CN113740385A - Determination method for detecting chip characteristic response - Google Patents
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- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 3
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- 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
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- 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 the characteristic response of a detection chip, which comprises the following steps: s110, cleaning the chip for a first preset time by using the background liquid A, adding the background liquid A into the chip, and performing first frequency scanning to obtain a first curve; s120, removing the background liquid A in the chip, cleaning the chip for a first preset time by using the background liquid B, adding the background liquid B after cleaning is finished, and performing second frequency scanning to obtain a second curve; s130, comparing the first curve with the second curve, judging whether coincidence exists, and if coincidence exists, determining a frequency band corresponding to the coincidence part of the first curve and the second curve as a characteristic response frequency band F1. By adopting the technical scheme of the invention, the characteristic response frequency band of the chip in the immunodetection can be determined, so that the universality of different chips is improved.
Description
Technical Field
The invention relates to the technical field of immunoassay detection, in particular to a method for determining the characteristic response of a detection chip.
Background
Immunodetection is a technique based on antigen-antibody binding for qualitative or quantitative analysis of specific biochemical substances. Antibodies recognize and bind to the corresponding antigen through an epitope on the surface of the antigen. This recognition also gives the immunoassay high specificity: for example, an AIDS antibody will only bind to AIDS antigen and will not react with other antigens.
In the conventional detection method, the binding of a target antibody or antigen with a corresponding antigen or antibody in a kit is completed by molecular diffusion motion and random Brownian motion, the whole process is passive, so the detection time is long, and the result obtained from the collection of a sample often needs tens of minutes to hours.
In order to increase the detection time, chip immunoassay technology has been developed, which coats antigens (or antibodies) on a chip, and simultaneously reacts with a sample to be detected or a biological specimen, so as to obtain the detection results of all known antigens (or antibodies) in the chip at one time. For example, CN104965081B discloses an antibody and antigen detection method based on a mobile device, in which an excitation signal is applied to a chip to generate a dielectrophoresis effect, an electrothermal effect and an electroosmosis effect in the chip, the dielectrophoresis effect can move a target antibody or antigen in a sample toward an electrode pad in the chip, so as to accelerate the binding of the target antibody or antigen with a corresponding antigen or antibody coated on the surface of the electrode pad, and the electrothermal effect and the electroosmosis effect can drive a liquid to flow, so as to bring the target object to the vicinity of the electrode, thereby accelerating detection and shortening detection time. However, the conventional chip/electrode is not universal because the process, material, and the like are not consistent, and the frequency points for characterizing the specific binding of substances are different when the chip/electrode is used for accelerated detection.
Therefore, a determination method for detecting a chip characteristic response is required to improve versatility.
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 immunoassay.
In order to solve the technical problem, the present application provides the following technical solutions:
a method for determining the response of the detection chip features comprises the following steps:
s110, cleaning the chip for a first preset time by using the background liquid A, adding the background liquid A into the chip, and performing first frequency scanning to obtain a first curve;
s120, removing the background liquid A in the chip, cleaning the chip for a first preset time by using the background liquid B, adding the background liquid B after cleaning is finished, and performing second frequency scanning to obtain a second curve;
s130, comparing the first curve with the second curve, judging whether coincidence exists, and if coincidence exists, determining a frequency band corresponding to the coincidence 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 the scheme, the background liquid A is used for cleaning the chip for a first preset time, and then the background liquid A is added into the chip, so that the chip is ensured to only contain the background liquid A, and the interference of other liquids or substances on the surface of the chip can be avoided during first frequency scanning. And cleaning the chip by using the background liquid B for a first preset time, and adding the background liquid B after cleaning is finished, so that the interference of other liquids or substances on the surface of the chip can be avoided during the second frequency scanning. By determining the frequency corresponding to the overlapped part of the first curve and the second curve as the characteristic response frequency band F1, the same part in the frequency scanning of the two different solutions is taken as the characteristic of the chip, and the correlation degree of the response value of the chip and the solution is small at the characteristic. By selecting a plurality of chips from a certain class or batch of chips and determining the characteristic response frequency band F1, the remaining chips can use the characteristic response frequency band F1, and the universality is good.
Further, in S130, if there is no overlap, the sample solution a is added to the chipsampleAnd performing frequency scanning, determining a frequency corresponding to an inflection point of a 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 is a, b and c ….
The extreme points of the phase are more reflective of the characteristic response than other electrical parameters.
Further, in S130, the feature point minus a preset value is used as a left end point, the feature point plus a 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 feature response frequency band F1 x.
Further, in S120, after the background liquid a in the chip is removed, the chip is washed with water for a first preset time.
By washing with water, the residual background liquid A on the chip can be removed.
Further, the frequency sweep is a complex impedance frequency sweep.
Further, the background liquid A is 1mM phosphate buffer salt solution, and the background liquid B is 1mM borate buffer solution.
Further, the first preset time is more than 30 seconds.
A method for determining the response of the detection chip features comprises the following steps:
s210, adding the sample solution A into the chipsamplePerforming a first frequency scanning to obtain a third curve;
s220, placing the chip for a second preset time;
s221, washing the chip by using the background liquid A, and adding the sample solution AsamplePerforming secondary frequency scanning; obtaining a fourth curve;
and S230, comparing the third curve with the fourth curve, judging whether the third curve is changed, and if so, determining the frequency band corresponding to the changed part of the fourth curve relative to the third curve as the characteristic response frequency band F2.
After the chip is subjected to biological modification, the characteristic points can be changed, and further, a universal excitation signal is difficult to determine according to the characteristic points. In the scheme, the sample adding solution is placed for a certain time after being subjected to first frequency scanning, the same sample solution is added after being cleaned for second frequency scanning, and frequency bands changing in a third curve and a fourth curve are respectively obtained after the frequency scanning twice, namely the characteristic response frequency band F2 reflecting the change generated after the surface reaction of the chip.
Further, the second preset time is 1-48 hours.
Further, the sample solution AsampleIs a background liquid A containing a sample to be tested.
Drawings
FIG. 1 is a flow chart of determining a characteristic response frequency band of a preset type chip by different background liquids;
FIG. 2 is a flow chart of determining the characteristic response frequency band of the chip by using a background solution and a sample solution;
FIG. 3 is a Baud plot from two frequency sweeps;
FIG. 4 is a graph of the percentage change in capacitance before and after acceleration at a particular frequency point for chip type I over time;
FIG. 5 is a graph of the percentage change in capacitance before and after acceleration of chip type I over time at a particular frequency band;
FIG. 6 is a graph of the percentage change in capacitance before and after acceleration at a particular frequency point for chip type II over time;
FIG. 7 is a graph of the percentage change in capacitance before and after acceleration of chip type II over time at a particular frequency band;
FIG. 8 is a schematic diagram showing an example of the negative and positive types of chips I;
FIG. 9 is a schematic diagram showing the differentiation between negative and positive types of the chip type II.
Detailed Description
The following is further detailed by way of specific embodiments:
example one
The method for determining the detection chip characteristic response comprises the following steps:
as shown in fig. 1, the characteristic response frequency band of a preset type chip (the preset type may be a certain type or a certain batch of chips) is determined by different background liquids:
and S110, cleaning the chip by using the background liquid A according to a first preset time, wherein the first preset time is more than 30 seconds in the embodiment, 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;
and S120, immediately removing the background liquid A in the chip after scanning, and cleaning the chip for a first preset time by using water, wherein the water is ultrapure water in the embodiment. Cleaning the sample by using the background liquid B for a first preset time, adding the background liquid B after cleaning, and performing second frequency scanning to obtain a second curve;
s130, comparing the first curve with the second curve, judging whether coincidence exists, and if coincidence exists, determining a frequency band corresponding to the coincidence part of the first curve and the second curve as a characteristic response frequency band F1. That is, the same portion in the above two different solution tests is measured as a 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 maps obtained by two frequency scans, and specifically, the data maps are bode maps. For example: using 1mMPBS (Phosphate Buffered Saline) as background liquid A, cleaning and adding the chip, and performing first frequency scanning;
using 1mMBBS (Borate buffer solution Borate Buffered Saline) as background solution B, cleaning and adding the chip, and performing secondary frequency scanning;
data as described in fig. 3 can be obtained: at 20kHz-50kHz, the two curves are superposed, namely the two curves have equal values, and the frequency band is the characteristic response frequency band F1. That is, when PBS and BBS are used, the response of the chip is not changed by the change of the solution at 20kHz-50 kHz.
If there is no overlap, add sample solution A to the chipsampleThe curve obtained by performing the first frequency sweep within 30 seconds (i.e., using the curve in S210)Third curve), subtracting a preset value from the characteristic point to obtain a left end point, adding a preset value to the characteristic point to obtain a right end point, and obtaining a frequency from the left end point to the right end point as a characteristic response frequency band F1x, where the preset value is 20kHz in this embodiment and the characteristic response frequency band F1x includes values of the left end point and the right end point. Where x is a, b, c, … (in this embodiment, the lowest is not lower than 1kHz and the highest is not higher than 1MHz), for example, there are several characteristic response bands. Labeled sequentially F1a, F1b …
As shown in fig. 2, the characteristic response 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 a sample solution A into the chipsampleNamely, background liquid A containing a sample to be tested is subjected to first frequency scanning within 30 seconds to obtain a third curve;
s220, placing the chip in a humid environment for a second preset time, wherein the second preset time is 1-48 hours, in the embodiment, 6 hours; in this example, the humidity range is 45% to 95% RH.
S221, washing with background liquid A, and adding sample solution AsamplePerforming secondary frequency scanning; obtaining a fourth curve;
and S230, comparing the third curve with the fourth curve, judging whether the third curve is changed, and if so, determining a frequency band corresponding to the changed part of the fourth curve relative to the third curve as a characteristic response frequency band F2. Namely, the sample solution is placed for a certain time after being subjected to the first frequency scanning, the sample solution is cleaned and then subjected to the second frequency scanning, and the frequency band changed after the two frequency scanning is the characteristic response frequency band F2 showing the change generated after the surface reaction of the chip.
To further illustrate the application of the characteristic response band in detection, the present embodiment further provides an antigen-antibody detection method, including:
the selection steps specifically comprise:
s310, determining whether the characteristic response frequency band F1 or F1x (x is a, b, c, …) overlaps with the characteristic response frequency band F2, if not, jumping to S510, if so, determining an overlapping segment or an overlapping point, and jumping to S410.
The detection step specifically comprises:
s410, adding a sample on a chip, selecting a specific frequency section or a specific frequency point based on a superposition section or a superposition point under a specific voltage, and measuring the voltage under the acceleration condition at any time, wherein the voltage under the acceleration condition is 0.01-30Vp-p(ii) a In this embodiment, tens of KHz before and after the coincidence point are selected as the specific frequency band, for example, 10KHz to 50KHz may be selected if the coincidence point is 30 KHz. Acceleration and measurement are simultaneously carried out, the acceleration is carried out by applying alternating current with larger voltage and certain frequency on the electrode, the adsorption force is generated at the moment, and the current response is also carried out at the same time, so the measurement is also finished.
S420, as shown in fig. 4 to 7, determining the content of the target object in the sample or qualitatively determining the content according to the change rate (for example, the slope obtained after straight line fitting, the coefficient of the quadratic term or the first term obtained after parabolic fitting, etc.) of the measured electrical signal value (for example, impedance, capacitance value or resistance value, etc.). In this embodiment, a least square method is used to fit a straight line.
For example, after a sample is added to a chip, under a specific voltage, frequency scanning is performed from a high frequency point of a specific frequency segment as a starting point to a low frequency point while accelerating for 5 to 240 seconds, a curve a of capacitance values changing with time is obtained after completion, and the slope of the curve a is calculated. If the mode of scanning the frequency from the low frequency point as the starting point to the high frequency point is adopted, the negative sign is added to the obtained slope.
S510, adding a sample, and performing frequency scanning based on a first preset frequency, wherein the first preset frequency is 1MHz-100Hz (from high scanning to low scanning), so as to obtain a curve B of response electric signals (such as capacitance values, impedance and resistance values); in this embodiment, the voltage range during frequency scanning is 1mVp-p-1Vp-p. The first preset frequency includes a first preset frequency segment and a first preset frequency point.
S520, accelerating the chip based on a second preset frequency; the range of the second preset frequency is 1kHz-1MHz, and the second preset frequency comprises a second preset frequency section and a second preset frequency point; second predetermined frequency pointIs a certain point value in the second preset frequency band. For example, the second predetermined frequency point is 100kHz and the voltage is 0.01-30Vp-p. In this embodiment, the second predetermined frequency is a theoretically calculated frequency that can be accelerated, and whether the factor reflecting the antigen-antibody binding is ignored during calculation.
And S530, performing frequency scanning again by using the parameters of the S510 to obtain a curve C responding to the electric signal.
And S540, calculating the integral area difference of the curve B and the curve C obtained by the two frequency scans, and determining the content of the target object in the sample or qualitatively judging the target object based on the integral area difference.
The calculation formula of the change rate of the capacitance integration is as follows:
(Cs2-Cs1)/Cs1*100％
wherein Cs1 is a capacitance value at a first predetermined frequency point on the curve B before acceleration, or an integrated area value at a first predetermined frequency band;
cs2 is the capacitance at the second predetermined frequency point on curve C after acceleration, or the integrated area at the second predetermined frequency band. As shown in FIGS. 8 to 9, in the detection of a myocardial infarction marker, negative and positive determinations can be made by integrating the area values, with the circled portion being positive.
In the embodiment, a plurality of chips are selected from the preset chips to be detected according to the method, the calibration is carried out according to the detection result, the rest chips are detected according to the same method, and the universality is high. The predetermined type may be a certain type or a certain batch of chips. For example, for a certain type of chip, the selected characteristic response frequency band F1 of the 2-5 core chip is overlapped with the characteristic response frequency band F2, and the rest chips can be directly detected according to the steps S410-S420. And for example, selecting 1 chip and adding a positive sample, wherein the response value of the obtained chip is 1, selecting another chip and adding a negative sample, and the response value of the obtained chip is 2, wherein the response value 1.5 of the rest chips can be divided into division lines, and is negative when the division line is more than 1.5 and is positive when the division line is less than 1.5.
When the difference between the integrated areas of the curve B and the curve C obtained by the two frequency scans is calculated in step S540, the range of the abscissa may be selected according to the actual situation, for example, the portion with the maximum negative-positive difference between the curve B and the curve C is selected, for example, the range of the abscissa corresponding to the circled portion in fig. 8-9 is selected, and for example, the portion with the maximum negative-positive difference between the curve B and the curve C is selected and then extends to the intersection. It should be noted that after the scaled chips are determined to have the range of the abscissa, the remaining chips are selected to have the same range of the abscissa.
In other embodiments, in performing step S520, measurements may also be performed over time during chip acceleration operations, and measurements may also be performed in step S540. And judging whether the measurement result of the step S520 is correlated with the measurement result of the step S540, if so, calibrating the measurement result of the step S520 based on the measurement result of the step S540, and directly determining the content of the target object in the sample or qualitatively judging the content of the target object by adopting the test method of the step S520 for the rest chips. The steps can be effectively simplified, and the detection efficiency is improved. Specifically, a group of positive samples with concentration gradients is measured, step S520 may obtain a change rate (e.g., a slope obtained after straight line fitting, a quadratic term or a first order term coefficient obtained after parabolic fitting, etc.) of an electrical signal value (e.g., an impedance, a capacitance, or a resistance) at each concentration, i.e., a response value, similarly, step S540 may also obtain a response value at each concentration after measurement, and these results and the concentrations will present a certain functional relationship, after detection by a plurality of groups of concentration gradient chips (usually greater than 3 groups), it is determined whether the measurement result of step S520 and the measurement result of step S540 have a correlation, if so, the measurement result of step S520 and the concentration may be related by a function, and then the content of the target object in the sample is determined or qualitatively determined. If not, it is described whether the measurement result of S520 cannot be directly applied or should be performed in step S540.
The above are merely examples of the present invention, and the present invention is not limited to the field related to this embodiment, and the common general knowledge of the known specific structures and characteristics in the schemes is not described herein too much, and those skilled in the art can know all the common technical knowledge in the technical field before the application date or the priority date, can know all the prior art in this field, and have the ability to apply the conventional experimental means before this date, and those skilled in the art can combine their own ability to perfect and implement the scheme, and some typical known structures or known methods should not become barriers to the implementation of the present invention by those skilled in the art in light of the teaching provided in the present application. It should be noted that, for those skilled in the art, without departing from the structure of the present invention, several changes and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.
Claims (10)
1. A method for determining a response of a test chip feature, comprising the steps of:
s110, cleaning the chip for a first preset time by using the background liquid A, adding the background liquid A into the chip, and performing first frequency scanning to obtain a first curve;
s120, removing the background liquid A in the chip, cleaning the chip for a first preset time by using the background liquid B, adding the background liquid B after cleaning is finished, and performing second frequency scanning to obtain a second curve;
s130, comparing the first curve with the second curve, judging whether coincidence exists, and if coincidence exists, determining a frequency band corresponding to the coincidence part of the first curve and the second curve as a characteristic response frequency band F1.
2. The method for determining a response to a test chip as claimed in claim 1, wherein: in the step S130, if no superposition exists, adding the sample solution A into the chipsampleAnd performing frequency scanning, determining a frequency corresponding to an inflection point of a 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 is a, b and c ….
3. The method for determining a response to a test chip as claimed in claim 2, wherein: in S130, subtracting a preset value from the feature point to obtain a left end point, adding the preset value to the feature point to obtain a right end point, and using the frequency from the left end point to the right end point as the characteristic response frequency band F1 x.
4. The method for determining a response to a test chip as claimed in claim 1, wherein: in S120, the background liquid a in the chip is removed and then the chip is cleaned with water for a first preset time.
5. The method for determining a response to a test chip as claimed in claim 1, wherein: the frequency sweep is a complex impedance frequency sweep.
6. The method for determining a response to a test chip as claimed in claim 1, wherein: the background liquid A is 1mM phosphate buffer solution, and the background liquid B is 1mM borate buffer solution.
7. The method for determining a response to a test chip as claimed in claim 1, wherein: the first preset time is greater than 30 seconds.
8. A method for determining a response of a test chip feature, comprising the steps of:
s210, adding the sample solution A into the chipsamplePerforming a first frequency scanning to obtain a third curve;
s220, placing the chip for a second preset time;
s221, washing the chip by using the background liquid A, and adding the sample solution AsamplePerforming secondary frequency scanning; obtaining a fourth curve;
and S230, comparing the third curve with the fourth curve, judging whether the third curve is changed, and if so, determining the frequency band corresponding to the changed part of the fourth curve relative to the third curve as the characteristic response frequency band F2.
9. The method for determining a response to a test chip as claimed in claim 8, wherein: the second preset time is 1-48 hours.
10. The method for determining a response to a test chip as claimed in claim 8, wherein: the sample solution AsampleIs a background liquid A containing a sample to be tested.
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| CN114660283A (en) * | 2022-05-24 | 2022-06-24 | 佛山微奥云生物技术有限公司 | Immunoassay plate type chip based on electrical acceleration and preparation method thereof |
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