KR101592499B1 - Ultramicroanalysis Methods of fluorescence - Google Patents

Abstract

A fluorescent particle detection method is provided. A fluorescent particle detecting method according to the present invention comprises: irradiating light of a first wavelength onto a substrate from a light source part spaced from the substrate; And measuring light of a second wavelength emitted from the fluorescent particles and the fluorescent particle-analyte combination on the substrate, respectively. The fluorescence particle-analyte conjugate may be formed by binding the fluorescence particle contained in the release pad with the analyte moving to the release pad. The fluorescence particle detection method can be performed by a time division detection method.

Description

Ultramicroanalysis Methods of fluorescence < RTI ID = 0.0 >

The present invention relates to fluorescence detection, and more particularly, to a fluorescence detection system and a fluorescence detection method using a time-division detection method using a lymphocyte fluorescent substance.

Fluorescence occurs when molecules absorb photons and are released by electron transfer when the excited material returns to the ground state. A fluorescent material absorbs energy of a specific wavelength and re-emits it to another wavelength. Fluorescent materials are used for fluorescent dyes such as fluorescent inks, fluorescent paints, and pigments, including inorganic materials and organic materials.

As the development of biomaterial diagnosis and detection technologies has progressed rapidly, studies are underway to apply fluorescent materials in biological and biomedical research. Fluorescent materials can be advantageous for the detection of trace samples due to their high sensitivity characteristics. However, most of the fluorescent dyes have a problem of improving the detection limit because the fluorescence duration is in the nanosecond range and the difference in the peak wavelength-fluorescence wavelength is very close to 10 to 20 nm. That is, since most of the fluorescent dyes have a very short fluorescence duration, conventional fluorescence detection is performed simultaneously with the light source introduction for excitation and the fluorescence measurement by emission. Such a fluorescence detection method is difficult to completely remove excitation light from the detected fluorescence, and background fluorescence signals such as autofluorescence are not removed from the object to be analyzed, resulting in limitations in detection sensitivity.

Domestic and overseas time division fluorescence analysis techniques are mostly approaches and attempts through background principles and theories. There is a limit to providing improved detection sensitivity over the existing detection system in the diagnostic kit area. Further, since the analyte undergoes enzymatic treatment and / or washing, the fluorescence detection method is complicated and takes a long time. Fluorescence detection systems have been difficult to use in small hospitals, emergency rooms and homes due to their large size and / or high cost.

An object of the present invention is to provide a detection system capable of detecting a trace amount of an analyte.

Another object of the present invention is to provide a method for easily detecting an analyte by a time division detection method.

Another object of the present invention is to provide a detection system that can easily detect whether or not an analyte is detected.

The problems to be solved by the present invention are not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

A fluorescence detection method according to the present invention provides a substrate comprising a preform, a sample pad, and a discharge pad, wherein the sample pad comprises an analyte, the discharge pad comprising fluorescent particles; Irradiating light of a first wavelength onto the substrate from a light source part spaced from the substrate; Measuring light of a second wavelength emitted from the fluorescent particles and the fluorescent particle-analyte combination on the substrate, respectively; Converting the measured intensity of light of the second wavelength into a signal; And displaying an image by receiving the signal in an image display unit spaced apart from the substrate, wherein the emission pad further comprises a stabilizer or a blocking agent, wherein the fluorescent particles include a lanthanide complex, The fluorescence particle-analyte conjugate may be formed by binding the fluorescent particles included in the release pad with the analyte moving to the release pad.

According to the embodiment, the measurement of the light of the second wavelength may be performed by a time division detection method.

According to the embodiment, the measurement of the light of the second wavelength may proceed in the off mode of the light source unit after the light of the first wavelength is irradiated onto the substrate.

According to an embodiment, the light source unit may include an LED.

According to an embodiment of the present invention, the measurement of the light of the second wavelength is performed a plurality of times, and the conversion of the intensity of the light of the second wavelength to the signal may be performed by measuring the intensity of the light of the second wavelength And converting the average value to the signal.

According to the present invention, the fluorescence detection system can use lanthanide complex particles to increase the intensity of emitted fluorescence. The lanthanide complex particles exhibit a long luminescence duration, so that fluorescence can be sustained when the light source is in the off mode. Thus, the background signal of the fluorescence measured is reduced, so that sensitivity and detection limit of fluorescence detection can be improved.

According to the fluorescence detection method of the present invention, conditions such as on-mode duration can be optimized, and the sensitivity of fluorescence detection can be improved. In addition, the control unit performs image processing, and the image display unit can display a clearer image than the image of the strip unit sensed by the sensing unit.

INDUSTRIAL APPLICABILITY The fluorescence detection system according to the present invention is capable of observing the fluorescence generated in the strip portion with the naked eye, and can easily determine the presence or absence of the analyte. The fluorescence detection system includes an image display unit, and fluorescence generated in the strip unit can be displayed as an image.

1A is a plan view showing a strip unit according to an embodiment of the present invention.
1B is a cross-sectional view taken along line AB of FIG. 1A.
2A to 2C are schematic views illustrating a method of detecting an analyte according to an embodiment of the present invention.
3 is a schematic diagram showing a fluorescence detection system according to an embodiment of the present invention.
4A to 4C show images displayed on the image display unit according to the embodiments of the present invention.
FIG. 5A shows the results of the strips of the experimental examples and the comparative example 1-1 when the light source part is in the ON mode. FIG.
FIG. 5B shows the results of the strip portions of Comparative Examples 1-2 to 1-9, respectively, when the light source portion is in the ON mode.
FIG. 6A is a photograph showing fluorescence emitted from the strips of Experimental Examples 2-1 to 2-3, respectively.
FIG. 6B shows the fluorescence emitted from the strips of Comparative Example 2-1 and Comparative Example 2-2.
7A and 7B are graphs showing fluorescence measurement results of Experimental Example 3 and Comparative Example 3, respectively.
FIG. 8 is a graph showing fluorescence intensities with time in Experimental Examples 4-1 to 4-4, Comparative Examples 4-1 and 4-2. FIG.
9 shows photographs and analyzed images of strip portions according to Experimental Example 5 and Comparative Example 5. Fig.
FIG. 10A is a photograph of the fluorescence emitted from the strips of Experimental Examples 6-1 to 6-6 when the light source portion is in the ON mode. FIG.
FIG. 10B shows the image display unit in which the fluorescence detection results of Experimental Examples 6-1 to 6-6 are displayed when the light source unit is in the off mode.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS The advantages and features of the present invention and the manner of achieving them will become apparent with reference to the embodiments described in detail below with reference to the accompanying drawings. However, the present invention is not limited to the embodiments described herein but may be embodied in different forms. Rather, the embodiments disclosed herein are provided so that the disclosure can be thorough and complete, and will fully convey the concept of the invention to those skilled in the art, and the invention is only defined by the scope of the claims. Like reference numerals refer to like elements throughout the specification.

The terminology used herein is for the purpose of illustrating embodiments and is not intended to be limiting of the present invention. In the present specification, the singular form includes plural forms unless otherwise specified in the specification. As used herein, the terms 'comprises' and / or 'comprising' mean that the stated element, step, operation and / or element does not imply the presence of one or more other elements, steps, operations and / Or additions. In addition, since they are in accordance with the preferred embodiment, the reference numerals presented in the order of description are not necessarily limited to the order.

In addition, the embodiments described herein will be described with reference to cross-sectional views and / or plan views, which are ideal illustrations of the present invention. In the drawings, the thicknesses of the films and regions are exaggerated for an effective description of the technical content. Thus, the shape of the illustrations may be modified by manufacturing techniques and / or tolerances. Accordingly, the embodiments of the present invention are not limited to the specific forms shown, but also include changes in the shapes that are generated according to the manufacturing process. For example, the area shown at right angles may be rounded or may have a shape with a certain curvature. Thus, the regions illustrated in the figures have schematic attributes, and the shapes of the regions illustrated in the figures are intended to illustrate specific types of regions of the elements and are not intended to limit the scope of the invention.

1A is a plan view showing a strip unit according to an embodiment of the present invention. 1B is a cross-sectional view taken along line A-B of FIG. 1A.

1A and 1B, a strip portion 10 may include a substrate 100, a transport layer 110, a sample pad 120, a discharge pad 130, and an absorbent pad 140.

The substrate 100 may support the strip portion 10. The substrate 100 may include a substance that reacts with the analyte and the complex of the analyte or does not chemically and physically interact with the analyte. The substrate 100 can include various materials, including, but not limited to, natural or synthetic organic and / or inorganic materials. As an example, the substrate 100 may be made of a material such as polyethylene, polyester, polypropylene, poly (4-methylbutec), polystyrene, polymethacrylate, poly (ethylene terephthalide), and / or nylon, poly Organic materials. As another example, the substrate 100 may comprise inorganic materials such as glass, ceramics, and / or metals. The substrate 100 may include non-aqueous and non-porous materials.

The sample pad 120 may be disposed adjacent to one side 110a of the conductive film 110 on the conductive film 110. [ An analyte (not shown) may be provided in the sample pad 120. The sample pad 120 may function to filter the insoluble particles contained in the analyte. For example, the sample pad 120 may include cellulose filters or glass fiber filters, but may include various materials.

Emissive pads 130 may be provided adjacent to the sample pads 120 on the transport membrane 110. The emissive pad 130 may be spaced apart from one side 110a of the transport membrane 110 than the sample pad 120. [ Emissive pad 130 may be provided with a fluorescent particle-labeled detector adsorbed thereon. The release pad 130 may include, but is not limited to, glass fiber, cellulose, and / or polyester. The release pad 130 may further comprise a stabilizer and / or a blocking agent. For example, the stabilizer may include saccharides such as sucrose and / or trehalose, and the blocking agent may include proteins such as bovine serum albumin, gelatin, casein, and / or skim milk. By the stabilizer, the intensity of the light of the second wavelength emitted from the fluorescence particle and the fluorescent particle-analyte complex to be described later in FIG. 2C can be increased.

A conductive film 110 may be provided on the substrate 100. [ As an example, the preform 110 may comprise natural rubber, such as cellulose, cellulose acetate, and / or cellulose nitrate. As another example, the preform 110 may comprise a variety of materials including, but not limited to, synthetic polymers such as polyacrylonitrile, polyamide, and / or polyether. The pre-exposure 110 may have a detection area T and a verification area C. The detection region T and the verification region C are interposed between the emission pad 130 and the absorption pad 140 and the verification region C can be adjacent to the emission pad 130 more than the detection region T . A plurality of detection regions T may be provided.

The absorption pad 140 may be provided adjacent to the other side 110b of the transport membrane 110 on the transport membrane 110. [ The other side 110b may be opposed to the other side 110a. The absorbing pad 140 may absorb the solvents provided to the sample pad 120 and the emissive pad 130. Accordingly, the analyte and the fluorescent particle-analyte complex can be moved to the detection region T and the control region C along the transition membrane 110 by the capillary force.

Hereinafter, a fluorescence detection method according to an embodiment of the present invention will be described.

2A to 2C are schematic views illustrating a method of detecting an analyte according to an embodiment of the present invention. Hereinafter, duplicated description will be omitted.

Referring to FIG. 2A, an analyte 200 may be provided on the sample pad 120. FIG. As used herein, the term " analyte 200 " may refer to a compound or composition to be analyzed included in the sample. In an embodiment, the analyte 200 may be a single kind or a plurality of kinds. The analyte 200 may include an antigen, an antibody, a nucleic acid, and / or a hapten.

Fluorescent particles P labeled with a detector 210 may be provided in the emission pad 130. The fluorescent particles (P) may comprise a lanthanide complex. For example, the fluorescent particles (P) may comprise a lanthanide element and ligands bonded to the lanthanide element. The lanthanide element may comprise europium (III), terbium (III), samarium (III), dysprosium, and / or combinations thereof. Lanthanide complexes can exhibit stronger fluorescence intensity than gold. According to the present invention, the fluorescent particles (P) include a lanthanide complex, so that the intensity of fluorescence generated from the fluorescent particles (P) can be improved. The fluorescent particles P may have a diameter of about 100 nm to 590 nm. When the fluorescent particles (P) have an average diameter of less than 100 nm, fluorescence may not be detected from the fluorescent particles (P). If the fluorescent particles P have a larger average diameter, it may be difficult for the fluorescent particles P to move along the transporting film 110 from the emitting pad 130.

The detector 210 may be determined according to the analysis object 200. For example, the detectors 210 may include antibodies, antigens, peptides, and / or proteins, but may include various materials.

2B, when the analyte 200 moves from the sample pad 120 to the emission pad 130, the fluorescence particle P contained in the emission pad 130 is coupled to the detector 210 can do. The fluorescent particles P can be combined with the analyte 200 by the detector 210 to form the fluorescent particle-analyte conjugate 250. Various kinds of analytes 200 are provided A plurality of kinds of fluorescence particles (P) labeled detectors 210 may be provided on the sample pad 120. In another example, in the case of various kinds of the analytes 200, Each of the sample pads 120 may include different types of fluorescent particles P and detectors 210 labeled with the fluorescent particles P. The fluorescent particles P can be selectively combined with the analyte 200 by the detectors 210 to form the fluorescent particle-analyte complexes 250. Hereinafter, A method of detecting a single number of analysis objects 200 is described, but the present invention is limited to a single number of analysis objects 200 No. The fluorescent particles (P) coupled to the detector 210 is provided in excess, some of the analyte 200 and the reaction and the other part may be left.

Referring to FIG. 2C, the fluorescence particle-analyte binding body 250 may reach the detection region T of the transflecting film 110. The detection region T may be provided between the emission pad 130 and the absorption pad 140 on the transport membrane 110 and may be a region including the first trapping material 220. The first capturing material 220 may interact with the fluorescence particle-analyte conjugate 250 so that the fluorescence particle-analyte conjugate 250 is fixed to the detection region T. [ The fluorescent particles P forming the fluorescent particle-analyte binding body 250 may not interact with the first capturing material 220. The remaining fluorescent particles P can pass through the detection region T and can move to the verification region C. [ A control region C may be provided between the detection region T and the absorption pad 140 and may include a second trapping material 230. The fluorescent particles P may interact with the second capturing material 230 of the contrast region C and be fixed to the contrast region C. [ The light of the first wavelength incident from the light source (not shown) can be irradiated to the detection area T and the verification area C. The light source unit may be spaced apart from the transport membrane 110 on the transport membrane 110. The fluorescent particle-analyte binding substance 250 on the detection region T and the fluorescent particles P on the control region C can absorb light of the first wavelength and emit light of the second wavelength. It can be confirmed whether or not the fluorescent particles P have moved to the check region C by the presence or absence of emission of the second wavelength in the check region C. [ The rate at which the fluorescent particles P and the fluorescent particle-analyte binding bodies 250 move on the transport membrane 110 may be the same or similar. The wavelength and intensity of the light emitted from the detection region T can be measured and a qualitative / quantitative analysis of the analysis object 200 can be performed. At this time, the qualitative / quantitative analysis of the analysis object 200 can be performed by measuring light emitted from the detection region T when the light of the second wavelength is emitted from the control region C.

According to the embodiment, fluorescence of the check region C and the detection region T (for example, whether or not light of the second wavelength is generated) can be visually observed. Thus, the presence or absence of the analysis object 200 can be easily confirmed visually.

Fluorescence measurement of the fluorescent particle-analyte binding body 250 according to the present invention can be performed by time-division detection. For example, the light source unit may repeat an on mode for providing light to the strip unit 10 and an off mode for not providing light to the strip unit 10. When the light of the second wavelength is measured in the on mode, the fluorescence particle-analyte complex 250 can be measured for autofluorescence generated in an exaggerated region. According to an embodiment, measurement of fluorescence may proceed in an off mode. As the fluorescent particles (P) of the present invention include a lanthanide complex, the fluorescence duration in the off mode may be long. For example, the fluorescent particles (P) may exhibit a fluorescence duration of 10 ms to 1000 ms. Accordingly, the wavelength and intensity of the light emitted by the fluorescent particle-analyte conjugate 250 in the off mode can be measured. In this case, the magnetic fluorescence of the fluorescent particle-analyte conjugate 250 is not detected, and the background signal can be reduced. According to the present invention, the detection sensitivity of the fluorescence particle-analyte binding body 250 can be improved.

Hereinafter, the fluorescence detection system of the present invention will be described.

3 is a schematic diagram showing a fluorescence detection system according to an embodiment of the present invention.

3, the fluorescence detection system 1 includes a strip unit 10, a light source unit 20, a sensing unit 30, a control unit 40, an image display unit 50, an input unit 51, 53).

The light source unit 20 can irradiate the strip unit 10 with light. The light emitted from the light source 20 may have a wavelength of about 200 to 500 nm. For example, when the fluorescent particles provided in the strip portion 10 are europium complexes, the light source portion 20 can irradiate the strip portion 10 with light having a wavelength of 333 nm.

The light source 20 may include a halogen lamp, a mercury lamp, a xenon bulb, an LED, and / or a diode laser. Since the LED has a small volume, the fluorescence detection system 1 using the LED as the light source 20 can be miniaturized. In addition, the fluorescence detection system 1 using the LED as the light source unit 20 can exhibit high energy conversion efficiency. The fluorescence detection system (1) of the present invention can use a time division detection method. The light source unit 20 may repeat an on mode for providing light to the strip unit 10 and an off mode for not providing light to the strip unit 10. [ The LED does not have afterglow in the on mode in the off mode and the fluorescent detection system 1 using the LED as the light source 20 can be adapted to the time division detection method.

The light provided in the light source unit 20 may not be stabilized if the duration of the ON mode of the light source unit 20 is too short (for example, less than 0.5 ms). Further, the fluorescence particle-analyte conjugate can be dispensed and divided to reach the excited state. Accordingly, the intensity of light emitted from the fluorescence particle and the fluorescence particle-analyte conjugate in the off mode can be reduced. According to an embodiment, the duration of the on mode may be approximately 0.5 ms or more.

The fluorescence detection system 1 can use the ray tracing simulation method to improve the detection strength. For example, the fluorescence detection system 1 may have an optical head structure including a condenser lens 21, a beam splitter 23, and an objective lens 25 which are adjacent to each other. With the optical module structure, the fluorescence detection system 1 can be further downsized. A beam splitter 23 may be provided between the light source unit 20 and the strip unit 10. [ For example, the light emitted from the light source unit 20 can be transmitted through the beam splitter 23 and irradiated onto the strip unit 10. The beam splitter 23 may be a diachronic filter. In this case, the beam splitter 23 can reflect light having a wavelength of 365 nm and can transmit light having a wavelength of 610 nm. Light can be uniformly provided to the strip portion 10 by the beam splitter 23. A condenser lens 21 may be further interposed between the beam splitter 23 and the light source 20. [ Light generated in the light source unit 20 can be converged on the beam splitter 23 by the condenser lens 21. The objective lens 25 can be further interposed between the beam splitter 23 and the strip portion 10. [ The numerical aperture of light provided by the light source unit 20 can be increased by the objective lens 25. [ According to the embodiment, the light source unit 20 and the sensing unit 30 are disposed adjacent to each other, so that the fluorescence detection system 1 can be further downsized.

Referring to FIG. 3 together with FIG. 1, the strip portion 10 may be the same as the strip portion 10 described in the example of FIG. The fluorescent particle-analyte complex included in the strip portion 10 can emit light of the second wavelength by receiving the light of the first wavelength in the light source portion 20 as described in FIGS. 2A to 2C. The strip portion 10 may be provided on the stage 11. The stage 11 can be moved so that misalignment of the strip portion 10 can be prevented. The light emitted from the light source 20 can be irradiated to the detection area T and the verification area C of the strip part 10. [

Referring again to FIG. 3, the sensing unit 30 may be disposed adjacent to the strip unit 10. The sensing unit 30 may include a CCD and / or a CMOS. The sensing unit 30 senses the wavelength and intensity of light emitted from the strip unit 10 and converts the sensed wavelength into an electrical signal.

The controller 40 may include a field programmable gate array (FPGA). The control unit 40 may amplify the electrical signal sensed by the sensing unit 30 and process the data. The control unit 40 may provide an electrical signal to the light source unit 20 to control the on and off modes of the light source unit 20. [ At this time, the control unit 40 may process the electrical signal received from the sensing unit 30 and transmit the processed electrical signal to the image display unit 50.

The control unit 40 may perform image processing of a signal received from the sensing unit 30. [ For example, fluorescence detection of a fluorescent particle-analyte can proceed multiple times under the same conditions. The control unit 40 can perform qualitative / quantitative analysis of the analysis object from the fluorescence measurement value that has been performed a plurality of times. Accordingly, the reliability of the qualitative / quantitative analysis of the analyte can be improved. At this time, the control unit 40 can adjust the integration time, frame, and gain of the sensing unit 30. [ The control unit 40 may convert the average value of the fluorescence measurement values that have been processed a plurality of times to a signal and transmit the converted signal to the image display unit 50. Accordingly, the image display unit 50 can display a fluorescent image that is clearer than the image of the captured fluorescence.

In this specification, the integration time may mean the time for opening the shutter of the sensing unit 30 to obtain an image. In the on mode, the longer the exposure time, the greater the intensity and amount of light being measured. According to an embodiment, when the light source unit 20 is in the off mode, the shutter of the sensing unit 30 may be opened. Thus, the intensity and amount of light measured relative to the exposure time can be relatively constant. Accordingly, the detection accuracy of the analyte due to the exposure time may not be affected.

In this specification, a frame may mean the number of cycles in which the fluorescence is repeatedly measured under the same conditions. The ON mode and the OFF mode of the light source unit 20 may be performed once for each cycle. The gain value can be defined as a value that controls an analog-to-digital converter (ADC). When the gain value is increased, the contrast can be improved. Thus, the detection accuracy can be improved.

According to the present invention, the fluorescence analysis system includes a delay time, an image sum, a start mode (beginning time), an exposure time, a frame, and a gain value of the ON mode of the light source unit 20, mode, and the total cut number. For example, the fluorescence measurement can be performed a plurality of times. At this time, the start mode means a result of each fluorescence measurement, and the total number of times of cutting can mean the total number of fluorescence measurements. The image sum may be an average value of the fluorescence measurement results of the start modes. By such optimization, the detection sensitivity of the fluorescent particle-analyte conjugate can be improved. The parameters that are adjusted and optimized in the fluorescence analysis system are not limited to these examples and may vary. For example, depending on the kind of the fluorescent particles and the kind of the analyte, the arrangement of the detection region and the capturing substance contained in the detection region can be adjusted. In addition, the intensity of light provided by the light source unit 20, the detection sensitivity of the sensing unit 30, and the signal amplification rate and peak detection method of the control unit 40 are adjusted to improve the detection sensitivity of the fluorescence analysis system .

The input unit 51 may perform a function of inputting a signal to the control unit 40. In one example, the input unit 51 may include a keyboard. The external device 53 may input a signal to the control unit 40 or may output a signal received from the control unit 40. [ The external device 53 may include a printer and / or a barcode reader.

The image display unit 50 receives data processed by the control unit 40 as a video signal, and can display the analysis values of the fluorescence image. The image display unit 50 may include an LED and / or an LCD.

4A to 4C show images displayed on the image display unit according to the embodiments of the present invention. Hereinafter, Fig. 3 will be described together.

4A, the plane of the strip unit 10 photographed when the light source unit 20 is in the OFF mode may be displayed on the image display unit 50. FIG. 4B and 4C, the fluorescence detection result analyzed by the control unit 40 can be displayed. FIG. 4B is a graph showing fluorescence detection results analyzed by the control unit 40 according to an example. 4B shows an example of the fluorescence detection result analyzed by the control unit 40 in the form of a table. As another example, the image display unit 50 can display two or more images in Figs. 4A to 4C. The image displayed on the image display unit 50 is not limited thereto and may be various.

Hereinafter, the fluorescence analysis system according to the present invention and the fluorescence detection method using the same will be described in more detail with reference to experimental examples and comparative examples.

1. Fabrication of Fluorescence Analysis System and Fluorescence Detection by Fluorescent Particle Type

Experimental Example  1-1 to 1-5

Using a lanthanide complex as the fluorescent particles, a fluorescence analysis system was prepared. In Experimental Examples 1-1 to 1-5, when fluorescence was observed in the control region using the Lanthanon fluorescence analysis system, the intensity of light measured in the detection region was measured. At this time, chlamydia antigen was used as an analyte. Experimental Examples 1-1 to 1-5 are results of using 40 ng, 4 ng, 1 ng, 0.54 ng, and 0.27 ng chlamydia antigens, respectively. Measurement of fluorescence proceeded in the ON mode and the OFF mode of the light source portion, respectively.

Comparative Example  1-1

Using the same fluorescence analysis system as in Experimental Example 1-1, fluorescence was measured by the same method. However, the sample to be analyzed is not provided in the sample pad in Comparative Example 1-1.

Comparative Example  1-2 Comparative Example  1-9

A fluorescence analysis system was fabricated using gold colloidal particles as fluorescent particles. In Comparative Examples 1-2 to 1-9, the analytes of 200 ng, 20 ng, 2 ng, 1 ng, 0.54 ng, 0.27 ng and 0.13 ng, and 0 mg (none) were measured using the Lanthanon fluorescence analysis system to be. At this time, chlamydia antigen was used as an analyte.

FIG. 5A shows the results of the strips of the experimental examples and the comparative example 1-1 when the light source part is in the ON mode. FIG. FIG. 5B shows the results of the strip portions of Comparative Examples 1-2 to 1-9, respectively, when the light source portion is in the ON mode. Figs. 5A and 5B photographs whether fluorescence is generated in the detection region T when fluorescence is observed in the control region C. Fig.

As shown in FIG. 5A, it was confirmed that the fluorescence of the detection region (T) was visually observed in Experimental Examples 1-1 (e11), 1-2 (e12) and 1-3 (e13) have. In Experimental Examples 1-4 (e14) and Experimental Examples 1-5 (e15), the fluorescence of the detection region (T) may be observed to be very low or hard to be visually recognized. 5B, fluorescence of the detection region T in Comparative Examples 1-2 (c12), Comparative Examples 1-3 (c13), Comparative Examples 1-4 (c14), and Comparative Example 1-5 (c15) Of the detection region T in Comparative Examples 1-6 (c16), Comparative Examples 1-7 (c17), Comparative Examples 1-8 (c18), and Comparative Example 1-9 (c19) Was not observed with the naked eye. The fluorescent particles of Experimental Examples e11, e12, e13, e14, and e15 include lanthanide complexes, and therefore, Comparative Examples 1-2 to 1-9 (c12, c12, c13, c14 , c15, c16, c17, c18, c19) can be detected.

Table 1 shows the results of analysis of the fluorescence detection results in the strip portions of the experimental examples and the comparative example 1-1 when the light source part is in the off mode. Hereinafter, Figs. 5A and 5B will be described together.

Experimental Example 1-1 Experimental Example 1-2 Experimental Example 1-3 Experimental Examples 1-4 Experimental Examples 1-5 Comparative Example 1-1 TRF Peak value
(AD) 1201.19 117.28 76.79 53.97 33.14 0

In Table 1, it can be confirmed that fluorescence is emitted from the detection region in each of Experimental Examples 1-1 to 1-5 (e11, e12, e13, e14, and e15). The detection limit of fluorescence in the off mode may be lower than the detection limit of fluorescence in the on mode observed through Figure 5a. The fluorescence detection apparatus of the present invention can measure the fluorescence in the ON mode, and can easily check the presence or absence of the analyte in the naked eye when the analyte is sufficient. If the analyte is small, fluorescence can be measured in off mode. Accordingly, it is possible to detect a small amount of the analyte.

2. Fabrication of Fluorescence Detection System by Fluorescent Particle Size and Fluorescence Detection

Experimental Example  2-1 to Experimental Example  2-3

A fluorescence detection system was provided in which europium complex particles were provided in the emissive pad. Experimental Examples 2-1 to 2-3 are fluorescence detection results measured using a fluorescence detection system in which europium complex particles having average diameters of 112 nm, 194 nm and 359 nm were provided in the emission pad. Silver (Ag) was used as the analyte.

Comparative Example  2-1 and 2-2

Fluorescence of the detection region and the control region was measured in the same manner as in Experimental Example 2-1. However, in the fluorescence measurement of Comparative Example 2-1, europium complex particles having an average diameter of 74.4 nm were provided to the emission pad. In the fluorescence measurement of Comparative Example 2-2, europium complex particles having an average diameter of 598 nm were provided to the emissive pad.

FIG. 6A is a photograph showing fluorescence emitted from the strips of Experimental Examples 2-1 to 2-3, respectively. FIG. 6B shows the fluorescence emitted from the strips of Comparative Example 2-1 and Comparative Example 2-2. 6A and 6B photographs whether or not fluorescence is generated in the detection region T when the light source portion is in the ON mode and fluorescence is observed in the control region C. Comparative Example 1-2 (e12) in FIG. 6B is a result of photographing the strip portion from which the outer case is removed.

Referring to FIG. 6A, it can be seen that the fluorescence is well emitted in the detection region when the fluorescence is emitted from the control region. Thus, the presence or absence of the analyte (Ag) can be visually confirmed easily. As shown in Fig. 6B, in Comparative Example 1-1, fluorescence was not observed in the entire region of the transcription film, as well as in the control region and the detection region, regardless of the presence or absence of the analyte. From the above, it can be confirmed that when fluorescent particles smaller than 100 nm are irradiated with light of the first wavelength in the light source portion, the fluorescent particles do not emit light of the second wavelength observable by the naked eye. In Comparative Example 1-2, fluorescence appears at a position adjacent to one side of the opening film. For example, in Comparative Example 1-2, fluorescence is observed in the emissive pad provided with fluorescent particles. When the fluorescent particles have an average diameter of about 590 nm, the fluorescent particles and the fluorescent particle-analyte conjugate may be difficult to move along the developing film due to the size of the fluorescent particles. In this case, the fluorescent particles may be difficult to use in a fluorescence detection system. According to the present invention, a fluorescence detection system and a fluorescence detection method using the same can use fluorescent particles having an average diameter of 100 nm to 590 nm.

3. Fluorescence detection by time division detection method

Experimental Example  3

An on mode in which the light source irradiates light onto the substrate and an off mode in which the light is not irradiated proceed. Detection of fluorescence was performed in the off mode of the light source part. Fluorescence was measured in the detection zone.

Comparative Example  3

Fluorescence was measured under the same conditions as in Experimental Example 3. However, fluorescence was measured when the light source part was in the ON mode.

7A and 7B are graphs showing fluorescence measurement results of Experimental Example 3 and Comparative Example 3, respectively. At this time, time-share analysis was used. 7A and 7B, the abscissa represents the width of the strip portion, the ordinate represents the intensity of fluorescence, and the portion indicated by the arrow indicates the detection region. In FIGS. 7A and 7B, the horizontal axis and the vertical axis are relative values converted by time-division analysis.

Experimental Example 3 shown in FIG. 7A shows a lower fluorescence peak in a region other than the detection region than that of Comparative Example 3 shown in FIG. 7B. The fluorescence analysis system of the present invention can use a time division detection method for measuring fluorescence in off mode. Accordingly, the fluorescence is measured under a condition in which the interference phenomenon of the light irradiated from the light source is reduced, so that, in the fluorescence measurement, the background noise can be reduced. For example, fluorescence can be measured after the scattered light in the transflective region other than the detection region and the reference region is extinguished. According to the fluorescence detection method of the present invention, detection accuracy and sensitivity are improved, and a small amount of analyte can be detected.

4. Light source  On mode  Fluorescence detection by time interval

Experimental Example  4-1 to Experimental Example  4-4

The on mode and the off mode of the light source unit are performed. The duration of the on mode was adjusted from 0.1 ms to 100 ms, and accordingly the light of 625 nm wavelength emitted from the strip portion in the off mode was measured. In Experimental Example 4-1, an analyte prepared by dissolving 0.1 ng of chlamydia antigen in 250 μL of a solvent is provided on a sample pad. Fluorescence was measured in the same manner as in Experimental Example 4-1, except that 1 ng, 10 ng, and 100 ng of Chlamydia antigen were used in Experimental Examples 4-2 to 4-4, respectively.

Comparative Example  4-1 and Comparative Example  4-2

In the comparative example, light of 625 nm emitted from the strip portion was measured in the same manner as in Experimental Example 1-1. However, in Comparative Example 4-1, chlamydia antigen was not provided in the sample pad. In the case of Comparative Example 4-2, 0.01 ng of chlamydia antigen is provided on the sample pad.

FIG. 8 is a graph showing fluorescence intensities with time in Experimental Examples 4-1 to 4-4, Comparative Examples 4-1 and 4-2. FIG. In FIG. 8, the vertical axis represents the intensity of fluorescence and is a relative value converted by time-division analysis.

8, in the case of Experimental Example 4-1 (e41), Experimental Example 4-2 (e42), Experimental Example 4-3 (e43), and Experimental Example 4-4 (e44) If the duration is less than 0.5 ms, it can be observed that the fluorescence signal is not measured. If the time interval of the on / off mode of the light source unit is less than 2 ms, the intensity of fluorescence increases according to the time interval of the on / off mode of the light source unit. The fluorescence of Experimental Examples 4-1 to 4-4 (e41, e42, e43, e44) is relatively constant when the time interval of the light source portion in the on / off mode is 2 ms or more. If the duration of the on mode of the light source is too short (e.g., less than 0.5 ms), the light generated in the light source may be less than the amount of light needed to emit fluorescence from the fluorescent particles. Accordingly, fluorescence may not be measured. According to the present invention, the ON mode of the light source portion can be maintained at 0.5 nm or more, and more preferably 2 nm or more. The on-mode time interval can be adjusted depending on the kind of fluorescent particle-analyte combination.

5. Fluorescence detection result by image processing

Experimental Example  5

An on mode in which the light source irradiates light onto the substrate and an off mode in which the light is not irradiated proceed. Detection of fluorescence was performed a plurality of times in the off mode of the light source part. Thereafter, the control unit performs image processing for processing / analyzing a plurality of images obtained from the measuring unit.

Comparative Example  5

Fluorescence was measured in the same manner as in Experimental Example 5. However, the image processing of the control unit has been omitted.

9 shows photographs and analyzed images of strip portions according to Experimental Example 5 and Comparative Example 5. Fig.

Referring to FIG. 9, it can be confirmed that the fluorescence image of Test Example 5 (e5) is clearer than that of Comparative Example 5 (c5). The control unit performs image processing to adjust the gain value, image sum, and frames so that the sensitivity of the measured fluorescence detection system can be improved.

6. Fluorescence detection according to the concentration of fluorescent particles

Experimental Examples  6-1 to 6-6

In Experimental Example 6-1, fluorescence was measured using europium complexes having a concentration of 0.75 μg / mL as fluorescent particles. An on mode in which the light source irradiates light onto the substrate and an off mode in which the light is not irradiated proceed. Measurement of fluorescence was performed in an on mode and an off mode of the light source, respectively. Experimental Examples 6-2 to 6-6 were carried out in the same manner as in Experimental Example 6 except that europium complexes having concentrations of 1.5 μg / mL, 3 μg / mL, 6 μg / mL, 25 μg / mL and 100 μg / Fluorescence was measured in the same manner as in Example 6-1.

FIG. 10A is a photograph of the fluorescence emitted from the strips of Experimental Examples 6-1 to 6-6 when the light source portion is in the ON mode. FIG. FIG. 10B shows the image display unit in which the fluorescence detection results of Experimental Examples 6-1 to 6-6 are displayed when the light source unit is in the off mode. The fluorescence measurement in Fig. 10B was carried out using a time-division detection method.

(E63), Experimental Example 6-4 (e64), Experimental Example 6-5 (e65), and Experimental Example 6-6 (e66) are performed when the light source portion is in the ON mode Fluorescence. In Examples 6-1 (e61) and 6-2 (e62), almost no fluorescence was observed. 10B, when the light source unit is in the OFF mode, the light intensity of the light emitted from each of the light emitting units of Experimental Example 6-1 (e61), Experimental Example 6-2 (e62), Experimental Example 6-3 (e63), Experimental Example 6-4 (e64) Fluorescence was observed in Examples 6-5 (e65) and 6-6 (e66). Particularly, Experimental Examples 6-1 (e61) and 6-2 (e62) show that the detection limit of fluorescence is improved when the light source is in the off mode. Experimental Examples 6-1 to 6-6 (e61 , e62, e63, e64, e65, and e66), fluorescence intensities increase as the concentration of the fluorescent particles increases. The concentration of the fluorescent particles can be controlled, and the detection limit and sensitivity of the fluorescence detection system can be improved.

Table 2 shows the result of analyzing the fluorescence image photographed in Fig. 11 using the time-divisional detection method. Hereinafter, FIGS. 10A and 10B will be described together.

Experimental Example 6-1 Experimental Example 6-2 Experimental Example 6-3 Experimental Example 6-4 Experimental Example 6-5 Experimental Example 6-6 TRF Peak value (A.D.) 84 217 502 845 2855 6663

In Table 2, it can be confirmed that fluorescence is emitted from each of Experimental Examples 6-1 to 6-6. As described above, the detection limit of fluorescence in the off mode may be lower than the detection limit of fluorescence in the ON mode observed through FIG. 10A. Further, by using the time-division detection method, the presence or absence of the fluorescent particles or the fluorescent particle-analyte binding substance can be more easily discriminated as the peak value is displayed numerically. Accordingly, it is possible to detect a small amount of the analyte.

While the present invention has been described in connection with what is presently considered to be practical exemplary embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, It will be understood. It is therefore to be understood that the above-described embodiments are illustrative and non-restrictive in every respect.

Claims (5)

Providing a substrate comprising a preform, a sample pad, and an emissive pad, the sample pad comprising an analyte, the emissive pad comprising fluorescent particles;
Irradiating light of a first wavelength onto the substrate from a light source part spaced from the substrate;
Measuring light of a second wavelength emitted from the fluorescent particles and the fluorescent particle-analyte combination on the substrate, respectively;
Converting the measured intensity of light of the second wavelength into a signal; And
And displaying the image by receiving the signal from the image display unit spaced apart from the substrate,
Wherein the release pad further comprises a stabilizer or a blocking agent,
The fluorescent particles include a lanthanide complex and have a diameter of 100 nm to 590 nm,
Wherein the fluorescence particle-analyte conjugate is formed by binding the fluorescence particle contained in the release pad with the analyte moved to the release pad.
The method according to claim 1,
And measuring light of the second wavelength is performed by a time division detection method.
The method according to claim 1,
Wherein measuring the light of the second wavelength is performed in an off mode of the light source unit after the light of the first wavelength is irradiated onto the substrate.
The method according to claim 1,
The light source unit includes a fluorescent detection method
The method according to claim 1,
Measuring the light of the second wavelength is performed a plurality of times,
Wherein the step of converting the measured intensity of light of the second wavelength into a signal includes converting an average value of the intensity of light of the second wavelength measured to the signal.

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