CN113740385B - Determination method for detecting chip characteristic response - Google Patents
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- 238000000034 method Methods 0.000 title claims abstract description 25
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- 239000000523 sample Substances 0.000 claims description 29
- 239000012488 sample solution Substances 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 2
- 239000008363 phosphate buffer Substances 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 22
- 238000003018 immunoassay Methods 0.000 abstract description 5
- 238000005259 measurement Methods 0.000 description 13
- 239000000427 antigen Substances 0.000 description 12
- 102000036639 antigens Human genes 0.000 description 12
- 108091007433 antigens Proteins 0.000 description 12
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- 238000004720 dielectrophoresis Methods 0.000 description 2
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- 238000005370 electroosmosis Methods 0.000 description 2
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- 230000009286 beneficial effect Effects 0.000 description 1
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
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- 208000010125 myocardial infarction Diseases 0.000 description 1
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- 238000004445 quantitative analysis Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/02—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/02—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance
- G01N27/04—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance by investigating resistance
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/02—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance
- G01N27/22—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance by investigating capacitance
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Abstract
The invention relates to the technical field of immunoassay detection, and particularly discloses a method for determining characteristic response of a detection chip, which comprises the following steps: s110, cleaning a chip for a first preset time by using background liquid A, adding the background liquid A into the chip, and performing first frequency scanning to obtain a first curve; s120, removing background liquid A in the chip, cleaning for a first preset time by using background liquid B, adding the background liquid B after cleaning, and performing frequency scanning for the second time to obtain a second curve; s130, comparing the first curve with the second curve, judging whether the first curve and the second curve are overlapped, and if so, determining a frequency band corresponding to the overlapped part of the first curve and the second curve as a characteristic response frequency band F1. The technical scheme of the invention can determine the characteristic response frequency band of the chip in the immunodetection, thereby improving the universality of different chips.
Description
Technical Field
The invention relates to the technical field of immunoassay detection, in particular to a method for determining characteristic response of a detection chip.
Background
Immunoassays are techniques that perform qualitative or quantitative analysis of specific biochemical substances based on antigen-antibody binding. Antibodies recognize the corresponding antigen through epitopes on the surface of the antigen and bind. This recognition also makes immunoassays highly specific: for example, an aids antibody will only bind to an aids antigen and will not react with other antigens.
In the conventional detection method, the binding of the target antibody or antigen and the corresponding antigen or antibody in the kit is completed by means of molecular diffusion motion and random brownian motion, the whole process is very passive, so that the detection time is long, and the time from the collection of a sample to the obtaining of a result often varies from tens of minutes to hours.
In order to increase the detection time, chip immunoassay technology has emerged, which coats antigens (or antibodies) on a chip, and reacts with a sample to be detected or a biological specimen at the same time, so that the detection result of all known antigens (or antibodies) in the chip can be obtained at one time. For example, CN104965081B discloses an antibody antigen detection method based on mobile equipment, by applying an excitation signal to a chip, dielectrophoresis effect, electrothermal effect and electroosmosis effect are generated in the chip, the dielectrophoresis effect can enable a target antibody or antigen in a sample to move towards an electrode plate in the chip, so that the combination of the target antibody or antigen and a corresponding antigen or antibody coated on the surface of the electrode plate is accelerated, the electrothermal effect and the electroosmosis effect can drive liquid to flow, and thus a target object can be brought to the vicinity of the electrode, thereby accelerating detection and shortening detection time. However, current chips/electrodes have a lack of versatility because of inconsistent processes, materials, etc., and different frequency points for characterizing specific binding of substances when used for accelerated detection.
For this reason, a determination method for detecting chip feature response that can improve versatility is required.
Disclosure of Invention
The invention provides a method for determining the characteristic response of a detection chip, which can improve the universality of a characteristic response section in chip immunodetection.
In order to solve the technical problems, the application provides the following technical scheme:
a determination method for detecting chip characteristic response comprises the following steps:
s110, cleaning a chip for a first preset time by using background liquid A, adding the background liquid A into the chip, and performing first frequency scanning to obtain a first curve;
s120, removing background liquid A in the chip, cleaning for a first preset time by using background liquid B, adding the background liquid B after cleaning, and performing frequency scanning for the second time to obtain a second curve;
s130, comparing the first curve with the second curve, judging whether the first curve and the second curve are overlapped, and if so, determining a frequency band corresponding to the overlapped part of the first curve and the second curve as a characteristic response frequency band F1.
The basic scheme principle and the beneficial effects are as follows:
in this scheme, wash chip with background liquid A and predetermine time, add background liquid A to the chip again, only contain background liquid A on the assurance chip, can ensure when first frequency scanning, can not receive the interference of other liquid or material on the chip surface. The background liquid B is used for cleaning the first preset time, and the background liquid B is added after the cleaning is finished, so that the interference of other liquid or substances on the surface of the chip can be avoided during the second frequency scanning. The frequency corresponding to the overlapping part of the first curve and the second curve is determined as the characteristic response frequency band F1, namely the same part in the frequency scanning of the two different solutions is used as the characteristic of the chip, and the association degree of the response value of the chip and the solution is small at the characteristic. After the characteristic response frequency band F1 is determined by selecting a plurality of chips from a certain class or batch of chips, the remaining chips can use the characteristic response frequency band F1, so that the universality is good.
Further, in S130, if there is no coincidence, the sample solution A is added to the chip sample And performing frequency scanning, determining the frequency corresponding to the inflection point of the phase obtained by the obtained curve as a characteristic point, and determining a characteristic response frequency band F1x based on the characteristic point, wherein x=a, b and c ….
The extreme points of the phase reflect the characteristic response more than other electrical parameters.
Further, in S130, the characteristic point minus the preset value is used as a left end point, the characteristic point plus the preset value is used as a right end point, and the frequency from the left end point to the right end point is used as the characteristic response frequency band F1x.
Further, in S120, the chip is cleaned with water for a first preset time after the background solution a is removed.
The residual background liquid A on the chip can be removed by washing with water.
Further, the frequency sweep is a complex impedance frequency sweep.
Further, the background solution A is 1mM phosphate buffer salt solution, and the background solution B is 1mM borate buffer solution.
Further, the first preset time is greater than 30 seconds.
A determination method for detecting chip characteristic response comprises the following steps:
s210, adding sample solution A into the chip sample Performing first frequency scanning to obtain a third curve;
s220, placing the chip for a second preset time;
s221, cleaning the chip with background solution A and then adding sample solution A sample Performing secondary frequency scanning; obtaining a fourth curve;
s230, comparing the third curve with the fourth curve, judging whether the third curve and the fourth curve have changes, and if the third curve and the fourth curve have changes, determining a frequency segment corresponding to the changed part of the fourth curve relative to the third curve as a characteristic response frequency band F2.
After the chip is subjected to biological modification, the characteristic points can be changed, and therefore, the excitation signals with universality are difficult to determine according to the characteristic points. In the scheme, a sample solution is added for carrying out first frequency scanning, then the sample solution is added for carrying out second frequency scanning after cleaning, and frequency bands which change in a third curve and a fourth curve are respectively obtained after the second frequency scanning, namely, a characteristic response frequency band F2 which shows that the surface of a chip changes after reaction is obtained.
Further, the second preset time is 1-48 hours.
Further, the sample solution A sample Is background liquid A containing the sample to be tested.
Drawings
FIG. 1 is a flow chart of determining characteristic response frequency bands of a chip of a preset type by different background liquids;
FIG. 2 is a flow chart for determining chip characteristic response frequency bands by background liquid and sample solution;
FIG. 3 is a Bode plot obtained from two frequency sweeps;
FIG. 4 is a graph showing the percentage of change of the capacitance value with time before and after acceleration of the chip type I at a specific frequency point;
FIG. 5 is a graph showing the percentage change of the capacitance value with time before and after acceleration of the chip type I in a specific frequency band;
FIG. 6 is a graph showing the percentage change of the capacitance value with time before and after acceleration of the chip type II at a specific frequency point;
FIG. 7 is a graph showing the percentage change of the capacitance value with time before and after acceleration of the chip type II in a specific frequency band;
FIG. 8 is a diagram showing examples of negative and positive differentiation of chip type I;
FIG. 9 is a schematic diagram showing negative and positive differentiation of chip type II.
Detailed Description
The following is a further detailed description of the embodiments:
example 1
The method for determining the response of the detection chip features of the embodiment comprises the following steps:
as shown in fig. 1, the characteristic response frequency bands of chips of a preset type (the preset type may be a certain type or a certain lot of chips) are determined by different background liquids:
s110, cleaning the chip with the background liquid A for a first preset time, wherein in the embodiment, the first preset time is more than 30 seconds, adding the background liquid A into the chip, and performing first frequency scanning to obtain a first curve. In this embodiment, the frequency sweep is a complex impedance frequency sweep;
s120, immediately removing background liquid A in the chip after scanning and cleaning the chip for a first preset time by using water, wherein in the embodiment, the water adopts ultrapure water. Then cleaning the first preset time by using the background liquid B, adding the background liquid B after cleaning, and performing frequency scanning for the second time to obtain a second curve;
s130, comparing the first curve with the second curve, judging whether the first curve and the second curve are overlapped, and if so, determining a frequency segment corresponding to the overlapped part of the first curve and the second curve as a characteristic response frequency band F1. I.e. the same part in the two different solution tests is measured as the characteristic of the chip where the correlation of the response value of the chip with the solution is small.
In this embodiment, a first curve and a second curve are obtained from data graphs obtained by two frequency scans, and the data graphs are baud graphs. For example: using 1mM BS (phosphate buffer solution Phosphate Buffered Saline) as background solution A, cleaning and adding the chip, and performing first frequency scanning;
using 1mM BS (borate buffer Borate Buffered Saline) as background liquid B, cleaning and adding the chip, and performing secondary frequency scanning;
data as described in fig. 3 can be obtained: in the graph, at 20kHz-50kHz, two curves are coincident, namely the values of the two curves are equal, and the frequency of the section is the characteristic response frequency band F1. I.e., the chip response at 20kHz-50kHz was not changed by the solution changes when PBS and BBS were used.
If there is no coincidence, add sample solution A to the chip sample The first frequency scan is performed within 30 seconds, the frequency corresponding to the inflection point of the phase of the obtained curve (i.e. the third curve in S210) is used as a characteristic point, the characteristic point is subtracted by a preset value to be used as a left endpoint, the characteristic point is added with the preset value to be used as a right endpoint, the frequency from the left endpoint to the right endpoint is used as a characteristic response frequency band F1x, the preset value in this embodiment is 20kHz, and the characteristic response frequency band F1x includes values of the left endpoint and the right endpoint. Where x=a, b, c, … (in this embodiment, the lowest is not lower than 1kHz and the highest is not higher than 1 MHz), for example, there are several characteristic response bands at this time. Marked as F1a, F1b … in turn
As shown in fig. 2, the characteristic response frequency band caused by the sample is determined by the background liquid and the sample solution (the sample is diluted in the background liquid):
s210, adding to the chipInto sample solution A sample Namely, carrying out first frequency scanning within 30 seconds to obtain a third curve when the background liquid A containing the sample to be tested is contained;
s220, placing the chip in a wet environment for a second preset time, wherein the second preset time is 1-48 hours, and in the embodiment, 6 hours; in this example, the humidity range is 45% to 95% RH.
S221, cleaning with background solution A, adding sample solution A sample Performing secondary frequency scanning; obtaining a fourth curve;
s230, comparing the third curve with the fourth curve, judging whether the third curve is changed or not, and if the third curve is changed, determining a frequency segment corresponding to the part of the fourth curve, which is changed relative to the third curve, as a characteristic response frequency band F2. Namely, a sample solution is added for carrying out first frequency scanning, then the sample solution is placed for a certain time, then the sample solution is cleaned, and then the same sample solution is added for carrying out second frequency scanning, and the frequency range which changes after the two frequency scanning is the characteristic response frequency range F2 which changes after the chip surface reaction is reflected.
In order to further explain the application of the characteristic response frequency band in detection, the embodiment also provides an antigen-antibody detection method, which comprises the following steps:
the selection step comprises the following steps:
s310, judging whether the characteristic response frequency band F1 or F1x (x=a, b, c, …) is overlapped with the characteristic response frequency band F2, if not, jumping to S510, if so, determining an overlapped section or an overlapped point, and jumping to S410.
The detection step specifically comprises the following steps:
s410, adding a sample on the chip, selecting a specific frequency band or a specific frequency point based on the coincident segment or the coincident point under a specific voltage, measuring with time under an acceleration condition, wherein the voltage under the acceleration condition is 0.01-30V p-p The method comprises the steps of carrying out a first treatment on the surface of the In this embodiment, tens of KHz before and after the coincidence point is selected as the specific frequency band, for example, the coincidence point is 30KHz, and then 10KHz-50KHz can be selected. Acceleration and measurement are performed simultaneously, and acceleration is performed by applying alternating current with a certain frequency and a larger voltage to the electrode, so that adsorption force is generated at the moment, and meanwhile, current response is generated, and the measurement is completed.
And S420, as shown in fig. 4-7, determining the content of the target object in the sample or qualitatively judging according to the change rate (such as the slope obtained after straight line fitting, the quadratic term or the first term coefficient obtained after parabolic fitting, and the like) of the measured electric signal value (such as impedance, capacitance value or resistance value). In this embodiment, a least square method is used to fit a straight line.
For example, after a sample is added on a chip, a frequency scan is performed from a high frequency point of a specific frequency band to a low frequency point under a specific voltage while accelerating for 5-240s, a curve A of a capacitance value changing with time is obtained after completion, and a slope of the curve A is calculated. If the mode of frequency scanning is adopted from the low frequency point to the high frequency point, the negative sign is added to the obtained slope finally.
S510, adding a sample, and performing frequency scanning based on a first preset frequency, wherein the first preset frequency is 1MHz-100Hz (from high to low), so as to obtain a curve B of a response electric signal (such as a capacitance value, impedance and resistance value); in the present embodiment, the voltage range during frequency sweep is 1mV p-p -1V p-p . The first preset frequency comprises a first preset frequency segment and a first preset frequency point.
S520, performing acceleration operation on the chip based on a second preset frequency; the second preset frequency ranges from 1kHz to 1MHz, and comprises a second preset frequency section and a second preset frequency point; the second preset frequency point is a certain point value in the second preset frequency section. For example, the second preset frequency point is 100kHz and the voltage is 0.01-30V p-p . In this embodiment, the second preset frequency is a theoretically calculated frequency capable of accelerating, and the factor of whether the antigen-antibody binding can be reflected is ignored in the calculation.
S530, frequency scanning is performed again by using the parameters of S510, and a curve C of the response electric signal is obtained.
S540, calculating the integral area difference between the curve B and the curve C obtained by two frequency scans, and determining the content of the target object in the sample or qualitatively judging the content of the target object based on the integral area difference.
The calculation formula of the change rate of the capacitance integral is as follows:
(Cs2-Cs1)/Cs1*100％
wherein Cs1 is a capacitance value at a first preset frequency point on the curve B before acceleration or an integral area value at a first preset frequency section;
cs2 is the capacitance value at the second preset frequency point on the curve C after acceleration, or the integral area value at the second preset frequency segment. As shown in FIGS. 8-9, in the detection of a myocardial infarction marker, the judgment of negative and positive can be made by integrating the area values, wherein the circled portion is positive.
In this embodiment, a plurality of chips are selected from the preset type chips to perform detection according to the method, calibration is performed according to the detection result, and the remaining chips are detected according to the same method, so that the universality is strong. The preset type may be a certain type or a certain lot of chips. For example, a chip of a certain class, the selected 2-5 chip characteristic response frequency bands F1 and the characteristic response frequency band F2 are all coincident, and the rest chips can be directly detected according to steps S410-S420. For example, 1 chip is selected to be added with a positive sample, the response value of the obtained chip is 1, another chip is selected to be added with a negative sample, the response value of the obtained chip is 2, and the rest chips can be used for defining the response value of 1.5 as a distinguishing line, and the response value is more than 1.5 and less than 1.5 and positive.
When the integral area difference between the curve B and the curve C obtained by two frequency scans is calculated in step S540, the abscissa range may be selected according to the actual situation, for example, the portion with the largest difference between the curve B and the curve C negative and positive is selected, for example, the abscissa range corresponding to the circled portion in fig. 8-9 is selected, for example, the portion with the largest difference between the curve B and the curve C negative and positive is selected, and then the abscissa range is extended to the intersection. It should be noted that after the scaled chips determine the abscissa range, the remaining chips need to select the same abscissa range.
In other embodiments, measurements may also be made over time as the chip accelerates as step S520 is performed, and measurements may also be made as step S540. Judging whether the measurement result of the step S520 and the measurement result of the step S540 have correlation, if so, calibrating the measurement result of the step S520 based on the measurement result of the step S540, and directly adopting the test method of the step S520 to determine the content of the target in the sample or qualitatively judging the content of the target. The steps can be effectively simplified, and the detection efficiency is improved. Specifically, a positive sample of a set of concentration gradients is measured, the change rate (for example, the slope obtained after straight line fitting, the quadratic term or the first term coefficient obtained after parabolic fitting, etc.) of the electrical signal value (for example, the impedance, the capacitance value or the resistance value, etc.) at each concentration can be obtained in step S520, that is, the response value, likewise, the response value at each concentration can also be obtained after measurement in step S540, and these results and the concentration will show a certain functional relationship, after detection by a plurality of sets of concentration gradient chips (usually more than 3 sets), it is determined whether the measurement result in step S520 has a correlation with the measurement result in step S540, if so, the measurement result in step S520 can be functionally correlated with the concentration, thereby determining the content of the target in the sample or determining qualitatively. If not, it is indicated whether the measurement result of S520 cannot be directly applied or should be performed in step S540.
The foregoing is merely an embodiment of the present invention, the present invention is not limited to the field of this embodiment, and the specific structures and features well known in the schemes are not described in any way herein, so that those skilled in the art will know all the prior art in the field before the application date or priority date, and will have the capability of applying the conventional experimental means before the date, and those skilled in the art may, in light of the teaching of this application, complete and implement this scheme in combination with their own capabilities, and some typical known structures or known methods should not be an obstacle for those skilled in the art to practice this application. It should be noted that modifications and improvements can be made by those skilled in the art without departing from the structure of the present invention, and these should also be considered as the scope of the present invention, which does not affect the effect of the implementation of the present invention and the utility of the patent. The protection scope of the present application shall be subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.
Claims (8)
1. A method of determining a response to a detected chip feature, comprising the steps of:
s110, cleaning a chip for a first preset time by using background liquid A, adding the background liquid A into the chip, and performing first frequency scanning to obtain a first curve;
s120, removing background liquid A in the chip, cleaning for a first preset time by using background liquid B, adding the background liquid B after cleaning, and performing frequency scanning for the second time to obtain a second curve;
s130, comparing the first curve with the second curve, judging whether the first curve and the second curve are overlapped, and if so, determining a frequency band corresponding to the overlapped part of the first curve and the second curve as a characteristic response frequency band F1; if the sample solution A is not overlapped, adding the sample solution A to the chip sample Frequency scanning is carried out, the frequency corresponding to the inflection point of the phase obtained by the obtained curve is determined to be a characteristic point, and a characteristic response frequency band F1x is determined based on the characteristic point, wherein x=a, b and c …;
or determining the characteristic response frequency band of the chip by the following steps:
s210, adding sample solution A into the chip sample Performing first frequency scanning to obtain a third curve;
s220, placing the chip for a second preset time;
s221, cleaning the chip with background solution A and then adding sample solution A sample Performing secondary frequency scanning; obtaining a fourth curve;
s230, comparing the third curve with the fourth curve, judging whether the third curve and the fourth curve have changes, and if the third curve and the fourth curve have changes, determining a frequency segment corresponding to the changed part of the fourth curve relative to the third curve as a characteristic response frequency band F2.
2. The method for determining a response of a detected chip feature of claim 1, wherein: in S130, the characteristic point minus the preset value is used as a left end point, the characteristic point plus the preset value is used as a right end point, and the frequency from the left end point to the right end point is used as a characteristic response frequency band F1x.
3. The method for determining a response of a detected chip feature of claim 1, wherein: in S120, the chip is cleaned with water for a first preset time after the background solution a is removed.
4. The method for determining a response of a detected chip feature of claim 1, wherein: the frequency sweep is a complex impedance frequency sweep.
5. The method for determining a response of a detected chip feature of claim 1, wherein: background solution A was 1mM phosphate buffer and background solution B was 1mM borate buffer.
6. The method for determining a response of a detected chip feature of claim 1, wherein: the first preset time is greater than 30 seconds.
7. The method for determining a response of a detected chip feature of claim 1, wherein: the second preset time is 1-48 hours.
8. The method for determining a response of a detected chip feature of claim 1, wherein: the sample solution A sample Is background solution A containing the sample.
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