Patent Description:
Recently, CMOS image sensors also have been used for biological or biochemical detection. For such an application, a biological or biochemical sample may be placed on a photodiode, and light emitted by the biological or biochemical sample may be directed to the photodiode. The color and intensity of fluorescence or chemiluminescence of the sample may be detected and distinguished by the photodiode. The color and intensity may be used to identify the interaction or properties of the biological or biochemical sample.

General methods to identify the color and intensity of light are stacking an organic color filter and/or dielectric interference filter on the photodiodes for color isolation. Although the organic color filter is not angle-sensitive, the color selection and the transmittance spectrum are less free. Therefore, dielectric interference filter and other angle-sensitive filters are getting popular to achieve the need for various color identification application.

When light irradiates an angle-sensitive filter at an angle of more than <NUM> degrees, the transmittance spectrum of the filter wavelength will shift. This phenomenon is called a spectrum shift. Then, the light received by a detector with a large incident angle under the angle-sensitive filter is not the same as the detector with a small incident angle. Therefore, the property of spectrum shift is considered a drawback in such fields as CMOS image sensors and the applications for cell behavior observation, DNA sequencing, quantitative polymerase chain reaction and DNA/protein microarray.

Although existing image sensors with optics integration have been adequate for their intended purposes, they have not been satisfactory in every respect. For example, a lot of effort has been made to reduce the spectrum shift phenomenon by including light angle directing components on the angle-sensitive filter, which results in a complicated and expensive fabrication process. Therefore, a novel biosensor is still needed.

<CIT> presents a fluorescence biosensor chip having a substrate, at least one electromagnetic radiation detection device arranged in or on the substrate, an optical filter layer arranged on the substrate, and an immobilization layer, which is arranged on the optical filter layer and immobilizes capture molecules. The electromagnetic radiation detection device, the optical filter layer, and the immobilization layer are integrated in the fluorescence biosensor chip.

<CIT> describes an integrated device and related instruments and systems for analyzing samples in parallel. The integrated device includes sample wells arranged on a surface of where individual sample wells are configured to receive a sample labeled with at least one fluorescent marker configured to emit emission light in response to excitation light.

The integrated device may further include photodetectors positioned in a layer of the integrated device, where one or more photodetectors are positioned to receive a photon of emission light emitted from a sample well. The integrated device further includes one or more photonic structures positioned between the sample wells and the photodetectors, where the one or more photonic structures are configured to attenuate the excitation light relative to the emission light such that a signal generated by the one or more photodetectors indicates detection of photons of emission light. <CIT> presents a sensor element for determination of concentration of substances by having integrated on the carrier layer at least one photosensitive element and its electric contact leads in planar arrangement, and by establishing optical contact between the indicator substance of the indicator layer stimulated by the excitation radiation, and the photosensitive elements.

<CIT> presents an apparatus for detecting fluorescent light emitted from a sample and that comprises: a light source, which is configured to emit excitation light of an excitation wavelength towards a sample comprising fluorophores such that fluorescent light is induced; a photo-detector for detecting light incident on the photo-detector; and an interference filter arranged on the photo-detector, wherein the interference filter is configured to selectively collect and transmit light towards the photo-detector based on an angle of incidence of the light towards the interference filter, wherein the interference filter is configured to selectively transmit supercritical angle fluorescence from the sample towards the photo-detector and suppress undercritical angle fluorescence from the sample.

The biosensors provided by the embodiments of the present disclosure utilize the property of spectrum shift caused by the angle-sensitive filter, which is considered a drawback in the prior art, to distinguish between different lights. Therefore, the embodiments of the present disclosure successfully deal with the property of spectrum shift in a novel way.

According to the invention, a biosensor according to claim <NUM> is provided. The biosensor includes a substrate, a first photodiode and a second photodiode, an angle-sensitive filter, and an immobilization layer. The first photodiode and the second photodiode are disposed in the substrate and defining a first pixel and a second pixel, respectively, wherein the first pixel and the second pixel receive a light. The angle-sensitive filter is disposed on the substrate. The immobilization layer is disposed on the angle-sensitive filter.

According to a second aspect of the invention, a method of distinguishing a light according to claim <NUM> is provided. The method includes:
placing an analyte on the aforementioned biosensor; making the analyte emit the light; obtaining a first signal intensity of the first portion of the light and a second signal intensity of the second portion of the light; and distinguishing the light according to the first signal intensity and the second signal intensity.

In some embodiments, a method of distinguishing a light is provided. The method includes: placing an analyte on the aforementioned biosensor, wherein the biosensor further includes a first color filter disposed adjacent to the angle-sensitive filter and corresponding to one of the pixels and the first color filter is irradiated by a third portion of the light; making the analyte emit the light; obtaining a first signal intensity of the first portion of the light, a second signal intensity of the second portion of the light and a third signal intensity of the third portion of the light; and distinguishing the light according to the first signal intensity, the second signal intensity and the third signal intensity.

The present disclosure can be more fully understood by reading the subsequent detailed description and examples with references made to the accompanying drawings, wherein:.

The biosensor of the present disclosure is described in detail in the following description. In the following detailed description, for purposes of explanation, numerous specific details and embodiments are set forth in order to provide a thorough understanding of the present disclosure. The specific elements and configurations described in the following detailed description are set forth in order to clearly describe the present disclosure. It will be apparent, however, that the exemplary embodiments set forth herein are used merely for the purpose of illustration, and the inventive concept may be embodied in various forms without being limited to those exemplary embodiments. In addition, the drawings of different embodiments may use like and/or corresponding numerals to denote like and/or corresponding elements in order to clearly describe the present disclosure. However, the use of like and/or corresponding numerals in the drawings of different embodiments does not suggest any correlation between different embodiments. In addition, in this specification, expressions such as "first material layer disposed on/over a second material layer", may indicate the direct contact of the first material layer and the second material layer, or it may indicate a non-contact state with one or more intermediate layers between the first material layer and the second material layer. In the above situation, the first material layer may not be in direct contact with the second material layer.

In addition, in this specification, relative expressions are used. For example, "lower", "bottom", "higher" or "top" are used to describe the position of one element relative to another. It should be appreciated that if a device is flipped upside down, an element that is "lower" will become an element that is "higher".

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It should be appreciated that, in each case, the term, which is defined in a commonly used dictionary, should be interpreted as having a meaning that conforms to the relative skills of the present disclosure and the background or the context of the present disclosure, and should not be interpreted in an idealized or overly formal manner unless so defined.

In the description, relative terms such as "lower," "upper," "horizontal," "vertical,", "above," "below," "up," "down," "top" and "bottom" as well as derivative thereof (e.g., "horizontally," "downwardly," "upwardly," etc.) should be construed as referring to the orientation as described or as shown in the drawing under discussion. These relative terms are for convenience of description and do not require that the apparatus be constructed or operated in a particular orientation. Terms concerning attachments, coupling and the like, such as "connected" and "interconnected," refer to a relationship wherein structures are secured or attached to one another either directly or indirectly through intervening structures, as well as both movable or rigid attachments or relationships, unless expressly described otherwise.

It should be understood that, although the terms first, second, third etc. may be used herein to describe various elements, components, regions, layers, portions and/or sections, these elements, components, regions, layers, portions and/or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer, portion or section from another element, component, region, layer or section. Thus, a first element, component, region, layer, portion or section discussed below could be termed a second element, component, region, layer, portion or section without departing from the teachings of the present disclosure.

The biosensors provided by the embodiments of the present disclosure utilize the property of spectrum shift caused by the angle-sensitive filter, which is considered a drawback in prior art, to distinguish different lights. Therefore, the embodiments of the present disclosure successfully deal with the property of spectrum shift in a novel way.

This description of the exemplary embodiments is intended to be read in connection with the accompanying drawings, which are to be considered part of the entire written description. The drawings are not drawn to scale. In addition, structures and devices are shown schematically in order to simplify the drawings.

<FIG> illustrates a top view of a biosensor <NUM> in accordance with some embodiments of the present disclosure, and <FIG> illustrate cross-sectional views of the biosensor <NUM> of <FIG> along line A-A' in accordance with some embodiments of the present disclosure. Referring to <FIG>, the biosensor <NUM> includes a substrate <NUM> and pixels <NUM>.

In some embodiments of the present disclosure, the substrate <NUM> is a bulk semiconductor substrate, such as a semiconductor wafer. For example, the substrate <NUM> is a silicon wafer. The substrate <NUM> may include silicon or another elementary semiconductor material such as germanium. In some other embodiments, the substrate <NUM> includes a compound semiconductor. The compound semiconductor may include gallium arsenide, silicon carbide, indium arsenide, indium phosphide, another suitable material, or a combination thereof.

In some embodiments, the substrate <NUM> includes a semiconductor-on-insulator (SOI) substrate. The SOI substrate may be fabricated using a separation by implantation of oxygen (SIMOX) process, a wafer bonding process, another applicable method, or a combination thereof. In some embodiments, the substrate <NUM> is an un-doped substrate.

As illustrated in <FIG>, the biosensor <NUM> includes photodiodes <NUM>, an angle-sensitive filter <NUM> and an immobilization layer <NUM>. The photodiodes <NUM> are disposed in the substrate <NUM>, and the photodiodes <NUM> define pixels <NUM>. The pixels <NUM> receive a light. The angle-sensitive filter <NUM> is disposed on the substrate <NUM>. The immobilization layer <NUM> is disposed on the angle-sensitive filter <NUM>. The angle-sensitive filter <NUM> can be a shortpass filter, a bandpass filter, a longpass filter or a multiple bandpass filter. The angle-sensitive filter <NUM> can be a dielectric interference filter with alternating deposition of dielectric materials with high and low refractive indices. If the refractive index is greater than about <NUM> at visible light wavelength, the refractive index is considered a high refractive index. The dielectric materials with a high refractive index may include Nb<NUM>O<NUM>, Ta<NUM>O<NUM>, TiO<NUM>, Si<NUM>N<NUM>, Al<NUM>O<NUM>, SiH, or a combination thereof. If the refractive index is smaller than about <NUM> at visible light wavelength, the refractive index is considered a low refractive index. The dielectric materials with a low refractive index may include SiO<NUM> Al<NUM>O<NUM>, organic polymer, air or a combination thereof. Alternatively, the angle-sensitive filter <NUM> can be a plasmonic filter or a dielectric metasurface structure.

Referring to <FIG>, in some embodiments, the biosensor <NUM> may include an excitation light rejection filter <NUM> disposed on the angle-sensitive filter <NUM>. To be specific, the excitation light rejection filter <NUM> is disposed between the angle-sensitive filter <NUM> and the immobilization layer <NUM>. The excitation light rejection filter <NUM> is an interference filter embedded with metal layers and abbreviated as metal multifilm. Since the metal layers are included in the interference filter, a thickness of the metal multifilm <NUM> may be thinner than the general dielectric interference filter without metal layers embedded. The thickness of metal multifilm may be from <NUM> to <NUM> and have a compatible optical density (OD) with the general dielectric interference filter. The metal layers may include Ag, Au, Al, Cu or a combination thereof.

Referring to <FIG>, in some embodiments, the biosensor <NUM> may include an aperture structure <NUM>. The aperture structure <NUM> is embedded in the immobilization layer <NUM>. The aperture structure <NUM> includes an opening so that the light can be controlled to irradiate certain areas of the substrate <NUM>, and cross-talk can be avoided. The opening corresponds to one pixel <NUM>. In some embodiments, the opening may not be located directly above the center of one pixel. It should be appreciated that the opening is located according to actual needs. The aperture structure <NUM> may include an opaque material. The opaque material may include Ag, Al, Au, Cu, Nb, Ni, Ti, W, an alloy thereof or a hybrid material thereof.

Referring to <FIG>, in some embodiments, the biosensor <NUM> may include a shielding layer <NUM> disposed on the substrate <NUM>. The shielding layer <NUM> surrounds the angle-sensitive filter <NUM> to isolate different areas of the angle-sensitive filter <NUM>. For example, the shielding layer <NUM> isolates the angle-sensitive filter <NUM> every two adjacent pixels <NUM>. The shielding layer <NUM> may include a material with a high reflective index. If the shielding layer can reflect a light greater than about <NUM>%, the material or structure of the shielding layer is considered to have a high reflective index. The material of the shielding layer <NUM> may include Ag, Al, Au, Cu, Nb, Ni, Ti, W, Ta<NUM>O<NUM>, Nb<NUM>O<NUM>, Al<NUM>O<NUM>, TiO<NUM>, SiH, Si<NUM>N<NUM>, air, vacuum, an alloy thereof or a hybrid material thereof.

Referring to <FIG>, in some embodiments, the biosensor <NUM> may include a waveguide <NUM>. The waveguide <NUM> is embedded in the immobilization layer <NUM>. The waveguide <NUM> corresponds to one pixel <NUM>. In some embodiments, the waveguide <NUM> may not be located directly above the center of one pixel. It should be appreciated that the waveguide is located according to actual needs. In these embodiments, the waveguide <NUM> is linear so that the light can be controlled to irradiate biosamples in sequence and sequentially generate emission light to the certain areas of substrate <NUM>, and cross-talk can be avoided. The waveguide <NUM> includes a material with a high refractive index. If the refractive index is greater than about <NUM> at visible light wavelength, the refractive index is considered a high refractive index. The material with a high refractive index may include Al<NUM>O<NUM>, Ta<NUM>O<NUM>, Nb<NUM>O<NUM>, TiO<NUM>, Si<NUM>N<NUM>, or polymer.

Referring to <FIG>, in some embodiments, the waveguide <NUM> may be disposed on the aperture structure <NUM>. To be specific, the aperture structure <NUM> and the waveguide <NUM> are embedded in the immobilization layer <NUM>. In these embodiments, the waveguide <NUM> is a continuous layer, but the aperture structure <NUM> confines the pathway of emission light. Therefore cross-talk can be also avoided.

<FIG> illustrates a top view of a biosensor <NUM> in accordance with some embodiments of the present disclosure. <FIG> illustrate cross-sectional views of the biosensor <NUM> of <FIG> along line A-A' in accordance with some embodiments of the present disclosure.

Referring to <FIG>, one of the differences between the biosensor <NUM> and biosensor <NUM> is that the biosensor <NUM> further includes a first color filter 120a disposed on the substrate <NUM>. To be specific, the first color filter 120a is disposed adjacent to the angle-sensitive filter <NUM> and corresponds to one pixel <NUM>. For example, two first color filters 120a are separated by the angle-sensitive filter <NUM> that covers two pixels <NUM> in a direction X. Two first color filters 120a are separated by the angle-sensitive filter <NUM> that covers one pixel <NUM> in a direction Y perpendicular to the direction X. The coverage area of the angle-sensitive filter <NUM> is equal to or larger than that of the first color filter 120a. In other words, an area of a projection of angle-sensitive filter <NUM> is equal to or larger than that of a projection of the first color filter 120a.

Referring to <FIG>, one of the differences between <FIG> and <FIG> is that the first color filter 120a is disposed on the substrate <NUM>.

Referring to <FIG>, one of the differences between <FIG> and <FIG> is that the first color filter 120a is disposed on the substrate <NUM>. The opening of the aperture structure <NUM> corresponds to a pixel <NUM> that is covered by the angle-sensitive filter <NUM>. Referring to <FIG>, one of the differences between <FIG> and <FIG> is that the first color filter 120a is disposed on the substrate <NUM>. The shielding layer <NUM> surrounds one first color filter 120a and one angle-sensitive filter <NUM> that corresponds to two adjacent pixels <NUM>.

The difference between the biosensor <NUM> and biosensor <NUM> is that the biosensor <NUM> includes a second color filter 120b disposed on the substrate <NUM>. To be specific, the second color filter 120b is disposed adjacent to the angle-sensitive filter <NUM> and corresponds to one of the pixels <NUM>. For example, the angle-sensitive filter <NUM> is sandwiched by the first color filter 120a and the second color filter 120b in the direction X. the angle-sensitive filter <NUM> is sandwiched by two first color filters 120a or two second color filters 120b in the direction Y. The coverage area of the angle-sensitive filter <NUM> is equal to or larger than that of the first color filter 120a or the second color filter 120b. In other words, an area of a projection of angle-sensitive filter <NUM> is equal to or larger than that of a projection of the first color filter 120a or the second color filter 120b.

Referring to <FIG>, one of the differences between <FIG> and <FIG> is that the second color filter 120b is disposed on the substrate <NUM>.

Referring to <FIG>, one of the differences between <FIG> and <FIG> is that the second color filter 120b is disposed on the substrate <NUM>. The opening of the aperture structure <NUM> corresponds to a pixel <NUM> that is covered by the angle-sensitive filter <NUM>. Referring to <FIG>, one of the differences between <FIG> and <FIG> is that the second color filter 120b is disposed on the substrate <NUM>. The shielding layer <NUM> is disposed between a set of the angle-sensitive filter <NUM> and the first color filter 120a and a set of the angle-sensitive filter <NUM> and the second color filter 120b in the cross-sectional views.

Referring to <FIG>, in some embodiments, the first color filter 120a and the second color filter 120b may have areas equal to or smaller than that of one of the pixels <NUM>. When the first and second color filters are smaller than the angle-sensitive filter, the organic filter material may be fully embedded in the surrounding angle-sensitive filter to provide a robust chemical and mechanical resistance. Moreover, the shrinked size of the organic filter may ensure that the emission light from a biosample can pass through the angle-sensitive filter to the diagonal photodiode without the discontinuity due to the first and second color filters.

According to the different compositions of the filter, the inventive concept is described in detail below.

In the aspect, the biosensor <NUM> includes the angle-sensitive filter <NUM>. <FIG> illustrates an arrangement of analytes 122a on the biosensor <NUM>. <FIG> illustrate a cross-sectional view of <FIG> along line A-A'. In the embodiment, it is assumed that the light emitted by one analyte can reach the pixels that are one pixel away from the pixel under the light. In other words, the closest eight pixels surround the pixel under the light. For example, as illustrated in <FIG>, the pixel <NUM> is under the light emitted by the analyte 122a1, and the pixels <NUM>-<NUM> are the pixels that are only one pixel away from the pixel <NUM>. The light emitted by the analyte 122a1 can reach the pixels <NUM>-<NUM>.

The analytes 122a are placed on the biosensor <NUM>. One analyte 122a has an area equal to or smaller than that of one pixel <NUM>.

In the case of fluorescence, an excitation light <NUM> irradiates the analytes 122a. The excitation light <NUM> may move from one side of the biosensor <NUM> to the opposite side so that the analyte 122a will be excited and emit a light L in order, but not simultaneously. For example, the excitation light <NUM> may move along a direction X. In the embodiment, the direction X is a direction from a left side to a right side of the biosensor <NUM> in top view, as shown in <FIG>.

Therefore, the analytes 122a may be placed on the biosensor <NUM> at one-pixel intervals to avoid cross-talk. In other words, any two adjacent analytes 122a are spaced apart from each other by one pixel <NUM>.

Referring to <FIG>, the analytes 122a are excited by the excitation light <NUM> and emit a light L. The excitation light rejection filter <NUM> is disposed to block the excitation light <NUM> so that the excitation light <NUM> can be prevented from entering the substrate <NUM> and being absorbed by the photodiodes <NUM>.

The term "incident angle" refers to an angle between the incident light and the normal line of the angle-sensitive filter. A first portion L1 of the light L enters the angle-sensitive filter <NUM> at a first incident angle θ1. A pixel that receives the first portion L1 of the light L is a first pixel 104a. The first incident angle θ1 is an angle from <NUM> degrees to the maximum incident angle of the light L that can be received in the first pixel 104a.

A second portion L2 of the light L enters the angle-sensitive filter <NUM> at a second incident angle θ2. A pixel that receives the second portion L2 of the light L is a second pixel 104b. The second incident angle θ2 is an angle from the maximum incident angle of the first incident angle θ1 to the maximum incident angle of the second portion L2 of the light L that can be received in the second pixel 104b. The first incident angle θ1 is smaller than the second incident angle θ2.

In some embodiments, the first incident angle θ1 is from <NUM> degrees to <NUM> degrees, and the second incident angle θ2 is from <NUM> degrees to <NUM> degrees. In some embodiments, the first incident angle θ1 is from <NUM> degrees to <NUM> degrees, and the second incident angle θ2 is from <NUM> to <NUM> degrees. Since the first incident angle θ1 is smaller than the second incident angle θ2, the second portion L2 will be spectrum-shifted more significantly than the first portion L1.

<FIG> illustrates a cross-sectional view of <FIG> in accordance with other embodiments of the present disclosure. The difference between the embodiments of <FIG> is that the analyte 122b has a greater area than that of one pixel <NUM>. The analyte 122b may be a cell, a tissue, an organ and so on.

Although the analyte 122b has a greater area than that of one pixel <NUM>, it should be understood that the same concept as recited in the embodiment of <FIG> and <FIG> can also be applied to the embodiment of <FIG> after reading the following exemplary embodiments, which will not be repeated for the sake of brevity. Furthermore, the arrangement is merely an example. One of ordinary skill in the art can place the analytes to meet practical needs.

<FIG> illustrates a top view of an arrangement of analytes 122a on the biosensor <NUM> in accordance with some embodiments of the present disclosure. <FIG> illustrate cross-sectional views of <FIG> along line A-A'. The difference between the embodiments of <FIG> and <FIG> is the arrangement of the analytes 122a.

In the case of bioluminescence, since the analytes 122a do not need to be excited by an excitation light, the excitation light rejection filter <NUM> can be omitted as shown in <FIG>. Since all the analytes 122a simultaneously emit the light L, the analytes 122a may be placed on the biosensor <NUM> at a lower density than that in the embodiment of <FIG> to avoid cross-talk. For example, as illustrated in <FIG>, the analytes 122a are arranged at two-pixel intervals along the direction X and at one-pixel intervals in a direction Y perpendicular to the direction X. The arrangement is merely an example. One of ordinary skill in the art can arrange the analytes to meet practical needs.

Although the analytes 122a are arranged at different intervals than those of <FIG> and <FIG> and the light L is generated by bioluminescence, it should be understood that the same concept as recited in the embodiment of <FIG> and <FIG> can also be applied to the embodiment of <FIG> after reading the following exemplary embodiments, which will not be repeated for the sake of brevity. Furthermore, the arrangement is merely an example. One of ordinary skill in the art can place the analytes to meet practical needs.

<FIG> illustrates a cross-sectional view of <FIG> in accordance with other embodiments of the present disclosure. The difference between the embodiments of <FIG> is that the analytes 122a generate the light L by fluorescence.

In the case of fluorescence, since an excitation light is needed to excite the analytes, the excitation light rejection filter <NUM> is disposed as shown in <FIG>. In the embodiment, since all the analyte 122a are excited simultaneously by the excitation light <NUM> and emit the light L simultaneously, the analytes 122a may be placed on the biosensor <NUM> as in <FIG>.

Although the analytes 122a are placed in a different arrangement than that of <FIG> and <FIG>, it should be understood that the same concept as recited in the embodiment of <FIG> and <FIG> can also be applied to the embodiment of <FIG> after reading the following exemplary embodiments, which will not be repeated for the sake of brevity. Furthermore, the arrangement is merely an example. One of ordinary skill in the art can place the analytes to meet practical needs.

<FIG> illustrates a top view of an arrangement of analytes 122a on the biosensor <NUM> in accordance with some embodiments of the present disclosure. <FIG> illustrate cross-sectional views of <FIG> along line A-A', wherein <FIG> represents an example of bioluminescence and <FIG> represents an example of fluorescence. The differences between the embodiments of <FIG> and <FIG> are that the biosensor <NUM> includes the aperture structure <NUM> and that the light L is emitted at the same time.

As illustrated in <FIG>, the aperture structure <NUM> includes openings that correspond to each analyte 122a, so that the light can be further controlled to irradiate only certain areas of the substrate <NUM>.

To be specific, referring to <FIG>, the aperture structure <NUM> prevents the light L emitted by the analyte 122a1 from reaching the pixel <NUM>, and allows the light L emitted by the analyte 122a2 adjacent to the analyte 122a1 to reach the pixel <NUM>. Therefore, the photodiode <NUM> in the pixel <NUM> only receives the light from analyte 122a2, thereby avoiding cross-talk. Therefore, the analytes 122a may be placed on the biosensor <NUM> at one-pixel intervals to avoid cross-talk. In other words, any two adjacent analytes 122a are spaced apart from each other by one pixel <NUM>.

In some embodiments, from the top view of <FIG>, the shape of the openings may be rectangular, circular, or triangular, but it is not limited thereto.

Referring to <FIG>, the difference between the embodiments of <FIG> is that the biosensor <NUM> of <FIG> further includes the excitation light rejection filter <NUM>.

Although the biosensor <NUM> of <FIG> includes the aperture structure <NUM>, it should be understood that the same concept as recited in the embodiment of <FIG> and <FIG> can also be applied to the embodiment of <FIG> after reading the following exemplary embodiments, which will not be repeated for the sake of brevity. Furthermore, the arrangement is merely an example. One of ordinary skill in the art can place the analytes to meet practical needs.

<FIG> illustrates a top view of an arrangement of analytes 122a on the biosensor <NUM> in accordance with some embodiments of the present disclosure. <FIG> illustrate cross-sectional views of <FIG> along line A-A', wherein <FIG> represents an example of bioluminescence and <FIG> represents an example of fluorescence. The differences between the embodiments of <FIG> and <FIG> are that the biosensor <NUM> includes the shielding layer <NUM> and that the light L is emitted at the same time.

Since the shielding layer <NUM> can reflect the light L, the first pixel 104a may receive the first portion L1' of the light L that enters the angle-sensitive filter <NUM> at a first incident angle θ1' greater than the first incident angle θ1 of the embodiments in <FIG>. The second pixel 104b may also receive the second portion L2' of the light L that enters the angle-sensitive filter <NUM> at a second incident angle θ2' greater than the second incident angle θ2 of the embodiments in <FIG>. For example, the first incident angle θ1' may be from <NUM> degrees to <NUM> degrees, and the second incident angle θ2' may be from <NUM> degrees to <NUM> degrees. However, it should be appreciated that the first incident angle θ1' and the second incident angle θ2' are determined according to the heights of the analyte, shielding layer and photodiode.

Although the biosensor <NUM> of <FIG> includes the shielding layer <NUM>, it should be understood that the same concept as recited in the embodiment of <FIG> and <FIG> can also be applied to the embodiments of <FIG> after reading the following exemplary embodiments, which will not be repeated for the sake of brevity. Furthermore, the arrangement is merely an example. One of ordinary skill in the art can place the analytes to meet practical needs.

<FIG> is analytical graphs of emission or transmission versus wavelength in accordance with some embodiments of the present disclosure. 104A represents the wavelength of the light that enters the angle-sensitive filter <NUM> at the first incident angle θ1 or θ1' and is received by the first pixel 104a. 104B represents the wavelength of the light that enters the angle-sensitive filter <NUM> at the second incident angle θ2 or θ2' and is received by the second pixel 104b. Alexa <NUM> and Alexa <NUM> are two dyes that emit two different lights. A first signal intensity of the first portion L1 of the light L is obtained from the first pixel 104a. A second signal intensity of the second portion L2 of the light L is obtained from the second pixel 104b. The overlapping area between the area under curve (AUC) of 104A and the AUC of Alexa <NUM> represents the first signal intensity of Alexa <NUM> while the overlapping area between the AUC of 104B and the AUC of Alexa <NUM> represents the second signal intensity of Alexa <NUM>.

For the same concept, the Alexa <NUM> has a first signal intensity and a second signal intensity. A first threshold can be set for the first signal intensities. A second threshold can be set for the second signal intensities. The first signal intensities are defined as Pass or No, depending on whether the first signal intensities are higher or lower than the first threshold, respectively. For example, when the first signal intensity of Alexa <NUM> is higher than the first threshold, the first signal intensity of Alexa <NUM> is defined as Pass. When the first signal intensity of Alexa <NUM> is lower than the first threshold, the first signal intensity of Alexa <NUM> is defined as No..

For the same concept, the second signal intensities can be defined as Pass or No depending on whether the second signal intensities are higher or lower than the second threshold, respectively.

The first threshold and the second threshold may be set according to actual situations. Table <NUM> below is made according to the concept described above and in <FIG>, in which the first signal intensity is denoted by 104a and the second signal intensity is denoted by 104b.

According to a combination of the definitions of the first signal intensity and the second signal intensity, the two dyes can be distinguished. For example, in the embodiment where the angle-sensitive filter is a shortpass filter, the first signal intensity of Alexa <NUM> is defined as Pass and the second signal intensity of Alexa <NUM> is defined as Pass, and the first signal intensity of Alexa <NUM> is defined as Pass and the second signal intensity of Alexa <NUM> is defined as No. When the combination of the first signal intensity and the second signal intensity is Pass and Pass (or Pass and No), it can be learned that the light is Alexa <NUM> (or Alexa <NUM>).

In the embodiment where the angle-sensitive filter is a bandpass filter, for the same concept as described above, the light can be distinguished.

In the embodiment where the angle-sensitive filter is a longpass filter, for the same concept as described above, the light can be distinguished.

Alternatively, first signal intensity ratios of the second signal intensity to the first signal intensity (denoted by 104b/104a) can also be calculated to distinguish the two different dyes, namely two different lights. The first signal intensity ratios are defined as H or L depending on whether the first signal intensity ratio is higher or lower than a predetermined ratio. For example, when the first signal intensity ratio of 104b/104a of Alexa <NUM> is higher than the predetermined ratio, the first signal intensity ratio of 104b/104a of Alexa <NUM> is defined as H. When the first signal intensity ratio of 104b/104a of Alexa <NUM> is lower than the predetermined ratio, the first signal intensity ratio of 104b/104a of Alexa <NUM> is defined as L.

Table <NUM> below is made according to the concept described above and in <FIG>, in which the first signal intensity is denoted by 104a and the first signal intensity ratio is denoted by 104b/104a.

According to the definitions of the first signal intensity ratio of 104b/104a, the two dyes can be distinguished. For example, in the embodiment where the angle-sensitive filter is a shortpass filter, the first signal intensity ratio of 104b/104a of Alexa <NUM> is higher than the predetermined ratio and the first signal intensity ratio of 104b/104a of Alexa <NUM> is defined as H. The first signal intensity ratio of 104b/104a of Alexa <NUM> is lower than the predetermined ratio and the first signal intensity ratio of 104b/104a of Alexa <NUM> is defined as L. As a result, if the definition of the first signal intensity ratio of 104b/104a is H, it can be learned that the light is emitted by Alexa <NUM>. If the definition of the first signal intensity ratio of 104b/104a is L, it can be learned that the light is emitted by Alexa <NUM>.

<FIG> is an analytical graph of emission or transmission versus wavelength in accordance with some embodiments of the present disclosure. 104A represents the wavelength of the light that enters the angle-sensitive filter <NUM> at the first incident angle θ1 or θ1' and is received by the first pixel 104a. 104B represents the wavelength of the light that enters the angle-sensitive filter <NUM> at the second incident angle θ2 or θ2' and is received by the second pixel 104b. EYFP, PE, FM2-<NUM> and eFluor <NUM> are four dyes that emit four different lights.

Table <NUM> below is made according to the concept previously discussed and <FIG>, in which the first signal intensity is denoted by 104a and the second signal intensity is denoted by 104b.

According to a combination of the definitions of the first intensity and the second intensity, the four dyes can be distinguished based on the same concept as described above.

Alternatively, the first signal intensity ratios of the second signal intensity to the first signal intensity (denoted by 104b/104a) can also be calculated to distinguish the four different dyes, namely four different lights. <FIG> is analytical graphs of emission or transmission versus wavelength in accordance with some embodiments of the present disclosure. The leftmost graph is an embodiment where the angle-sensitive filter is a shortpass filter. The middle graph is an embodiment where the angle-sensitive filter is a bandpass filter. The rightmost graph is an embodiment where the angle-sensitive filter is a longpass filter. 104A represents the wavelength of the light that enters the angle-sensitive filter <NUM> at the first incident angle θ1 or θ1' and is received by the first pixel 104a. 104B represents the wavelength of the light that enters the angle-sensitive filter <NUM> at the second incident angle θ2 or θ2' and is received by the second pixel 104b. Alexa <NUM>, eFluor <NUM>, Alexa <NUM> and Alexa <NUM> are four dyes that emit four different lights. The first signal intensity ratios of 104b/104a are defined as H or L depending on whether the first signal intensity ratio is higher or lower than a predetermined ratio. Then in the groups which are defined as H, the first signal intensities are defined as H or L depending on whether the first signal intensity is higher or lower than a first threshold, or the second signal intensities are defined as H or L depending on whether the second signal intensity is higher or lower than a second threshold.

Table <NUM> below is made according to the concept described above and in <FIG>, in which the first signal intensity is denoted by 104a, the second signal intensity is denoted by 104b, and the first signal intensity ratio is denoted by 104b/104a.

According to the definition of the first signal intensity ratio of 104b/104a and the definition of the first signal intensity or the second signal intensity, the four dyes can be distinguished. For example, in the embodiment where the angle-sensitive filter is a shortpass filter, the first signal intensity ratio of 104b/104a of Alexa <NUM> and the first signal intensity ratio of eFluor <NUM> are higher than the predetermined ratio and the first signal intensity ratio of 104b/104a of Alexa <NUM> and the first signal intensity ratio of eFluor <NUM> are defined as H. The first signal intensity ratio of 104b/104a of Alexa <NUM> and the first signal intensity ratio of 104b/104a of Alexa <NUM> are lower than the predetermined ratio and the first signal intensity ratio of 104b/104a of Alexa <NUM> and the first signal intensity ratio of 104b/104a of Alexa <NUM> are defined as L. Then in Group H, the first signal intensity of Alexa <NUM> is higher than the first threshold and the first signal intensity of Alexa <NUM> is defined as H. The first signal intensity of eFluor <NUM> is lower than the first threshold and the first signal intensity of eFluor <NUM> is defined as L. In Group L, the first signal intensity of Alexa <NUM> is higher than the other first threshold and the first signal intensity of Alexa <NUM> is defined as H. The first signal intensity of Alexa <NUM> is lower than the other first threshold and the first signal intensity of Alexa <NUM> is defined as L. As a result, if the definition of the first signal intensity ratio of 104b/104a is H and the definition of the first signal intensity is H, it can be learned that the light is emitted by Alexa <NUM>. Therefore, the light can be distinguished according to the same concept as described above.

Therefore, one kind of a filter is sufficient to distinguish at most four different lights. On the contrary, the existing biosensor still required more than one kind of a filter to distinguish different lights.

In the aspect, the biosensor <NUM> includes the angle-sensitive filter <NUM> and the first color filter 120a. <FIG> illustrates an arrangement of analytes 122a on the biosensor <NUM>. <FIG> illustrate a cross-sectional view of <FIG> along line A-A'. In the embodiment, it is assumed that the light emitted by one analyte can reach the pixels that are one pixel away from the pixel under the light. In other words, the closest eight pixels surround the pixel under the light. For example, as illustrated in <FIG>, the pixel <NUM> is under the light emitted by the analyte 122a1, and the pixels <NUM>-<NUM> are the pixels that are only one pixel away from the pixel <NUM>. The light emitted by the analyte 122a1 can reach the pixels <NUM>-<NUM>.

As shown in <FIG>, the first color filters 120a are separated by two pixels that are covered by the angle-sensitive filter <NUM> in a direction X, and the first color filters <NUM> are separated by one pixel that is covered by the angle-sensitive filter <NUM> in a direction Y perpendicular to the direction X.

The analytes 122a are placed on the biosensor <NUM>. One analyte 122a has an area equal to or smaller than that of one pixel.

In the case of fluorescence, an excitation light <NUM> irradiates the analytes 122a or 122b (not shown). The excitation light <NUM> moves from one side of the biosensor <NUM> to the opposite side so that the analyte 122a will be excited and emit a light L in order, but not simultaneously. For example, the excitation light <NUM> may move along the direction X. In the embodiment, the direction X is a direction from a left side to a right side of the biosensor <NUM> in top view, as shown in <FIG>.

Referring to <FIG>, the analytes 122a are excited by the excitation light <NUM> and emit a light L. The excitation light rejection filter <NUM> is disposed to block the excitation light <NUM> so that the excitation light <NUM> can be prevented from entering the substrate <NUM> and being absorbed by the photodiodes <NUM>. A first portion L1 of the light L enters the angle-sensitive filter <NUM> at a first incident angle θ1. A pixel that receives the first portion L1 of the light L is a first pixel 104a. The first incident angle θ1 is an angle from <NUM> degrees to the maximum incident angle of the first portion L1 of the light L that can be received in the first pixel 104a.

A second portion L2 of the light L enters the angle-sensitive filter <NUM> at a second incident angle θ2. A pixel that receives the second portion L2 of the light L is a second pixel 104b. The second incident angle θ2 is an angle from the maximum incident angle of the first portion L1 of the light L that can be received in the first pixel 104a to the maximum incident angle of the second portion L2 of the light L that can be received in the second pixel 104b. The first incident angle θ1 is smaller than the second incident angle θ2.

A third portion L3 of the light L enters the first color filter 120a at a third incident angle θ3. A pixel that receives the third portion L3 of the light L is a third pixel 104c. The third incident angle θ3 is angle from the maximum incident angle of the first portion L1 of the light L that can be received in the first pixel 104a to the maximum incident angle of the third portion L3 of the light L that can be received in the third pixel 104c.

In some embodiments, the first incident angle θ1 is from <NUM> degrees to <NUM> degrees, the second incident angle θ2 is from <NUM> degrees to <NUM> degrees, and the third incident angle θ3 is from <NUM> degrees to <NUM> degrees. In some embodiments, the first incident angle θ1 is from <NUM> to <NUM> degrees, the second incident angle θ2 is from <NUM> degrees to <NUM> degrees, and the third incident angle θ3 is from <NUM> degrees to <NUM> degrees. Since the first incident angle θ1 is smaller than the second incident angle θ2, the second portion L2 will be spectrum-shifted more significantly than the first portion L1.

<FIG> illustrates a top view of an arrangement of analytes 122a on the biosensor <NUM> in accordance with some embodiments of the present disclosure. <FIG> illustrate cross-sectional views of <FIG> along line A-A', wherein <FIG> represents an example of bioluminescence and <FIG> represents an example of fluorescence. Some of the differences between the embodiments of <FIG> and <FIG> are that the biosensor <NUM> includes the aperture structure <NUM> and that the analytes 122a emit the light L at the same time.

As illustrated in <FIG>, the aperture structure <NUM> includes openings that correspond to one analyte 122a, respectively, so that the light L can be further controlled to irradiate only certain areas of the substrate <NUM>.

To be specific, referring to <FIG>, the aperture structure <NUM> prevents the light L emitted by the analyte 122a2 from reaching the pixel 104b, and allows the light L emitted by the analyte 122a1 adjacent to the analyte 122a2 to reach the pixel 104b. Therefore, the photodiode <NUM> in the pixel 104b only receives the light from analyte 122a1, thereby avoiding cross-talk. Therefore, the analytes 122a may be placed on the biosensor <NUM> at two-pixel intervals to avoid cross-talk in the X-direction. The aperture opening in the Y-direction may be smaller than that in the X-direction, and therefore the analytes 122a may be placed on the biosensor <NUM> at one-pixel intervals to avoid cross-talk in the Y-direction. In other words, any two adjacent analytes 122a are spaced apart from each other by one pixel <NUM> in the Y-direction and by two pixels <NUM> in the X-direction.

In some embodiments, from the top view of <FIG>, the shape of the openings may be a rectangle, a circle, or a triangle, but it is not limited thereto.

Although the biosensors <NUM> of <FIG> include the aperture structure <NUM>, it should be understood that the same concept as recited in the embodiment of <FIG> and <FIG> can also be applied to the embodiments of <FIG>and <FIG>, which will not be repeated for the sake of brevity.

<FIG> illustrates a top view of an arrangement of analytes 122a on the biosensor <NUM> in accordance with some embodiments of the present disclosure. <FIG> illustrate cross-sectional views of <FIG> along line A-A', wherein <FIG> represents an example of bioluminescence and <FIG> represents an example of fluorescence. In the embodiment, it is assumed that the light emitted by the analytes can reach the pixels that are one pixel away from the pixel under the light. For example, as illustrated in <FIG>, the pixel <NUM> is under the light L emitted by the analyte 122a1, and the pixels <NUM>-<NUM> are the pixels that are only one pixel away from the pixel <NUM>. Some of the differences between the embodiments of <FIG> and <FIG> are that the biosensor <NUM> includes the shielding layer <NUM> and that the analytes 122a emit the light L at the same time.

Since the shielding layer <NUM> can reflect the light L, the second pixel 104b may receive the second portion L2' of the light L that enters the angle-sensitive filter <NUM> at a second incident angle θ2' greater than the second incident angle θ2 of the embodiments in <FIG>. The third pixel 104c may also receive the third portion L3' of the light L that enters the first color filter 120a at a third incident angle θ3' greater than the third incident angle θ3 of the embodiments in <FIG>. For example, the first incident angle θ1 may be from <NUM> degrees to <NUM> degrees. The second incident angle θ2' may be from <NUM> degrees to <NUM> degrees. The third incident angle θ3' may be from <NUM> degrees to <NUM> degrees. However, it should be appreciated that the first incident angle <NUM>', the second incident angle θ2' and the third incident angle θ3' are determined according to the heights of the analyte, shielding layer and photodiode.

<FIG> is analytical graphs of emission or transmission versus wavelength in accordance with some embodiments of the present disclosure. 104A represents the wavelength of the light that enters the angle-sensitive filter <NUM> at the first incident angle θ1 or θ1' and is received by the first pixel 104a. 104B represents the wavelength of the light that enters the angle-sensitive filter <NUM> at the second incident angle θ2 or θ2' and is received by the second pixel 104b. 104C represents the wavelength of the light that enters the first color filter at the third incident angle θ3 or θ3' and is received by the third pixel 104c. Alexa <NUM>, Alexa <NUM> and Alexa <NUM> are three dyes that emit three different lights.

For the same concept as described previously, the signal intensities can be defined as Pass or No. The first threshold, the second threshold and the third threshold may be set according to actual situations. Table <NUM> below is made according to the concept described above and in <FIG>, in which the first signal intensity is denoted by 104a, the second signal intensity is denoted by 104b, and the third signal intensity is denoted by 104c.

According to a combination of the definitions of the first signal intensity, the second signal intensity and the third signal intensity, the three dyes can be distinguished as previously described.

Although there are only three dyes in the embodiments, since there are eight combinations of definitions of the first signal intensity, the second signal intensity and the third signal intensity, it should be understood that at most eight dyes can be distinguished.

Alternatively, first signal intensity ratios of the first signal intensity to the third signal intensity (denoted by 104a/104c), and second signal intensity ratios of the second signal intensity to the third signal intensity (denoted by 104b/104c) can also be calculated to distinguish the three different dyes, namely three different lights. A graph is plotted according to the first signal intensity ratio and the second signal intensity ratio. To be specific, a cluster distribution graph of the first signal intensity ratio vs. the second signal intensity ratio is plotted. For example, <FIG> is analytical graphs of the first signal intensity ratio vs. the second signal intensity ratio plotted in accordance with some embodiments of the present disclosure. The X-axis represents the first signal intensity ratio. The Y-axis represents the second signal intensity ratio. Alexa <NUM> is represented by diamond. Alexa <NUM> is represented by square. Alexa <NUM> is represented by triangle.

In the embodiment where the angle-sensitive filter is a shortpass filter, the data points of Alexa <NUM>, Alexa <NUM> and Alexa <NUM> are clustered at different positions in the graph, respectively. As a result, if a dye is located at the position where the data points of Alexa <NUM> are clustered, it can be learned that the light is emitted by Alexa <NUM>. If a dye is located at the position where the data points of Alexa <NUM> are clustered, it can be learned that the light is emitted by Alexa <NUM>. If a dye is located at the position where the data points of Alexa <NUM> are clustered, it can be learned that the light is emitted by Alexa <NUM>.

In the embodiment where the angle-sensitive filter is a bandpass filter, the data points of Alexa <NUM>, Alexa <NUM> and Alexa <NUM> are clustered at different positions at the graph, respectively. As a result, for the same concept as described above, a dye can be distinguished depending on where the dye is located in the cluster distribution plot.

In the embodiment where the angle-sensitive filter is a longpass filter, the data points of Alexa <NUM>, Alexa <NUM> and Alexa <NUM> are clustered at different positions at the graph, respectively. As a result, for the same concept as described above, a dye can be distinguished depending on where the dye is located in the cluster distribution plot.

Although there are only three dyes in the embodiments, it should be understood that more than three dyes can be distinguished.

In the aspect, the biosensor <NUM> includes the angle-sensitive filter <NUM>, the first color filter 120a and the second color filter 120b. <FIG> illustrates an arrangement of analytes 122a on the biosensor <NUM>. <FIG> illustrates a cross-sectional view of <FIG> along line A-A'. <FIG> illustrates a cross-sectional view of <FIG> along line B-B'. <FIG> illustrates a cross-sectional view of <FIG> along line C-C'. In the embodiment, it is assumed that the light emitted by one analyte can reach the pixels that are one pixel away from the pixel under the light. In other words, the closest eight pixels surround the pixel under the light. For example, as illustrated in <FIG>, the pixel <NUM> is under the light emitted by the analyte 122a1, and the pixels <NUM>-<NUM> are the pixels that are only one pixel away from the pixel <NUM>. The light emitted by the analyte 122a1 can reach the pixels <NUM>-<NUM>.

As shown in <FIG>, the first color filters 120a, the angle-sensitive filter <NUM>, the second color filters 120b are disposed on the substrate <NUM> in an order of first color filter/angle-sensitive filter/second color filter/angle-sensitive filter in a direction X. The first color filters 120a are separated by one pixel that is covered by the angle-sensitive filter <NUM> in a direction Y perpendicular to the direction X. The second color filters 120b are separated by one pixel that is covered by the angle-sensitive filter <NUM>. In other words, the first color filters 120a are disposed on the substrate <NUM> in an order of first color filter/angle-sensitive filter in the direction Y, and the second color filters 120b are disposed on the substrate <NUM> in an order of second color filter/angle-sensitive filter in the direction Y. A set of pixels S that includes one pixel that is covered by the first color filter 120a, one pixel that is covered by the second color filter 120b, and two pixels that are covered by the angle-sensitive filter <NUM>. The two pixels that are covered by the angle-sensitive filter are not separated by the first color filter 120a or the second color filter 120b.

Referring to <FIG>, the analytes 122a are excited by the excitation light <NUM> and emit a light L. The excitation light rejection filter <NUM> is disposed to block the excitation light <NUM> so that the excitation light <NUM> can be prevented from entering the substrate <NUM> and being absorbed by the photodiodes <NUM>. A first portion L1 of the light L enters the angle-sensitive filter <NUM> at a first incident angle θ1, as shown in <FIG>.

A pixel that receives the first portion L1 of the light L is a first pixel 104a. The first incident angle θ1 is an angle from <NUM> degrees to the maximum incident angle of the first portion L1 of the light L that can be received in the first pixel 104a.

A fourth portion L4 of the light L enters the second color filter 120b at a fourth incident angle θ4, as shown in <FIG>. A pixel that receives the fourth portion L4 of the light L is a fourth pixel 104d. The fourth incident angle θ4 is an angle from the maximum incident angle of the first portion L1 of the light L that can be received in the first pixel 104a to the maximum incident angle of the fourth portion L4 of the light L that can be received in the fourth pixel 104d.

In some embodiments, the first incident angle θ1 is from <NUM> degrees to <NUM> degrees, the second incident angle θ2 is from <NUM> degrees to <NUM> degrees, the third incident angle θ3 is from <NUM> degrees to <NUM> degrees, and the fourth incident angle θ4 is from <NUM> degrees to <NUM> degrees. In some embodiments, the first incident angle θ1 is from <NUM> to <NUM> degrees, the second incident angle θ2 is from <NUM> degrees to <NUM> degrees, the third incident angle θ3 is from <NUM> degrees to <NUM> degrees, and the fourth incident angle θ4 is from <NUM> degrees to <NUM> degrees. Since the first incident angle θ1 is smaller than the second incident angle θ2, the second portion L2 will be spectrum-shifted more significantly than the first portion L1.

<FIG> illustrates a top view of an arrangement of analytes 122a on the biosensor <NUM> in accordance with some embodiments of the present disclosure. <FIG> and <FIG> illustrate cross-sectional views of <FIG> along line A-A'. <FIG> and <FIG> illustrate cross-sectional view of <FIG> along line B-B'. <FIG> and <FIG> illustrate cross-sectional views of <FIG> along line C-C'. <FIG> represent an example of bioluminescence and <FIG> represent an example of fluorescence. Some of the differences between the embodiments of <FIG> and <FIG> are that the biosensor <NUM> includes the aperture structure <NUM> and that the analytes 122a emit the light L at the same time.

As illustrated in <FIG>, the aperture structure <NUM> includes openings that correspond to one analyte 122a, so that the light L can be further controlled to irradiate only certain areas of the substrate <NUM>.

To be specific, referring to <FIG>, the aperture structure <NUM> prevents the light L emitted by the analyte 122a1 from reaching the pixel <NUM>. Referring to <FIG>, the aperture structure <NUM> prevents the light L emitted by the analyte 122a1 from reaching the pixel <NUM>. Referring to <FIG>, the aperture structure <NUM> prevents the light L emitted by the analyte 122a1 from reaching the pixel <NUM>. Therefore, cross-talk can be avoided. Therefore, the analytes 122a may be placed on the biosensor <NUM> at one-pixel intervals to avoid cross-talk. In other words, any two adjacent analytes 122a are spaced apart from each other by one pixel <NUM>.

In some embodiments, from the top view of <FIG>, the openings may be shapes like a rectangle, a circle, or a triangle, but they are not limited thereto.

Referring to <FIG>, the difference between the embodiments of <FIG> and <FIG> is that the biosensor <NUM> further includes the excitation light rejection filter <NUM>.

Although the biosensors <NUM> of <FIG> and <FIG> include the aperture structure <NUM>, it should be understood that the same concept as recited in the embodiment of <FIG> can also be applied to the embodiments of <FIG> and <FIG>, which will not be repeated for the sake of brevity.

<FIG> illustrates a top view of an arrangement of analytes 122a on the biosensor <NUM> in accordance with some embodiments of the present disclosure. <FIG> and <FIG> illustrate cross-sectional views of <FIG> along line A-A'. <FIG> and <FIG> illustrate cross-sectional view of <FIG> along line B-B'. <FIG> and <FIG> illustrate cross-sectional views of <FIG> along line C-C'. <FIG> represent an example of bioluminescence and <FIG> represent an example of fluorescence. Some of the differences between the embodiments of <FIG> and <FIG> are that the biosensor <NUM> includes the shielding layer <NUM> and that the analytes 122a emit the light L at the same time.

Since the shielding layer <NUM> can reflect the light L, the first pixel 104a may receive the first portion L1' of the light L that enters the angle-sensitive filter <NUM> at a first incident angle θ1' (as shown in <FIG> and <FIG>) greater than the first incident angle θ1 of the embodiments in <FIG>. The second pixel 104b may also receive the second portion L2' of the light L that enters the angle-sensitive filter <NUM> at a second incident angle θ2' (as shown in <FIG> and <FIG>) greater than the second incident angle θ2 of the embodiments in <FIG>. The third pixel 104c may also receive the third portion L3' of the light L that enters the first color filter 120a at a third incident angle θ3' (as shown in <FIG> and <FIG>) greater than the third incident angle θ3 of the embodiments in <FIG>. The fourth pixel 104d may also receive the fourth portion L4' of the light L that enters the second color filter 120d at a fourth incident angle θ4' (as shown in <FIG> and <FIG>) greater than the third incident angle θ4 of the embodiments in <FIG>. For example, the first incident angle θ1' may be from <NUM> degrees to <NUM> degrees. The second incident angle θ2' may be from <NUM> degrees to <NUM> degrees. The third incident angle θ3' may be from <NUM> degrees to <NUM> degrees. The fourth incident angle θ4' may be from <NUM> degrees to <NUM> degrees. However, it should be appreciated that the first incident angle <NUM>', the second incident angle θ2', the third incident angle θ3' and the fourth incident angle θ4' are determined according to the heights of the analyte, shielding layer and photodiode.

<FIG> is analytical graphs of emission or transmission versus wavelength in accordance with some embodiments of the present disclosure. 104A represents the wavelength of the light that enters the angle-sensitive filter <NUM> at the first incident angle θ1 or θ1' and is received by the first pixel 104a. 104B represents the wavelength of the light that enters the angle-sensitive filter <NUM> at the second incident angle θ2 or θ2' and is received by the second pixel 104b. 104C represents the wavelength of the light that enters the first color filter at the third incident angle θ3 or θ3' and is received by the third pixel 104c. 104D represents the wavelength of the light that enters the second color filter at the fourth incident angle θ4 or θ4'. Alexa <NUM>, Alexa <NUM>, eFluor <NUM> and Alexa <NUM> are four dyes that emit four different lights. A first signal intensity of the first portion L1 or L1' of the light L is obtained from the first pixel 104a. A second signal intensity of the second portion L2 or L2' of the light L is obtained from the second pixel 104b. A third signal intensity of the third portion L3 or L3' of the light L is obtained from the third pixel 104c. A fourth signal intensity of the fourth portion L4 or L4' of the light L is obtained from the fourth pixel 104d. The overlapping area between the area under curve (AUC) of 104A and the AUC of Alexa <NUM> represents the first signal intensity of Alexa <NUM>, the overlapping area between the AUC of 104B and the AUC of Alexa <NUM> represents the second signal intensity of Alexa <NUM>, the overlapping area between the AUC of 104C and the AUC of Alexa <NUM> represents the third signal intensity of Alexa <NUM> and the overlapping area between the AUC of 104D and the AUC of Alexa <NUM> represents the fourth signal intensity of Alexa <NUM>. For the same concept, Alexa <NUM>, eFluor <NUM> and Alexa <NUM> have first signal intensities, second signal intensities, third signal intensities and fourth signal intensity.

For the same concept as described previously, the signal intensities can be defined as Pass or No. Table <NUM> below is made according to the concept described above and in <FIG>, in which the first signal intensity is denoted by 104a, the second signal intensity is denoted by 104b, the third signal intensity is denoted by 104c, and the fourth signal intensity is denoted by 104d.

According to a combination of the definitions of the first signal intensity, the second signal intensity, the third signal intensity and the fourth signal intensity, the four dyes can be distinguished as previously described.

Although there are only four dyes in the embodiments, since there are sixteen combinations of definitions of the first signal intensity, the second signal intensity, the third signal intensity and the fourth signal intensity, it should be understood that at most sixteen dyes can be distinguished.

Alternatively, first signal intensity ratios of the first signal intensity to the fourth signal intensity (denoted by 104a/104d), second signal intensity ratios of the second signal intensity to the fourth signal intensity (denoted by 104b/104d) and third signal intensity ratios of the third signal intensity to the fourth signal intensity (denoted by 104c/104d) can also be calculated to distinguish the four different dyes, namely four different lights. A cluster distribution graph is plotted according to the first signal intensity ratio, the second signal intensity ratio and the third signal intensity ratio. To be specific, a cluster distribution graph of the first signal intensity ratio vs. the second signal intensity ratio vs. the third signal intensity ratio is plotted.

For example, <FIG> is analytical graphs of the first signal intensity ratio vs. the second signal intensity ratio vs. the third signal intensity ratio plotted in accordance with some embodiments of the present disclosure. The X-axis represents the first signal intensity ratio. The Y-axis represents the third signal intensity ratio. The Z-axis represents the second signal intensity ratio. Alexa <NUM> is represented by a pentagon. Alexa <NUM> is represented by triangle. eFluor <NUM> is represented by square. Alexa <NUM> is represented by a star.

In the embodiment where the angle-sensitive filter is a shortpass filter, the data points of Alexa <NUM>, Alexa <NUM>, eFluor <NUM> and Alexa <NUM> are clustered at different positions in the graph, respectively. As a result, a dye can be distinguished depending on where the dye is located in the cluster distribution plot.

In the embodiment where the angle-sensitive filter is a bandpass filter, the data points of Alexa <NUM>, Alexa <NUM>, eFluor <NUM> and Alexa <NUM> are clustered at different positions in the graph, respectively. As a result, a dye can be distinguished depending on where the dye is located in the cluster distribution plot.

In the embodiment where the angle-sensitive filter is a longpass filter, the data points of Alexa <NUM>, Alexa <NUM>, eFluor <NUM> and Alexa <NUM> are clustered at different positions in the graph, respectively. As a result, a dye can be distinguished depending on where the dye is located in the cluster distribution plot.

Although there are only four dyes in the embodiments, it should be understood that more than four dyes can be distinguished.

Although the embodiments of the analyte 122b are not illustrated in the drawings, it can be appreciated that the analyte 122b can also be placed on the biosensor provided by the embodiments of the present application.

To sum up, the advantages of the biosensors and the methods of distinguishing a light which are provided by the embodiments of the present disclosure at least include:.

Claim 1:
A biosensor (<NUM>), comprising:
a substrate (<NUM>);
a first photodiode (<NUM>) and a second photodiode (<NUM>) disposed in the substrate (<NUM>) and defining a first pixel (104a) and a second pixel (104b), respectively, wherein the first pixel (104a) and the second pixel (104b) are configured to receive a light (L) emitted from an analyte (<NUM>);
an angle-sensitive filter (<NUM>) disposed on the substrate (<NUM>); and
an immobilization layer (<NUM>) disposed on the angle-sensitive filter (<NUM>) for immobilizing an analyte (<NUM>);
wherein the biosensor further comprising:
- an aperture structure (<NUM>) embedded in the immobilization layer (<NUM>), wherein the aperture structure (<NUM>) includes an opening so that the light can be controlled to irradiate certain areas of substrate (<NUM>) to avoid cross-talk; and/or
- a waveguide (<NUM>) embedded in the immobilization layer (<NUM>), wherein the waveguide (<NUM>) is linear so that the light can be controlled to irradiate analyte in sequence and sequentially generate emission light to certain areas of substrate (<NUM>) to avoid cross-talk; and/or
- a shielding layer (<NUM>) surrounding the angle-sensitive filter (<NUM>) to isolate different areas of the angle-sensitive filter (<NUM>);
such that the first pixel (104a) receives a first portion (L1) of the light (L) and the second pixel (104b) receives a second portion (L2) of the light (L), and the first portion (L1) enters the angle-sensitive filter (<NUM>) at a first incident angle (θ1), the second portion (L2) enters the angle-sensitive filter (<NUM>) at a second incident angle (θ2), and the first incident angle (θ1) is smaller than the second incident angle (θ2);
wherein the angle-sensitive filter (<NUM>) is configured to cause a spectrum shift, wherein the second portion (L2) is spectrum-shifted more significantly than the first portion (L1); such that the analyte can be recognized:
- based on comparing a first signal intensity of the first light (L1) to a first threshold and a second signal intensity of the second light (L2) to a second threshold, or
- based on a ratio of a first signal intensity of the first light (L1) and a second signal intensity of the second light (L2).