Detection method and detection system for detecting biological sample

Abstract

The invention discloses a detection method and a detection system, which are used for detecting a target object in a biological sample. The detection method comprises the following steps: providing a detection kit which comprises a detection carrier and a report molecule, the detection carrier comprises a fixation protein, the fixation protein has an identification area combined with a target object, and the report molecule comprises a spin fluorescent material; mixing the report molecules and the biological sample, and placing the mixture into a detection carrier; placing the detection carrier with the report molecules and the biological sample in a time-varying physical field; irradiating the detection carrier placed in the time-varying physical field with exciting light to excite the spin fluorescent material to generate a fluorescence signal regulated by the time-varying physical field; and receiving and analyzing the fluorescence signal.

Description

Technical Field

[0001] This invention relates to a detection method and a detection system, and more particularly to a detection method and a detection system for detecting biological samples. Background Technology

[0002] For the detection of specific targets in biological samples, such as antigens (e.g., DNA, proteins, or microorganisms) or antibodies, immunoassays are frequently used. Depending on the specific needs and context, many different testing products using immunoassays are available on the market.

[0003] For quick results, immunochromatographic (ICT) test strips are typically used. This method involves attaching antibodies that bind to the target antigen to the test strip, forming the test line. The biological sample is then mixed with the color-labeled antibody. Common color-labeled substances include colored or stained latex, colloidal gold, or other dyes. When the target antigen is present in the biological sample, the color-labeled antibody binds to it. As the sample flows through the test line, the target antigen also binds to the antibody on the test line, resulting in a color change. However, this color-changing method relies on visual observation and has poor sensitivity, making it unsuitable for detecting trace amounts of the target antigen.

[0004] Immunoassays for detecting trace amounts of analytes commonly utilize enzymes to amplify signals, such as enzyme-linked immunosorbent assays (ELISA). In addition to antibodies that bind to antigens, ELISA requires a secondary antibody linked to an enzyme and a chromogenic receptor (chromogenic agent). The concentration of the target analyte in the biological sample is quantified by detecting the absorbance of the chromogenic receptor at a specific wavelength. However, ELISA is a complex procedure, requiring steps such as obtaining the enzyme-linked secondary antibody and washing to remove unbound secondary antibody. Incomplete washing or excessively high concentrations of the secondary antibody can also lead to high background values. Summary of the Invention

[0005] In view of the above-mentioned problems, the main objective of the present invention is to provide a detection method and system for detecting biological samples. By using spin fluorescent materials as reporter molecules in immunoassays and taking advantage of the time-varying physical field modulation properties of spin fluorescent materials, the present invention solves the problems of poor sensitivity or cumbersome operation in conventional immunoassays.

[0006] To achieve the above objectives, the present invention provides a detection method for detecting a target analyte in a biological sample. The detection method includes the following steps: providing a detection kit comprising a detection carrier and a reporter molecule; the detection carrier comprising a fixation protein having a recognition region for binding the target analyte; the reporter molecule comprising a spin fluorescent material; mixing the reporter molecule with the biological sample and placing it into the detection carrier; placing the detection carrier containing the reporter molecule and the biological sample in a time-varying physical field; irradiating the detection carrier placed in the time-varying physical field with an excitation light to excite the spin fluorescent material to generate a fluorescence signal modulated by the time-varying physical field; and receiving and analyzing the fluorescence signal.

[0007] According to one embodiment of the present invention, a reporting molecule labels a target in a biological sample, and the reporting molecule binds to the anchoring protein via the target.

[0008] According to one embodiment of the present invention, before the step of placing the detection carrier in the time-varying physical field, the method further includes the step of removing reporter molecules that are not bound to the fixation protein.

[0009] According to one embodiment of the present invention, the reporter molecule further includes a capture antibody having a recognition region for binding to the target. The capture antibody binds to a spin fluorescent material, and the reporter molecule labels the target in the biological sample by capturing the antibody.

[0010] According to one embodiment of the present invention, the spin fluorescent material includes a fluorescent diamond nanoparticle, a fluorescent magnetic bead, or a rare earth metal magnetic bead.

[0011] According to one embodiment of the present invention, the spin fluorescent material comprises a plurality of spin fluorescent particles, each of which has a diameter between 1 nanometer and 1 millimeter.

[0012] According to one embodiment of the present invention, the detection carrier comprises a solid matrix.

[0013] According to one embodiment of the present invention, the time-varying physical field includes an alternating electromagnetic field or a microwave field.

[0014] According to one embodiment of the present invention, the frequency of the alternating electromagnetic field is between 1 Hz and 1 MHz, and the root mean square value of the amplitude is between 1 G and 10000 G. The frequency of the microwave field is between 0.1 MHz and 10 GHz.

[0015] According to one embodiment of the present invention, the step of analyzing the fluorescence signal further includes: comparing the fluorescence signal with a certain amount of standard curves to quantify the concentration of the target analyte.

[0016] To achieve the above objectives, the present invention further provides a detection system used in conjunction with a detection kit for detecting a target analyte in a biological sample. The detection kit includes a detection carrier and a reporter molecule. The detection carrier includes a fixation protein having a recognition region for binding the target analyte. The reporter molecule includes a spin fluorescent material. The reporter molecule is mixed with the biological sample and then placed into the detection carrier. The detection system includes a time-varying physical field, a light source, a signal collection component, and a processing device. The detection carrier containing the reporter molecule and the biological sample is placed in the time-varying physical field. The light source provides excitation light to irradiate the detection carrier placed in the time-varying physical field, thereby exciting the spin fluorescent material to generate a fluorescence signal modulated by the time-varying physical field. The signal collection component receives the fluorescence signal. The processing device is electrically connected to the time-varying physical field, the light source, and the signal collection component. The processing device receives the fluorescence signal from the signal collection component and analyzes the fluorescence signal.

[0017] To achieve the above objectives, the present invention provides a detection system for detecting a target analyte in a biological sample. The detection system includes a detection kit, a time-varying physical field, a light source, a signal collection component, and a processing device. The detection kit includes a detection carrier and a reporter molecule. The detection carrier includes a fixation protein having a recognition region for binding the target analyte. The reporter molecule includes a spin fluorescent material. The reporter molecule is mixed with the biological sample and then placed into the detection carrier. The detection carrier containing the reporter molecule and the biological sample is placed in the time-varying physical field. The light source provides excitation light to irradiate the detection carrier placed in the time-varying physical field, thereby exciting the spin fluorescent material to generate a fluorescence signal modulated by the time-varying physical field. The signal collection component receives the fluorescence signal. The processing device is electrically connected to the time-varying physical field, the light source, and the signal collection component. The processing device receives the fluorescence signal from the signal collection component and analyzes the fluorescence signal.

[0018] According to one embodiment of the present invention, the time-varying physical field includes an alternating electromagnetic field or a microwave field.

[0019] According to one embodiment of the present invention, the frequency of the alternating electromagnetic field is between 1 Hz and 1 MHz, and the root mean square value of the amplitude is between 1 G and 10000 G. The frequency of the microwave field is between 0.1 MHz and 10 GHz.

[0020] According to one embodiment of the present invention, the processing device compares the fluorescence signal with a certain number of standard curves to quantify the concentration of the target analyte.

[0021] According to one embodiment of the present invention, the signal collection assembly includes a photomultiplier tube, an objective lens, a filter, and a data acquisition element.

[0022] As described above, the detection method and system of the present invention can be used to detect the presence or characteristics of a target analyte in a biological sample. The detection method includes providing a detection kit comprising a detection carrier and a reporter molecule. The detection carrier has a fixation protein with a recognition region for binding the target analyte, while the reporter molecule contains a spin fluorescent material. Therefore, in operation, it is only necessary to mix the reporter molecule with the biological sample, place it in the detection carrier, and then place it in a time-varying physical field provided by the detection system, achieving a convenient operation. Furthermore, by utilizing the characteristic that the spin fluorescent material generates a fluorescence signal when irradiated with excitation light, the target analyte bound to the reporter molecule can be further analyzed. The fluorescence signal generated by the spin fluorescent material under the modulation of the time-varying physical field can avoid background interference, increasing its sensitivity compared to conventional immunoassay methods. Attached Figure Description

[0023] Figure 1 This is a flowchart of a detection method according to an embodiment of the present invention.

[0024] Figure 2A This is a schematic diagram of the detection kit according to the first embodiment of the present invention.

[0025] Figure 2B This is a schematic diagram of the detection kit according to the second embodiment of the present invention.

[0026] Figure 3 This is a schematic diagram of a detection system according to an embodiment of the present invention.

[0027] Figure 4A The electronic signal generated by the time-varying physical field of fluorescent nanodiamonds deposited on nitrocellulose membrane.

[0028] Figure 4B The fluorescence intensity changes of the nitrocellulose membrane and fluorescent nanodiamonds when an alternating electromagnetic field is switched on and off.

[0029] Figure 4C The correlation between the fluorescence intensity of fluorescent nanodiamonds on nitrocellulose membranes and magnetic field strength.

[0030] Figure 5A This is a distribution diagram of the fluorescence signal generated by bovine serum albumin-fluorescent nanodiamonds deposited on a nitrocellulose membrane.

[0031] Figure 5B This is a graph showing the results of ultrasensitive detection of bovine serum albumin-fluorescent nanodiamonds deposited on nitrocellulose membranes.

[0032] Figure 6A This is a distribution diagram of the fluorescence signal generated by the binding of neutral avidin to biotinylated bovine serum albumin-fluorescent nanodiamonds on nitrocellulose membranes.

[0033] Figure 6B The image shows the integrated intensity of neutral avidin on nitrocellulose membranes bound to different concentrations of biotinylated bovine serum albumin-fluorescent nanodiamonds.

[0034] Figure 6C The integral intensity diagram shows the results of the binding of anti-βhCG(D) on nitrocellulose membranes with different concentrations of anti-βhCG(B)-FND.

[0035] Figure 7 The image shows the results of applying fluorescent nanodiamonds to an antibody pair against human chorionic gonadotropin.

[0036] In the attached figures, the following labels are used:

[0037] Test kit 1, 1a; Test carrier 11, 11a

[0038] Fixation proteins 111 and 111a; reporter molecules 12 and 12a

[0039] Spin fluorescent material 121 Capture antibody 122

[0040] Detection System 2 Time-varying Physical Field 21

[0041] Coil 211 Current Amplifier 212

[0042] Displacement stage 213 Stepper motor 214

[0043] Light source 22 Signal collection component 23

[0044] Photomultiplier tube 231, objective lens 232

[0045] Filter 233 Data acquisition element 234

[0046] Processing device 24, Reflector 25

[0047] Targets O1 and O2; biological samples S1 and S2

[0048] Steps S10 to S50 Detailed Implementation

[0049] To better understand the technical content of the present invention, preferred embodiments are described below.

[0050] In this specification, references such as "an embodiment" and "in one embodiment" indicate that the described embodiment may include a specific appearance, feature, structure, or characteristic, but do not limit every embodiment to include that specific appearance, feature, structure, or characteristic. Furthermore, this terminology may, but is not necessarily, refer to the same embodiment mentioned in other parts of the specification. Also, when a specific module, appearance, feature, structure, or characteristic is described and combined into an embodiment, whether explicitly described in the specification or not, those skilled in the art can combine that module, appearance, feature, structure, or characteristic into other embodiments. In other words, any module, element, or feature can be combined with other elements or features in different embodiments, unless there are obvious or inherent incompatibilities, or those specifically excluded.

[0051] First, the detection method and system provided by this invention combine immunoassay and optical methods to detect a target analyte in a biological sample, and can further perform qualitative or quantitative analysis on the target analyte. The target analyte can be an antigen or an antibody. Furthermore, antigens can include biomolecules such as nucleic acids, proteins, or carbohydrates, and can also include microorganisms such as viruses or bacteria. In some embodiments, the biological sample can be a clinical specimen, allowing the detection method and system to detect the presence of the target analyte in the specimen, and further quantify the target analyte. In another embodiment, the biological sample can also be different (monoclonal) antibody solutions, and can be used for antibody screening to perform qualitative tests on the target analyte (antibody).

[0052] Figure 1 This is a flowchart of a detection method according to an embodiment of the present invention. Please also refer to... Figure 1 As shown. The detection method of this embodiment includes the following steps: providing a detection kit, which includes a detection carrier and a reporter molecule (step S10); mixing the reporter molecule with a biological sample and placing it into the detection carrier (step S20); removing the reporter molecule that is not bound to the fixation protein (step S22); placing the detection carrier containing the reporter molecule and the biological sample in a time-varying physical field (step S30); irradiating the detection carrier placed in the time-varying physical field with an excitation light to excite the spin fluorescent material to generate a fluorescence signal regulated by the time-varying physical field (step S40); and receiving and analyzing the fluorescence signal (step S50).

[0053] Figure 2A This is a schematic diagram of the detection kit 1 according to the first embodiment of the present invention. Figure 2B This is a schematic diagram of the detection kit 1a according to the second embodiment of the present invention. Figure 2A and Figure 2B Both can be used for explanation Figure 1 Steps S10 to S30 are shown below. The following description uses the detection kit 1 of the first embodiment as an example; please refer to... Figure 1 and Figure 2A As shown. In step S10, a detection kit 1 is first provided. The detection kit 1 includes a detection carrier 11 and a reporter molecule 12. The detection carrier 11 includes a coating protein 111. The coating protein 111 is a protein pre-coated onto the detection carrier 11, used to recognize and bind to the target analyte O1. The coating protein 111 has a recognition site for binding to the target analyte O1.

[0054] Specifically, fixation protein 111 can be an antibody or an antigen, depending on the desired experiment or the type of target analyte O1. Figure 2A The fixation protein 111 in the test kit 1 shown is an antibody, while Figure 2B The fixation protein 111a of the test kit 1a shown is used as the antigen. Figure 2A For example, detection kit 1 can be used to detect specific foreign substances in biological sample S1, i.e., target substance O1 is an antigen. In this case, the fixation protein 111 can be an antibody. In this embodiment, the recognition region of fixation protein 111 is a paratope, which can recognize and bind to the epitope of target substance O1 (antigen).

[0055] by Figure 2B For example, the detection kit 1a of the second embodiment can be used in qualitative experiments for antibody screening, where the target substance O2 can be the antibody to be screened, to detect whether the antibody in the sample can bind to a specific antigen. In this case, the fixation protein 111a can be the specific antigen. In this embodiment, the recognition region of the fixation protein 111a is an epitope, which can be bound to the paratope of the target substance O2 (monoclonal antibody).

[0056] Additionally, the detection carriers 11 and 11a of the first and second embodiments may include a solid matrix, such as a membrane for protein binding, commonly a nitrocellulose membrane, also known as an NC membrane. NC membranes are frequently used in immunoassay strips. Therefore, the detection kits 1 and 1a of the first and second embodiments can be used in lateral flow immunoassay (LFIA) tests. In other embodiments, the detection carrier may also include a liquid matrix for use in enzyme-linked immunosorbent assay (ELISA). Specifically, the solid matrix of the detection carrier may be an ELISA microplate with a bottom for attaching proteins. The liquid matrix may be a substrate solution.

[0057] Reporter molecule 12 is a substance used to label the target analyte O2 and can be photoexcited to emit fluorescence. First, let's look at the first embodiment (e.g.) Figure 2A For example, reporter molecule 12 includes a spin fluorescent material 121 and a capture antibody 122. The spin fluorescent material 121 is a material that can be modulated by a magnetic field and emits fluorescence of different wavelengths upon stimulation by light of a specific wavelength. The spin fluorescent material 121 can be, for example, but not limited to, a fluorescent nanodiamond (FND), an organic dye-labeled magnetic bead, or a rare-earth metal ion doped magnetic bead. Furthermore, the spin fluorescent material 121 includes multiple spin fluorescent particles. Preferably, the diameter of each spin fluorescent particle is between 1 nanometer and 1 millimeter.

[0058] In this embodiment, the spin fluorescent material 121 is an example of fluorescent nanodiamond (FND). When the fluorescent nanodiamond (spin fluorescent material 121) is irradiated with yellow-green light (wavelength 500-600 nm), it emits red light with a wavelength of approximately 700 nm. Therefore, the intensity of the red light signal can be analyzed to quantitatively or qualitatively identify the target substances O1 and O2 (to be further explained later). In addition, functional groups, such as carboxyl groups (-COOH) or amino groups (-NH2), can be easily derived on the surface of the fluorescent nanodiamond, allowing it to link with biomolecules such as nucleic acids, proteins, or carbohydrates.

[0059] To increase the specificity of reporter molecule 12 in labeling target analyte O1, a complex can be formed in which spin fluorescent material 121 and capture antibody 122 conjugate, allowing reporter molecule 12 to label target analyte O1 in biological sample S1 via capture antibody 12. Specifically, capture antibody 122 and immobilization protein 111 can be the same antibody, both possessing a recognition region for binding to target analyte O1. That is, capture antibody 122 has a paratope to recognize and bind to the epitope of target analyte O1 (antigen). Therefore, reporter molecule 12 includes a structure of spin fluorescent material 121 and capture antibody 122 (e.g., ...). Figure 2A It is suitable for use in detecting and quantifying antigens (target O1) in biological samples S1.

[0060] In step S20, reporter molecule 12 is mixed with biological sample S1 and placed into detection carrier 11. Specifically, after mixing reporter molecule 12 with biological sample S1, if target substance O1 (antigen) is present in biological sample S1, the capture antibody 122 of reporter molecule 12 can specifically capture target substance O1 through antibody-antigen specificity. Next, the mixture of reporter molecule 12 and biological sample S1 is added to detection carrier 11. At this time, the fixation protein 111 (antibody) on detection carrier 11 can also specifically capture target substance O1 (antigen) labeled with reporter molecule 12 through antibody-antigen specificity. In other words, reporter molecule 12 can bind to fixation protein 111 through target substance O1.

[0061] In the second embodiment (e.g.) Figure 2B For example, reporter molecule 12a only possesses spin fluorescent material 121. Since spin fluorescent material 121 is also a fluorescent nanodiamond as in the aforementioned embodiment, it is referred to by the same designation. The biological sample S2 used in the second embodiment can be a (monoclonal) antibody solution, and multiple biological samples S2 with different (monoclonal) antibodies can be prepared for antibody screening. In step S20, when reporter molecule 12a is mixed with biological sample S2, spin fluorescent material 121 can directly bind to the (monoclonal) antibody to be detected / screened. In other words, spin fluorescent material 121 directly binds to the target analyte O2.

[0062] It should also be noted that when the spin fluorescent material 121 is a fluorescent magnetic bead or a rare earth metal magnetic bead, the capture antibody 122 or the antibody to be detected (target O2) can be bound using conventional methods for preparing magnetic bead antibodies.

[0063] Next, in step S22, reporter molecules 12 and 12a that are not bound to fixation proteins 111 and 111a are removed. Since the detection kits 1 and 1a of the first and second embodiments are applied to lateral flow immunoassay strips (LFIA strips), the reporter molecules 12 and 12a that are not bound to fixation proteins 111 and 111a can be removed by their flow characteristics.

[0064] In the first embodiment, if the biological sample S1 contains the target substance O1 (antigen), the mixture of biological sample S1 and reporter molecule 12 flows through the anchoring protein 111 (generally referred to as the test line). The target substance O1 (labeled by reporter molecule 12) can bind to the anchoring protein 111 and remain on the test line. Reporter molecules 12 that do not bind to the anchoring protein 111, that is, reporter molecules 12 that do not bind to the target substance O1, can flow to locations outside the test line, such as the end of the test strip, to avoid affecting the quantification of the target substance O1.

[0065] In the second embodiment, if the target analyte O2 (the antibody to be detected / screened) can bind to the fixation protein 111a (a specific antigen), then the target analyte O2 labeled by the reporter molecule 12a can remain at the test line (fixation protein 111a). The reporter molecule 12a that does not bind to the fixation protein 111a indicates that the target analyte O2 it binds to is not specific to the fixation protein 111a. Removing the reporter molecule 12a bound to the fixation protein 111a by the flow property to screen for the target analyte O2 (the antibody to be detected / screened) is also a qualitative test for the target analyte O2.

[0066] In other embodiments, when the detection kit is used in enzyme-linked immunosorbent assay (ELISA), reporter molecules that are not bound to the fixation protein can be removed through a washing step.

[0067] Figure 3 This is a schematic diagram of a detection system according to an embodiment of the present invention. Please refer to... Figure 1 , Figure 2A , Figure 2B and Figure 3 As shown. In the first or second embodiment, detection kits 1 and 1a can both be placed in detection system 2 for subsequent steps S30 to S50. In other words, detection system 2 can be used in conjunction with detection kits 1 and 1a to detect target substances O1 and O2 in biological samples S1 and S2, and can quantify or qualitatively analyze target substances O1 and O2. In this embodiment, detection system 2 includes a time-varying physical field 21, a light source 22, a signal collection component 23, and a processing device 24. The processing device 24 is electrically connected to the time-varying physical field 21, the light source 22, and the signal collection component 23 to control their operation and receive fluorescence signal information.

[0068] Taking the detection kit 1 of the first embodiment as an example, in step S30, the detection carrier 11 containing the mixture of reporter molecule 12 and biological sample S1 is placed in the time-varying physical field 21. The time-varying physical field 21 can be, for example, but not limited to, an alternating current (AC) magnetic field or a microwave field. In this embodiment, the time-varying physical field 21 is an AC electromagnetic field, and includes a coil 211 and a current amplifier 212, which are electrically connected to each other. Furthermore, the current amplifier 212 is electrically connected to the processing device 24 through the signal collection component 23 (details are further explained in step S50), allowing the processing device 24 to drive the current amplifier 212 to provide AC current to the coil 211. The AC electromagnetic field generated when AC current passes through the coil 211 can also be considered as the time-varying electromagnetic field (time-varying physical field 21). Depending on the experimental design, the frequency of the AC electromagnetic field can be between 1 Hz and 1 MHz, and the root mean square value of the amplitude can be between 1 G and 10000 G. In addition, when the time-varying physical field 21 is a microwave field, the frequency of the microwave field is preferably between 0.1MHz and 10GHz.

[0069] In addition, the time-varying physical field 21 also includes a translation stage 213, which is adjacent to the coil 211. The detection carrier 11 is disposed on the translation stage 213 and can therefore be adjusted by the time-varying magnetic field generated by the coil 211. Preferably, the translation stage 213 can be driven by a stepper motor 214. The stepper motor 214 is electrically connected to the processing device 24, so that the processing device 24 can control the stepper motor 214 to drive the translation stage 213 to move along a default path to scan and detect the detection carrier 11.

[0070] In step S40, the detection carrier 11 placed in the time-varying physical field 21 is irradiated with excitation light to excite the spin fluorescent material 121 to generate a fluorescence signal modulated by the time-varying physical field 21. Specifically, the excitation light is provided by the light source 22 to irradiate the detection carrier 11 located in the time-varying physical field 21. The light source 22 can be a laser light source and can emit laser light (i.e., excitation light) with a wavelength between 500 nm and 600 nm to excite the fluorescent nanodiamond (spin fluorescent material 121) to emit red light with a wavelength of approximately 700 nm. In other embodiments, depending on the characteristics of the fluorescent magnetic beads or rare earth metal magnetic beads, different wavelengths of laser light can be irradiated, with a wavelength range of 400 nm to 800 nm.

[0071] Additionally, the detection system 2 may include at least one reflector 25 positioned in the illumination path between the light source 22 and the displacement stage 213 to guide the excitation light to irradiate the detection carrier 11 placed on the displacement stage 213. Furthermore, since the spin fluorescent material 121, along with the detection carrier 11, is placed in the time-varying physical field 21, it can be modulated by the time-varying physical field 21 to emit a fluorescence signal. The fluorescence signal generated by the time-varying physical field 21, after data analysis in the subsequent step S50, can be determined to be the fluorescence signal generated by the spin fluorescent material 121, thereby achieving the effect of reducing background interference.

[0072] In step S50, the fluorescence signal is received and analyzed by the signal collection component 23 and the processing device 24, respectively. In this embodiment, the signal collection component 23 includes a photomultiplier tube 231, an objective lens 232, a filter 233, and a data acquisition element 234. The objective lens 232 is positioned on the detection carrier 11 and corresponds to the test line (binding site of the fixation protein 111) on the detection carrier 11 to receive the fluorescence signal generated by the spin fluorescent material 121 of the reporter molecule 12. The photomultiplier tube 231 corresponds to the objective lens 232 to enhance the received fluorescence signal. The filter 233 is disposed between the photomultiplier tube 231 and the objective lens 232. In this embodiment, the wavelength of the filter 233 is higher than the excitation wavelength to avoid interference from the excitation light.

[0073] The data acquisition element 234 is electrically connected to the photomultiplier tube 231, the processing device 24, and the aforementioned current amplifier 212. After receiving the fluorescence signal generated by the spin fluorescent material 121 from the photomultiplier tube 231, the data acquisition element 234 performs digital-to-analog signal conversion. Furthermore, the design of the data acquisition element 234 electrically connected to the processing device 24 and the current amplifier 212 allows it to function simultaneously as a digital lock-in amplifier and a function generator. The processing device 24 first generates a digital sequence of voltages, which resembles a voltage sine wave of a selected frequency. The current amplifier 212 is electrically connected to the processing device 24 via the data acquisition element 234, enabling it to output an infinitely repeating voltage sine wave. Simultaneously, the data acquisition element 234 measures the input voltage and transmits digital signals to the processing device 24. The input and output electronic or digital signals undergo mathematical operations to generate lock-in signals.

[0074] For example, by analyzing the acquired signal using a fast Fourier transform or lock-in amplification detection, the signal generated by the spin fluorescent material 121 (e.g., a fluorescent nanodiamond signal) can be obtained. Specifically, the electronic signal with fluorescence information can be input into a conventional lock-in amplifier or a data acquisition card (i.e., the signal collection component 23 of this invention) for computational phase-sensitive detection. When the mode described later is executed, the detected signal (V m First, multiply by two reference signals (2πft and cos(2πft)), as shown below:

[0075] V x =V m (2πft)

[0076] V y =V m (2πft).

[0077] Next, record V x Value and V y The root mean square (rms) of the values ​​is used to generate X and Y values, which are then used to calculate the R value. The R value is defined as X... 2 and Y 2 The sum of the square roots (as shown below) is linearly proportional to the concentration of the reported molecule 12 or 12a.

[0078]

[0079] In addition, the phase angle (θ) is also a useful indicator for phase-locked detection, and its definition is as follows:

[0080] θ = tam -1 (Y / X)

[0081] The above calculations enable the signal of the spin fluorescent material 121 (in this embodiment, fluorescent nanodiamond (FND)) to be identified and separated from the background fluorescence signal because, compared to other fluorescent groups, the fluorescence intensity of the signal generated by the spin fluorescent material 121 can be magnetically modulated. Collecting the measured phase angles constructs a phase angle distribution, and a constant angle can be observed in the distribution plot, indicating that the signal is not random noise but a phase-locked signal, which is information about the fluorescence signal of the spin fluorescent material 121.

[0082] In the first embodiment, the processing device 24 receives and analyzes the fluorescence signal from the signal collection component 23. If the fluorescence signal of the spin fluorescent material 121 is detected, it indicates that the biological sample S1 contains the target substance O1 (antigen), and thus can be used to determine whether the biological sample S1 contains the target substance O1. Preferably, the detection method of the first embodiment may further include the step of: the processing device 24 comparing the fluorescence signal with a certain amount of standard curve to quantify the concentration of the target substance O1. Specifically, multiple sample solutions containing different concentrations of reporter molecule 12 (spin fluorescent material 121) can be prepared by serial dilution, and the fluorescence signal of each concentration of sample solution can be measured to generate a quantitative standard curve. Subsequently, the concentration of the target substance O1 in the biological sample S1 can be calculated by comparing with the quantitative standard curve.

[0083] In the second embodiment, if a fluorescence signal of spin fluorescent material 121 is detected, it indicates that the target substance O2 (antibody) of biological sample S2 and the fixation protein 111a (antigen) on the detection carrier 11 are specific and can bind, so it can be used for antibody screening test.

[0084] Experimental Example 1: Preparation of detection carrier and establishment of detection system.

[0085] The spin fluorescent material 121 in this experiment is composed of 100 nm fluorescent diamond nanoparticles (hereinafter referred to as FND). It was prepared by electron irradiation of synthetic diamond powder (element six), followed by thermal annealing, air oxidation, and strong oxidizing acid cleaning.

[0086] This experimental example uses lateral flow immunoassay (LFIA) as an example. The test strip used for lateral flow immunoassay (LFIA) consists of a 4 mm wide nitrocellulose (NC) membrane, with the test line located approximately 45 mm from the bottom of the test strip.

[0087] The detection system architecture of this experiment is as follows: Figure 3 As shown. The computer program executed by the processing device 24 can simultaneously process the data acquisition program and the trigger current amplifier 212 to generate an AC electromagnetic field with a frequency of 102.4Hz switching on / off and an intensity (B) of 40mT (the root mean square value of the amplitude is 400G), thereby controlling the fluorescence signal generated by the FND (refer to the previous section). Figure 4A and Figure 4B (As shown).

[0088] The light source 22 is a green laser device (Coherent, Obis), whose generated green light can excite the solid surface of FND particles located in an alternating electromagnetic field. The objective lens 232 used in this experiment is a long working distance objective lens (Mitutoyo, 20x objective lens) used to receive the second-harmonic fluorescence signal generated by the FND particles (tuned to 204.8 Hz), filtered by filter 233 (Semrock filter, 740LP), and detected by photomultiplier tube 231 (Thorlabs, PMT1001). The detected electronic signal is input to processing device 24 via a data acquisition card (National Instruments, USB-4431) and analyzed by a lock-in amplifier. Details of the analysis can be found above and will not be repeated here.

[0089] Experimental Example 2: Pretreatment of biological samples and reporter molecules.

[0090] Proteins (antibodies or antigens) are non-covalently linked to FND in phosphate-buffered saline (PBS) at pH 7.4. First, a solution containing acid-treated FND (1 mg / mL) and the target protein (1 mg / mL) is mixed at room temperature for 10 minutes. It should be noted that the target protein can be the capture antibody 122 described in the first embodiment or the target substance O2 (antibody used for antibody screening) in the biological sample S2 of the second embodiment. Depending on the application, the weight ratio of FND to target protein can be between 10:1 and 100:1. Next, the mixture is centrifuged at 20,000 × g for 5 minutes to form a pellet. After removing the supernatant, the protein-bound FND (i.e., the pellet) is reconstituted in phosphate-buffered saline (PBS) containing 3% bovine serum albumin (BSA) and stored at 4°C for use in lateral flow immunoassay (LFIA) and enzyme-linked immunosorbent assay (ELISA).

[0091] A mixture of biological samples S1 and S2 with reporter molecules 12 and 12a is dropped onto the sample pad position and then slowly moved through the test line. Alternatively, the test strip can be directly immersed in the aforementioned mixture in a 96-well pan to achieve the same purpose. Furthermore, lateral flow immunoassay can be performed at room temperature.

[0092] Experimental Example 3: Detection of fluorescence signal in fluorescent nanodiamonds (FND).

[0093] In this experimental example, the 100nm fluorescent nanodiamond (FND) prepared in Experiment 1 above was directly deposited on a nitrocellulose (NC) membrane, placed in an AC electromagnetic field (intensity of 40mT, modulation frequency of 102.4Hz), and its electronic signal was detected to confirm that the FND can be modulated by a time-varying physical field.

[0094] Figure 4A Electronic signals generated by fluorescent nanodiamonds (FND) deposited on nitrocellulose (NC) membranes and modulated by time-varying physical fields; Figure 4B The fluorescence intensity changes of nitrocellulose (NC) membranes and fluorescent nanodiamonds (FND) under switched AC electromagnetic fields. Figure 4A It is known that the electronic signal containing fluorescent information generated by the FND can change accordingly with the intensity of the alternating electromagnetic field; therefore, the fluorescent signal generated by the FND can be modulated by a time-varying physical field. Furthermore... Figure 4B It is known that when the alternating electromagnetic field is switched on and off (ON / OFF), although the signal of the NC film (control group) also changes, the change in signal intensity of the NC film is less than 1 / 1000 of the change in signal intensity of the FND. Therefore, using the FND as the spin fluorescent material 121 for reporter molecules 12 and 12a can indeed distinguish it from the background value (noise).

[0095] Figure 4C The correlation between the fluorescence intensity of fluorescent nanodiamonds (FND) on nitrocellulose (NC) membranes and magnetic field strength was investigated. Similarly, 100 nm fluorescent nanodiamonds (FND) were deposited on nitrocellulose (NC) membranes, placed in an AC electromagnetic field, and the correlation between the output electronic signal (voltage) and magnetic field strength was analyzed using lock-in detection. Figure 4C The intensity (root mean square value of amplitude) of the displayed (alternating current) magnetic field is between 1G and 300G.

[0096] Experimental Example 4: Absolute quantification of fluorescent nanodiamonds (FND) on a side-flow immunoassay strip (LFIA strip).

[0097] Following the aforementioned Experimental Example 2, but using bovine serum albumin (BSA) as the target protein, an aqueous solution of BSA-bound FND (hereinafter referred to as BSA-FND) was prepared. Next, 0.5 μL drops of each of the BSA-FND aqueous solution containing 10 ng of FND and 100 ng of FND were respectively placed on LFIA test paper and dried in air. Then, the LFIA test paper was placed in the aforementioned alternating electromagnetic field, and the fluorescence signal generated by BSA-FND was detected and analyzed, such as... Figure 5A As shown. Figure 5AThis is a distribution diagram of the fluorescence signal generated by bovine serum albumin-fluorescent nanodiamonds (FND) deposited on nitrocellulose (NC) membranes.

[0098] Next, 0.5 μL of different concentrations of BSA-FND aqueous solution and phosphate buffer solution containing 3% BSA (as blank values) were dropped onto LFIA test paper, and ultrasensitive detection was performed under conditions with and without magnetic field regulation (MM). Figure 5B This is a graph showing the results of ultrasensitive detection of bovine serum albumin-fluorescent nanodiamonds (FND) deposited on nitrocellulose (NC) membranes. Figure 5B Displayed in an environment with a small amount of FND (10 -2 ng / mm 2 Up to 10 3 ng / mm 2 The signal-to-blank ratio of the magnetically regulated FND changes linearly with the concentration of BSA-FND, and the detection dynamic range exceeds 5 orders of magnitude (10). 5 This indicates that the detection method and system of the present invention are very suitable for the purpose of quantifying target objects.

[0099] Furthermore, the measured limit of detection (LOD) achieved by alternating electromagnetic field modulation (with MM) is 0.01 ng / mm². 2 Or 5×10 4 FND particles / mm 2 Furthermore, the fluorescence detection sensitivity modulated by an alternating electromagnetic field is approximately 1000 times higher than that without magnetic field modulation.

[0100] Experimental Example 5: Fluorescent nanodiamonds (FND) applied in quantitative analysis.

[0101] This experimental example uses two systems, biotin / avidin and human chorionic gonadotropin (hCG), to demonstrate that the detection method and system of the present invention can be applied to lateral flow immunoassay strips with (fixed) antibodies for antigen detection (i.e., the state of the first embodiment mentioned above) and can quantify the antigen.

[0102] First, a 1.5 μL solution of neutral avidin (5 mg / mL) was dropped into the center of the NC membrane test paper and fixed to the absorbent pad to form a band across the NC membrane test paper. Neutral avidin served as the fixation protein 111. Next, referring to Experiment 2, biotinylated bovine serum albumin (hereinafter referred to as B-BSA) and FND were mixed at a ratio of 1:10 in phosphate-buffered saline (PBS) for 10 minutes to form an FND-bound B-BSA complex (hereinafter referred to as B-BSA-FND). Unbound B-BSA was removed by centrifugation, and the flaky precipitate was resuspended in phosphate-buffered saline containing 3% BSA to obtain a B-BSA-FND suspension.

[0103] Furthermore, the NC membrane test paper containing neutral avidin prepared above was immersed in 100 μL of B-BSA-FND suspension. After drying, the test paper was placed in the detection system of Experimental Example 1 to detect its electronic signal. Figure 6A This is a fluorescence signal distribution diagram showing the binding of neutral avidin to biotinylated bovine serum albumin-fluorescent nanodiamonds (B-BSA-FND) on a nitrocellulose (NC) membrane. Figure 6A It is known that neutral avidin on nitrocellulose (NC) membranes can bind to B-BSA-FND through its specificity to biotin, and biotin can be detected and quantified through the characteristics of FND.

[0104] Specifically, the fluorescence intensity can be detected using B-BSA-FND solutions of different concentrations in the manner described above, and the fluorescence intensity can be integrated to obtain a curve that can be used for subsequent quantification. Figure 6B As shown. Figure 6B The integral intensity map shows the binding of neutral avidin on nitrocellulose (NC) membranes with different concentrations of biotinylated bovine serum albumin-fluorescent nanodiamonds (B-BSA-FND).

[0105] In the human chorionic gonadotropin (hCG) experiment, anti-hCG antibody pairs were prepared, purchased from Fitzgerald under the product numbers 10-C25B (anti-βhCG(B)) and 10-C25D (anti-βhCG(D)). First, 1.5 μL of anti-βhCG(D) solution was deposited onto NC membrane test paper, using anti-βhCG(D) as a fixation protein 111. Next, different concentrations of anti-βhCG(B)-FND complexes were prepared according to the aforementioned experimental example. The fluorescence intensity was detected as described above, and the fluorescence intensity was integrated to obtain a curve suitable for subsequent quantification, as shown below. Figure 6C As shown. Figure 6C This is an integrated intensity map showing the binding of anti-βhCG(D) to different concentrations of anti-βhCG(B)-FND on a nitrocellulose (NC) membrane. Figure 6C It is known that the limit of detection (LOD) for anti-βhCG(B) is approximately 0.01 ng / mL.

[0106] Experimental Example 6: Fluorescent nanodiamonds (FND) are used in antibody screening analysis.

[0107] This experimental example uses the human chorionic gonadotropin (hCG) system to demonstrate that the detection method and system of the present invention can be applied to antibody screening (i.e., the sample of the aforementioned second embodiment). It should be noted that the second embodiment illustrates a possible scenario for practical application, and therefore detects the deposited antigen (fixed protein 111a) before detecting the carrier 11a. The experimental design of Example 6 also utilizes anti-hCG antibody pairs, thus using the antibody as the fixed protein 111a.

[0108] The antibodies used in this experiment were also purchased from Fitzgerald, with part numbers 10-C25B (anti-βhCG(B)), 10-C25D (anti-βhCG(D)), and 10-C25E (anti-βhCG(E)). First, 1.5 μL solutions containing anti-βhCG(B), anti-βhCG(D), and anti-βhCG(E) were deposited onto different NC membrane test strips, with at least two aliquots prepared for each antibody (B, D, E). Next, following the previously described experimental example, 10 ng of anti-βhCG(B)-FND detection solution, 10 ng of anti-βhCG(D)-FND detection solution, and 10 ng of anti-βhCG(E)-FND detection solution were prepared.

[0109] The aforementioned NC membrane test strips were immersed in detection solutions with different antibodies, and after drying, they were placed into the detection system of Experimental Example 1 to detect and analyze the fluorescence signals, thereby analyzing the binding of the antibody pairs. The results are as follows: Figure 7 As shown. Figure 7 This image shows the results of applying fluorescent nanodiamonds to an antibody pair against human chorionic gonadotropin (hCG). Figure 7 The first letter of the labeled antibody pair indicates the antibody deposited on the NC membrane. For example, Figure 7 The "BD" designation indicates that anti-βhCG(B) is deposited on the NC membrane, and the detection solution contains anti-βhCG(D)-FND.

[0110] For example, two NC membrane test strips deposited with anti-βhCG(B) are immersed in anti-βhCG(D)-FND detection solutions and anti-βhCG(E)-FND detection solutions, respectively. After the NC membrane test strips are dried, they are placed into a detection system to detect the fluorescence signal of anti-βhCG(D)-FND or anti-βhCG(E)-FND bound by anti-βhCG(B) (fixing protein). Figure 7 It can be seen that the antibody's specific binding to anti-βhCG(B) and anti-βhCG(D) is better than that to anti-βhCG(B) and anti-βhCG(E).

[0111] In summary, the detection method and system of the present invention can be used to detect the presence or characteristics of a target analyte in a biological sample. The detection method includes providing a detection kit comprising a detection carrier and a reporter molecule. The detection carrier has a fixation protein with a recognition region for binding the target analyte, while the reporter molecule contains a spin fluorescent material. Therefore, operationally, it is only necessary to mix the reporter molecule with the biological sample, place it into the detection carrier, and then place it into a time-varying physical field provided by the detection system, achieving a convenient operation. Furthermore, the characteristic of the spin fluorescent material to generate a fluorescence signal when irradiated with excitation light allows for further analysis of the target analyte bound to the reporter molecule. The fluorescence signal generated by the spin fluorescent material under the modulation of the time-varying physical field further avoids background interference, increasing its sensitivity compared to conventional immunoassay methods.

[0112] However, the present invention may have other various embodiments. Without departing from the spirit and essence of the present invention, those skilled in the art can make various corresponding changes and modifications according to the present invention, but these corresponding changes and modifications should all fall within the protection scope of the appended claims.

Claims

1. A detection method for detecting a target analyte in a biological sample, characterized in that, The detection method includes the following steps: A detection kit is provided, comprising a detection carrier and a reporter molecule, the detection carrier comprising a fixation protein having a recognition region for binding the target, and the reporter molecule comprising a spin fluorescent material. The reporter molecule was mixed with the biological sample and placed into the detection vector; The detection vector containing the reporter molecule and the biological sample is placed in a time-varying physical field; The detection carrier placed in the time-varying physical field is irradiated with an excitation light to excite the spin fluorescent material to generate a fluorescence signal modulated by the time-varying physical field; as well as The fluorescence signal is received and analyzed.

2. The detection method according to claim 1, characterized in that, The reporter molecule tags the target in the biological sample, and the reporter molecule binds to the anchoring protein through the target.

3. The detection method according to claim 2, characterized in that, The step of placing the detection carrier in the time-varying physical field also includes the following step: Remove the reporter molecule that is not bound to the anchoring protein.

4. The detection method according to claim 1, characterized in that, The reporter molecule also includes a capture antibody having a recognition region that binds to the target, the capture antibody binding to the spin fluorescent material, and the reporter molecule using the capture antibody to label the target in the biological sample.

5. The detection method according to claim 1, characterized in that, The spin fluorescent material includes a fluorescent nanodiamond, a fluorescent magnetic bead, or a rare earth metal magnetic bead.

6. The detection method according to claim 5, characterized in that, The spin fluorescent material contains multiple spin fluorescent particles, each with a diameter between 1 nanometer and 1 millimeter.

7. The detection method according to claim 1, characterized in that, The detection carrier comprises a solid matrix.

8. The detection method according to claim 1, characterized in that, The time-varying physical field includes an alternating electromagnetic field or a microwave field.

9. The detection method according to claim 8, characterized in that, The frequency of the alternating electromagnetic field is between 1 Hz and 1 MHz, and the root mean square value of the amplitude is between 1 G and 10000 G. The frequency of the microwave field is between 0.1 MHz and 10 GHz.

10. The detection method according to claim 1, characterized in that, The steps for analyzing the fluorescence signal also include comparing the fluorescence signal with a certain number of standard curves to quantify the concentration of the target analyte.

11. A detection system, used in conjunction with a detection kit, for detecting a target analyte in a biological sample, characterized in that, The detection kit includes a detection carrier and a reporter molecule. The detection carrier contains a fixation protein having a recognition region for binding the target. The reporter molecule contains a spin fluorescent material. The reporter molecule is mixed with the biological sample and then placed into the detection carrier. The detection system includes: A time-varying physical field, in which the detection carrier containing the reporter molecule and the biological sample is placed; A light source provides an excitation light to irradiate the detection carrier placed in the time-varying physical field, so as to excite the spin fluorescent material to generate a fluorescence signal modulated by the time-varying physical field; A signal collection component receives the fluorescence signal; and A processing device is electrically connected to the time-varying physical field, the light source, and the signal collection component, receives the fluorescence signal from the signal collection component, and analyzes the fluorescence signal.

12. The detection system according to claim 11, characterized in that, The time-varying physical field includes an alternating electromagnetic field or a microwave field.

13. The detection system according to claim 12, characterized in that, The frequency of the alternating electromagnetic field is between 1 Hz and 1 MHz, and the root mean square value of the amplitude is between 1 G and 10000 G. The frequency of the microwave field is between 0.1 MHz and 10 GHz.

14. The detection system according to claim 11, characterized in that, The processing device compares the fluorescence signal with a certain number of standard curves to quantify the concentration of the target analyte.

15. The detection device according to claim 11, characterized in that, The signal collection assembly includes a photomultiplier tube, an objective lens, a filter, and a data acquisition element.

16. A detection system for detecting a target analyte in a biological sample, characterized in that, The detection system includes: A detection kit includes a detection carrier and a reporter molecule. The detection carrier contains a fixation protein having a recognition region for binding the target. The reporter molecule contains a spin fluorescent material. The reporter molecule is mixed with the biological sample and then placed into the detection carrier. A time-varying physical field, in which the detection carrier containing the reporter molecule and the biological sample is placed; A light source provides an excitation light to irradiate the detection carrier placed in the time-varying physical field, so as to excite the spin fluorescent material to generate a fluorescence signal modulated by the time-varying physical field; A signal collection component receives the fluorescence signal; and A processing device is used to analyze the fluorescence signal.

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