TWI601948B - Method for obtaining binding kinetic rate constants using fiber optics particle plasmon resonance (foppr) sensor - Google Patents

Description

Estimation method of dynamic constant using fiber-optic particle plasma resonance sensor

The invention relates to a method for estimating the dynamic constant of a fiber-optic particle plasma resonance sensor, in particular to a power that can make the process of estimating the dynamic constant very simple and rapid using the fiber-optic particle plasma resonance sensor. Learning constant estimation method.

Chemical Thermodynamics is a study of the equilibrium nature of chemical reactions, with a primary focus on the initial and final state of the chemical reaction. Chemical Kinetics studies the rate of reaction during chemical reactions. Chemical kinetics are often applied to the discussion of biomolecular binding capabilities. The affinity constant K _f , the binding rate constant k _a and the decomposition rate constant k _d are important parameters in chemical kinetics. For the chemical kinetics of biomolecules, the binding rate constant represents the rate at which the molecular complex is formed, and the decomposition rate constant represents the stability of the molecular complex.

Biosensors are devices used to detect chemical analytes. Biosensors also have the ability to monitor the amount of variation in chemical reactions and convert the amount of variation into a specific signal for easy viewing. Chemical kinetics related studies can be performed by observing specific signals, such as using specific signal values to calculate decomposition rate constants or association rate constants in chemical kinetics.

However, the aforementioned biosensor needs to use a fluorescent mechanism to mark the analyte to be detected, which affects the characteristics of the analyte to be detected. In addition, the existing plasma resonance sensor (for example, the Biacore system) is designed such that the combination of the rate constant k _a and the decomposition rate constant k _d is subject to the flow rate of the solution to be detected. All of the above are technical issues to be solved.

In view of the problems of the prior art, the present inventors have proposed a method for estimating the dynamic constant of a fiber-optic particle plasma resonance sensor based on years of research and development and many practical experiences, as an implementation method for improving the above disadvantages. in accordance with.

It is an object of the present invention to provide a method for estimating the kinetic constant of a fiber-optic particle plasma resonance sensor that is simple and rapid in the process of estimating the kinetic constant.

Another object of the present invention is to provide a method for estimating the kinetic constant of a fiber-optic particle plasma resonance sensor using a fluorescent mechanism for marking an analyte to be detected.

It is still another object of the present invention to provide a method for estimating the kinetic constant of a fiber-optic particle plasma resonance sensor by estimating the kinetic constant by simply obtaining the optical signal intensity value corresponding to the initial time of the reaction.

According to the above object of the present invention, the present invention provides a method for estimating the kinetic constant using a fiber-optic particle plasma resonance sensor, which is suitable for a solution having at least two different concentrations to be detected, and is also suitable for having a plurality of different concentrations. Detecting the solution, the steps of which are as follows: providing a fiber-optic particle plasma resonance sensor, the fiber-optic particle plasma resonance sensor comprising at least: a light source that emits light; and a light-receiving element, such as a photodetector; And a fiber sensing chip, the fiber sensing chip is located between the light source and the light receiving component, the fiber sensing chip comprises: an optical fiber, the optical fiber is divided into a first area and a second area, and the first area is located in the second area. On the side, the first region is sequentially composed of a core, a shell, and a protective layer. The refractive index of the material of the core is greater than the refractive index of the material of the shell, so that the light travels in the core, and the second region From the inside to the outside, the core, the shell, the nano particle layer and the detecting layer; the first plate body, the first plate body has a groove, the groove is for the corresponding placement of the optical fiber; and the second plate a first tube body and a second tube body are longitudinally disposed on one side of the second plate body, the first tube system is hollow and has a first opening, the second tube system is hollow and has a second opening, the first tube body and The second tube system is in communication with the second plate body, the second plate body is different from the first tube body and the other side of the second tube body is opposite to the first plate body, so that the optical fiber is located in the first plate body and the first plate body Between the two plates, the optical fibers are correspondingly placed in the grooves of the first plate body, and then the second plate body and the first plate body are face-to-face corresponding to each other and packaged, so that the assembly of the fiber sensing wafer can be completed; Turning on the light source of the fiber-optic particle plasma resonance sensor, the light enters the fiber of the fiber-sensing chip, the light travels in the core due to total reflection, and the light-receiving element of the fiber-optic particle plasma resonance sensor Starting to receive the optical signal; sequentially injecting at least N solutions to be detected into the first tube body as the first opening of the inflow port, so that the solution to be detected flows into the fiber sensing wafer through the first tube body in sequence, in order The injected solution to be tested has different Concentration C, and the number of N is greater than or equal to 2; the fiber-optic particle plasma resonance sensor converts the optical signal received by the light-receiving element into a curve relationship between time and optical signal intensity value, and a curve in a curve relationship diagram There are the same paragraphs as the number of solutions to be tested, and these paragraphs correspond to the optical signal intensity generated by the sequentially injected detection solutions; respectively, the optical signal intensity values corresponding to the initial time of the reaction of all the paragraphs in the curve relationship diagram are obtained. _t , the optical signal intensity value I _eq when the reaction reaches the dynamic balance in these paragraphs and the reference optical signal intensity I ₀ obtained by detecting the reference blank solution; the time after each detection solution is filled with the first pipe body Initially, the optical signal intensity value I _t obtained when a static state is present is brought into the formula ln[(I _t -I _eq )/(I _o -I _eq )] to calculate a plurality of fractional logarithms, And linearly regressing the logarithm of the scores with respect to the time to obtain a linear relationship diagram corresponding to the number of the above paragraphs; respectively obtaining a slope S of one of the straight lines in the linear relationship diagram; And the corresponding concentration C S is the slope of the linear regression of another to obtain the slope and intercept of the regression line, the concentration carried out by the slope of the linear relationship _{S (C i) = k a} C i + k d ratio Yes, to obtain the combination rate constant k _a and the decomposition rate constant k _d .

The method for estimating the kinetic constant of the fiber-optic particle plasma resonance sensor provided by the present invention obtains N intensity optical signal intensity (time) curves by N solutions to be detected, and obtains corresponding N slopes after regression. Then, the N slope values are subjected to another regression for the N concentration values, and finally k _a value and k _d value are obtained, wherein the N value is greater than or equal to two.

The invention does not need to use a fluorescent mechanism to label the analyte to be detected, and does not affect the characteristics of the analyte to be detected. In addition, the present invention rapidly measures the change of the intensity of the optical signal with time after being injected into the solution to be detected. Unlike existing continuous flow type plasma resonance sensors (such as the Biacore system), the upper limit of the combined rate constant k _a and the decomposition rate constant k _d is not subject to the injection flow rate.

For a better understanding and understanding of the technical features and the efficacies of the present invention, the preferred embodiments and the detailed description are as follows.

1‧‧‧Fiber sensing chip

11‧‧‧ first board

111‧‧‧ Groove

12‧‧‧Second plate

121‧‧‧First tube

1211‧‧‧ first opening

122‧‧‧Second body

1221‧‧‧ second opening

13‧‧‧Fiber

131‧‧‧Silicon

132‧‧‧Stained shell

133‧‧‧protection layer

134‧‧‧ nano particle layer

135‧‧‧Detection layer

2‧‧‧Light source

3‧‧‧Light receiving components

4‧‧‧Power supply unit

5‧‧‧Signal processing device

6‧‧‧ computer

7‧‧‧Testables

100~200‧‧‧Steps

400~900‧‧‧Steps

301~303‧‧‧Steps

A1‧‧‧ first area

A2‧‧‧Second area

First paragraph of B1‧‧

B2‧‧‧Second paragraph

B3‧‧‧3rd paragraph

Fourth paragraph of B4‧‧

C1‧‧‧first concentration

C2‧‧‧second concentration

S1‧‧‧ first slope

S2‧‧‧second slope

1 is a perspective view of a fiber-optic particle plasma resonance sensor using a method for estimating a dynamic constant of a fiber-optic particle plasma resonance sensor according to the present invention; and FIG. 2 is a fiber-optic particle device of the present invention. 3D is a schematic exploded perspective view of a fiber sensing chip of a slurry resonance sensor; FIG. 3A is a side cross-sectional view of a first region of an optical fiber of the fiber sensing chip of the present invention; 3B is a side cross-sectional view of a second region of an optical fiber of the optical fiber sensing chip of the present invention; FIG. 4 is a schematic diagram showing steps of a method for estimating a dynamic constant of a fiber-optic particle plasma resonance sensor according to the present invention; Figure 5 is a graph showing the relationship between egg protein (OVA) as the detection layer and egg-protein antibody (anti-OVA) as the solution to be detected and the kinetic constant estimation method according to the present invention; The first linear relationship diagram obtained by using egg protein (OVA) as a detection layer and egg-protein antibody (anti-OVA) as a solution to be detected and estimating the kinetic constant according to the present invention; FIG. 6B is an egg The protein (OVA) is used as the detection layer and the second linear relationship diagram obtained by using the egg-protein antibody (anti-OVA) as the solution to be detected and estimating the kinetic constant according to the present invention; FIG. 7A is the picture in FIG. Residual analysis of sample points and linear regression lines.

Figure 7B is a residual analysis of the sampling points and linear regression lines in Figure 6B.

Figure 8 is a graph showing the relationship between mouse immunoglobulin (mouse IgG) as a detection layer and mouse immunoglobulin antibody (anti-mouse IgG) as a solution to be tested and according to the kinetic constant estimation method of the present invention. Figure; and Figure 9 is a schematic diagram of the binding reaction between the solution to be detected and the detection layer.

The kinetic constant estimation method using the fiber-optic particle plasma resonance sensor of the present invention will be described below with reference to the related drawings. For ease of understanding, the same components in the following embodiments are denoted by the same reference numerals.

First, please refer to FIG. 1 , which is a perspective view of a fiber-optic particle plasma resonance sensor using the method for estimating the dynamic constant of a fiber-optic particle plasma resonance sensor according to the present invention. Figure. The fiber-optic particle plasma resonance sensor of the present invention comprises at least a fiber-optic sensing wafer 1, a light source 2, and a light-receiving element 3. The fiber sensing wafer 1 is located between the light source and the light receiving element 3. The light source is a single-frequency light such as a laser light or a narrow-band light such as a light-emitting diode.

The fiber-optic particle plasma resonance sensor of the present invention more selectively includes a power supply device 4, a signal processing device 5, and a computer 6. The power supply device can be any type of power supply device, such as a signal waveform generator, and the signal processing device can be any signal processing device, such as a lock-in amplifier. The power supply device 4 is disposed on one side of the light source 2 that is different from the fiber sensing wafer 1. The signal processing device 5 is provided on the side of the light receiving element 3 which is different from one side of the fiber sensing wafer 1. The computer 6 is provided on the side of the signal processing device 5 which is different from one side of the light receiving element 3. The power supply device 4 is configured to generate a square wave of a fixed frequency to the light source 2, and the power supply device 4 generates a reference signal to the signal processing device 5. The signal processing device 5 receives the optical signal from the light receiving element 3 and processes the optical signal and the reference signal to generate a processed signal. The computer 6 receives the processed signal from the signal processing device 5 and displays it for reading. The power supply device 4 and the signal processing device 5 are arranged to increase the signal-to-noise ratio (S/N ratio) of the optical signal.

Please refer to FIG. 2 again, which is a perspective exploded view of the fiber sensing wafer of the fiber-optic particle plasma resonance sensor of the present invention. The fiber optic sensing wafer of the present invention includes a first plate body 11, a second plate body 12, and an optical fiber 13. The first plate body 11 has a recess 111 for the optical fiber 13 to be placed therein. The first tube body 121 and the second tube body 122 are longitudinally disposed on one side of the second plate body 12. The first tube body 121 is hollow and has a first opening 1211. Similarly, the second tube body 122 is hollow and There is a second opening 1221. The first tube body 121 and the second tube body 122 are in communication with the second plate body 12. The second board body 12 is different from the first tube body 121 and the other side of the second tube body 122 and the first board body 11 face each other so that the optical fiber 13 is located between the first board body 11 and the second board body 12. between. If the fiber 13 Correspondingly placed in the groove 111 of the first plate body 11, the second plate body 12 and the first plate body 11 are face-to-face corresponding to each other and packaged, so that the assembly of the fiber sensing wafer 1 can be completed [see the Figure 1 shows the assembled fiber optic sensing wafer]. The first plate body 11 or the second plate body 12 is, for example, a plastic plate body.

Please refer to FIG. 3A again, which is a side cross-sectional view showing the first region of the optical fiber of the optical fiber sensing chip of the present invention. The optical fiber 13 of the present invention is divided into a first area A1 and a second area A2. The first area A1 is located on the corresponding two sides of the second area A2. The first region A1 of the optical fiber 13 of the present invention is sequentially composed of a core 131, a shell 132 and a protective layer 133 from the inside to the outside. The material of the aforementioned core 131 is, for example, cerium oxide. The material of the shell 132 is, for example, a polymer material. The refractive index of the material of the core 131 is greater than the refractive index of the material of the shell 132 such that light travels within the core 131 due to total reflection.

Please refer to FIG. 3B again, which is a side cross-sectional view showing a second region of the optical fiber of the optical fiber sensing chip of the present invention. The second region A2 of the optical fiber 13 of the present invention is sequentially composed of a core 131, a shell 132, a nanoparticle layer 134, and a detection layer 135 from the inside to the outside. The material of the aforementioned core 131 is, for example, cerium oxide. The material of the shell 132 is, for example, a polymer material. The refractive index of the material of the core 131 is greater than the refractive index of the material of the shell 132. The material of the nanoparticle layer 134 is, for example, nano gold or nano silver. The nanoparticle layer 134 is composed of a plurality of noble metal nanospheres, a plurality of noble metal nanorods or a plurality of noble metal nanoshells. The surface of the nanoparticle layer 134 can be modified with various identification units to produce the detection layer 135. The detection layer 135 is an antibody such as an anti-mouse IgG, an antigen such as ovalbumin (OVA), a lectin, and a hormone receptor ( Hormone receptor), a nucleic acid (nucleic acid) or a saccharide, the detection layer 135 is used to sense an antigen, a cytokine, an antibody, a hormone receptor, a growth factor ( Growth factor), neuropeptide, hemoglobin, plasma protein, nucleic acid, carbohydrate, glycoprotein (glycoprotein), fatty acid, phosphatidic acid, sterol, antibiotic or toxin. It should be noted that, for ease of understanding, the nanoparticle layer 134 and the detection layer 135 in the figure are enlarged, not actual size.

Referring to FIG. 4, it is a schematic diagram showing the steps of the method for estimating the kinetic constant of the fiber-optic particle plasma resonance sensor of the present invention. The method for estimating the kinetic constant of the present invention is applicable to N solutions to be detected, and the number of N is greater than or equal to 2. In other words, the present invention is applicable to a solution to be detected having at least two different concentrations, and is also applicable to a solution to be detected having a plurality of different concentrations. Taking two examples of the solution to be tested, the steps are as follows:

Step 100: Providing a fiber-optic particle plasma resonance sensor as described above.

Step 200: Turn on the light source 2 of the fiber-optic particle plasma resonance sensor, so that the light enters the optical fiber 13 of the fiber-sensing wafer 1. The light travels in the core 131 due to total reflection, and causes the fiber-optic particle to resonate. The light receiving element 3 of the sensor begins to receive the optical signal.

Step 301: Injecting a reference blank solution into the first tube 121 by using the first opening 1211 as an inflow port, for example, deionized water or a buffer solution.

Step 302: The first solution to be detected is rapidly injected into the first tube 121 by the first opening 1211 as an inflow port, so that the first solution to be detected flows into the fiber sensing wafer 1 via the first tube 121. The first solution to be detected has a first concentration C ₁ and a detection time of 10 seconds.

Step 303: The second solution to be detected is rapidly injected into the first tube 121 by the first opening 1211 as an inflow port, so that the second solution to be detected flows into the fiber sensing wafer 1 via the first tube 121. The second solution to be detected has a second concentration C ₂ , and the second concentration C _{2 is} higher than the first concentration C ₁ described above, and the detection time is 10 seconds.

Step 400: The fiber-optic particle plasma resonance sensor converts the optical signal received by the light-receiving element 3 into a relationship diagram between the time and the optical signal intensity value, and the curve in the curve relationship diagram has the same as the solution to be detected. The same number of paragraphs, and the paragraphs correspond to the optical signal intensity generated by the detection solutions sequentially injected. Taking the number of dissolved solutions as two as an example, the curves in the curve relationship diagram are the first paragraph B1 and the second paragraph B2, respectively, and the first paragraph B1 is the light signal intensity value generated by the first solution to be detected, the second paragraph B2 is the optical signal intensity value generated by the second solution to be tested.

Step 500: Obtaining the optical signal intensity values (I ₁ ) and (I ₂ ) corresponding to the initial time of the reaction of the first paragraph and the second paragraph in the curve relationship diagram, and the dynamic balance in the first paragraph and the second paragraph The optical signal strength values (I _eq1 ) and (I _eq2 ) and the reference optical signal strength I ₀ . Please refer to the following 5th or 8th figure for the above relationship diagram.

Step 600: At the beginning of the time after the first or second detection solution of the solution to be detected is filled with the first tube body, the optical signal intensity value obtained when the static state is present is brought into the formula by the pseudo first-order reaction rate equation (pseudo- First order reaction rate equation) Assuming the formula ln[(I _t -I _eq )/(I ₀ -I _eq )] derived from the model constructed below, the formula of the formula is the fractional function of the optical signal intensity value. For the logarithmic linear relationship of time, the complex fractional logarithmic value is calculated, and the linear regression of the fractional value versus time is performed to obtain a first linear relationship diagram corresponding to the first paragraph B1 and corresponding to the second The second straight line diagram of paragraph B2. Please refer to the following figure 6A for the first line relationship diagram. Please refer to the following 6B picture for the second line relationship diagram.

Step 700: Obtain a first slope (S ₁ ) of the straight line in the first straight line relationship diagram and a second slope (S ₂ ) of the straight line in the second straight line relationship diagram, respectively.

Step 800: Perform a linear regression between the first concentration (C ₁ ) and the corresponding first slope (S ₁ ) and the second concentration (C ₂ ) and the corresponding second slope (S ₂ ) to obtain a regression line Slope and intercept.

Step 900: Perform an alignment with _a linear relationship between the concentration and the slope S(C _i )=k _a C _i +k _d to obtain an estimated value of the combination rate constant k _a and the decomposition rate constant k _d , respectively.

The above steps are exemplified by using two concentrations of the solution to be detected, but not limited thereto, and the same type of solution to be detected exceeding two or more different concentrations is also suitable for detection and analysis using the above steps.

For example, please refer to FIG. 5 again, which shows egg protein (OVA) as a detection layer and four concentrations of egg protein antibody (anti-OVA) as a solution to be tested and according to the present invention. A graph of the relationship between the kinetic constant estimation methods. The curve in the curve relationship diagram is divided into a first paragraph B1, a second paragraph B2, a third paragraph B3, and a fourth paragraph B4, wherein the third paragraph B3 and the fourth paragraph B4 are other different concentrations of the same kind of solution to be detected. Obtaining the optical signal intensity values (I ₁ ) and (I ₂ ) corresponding to the initial time of the reaction of the first paragraph B1 and the second paragraph B2 in the curve relationship diagram, and the dynamic balance in the first paragraph and the second paragraph Optical signal strength values (I _eq1 ) and (I _eq2 ) and reference optical signal strength (I ₀ ). The concentrations of the anti-OVA to be detected solutions used in the first paragraph B1, the second paragraph B2, the third paragraph B3, and the fourth paragraph B4 in Fig. 5 were 67 nM, 134 nM, 268 nM, and 536 nM, respectively.

Please refer to FIG. 6A and FIG. 6B again, which respectively show egg white protein (OVA) as a detection layer and egg-protein antibody (anti-OVA) as a solution to be tested and according to the kinetics of the present invention. The first straight line relationship diagram obtained by the constant estimation method and the second straight line relationship diagram. The obtained optical signal intensity value is brought into the formula ln[(I _t -I _eq )/(I ₀ -I _eq )] to calculate a plurality of fractional logarithmic values, and the fractional value versus time is obtained by the fractions A linear regression is performed to obtain a first straight line relationship map corresponding to the first paragraph B1 and a second straight line relationship map corresponding to the second paragraph B2. The egg-protein antibody (anti-OVA) used in the 6A and 6B images was used as the solution concentration of 67 nM and 134 nM, respectively, and the correlation coefficient of the data points in the 6A and 6B images were 0.97.

Please refer to FIG. 7A and FIG. 7B again, which respectively show residual analysis of sampling points and linear regression lines in FIGS. 6A and 6B. As can be seen from the figure, the sampling points obtained by the present invention have high accuracy.

Please refer to Table 1 below, which is a combination of egg protein (OVA) as a detection layer and egg-protein antibody (anti-OVA) as a solution to be tested and according to the kinetic constant estimation method of the present invention. Rate constant (k _a ) and decomposition rate constant (k _a ) and data sheets compared to the references. As can be seen from the figure, the present invention can indeed estimate the binding rate constant (k _a ) and the decomposition rate constant (k _d ) of egg protein (OVA) and when reacted with an egg protein antibody (anti-OVA).

Table 1 shows the binding rate constant (k _a ) and the decomposition rate constant obtained by using egg protein (OVA) as the detection layer and egg-protein antibody (anti-OVA) as the solution to be detected and estimating the kinetic constant according to the present invention. (k _a ) and data compared to references.

*Reference 1: Masayuki Oda, S. U., Carol V. Robinson, Kiichi Fukui, Yuji Kobayashi, and Takachika Azuma FRBS Joumal 2006, 273, 1476.

*Reference 2: Brogan, K. L.; Shin, J. H.; Schoenfisch, M. H. Langmuir 2004, 20, 9729.

For example, please refer to Figure 8, which shows that mouse IgG is used as the detection layer and four concentrations of mouse immunoglobulin antibody (anti-mouse IgG) are to be detected. The relationship between the solution and the kinetic constant estimation method of the present invention is obtained. The curve in the curve relationship diagram is divided into a first paragraph B1, a second paragraph B2, a third paragraph B3, and a fourth paragraph B4, wherein the third paragraph B3 and the fourth paragraph B4 are other different concentrations of the same kind of solution to be detected. Obtaining the optical signal intensity values (I ₁ ) and (I ₂ ) corresponding to the initial time of the reaction of the first paragraph B1 and the second paragraph B2 in the curve relationship diagram, and the dynamic balance in the first paragraph and the second paragraph Optical signal strength values (I _eq1 ) and (I _eq2 ) and reference optical signal strength (I ₀ ). The concentration of the test solution of the mouse immunoglobulin antibody (anti-mouse IgG) used in the first paragraph B1, the second paragraph B2, the third paragraph B3, and the fourth paragraph B4 in Fig. 5 was 1.3 nM, 5.2 nM, and 10.4, respectively. nM and 20.8 nM. The data point sampled by the curve of Fig. 8 for subsequent analysis has a correlation coefficient of 0.97.

Please refer to Table 2 below for the detection of mouse immunoglobulin (mouse IgG) and the use of mouse immunoglobulin antibody (anti-mouse IgG) as the solution to be tested and according to the kinetics of the present invention. The data of the binding rate constant (k _a ) obtained by the constant estimation method and the decomposition rate constant k _d . As can be seen from the figure, the present invention can indeed estimate the binding rate constant (k _a ) and the decomposition rate constant (k _d ) of mouse immunoglobulin (mouse IgG) and mouse immunoglobulin antibody (anti-mouse IgG). ).

Table 2 shows the binding rate constant obtained by using mouse immunoglobulin (mouse IgG) as a detection layer and mouse immunoglobulin antibody (anti-mouse IgG) as a solution to be detected and according to the kinetic constant estimation method of the present invention. _a ) and the data of the decomposition rate constant k _d .

*Reference 1: Masayuki Oda, S. U., Carol V. Robinson, Kiichi Fukui, Yuji Kobayashi, and Takachika Azuma FRBS Journal 2006, 273, 1476.

*Reference 2: Brogan, K. L.; Shin, J. H.; Schoenfisch, M. H. Langmuir 2004, 20, 9729.

Please refer to FIG. 9 again, which is a schematic diagram showing the binding reaction between the solution to be detected and the detection layer. It should be specially noted that when the analyte 7 to be detected in the solution to be detected is combined with the detection layer 135, the nanoparticle layer 134 undergoes a particle plasma resonance phenomenon due to the binding reaction, and the particle plasma resonance phenomenon further causes the optical signal intensity. The value changes. Accordingly, as long as the change in the intensity value of the optical signal is measured, the kinetic constant can be estimated.

It should be further noted that the nanoparticle layer 134 is excited by light to produce a characteristic extinction spectrum, which is called a particle plasmon resonance (PPR) band. The basic sensing principle of the particle plasma resonance sensing system is: when the nano particle layer 134 feels When the refractive index of the environment changes, the peak wavelength and the extinction cross-section of the particle plasma resonance band will also change. In the waveguide phenomenon, at a specific reflection interface, light of a specific frequency acts on the PPR phenomenon of the nanoparticle layer 134. Therefore, when the number of reflections is more than one time, the incident light is totally totally internally reflected, so that the light-emitting signal of the optical fiber is weakened. In general, the total internal reflection phenomenon can accumulate the amount of change in the PPR signal, so that the sensitivity of the sensing can be improved.

It should be specially stated that the first and second to-be-tested solutions are respectively filled with the first tube body and then the solution is stopped, and the solution is in a static state; the time for the solution to be detected to fill the first tube body is much smaller than that of the first tube body. The time at which the solute to be detected in the surrounding corner is diffused and combined with the probe molecules on the detection layer. The injection time used refers to the following criteria: the injection time is less than the acceptance light intensity I, which is one-half of the time when the [(I _t -I _eq )/(I ₀ -I _eq )] fractional value is 0.4, where I ₀ and I _eq are the light intensity signals of the reference solution light intensity signal and the dynamic balance, respectively. However, when the solution to be tested is injected into the first tube body, that is, in the non-resting state, the received light intensity signal, and the ratio of [(I _t -I _eq )/(I ₀ -I _eq )] is greater than 0.4 region. The luminosity signal is not used. Under the assumption of the pseudo-first order reaction rate equation, the concentration of the complex changes with time, which is the rate of binding of the solute to the detection layer to be detected, minus the decomposition rate of the complex. When the concentration of the complex is proportional to the intensity of the photometric signal, we derive a linear relationship between the logarithm of [(I _t -I _eq )/(I ₀ -I _eq )] and time. In the foregoing FIG. 6A, the last signal value used is selected by the first paragraph B1 and the second paragraph B2, and the ratio calculated by substituting [(I _t -I _eq ) / (I ₀ -I _eq )] is 0.1. Less than the aforementioned reference value of 0.4.

In summary, the method for estimating the dynamic constant of the fiber-optic particle plasma resonance sensor of the present invention has at least the following advantages: the method for estimating the dynamic constant of the fiber-optic particle plasma resonance sensor of the present invention N light intensity (on time) curves are obtained with N solutions to be detected, and the corresponding N slopes are obtained after regression, and then the N slope values are returned to N concentration values, and finally k _a value is obtained. And k _d values, where the N value is greater than or equal to two.

Accordingly, the present invention does not require the use of a fluorescent mechanism to label the analyte to be detected, and does not affect the characteristics of the analyte to be detected. In addition, the present invention rapidly measures the change of the intensity of the optical signal with time after being injected into the solution to be detected. Unlike existing continuous flow type plasma resonance sensors (such as the Biacore system), the upper limit of the combined rate constant k _a and the decomposition rate constant k _d is not subject to the injection flow rate.

The above is intended to be illustrative only and not limiting. Any equivalent modifications or alterations to the spirit and scope of the invention are intended to be included in the scope of the appended claims.

400~900‧‧‧Steps

Claims (10)

A method for estimating a dynamic constant of a fiber-optic particle plasma resonance sensor, comprising the steps of: providing a fiber-optic particle plasma resonance sensor, the fiber-optic particle plasma resonance sensor comprising: at least one a light source; a light receiving element; and a fiber sensing chip, the fiber sensing chip being located between the light source and the light receiving element, the fiber sensing chip comprising: an optical fiber, the fiber is divided into a first area And a second area, the first area is located on opposite sides of the second area, and the first area is sequentially composed of a core, a fiber shell and a protective layer, and the material of the core The refractive index is greater than the refractive index of the material of the shell, such that the light travels within the core, the second region being sequentially from the inside to the outer core, the shell, the nanoparticle layer, and a detecting layer; a first plate body, the first plate body having a groove for the corresponding placement of the optical fiber; and a second plate body, the side of the second plate body being vertically vertical There is a first tube body and a second tube body, The first tube system is hollow and has a first opening, the second tube system is hollow and has a second opening, the first tube body and the second tube system are in communication with the second plate body, the second plate The body is different from the first tube body and the other side of the second tube body and the first board body face each other so that the optical fiber is located between the first board body and the second board body, so that Correspondingly, the optical fiber is placed in the groove of the first plate body, and the second plate body and the first plate body are correspondingly facing each other and packaged; and the light source of the fiber-optic particle plasma resonance sensor is turned on. The light enters the fiber of the fiber sensing chip, the light travels in the core due to total reflection, and causes the light receiving component of the fiber particle plasma resonance sensor to start receiving an optical signal; The first opening as the inflow port is rapidly injected into the first pipe body; and the first opening as the inflow port sequentially injects at least N to be detected solutions into the first pipe body, Causing the solution to be detected to flow to the light through the first tube body in sequence Sensing one of the wafers on the detection layer and presenting a standing state in the groove until the next injection, the solutions to be detected have different concentrations C, and the number of N is greater than or equal to 3; using the fiber type The particle plasma resonance sensor converts the optical signal received by the light receiving component into a relationship diagram between time and optical signal intensity values, and the curve in the curve relationship diagram has the same number of the solutions to be detected. And the paragraphs respectively corresponding to the optical signal intensity generated by the detection solutions sequentially injected; respectively obtaining the optical signal intensity values I _t corresponding to the initial time of the reaction of the paragraphs in the curve relationship diagram, The light signal intensity value I _eq and the reference light signal intensity I ₀ when the reaction reaches the dynamic balance in the paragraph; the light obtained when the state is left after the time when each of the to-be-detected solutions is filled with the groove The signal strength value I _{t is} taken into the formula ln[(I _t -I _eq )/(I ₀ -I _eq )] to calculate a plurality of fractional logarithmic values, and the scores are plotted against the time Sexual regression to get the corresponding a linear relationship diagram of the number of paragraphs; respectively obtaining a slope S of one of the straight lines in the linear relationship diagram; and performing another linear regression on the concentration C and the corresponding slopes S to obtain a slope of a regression line And an intercept, by performing a correlation coefficient analysis with a linear relationship S(C _i )=k _a C _i +k _{d of} a concentration and a slope, to obtain a combination rate constant k _a and a decomposition rate constant k _d . The method for estimating a dynamic constant of a fiber-optic particle plasma resonance sensor according to claim 1, wherein the light source is a single-frequency light or a narrow-band light. The method for estimating a dynamic constant of a fiber-optic particle plasma resonance sensor according to the first aspect of the invention, wherein the first plate body or the second plate body is a plastic plate body. The method for estimating a dynamic constant of a fiber-optic particle plasma resonance sensor according to claim 1, wherein the material of the core is ceria, and the material of the shell is a polymer material. The method for estimating the kinetic constant of a fiber-optic particle plasma resonance sensor according to claim 1, wherein the detection layer is an antibody, an antigen, a lectin, a hormone receptor, a nucleic acid or a saccharide. The method for estimating the kinetic constant of a fiber-optic particle plasma resonance sensor according to claim 1, wherein the material of the nano particle layer is nano gold or nano silver. The method for estimating the kinetic constant of a fiber-optic particle plasma resonance sensor according to the first aspect of the patent application, wherein the nano particle layer is composed of a plurality of precious metals It consists of a nanosphere, a plurality of precious metal nanorods or a plurality of precious metal nanoshells. The method for estimating a dynamic constant of a fiber-optic particle plasma resonance sensor according to the first aspect of the invention, wherein the fiber-optic particle plasma resonance sensor further comprises: a power supply device, the power supply device The power supply device is configured to generate a fixed frequency and a fixed voltage as a function of driving a signal to drive the light source to generate the optical signal; a signal processing device is disposed on the side of the light source. The signal processing device is disposed on a side of the optical receiving component different from the optical fiber sensing chip, the power supply device generates a reference signal to the signal processing device, and the signal processing device receives the light receiving component from the optical receiving component An optical signal, and the optical signal and the reference signal are processed to generate a processed signal; and a computer disposed on the side of the signal processing device different from the light receiving component, the computer receiving The processed signal of the signal processing device is displayed and displayed for reading. The method for estimating a dynamic constant of a fiber-optic particle plasma resonance sensor according to claim 1, wherein the concentration of the solution to be tested injected into the first tube is gradually increased according to the order of injection. . A method for estimating a dynamic constant of a fiber-optic particle plasma resonance sensor according to the invention of claim 8, wherein the power supply device is a waveform signal generator and the signal processing device is a lock-in amplifier.