosorbent assay (ELISA) sample liquid absorbance detection instrument and detection method Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
Abstract
The invention provides a portable enzyme-linked immunosorbent assay (ELISA) sample liquid absorbance detection instrument, which comprises (1) an absorbance detection module; (2) a data processing module; (3) and a power supply module. The invention further provides a 'fool' analysis method, which can directly detect the sample only by pressing a start key without setting instrument parameters, directly give a detection result whether the detected object exceeds the safety limit, and realize the on-site rapid detection of bacteria, viruses, diseases, toxic and harmful components in food and environment.
Description
Technical Field
The invention belongs to the field of rapid detection, and particularly relates to a portable detection instrument and a detection method for obtaining an enzyme-linked immunosorbent assay quantitative detection result.
Background
Enzyme-linked immunosorbent assay is widely applied to diagnosis of bacteria, viruses and diseases, detection of toxic and harmful components in food and environment and the like, and is one of the most widely applied detection methods at present. The method has the advantages of simple and convenient operation, high sensitivity, high analysis speed and the like, and is an excellent rapid detection method. However, analysis of the detection result is complex, which becomes a bottleneck in field use, market use and family use, and limits application of the detection result in field detection.
The enzyme-linked immunosorbent assay uses enzymatic reaction to generate a colored product, which usually shows blue or yellow, and then the content of the detected substance is obtained according to the color development degree of a sample. Therefore, the result analysis of the enzyme-linked immunoassay depends on a spectrophotometer or an enzyme-linked immunosorbent assay, and the absorbance of a light source with a specific wavelength after the light source penetrates through a color development sample is determined according to the Lambert-beer law. However, such absorbance detection instruments mainly aim at laboratory analysis, adopt light sources capable of generating different wavelengths, and use different filters or gratings to obtain the required specific wavelength from the light source during detection, and have the disadvantages of complex instrument structure, more parts, higher energy consumption, large volume and weight, inconvenience in carrying, and difficulty in field detection. In addition, the spectrophotometer or the microplate reader has many setting parameters, at least the detection wavelength and the zero setting operation need to be set before use, corresponding input keyboards are needed, the complexity of the structure of the instrument is increased, the operation steps during use are increased, users need to train to master the operation method of the instrument, and the work efficiency is reduced due to the excessive operation steps.
Therefore, the existing instrument for detecting the enzyme-linked immunosorbent assay and the use method thereof can not meet the requirement of rapid detection in the scenes without laboratory analysis conditions, such as fields, markets, families and the like.
Disclosure of Invention
The invention aims to provide a portable enzyme-linked immune sample liquid absorbance detection instrument and further provides a 'fool' analysis method, so that a sample can be directly detected only by pressing a power-on key without setting instrument parameters, a detection result of whether a detected object exceeds a safety limit is directly given, and the field rapid detection of toxic and harmful components in bacteria, viruses, diseases, food and the environment is realized.
In order to achieve the above object, according to one aspect of the present invention, the present invention provides a portable absorbance detection apparatus for enzyme-linked immune sample solution, comprising (1) an absorbance detection module; (2) a data processing module; (3) a power supply module;
the absorbance detection module is used for acquiring the absorbance of the sample liquid, and comprises a light source for emitting detection light, a photoelectric conversion sensor for detecting the intensity of the light, and an absorbance detection module shell for isolating the interference of environmental light, placing the light source, the photoelectric conversion sensor, a detected sample and other components;
the data processing module processes and calculates the signals obtained by the absorbance detection module and outputs results;
the power module provides power for the absorbance detection module and the data processing module.
Preferably, the light source of the detection module uses a blue or red light source.
Further preferably, the light source uses blue or red light emitting diodes.
Preferably, the housing of the absorbance detection module is divided into three regions, namely, a light source region, a sample region and a detection region; the three regions are connected by a light entrance hole and a light exit hole. The light emitting diode is positioned in the detection area, and the emitted light enters the sample area through the incident hole, is partially absorbed by the sample, then enters the detection area through the emergent hole on the other side, and is captured by the photoelectric conversion sensor.
The light source region and the detection region are distributed on both sides of the sample region.
The sample area is provided with a movable hatch cover so as to be conveniently placed into a cuvette containing the tested sample.
Further preferably, the bottom of the sample area is provided with a groove for fixing the position of the cuvette. The groove dimensions are preferably 1.2-1.3cm wide, 1.2-1.3cm long, and 0.1-0.6cm deep to accommodate the most commonly used types of cuvettes having a 1cm optical path.
Preferably, the data processing module comprises an analog-to-digital converter, a data calculation module, a display module and/or a sound generation module. The input end of the analog-to-digital converter is connected with the photoelectric conversion sensor and converts the signal obtained by the absorbance detection module into a digital signal. The input end of the data calculation module is connected with the output end of the analog-to-digital converter, and the output end of the data calculation module is connected with the input end of the display module. The data calculation module calculates signals input by the analog-to-digital converter according to the Lambert beer theorem to obtain absorbance, and transmits results to the display module and/or the sound production module.
Preferably, the display module displays an absorbance of a = ax-A1(ii) a Wherein A is a display numerical value, AxThe data calculation module calculates the current absorbance of the sample A1The absorbance of the first sample tested after start-up.
Further preferably, the data calculation module waits for several seconds after starting up, and displays a sample adding prompt on a screen to remind an operator to place the cuvette containing the sample to be detected in the detection area. After the sample adding waiting is finished, the calculation module calculates the absorbance of the sample and records the absorbance as A1And displaying the absorbance A on a display module, wherein A = Ax-A1. For the first sample tested after start-up, AxAnd A1Equal, so the screen shows an absorbance of 0 or its code. When detecting the subsequent sample, if the absorbance of the sample is more than A1The value is shown to be positive, if the absorbance is less than A1The value is shown as negative and a minus sign is shown before the absorbance value. The above equation can also be: a = A1-AxThe judgment mode of the result is opposite.
Further preferably, the setting range of the sample adding waiting time after the startup is 5-60 seconds.
Preferably, the power module comprises a rechargeable battery and a charging and discharging control component, and provides power for the absorbance detection module and the data processing module.
Further preferably, the charging voltage of the rechargeable battery is 5V.
According to another aspect of the present invention, there is also provided a "fool" enzyme-linked immune rapid detection method, wherein the sample after enzyme-linked immune reaction is placed in the detection area, and the instrument can automatically determine whether the detected sample meets the safety limit standard, the method comprising the following steps:
1) putting a first color sample with the content of the measured object as the limit standard into the detection area, pressing a start key, or pressing the start key first, and putting the sample into the detection area within a waiting period; the photoelectric conversion sensor captures the light absorbed by the first chromogenic sample, and the data calculation module calculates the absorbance of the first chromogenic sample;
2) after the waiting time period is finished, the sample to be detected is placed in the detection area, the photoelectric conversion sensor captures light rays absorbed by the sample to be detected, the absorbance of the sample is calculated by the data calculation module and is compared with the absorbance of the first color development sample, and the comparison result is output.
The comparison result output can be result display or voice prompt, stored data or data output and the like.
Preferably, the result is displayed through a display screen, and whether the measured sample exceeds the safety limit standard can be judged according to whether the numerical value of the display screen has a negative sign < - >.
When measuring a plurality of samples, the samples are sequentially placed in the detection region.
In the method, the first chromogenic sample and the sample to be detected are samples after reaction according to an enzyme-linked immunosorbent assay kit, and preferably are both yellow samples or both blue samples. The enzyme linked immunosorbent assay realizes the capture of a target object by utilizing the specific recognition reaction between an antigen and an antibody, generates a detection signal by using enzyme as a marker, and realizes the detection of a detected object according to the generation amount of a colored product after an enzymatic reaction substrate is added. Commonly used labeling enzymes in enzyme-linked immunoassays are horseradish peroxidase (HRP) and alkaline phosphatase. Using horseradish peroxidase as a labeling enzyme, it catalyzes the reaction of Tetramethylbenzidine (TMB) with hydrogen peroxide to produce a blue product which is acidified to a yellow color. When alkaline phosphatase is used as a labeling enzyme, the phosphate compound can be hydrolyzed to produce a colored product, for example, p-nitrophenyl phosphate is hydrolyzed to produce a yellow product; in addition, phosphate ions are released after the phosphate ester is hydrolyzed, and a blue product can be produced by a color developing reagent. The yellow and blue products can be used for detection, and the content of the detected object can be obtained according to the generation amount of the colored product.
The technical scheme of the invention has the following advantages:
(1) the invention provides an absorbance detection module which has simple structure, light weight and small volume and is used for enzyme-linked immunoassay. The spectrophotometer and other instruments need to use different filters or gratings to obtain the required specific wavelength from the light source and then can be used for enzyme-linked immunosorbent assay, so the instrument has mechanical movable parts, precise and complex structure and large volume and weight. In contrast, the invention directly uses red or blue light sources, does not provide filters or gratings, and does not need a complex light splitting system. The enzyme-linked immunosorbent assay module generally enables a sample to be blue or yellow and respectively has strong absorption to red or blue light, and the invention finds that a red or blue light-emitting diode can be used as a light source, and the absorbance of the enzyme-linked immunosorbent assay solution can be directly measured without screening out light with specific wavelength or using a reference light source, so that the absorbance detection module has few parts, simple structure and no mechanical movable part. These advantages not only reduce the volume and weight of the instrument, but also reduce the cost and failure rate of the instrument.
(2) The invention provides a portable, low-cost and convenient-to-use instrument, which realizes the field detection of enzyme-linked immunity. Firstly, the absorbance detection module of the invention has good portability and the light emitting diode has low energy consumption, and the battery can supply power for the absorbance detection module. Secondly, the invention uses the battery to supply power for all working parts, not only gets rid of the restriction of an external 220V power supply on the portability of the instrument, but also does not need to consider the conversion of alternating current and direct current and the influence of the voltage fluctuation of the external power supply on the detection, and does not need to design a voltage reduction, voltage stabilization and source filter circuit, so that the circuit of the instrument is simpler, the weight of the whole machine is lighter and the volume is smaller.
(3) The invention provides a 'fool' operation method for rapidly judging the safety of a detected sample aiming at the field rapid screening requirement. The whole instrument only needs one button, namely a starting button, and only needs to put a sample into the detection zone after starting without setting parameters. The instrument screen can not only display the detected absorbance value, but also directly draw the conclusion whether the sample exceeds the standard. The absorbance of the first sample is automatically recorded after the computer is started, and the absorbance value displayed on the screen is the difference value between the absorbance of the current sample and the absorbance of the first sample after the computer is started, so that the absorbance value of the first sample to be detected is displayed as 0. And (3) taking the color development sample with the content of the measured object as the limit standard as a first detection sample, and judging whether the measured object in the detected sample exceeds the standard or not according to the fact that the absorbance value measured by the subsequent sample is positive or negative. In addition, the judgment can be made according to whether the sound production module produces sound. The 'fooled' operation method can be used after starting up without setting instrument parameters, and is suitable for on-site rapid screening of a large number of samples.
Drawings
FIG. 1 is a schematic diagram of a portable ELISA absorbance detection instrument according to the present invention.
FIG. 2 is a perspective view of an embodiment of a housing of the absorbance detection module of the portable detecting device according to the present invention; wherein, 1 is detection area casing cang gai, 2 is sample district casing, and 3 and 4 of symmetric distribution in sample district casing both sides are regarded as detection area casing and light source district casing respectively, 5 are the unthreaded hole incidence and the exit aperture of sample district casing respectively, 6 are sample district casing bottom recess. The above 1-4 parts may have any suitable shape or form; the 3 and 4 symmetrical to the two sides of the detection zone, respectively, may have the same shape or different shapes.
FIG. 3 is a data diagram of the evaluation of the detection performance of the portable ELISA absorbance detection instrument according to the present invention; the standard instrument used was an Shimadzu UV mini1240 ultraviolet visible spectrophotometer.
FIG. 4 is a data diagram of aflatoxin content detection obtained by the "foolishing" detection method of the present invention; a value less than 0 indicates a content exceeding 0.12 ppb.
Detailed Description
The invention is described in further detail below with reference to the figures and the embodiments.
FIG. 1 is a schematic structural diagram of a portable enzyme-linked immunoassay instrument, which is composed of an absorbance detection module, a data processing module and a power supply module. The absorbance detection module comprises a light source, a photoelectric conversion sensor and a shell, wherein the shell comprises a sample area shell 2, a detection area shell 3 and a light source area shell 4. The data processing module comprises an analog-to-digital converter, a data calculation module and a display module. The power module comprises a rechargeable battery and a charging and discharging control component.
Fig. 2 is a schematic structural diagram of an absorbance detection module disclosed by the present invention, wherein a light source region housing 4 and a detection region housing 3 are located at two sides of a sample region housing 2, the sample region housing 2 is provided with a movable bin cover 1, and the bottom of the sample region housing 2 is provided with a groove 6 for fixing a cuvette for measuring a sample.
The light source area shell 4 is internally provided with a light emitting diode as a light source, and detection light emitted by the light emitting diode enters the sample area shell from the light hole, is partially absorbed by a sample in the cuvette and then enters the detection area shell 3 from another light hole. The photoelectric conversion sensor located in the detection zone housing 3 converts the intensity of light entering the detection zone housing into an electrical signal.
The analog-to-digital converter is connected with the photoelectric conversion sensor and converts a detection signal of the photoelectric conversion sensor into a digital signal. The data calculation module can use a single chip microcomputer to convert the signal of the analog-to-digital converter into absorbance and control the display module and/or the sound generation module to output a detection result. The display module can use a liquid crystal display screen, and the calculation formula of the absorbance A displayed on the screen is A = Ax-A1Or A = A1-Ax(ii) a Wherein A is a screen display numerical value, AxThe value measured for the current sample, A1The detection value of the first sample is detected after the power-on. Therefore, the blank sample is used as the first detection sample, and the actual absorbance of the subsequent sample can be measured; if the sample with the content of the measured object as the maximum allowable residual quantity of the substance is taken as the first sample, whether the measured sample exceeds the limit standard or not can be quickly judged according to the positive and negative properties of the display value A when the subsequent samples are detected. The sounding module can use a buzzer or a loudspeaker to send out alarm prompt sound according to the positive and negative of the A obtained through calculation.
FIG. 3 is a data diagram of the evaluation of the detection performance of the portable ELISA absorbance detection instrument according to the present invention. The sample to be detected is a yellow product obtained by catalyzing TMB with horseradish peroxidase to react with hydrogen peroxide and then acidifying. As can be seen from the graph, the two results have good correlation (R)2= 0.999), the slope is 0.995, which indicates that the two groups of detection results have almost no difference, and the portable enzyme-linked immune sample liquid absorbance detection instrument of the invention has accurate measurement results.
The operation method for detecting the enzyme-linked immunosorbent assay quantitative detection by using the invention is as follows:
1. and (3) taking a sample to be detected and a group of standard samples with known concentrations, operating according to the operation instruction of the kit to obtain sample liquids with different color development degrees, and placing the sample liquids into a cuvette.
2. The cuvette with the blank is placed in the detection zone of the instrument and then turned on. Or starting the machine first and then placing the cuvette, and finishing the lofting operation in the waiting period.
3. And after the instrument displays that the absorbance is 0, sequentially putting the standard sample groups into the detection area, and recording the absorbance. Then, the sample to be measured was placed, and the absorbance was recorded.
4. And (5) drawing a concentration-absorbance curve of the standard sample, and calculating the concentration of the measured object in the sample to be measured according to a regression equation.
The operation method for carrying out 'fool' quick screening on sample safety by using the invention is as follows:
1. and obtaining a color development sample. And (3) taking a sample to be detected and a standard sample with the content of the detected object being the maximum allowable residual quantity of the substance, operating according to the operation instruction of the enzyme linked immunosorbent assay kit to obtain sample solutions with different color development degrees, and placing the sample solutions into a cuvette.
2. And acquiring the absorbance of the first sample. The cuvette with the standard sample is placed in the detection zone of the instrument and then turned on. Or starting up the machine first and finishing the lofting operation in the waiting time period. When the instrument shows that the absorbance development is 0, the completion of the step is indicated.
3. And screening the tested sample. And (3) putting the sample to be detected into a detection area, and judging whether the detected object exceeds the standard or not according to the positive and negative values and/or whether an alarm prompt sound is given or not. When a plurality of samples to be detected exist, the samples are sequentially placed in the detection area, and no additional operation is needed.
Examples 1
The absorbance detection module uses a 3mm blue light emitting diode as a detection light source, and the light emitting diode can emit blue light for detecting yellow sample liquid after enzyme-linked immunosorbent assay. The photoelectric conversion sensor uses a cadmium sulfide (CdS) photoresistor, and the resistance value of the photoelectric conversion sensor changes along with the change of the light intensity. The groove at the bottom of the sample area has the length of 1.2cm, the width of 1.2cm and the depth of 0.1 cm. The appearance of the light source area and the detection area is a cylinder, the inner diameter is 5mm, and the incident light and the emergent light hole are round holes with the diameter of 2 mm.
The model of an analog-to-digital converter in the data processing module is ADS1115, the singlechip used by the data calculation module is AT89C52 singlechip produced by Atmel company, the model of a screen used by the display module is L CD1602, the sample adding waiting time after starting up is set to 60 seconds, the screen displays 60 seconds countdown in the period, the absorbance calculation is started after the countdown is finished, and the first value is recorded as A1And displaying the absorbance A on a screen, wherein A = Ax-A1AxThe power module uses L P803450x5V type batteries integrated with a boost protection board and a lithium battery and manufactured by New energy batteries Limited, and uses a Micro USB charging interface.
The method of use of this embodiment is as follows:
1. the aflatoxin B1 enzyme-linked immunoassay kit produced by Fuzhou Jia atlas biotechnology limited, the product number of which is E030105, is used for carrying out enzyme-linked immunosorbent assay to obtain a sample to be detected, and the kit mainly comprises an ELISA plate pre-coated with an antigen, aflatoxin standard solutions (0, 0.03, 0.06, 0.12, 0.24 and 0.48 ppb) of 1m L respectively, an enzyme marker of 5.5m L, an antibody working solution of 5.5m L, substrates A and B of 6m L respectively, a stop solution of 6m L and 20X concentrated washing solution of 40m L, and according to the use specification, the sample developing step mainly comprises the following steps:
(1) adding sample for reaction, respectively adding Aspergillus flavus standard or sample 50 μ L/hole into each micropore, adding enzyme marker 50 μ L/hole and antibody working solution 50 μ L/hole, standing for reaction for 30 min;
(2) washing, removing reaction liquid, and washing for 5 times by using washing liquid;
(3) developing, adding 50 mu L of each developing substrate A and B into each hole, performing enzymatic reaction to generate a blue product, and reacting for 15 min;
(4) the reaction was stopped and 50. mu. L of stop solution was added to each well and the color turned yellow.
2. Background was subtracted. Adding an undeveloped control sample into a micro cuvette with an optical path of 1cm, and placing the sample into an instrument detection area within 60 seconds after starting up the instrument for deducting the absorbance of the cuvette, water and the reagent so as to enable the absorbance displayed on a screen of the instrument to be 0.
3. And (4) sequentially placing the cuvettes containing the developed standard substance and the sample solution into a detection area, and recording the absorbance.
4. And calculating the content of the aflatoxin according to the concentration-absorbance regression equation of the aflatoxin and the absorbance of the detected sample.
EXAMPLES example 2
The absorbance detection module uses a 5mm blue light emitting diode as a detection light source, and the light emitting diode can emit blue light for detecting yellow sample liquid after enzyme-linked immunosorbent assay. The photoelectric conversion sensor uses a CdS photosensitive resistor, and the resistance value of the photoelectric conversion sensor changes along with the change of the light intensity. The groove at the bottom of the sample area has the length of 1.3cm, the width of 1.2cm and the depth of 0.6 cm. The shapes of the light source area and the detection area are cylinders with the side length of 8mm, and the incident light holes and the emergent light holes are round holes with the diameter of 5 mm.
NE555 is used by an analog-to-digital converter in the data processing module, AT89C52 produced by Atmel company is used as a singlechip used by the data calculating module, the screen model used by the display module is L CD12864, the sample adding waiting time after startup is set to be 5 seconds, the countdown of 5 seconds is displayed on the screen in the period, the absorbance is calculated after the countdown is finished, and the first value is recorded as A1And displaying the absorbance A on a screen, wherein A = Ax-A1AxThe value measured for the current sample. Power module using sequenceAn L P803450x5V type battery which is produced by new energy batteries and integrates a boosting protection plate and a lithium battery is used as a charging interface of a Micro USB.
The method of use of this embodiment is as follows:
1. and carrying out enzyme-linked immunosorbent assay to obtain a sample to be detected. The sample solution to be measured and a standard solution having an aflatoxin content of 0.12ppb were subjected to color development according to the kit and the sample color development procedure used in example 1.
2. And (3) placing the cuvette filled with 0.12ppb aflatoxin color development liquid into an instrument detection area, and then starting up the instrument.
3. And after the countdown of 5 seconds is finished, taking out the standard product, putting the sample to be detected, and judging whether the content of the aspergillus flavus exceeds 0.12ppb or not according to the positive and negative values. Because the absorbance value of the sample obtained by the kit is in negative correlation with the content of aflatoxin contained in the sample, the screen display value is more than 0, which indicates that the content of aflatoxin is less than 0.12ppb, and if the value is less than 0, the content of aflatoxin is higher than 0.12 ppb.
FIG. 4 is a data diagram obtained by measuring aflatoxin with different contents by using the method, and the data diagram shows that the method can accurately judge whether the content of the measured substance exceeds the limit.
EXAMPLE 3
The absorbance detection module uses a 3mm red light emitting diode as a detection light source, and the light emitting diode can emit red light for detecting blue sample liquid after enzyme-linked immunosorbent assay. The photoelectric conversion sensor uses a CdS photosensitive resistor, and the resistance value of the photoelectric conversion sensor changes along with the change of the light intensity. The groove at the bottom of the sample area has the length of 1.3cm, the width of 1.3cm and the depth of 0.6 cm. The light source region shape is the cylinder, and internal diameter 10mm, incident unthreaded hole are the round hole of diameter 3mm, and the detection zone appearance is the cube, and length of side is 7mm, and the exit unthreaded hole is square, and length of side is 4 mm.
The STC5A60S2 single chip microcomputer with an analog-to-digital conversion function produced by STC company is used, the screen model used by a display module is L CD12864. the sample adding waiting time after the startup is set to be 10 seconds, the screen displays countdown for 10 seconds in the time period, the absorbance is calculated after the countdown is finished, and the first value is recorded as A1And displaying the light absorption on the screenDegree A, wherein A = Ax-A1AxThe value measured for the current sample. The power module uses 18650 type lithium batteries and Shenzhen Xintai micro-technology Limited's 5V integrated module of charging and discharging, uses DC-0023.5-1.1 mm type power interface.
The method of use of this embodiment is as follows:
1. using human original cadherin 1 enzyme linked immunosorbent assay kit produced by R & D Systems, the product number YX-160304H, mainly comprises a micropore ELISA plate, 6 standard substances with the concentration of 0-160pg/m L, 0.3m L respectively, a detection antibody-HRP 10m L, 20X washing buffer solution 25m L, substrates A and B, 6m L respectively and stop solution 6m L, and the sample developing step mainly comprises:
(1) adding sample for reaction, adding the sample to be detected and the standard substance 50 mu L of 40 pg/m L into the micropores respectively, adding 100 mu L of detection antibody marked by horseradish peroxidase into each hole, and standing for reaction for 60 min;
(2) washing, removing liquid, and washing with washing liquid for 5 times;
(3) and (3) developing, adding 50 mu L of substrate A, B into each well to generate a blue product, and transferring the reaction solution into a microcuvette with the optical path of 1cm after reacting for 15 min.
2. The cuvette containing 40 pg/m L standard was placed in the detection zone of the instrument and then turned on.
3. And after the countdown of 10 seconds is finished, taking out the standard product, putting the sample to be detected into the standard product, and judging whether the content of the detected object exceeds 40 pg/m L according to the positive and negative values of the value, wherein the absorbance value of the sample obtained by the kit is in positive correlation with the content of the detected object, so that the screen display value is less than 0, which indicates that the content of the detected object is less than 40 pg/m L, and if the value is more than 0, the content of the detected object is more than 40 pg/m L.
EXAMPLE 4
Screen display absorbance a calculation formula a = a1-AxAs in example 3, the screen display indicates a value less than 0 indicating a level greater than 40 pg/m L, and if the value is greater than 0, the level of the test substance is less than 40 pg/m L.
EXAMPLE 5
The buzzer is used as the sound production module, and the rest is the same as the embodiment 4. when the display value is larger than 0, namely the content is higher than 40 pg/m L, the sound prompt is produced.
EXAMPLE 6
The hydrogen peroxide concentration was measured using the same measuring instrument as in example 1, using the following method:
1. taking 0.1M L sample to be tested, adding the sample to be tested into a cuvette, adding 10U horseradish peroxidase and 0.3M L mM TMB, uniformly mixing, standing for 15min, adding 0.1M L mM sulfuric acid, and preparing a standard color developing solution by using 0-320 mu M hydrogen peroxide standard sample according to the same method.
2. Background was subtracted. And adding the standard color developing solution with the content of 0 into a micro cuvette with the optical path of 1cm, and putting the cuvette into an instrument detection area within 60 seconds after starting up the cuvette for background subtraction so as to enable the absorbance displayed on the screen of the instrument to be 0.
3. And detecting the content of the substance to be detected. And sequentially placing the cuvettes containing the developed standard substances with different concentrations and the sample solution into a detection area, and recording the absorbance. And calculating the concentration of the sample to be detected according to the concentration-absorbance regression equation and the absorbance of the sample to be detected.
EXAMPLES example 7
The phosphate content was measured using the same measuring instrument as in example 3, using the following method:
1. and (3) obtaining a color development sample, namely respectively taking a sample to be detected at 0.5M L and a standard sample with the phosphate ion content of 0-2 mM, and mixing the samples with a color development agent with the components of 21mM ammonium molybdate, 2mM antimony potassium tartrate, 5.4M sulfuric acid and 3.3% ascorbic acid at 0.5M L to obtain a blue sample.
The background subtraction and the content detection of the analyte were performed as in example 6.
EXAMPLES example 8
Horseradish peroxidase activity was measured using the same detection instrument as in example 3, using the following method:
1. taking 0.05m L enzyme solution to be detected, putting the enzyme solution into a cuvette, adding 2.8 m L phosphate buffer solution with pH 7.0, 1mM TMB with concentration of 0.2 m L and 4mM hydrogen peroxide with concentration of 0.05m L, mixing uniformly, and quickly putting the mixture into a detection area.
2. And starting the instrument, displaying the sample adding waiting screen as 0, timing, and recording the display value after 2min to obtain the increase of the absorbance within 2 min.
3. And (3) using a horseradish peroxidase standard sample with the enzyme activity range of 0.1-1.6U, repeating the steps 1 and 2, drawing an enzyme activity-absorbance increase standard curve, and calculating the enzyme activity according to the absorbance value of the sample liquid to be detected.
The invention is described above by taking enzyme-linked immunosorbent assay of aflatoxin and human crude cadherin 1, horse radish peroxidase activity, and detection of hydrogen peroxide and phosphate ion content as examples. The invention can be applied to other samples which are yellow or blue after color development, and the 'foolproof' quick judgment method for judging whether the detected object exceeds the standard can be applied to the detection of other components in spectrophotometry analysis. The components and parameters of the absorbance detection module, the data processing module and the power supply module of the detection apparatus are only preferred embodiments of the present invention. The above is not meant to limit the invention in any way. On the contrary, many modifications, variations and changes will occur within the scope of the present invention.
Claims (10)
1. A portable enzyme-linked immunosorbent assay (ELISA) sample liquid absorbance detection instrument comprises (1) an absorbance detection module; (2) a data processing module; (3) a power supply module;
the absorbance detection module comprises a light source for emitting detection light, a photoelectric conversion sensor and an absorbance detection module shell for isolating ambient light interference and placing the light source, the photoelectric conversion sensor and a detected sample;
the data processing module processes and calculates the signals obtained by the absorbance detection module and outputs results;
the power module provides power for the absorbance detection module and the data processing module.
2. The portable enzyme-linked immunosorbent assay instrument for detecting absorbance of a sample solution according to claim 1, wherein the light source of the absorbance detection module is a blue or red light source.
3. The portable enzyme-linked immunosorbent assay instrument for detecting absorbance of a sample solution according to claim 1, wherein the light source of the absorbance detection module uses a blue or red light emitting diode.
4. The portable enzyme-linked immunosorbent assay device of claim 1, wherein the housing of the absorbance detection module is divided into three areas, namely a light source area, a sample area and a detection area; the three regions are connected through the light incident hole and the light exit hole, the light source is arranged in the light source region, and the photoelectric conversion sensor is arranged in the detection region.
5. The portable enzyme-linked immunosorbent assay device of claim 4, wherein the light source region and the detection region are disposed on both sides of the sample region.
6. The portable enzyme-linked immunoassay sample solution absorbance detecting instrument according to claim 4, wherein the bottom of said sample region has a groove for fixing the position of the cuvette.
7. The portable enzyme-linked immunosorbent assay device of claim 1, wherein said data processing module comprises an analog-to-digital converter, a data calculating module, a display module and/or a sound generating module; the input end of the analog-to-digital converter is connected with the photoelectric conversion sensor and converts the signal obtained by the absorbance detection module into a digital signal, the input end of the data calculation module is connected with the output end of the analog-to-digital converter, and the output end of the data calculation module is connected with the input end of the display module; the data calculation module calculates signals input by the analog-to-digital converter according to the Lambert beer theorem to obtain absorbance, and transmits results to the display module and/or the sound production module.
8. The method of claim 7A portable enzyme-linked immunosorbent assay instrument for detecting absorbance of a sample solution is characterized in that the absorbance displayed by a display module is A = Ax-A1Or A = A1-Ax(ii) a Wherein A is a display numerical value, AxThe absorbance, A, of the current sample calculated by the data calculation module1The absorbance of the first sample tested after start-up.
9. The portable enzyme-linked immunosorbent assay device of claim 1, wherein said power module comprises a rechargeable battery and a charging/discharging control unit for providing power to said absorbance detection module and said data processing module.
10. An enzyme-linked immunosorbent assay rapid detection method is characterized by comprising the following steps:
1) placing a first color development sample with the content of the measured object being the limit standard into a detection area, capturing the light absorbed by the first color development sample by a photoelectric conversion sensor, and calculating the absorbance of the first color development sample by a data calculation module;
2) and putting a sample to be detected into the detection area, capturing the light absorbed by the sample to be detected by the photoelectric conversion sensor, calculating the absorbance of the sample by the data calculation module, comparing the absorbance with the absorbance of the first color sample, outputting the comparison result by a display screen and/or sound, and judging whether the sample to be detected exceeds the safety limit standard according to whether the numerical value of the display screen has a negative sign < - > and/or whether an alarm sound is given.
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