TWI726446B - Analytical system and analytical method thereof - Google Patents

Abstract

An analytical method includes obtaining a first data and performing curve fitting on the first data according to at least one basis to generate at least one coefficient corresponding to the at least one basis. A function pattern corresponding to one of the at least one basis has asymmetry.

Description

Analysis system and its analysis method

The present invention refers to an analysis system and an analysis method thereof, especially an analysis system and an analysis method that have high analysis accuracy and analysis efficiency and are beneficial to size miniaturization.

The DNA amplification reaction of polymerase chain reaction (PCR) is an important technology of molecular biology. Among them, real-time polymerase chain reaction refers to simultaneous DNA amplification reaction and quantitative analysis after DNA amplification in the same container of the test substance. Quantitative analysis after DNA amplification is to separate the excited fluorescent signal of the test object into different multiple light paths, and filter it into multiple signals of different frequency bands through multiple bandpass filters for analysis. However, signals in adjacent frequency bands will cause crosstalk problems, and the crosstalk problems will vary with solution concentration or temperature, which affects analysis accuracy and analysis efficiency. Furthermore, the excitation efficiency of using a wide-band Xenon light to excite the analyte is low, so it is not conducive to detecting low-concentration analytes or a variety of analytes, and the configuration is complicated and bulky. Optical system.

Therefore, the present invention mainly provides an analysis system and an analysis method thereof to improve analysis accuracy and analysis efficiency and facilitate size miniaturization.

The present invention discloses an analysis method, which includes obtaining a first data, and performing curve fitting on the first data according to at least one base to generate at least one coefficient corresponding to the at least one base. Wherein, a function graph corresponding to one of the at least one base has asymmetry.

The present invention further discloses an analysis system, which includes a storage unit for storing a program code, and a processing unit for executing the program code. An analysis method compiled into the program code includes obtaining a first data, and performing curve fitting on the first data according to at least one base to generate at least one coefficient corresponding to the at least one base. Wherein, a function graph corresponding to one of the at least one base has asymmetry.

10.70: Analysis system

100: light source

120: DUT

150: processing circuit

160: storage device

110, 130, 710, 730: optical components

140, 740: detector

BS1~BS3: base

BS1w~BS3w: data component

DTr, DTf, DTp, DTn: data

FS: Fluorescent signal

L1, L2: lower bound of integral

LB: beam

U1, U2: integral upper bound

WR1, WR2: Band range

Figure 1 is a schematic diagram of an analysis system in an embodiment of the present invention.

Figure 2 is a flowchart of an analysis method according to an embodiment of the present invention.

Figure 3 is a schematic diagram of the noise removal step of the analysis method of Figure 2.

FIG. 4 is a schematic diagram of the partial extraction steps of the analysis method of FIG. 2. FIG.

Figure 5 is a schematic diagram of the substrate used in the analysis method of Figure 2.

Figure 6 is a schematic diagram of the curve fitting steps of the analysis method in Figure 2.

Figure 7 is a schematic diagram of an analysis system in an embodiment of the present invention.

Please refer to Figure 1, which is a schematic diagram of an analysis system 10 in an embodiment of the present invention. The analysis system 10 can be used to detect and analyze fluorescent signals in real time. The analysis system 10 includes a light source 100, optical elements 110, 130, a test object 120, a detector 140, a processing circuit 150, and a Storage device 160. The light beam LB emitted by the light source 100 is guided by the optical element 110 to the test object 120 and excites the test object 120 to generate a fluorescent signal FS (also referred to as a first fluorescent signal). The fluorescent signal FS can be guided to the detector 140 by the optical element 130. The detector 140 receives the fluorescent signal FS and measures the corresponding relationship between the intensity of the fluorescent signal FS and the wavelength, and outputs the measured data DTr to Processing circuit 150. The data DTr can be spectral data, the processing circuit 150 can analyze the spectral data transmitted by the detector 140 according to an analysis method compiled into a program code, and the storage device 160 can be used to store spectral data, analysis method code or other information.

In short, the test object 120 may include at least one known substance. The light beam LB emitted by the light source 100 can excite the known substance in the test object 120 to emit a fluorescent signal FS. Since the corresponding relationship between the (light) intensity and the wavelength of the fluorescent signal (also known as the second fluorescent signal) excited by each known substance is known, the processing circuit 150 can analyze that the test object is excited The corresponding relationship between the (light) intensity of the fluorescent signal FS and the wavelength is used to determine the concentration of each known substance in the test object 120.

Further, please refer to Figures 2 to 6 together. Figure 2 is a flowchart of an analysis method 20 according to an embodiment of the present invention. Figure 3 is a schematic diagram of the noise removal steps of the analysis method 20 of Figure 2 and Figure 4 is a partial extraction of the analysis method 20 of Figure 2 The schematic diagram of the steps, Figure 5 is a schematic diagram of the substrates BS1 to BS3 used in the analysis method 20 of Figure 2, and Figure 6 is a schematic diagram of the curve fitting steps of the analysis method 20 of Figure 2. The analysis method 20 can be compiled into a program code and executed by the processing circuit 150 in FIG. 1, which can include the following steps:

Step 200: Start.

Step 202: Obtain a data DTr.

Step 204: Perform a data processing step to remove noise from the data DTr, and obtain a data DTf.

Step 206: Perform another data processing step to retrieve part of the data DTf to obtain a data DTp.

Step 208: Perform another data processing step to normalize the data DTp to obtain a data DTn.

Step 210: Perform curve fitting on the data DTn according to at least one basis BS1~BS3 to generate at least one coefficient corresponding to at least one basis BS1~BS3, wherein at least one of the bases BS1~BS3 The graph of a function corresponding to one has asymmetry.

Step 212: End.

Specifically, in step 202, the processing circuit 150 receives the data DTr transmitted by the detector 140. The data DTr may be spectral data, which provides the corresponding relationship between the (light) intensity and the wavelength of the fluorescent signal FS excited by the test object 120.

Step 204 can be a noise removal step to improve the accuracy of subsequent data analysis. In other words, the processing circuit 150 can perform a data processing step to remove noise (such as background noise) of the data DTr, and convert the data DTr into the data DTf. In some embodiments, the processing circuit 150 may perform noise filtering calculations for the time domain or the frequency domain. In some embodiments, the noise can be filtered by a low-pass filter, but it is not limited to this. In some embodiments, a hardware filter may be used to perform noise filtering or a software filter may be used to perform noise filtering calculations, but it is not limited to this. As shown in Figure 3, the function graph corresponding to the data DTf is a smoother curve compared to the function graph corresponding to the data DTr.

Step 206 may be a part of the capturing step to reduce the interference caused by the light beam LB emitted by the light source 100 or to avoid interference bands of other light sources. In other words, the processing circuit 150 can perform a data processing The step is to capture the data DTf of a part of the wavelength interval, and convert the data DTf to the data DTp. Therefore, the processing circuit 150 can analyze the spectrum data of the entire wavelength range or a specific wavelength range. In some embodiments, part of the capture step can be performed by hardware or software, but it is not limited to this. As shown in Figure 4, the function graph corresponding to the data DTf only overlaps the function graph corresponding to the data DTp on the right half.

Step 208 can be a normalization step to improve the efficiency of subsequent data analysis. In other words, the processing circuit 150 can perform a data processing step to normalize the data DTp and convert the data DTp into the data DTn. In some embodiments, the processing circuit 150 can normalize the (light) intensity of the data DTp, for example, normalize the maximum light intensity. In some embodiments, the normalization can be performed according to the formula DTn(λ)=(DTp(λ)-min(DTp))/(max(DTp)-min(DTp)), where max(DTp) is the data The maximum light intensity value of DTp, min(DTp) is the minimum light intensity value of the data DTp, DTp(λ), DTn(λ) are the light intensity values corresponding to the data DTp and DTn at a certain wavelength, respectively.

In step 210, the processing circuit 150 may perform curve fitting on the data DTn (also referred to as the first data) according to the bases BS1 to BS3 to generate coefficients corresponding to the bases BS1 to BS3. Among them, the substrates BS1 to BS3 can respectively be spectral data, and respectively provide a corresponding relationship between (light) intensity and wavelength of a fluorescent signal (also called a second fluorescent signal) excited by a known substance. As shown in FIG. 6, the data components BS1w~BS3w respectively represent the corresponding relationship between the (light) intensity and the wavelength of the fluorescent signal excited by a certain known substance in the test object 120. Among them, the total light intensity of the data components BS1w~BS3w at a certain wavelength is equal to the light intensity of the data DTn at this wavelength, and the ratio of the data component BS1w (or data components BS2w, BS3w) to the base BS1 (or base BS2, BS3) It is the coefficient corresponding to the base BS1 (or base BS2, BS3). In other words, DTn(λ)=BS1w(λ)+BS2w(λ)+BS3w(λ)=c1*BS1(λ)+c2*BS2(λ)+c3*BS3(λ), BS1w(λ)~ BS3w(λ) are the light intensity values corresponding to the data components BS1w~BS3w at a certain wavelength, BS1(λ)~BS3(λ) are the light intensity values corresponding to the substrate BS1~BS3 at a certain wavelength, and c1~c3 are the coefficients corresponding to the substrate BS1~BS3. The processing circuit 150 can determine the corresponding relationship between the (light) intensity and the wavelength of the fluorescent signal excited by each known substance in the test object 120 by using the coefficients corresponding to the substrates BS1~BS3 (that is, as shown in Figure 6 Data components BS1w~BS3w), and determine the concentration of each known substance in the test object 120.

According to step 210, even if the distance between the data components BS1w~BS3w is relatively short (for example, when the difference between the wavelengths corresponding to the local maximums of the data components BS1w~BS3w is small or the cross-interference problem is severe), or even if When the excitation efficiency of different known substances differs greatly, or even when the concentration or excitation efficiency of a certain known substance is low, the data components BS1w~BS3w of each known substance can still be effectively analyzed. In some embodiments, the analyte 120 performs real-time polymerase chain reaction (PCR), and the processing circuit 150 determines the concentration of each known substance in the analyte 120. Calculate the amount of DNA after amplification (amplification). Among them, the fluorescent signal FS can be a real-time polymerase chain reaction (PCR) DNA amplification reaction, in which the reagent (bonded to the DNA double strand) in the test substance 120 is excited Fluorescent signal. In this case, the number of amplified DNA can be calculated by the coefficients corresponding to the base BS1~BS3.

In some embodiments, curve fitting is based on least square fit, multiple linear regression, principal component analysis, and point-wise cross-correlation function. cross-correlation, least absolute deviation regression or wavelet transfer. In some embodiments, the substrates BS1 to BS3 have respectively completed the normalization calculation of (light) intensity, for example, normalization of the maximum light intensity. In some embodiments, as shown in FIG. 6, the function pattern corresponding to the substrate BS1 (or the substrate BS2, BS3) may have asymmetry. In some embodiments, as shown in Figure 5, the definite integral of the function pattern corresponding to the base BS1 (or the base BS2, BS3) in a first wavelength interval may be smaller than the definite integral of the function pattern in a first wavelength interval. Definite integral of two wavelength intervals. In other words,

Among them, L1 is the lower integral of the first wavelength interval and corresponds to a first local minimum of the function graph, and U2 is the integral upper bound of the second wavelength interval and it corresponds to a second local minimum of the function graph , U1 and L2 are respectively the upper integral bound of the first wavelength interval and the lower integral bound of the second wavelength interval, and both of them correspond to a local maximum value of the function graph. In some embodiments, the function graph corresponding to the base BS1 (or the base BS2, BS3) may have more than one local maximum. In some embodiments, the function graph corresponding to the substrate BS1 (or the substrate BS2, BS3) can be determined by experiment or theoretical calculation, for example, the excitation spectrum or emission spectrum of a specific known substance is measured.

It should be noted that the analysis system 10 is an embodiment of the present invention, and those with ordinary knowledge in the art can make various changes and modifications accordingly. For example, please refer to FIG. 7, which is a schematic diagram of an analysis system 70 according to an embodiment of the present invention. The structure of the analysis system 70 is similar to that of the analysis system 10, so the same components are represented by the same symbols. The optical elements 110 and 130 and the detector 140 of the analysis system 10 can be implemented by the optical elements 710 and 730 and the detector 740 of the analysis system 70.

In detail, the light source 100 may be a light source with a high light intensity and a narrow wavelength band. In this way, even if the distance between the data components BS1w~BS3w is relatively short (for example, when the difference between the wavelengths corresponding to the local maximums of the data components BS1w~BS3w is small or the cross-interference problem is serious), or even if the When the difference in the excitation efficiency of known substances is large, or even when the concentration or excitation efficiency of a certain known substance is low, each known substance can still be effectively excited. For example, in some embodiments, the light intensity of the light source 100 may range from ^{10 milliwatts (milliWatt, mW) per square millimeter (millimeter, mm 2} ) to 500 milliwatts per square millimeter (ie, 500 milliwatts). /Square millimeter (mW/mm ² )), therefore, the light source 100 is relatively a high light intensity light source. In some embodiments, the (instantaneous) output power of the light source 100 may be between 1 mW and 500 mW, but is not limited to this.

In some embodiments, the wavelength range (or bandwidth) of the light source 100 is less than one-tenth of the wavelength range (or bandwidth) of the fluorescent signal excited by at least one known substance. Therefore, the light source 100 is relatively narrow. Band light source. Among them, the band range (or bandwidth) can be defined by the full width at half maximum (FWHM). In some embodiments, as shown in Figure 6, the wavelength range WR1 of the light source 100 is smaller than the wavelength range WR2 of the substrate BS1 (or the substrate BS2, BS3). For example, the wavelength range WR1 of the light source 100 is smaller than the wavelength range WR1 of the substrate BS1 (or substrate BS1). BS2, BS3) one-tenth of the band range WR2. In some embodiments, the wavelength range of the light source 100 may be between 0.1 nanometer (nm) and 2 nanometers, but it is not limited thereto. In some embodiments, the light source 100 may be a laser light source. Further, the light source 100 may be a diode laser light source or a semiconductor laser light source, such as Fabry-Perot. (Fabry-Perot, FP) Semiconductor laser, Distributed Feedback Laser (DFB) or Vertical-Cavity Surface-Emitting Laser (VCSEL), but not limited to this . In some embodiments, the light source 100 may be a semiconductor laser light source group composed of more than one semiconductor laser light source, and all or part of the semiconductor laser light sources may have different center wavelengths. In some embodiments, the light source 100 may be a light-emitting diode with a divergence angle less than or equal to 10 degrees, but it is not limited to this. In some embodiments, the light source 100 may be a light-emitting diode light source group composed of more than one light-emitting diode light source, and all or part of the light-emitting diode light sources may have different central wavelengths. In some embodiments, the center wavelength of the light source 100 may be between 405 nm and 660 nm, but is not limited to this.

The analyte 120 may include at least one reactant or reagent, and the reagent may include at least one fluorescent probe or fluorescent dye, but it is not limited thereto. In some embodiments, the fluorescent probe or fluorescent dye can be FAM, VIC, HEX, ROX, CY3, CY5, CY5.5, JOE, TET, SyBR, Texas Red, TAMRA, NED, Quasar705, Alexa488, Alexa546, Alexa594, Alexa633, Alexa643, Alexa680 or other fluorescent probes or fluorescent dyes. In some embodiments, the center wavelength of the fluorescent probe or fluorescent dye is between 340 nanometers and 850 nanometers, but not limited to this. Among them, the band range can be defined by the full width at half maximum. In addition, the test object 120 can be loaded in a test object container.

The optical elements 710 and 730 can be used to adjust the beam width, for example, to converge or diverge the beam, or the optical elements 710 and 730 can be further used to adjust the direction of the beam. In some embodiments, the optical element 710 or the optical element 730 may include at least one polymer lens, but it is not limited thereto. In some embodiments, the optical element 710 or the optical element 730 may include bi-convex lenses, plano-convex lenses, double lenses, aspheric lenses, achromatic lenses, aberration lenses, Fresnel lenses, plano-concave lenses, bi-concave lenses, and positive lenses. /At least one of a negative meniscus lens, an axial lens, a gradient index lens, a microlens array, a cylindrical lens, a diffractive optical element, a parabolic mirror, a holographic optical element, or an optical waveguide element. In some embodiments, the configuration of the optical element 710 or the optical element 730 may be omitted in the analysis system 70.

In some embodiments, the detector 740 may be a spectrometer. In some embodiments, the detector 740 may be a miniature spectrometer to facilitate the miniaturization of the size of the analysis system 70. For example, the detector 740 is a Microelectromechanical Systems (MEMS) spectrometer, but it is not limited to this. In some embodiments, the wavelength range of the detector 740 may be between 340 nm and 850 nm, but is not limited to this.

The processing circuit 150 can analyze the spectral data transmitted by the detector 140 according to the analysis method 20 compiled into a program code. In some embodiments, the processing circuit 150 may be a central processing unit (CPU), a microprocessor, or an application-specific integrated circuit (ASIC). In some embodiments, the processing circuit 150 can control the light source 100 to be turned on or off at a fixed time interval. Moreover, when the light source 100 is turned on, the detector 140 synchronously detects the fluorescent signal FS. In some embodiments, the processing circuit 150 can control the light source 100 to be turned on or off at a fixed time interval by controlling the operating current of the light source 100 or by controlling a light path switch. Among them, the optical path switch may be a mechanical optical path switch or an electronic optical path switch.

In some embodiments, the storage device 160 can be any data storage device for storing a program code, and the processing circuit 150 can read and execute the program code through the storage device 160. For example, the storage device 160 may be a Subscriber Identity Module (SIM), a read-only memory (Read-Only Memory, ROM), a flash memory (flash memory), and a random access memory (SIM). Random-Access Memory (RAM), hard disk (hard disk), optical data storage device (optical data storage device), non-volatile storage device (non-volatile storage device), non-transitory computer readable media (non- transitory computer-readable medium), etc., but not limited to this.

In addition, the analysis method 20 is also an embodiment of the present invention, and those with ordinary knowledge in the art can make various changes and modifications accordingly. For example, in the case of low noise, step 204 in the analysis method 20 can be optionally omitted. Therefore, in step 206, part of the data DTr can also be retrieved to obtain the data DTp. In the absence of interference from other light sources, step 206 in the analysis method 20 can be optionally omitted. Therefore, in step 208, part of the data DTf can also be captured to obtain the data DTn. In the case that the normalization can be omitted, step 208 in the analysis method 20 can be optionally omitted, so the data DTp can also be curve-fitted in step 210. Similarly, in the case where step 204 or step 206 is omitted, the data can also be Data DTr or data DTf for curve fitting. Also, the order of step 204, step 206, or step 208 may be reversed.

In summary, the present invention does not use a wide-band xenon lamp, but uses a high-intensity and narrow-band light source 100, so it can detect low-concentration test objects 120, and can reduce the complexity and complexity of the analysis system 10 Volume to meet the requirements of instant detection. Moreover, the present invention does not separate the fluorescent signal FS excited by the test object 120 into different multiple optical paths and filter them into multiple signals of different frequency bands, but performs curve fitting on the data DTn corresponding to the fluorescent signal FS ( curve fitting) to generate coefficients corresponding to the substrates BS1~BS3 to determine the (light) intensity of the fluorescent signal and the wavelength corresponding to each known substance in the test object 120 (ie, the data components BS1w~BS3w ), and determine the concentration of each known substance in the analyte 120. In this way, even if there is a cross-interference problem, or even if the excitation efficiency of different known substances differs greatly, each known substance can still be effectively excited and analyzed, and the analysis accuracy and efficiency can be improved. In addition, the present invention can improve the signal-to-noise ratio (SNR) through data processing steps.

The foregoing descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made in accordance with the scope of the patent application of the present invention should fall within the scope of the present invention.

BS1w~BS3w: data component

DTn: Information

WR1: Band range

Claims (20)

An analysis method includes: obtaining a first data; and performing curve fitting on the first data according to at least one base to generate at least one coefficient corresponding to at least one base, wherein one of the at least one base The corresponding function graph has asymmetry. The definite integral of the function graph in a first wavelength interval is smaller than the definite integral of the function graph in a second wavelength interval. The lower bound of the integral of the first wavelength interval corresponds to a function graph. The first local minimum, the upper integral of the second wavelength interval corresponds to a second local minimum of the function graph, and the upper integral of the first wavelength interval and the lower integral of the second wavelength interval both correspond to the function A local maximum of the graph. The analysis method according to claim 1, wherein the first data is the corresponding relationship between the intensity and wavelength of a first fluorescent signal excited by an object under test, and each of the at least one substrate is a Know the corresponding relationship between the intensity of a second fluorescent signal excited by the substance and the wavelength. The analysis method according to claim 1, wherein the curve fitting is based on the least square method, the complex linear regression method, the principal component analysis method, the point-by-point cross-correlation function method, the least absolute difference recursive method, or the wavelet transformation method. get on. The analysis method according to claim 1, further comprising: obtaining a second data; and performing a data processing step to remove noise from the second data, and obtain the first data. The analysis method described in claim 1 further includes: obtaining a second data; and performing a data processing step to retrieve part of the second data to obtain the first data. The analysis method described in claim 1 further includes: obtaining a second data; and performing a data processing step to normalize the second data to obtain the first data. The analysis method according to claim 1, further comprising: directing a light beam emitted by a light source to an object to be measured, so as to obtain the first data by measuring a first fluorescent signal excited by the object to be measured . The analysis method according to claim 7, wherein the light source is a laser light source, the bandwidth of the light source is less than one-tenth of the bandwidth of one of the at least one substrate, and the bandwidth can be determined by the full width at half maximum To define. The analysis method according to claim 7, wherein the light source is a light-emitting diode light source with a divergence angle less than or equal to 10 degrees. The analysis method according to claim 7, wherein the analyte is subjected to real-time polymerase chain reaction. An analysis system includes: a storage unit for storing a program code; and A processing unit for executing the program code, wherein an analysis method compiled into the program code includes: obtaining a first data; and performing curve fitting on the first data according to at least a base to generate a corresponding At least one coefficient of at least one base, wherein a function pattern corresponding to one of the at least one base has asymmetry, and the definite integral of the function pattern in a first wavelength interval is smaller than that of the function pattern in a second wavelength interval The lower bound of the integral of the first wavelength interval corresponds to a first local minimum value of the function graph, the upper bound of the integral of the second wavelength interval corresponds to a second local minimum value of the function graph, and the first wavelength The upper bound of the integral of the interval and the lower bound of the integral of the second wavelength interval both correspond to a local maximum value of the function graph. The analysis system according to claim 11, wherein the first data is the corresponding relationship between the intensity and wavelength of a first fluorescent signal excited by an object to be measured, and each of the at least one substrate is a Know the corresponding relationship between the intensity of a second fluorescent signal excited by the substance and the wavelength. The analysis system according to claim 11, wherein the curve fitting is based on the least square method, the complex linear regression method, the principal component analysis method, the point-by-point cross-correlation function method, the least absolute difference recursive method, or the wavelet transformation method get on. The analysis system according to claim 11, wherein the analysis method further includes: obtaining a second data; and performing a data processing step to remove noise from the second data and obtain the first data. The analysis system according to claim 11, wherein the analysis method further includes: obtaining a second data; and performing a data processing step to extract part of the second data to obtain the first data. The analysis system according to claim 11, wherein the analysis method further includes: obtaining a second data; and performing a data processing step to normalize the second data to obtain the first data. The analysis system according to claim 11, wherein the analysis method further comprises: directing a light beam emitted by a light source to an object to be measured, so as to measure a first fluorescent signal excited by the object to be measured Obtain the first data. The analysis system according to claim 17, wherein the light source is a laser light source, the bandwidth of the light source is less than one-tenth of the bandwidth of one of the at least one substrate, and the bandwidth can be determined by the full width at half maximum To define. The analysis system according to claim 17, wherein the light source is a light emitting diode light source with a divergence angle less than or equal to 10 degrees. The analysis system according to claim 17, wherein the analyte is subjected to real-time polymerase chain reaction.

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