阅读说明：本技术 一种分子检测方法 (Molecular detection method ) 是由 刘晓竹 徐海 李俊 马良 杨黎华 林杰 童立 宋娜 于 2021-09-03 设计创作，主要内容包括：本发明涉及免疫分析检测技术领域,具体公开了一种分子检测方法,包括如下步骤：步骤A1、获取芯片的特定频率段或特定频率点；步骤A2、在芯片上加入样品,基于特定频率段或特定频率点对芯片进行加速；步骤A3、在加速时进行测量得到电信号值；步骤A4、计算电信号值的变化率,基于变化率确定样品中目标物含量或者定性判断。采用本发明的技术方案能够快速确定样品中目标物含量或者定性。(The invention relates to the technical field of immunoassay detection, and particularly discloses a molecular detection method, which comprises the following steps: a1, acquiring a specific frequency band or a specific frequency point of a chip; step A2, adding a sample on a chip, and accelerating the chip based on a specific frequency band or a specific frequency point; step A3, measuring during acceleration to obtain an electric signal value; and A4, calculating the change rate of the electric signal value, and determining the content of the target object in the sample or qualitatively judging based on the change rate. By adopting the technical scheme of the invention, the content or the qualification of the target object in the sample can be rapidly determined.)

1. A molecular detection method is characterized by comprising the following steps:

a1, acquiring a specific frequency band or a specific frequency point of a chip;

step A2, adding a sample on a chip, and accelerating the chip based on a specific frequency band or a specific frequency point;

step A3, measuring during acceleration to obtain an electric signal value;

and A4, calculating the change rate of the electric signal value, and determining the content of the target object in the sample or qualitatively judging based on the change rate.

2. The molecular detection method according to claim 1, characterized in that: the rate of change comprises a slope, a quadratic term, or a first order coefficient; the electrical signal values include impedance, capacitance or resistance values.

3. The molecular detection method according to claim 2, characterized in that: in the step a2, under a specific voltage, an acceleration operation is performed from a high frequency point of a specific frequency band as a starting point to a low frequency point or from a low frequency point of the specific frequency band as a starting point to a high frequency point;

in the step A3, frequency scanning is carried out during acceleration to obtain a curve A of capacitance value changing along with time;

in step A4, the slope of curve A is calculated, and the content of the target in the sample is determined or a qualitative judgment is made based on the slope.

4. The molecular detection method according to claim 3, characterized in that: in the step A2, the specific voltage is 0.01-30V_p-p。

5. The molecular detection method according to claim 3, characterized in that: in the A3, the duration of the frequency sweep is 5-240 s.

6. The molecular detection method according to claim 1, characterized in that: the method further comprises the step A0 of obtaining a characteristic response frequency band F1 and a characteristic response frequency band F2; judging whether the characteristic response frequency band F1 is overlapped with the characteristic response frequency band F2 or not; if the superposition exists, determining a superposition section or a superposition point; a particular frequency segment or a particular frequency point is selected based on the coincidence segment or point and a jump is made to step a 1.

7. The molecular detection method according to claim 6, characterized in that: in the step A0, if the overlapping does not exist, jumping to the step B1;

step B1, adding a sample, and performing frequency scanning based on a first preset frequency to obtain a curve B of the electric signal;

step B2, accelerating the chip based on a second preset frequency;

step B3, frequency scanning is carried out again to obtain a curve C of the electric signal;

and step B4, 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 target object based on the integral area difference.

8. The molecular detection method according to claim 7, characterized in that: in the step B1, during frequency scanning, the first preset frequency is 1MHz-100Hz, and the voltage is 1mV_p-p-1V_p-p。

9. The molecular detection method according to claim 7, characterized in that: in the step B2, the second preset frequency is in the range of 1MHz to 1 kHz.

10. The molecular detection method according to claim 7, characterized in that: in the step B2, the measurement is also performed over time during the acceleration operation, whether the measurement result in the step B2 has a correlation with the measurement result in the step B4 is determined, if so, the measurement result in the step B2 is calibrated based on the measurement in the step B4, and after the calibration, the content of the target substance in the sample is determined or the qualitative determination is performed in the measurement mode in the step B2.

Technical Field

The invention relates to the technical field of immunoassay detection, in particular to a molecular detection method.

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 improve the detection efficiency, 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.

After the target antibody or antigen is combined with the corresponding antigen or antibody coated on the surface of the electrode slice, how to quickly determine the content of the target object in the sample or qualitatively judge becomes a problem to be solved.

For this reason, there is a need for a molecular detection method that can rapidly determine the amount of a target in a sample or that can be characterized.

Disclosure of Invention

The invention provides a molecular detection method, which can rapidly determine the content or the nature of a target object in a sample.

In order to solve the technical problem, the present application provides the following technical solutions:

a molecular detection method comprises the following steps:

a1, acquiring a specific frequency band or a specific frequency point of a chip;

step A2, adding a sample on a chip, and accelerating the chip based on a specific frequency band or a specific frequency point;

step A3, measuring during acceleration to obtain an electric signal value;

and A4, calculating the change rate of the electric signal value, and determining the content of the target object in the sample or qualitatively judging based on the change rate.

The basic scheme principle and the beneficial effects are as follows:

in the scheme, the chip is accelerated based on the specific frequency band or the specific frequency point, so that the combination of the target antibody or antigen in the sample and the corresponding antigen or antibody coated on the surface of the chip can be accelerated. The measurement is carried out while the acceleration is carried out, and the detection result can be obtained after the acceleration is finished, so that the detection time can be shortened. And finally, determining the content of the target object in the sample or qualitatively judging the content of the target object through the change rate of the electric signal value, so that a detection result can be quickly obtained, and the detection sensitivity is improved.

Further, the rate of change comprises a slope, a quadratic term, or a first order coefficient; the electrical signal values include impedance, capacitance or resistance values.

Further, in the step a2, under a specific voltage, an acceleration operation is performed from a high frequency point of a specific frequency band as a starting point to a low frequency point or from a low frequency point of the specific frequency band as a starting point to a high frequency point;

in the step A3, frequency scanning is carried out during acceleration to obtain a curve A of capacitance value changing along with time;

in step A4, the slope of curve A is calculated, and the content of the target in the sample is determined or a qualitative judgment is made based on the slope.

In a baud chart obtained by frequency scanning, the curve A can intuitively reflect the change condition of the capacitance value along with time.

Further, in the step A2, the specific voltage is 0.01-30V_p-p。

Further, in the A3, the duration of the frequency scanning is 5-240 s.

Further, the method comprises the step A0 of obtaining a characteristic response frequency band F1 and a characteristic response frequency band F2; judging whether the characteristic response frequency band F1 is overlapped with the characteristic response frequency band F2 or not; if the superposition exists, determining a superposition section or a superposition point; a particular frequency segment or a particular frequency point is selected based on the coincidence segment or point and a jump is made to step a 1.

At the characteristic response frequency band F1, the degree of correlation between the response value of the chip and the solution is small, that is, the part of the chip with the small degree of correlation between the response value and the solution is determined, and at the part of the characteristic response frequency band F1, which is overlapped with the characteristic response frequency band F2 caused by the sample, the overlapped part is the part of the chip with the small degree of correlation with the solution and affected by the sample. On the basis of selecting a specific frequency band or a specific frequency point on the basis of the coincidence segment or the coincidence point, the sensitivity is higher, the speed of combining the target antibody or antigen with the corresponding antigen or antibody coated on the surface of the chip is higher, the detection time can be effectively shortened, and the universality of the chip is better.

Further, in the step a0, if there is no overlap, jumping to the step B1;

step B1, adding a sample, and performing frequency scanning based on a first preset frequency to obtain a curve B of the electric signal;

step B2, accelerating the chip based on a second preset frequency;

step B3, frequency scanning is carried out again to obtain a curve C of the electric signal;

and step B4, 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 target object based on the integral area difference.

Whether the target object exists on the surface of the chip before and after acceleration causes the change of the electric signal, and the change can be obtained through the integral area difference of the curve B and the curve C, so that whether the target object is combined on the chip through acceleration can be deduced.

The characteristic response frequency band F1 is not overlapped with the characteristic response frequency band F2, which shows that besides the change caused by antigen-antibody combination, the change caused by other influences such as solution and the like also exists, the integral area difference is used for calculation, and the change caused by other influences can be removed by deducting a background.

Further, in the step B1, during the frequency scanning, the first preset frequency is 1MHz-100Hz, and the voltage is 1mV_p-p-1V_p-p。

Further, in the step B2, the second preset frequency is in a range of 1kHz to 1 MHz.

Further, in the step B2, during the acceleration operation, the measurement is also performed over time, it is determined whether the measurement result of the step B2 has a correlation with the measurement result of the step B4, if so, the measurement result of the step B2 is calibrated based on the measurement of the step B4, and after the calibration, the content of the target substance in the sample is determined or qualitatively determined by the measurement method of the step B2.

Drawings

FIG. 1 is a flow chart of a method of molecular detection;

FIG. 2 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. 3 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. 4 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. 5 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. 6 is a schematic diagram showing an example of negative and positive types of chips I;

FIG. 7 is a schematic diagram showing the differentiation between negative and positive types of chip type II;

fig. 8 is a bode diagram obtained by two frequency scans in the characteristic response band determination method.

Detailed Description

Example one

As shown in fig. 1, the molecular detection method of the present embodiment includes the following steps:

a0, acquiring a characteristic response frequency band F1, F1x (x is a, b, c, …) and a characteristic response frequency band F2, judging whether the characteristic response frequency band F1 or F1x (x is a, b, c, …) is overlapped with the characteristic response frequency band F2, and if so, determining an overlapped section or an overlapped point; selecting a specific frequency segment or a specific frequency point based on the superposition segment or the superposition point, and jumping to the step A1; if there is no coincidence, go to step B1. In this embodiment, tens of kHz before and after the coincidence point are selected as the specific frequency segment, for example, 10kHz to 50kHz may be selected when the coincidence point is 30 kHz.

A1, acquiring a specific frequency band or a specific frequency point of a chip;

step A2, adding a sample on a chip, and accelerating the chip based on a specific frequency segment or a specific frequency point under a specific voltage;

step A3, measuring the voltage of the acceleration condition at 0.01-30V_p-p(ii) a 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.

Step a4, as shown in fig. 2-5, determines the content of the target object in the sample or qualitatively determines the content according to the change rate (e.g. slope obtained after straight line fitting, quadratic term or first term coefficient obtained after parabolic fitting, etc.) of the measured electrical signal value (e.g. impedance, capacitance 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 5-240s, a curve A of the obtained capacitance value changing with time is calculated, 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.

Step B1, 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 an electric signal value (such as a capacitance value, an impedance value or a resistance value); in this embodiment, the voltage range during frequency scanning is 1mV_p-p-1V_p-p. The first preset frequency includes a first preset frequency segment and a first preset frequency point.

Step B2, 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; the second preset frequency point is a certain point value in the second preset frequency section. For example, the second predetermined frequency point is 100kHz and the voltage is 0.01-30V_p-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 B3, performing frequency scanning again by using the parameters of the step B1 to obtain a curve C of the electric signal.

And step B4, calculating the integral area difference of 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 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. 6 to 7, 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 characteristic response frequency band F1 of the selected 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 A1-A4. 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 B4, the range of the abscissa may be selected according to the actual situation, for example, the portion of the curve B with the maximum negative-positive difference from the curve C is selected, for example, the range of the abscissa corresponding to the circled portion in fig. 6-7 is selected, and for example, the portion of the curve B with the maximum negative-positive difference from the curve C is selected to extend back and forth 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, measurements may also be taken over time during chip acceleration operations, as well as measurements taken at step B4, while performing step B2. And B2, judging whether the measurement result in the step B2 has correlation with the measurement result in the step B4, if so, calibrating the measurement result in the step B2 based on the measurement result in the step B4, and directly adopting the test method in the step B2 to determine the content of the target substance in the sample or qualitatively judging the content of the target substance in the sample. The steps can be effectively simplified, and the detection efficiency is improved. Specifically, a group of positive samples with concentration gradients is measured, step B2 may obtain the change rate (e.g., slope obtained after straight line fitting, quadratic term or first term coefficient obtained after parabolic fitting, etc.) of the electrical signal value (e.g., impedance, capacitance or resistance value, etc.) at each concentration, i.e., the response value, similarly, the response value at each concentration may also be obtained after step B4 measurement, and these results and the concentrations will present a certain functional relationship, after several groups of concentration gradient chips (usually greater than 3 groups) are used for detection, it is determined whether the measurement result of step B2 and the measurement result of step B4 have a correlation, if so, the measurement result of step B2 and the concentrations may be related by a function, and the content of the target object in the sample may be determined or qualitatively determined. If not, it indicates that the measurement result of B2 cannot be directly applied, or should be performed according to step B4.

The embodiment also provides a method for determining a characteristic response frequency band, which specifically includes:

determining the characteristic response frequency band of the preset type chip through 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 the frequency 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. 8 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 the phase does not coincide with the phase, the frequency corresponding to the inflection point of the phase obtained by the third curve in S210 is used as a feature point, a preset value is subtracted from the feature point to be used as a left end point, a preset value is added to the feature point to be 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, wherein the preset value is 20kHz in the 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 …

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 a sample solution A into the chip_sampleNamely, carrying out first frequency scanning on the background liquid A containing the sample 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 A_samplePerforming 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 corresponding to the changed part of the fourth curve relative to the third curve as the 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.

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.