KR20100091843A - Integrated bio-chip and method of fabricating the integrated bio-chip - Google Patents

Abstract

PURPOSE: An integrated bio-chip and a method for manufacturing the same are provided to reflect fluorescence to a sample detection unit by a window and to improve light use efficiency. CONSTITUTION: An integrated bio-chip comprises: a sample detection unit(200) having a light receiving device for detecting fluorescence; a light transfer unit(100) which is prepared on the upper side of the sample detection unit and transfers fluorescence to the sample detection unit; a sample reaction unit(140) comprising a reaction regions; and a window having a fluorescence reflection film which reflects fluorescence emitted from the sample.

Description

Integrated bio-chip and method of fabricating the integrated bio-chip

The present invention relates to an integrated biochip and a method for manufacturing the same, and more particularly, to an integrated biochip and a method for manufacturing the same used for detecting a sample by a spectroscopic method.

Bio-chips generally have a structure in which very small cells of micro units are arranged in a matrix form on a substrate, and such cells have a bio-material such as nucleic acid or protein. The probe bio-material on the biochip's substrate functions as a biological receptor for the target biomaterial.

Biochips detect target organisms by using interactions between such organisms, such as hybridization of nucleic acids or antigen-antibody reactions. Such a biochip can be used as a tool for analyzing gene function, searching for disease-related genes, gene expression, protein distribution, and the like by detecting biomaterials such as nucleic acids or proteins having a specific base sequence.

Detection of interactions between biomass mainly uses fluorescence detection methods. This fluorescence detection method is a spectroscopic method of detecting a fluorescence image obtained by irradiating predetermined excitation light onto a fluorescent substance labeled on a biomaterial. The detection of the fluorescence image is made through an optical detection device such as a charge coupled device (CCD) scanner or a CMOS image sensor (CIS) scanner.

Since the fluorescence obtained by irradiating predetermined excitation light to the fluorescent substance labeled on the biomaterial is very weak light compared to the excitation light to be irradiated, it is necessary to remove the excitation light. Furthermore, since fluorescence itself is very weak light, it is necessary to detect it effectively. On the other hand, the current detection device using a commercially available biochip uses a complex and expensive scanner equipment as a photodetector, a more compact biochip and its manufacturing method is required.

Embodiments of the present invention provide an integrated biochip and a method for manufacturing the same, which can react a sample such as a biomaterial in an integrated chip and optically detect the sample.

Integrated biochip according to an embodiment of the present invention comprises a sample detection unit provided with at least one light receiving element for detecting fluorescence; A phototransmitter provided on an upper surface of the sample detector to transmit fluorescence emitted from the sample to the sample detector; A sample reaction part provided on an upper surface of the light transmitting part and including at least one reaction area to which a sample may be attached; And a window disposed to be spaced apart from the sample reaction part and having a fluorescence reflecting film reflecting fluorescence emitted from the sample.

The fluorescent reflection film may be a dichroic mirror having a wavelength band of fluorescence as a reflection band or a band cut filter having a wavelength band of fluorescence as a blocking band. In addition, such a fluorescent reflection film can be the pass band of the wavelength band of the excitation light.

An excitation light reflection prevention film for preventing reflection of the excitation light may be provided on the upper surface of the window.

The window focuses fluorescence emitted from the sample so that the reflector may comprise at least one micromirror. The micromirror may be provided in a concave shape on the lower surface facing the sample reaction part of the window.

The micromirror may correspond one-to-one or one-to-many with the reaction zone of the sample reaction part.

An alignment mark for allowing the micromirror to match the reaction region of the sample reaction unit may be provided in at least one of the window and the sample reaction unit.

In addition, a microlens for condensing fluorescence may be provided on the optical path between the at least one reaction region and the light receiving element.

The window may include a lead portion protruding downward to be spaced apart from the sample reaction portion. The lead-out portion may be formed at at least a portion of the outer periphery of the window. Alternatively, the sample reaction part and the window may be accommodated spaced apart from each other in the frame.

The light transmitting unit may include at least one excitation light absorption waveguide provided under the at least one reaction region to absorb excitation light for exciting the sample and transmit fluorescence emitted from the sample.

The material surrounding the excitation light absorption waveguide of the light transmitting unit may be a black material that absorbs the excitation light and the fluorescence.

The material surrounding the excitation light absorption waveguide of the light transmitting unit may have a refractive index lower than that of the excitation light absorption waveguide.

The light transmitting unit may include a monochromatic color filter provided under the sample reaction unit to absorb excitation light to excite the sample and transmit fluorescence emitted from the sample.

The sample detection unit may be a charge coupled device (CCD) or a CMOS image sensor (CIS).

In accordance with another aspect of the present invention, there is provided a method of manufacturing an integrated biochip, the method comprising: providing a sample detection unit provided with at least one light receiving element for detecting fluorescence; Forming a light transmission unit configured to transfer fluorescence emitted from the sample to an upper surface of the sample detection unit; Forming a sample reaction unit including at least one reaction region to which a sample is attached to an upper surface of the light transmitting unit; Providing a window having a fluorescent reflecting film; And disposing a window spaced apart from the sample reaction unit.

The preparing of the window may include concentrating fluorescence emitted from the sample on the window to provide at least one micromirror with the reflector.

In the step of preparing the window, an excitation light reflection prevention film for preventing reflection of the excitation light may be provided on the rear surface of the surface on which the micromirror of the window is provided.

The preparing of the window may further include preparing an alignment mark for alignment with the sample reaction part in the window.

The forming of the light transmitting unit may include applying an excitation light absorbing material on the upper surface of the sample detector to absorb excitation light and absorb fluorescence; Forming an excitation light absorption waveguide by forming a trench in the excitation light absorption material applied to expose the upper portion of the sample detection unit in the remaining region except for the region where the light receiving element is located; And filling the trench with a dielectric material having a refractive index smaller than that of the excitation light absorbing material, thereby forming a light blocking portion.

Alternatively, the forming of the light transmitting unit may include applying an excitation light absorbing material to absorb the excitation light and to absorb the fluorescence on the upper surface of the sample detection unit; Forming an excitation light absorption waveguide by forming a trench in the excitation light absorption material applied to expose the upper portion of the sample detection unit in the remaining region except for the region where the light receiving element is located; And filling the trench with a black material that absorbs both excitation light and fluorescence, thereby forming a light blocking portion.

The method may further include forming a microlens on an upper side of a region where the light receiving element is located. In the case of forming the microlenses, the phototransmitter may be formed using only an excitation light absorbing material layer without forming a structure such as a trench, a light absorbing material, a light blocking part, and a waveguide.

The method may further include forming a fluorescent anti-reflection film on the microlens.

According to embodiments of the present invention, by integrating a portion, such as a biomaterial, to which a sample is attached and a portion that detects fluorescence emitted from the sample into a single chip, a biochip and a biomaterial detection apparatus using the same can be made more compact. have.

In addition, since the fluorescence directed upward among the fluorescence emitted from the sample is reflected toward the sample detection unit by the window, the light utilization efficiency can be improved.

Further, by effectively transmitting only the fluorescence emitted from the sample to the sample detection unit can improve the sample detection performance.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the examples exemplified below are not intended to limit the scope of the present invention, but are provided to fully explain the present invention to those skilled in the art. In the drawings, like reference numerals refer to like elements, and the size of each element in the drawings may be exaggerated for clarity and convenience of description.

1 schematically illustrates an integrated biochip in accordance with an embodiment of the present invention.

Referring to FIG. 1, the integrated biochip of this embodiment includes a sample reaction detection unit and a window 300. The sample reaction detection unit includes a sample reaction unit 140 to which a sample is attached, a sample detection unit 200 to detect fluorescence emitted from the sample, and light located between the sample reaction unit 140 and the sample detection unit 200. It has a delivery unit (100). Here, the expression "integrated" means that the sample reaction unit 140, the light transmitting unit 100, and the sample detection unit 200 constituting the sample reaction detection unit are integrated into an integrated chip.

The sample 190 attached to the integrated biochip of this embodiment is a sample that can be detected by a fluorescence detection method, and may be, for example, a biomaterial such as a fluorescently labeled nucleic acid. The biomaterial attached to the reaction region 140a of the sample reaction unit 140 may be a probe biomaterial or a target biomass combined with the biomaterial for detection. Detection biomaterials are molecules that can interact with the target biomaterial, such as a hybridization reaction of a nucleic acid or an antigen-antibody reaction, for example, a nucleic acid molecule having a base sequence complementary to the nucleic acid molecule to be detected. have. On the other hand, the target biological material may be biological organisms such as enzymes, proteins, antibodies, nucleic acids, microorganisms, animal and plant cells and organs, nerve cells of the organism. For example, in the case of a DNA chip, a plurality of probe nucleic acids are attached to a predetermined reaction region 140a of the sample reaction unit 140 in the form of a single spiral as a detection biomaterial in the manufacturing step. When a target nucleic acid (eg, mRNA) having a base sequence complementary to those of these probe nucleic acids is hybridized with the probe nucleic acid, the fluorescence is emitted by the labeled fluorescent substance. Since the positions of the probe nucleic acids are predetermined, it is possible to determine the presence or absence of a plurality of target nucleic acids simultaneously through the detected two-dimensional fluorescence image.

The window 300 is a capping member that protects the sample reaction unit 140 to which the sample to be detected is attached, and is spaced apart from the sample reaction unit 140 by a predetermined distance. The window 300 includes a transparent body 310, a fluorescent reflection film 320 provided on a lower surface of the transparent body 310, and an excitation light reflection prevention film 330 provided on an upper surface of the transparent body 310. The transparent body 310 of the window 300 is formed of a material transparent to the excitation light.

The fluorescent reflection film 320 passes excitation light and reflects fluorescence, and may be formed by being coated on the lower surface of the transparent body 310. In general, since the wavelength of the fluorescence is longer than the wavelength of the excitation light that excites fluorescence, by properly designing the fluorescent reflection film 320, the light in the fluorescence wavelength band can be reflected and the light in the excitation light wavelength band can be transmitted. As the fluorescent reflection film 320, a dichroic mirror having a wavelength band of fluorescence or a band stop filter having a wavelength band of fluorescence may be employed. As the band cut filter, for example, a notch filter capable of reflecting only a wavelength band of fluorescence may be used.

The fluorescent reflection film 320 reflects the fluorescence directed upward among the fluorescence emitted from the sample 190 of the sample reaction unit 140 to be used for the detection of the sample detection unit 200, thereby greatly improving the detection efficiency of the sample. Can be. In addition, since the band blocking characteristic of the fluorescent reflection film 320 may be effective in both directions, when white light is used as excitation light, noise may be reduced by removing a wavelength band of fluorescence contained in white light.

At least one micromirror 325 may be provided on the bottom surface of the transparent body 310. As shown in FIG. 1, the micromirror 325 may include a fluorescent reflection film 320 coated on a concave surface 310a formed in a hemispherical shape concave into the transparent body 310. Since the fluorescent reflector 320, that is, the micromirror 325 coated on the concave surface 310a of the transparent body 310 has a curved surface according to the shape of the concave surface 310a, it has a focusing force on the reflected fluorescence. do. The micromirror 325 may correspond to one-to-one or one-to-many with the reaction region 140a of the sample reaction unit 140. The upward fluorescence may be effectively focused on the reaction region 140a of the sample reaction unit 140 by the micromirror 325. The shape of the micromirror 325 is not limited to the concave hemispherical shape shown in FIG. The shape of the cross section of the micromirror 325 may vary depending on the shape of the reaction region 140a of the sample reaction unit 140 to be described later. For example, the micromirror 325 may have a circular or polygonal shape. The curved surface of the micromirror 325 may vary depending on the distance between the bottom surface of the window 310 and the reaction region 140a of the sample reaction unit 140.

An excitation light reflection prevention layer 330 may be provided on the upper surface of the window 300 to prevent reflection of the excitation light. Furthermore, the excitation light reflection prevention layer 330 may be designed to have a blocking characteristic with respect to the wavelength band of fluorescence.

The sample reaction unit 140 includes a plurality of reaction regions 140a to which the sample 190 may be attached. The plurality of reaction regions 140a may be provided on the upper surface of the light transmitting part 100 and may correspond to the excitation light absorption waveguide 120 in one-to-one correspondence. The plurality of reaction zones 140a may be spaced apart from each other, and may have, for example, a diameter of sub μm to several μm and may be arranged in two dimensions such as a matrix. The reaction region 140a may be formed in a circular shape or a polygonal shape such as a quadrangle. In the integrated biochip of the present embodiment, a case where a plurality of reaction regions 140a are provided is described as an example. However, in some cases, only one reaction region 140a may be provided. For example, when the integrated biochip of this embodiment is used as a DNA chip, each of the reaction regions 140a is a minimum unit of a region to which a plurality of homologous nucleic acids are attached, and excitation light is used to detect a target nucleic acid. Is the minimum pixel of the fluorescence image obtained when illuminating the DNA chip.

The light transmitting unit 100 transmits the fluorescence emitted from the sample 190 to the sample detecting unit 200 located below, and includes a plurality of excitation light absorption waveguides surrounded by the light blocking unit 110 and the light blocking unit 110. 120.

The light blocking unit 110 surrounds each of the excitation light absorption waveguides 120 and may be formed of a black material that absorbs both the excitation light for exciting the sample 190 and the fluorescence emitted from the sample 190.

The excitation light absorption waveguide 120 guides the fluorescence emitted from the sample 190 to the sample detection unit 200 and is an optical path that absorbs the excitation light. In general, since the wavelength of the fluorescence is longer than the wavelength of the excitation light for exciting the fluorescence, the excitation light absorption waveguide 120 may be formed of a color filter material that transmits light in the fluorescence wavelength band and absorbs light in the excitation light wavelength band. The color filter material forming the excitation light absorption waveguide 120 may have a refractive index greater than that of the material forming the light blocking unit 110. Alternatively, a cladding layer (not shown) surrounding the excitation light absorption waveguide 120 may be further provided with a material having a refractive index smaller than that of the material of the excitation light absorption waveguide 120. Since the refractive index relationship is the same as the waveguide structure of the optical fiber, the fluorescence emitted from the sample 190 will pass through the total reflection in the excitation light absorption waveguide 120.

One end of the excitation light absorption waveguide 120 is placed in the reaction region 140a of the sample reaction unit 140 to which the sample is attached, and the other end of the excitation light absorption waveguide 120 is in contact with the sample detection unit 200. . The cross section of the excitation light absorption waveguide 120 may have a shape corresponding to the shape of the reaction region 140a and may have, for example, a circular or polygonal shape. The reaction regions 140a may be spaced apart from each other in two dimensions, and thus, the excitation light absorption waveguide 120 may also be arranged spaced apart from each other in two dimensions. The cross section or arrangement of the excitation light absorption waveguide 120 is not limited to this embodiment.

The light blocking unit 110 and the excitation light absorption waveguide 120 may be formed on the same layer. The thickness of the light blocking unit 110 and the excitation light absorption waveguide 120 is appropriately designed according to the degree of excitation light absorption of the material so that the excitation light can be sufficiently blocked. For example, the light blocking unit 110 and the excitation light absorption waveguide 120 may have a thickness of several μm to several tens of μm.

The microlens 160 may be provided on the upper surface of the excitation light absorption waveguide 120, that is, the surface of the sample reaction part 140 of the end of the excitation light absorption waveguide 120.

The microlens 160 corresponds to the end of the excitation light absorption waveguide 120 one-to-one, and the surface of the microlens 160 becomes a sample reaction unit 140 to which a sample is attached. If the focal length of the microlens 160 is properly designed, the fluorescence may be sufficiently induced to the photodiode 210 even if the light transmitting unit 100 does not have a waveguide structure. That is, even when the light blocking unit 110 is formed of the same material as the excitation light absorption waveguide 120, the microlens 160 may pass through the light transmitting unit 100 and the wiring line unit 230 without fluorescence being diffused. ) Can be designed.

The surface of the microlens 160 or the outside of the region in which the microlens 160 is provided may be surface-treated so that the sample may be attached to only the surface of the microlens 160. For example, the microlens 160 may be surface treated to have affinity for the sample 190 or the liquid in which the sample 190 is dispersed. This surface treatment may vary depending on the sample 190 to be detected. For example, the reaction zone 140a may be locally oxidized, an oxide may be applied, or a hydrophilic material may be applied to the reaction zone 140a to have hydrophilic characteristics. Specific examples of such surface treatment do not limit the present embodiment. Depending on the sample 190, an ion exchange surface, an immobilized metal surface, or other various surface treatment methods may be applied. The plurality of reaction regions 140a may be regions in which a probe bio-material is fixed to a target biomaterial. In this case, the biomaterial serving as the probe may be fixed to the sample reaction unit 140 through a semiconductor process. In the case of the microlens 160 formed or surface-treated with a material having an affinity for the sample 190 as described above, the surface itself of the microlens 160 may be the reaction region 140a.

A fluorescence antireflection film 165 may be further provided on the surface of the microlens 160 to suppress fluorescence from being lost on the surface of the microlens 160. The anti-reflection film 165 may be formed of a material that is friendly to a sample or a liquid in which the sample is dispersed.

The microlens 160 has a convex shape and has a refractive power for condensing fluorescence emitted from a sample. Since the surface of the microlens 160 is convex, its width is wider than that of the flat case. Since the surface itself of the microlens 160 becomes the reaction region 140a to which the sample is attached, when the convex microlens 160 is adopted as in the present embodiment, more samples can be attached to the same cross-sectional area. Therefore, the intensity of fluorescence emitted from the sample to be detected may be increased.

In addition, the fluorescence incident on the excitation light absorption waveguide 120 may be transmitted while minimizing light loss when the total reflection condition is satisfied in the excitation light absorption waveguide 120. In the present exemplary embodiment, since the incident fluorescence is refracted by the microlens 160, the fluorescence incident more obliquely than the case without the microlens 160 satisfies the total reflection condition in the excitation light absorption waveguide 120 and is transmitted. Can be. Accordingly, a greater amount of fluorescence emitted from the sample may be efficiently transmitted to the excitation light absorption waveguide 120.

In the present embodiment, the microlens 160 has a convex hemispherical shape as an example, but is not limited thereto. The external environment of the sample reaction unit 140 may be larger than the refractive index of the microlens 160. For example, when the liquid is filled between the sample reaction unit 140 and the window 300, and the refractive index of the liquid is relatively higher than that of the microlens 160, the macrolens 160 may absorb the excitation light. The end of the waveguide 120 may have a concave hemispherical shape.

In the present embodiment, the case in which the microlens 160 is formed on the sample reaction part 140 is described as an example, but is not limited thereto. The microlens 160 may be additionally or independently positioned between the sample reaction unit 140 and the photodiode unit 210. For example, it may be provided at a boundary between the light transmitting unit 100 and the sample detecting unit 200, or may be provided inside the sample detecting unit 200.

In addition, the present embodiment has been described in the case where the light transmitting unit 100 includes both the light blocking unit 110 and the excitation light absorption waveguide 120 as an example, but is not limited thereto. For example, the light transmitting unit 100 may be formed of only an excitation light absorbing material layer made of a material that absorbs excitation light and transmits fluorescence without a separate light blocking unit 110, that is, a monochromatic color filter. As described above, if the focal length of the microlens 160 is properly designed, the fluorescence may be sufficiently induced to the photodiode 210 even without the waveguide structure.

The sample detecting unit 200 includes a photodiode 210 provided with a plurality of photodiodes 220, and a wiring line unit 230 provided with a control circuit and a wiring line 239 for signal processing. The photodiode 220 is an example of a light receiving element. The sample detector 200 may further include a signal processor (not shown) capable of processing a signal of the detected fluorescent image.

The photodiode 220 of the sample detecting unit 200 may be disposed about the Rayleigh length from the lower surface of the light transmitting unit 100. Rayleigh length means the distance from the point where the light beam is focused and its cross-sectional area is minimum to the point where its cross-sectional area is doubled. The fluorescence progresses in the excitation light absorption waveguide 120 and then leaves the excitation light absorption waveguide 120, so that the cross-sectional area of the light beam is gradually increased. Therefore, by designing the sample detector 200 such that the photodiode 220 of the sample detector 200 is disposed about the Rayleigh length from the lower end of the excitation light absorption waveguide 120, the fluorescence is emitted at the position where the luminous flux is focused at the maximum. It is possible to increase the fluorescence detection efficiency by making it detected.

The sample detector 200 may be, for example, a substrate provided with an image sensor such as a charge coupled device (CCD) or a CMOS image sensor (CIS). CCD (Charge Coupled Device) or CIS (CMOS Image Sensor) is not limited to the front lighting method or the back lighting method. Since such an image sensor itself is well known in the art, a description thereof will be omitted.

1 illustrates a case in which the micromirror 325, the microlens 140, the excitation light absorption waveguide 120, and the photodiode 220 correspond one-to-one, but are not limited thereto. For example, the plurality of micromirrors 325 may face each other with respect to one reaction region 140a of the sample reaction unit 140, and the plurality of reaction regions 140a may be one pixel unit. Can be. In addition, the photodiode 220 may be arranged so as to correspond one-to-one or one-to-many with the end of the excitation light absorption waveguide 120 in which fluorescence is emitted.

2 schematically shows the optical path of fluorescence emitted from a sample in an integrated biochip according to this embodiment.

Referring to FIG. 2, the fluorescence L ′ emitted from the sample is generated by the excitation light L, and the downward fluorescence L ′ is incident toward the microlens 140. The fluorescence L ′ incident on the microlens 140 is directed to the sample detection unit 200 of FIG. 1 via the excitation light absorption waveguide 120. Since the refractive index of the excitation light absorption waveguide 120 is larger than the refractive index of the light blocking portion 110, the fluorescence incident on the excitation light absorption waveguide 120 is the interface between the excitation light absorption waveguide 120 and the light blocking portion 110. Proceed with total reflection at. Since the fluorescence passes through the excitation light absorption waveguide 120 while performing total reflection, light loss can be minimized. If the refractive index of the excitation light absorption waveguide 120 is less than or equal to the refractive index of the light blocking unit 110, a portion of the fluorescence L ′ passing through the excitation light absorption waveguide 120 may be lost. Since the thickness of the light transmitting part 100 is, for example, about several micrometers to several tens of micrometers, this loss may be neglected.

By the way, the fluorescence L 'generated by the excitation light L is omnidirectional and therefore diverges in all directions. Therefore, a considerable amount of fluorescence L 'generated by the excitation light L is directed upward. As described above, the upward fluorescence L ′ is reflected by the fluorescent reflection film 310 and directed toward the microlens 140. By properly designing the focusing force of the fluorescent reflection film 310 as described above, the fluorescence L 'can be focused on the microlens 140, so that the upward fluorescence L' is also used for sample detection. The fluorescence (L ') lost can be minimized. The window 300 is disposed adjacent to the sample 190 within a range that does not damage the sample 190, thereby covering the sample 190 in the reaction region 140a to which the micromirror 325 corresponds, thereby utilizing light. The efficiency can be improved. Since the fluorescence L ′ emitted upward may be focused to the reaction region 140a in which the sample emitted the fluorescence by the micromirror 325, the fluorescence L ′ emitted upward above the neighboring sample 190. Can be prevented from confusing or blurring the fluorescent image.

As described above, since the excitation light absorption waveguide 120 has a waveguide structure of an optical fiber, most of the fluorescence is transmitted through total reflection in the excitation light absorption waveguide 120. Some of the fluorescence incident outside the predetermined angle may be directed toward the light blocking unit 110 through the excitation light absorption waveguide 120, but the fluorescence is absorbed while passing through the light blocking unit 110. In addition, the light incident toward the light blocking portion 110 among the downward fluorescence L ′ is reflected on the surface of the light blocking portion 110 or absorbed while passing through the light blocking portion 110. Therefore, the fluorescence L 'is guided only through the excitation light absorption waveguide 120 in the lower portion of the sample reaction section 140 on which the sample 190 that emits itself is emitted and is emitted below the neighboring sample 190. It is possible to prevent the fluorescence image from being confused or blurred by the fluorescence L '. On the other hand, the excitation light L directed downward is reflected on the surface of the light transmitting unit 100 or absorbed while passing through the light transmitting unit 100, and does not leak to the lower side of the light transmitting unit 100.

Since the excitation light absorption waveguide 120 may correspond to one-to-one or one-to-many with the reaction region 140a, the image by the fluorescence emitted from the sample 190 may have the surface of the sample detection unit 200 of the light transmitting unit 100. Will appear as is. Therefore, the sample detecting unit 200 may immediately read an image of the fluorescence emitted from the sample 190 without an additional optical member. Thus, since the fluorescent image can be read directly from the chip itself, when using the integrated biochip of the present embodiment, a separate detection optical system is not required, and the biodetection device can be miniaturized.

In the present embodiment, the color filter material forming the excitation light absorption waveguide 120 has a refractive index larger than the refractive index of the material constituting the light blocking unit 110 as an example. However, the present invention is not limited thereto. no. Even when the refractive index of the color filter material forming the excitation light absorption waveguide 120 is less than or equal to the refractive index of the material constituting the light blocking portion 110, the light blocking portion 110 may generate the excitation light L and the fluorescence ( L ′), and the excitation light absorption waveguide 120 passes only the fluorescent light L ′, and thus the function of the light transmitting unit 100 may be performed as it is.

3 illustrates an integrated biochip according to another embodiment of the present invention. The integrated biochip of this embodiment is the same as the above embodiment except that the microlens 160 is excluded.

Referring to FIG. 3, the anti-reflective film 150 is provided on the side of the sample reaction part 140 of the end of the excitation light absorption waveguide 120. The antireflection film 150 prevents the fluorescence emitted from the sample 190 from being reflected, thereby improving the fluorescence detection efficiency. The fluorescent antireflection film 150 may be formed to completely cover the end of the exposed excitation light absorption waveguide 120. The anti-reflection film 150 may be formed of a material having affinity for the sample 190 or the liquid in which the sample 190 is dispersed so that the sample 190 may be attached well. When the anti-reflection film 150 is formed of a material having an affinity for the sample 190, the region itself on which the anti-reflection film 150 is formed becomes the reaction area 140a. The anti-reflection film 150 may be separately provided on the lower side of the reaction region 140a. The anti-reflective film 150 is not an essential component of the present embodiment and may be omitted. When the anti-reflective film 150 is omitted, the surface of the reaction region 140a may be surface treated to have affinity for the sample 190 or the liquid in which the sample 190 is dispersed. This surface treatment may vary depending on the sample 190 to be detected. For example, the reaction region 140a may be locally oxidized, an oxide may be applied, or a hydrophilic material may be applied to the reaction region 140a to have hydrophilic characteristics. Specific examples of such surface treatment do not limit the present embodiment.

4 illustrates an integrated biochip in accordance with another embodiment of the present invention. This embodiment shows an example in which the window is coupled to the sample reaction detection unit.

Referring to FIG. 4, the sample reaction unit 140, the phototransmitter 100, and the sample detection unit 200 form a sample reaction detection unit, and the sample reaction detection unit is manufactured in units of wafers through a semiconductor process as described below. Can be. At this time, the outside of the sample reaction detection unit constituting the individual chip becomes a space for signal processing or electrical connection of the sample detection unit 200. The upper surface of the peripheral portion 400 of the sample reaction detection unit may be used for coupling with the window 301.

In the transparent body 311 of the window 301, the side wall 311b protrudes downward from the outer circumference thereof and contacts the upper surface of the peripheral part 400 of the sample reaction detection unit. The side wall 311b may be provided only at a part of the outer periphery of the transparent body 311. The window 310 is provided with an inner space by the side wall 311b so that the sample attached to the sample reaction unit 140 of the sample reaction detection unit is not damaged. An alignment mark (not shown) is provided on the upper surface or the window 310 of the sample reaction detection unit, and when the window 310 and the sample reaction detection unit are combined, the micromirror 311a and the sample reaction unit 140 of the window 310 are provided. It is possible to ensure that the reaction zones of are properly aligned.

The combination of the window 310 and the sample reaction detection unit may be made after the sample 190 is attached to the sample reaction unit 140, and thus may be performed in individual chip units.

5 illustrates an integrated biochip in accordance with another embodiment of the present invention. Referring to FIG. 5, the integrated biochip 500 of the present embodiment is packaged, and a sample reaction detector 510 and a window 530 are mounted on the frame 540. The upper surface of the sample reaction detection unit 510 is a surface provided with a sample reaction unit (140 of FIG. 1) to which a sample may be attached, and may be exposed to the outside. For example, in the case of a DNA chip, a plurality of probe nucleic acids are fixed to the sample reaction unit 140 of the sample reaction detection unit 510 through a semiconductor process or the like, and when the liquid containing the target nucleic acid flows on the surface of the DNA chip, The probe nucleic acid having a base sequence complementary to the base sequence of the target nucleic acid is hybridized with the target nucleic acid, and the nucleic acids not combined with the probe nucleic acids of the DNA chip are washed out. Since the nucleic acid subjected to the hybridization reaction emits fluorescence by the labeled fluorescent substance, the presence or absence of the target nucleic acid to be detected is determined by detecting the position where the fluorescence is emitted.

The window 530 is provided with a fluorescent reflection film 520 and is installed in the frame 540. The window 530 may be a cover glass that protects the front surface of the biochip 500. When the sample is attached to the sample reaction unit 140, the window 530 is covered with the sample spaced above the sample reaction detection unit 510. Alignment marks (not shown) may be provided in the window 530 and the sample reaction detection unit 510 or the frame 540. This alignment mark allows the window 530 to be properly aligned when attached to the frame 540.

As described above, the biochip of the present embodiment can detect the emitted fluorescence on the chip itself, and thus it is possible to determine whether the sample is detected through the electrical signal output from the packaged integrated biochip 500.

Next, the manufacturing process of the integrated biochip according to an embodiment of the present invention will be described.

6A to 6F illustrate a manufacturing process of a sample reaction detection unit of a biochip.

Referring to FIG. 6A, a sample detector 200 is provided. For example, a substrate provided with a CMOS image sensor may be used as the sample detection unit 200. The photodiode 220 is formed on the silicon substrate by a frontend process, and the wiring line unit 230 having the CMOS circuit and the wiring lines 239 is formed thereon. Next, in a backend process, vertical and horizontal passive metal layers connecting the circuits formed in the photodiode unit 210 are formed to manufacture a CMOS image sensor substrate. In a typical CMOS image sensor manufacturing process, a process of forming a color filter on the CMOS image sensor substrate is further added, but the sample detecting unit 200 of the present embodiment does not further include a process of forming such a color filter. Since the sample detecting unit 200 of the present embodiment is substantially the same as a conventional image sensor and can be manufactured through the same process, detailed description thereof will be omitted. For example, in the CCD, a light receiving area for generating electrons, that is, a photodiode 220 and a horizontal vertical metal layer for transmitting electrons are connected through the wiring line unit 230. The photoelectron is transported to the periphery of the sensor through the field transfer device of the metal layer and then converted into a voltage.

Next, referring to FIG. 6B, the excitation light absorbing material is deposited on the sample detecting unit 200 by sputtering, chemical vapor deposition (CVD), or spin-coating. Form layer 120 '. The excitation light absorbing material is a material that absorbs light in the excitation light band and passes light in the fluorescent light band. For example, a transparent semiconductor, polymer or dielectric material is dyed with a dye belonging to the wavelength band of fluorescence, or a semiconductor of transparent material. It can be formed by electrodepositing or dispersing a pigment belonging to the wavelength range of fluorescence in the polymer or dielectric material. Since such dyes and pigments are well known in the display technology or optical field, detailed descriptions thereof will be omitted.

Next, referring to FIG. 6C, the photodiode 220 is etched into the excitation light absorbing material layer (120 ′ in FIG. 6B) applied to expose the upper portion of the sample detecting unit 200 in the remaining region except for the region where the photodiode 220 is located. The trench 120a is formed. The trench 120a may be formed using, for example, a dry etching method. As such, the excitation light absorbing material layer 120 ′ in which the trench 120a is formed forms an excitation light absorption waveguide 120.

Next, referring to FIG. 6D, the light blocking unit 110 is formed by filling a black material in the trench 120a between the excitation light absorption waveguides 120. These black materials are materials that absorb both excitation light and fluorescence. The black material may be a material having a refractive index lower than that of the excitation light absorbing material, but is not limited thereto.

Next, referring to FIG. 6E, after the top surfaces of the excitation light absorption waveguide 120 and the light blocking unit 110 are planarized through a process such as chemical mechanical polishing (CMP), the excitation light absorption waveguide 120 is The microlens 160 is formed in the exposed area. The microlens 160 may be formed of a material to which the sample may be attached well. Alternatively, the microlens 160 may be surface treated so that the sample may be attached well. The microlens 160 may be formed by, for example, forming a pattern of a microlens array using photoresist and then deforming the columnar patterned photoresist into a curved photoresist through a reflow process. In addition, the anti-reflection film 165 may be formed on the surface of the microlens 160, and the surface treatment may be performed so that the sample adheres well.

Next, referring to FIG. 6F, a process of attaching the sample 190 to the microlens 160 is performed. The sample 190 may be a probe for detecting a biomaterial that can interact with a target biomaterial to be detected. For example, DNA bases such as A (adenine), G (guanine), C (cytosine), and T (thymine) are stacked in a different order for each reaction region by using a photo-lithography process to have a predetermined base sequence. DNA chips to which probe nucleic acids are attached can be prepared.

The manufacturing process described with reference to FIGS. 6A through 6F may be performed on a wafer basis. Next, by dividing each chip (dicing), mounted on a frame and performing a wire bonding process, it may be shipped in a biochip state in which the target sample is not attached.

The manufacturing process of the sample reaction detection unit described above with reference to FIGS. 6A to 6F is merely one example, and is not limited thereto. For example, the light transmitting unit 100 may be manufactured through a separate process, and then bonded to the sample detecting unit 200 through a wafer bonding process. In addition, the microlenses may be additionally or independently formed between the light transmitting unit 100 and the wiring line unit 230 as well as the upper surface of the light transmitting unit 100. In addition, when the microlens 150 is provided in the light transmission unit 100 as described above, the waveguide structure by the excitation light absorption waveguide 120 is not essential, the light transmission unit 100 simply provides excitation light. It may be applied by absorbing material to form an excitation light absorption layer.

Next, the manufacturing process of a window is shown with reference to FIGS. 7A-7D.

Referring to FIG. 7A, first, a convex microlens mold 390 is prepared. For example, the microlens mold 390 forms a photoresist having a columnar shape through a photoresist process and an etching process on a substrate, and then forms a photoresist having a columnar shape through a reflow reflow process. It can be formed by deforming into a round photoresist. 7B and 7C, the transparent body 310 of the window is manufactured using the microlens mold 390 as a mold. As a material of the transparent body 310, a material that can be easily separated from the microlens mold 390 may be selected. The transparent body 310 has a concave surface 310a formed in a reversed phase of the microlens mold 390.

Next, referring to FIG. 7D, the window is completed by forming a microlens by coating the fluorescent reflection film 320 on the concave surface 310a of the transparent body 310.

The manufacturing process of the window described above has been described taking the case of manufacturing the transparent body 310 using an injection process as an example, but various other methods are possible. For example, a hemispherical concave surface may be directly formed on the transparent substrate through an etching process.

The window covers the sample reaction part of the biochip while the sample is attached to the biochip, and the biochip entering the detection step is completed. As described above, an alignment mark may be provided on the window to the upper side of the biochip, so that the window may be properly aligned using a high magnification optical device when the window is coupled to the biochip.

Hereinafter, a detection apparatus using an integrated biochip according to the above embodiments will be described.

8 shows a schematic configuration of a bio detection device according to an embodiment of the present invention.

Hereinafter, a detection apparatus using an integrated biochip according to the above embodiments will be described.

8 illustrates a schematic configuration of a bio detection device according to an embodiment of the present invention. Referring to FIG. 8, the biodetection apparatus of the present embodiment includes a light source 601, a light diffusing element 602, a collimating lens 604, and a condenser lens, which are illumination optical systems that irradiate excitation light to the integrated biochip 610. 607. Reference numeral 620 denotes a stage in which the integrated biochip 610 is detachably installed.

The light source 601 emits excitation light L. There is no limitation as long as the excitation light L can be emitted by the light source 601. For example, a semiconductor laser diode, an LED, a white light source, or the like can be used as the light source 601. The wavelength of the excitation light L emitted from the light source 501 may be shorter than the fluorescence. If a white light source is used as the light source 601, an excitation filter (not shown) that blocks light of a wavelength band such as fluorescence on the optical path between the light source 601 and the integrated biochip 610 is used. To place. As described above, in the biochips of the embodiments, since the fluorescent reflection layer is provided in the window, the excitation filter may be omitted.

The excitation light L is light for exciting a fluorescent substance attached to a sample in the integrated biochip 610. Typically, light having a wavelength of approximately 500 nm is used as the excitation light, but depending on the fluorescent material to be labeled, the wavelength of the excitation light L may vary.

The light diffusing element 602 diffuses the excitation light L evenly to have a uniform intensity through its entire cross section, and may be, for example, a bar-shaped light integrator. The excitation light L has an overall uniform intensity for illuminating light of the same intensity over a portion or the entire region of the integrated biochip 610.

The collimating lens 604 forms the excitation light L in parallel. Although FIG. 8 is shown to be disposed between the light scattering element 602 and the condenser lens 607, the collimating lens 604 may be disposed between the light source 601 and the light diffusing element 602. Further, when the divergence of the excitation light L emitted from the light source 601 is not large and the excitation light L can be sufficiently collected by the condenser lens 607, the collimating lens 604 is used. You may not.

The condenser lens 607 collects the excitation light L to provide a light spot having a predetermined diameter on the integrated biochip 610. The diameter of the light spot may be large enough to illuminate a part or the whole area of the integrated biochip 610.

When the spot of the excitation light L covers the entire surface of the integrated biochip 610, the integrated biochip 610 may simultaneously detect the fluorescent image emitted from the sample. If the spot of the excitation light L covers only a part of the integrated biochip 610, the stage 620 or the illumination optical system moves to cover the entire surface of the biochip 610 in which the excitation light L is illuminated by moving. In order to cover sequentially, the integrated biochip 610 acquires fluorescence images of the entire reaction region to which the sample is attached by synthesizing the images of fluorescence emitted sequentially.

The integrated biochip 610 of this embodiment directly detects the fluorescence emitted from the sample by the illuminated excitation light L, and the signal for the detected fluorescence is transmitted to the signal processing system not shown through the stage 620. do. The stage 620 itself may be a measurement board incorporating a signal processing circuit.

The biodetector of the present embodiment can read out an image of fluorescence emitted from a sample as the integrated biochip 610 itself without having to provide a separate detection optical system. Therefore, since the optical system of the bio-detection device is sufficient only as the illumination optical system, it is easy to manufacture portable, and it is possible to analyze and detect samples such as biomaterials regardless of the place.

The present inventors integrated biochip and its manufacturing method have been described with reference to the embodiments shown in the drawings for clarity, but this is merely illustrative, and those skilled in the art will appreciate that various modifications and equivalents therefrom It will be appreciated that other embodiments are possible. Therefore, the true technical protection scope of the present invention will be defined by the appended claims.

1 is a schematic cross-sectional view of an integrated biochip according to an embodiment of the present invention.

FIG. 2 illustrates a light path in the integrated biochip of FIG. 1.

3 is a schematic cross-sectional view of an integrated biochip according to another embodiment of the present invention.

4 is a schematic perspective view of an integrated biochip according to another embodiment of the present invention.

5 is a schematic cross-sectional view of an integrated biochip according to another embodiment of the present invention.

6A-6F and 7A-7D illustrate a method of manufacturing an integrated biochip according to an embodiment of the present invention.

8 is a schematic configuration diagram of a bio detection device according to an embodiment of the present invention.

<Description of the symbols for the main parts of the drawings>

100 ... light transmitting part 110 ... light blocking part

120.Excited light absorption waveguide 140.

150,165 ... Fluorescent reflector 160 ... Microlens

190.Drugs 200 Sample detection unit

210 ... photodiode section 220 ... photodiode

230 ... wiring line part 300, 301, 530 ... Windows

310,311 ... transparent body 310a, 311a ... micromirror

311b ... side wall 320 ... fluorescent reflector

330 Excitation light shielding film 400 ... periphery

500,610 Biochip 610 Sample reaction detection unit

601 ... light source 602 ... light diffusing element

604 Collimating Lens 607 Condensing Lens

620 stage

Claims (26)

A sample detector provided with at least one light receiving element for detecting fluorescence; An optical transmission unit provided on an upper surface of the sample detection unit to transfer fluorescence emitted from a sample to the sample detection unit; A sample reaction part provided on an upper surface of the light transmitting part and including at least one reaction area to which a sample may be attached; And And a window having a fluorescent reflecting film disposed to be spaced apart from the sample reaction part to reflect fluorescence emitted from the sample. According to claim 1, The fluorescent reflector is an integrated biochip that is a dichroic mirror having a wavelength band of fluorescence as a reflection band or a bandpass filter having a band of fluorescence as a blocking band. The method of claim 2, The fluorescent reflection film is an integrated biochip having a pass band of the wavelength band of the excitation light. According to claim 1, An integrated biochip having an excitation light reflection prevention film provided on the upper surface of the window to prevent reflection of the excitation light. The method according to any one of claims 1 to 4, And the window focuses fluorescence emitted from the sample so that the reflector includes at least one micromirror. 6. The method of claim 5, The micromirror is an integrated biochip provided in a concave shape on the lower surface facing the sample reaction portion of the window. 6. The method of claim 5, The micromirror is an integrated biochip corresponding one-to-one or one-to-many with the reaction region of the sample reaction unit. The method of claim 7, wherein And an alignment mark arranged at at least one of the window and the sample reaction unit to allow the micromirror to match the reaction region of the sample reaction unit. 6. The method of claim 5, And a microlens for condensing fluorescence provided on an optical path between the at least one reaction region and the light receiving device. The method according to any one of claims 1 to 4, The window includes an integrated biochip protruding downward to be spaced apart from the sample reaction unit. The method of claim 10, And the lead-out part is formed in at least a portion of an outer circumference of the window. The method according to any one of claims 1 to 4, Biochip comprising a frame for receiving the sample reaction portion and the window spaced apart from each other. The method according to any one of claims 1 to 4, And the light transmitting unit includes at least one excitation light absorption waveguide provided under the at least one reaction region to absorb excitation light for exciting the sample and transmit fluorescence emitted from the sample. The method of claim 13, And a material surrounding the excitation light absorption waveguide of the light transmitting part is a black material absorbing the excitation light and fluorescence. The method of claim 13, And a material surrounding the excitation light absorption waveguide of the light transmitting part has a refractive index lower than that of the excitation light absorption waveguide. The method according to any one of claims 1 to 4, And the light transmitting unit is provided under the sample reaction unit, and includes a monochromatic color filter which absorbs excitation light to excite the sample and transmits fluorescence emitted from the sample. The method according to any one of claims 1 to 4, The sample detection unit is an integrated biochip that is a charge coupled device (CCD) or a CMOS image sensor (CIS). Providing a sample detection unit provided with at least one light receiving element for detecting fluorescence; Forming a light transmission unit configured to transfer fluorescence emitted from a sample on an upper surface of the sample detection unit; Forming a sample reaction part including at least one reaction region to which a sample is attached to an upper surface of the light transmitting part; Providing a window having a fluorescent reflecting film; And And disposing the window spaced apart from the sample reaction unit. 19. The method of claim 18, Preparing the window, And concentrating the fluorescence emitted from the sample to the window to provide at least one micromirror with a reflector key. 19. The method of claim 18, Preparing the window, And providing an excitation light reflection prevention film to prevent reflection of the excitation light on the back surface of the window provided with the micromirror of the window. 19. The method of claim 18, Preparing the window, And providing an alignment mark for alignment with the sample reaction part in the window. The method according to any one of claims 18 to 21, Forming the light transmitting unit, Applying an excitation light absorbing material to the upper surface of the sample detector to absorb the excitation light and absorb the fluorescence; Forming an excitation light absorption waveguide by forming a trench in the applied excitation light absorption material so that the upper portion of the sample detection unit in the remaining area except the area where the light receiving element is located is exposed; And And filling the trench with a dielectric material having a refractive index smaller than the refractive index of the excitation light absorbing material, thereby forming a light blocking portion. The method according to any one of claims 18 to 21, Forming the light transmitting unit, Applying an excitation light absorbing material to the upper surface of the sample detector to absorb the excitation light and absorb the fluorescence; Forming an excitation light absorption waveguide by forming a trench in the applied excitation light absorption material so that the upper portion of the sample detection unit in the remaining area except the area where the light receiving element is located is exposed; And And filling the trench with a black material that absorbs both excitation light and fluorescence, thereby forming a light blocking portion. The method according to any one of claims 18 to 21, And forming a microlens on an upper side of a region in which the light receiving element is located. The method of claim 24, The light transmitting unit is a biochip manufacturing method of forming an excitation light absorbing material layer. The method according to any one of claims 18 to 21, And forming a fluorescent anti-reflection film on the microlenses.

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