Patent Application
20230119978
The present invention relates to a biomolecular image sensor and method thereof for detecting biomolecule, and more particularly to a biomolecular image sensor with a plurality of microstructures repeatedly arranged on a surface of an image sensing element, and method thereof for detecting biomolecule.
Enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immunoassay (EIA) is the specific antigen-antibody reaction tests. The specific binding properties between antigens and antibodies are used to detect the molecules in samples. The presence of specific antigens or antibodies may be shown by the color reaction with enzymes. Quantitative analysis may be carried out by the depth of color to achieve the detection and screening.
Biochips are micro devices that use biological materials on a substrate to produce specific biochemical reactions with the biomolecules, and may be quantified by a highly sensitive detection system. Biochips provide fast, accurate, and low cost bioanalytical testing capabilities. Biochips are basically miniaturized substrates that may perform hundreds or thousands of biochemical reactions simultaneously.
However, traditional biochemical tests such as on tissue sections require large and expensive equipment to receive optical or electronic signals for analyzing the status of biochemical molecular reactions, for example, observing with microscopes and capturing images with additional photo equipment for further analysis, which require time and manual operations. On the other hand, traditional biochips have to be additionally equipped with other expensive and large image capture systems or equipment to detect and capture the luminescent images of the biochips after the biochemical detection process for subsequent analysis. Further, traditional ELISA has to be equipped with an ELISA reader to detect the absorbance in each well of the micro-well plate after the operation with ELISA kit for quantification.
With the increasing popularity of the point of care testing (POCT), a personalized health test with a short analysis time and simple operation, it is necessary to develop a more sensitive and simpler detection device and method to overcome the problems from large equipment and complex biochemical detection processes in traditional biomolecule detection methods.
One objective of the present invention is to provide a biomolecular image sensor, comprising: an image sensing element, containing a plurality of unit pixels disposed in an array on a substrate, wherein each of the plurality of unit pixels contains at least one photoelectric conversion element, the photoelectric conversion element receives an incident light to generate electrons, and a surface of the image sensing element receiving the incident light is defined as a light receiving surface; and a microstructure layer, disposed on the light receiving surface of the image sensing element and having a plurality of microstructures arranged in a specific shape repeatedly, wherein each of the plurality of microstructures corresponds to at least one unit pixel.
In the preferred embodiment of the present invention, the microstructure layer is an array formed by a plurality of inverted pyramidal or honeycomb microstructures.
In the preferred embodiment of the present invention, each of the microstructures is formed on the image sensor element by photolithography process or imprint lithography process.
The further objective of the present invention is to provide a biomolecular image sensor, comprising: an image sensing element, containing a plurality of unit pixels disposed in an array on a substrate, wherein each of the plurality of unit pixels contains at least one photoelectric conversion element, the photoelectric conversion element receives an incident light to generate electrons, and a surface of the image sensing element receiving the incident light is defined as a light receiving surface; and a microstructure layer, disposed on the light receiving surface of the image sensing element and being an array formed by a plurality of microlenses, wherein a plurality of microstructures are formed between the plurality of microlenses, and each of the plurality of microstructures corresponds to at least one unit pixel.
In the preferred embodiment of the present invention, the biomolecular image sensor further comprises at least one readout circuit coupled to the unit pixels, and the readout circuit generates a voltage signal based on the number of the electrons.
In the preferred embodiment of the present invention, each of the microlenses is provided to accommodate at least one biomolecule.
In the preferred embodiment of the present invention, the incident light is a light emitted by a fluorescent marker or a chemiluminescent marker on the biomolecule.
In the preferred embodiment of the present invention, the biomolecular image sensor further comprises a carrier carrying the biomolecule, and the carrier is accommodated in the microstructure, and the carrier may be a microparticle. Further, one microstructure accommodates one carrier chemically bonded with multiple biomolecules.
In the preferred embodiment of the present invention, the bottom portions and the side portions of the microstructures are composed of different materials, and each of the microlenses is formed on the image sensor element by photolithography process or imprint lithography process.
The other objective of the present invention is to provide a method of detecting a biomolecule, comprising: (a) providing the biomolecular image sensor; (b) accommodating the biomolecule in a sample in each of the plurality of microstructures; (c) detecting an incident light in each of the plurality of microstructures by each of the plurality of unit pixels; (d) generating electrons from the incident light detected by each of the plurality of unit pixels by the photoelectric conversion element; (e) generating a voltage signal based on a number of the electrons by a readout circuit coupled to each of the plurality of unit pixels; and (f) analyzing a presence and/or a concentration of the biomolecule based on the voltage signal; wherein, a fluorescent marker or a chemiluminescent marker is added on the biomolecule before or after (b), and the incident light comprises a light emitted by the fluorescent marker or the chemiluminescent marker.
The present invention is a completely innovative image sensor for detecting biomolecules prepared by a semiconductor process. The image sensor of the present invention, which is smaller than a coin, may directly detect the presence of specific biomolecules in samples and quantify concentration thereof, but does not require additional large equipment.
On the other hand, compared with the traditional biochip, the present invention does not require additional image capture system or equipment. That is, the biomolecular image sensor of the present invention provides the functions of biological or chemical analysis and image capture and interpretation. That is, the detection process may be directly operated on the biomolecule image sensor of the present invention, and corresponding detection results may be obtained in real time.
On the other hand, compared with the traditional methods of quantifying biomolecules, the biomolecule image sensor of the present invention independently detects the incident light in the corresponding microstructure through each unit pixel, and compares each measured signal readout with the thresholds independently, and therefore, even when the concentration of the biomolecules in the sample is extremely low, the presence and intensity of chemiluminescent or fluorescent signals of the biomolecules would still be accurately interpreted, so as to increase the detection sensitivity.
Further, because the microstructure in the biomolecule image sensor of the present invention enables the biomolecules to be dispersed evenly on the light receiving surface, the photoelectric conversion element would easily determine the incident light really from the luminescent or fluorescent markers on the biomolecules. The biomolecule image sensor of the present invention provides the quantitative mode of analog colorimetric method and digital method, and could be switched according to different concentrations of biomolecules, so as to maximize the detection range.
In order to enable one with ordinary skill in the art to understand the purpose, features, and functions of the present invention, the invention is described in detail below by means of the following specific embodiments and with the attached drawings.
The embodiments of the present invention are further described with the following drawings. The following embodiments are given to illustrate the present invention and are not intended to limit the scope of the present invention, and one with ordinary skill in the art may make some modifications and refinements without departing from the spirit and scope of the present invention. Therefore, the scope of the present invention is defined by the scope of the appended claims.
The terms used herein are only for describing the embodiments, and are not intended to limit the present invention. Unless otherwise defined, the terms have meanings commonly understood by one with ordinary skill in the art to which the present invention belongs. As used herein, the singular forms “a” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.
Similarly, when an element is referred to be “on” another element, it would be understood that the element may be directly on the other element or intermediate elements may be present. In contrast, the term “directly” represents that there is no intermediate element. The term “comprising” used herein should be understood to indicate the presence of stated features, integers, steps, operations, elements, and/or components, but not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or any combination thereof.
According to the present invention, the operating procedures and parameter conditions for enzyme-linked immunosorbent assay (ELISA) are within the professional literacy and routine techniques of one with ordinary skill in the art.
According to the present invention, the fluorescent molecules may be, but not limited to, FITC, HEX, FAM, TAMRA, Cy3, Cy5, quantum dot, or the like, and may be used with a quencher dye.
More specifically, the image sensing element 10 in the biomolecular image sensor 100 according to the present invention may be a back-illuminated complementary metal-oxide-semiconductor (CMOS) image sensor or a front-illuminated CMOS image sensor; however, the present invention is not limited thereto.
In the embodiments of the present invention, each of the unit pixels 11 may include at least one photoelectric conversion element, wherein the photoelectric conversion element may generate electrons after receiving an incident light, and the photoelectric conversion element also contains the ability to accumulate the above-mentioned electrons; however, the present invention is not limited to thereto.
Further, the photoelectric conversion element may be an element that generates and accumulates electrons corresponding to the incident light. For example, the photoelectric conversion element may be a photodiode, a photo transistor, a photo gate, a pinned photo diode (PPD), an avalanche photodiode (APD), a single-photon avalanche diode (SPAD), a photomultiplier tube (PMT), or any combination thereof.
In the embodiments of the present invention, a surface of the image sensing element 10 receiving the incident light may be defined as a light receiving surface, and the microstructure layer 20 may be disposed on the light receiving surface. In the preferred embodiments of the present invention, each of the microstructures 21 in the microstructure layer 20 may correspond to the unit pixel 11 respectively. In this way, since the microstructures 21 have a corresponding relationship with the unit pixels 11, the photoelectric conversion elements in the unit pixels 11 may receive the incident light corresponding to the microstructures 21.
The biomolecular image sensor 100 of the present invention may be formed by a semiconductor process. More specifically, the microstructures 21 may be formed on the image sensing element 10 by photolithography process or imprint lithography process, and not just general by surface treatment or coatings.
In the embodiments of the present invention, the readout circuits may be coupled to the unit pixels 11 and generate a voltage signal, which is used as the signal readout, according to the number of the electrons generated after the photoelectric conversion element receives the incident light.
The biomolecular image sensor 100 of the present invention is not limited to any specific applications. In one preferred embodiment, the biomolecule image sensor 100 of the present invention is used for biological or chemical analysis, such as detecting the presence and/or concentration of a biomolecule A in a sample. That is, the incident light may be a light emitted by a marker, a fluorescent marker, a reporter marker, or a chemiluminescent marker of the biomolecule A. In other words, the presence and concentration of the biomolecule A may be analyzed by detecting the markers. More specifically, when the biomolecule image sensor 100 of the present invention is used to detect the biomolecule A, in the biological or chemical analysis process, the biomolecule A may undergo luminescence reaction with other molecules and emit an incident light such as chemiluminescence or fluorescence. Further, the biomolecule A may be a protein, a peptide, an antibody, a nucleic acid, or the like.
More specifically, the microstructures 21 may be disposed to accommodate the biomolecules A, and the biomolecules A in the sample may be dispersed in a plurality of the microstructures 21. Since the single microstructure 21 corresponds to the single unit pixel 11, the photoelectric conversion element in the single unit pixel 11 would only receive the light emitted by the biomolecules A in the single microstructure 21. Therefore, the sensitivity of detecting the specific biomolecule A in the sample would be improved.
Further, the biomolecule image sensor 100 of the present invention may be used with a carrier 22 for carrying the biomolecule A, so that the biomolecule A may be more easily accommodated in the microstructures 21, and the luminescence reaction of the biomolecule A may be easily performed. For example, the carrier 22 may be a microparticle, and the microparticle may be used for performing ELISA. The biomolecule A is linked on the microparticle for performing the luminescence reaction to generate a chemiluminescence, and the chemiluminescence is the incident light received by the photoelectric conversion element in the unit pixel 11. The biomolecule A such as an antibody may be linked to the microparticle by forming an amide bond through the EDC/NHS reaction.
More specifically, the microparticle is preferably a magnetic bead with 1 to 3 µm diameter. The magnetic bead may be prepared by magnetic materials of magnetic elements such as Fe, Ni, Co, ferromagnetic alloys such as Nb-Fe-B, or iron oxides such as Fe3O4, Fe2O3, FeO. Alternatively, the microparticle may also be prepared by non-magnetic materials such as Au, sepharose, polystyrene, and SiO2.
More specifically, the single microstructure 21 has to accommodate only one carrier 22 as much as possible, so the width/diameter and height/depth of the microstructures 21 have to match the carrier 22. More specifically, the diameter of the microstructure is preferably 1.3 to 1.8 times larger than the diameter of the microparticle, the depth is preferably 1.2 to 1.3 times larger than the diameter of the microparticle, and the aspect ratio is preferably 1 to 1.2 times. For example, when the carrier 22 is a microparticle with 2 µm diameter, the microstructures 21 may be grooves with 2.5 to 3 µm diameter and 2.5 µm depth, and the spacing between each microstructure is 2-3 µm. To avoid the molecular forces between the carriers 22 to cause them stack on each other and fail to disperse into a single one, which further causes the single carrier 22 difficult to accommodate in one single microstructure 21, surfactant, external magnetic field, or vibration to destroy the force between the carriers 22 may be used to improve the degree of dispersion thereof.
Alternatively, the biomolecule A may be detected by an antibody or aptamer labeled with a fluorescent molecule. A radiated light is generated by irradiating excitation light with a specific wavelength, and the radiated light is the incident light received by the photoelectric conversion element in the unit pixel 11. Antibodies or aptamers labeled with different fluorescent molecules may be contained on the same carrier 22, so that the detection of various target biomolecules may be performed on the same sample during one single detection. Further, a CMOS image sensor with RGB technology may be used for antibodies or aptamers containing multiple fluorescent labels.
In the biomolecular image sensor 100 of the present invention, the bottom portions and the side portions of the microstructures 21 may be composed of the same or different materials. For example, a surface of the microstructures 21 in contact with the light receiving surface (i.e., the bottom portions) may be composed of light-transmitting materials such as SiO2, while a surface of the microstructures 21 not in contact with the light receiving surface (i.e., the side portions) may be composed of opaque materials such as silicon. Alternatively, the bottom portions and the side portions of the microstructures 21 may both be composed of SiO2.
Alternatively, the microstructures 21 may be implemented in different embodiments to allow the carriers 22 to be more easily accommodated in the microstructures 21, taking into account the influence of air pressure or hydrodynamics on the carriers 22 during operation.
For example,
More specifically, as shown in
As shown in
Alternatively, when the biomolecule image sensor of the present invention is used to detect a biomolecule in a sample, the biomolecule containing fluorescent makers or chemiluminescent markers may also be placed on a substrate such as a glass slide, traditional biochips, or the like. The substrate is then placed on the biomolecule image sensor of the present invention, so that the unit pixels would detect an incident light from the corresponding position respectively. Then, electrons are also generated from the incident light through the photoelectric conversion element, and a voltage signal, which is used as the signal readout, is generated according to the number of the electrons through the readout circuits. The presence and/or concentration of the biomolecule are then analyzed according to the voltage signal.
When the biomolecule image sensor of the present invention is used to analyze the presence and/or concentration of the biomolecule, the quantification thereof may be not only performed by analog colorimetric method, that is, the incident light in one single microstructure received by the unit pixels is one single readout to determine the presence of the biomolecule (S151) or further compare with the standard curve for obtaining the concentration of the biomolecule (S161), but also by digital method, that is, according to the predetermined threshold (S152), the unit pixels with the signal readout exceeding the threshold are defined as 1 (S153) and the unit pixels with the signal readout not exceeding the threshold are defined as 0 (S154), and the number of unit pixels defined as 1 is calculated and compared with the standard curve (S162) to obtain the concentration of the biomolecule more accurately.
Further, traditional ELISA utilizes large and expensive equipment to receive optical or electrical signals after performing complex biochemical detection procedures to analyze the status of biochemical molecular reactions. The present invention is a completely innovative image sensor for detecting biomolecules prepared by a semiconductor process. The image sensor of the present invention, which is smaller than a coin, may directly detect the presence of specific biomolecules in samples and quantify concentration thereof, but does not require additional large equipment.
On the other hand, compared with the traditional biochip, the present invention does not require additional image capture system or equipment. That is, the biomolecular image sensor of the present invention provides the functions of biological or chemical analysis and image capture and interpretation. That is, the detection process may be directly operated on the biomolecule image sensor of the present invention, and corresponding detection results may be obtained in real time.
On the other hand, when quantifying biomolecular images by traditional methods, such as observation with a microscope, if the concentration of the biomolecules is extremely low, the biomolecules directly placed on the surface of the glass slide would distribute extremely uneven, resulting that only a small part of the area (i.e., the unit pixel) contains chemiluminescent or fluorescent signals, and therefore, the presence and intensity of the optical signals cannot be effectively interpreted within the overall field of view, so as to decrease the detection sensitivity. However, the biomolecule image sensor of the present invention independently detects the incident light in the corresponding microstructure through each unit pixel, and compares each measured signal readout with the thresholds independently, and therefore, even when the concentration of the biomolecules in the sample is extremely low, the presence and intensity of chemiluminescent or fluorescent signals of the biomolecules would still be accurately interpreted, so as to increase the detection sensitivity. Further, because the microstructure in the biomolecule image sensor of the present invention enables the biomolecules to be dispersed evenly on the light receiving surface, the photoelectric conversion element would easily determine the incident light really from the luminescent or fluorescent markers on the biomolecules. The biomolecule image sensor of the present invention provides the quantitative mode of analog colorimetric method and digital method, and could be switched according to different concentrations of biomolecules, so as to maximize the detection range.
This application claims priority of U.S. provisional application No. 63/255,446, filed on Oct. 14, 2021, the content of which is incorporated herein in its entirety by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63255446 | Oct 2021 | US |
