High-sensitivity molecule or particle detection method

Abstract

The invention relates to a high-sensitivity molecule or particle detection method, which comprises the following steps of: 1, drawing time-varying curves of light-emitting intensity values of molecules or particles under different concentrations to obtain a relation coefficient K between the light-emitting intensity values and time in each curve; 2, mapping each coefficient K value and the concentration, and drawing a standard curve of the relationship between the K value and the concentration; 3, detecting an actual sample, and drawing a curve of luminous intensity values changing along with time to obtain a relation coefficient K'between the luminous intensity values and the time; and 4, finding the corresponding concentration in the standard curve obtained in the step 2 according to the K'value, namely the concentration value of the actual sample. According to the method, the concentration of the molecules or the particles in the sample can be measured at high precision without specific statistics on the number of the molecules or the particles in the sample, a digital signal result can be obtained by adopting an analog signal mode, equipment required in a current imaging type molecule detection technology is greatly simplified, and the detection cost is reduced.

Description

Technical Field

[0001] This invention relates to a method for detecting molecules or particles, and more specifically to a method for detecting molecules or particles with high sensitivity. Background Technology

[0002] In numerous fields such as life science research, medical diagnostics, and environmental monitoring, the detection of molecular or particle concentrations plays a crucial role, and the accuracy and reliability of the detection results directly affect subsequent research decisions and application outcomes. With the rapid development of related fields, increasingly higher demands are being placed on the sensitivity, accuracy, and cost of molecular or particle detection methods.

[0003] Currently, molecular or particle detection technologies can be mainly divided into two categories: traditional biochemical detection technologies and modern imaging detection technologies. Traditional biochemical detection technologies, such as enzyme-linked immunosorbent assays (ELISA), can achieve molecular detection to a certain extent, but these technologies have low sensitivity for low-concentration samples, making it difficult to meet the needs of trace molecular detection.

[0004] Modern imaging molecular detection technologies, such as fluorescence imaging and confocal microscopy, have gained widespread attention in the field of high-sensitivity detection due to their ability to directly visualize the distribution of molecules or particles. These technologies label molecules or particles with fluorescent substances and use imaging equipment to capture the fluorescence signals, thereby enabling the detection and analysis of molecules or particles. However, existing imaging molecular detection technologies still face several bottlenecks in practical applications. On the one hand, achieving accurate counting of molecules or particles requires high-precision imaging equipment and complex image analysis systems, which keeps the overall cost of the detection equipment high, limiting its widespread application in small and medium-sized laboratories and grassroots testing institutions. On the other hand, during the detection process, image acquisition and analysis are easily affected by external environmental factors (such as light fluctuations and background noise) and the characteristics of the sample itself, which can lead to large errors in the counting of molecules or particles, making it difficult to guarantee detection accuracy.

[0005] In summary, the current field of molecular or particle detection urgently needs a detection method that can balance high sensitivity, high accuracy, and low cost. In particular, it needs to solve the problems of complex equipment, high cost, and susceptibility to interference in detection accuracy in existing technologies through innovative concentration calculation methods, so as to meet the needs of different fields for molecular or particle concentration detection and promote the further development and widespread application of detection technologies in related fields. Summary of the Invention

[0006] To address the aforementioned problems, this invention proposes a highly sensitive method for detecting molecules or particles. This method utilizes the difference between the concentration and luminescence intensity change curves to identify the concentration of the analyte molecules or particles, offering high accuracy and low cost.

[0007] The technical means adopted by this invention to solve the above problems is as follows: a high-sensitivity molecular or particle detection method, comprising the following steps: First, plotting curves showing the change of luminescence intensity values ​​of molecules or particles over time at different concentrations, obtaining the relationship coefficient K between luminescence intensity values ​​and time in each curve; Second, mapping each coefficient K value to concentration, plotting a standard curve showing the relationship between K values ​​and concentration; Third, detecting actual samples, plotting curves showing the change of luminescence intensity values ​​over time, obtaining the relationship coefficient K' between luminescence intensity values ​​and time; Fourth, finding the corresponding concentration in the standard curve obtained in the second step based on the K' value, which is the concentration value of the actual sample. High-precision detection of molecules or particles is achieved using luminescence intensity.

[0008] Furthermore, when plotting the curves of luminous intensity values ​​over time in the first and third steps, images were collected at different time periods under the same wavelength of light to obtain the luminous intensity values ​​corresponding to each image.

[0009] Furthermore, in the first and third steps, the curve of the luminous intensity value changing with time is a linear function, with coefficient K being the slope; or the curve of the luminous intensity value changing with time is another functional relationship, with coefficient K being the integral area.

[0010] Furthermore, in the first step, when plotting the curves of the change of luminescence intensity values ​​of molecules or particles with time at different concentrations, the detection experiment at the same concentration is conducted more than twice, and the relationship between the luminescence intensity values ​​and time is calculated based on the two or more luminescence intensity values.

[0011] Furthermore, in the third step, when plotting the curve of the sample's luminescence intensity value changing with time, the concentration detection experiment is performed more than twice, and the relationship between the two or more luminescence intensity values ​​and time is calculated.

[0012] Furthermore, the number of tests performed on the sample in the third step is the same as the number of tests performed on the same concentration in the first step.

[0013] Furthermore, for two or more luminescence intensity values ​​at the same concentration and time, the average value of each luminescence intensity value is used to plot the curve of luminescence intensity value changing with time.

[0014] Furthermore, for two or more luminescence intensity values ​​at the same concentration and time, the total intensity value of each luminescence intensity value is used to plot the curve of luminescence intensity value changing with time.

[0015] The beneficial effects of this invention are: 1. This invention uses known molecular or particle concentrations to derive a standard curve showing the relationship between luminescence intensity and time (K) and concentration. Then, it detects the relationship coefficient K' between luminescence intensity and time in the sample, and reads the sample concentration from the standard curve based on the K' value. This method can accurately measure the concentration without requiring specific statistics on the number of molecules or particles in the sample. It uses analog signals to obtain digital signal results, greatly simplifying the equipment required in current imaging molecular detection technologies and reducing detection costs.

[0016] 2. This invention can improve the accuracy of the relationship curve between luminescence intensity and time by controlling the time gradient, and can also improve the accuracy of the standard curve by designing a known concentration gradient, thereby improving the precision of sample detection. Attached Figure Description

[0017] Figure 1 A flowchart for drawing the standard curve in Example 1; Figure 2 This is a flowchart of the sample concentration detection process in Example 2; Figure 3 This shows the change in luminescence intensity over time at the same concentration in Example 1. Figure 4 This is the curve showing the change in luminescence intensity over time at the same concentration in Example 1; Figure 5 The curves showing the change in luminescence intensity over time at different concentrations in Example 1 are shown. Figure 6 This is a standard curve showing the relationship between coefficient K and concentration in Example 1; Figure 7 The curve showing the change in luminescence intensity of a sample concentration over time is shown in the example. Figure 8 The corresponding values ​​of the sample concentration in the example on the standard curve. Detailed Implementation Example 1

[0018] A highly sensitive method for detecting molecules or particles, such as Figure 1 and Figure 2 As shown, the specific steps include (where the concentration unit is pg / mL, which is omitted in the following description): S1: Image acquisition with known concentration of molecules or particles Multiple detection experiments are conducted on molecules or particles of known concentration. At a fixed wavelength, multiple emission images are acquired at different time periods. In this embodiment, for example... Figure 3 As shown, four detections were performed at one concentration, with images taken every second for a total of 9 seconds, resulting in four luminescent images within each time interval.

[0019] S2: Plot the curve of luminous intensity changing with time. Image processing techniques are used to obtain the luminous intensity values ​​for each time period. Two methods can be used to calculate the luminous intensity value for each time period: one is to calculate the average luminous intensity of all luminous images within the same time period; the other is to add the luminous intensities of all luminous images within the same time period and take the overall value. This embodiment uses the average value, such as... Figure 4 As shown, a curve of the luminescence intensity value changing with time at this concentration is plotted, and the relationship coefficient K between the luminescence intensity value and the curve is obtained. In this embodiment, the curve is a linear function, and the coefficient K is the slope.

[0020] Design a suitable concentration gradient, such as Figure 5 As shown, following the steps above, curves showing the change of luminescence intensity values ​​over time at different concentrations were plotted, and the relationship coefficient K between each luminescence intensity value and the curve was obtained.

[0021] S3: Plot the standard curve Mapping each coefficient K value to the concentration, such as Figure 6 As shown, a standard curve is plotted between the coefficient K and the concentration value, and the coefficient K is used to describe the characteristics of the curve.

[0022] S4: Plot the curve of the sample's luminescence intensity over time. Four identical samples were taken for testing. Images were taken every second for nine seconds, and the average luminous intensity for each time period was calculated from the images as the luminous intensity value. Figure 7 As shown, the curve of the sample luminescence intensity value changing with time was plotted, and its coefficient K (i.e., slope) was found to be 2.5.

[0023] S5: Obtain the sample concentration like Figure 8 As shown, substituting the sample coefficient K into the standard curve yields a sample concentration of 0.9.

[0024] It should be noted that since the technical improvement of this invention lies in the calculation process of molecular or particle concentration after enzyme-linked immunosorbent assay or other luminescent assay, the operation process before concentration calculation is not described in detail and can be implemented using existing methods.

[0025] In the above embodiment, the curve of luminous intensity changing with time is a linear function, and the coefficient K is the slope. In practical applications, the curve of luminous intensity changing with time may also be other functional relationships, such as a quadratic function. In this case, the coefficient K can be represented by the area of ​​integration.

[0026] The above embodiments are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can make various changes or modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions should also fall within the protection scope of the invention, which should be defined by the claims.

Claims

1. A highly sensitive method for detecting molecules or particles, characterized in that: Includes the following steps: The first step is to plot the curves showing the change of luminescence intensity values ​​of molecules or particles over time at different concentrations, and obtain the relationship coefficient K between luminescence intensity values ​​and time in each curve. The second step is to map each coefficient K value to the concentration and plot a standard curve showing the relationship between K value and concentration. The third step is to test the actual sample and plot the curves showing the change of luminescence intensity values ​​over time, and obtain the relationship coefficient K' between luminescence intensity values ​​and time. The fourth step is to find the corresponding concentration in the standard curve obtained in the second step based on the K' value, which is the concentration value of the actual sample.

2. The high-sensitivity molecular or particle detection method as described in claim 1, characterized in that: In the first and third steps, when plotting the curves of luminous intensity values ​​over time, images were collected at different time periods under the same wavelength of light to obtain the luminous intensity values ​​corresponding to each image.

3. The high-sensitivity molecular or particle detection method as described in claim 1, characterized in that: In the first and third steps, the curve of the luminous intensity value changing with time is a linear function, and the coefficient K is the slope; or the curve of the luminous intensity value changing with time is another functional relationship, and the coefficient K is the integral area.

4. The high-sensitivity molecular or particle detection method as described in claim 1, characterized in that: In the first step, when plotting the curves of the change of luminescence intensity values ​​of molecules or particles with time at different concentrations, the detection test at the same concentration is carried out more than twice, and the relationship between the luminescence intensity values ​​and time is calculated based on the two or more luminescence intensity values.

5. The high-sensitivity molecular or particle detection method as described in claim 4, characterized in that: In the third step, when plotting the curve of the sample's luminescence intensity value changing with time, the concentration detection experiment is performed more than twice, and the relationship between the two or more luminescence intensity values ​​and time is calculated.

6. The high-sensitivity molecular or particle detection method as described in claim 5, characterized in that: The number of tests performed on the sample in the third step is the same as the number of tests performed on the same concentration in the first step.

7. The high-sensitivity molecular or particle detection method as described in claim 5, characterized in that: For two or more luminescence intensity values ​​at the same concentration and time, the average value of each luminescence intensity value is used to plot the curve of luminescence intensity value changing with time.

8. The high-sensitivity molecular or particle detection method as described in claim 6, characterized in that: For two or more luminescence intensity values ​​at the same concentration and time, the total intensity value of each luminescence intensity value is used to plot the curve of luminescence intensity value changing with time.

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