High sensitivity biosensor using pixel analysis of CMOS image sensor

Abstract

Provided are a method of analyzing a target substance to be measured, the method including: dividing a surface of a measuring unit of a CMOS image sensor into a plurality of pixels, directly fixing a bioreceptor onto the surface of the measuring unit of the CMOS image sensor, and measuring chemiluminescent signals depending on concentrations of the target substance to be measured, and a CMOS image sensor applied to the same.

Claims

1. A method of measuring a concentration of a target substance to be measured by using a CMOS image sensor, the method comprising: dividing a surface of a measuring unit of the CMOS image sensor into a plurality of pixels; fixing a bioreceptor onto the surface of the measuring unit of the CMOS image sensor; analyzing a chemiluminescent signal at each pixel; determining the number of pixels that receive the chemiluminescent signal equal to or stronger than a predetermined cut-off value; and determine the concentration of the target substance based on the determined number of pixels that receive the chemiluminescent signal equal to or stronger than the predetermined cut-off value.

2. The method of claim 1, wherein analyzing the chemiluminescent signal at each pixel comprises determining a difference in chemiluminescent signal strength before and after a chemiluminescent reaction.

3. The method of claim 1, wherein the pixel has a size of 0.01 to 100 μm.sup.2.

4. The method of claim 1, wherein the target substance to be measured is at least one kind selected from the group consisting of enzymes, proteins, DNAs, RNAs, microorganisms, animal and plant cells and organs.

5. The method of claim 1, wherein the bioreceptor is at least one kind selected from the group consisting of antibodies, DNAs, enzymes, aptamers, peptide nucleic acids (PNAs), and ligands.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a schematic diagram of a CMOS image sensor according to the present invention.

(2) FIG. 2 shows a process measuring a chemiluminescent signal by fixing streptavidin-horseradish peroxidase (STA-HRP) in Example 1.

(3) FIG. 3 shows a sensing result obtained by confirming pixels indicating chemilumuminescent signal(s) having a cut-off value or more in Example 1.

(4) FIG. 4 is a graph showing a result obtained by comparing accuracy between an analysis method of Example 1 and a method of measuring an average intensity of pixels depending on concentrations of streptavidin-horseradish peroxidase (STA-HRP).

(5) FIG. 5 shows a process of fixing IL-5 in Example 2 and measuring a chemiluminescent signal.

(6) FIG. 6 shows a sensing result obtained by three-dimensionally confirming pixels indicating chemiluminescent signals having a cut-off value or more in Example 1.

(7) TABLE-US-00001 [Detailed Description of Main Elements] 10: CMOS Image Sensor 11: Measuring Unit 12: Connecting Unit 21: STA-HRP 22: IL-5 Capture Antibody 23: IL-5 Antigen 24: IL-5 Detection Antibody 25: HRP

BEST MODE

(8) The present invention is characterized by a method of measuring a concentration of a target substance to be measured by using a CMOS image sensor, including dividing a surface of a measuring unit of a CMOS image sensor into a plurality of pixels, fixing a bioreceptor onto the surface of the measuring unit of the CMOS image sensor, and analyzing a chemiluminescent signal at each pixel.

(9) In addition, the present invention is characterized by a CMOS image sensor including a measuring unit having a bioreceptor fixed onto a surface divided into a plurality of pixels.

(10) FIG. 1 is a schematic diagram of a strip CMOS image sensor according to an exemplary embodiment of the present invention. As shown in FIG. 1, the CMOS image sensor 10 includes the measuring unit 11 and a sensor connecting unit 12. The measuring unit 11 serves to sense a chemiluminescent signal, and the sensor connecting unit 12 serves to connect a measuring instrument with a sensor.

(11) The surface of the measuring unit is divided into a plurality of pixels, wherein the pixel preferably has a size of 0.01 to 100 μm.sup.2, and more preferably, a size of 0.1 to 50 μm.sup.2, but the number and the size of pixels may be appropriately adjusted depending on substances to be measured.

(12) In addition, a reactor may be introduced onto the surface of the measuring unit by various methods, and various kinds of bioreceptors may be fixed onto the surface of the sensor into which the reactor is introduced to thereby be used as a biochip. As the bioreceptor, at least one kind selected from the group consisting of antibodies, DNAs, enzymes, aptamers, peptide nucleic acids (PNAs), and ligands may be used.

(13) Meanwhile, in the present invention, a target substance to be measured may be at least one kind selected from the group consisting of enzymes, proteins, DNAs, RNAs, microorganisms, animal and plant cells and organs, wherein the enzymes may be peroxidase, phosphatase, or luciferase.

(14) A chemiluminescent reaction is used in order to analyze a signal of the target substance reacting with the bioreceptor fixed onto the surface of the measuring unit. In order to induce the chemiluminescent reaction, the chemiluminescent substance may be preferably luminol, isoluminol, luciferin, lucigenin, 3-(2′-Spiroadamantane)-4-methoxy-4-(3″-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD), disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2′-(5′-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate (CSPD), and the like, but the present invention is not limited thereto. The chemiluminescent substance may be appropriately selected by a person skilled in the art.

(15) The concentration of the target substance to be measured is determined by analyzing the chemiluminescent signal at each pixel on the surface of the measuring unit.

(16) Here, 1) the concentration of the target substance to be measured may be determined by selecting a cut-off value of the chemiluminescent signal, and measuring the number of pixels indicating signals having the cut-off value or more.

(17) In addition, 2) the concentration of the target substance to be measured may be determined by selecting a cut-off value of the chemiluminescent signal, and measuring the number of pixels showing signals in which a difference in signal intensity between before and after performing a chemiluminescent reaction has the cut-off value or more.

(18) The cut-off value may be appropriately adjusted and selected depending on the substance to be measured, measurement items or measurement methods.

(19) As compared to general methods of measuring chemiluminescent intensity, the measurement method using the CMOS image sensor of the present invention may maximize the detection sensitivity, and a distance between the chemiluminescent signal of the target substance reacted with the fixed bioreceptor and the sensor is about several nm, such that the sensor is advantageous to induce the chemiluminescent signal.

(20) In particular, the measurement method using the CMOS image sensor is characterized by analyzing the number of pixels indicating signals having the cut-off value or more at each pixel. For example, in the case in which a difference in signal intensity between before and after performing the chemiluminescent reaction is shown in 1 to 2 pixels among 1000 pixels, when converting the difference into an average light intensity of 1000 pixels, the converted value is not meaningless; however, when analyzing the number of each pixel having difference in signal, the number is meaningful.

(21) The above described aspects and additional aspects of the present invention will be more clearly described by preferred exemplary embodiments with reference to the accompanying drawings. Hereinafter, the present invention will be described in detail by these exemplary embodiments so that a person of ordinary skilled in the art can easily understand and realize the present invention.

(22) However, the following examples are provided only for exemplifying the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not construed to be limited to these examples.

Example 1 Measurement of Streptavidin-Horseradish Peroxidase (STA-HRP)

(23) 46000 pixels having each width of about 4 μm and each length of about 4 μm, were formed on the surface of the measuring unit 11 of the CMOS image sensor of FIG. 1. A solution (concentration of 0, 1, 100, 10000 fg/ml) obtained by dissolving streptavidin-horseradish peroxidase (STA-HRP) manufactured by Calbiochem, USA in 10 mM PB buffer (pH 7.0) was reacted onto the surface of the measuring unit modified by a biotin group at room temperature for 30 minutes, to fix enzymes onto the surface by an adsorption method. Then, the surface was washed with 10 mM PB buffer, distilled water, and 0.1M carbonate buffer (pH 9.0) in sequence.

(24) Next, the measuring unit was treated with 2 mM luminol dissolved in 0.1M carbonate buffer (pH 9.0) and 2 mM hydrogen peroxide solution.

(25) The process of forming the chemiluminescent signal according to the treatment of the luminol and the hydrogen peroxide solution was shown in FIG. 2.

(26) After 1 minute elapsed after the treatment, the chemiluminescent signal was measured in a pixel unit, and the cut-off value per each pixel was selected as 10 digits. That is, the number of pixels indicating the chemiluminescent signals having 10 digits or more was measured, and the pixels indicating the chemiluminescent signal having 10 digits or more determined by the measurement method represented as a black color and were shown in FIG. 3.

(27) When the STA-HRP was treated at a concentration of 100 fg/ml, the signal having 10 digits or more was shown in 3,419 pixels, and when the STA-HRP was treated at a concentration of 10,000 fg/ml (10 pg/ml), the signal having 10 digits or more was shown in 19,248 pixels.

(28) In addition, a graph showing a comparison result of average digit values in a measurement region depending on concentrations of streptavidin-horseradish peroxidase (STA-HRP) was shown in FIG. 4.

(29) As shown in the graph of FIG. 4, it could be confirmed that the concentration of the STA-HRP could be measured at high sensitivity by the method of the present invention.

Example 2 Measurement of IL-5

(30) The measuring unit 11 of the CMOS image sensor of FIG. 1 was ultrasonic-washed in 0.1N NaOH solution for 15 minutes, then washed with ethanol three times, and APTMS ((3-Aminopropyl)trimethoxysilane) having 1% concentration dissolved in ethanol was reacted for about 2 hours. Then, the reaction product was washed with ethanol about two times, then ultrasonic washed in ethanol for five minutes again, and dried with high purity nitrogen gas. In addition, in order to prepare a surface reacting with an antibody, 2 vol % of glutaraldehyde dissolved in 10 mM PB buffer was reacted for about 4 hours to finally modify the surface of the measuring unit with an aldehyde group.

(31) Then, a 0.1 mg/ml IL-5 capture antibody was reacted onto the modified surface for about 1 hour, followed by reaction with 1% BSA solution for about 2 hours in order to remove non-specific reaction. In addition, IL-5 antigen dissolved in PBS buffer and biotinylated 5 μg/ml IL-5 detection antibody were mixed and reacted for 10 minutes, then reacted on the surface of the measuring unit for 30 minutes to allow an antigen-antibody reaction to proceed, and the surface was washed with PBS buffer and diluted water in sequence. Then, STA-HRP 5 μg/ml dissolved in the PBS buffer was added thereto, followed by reaction for 30 minutes, and washed with PBS buffer, 0.1 M carbonate buffer (pH 9.0) in sequence.

(32) Next, the surface of the measuring unit was treated with 2 mM luminol dissolved in 0.1M carbonate buffer (pH 9.0) and 2 mM hydrogen peroxide solution.

(33) The process of forming the chemiluminescent signal depending on treatment of the luminol and the hydrogen peroxide solution was shown in FIG. 5.

(34) After 1 minute elapsed after the treatment, the difference in the chemiluminescent signal between before and after treating the luminol and the hydrogen peroxide solution was measured. The cut-off value per pixel was selected as 2 digits, and the number of pixels in which a difference in signal intensity between before and after performing the chemiluminescent reaction had the cut-off value or more was measured. FIG. 6 is a three-dimensional image of pixels having the cut-off value or more, an increase in signal having 2 digits or more was shown in 3 pixels when the concentration of the IL-5 antigen is 0, in 11 pixels when the concentration of the IL-5 antigen is 100 fg/ml, and in 4069 pixels when the concentration thereof is 10,000 fg/ml (10 pg/ml).

(35) Therefore, it could be confirmed that quantitative analysis of IL-5 could be conducted by the method of the present invention.

(36) Although specific embodiments of the present invention are described in detail as described above, it will be apparent to those skilled in the art that the specific description is merely desirable exemplary embodiment and should not be construed as limiting the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the accompanying claims and equivalent thereof.