TWI677684B - Biosensor and method for measuring antigen concentration - Google Patents

Abstract

The invention provides a biomedical sensing device and its application. The biomedical sensing device includes a substrate, a first polymer layer and a second polymer layer. The first polymer layer includes a composite antibody, which includes the first antibody and a labeling molecule. The second polymer layer has an inverse opal photonic crystal structure, in which gold nano particles and a second antibody are distributed. The complex antibody, the antigen, and the second antibody form a complex in the second polymer layer, and the antigen content can be obtained by changing the fluorescence intensity, redshift amount or visual color of the biomedical sensing device.

Description

Biomedical sensing device and method for detecting antigen content

The invention relates to a biomedical sensing device and its application, and in particular to a biomedical sensing device containing a photonic crystal structure and a method for detecting an antigen concentration using the device.

Common detection methods for diabetic retinopathy include optical coherence tomography or fundus vascular fluorescence. Not only are these instruments expensive, they also perform tests in an invasive manner. In addition, the detection of these instruments still has the disadvantages of being time-consuming, difficult to judge early or insignificant, and single-point sampling.

Lipocalin-1 (LCN1) is currently known as a biological indicator of diabetic retinopathy. Generally speaking, the content of LCN1 in the tears of ordinary people is about 1 to 2 mg / ml, and the LCN1 content of patients with diabetic retinopathy may be several times the normal value. Therefore, changes in LCN1 content can be used to detect diabetic retinopathy. However, the above-mentioned content changes are very small, and how to detect the above-mentioned minute changes is one of the difficult problems to be overcome at present.

The structure of the inverse opal photonic crystal is often used to improve the optical properties (such as fluorescence intensity) during detection, and its refractive index and reflectance change when the structure is changed. The analyte content can be detected by the change of the reflection peak.

One method is to set hydrogel microspheres with inverse opal photonic crystal structures that can detect glucose, pH, and potassium ion concentrations on contact lenses to observe changes in the above three values in tears, respectively. This method is mainly used to change the spacing between particles by the expansion or contraction of the hydrogel, causing the wavelength range and color to change, but the above changes are not significant under low concentration analytes. In addition, the above methods cannot detect proteins.

There is still a method for attaching photonic crystal gel films with different codes to different antibodies, which correspond to different analytes. Then, the individual reflection peaks of the photonic crystal gel films with different codes are detected. Then, after the analyte and the photonic crystal gel film are reacted and bonded, the reflection peak is detected again. The reflectance of the analyte-attached photonic crystal gel film changes. Although the above method can be used for protein detection, it still cannot overcome the limitation of low concentration analytes.

A further method is similar to the method of attaching antibodies described above, but after reacting with the analyte, this method further adds a secondary antibody to the analyte to increase the refractive index of the photonic crystal. However, the above method is not only cumbersome to operate, but the refractive index increased by using secondary antibodies is not enough to overcome the limitation of low concentration analytes.

In view of the above-mentioned shortcomings, there is an urgent need to propose a biomedical sensing device and an antigen concentration detection method that can detect low concentration antigens, have high sensitivity, can sample evenly, and can monitor the antigen concentration for a long time.

Therefore, one aspect of the present invention is to provide a biomedical sensing Device, which can effectively increase the fluorescence intensity and red shift, so that low-concentration antigens can be detected, and / or the content of the antigen can be directly determined by the color change of the biomedical sensing device.

Another aspect of the present invention is to provide a method for detecting an antigen content, which is performed using the above-mentioned biomedical sensing device.

According to the aspect of the present invention, a biomedical sensing device is first proposed. In some embodiments, the biomedical sensing device includes a substrate, a first polymer layer, and a second polymer layer. The substrate includes a first region and a second region adjacent to each other, and the second region is located on one side of the first region. The first polymer layer is disposed on the first region. A plurality of complex antibodies are distributed in the first polymer layer, and each of these complex antibodies includes a labeling molecule and a connected first antibody. The second polymer layer is disposed on the second region. The second polymer layer has an inverse opal photonic crystal structure, and the inverse opal photonic crystal structure includes a plurality of holes, and a plurality of gold nanoparticle and a plurality of second holes are provided on the pore wall of each of the holes. Antibodies, and the first antibody and the second antibody system recognize the same antigen.

According to some embodiments of the invention, the second region is located above the first region.

According to some embodiments of the present invention, the first region and the second region are arranged concentrically around the center of the substrate from outside to inside in order.

According to some embodiments of the present invention, the bottom surface of the first region has a recessed section, and the recessed section has a first depth. In addition, the bottom surface of the second region has an asymmetric U-shaped recessed section, and the asymmetric U-shaped recessed section has a second depth, and the second depth is greater than the first depth.

According to some embodiments of the present invention, the inverse opal photonic crystal structure is a reverse structure of a face-centered cubic stack of nano beads, and the nano beads have The particle diameter is from 100 nanometers to 1000 nanometers, and gold nano particles are distributed on the surface of the nano beads.

According to some embodiments of the present invention, the labeling molecule includes a fluorescent molecule, and the gold nanoparticle has a particle diameter of 5 nm to 80 nm.

According to some embodiments of the present invention, the biomedical sensing device further includes a third polymer layer disposed above the first polymer layer.

According to some embodiments of the present invention, the biomedical sensing device is a contact lens, the center of the substrate is the optical area of the contact lens, and the first area and the second area are distributed in the non-optical area of the contact lens, at least the second The molecular layer is exposed from one side of the contact lens, and this side is the opposite side of the direct contact surface of the eyeball.

According to the above aspect of the present invention, a method for detecting an antigen content is proposed. First, a biomedical sensing device as described above is provided, wherein the substrate of the biomedical sensing device includes a first region and a second region that are in communication, and the second region is located on one side of the first region. The biological fluid sample containing the antigen is caused to flow from the second region to the first region of the biomedical sensing device to release at least one of a plurality of complex antibodies, and at least one of the complex antibodies, the antigen, and At least one of the plurality of second antibodies reacts for a specific time to form a complex in the second region. Next, the optical property of the complex in the second region of the biomedical sensing device is detected by a light source with a specific wavelength, wherein the optical property includes fluorescence intensity or coloration. Then, the content of the antigen is obtained based on this optical property.

According to some embodiments of the present invention, the specific wavelength is 200 nm to 700 nm, and the inverse opal photonic crystal structure of the second region increases the fluorescence intensity by at least 2 times.

100, 200, 300, 400, 500, 600‧‧‧Biomedical sensing devices

110, 210, 310, 410, 510, 610‧‧‧ substrate

112, 212, 312, 412, 512, 612‧‧‧ District 1

114, 214, 314, 414, 514, 614‧‧‧ District 2

120, 220, 320, 420, 520, 620‧‧‧ first polymer layer

121‧‧‧ complex antibody

123‧‧‧ labeled molecules

125‧‧‧ primary antibody

130, 230, 330, 430, 530, 630‧‧‧ second polymer layer

130A‧‧‧ Opal Crystal Structure

130’‧‧‧second polymer material layer

131‧‧‧ Hole

131S‧‧‧hole wall

133‧‧‧Gold nano particles

135‧‧‧Second antibody

140‧‧‧ third polymer layer

340, 440, 540‧‧‧ Center

422‧‧‧ Sag profile

432‧‧‧Asymmetric U-shaped depression profile

434, 436‧‧‧ sidewall

700‧‧‧ Face-centered Cubic Stack

702‧‧‧ Nano beads

710‧‧‧ Silicon dioxide etchant

720‧‧‧aminoalkylalkoxysilane compound

800‧‧‧ Method

810‧‧‧ provides biomedical sensing device

820‧‧‧ causes a biological liquid sample containing an antigen to flow from the second area of the biomedical sensing device to the first area to release at least one of a plurality of complex antibodies and form a complex in the second area

830‧‧‧ Detecting the optical properties of the complex in the second region of the biomedical sensing device with a light source with a specific wavelength

840‧‧‧Acquiring antigen content based on said optical properties

900‧‧‧ Complex

901‧‧‧antigen

D1‧‧‧First Depth

D2‧‧‧Second Depth

1001, 1003, 1005‧‧‧‧ Bar graph

A better understanding of the aspects of the present invention can be obtained from the following detailed description in conjunction with the accompanying drawings. It should be noted that, according to industry standard practice, features are not drawn to scale. In fact, to make the discussion clearer, the dimensions of each feature can be arbitrarily increased or decreased.

1A is a cross-sectional view of a biomedical sensing device according to an embodiment of the present invention.

[Fig. 1B] A schematic cross-sectional view of a first polymer layer.

1C is a schematic cross-sectional view of a second polymer layer.

[FIG. 2] A sectional view of a biomedical sensing device according to another embodiment of the present invention.

[FIG. 3A] and [FIG. 3B] are a cross-sectional view and a top view of a biomedical sensing device according to some embodiments of the present invention.

4 is a cross-sectional view of a biomedical sensing device according to another embodiment of the present invention.

5A and 5B are a cross-sectional view and a top view of a biomedical sensing device according to still another embodiment of the present invention.

[FIG. 6A] and [FIG. 6B] A cross-sectional view and a top view of a biomedical sensing device according to other embodiments of the present invention.

[FIG. 7A] to [FIG. 7E] A method for forming a second polymer layer.

8 is a schematic flowchart of a method for detecting an antigen content according to an embodiment of the present invention.

9 is a schematic cross-sectional view of a second polymer layer forming a composite.

Fig. 10 is a bar graph showing the fluorescence intensity of Examples 1 and 2 of the present invention.

FIG. 11 is a bar graph of the fluorescence intensity in Example 3 of the present invention.

[FIG. 12A] and [FIG. 12B] Bar graphs of the fluorescence intensity and the fluorescence magnification magnification in Example 4 of the present invention.

An object of the present invention is to provide a biomedical sensing device capable of detecting the content of antigen. In a polymer layer with an inverse opal photonic crystal structure and gold nanoparticle distribution, the composite antibodies, antigens, and antibodies form a sandwich immune structure to improve the optical properties of the biomedical sensing device (such as fluorescence intensity or redshift) the amount). The biomedical sensing device can detect the content of antigen at a low concentration, or the change in the content of the antigen can be obtained from the color change of the biomedical sensing device. In addition, this biomedical sensing device is sampled on average and is suitable for long-term monitoring of changes in antigen concentration. Furthermore, the biomedical sensing device is a purely optical device and does not need to drive the device.

First, please refer to FIG. 1A, which is a cross-sectional view of a biomedical sensing device according to an embodiment of the present invention. As shown in FIG. 1A, the biomedical sensing device 100 includes a substrate 110, a first polymer layer 120 and a second polymer layer 130. The substrate 110 includes a first region 112 and a second region 114 adjacent to each other, and the second region 114 is located on one side of the first region 112. The first polymer layer 120 is disposed in the first region 112 and the second polymer layer 130 is disposed in the second region 114.

Please refer to FIG. 1B, which is a schematic cross-sectional view of a first polymer layer. As shown in FIG. 1B, a plurality of complex antibodies 121 are distributed in the first polymer layer 120. Each composite antibody 121 includes a labeling molecule 123 and a first antibody 125 connected thereto. In some embodiments, the first polymer layer 120 includes micropores (not shown), so that the composite antibody 121 can flow into the second polymer layer 130 through the micropores. The aforementioned micropores may, for example, have an average pore diameter greater than 5 nm, and preferably, the aforementioned micropores may be, for example, greater than 10 nm. In some embodiments, the above-mentioned labeling molecule 123 may include, but is not limited to, a fluorescent molecule. In one example, the fluorescent molecule may have an excitation wavelength of 200 nm to 700 nm, for example. In another example, this fluorescent molecule may, for example, have a divergent wavelength from 300 nm to 1000 nm. In a specific example, coumarin, luciferin, cyanine dye, tetramethylrhodamine ethylammonate, phycoerythrin, phycocyanin, trade names are Alexa Fluor 350, 405, 488, 532, 546, 555, 568, 594, 647, 680, or 750 (manufactured by Invitrogen) as the fluorescent molecules, but the fluorescent molecules of the present invention are not limited thereto.

FIG. 1C is a schematic cross-sectional view of a second polymer layer. As shown in FIG. 1C, the second polymer layer 130 has an inverse opal photonic crystal structure 130A. The inverse opal photonic crystal structure 130A includes a plurality of holes 131. A plurality of gold nanoparticle 133 and a plurality of second antibodies 135 are disposed on the pore wall 131S of each pore 131. These second antibodies 135 and the aforementioned first antibodies 125 recognize the same antigen. For example, the first antibody 125 may be a multiple antibody against a specific antigen, and the second antibody 135 may be a single antibody against the specific antigen. In some embodiments, the second polymer layer 130 may also include micropores such as the first polymer layer 120. In some embodiments, the particle size of the gold nano particles 133 may be, for example, 5 nanometers to 80 nanometers. In one example, the particle size of the gold nano particles 133 may be 40 nanometers. If the particle size of the nano-particles 133 is too large, the formation of the inverse opal photonic crystal structure 130A will be affected, and the detection performance of the biomedical sensing device 100 cannot be effectively improved.

When the biomedical sensing device 100 is used to detect the antigen content, the composite antibody 121 and the second polymer layer in the first polymer layer 120 are used. The second antibody 135 in 130 will form a sandwich immune structure in the inverse opal photonic crystal structure 130A, thereby increasing the refractive index of the light source and enhancing the fluorescence intensity and redshift. On the other hand, the surface of the nano-particles 133 in the second polymer layer 130 has surface plasmon resonance, which increases the reflectance of the light source and further enhances the fluorescence intensity of the labeled molecules 123. In addition, when the content of the antigen is high, the change in the content of the antigen can also be known by the color change of the biomedical sensing device 100.

Please refer to FIG. 1A again. In some embodiments, the biomedical sensing device 100 may further include a third polymer layer 140 disposed above the first polymer layer 120. The third polymer layer 140 may include a plurality of micropores (not shown), and the average pore diameter of the micropores is not greater than 5 nm. The third polymer layer 140 can prevent the complex antibody 121 from escaping from the top surface of the first polymer layer 120, and can control the flow direction of the complex antibody 121 toward the second polymer layer 130 when it contacts the liquid sample.

In some embodiments, the material of the first polymer layer 120 and the second polymer layer 130 may include, but is not limited to, a hydrogel having micropores larger than 5 nm as described above. In some embodiments, the hydrogel may be, for example, a polyalkylene glycol, a polyacrylate, or a copolymer comprising two or more polyalkylene glycols. In a specific example, the first polymer layer 120 may be poly (hydroxyethyl methacrylate; pHEMA), Pluronic F-127, polyethylene glycol, or polyethylene glycol diacrylate. In a specific example, the second polymer layer 130 may be polyethylene glycol or polyethylene glycol diacrylate to provide sufficient hardness to form the inverse opal photonic crystal structure 130A. The hydrogel will absorb water and swell and expand the micropores to facilitate the formation of sandwich immune structures. In other embodiments, the material of the substrate 110 and the third polymer layer 140 may be, for example, polymethyl methacrylate.

Please refer to FIG. 2, which is a cross-sectional view of a biomedical sensing device according to another embodiment of the present invention. As shown in FIG. 2, the biomedical sensing device 200 includes a substrate 210, a first polymer layer 220 and a second polymer layer 230. The substrate 210 includes a first region 212 and a second region 214 adjacent to each other, and the second region 214 is located above the first region 212. The first polymer layer 220 is disposed in the first region 212, and the second polymer layer 230 is disposed in the second region 214.

Please refer to FIG. 3A and FIG. 3B, which are respectively a cross-sectional view and a top view of a biomedical sensing device according to some embodiments of the present invention. In the biomedical sensing device 300, the first region 312 and the second region 314 are concentrically arranged from the outside to the center 340 of the substrate 310, and each of the first region 312 and the second region 314 is an annular region. The first polymer layer 320 and the second polymer layer 330 are respectively disposed on the ring-shaped first region 312 and the second region 314 to form two concentric rings, as shown in FIG. 3B.

4 is a cross-sectional view of a biomedical sensing device according to another embodiment of the present invention. In the biomedical sensing device 400, the substrate 410 includes a first region 412 and a second region 414. The first region 412 has a recessed section 422, and the recessed section 422 has a first depth D1. The second region 414 has an asymmetric U-shaped depression profile 432, and the asymmetric U-shaped depression profile 432 has a second depth D2, and the second depth D2 is greater than the first depth D1.

In some embodiments, the slope of one sidewall 436 of the asymmetric U-shaped depression profile 432 adjacent to the center 440 is greater than the slope of the other sidewall 434 of the asymmetric U-shaped depression profile 432 adjacent to the first region 412. With the arrangement shown in FIG. 4, the composite antibody in the first polymer layer 420 is facilitated to flow into the second polymer layer 430, and the optical properties of the biomedical sensing device 400 are effectively improved. The top view of FIG. 4 is similar to that of FIG. 3B, so it will not be described here.

Please refer to FIG. 5A and FIG. 5B, which are respectively a cross-sectional view and a top view of the biomedical sensing device according to some embodiments of the present invention. In the biomedical sensing device 500, the second region 514 is located above the first region 512. From the top view of FIG. 5B, the first polymer layer 520 and the second polymer layer 530 are two concentric rings, and the ring is set at the center 540 of the substrate 510. However, unlike FIG. 3B, the second polymer layer 530 is located in the middle of the first polymer layer 520, so that the complex antibody in the first polymer layer 520 can be uniformly diffused from the periphery to the second polymer 530 layer. in. The arrangement of FIG. 5A and FIG. 5B can greatly increase the space of the first polymer layer 520 containing the composite antibody (such as the composite antibody 121 shown in FIG. 1B). More complex antibodies can further improve the detection performance of the biomedical sensing device 500, for example, it can increase the detected fluorescence intensity.

In some embodiments, the biomedical sensing devices 300, 400, and 500 may be contact lenses. The centers 340, 440, and 540 are optical regions of the contact lens, and the first regions 312, 412, and 512 and the second regions 314, 414, and 514 are distributed in the non-optical region of the contact lens. In addition, at least the second polymer layer 330, 430, and 530 are exposed from one side of the contact lens, and this side is the opposite side of the direct contact surface of the eyeball of the contact lens.

6A and 6B, which are a cross-sectional view and a top view of a biomedical sensing device according to other embodiments of the present invention. As shown in FIGS. 6A and 6B, the biomedical sensing device 600 includes a substrate 610, a first polymer layer 620 and a second polymer layer 630. The first region 612 and the second region 614 of the substrate 610 are concentrically arranged in order from outside to inside. The first polymer layer 620 and the second polymer layer 630 are respectively disposed in the first region 612 and the second region. 614. The center of the biomedical sensing device 600 (such as the optical region described in FIGS. 5A and 5B) is composed of a second polymer Covered by layer 630. In some embodiments, the biomedical sensing device 600 may be a detection wafer. One or more of the detection wafers can be set in a sample tray to perform the detection of multiple groups of samples at the same time or to perform repeatable experiments on the same sample.

The first polymer layers 220, 320, 420, 520, and 620 described in FIGS. 2 to 6B are the same as the first polymer layer 120 shown in FIG. 1B, and the second polymer layers 230, 330, 430, 530, and 630 is the same as the second polymer layer 130, so it will not be repeated here.

A method for manufacturing the biomedical sensing device of the present invention is described below. To simplify the diagram, only the biomedical sensing device 100 is taken as an example. However, those having ordinary knowledge in the technical field should understand that the biomedical sensing devices 200, 300, 400, 500 and 500 shown in FIGS. 2 to 6B and 600 can also be made in a similar manner.

The biomedical sensing device 100 first uses a mold to manufacture a substrate 110 having a first region 112 and a second region 114 communicating with each other as shown in FIG. 1A. The mold can be manufactured, for example, using 3D printing. Next, a second polymer layer 130 is formed before the second region 114.

Please refer to FIGS. 7A to 7E, which illustrate a method for forming a second polymer layer. As shown in FIG. 7A, first, a nanobead mixture is applied to the substrate 110 of the second region 114, so that the nanobeads 702 form a three-dimensional structure of a face-centered cubic stack 700 in a fluid self-assembly manner. For the sake of simplicity, the second region 114 is represented by a planar substrate only. This nano-bead mixture contains silica nano-beads 702 and the above-mentioned gold nano-particles 133 dispersed in a solution. When the face-centered cubic stack 700 is formed, the gold nano particles 133 are adsorbed on the surface of the silicon dioxide nano beads 702. In one example, the particle diameter of the silica nano-beads 702 is 100 nm to 1000 nm. For example, the particle size of nanometer silica beads can be 300nm. In an embodiment In the nano-particle mixture, the weight ratio of the silica nano-beads to the nano-particles is 10: 1 to 30: 1. If the content of the nano-particles is too large, the nano-particles will self-aggregate and make the particle size larger than a predetermined range. In other embodiments, the nano-beads may be formed of a polymer material (such as polystyrene) or a metallic material, and the material may be etched by an organic solvent or an acid / base solution.

Next, as shown in FIG. 7B, a second polymer material layer 130 'is formed on the face-centered cubic stack 700, so that the second polymer material layer 130' surrounds the face-centered cubic stack 700. The second polymer material layer 130 'is formed by polymer material on the second polymer material layer 130', and then irradiated with ultraviolet light to cure the polymer material to form a second polymer material layer 130 '. In some embodiments, the polymer material may further include a photoinitiator. Alternatively, the second polymer material layer 130 'may be hardened only by drying the polymer material on the face-centered cubic structure 700. In one example, the polymer material is polyethylene glycol diacrylate. Then, as shown in FIG. 7C, a silicon dioxide etchant (Buffered Oxide Etch; BOE) 710 is applied to the second polymer material layer 130 'and the face-centered cubic stack 700 to remove the silicon dioxide nanometer beads 702. Thereby, the inverse opal photonic crystal structure 130A of the second polymer layer 130 is formed (FIG. 1C). Since the nano-particles 133 are not etched by the silicon dioxide etchant 710, the nano-particles 133 can be retained and distributed in the holes of the inverse opal photonic crystal structure 130A after removing the silicon dioxide nano-beads 702. The hole wall 131S of 131 is shown in FIG. 1C.

To simplify the drawing, FIG. 7C and FIG. 7D only show a part of the inverse opal photonic crystal structure 130A and the hole wall 131S of the hole. As shown in FIG. 7D, the aminoalkylalkoxysilane compound 720 is covalently bonded to the pore wall 131S with a silicon-oxygen bond. Thereafter, as shown in FIG. 7E, the aminoalkylalkoxysilane is compounded. The amino group of the substance 720 is bonded to the carboxylic acid group of the second antibody 135 to fix the second antibody 135 on the pore wall 131S through the amidine bond to form the second polymer layer 130 as shown in FIG. 1C. In one example, 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and N- The reaction of N-hydroxysuccinimide (NHS) causes the aminoalkylalkoxysilane compound 720 to bond with the second antibody 135. In one example, the aminoalkylalkoxysilane compound 720 is 3-Aminopropyltriethoxysilane (APTES).

After the second polymer layer 130 is formed, a mixture of another polymer material and the complex antibody 121 shown in FIG. 1B is filled in the first region 112, and the other polymer material is cured by irradiation with ultraviolet light, and then formed. First polymer layer 120. In some embodiments, the polymer material includes a photoinitiator. Alternatively, the first polymer layer 120 may be hardened only by drying the polymer material. The polymer material of the first polymer layer 120 may be the same as or different from the polymer material of the second polymer layer 130. For example, the first polymer layer 120 may be polyethylene glycol. In a preferred embodiment, a third polymer layer 140 of polymethyl methacrylate can be formed on the first polymer layer 120, as shown in FIG. 1A.

In other embodiments, a first polymer layer may be formed in the first region, and then a second polymer layer may be formed in the second region. For example, the biomedical sensing device 200 first forms the first polymer layer 220 in the first region 212, and then forms the second polymer layer 230 in the second region 214 on the first polymer layer 220.

Another object of the present invention is to provide a method for detecting an antigen content, which can be performed using any of the aforementioned biomedical sensing devices. Following with Figure 8 This will be described with reference to FIG. 9. FIG. 8 is a schematic flowchart of a method 800 for detecting an antigen content according to an embodiment of the present invention. FIG. 9 is a schematic cross-sectional view of a second polymer layer forming a composite. Although FIG. 9 illustrates the second polymer layer 130 of the biomedical sensing device 100, it may also be the second polymer layer of the biomedical sensing device 200, 300, 400, 500, or 600.

As shown in FIG. 8 and FIG. 9, in step 810, a biomedical sensing device 100 is provided. The substrate 110 of the biomedical sensing device 100 includes a first region 112 and a second region 114 that are in communication with each other. The area 114 is located on one side of the first area 112. Next, in step 820, a biological liquid sample containing the antigen 901 is caused to flow from the second region 114 of the biomedical sensing device 100 into the first region 112 to release the complex antibody 121 of the first region 112. Specifically, the micropores of the first polymer layer 120 and the second polymer layer 130 which are swelled by absorbing the liquid are enlarged, so that at least one of the plurality of complex antibodies 121 can flow from the first region 112 to the second region. 114. At least one of the complex antibodies 121, the antigen 901, and at least one of the plurality of second antibodies 135 are allowed to react for a specific time to form a complex 900 in the second region. In some embodiments, the biological fluid sample may be, for example, tears, urine, blood, or other similar. In one embodiment, the antigen content in the biological liquid sample is at least 10 μg / ml. In one embodiment, when a contact lens such as a biomedical sensing device 300, 400, or 500 is used, the contact lens can be directly worn to detect the tear fluid secreted by the eyes of people passing through the first and second regions. Specific antigen content. In one embodiment, the contact lens can be worn for 8 hours to monitor the change in the concentration of a specific antigen 901 for a long period of time. In one example, the antigen 901 may be, for example, lipocalin 1 (LCN1), the first antibody 125 (FIG. 1B) is a monoclonal antibody to LCN1, and the second antibody 135 is Multiple antibodies against LCN1.

Next, as shown in step 830, the optical property of the complex in the second region of the biomedical sensing device is detected by a light source with a specific wavelength. Specifically, when the aforementioned labeling molecule 123 is a fluorescent molecule, the optical properties referred to herein may be, for example, fluorescence intensity, redshift amount, coloration, and the like. In some embodiments, the specific wavelength is 200 nm to 700 nm. The light source may be, for example, a mobile phone or other light emitting device. In one example, a mobile phone light source can be used in combination with a fluorescent filter to obtain the above-mentioned specific wavelength.

Next, as shown in step 840, the content of the antigen is obtained according to the optical properties. In some embodiments, a mobile phone application can be used to detect changes in the fluorescence intensity of the biomedical sensing device for analysis and quantification and to obtain the antigen content. In other embodiments, other currently-known fluorescence detection devices (such as spectrometers) can also be used to detect changes in the fluorescence intensity of the biomedical sensing device to obtain the antigen content. In still other embodiments, the change in the antigen content can be determined directly by the color change of the biomedical sensing device.

In some embodiments, due to the photonic crystal structure in the second polymer layer, the fluorescence intensity of the biomedical sensing device 100, 200, 300, 400, 500, and 600 can be increased by at least 2 times (compared with no photon Crystal structure).

Please refer to FIG. 10, which is a bar graph of the fluorescence intensity of Examples 1 and 2 of the present invention. Examples 1 and 2 use a photonic crystal structure without a photonic crystal structure (bar graph 1001), a photonic crystal structure made of 200 nm silicon dioxide (bar graph 1003), and a photon made of 300 nm silicon dioxide. The biomedical sensing device with a crystal structure (bar graph 1005), and the LCN1 antibodies with fluorescent molecules attached are flowed into each of the above biomedical sensing devices for testing to compare photonic crystals Effect of structure on fluorescence intensity. In Examples 1 and 2, Alexa Fluor_488 was used as the fluorescent molecule. The concentration of the LCN1 polyclonal antibody in Example 1 was 100 μg / ml, and the concentration of the LCN1 polyclonal antibody in Example 2 was 1 mg / ml.

It can be seen from FIG. 10 that even if a relatively low concentration of fluorescent molecules is used, the photonic crystal structure can effectively improve the intensity of the fluorescent molecules (for example, 2 to 3 times), especially using a 300 nm photonic crystal structure. When using a higher concentration of fluorescent molecules as in Example 2, the difference in fluorescence intensity with or without a photonic crystal structure is even more significant. In addition, it is also known from the results of Examples 1 and 2 that if the biomedical sensing device does not use a photonic crystal structure, even if the concentration of fluorescent molecules is increased, the fluorescence intensity cannot be greatly improved.

Then, please refer to FIG. 11, which is a bar graph showing the fluorescence intensity of Embodiment 3 of the present invention. The bar graph 1101 in Embodiment 3 represents the fluorescence intensity before the reaction of the biomedical sensing device (such as the device 200 in FIG. 2) of the present invention, and the bar graph 1102 represents the barometric 1101 / biomedical sensing device forming a sandwich. The fluorescence intensity after the immune structure, the bar graph 1103 represents the fluorescence intensity before the reaction of the biomedical sensing device without a photonic crystal, and the bar graph 1104 represents the biomedical sensing device of the bar graph 1103 forming a sandwich immune structure. After fluorescence intensity. Both of the above biomedical sensing devices use 0.1 mg / ml of fluorescent molecules attached with multiple antibodies of LCN1 (wherein the concentration ratio of multiple antibodies to fluorescent molecules is 1: 2), and 0.1 mg / ml LCN1 monoclonal antibody. The three groups A, B, and C in Example 3 used liquid samples containing LCN1 at concentrations of 0.1 mg / ml, 0.05 mg / ml, and 0.01 mg / ml, respectively, and reacted for 8 hours for the biomedical sensing device. Sandwich immune structure is formed. As shown in FIG. 11, a biomedical sensing device with a photonic crystal can effectively increase the fluorescence intensity. Further, the rate of increase in Group A is 33% (i.e., the bar graph The difference between 1101 and bar graph 1102), the increase rate of group B is 16%, and the increase rate of group C is 12%.

Next, please refer to FIG. 12A and FIG. 12B, which are bar graphs showing the fluorescence intensity and the fluorescence magnification of Embodiment 4 of the present invention, respectively. In Example 4, the control group used the biomedical sensing device before the liquid sample flowed in to measure the fluorescence intensity, and the experimental group used the liquid sample to flow into each biomedical sensing device for 3 hours after the fluorescence intensity was measured. The bar graph 1201 represents the fluorescence intensity of the biomedical sensing device (such as the device 200 in FIG. 2) of the present invention, and the bar graph 1202 represents the fluorescence intensity of the biomedical sensing device without the LCN1 in the liquid sample, and the bar Figure 1203 represents the fluorescence intensity of a biomedical sensing device with no antibody attached to the photonic crystal. The liquid samples flowing into the biomedical sensing devices of the bar graphs 1201 and 1203 contained 0.1 mg / ml of LCN1, and the biosensor devices of the bar graphs 1201, 1202, and 1203 were prepared under conditions similar to those of Example 3. As shown in FIG. 12A, the fluorescence intensity (bar graph 1201) of the biomedical sensing device of the present invention is significantly different from the non-specific adsorption bar graphs 1202 and 1203. In addition, as shown in FIG. 12B, the fluorescence intensity of the biomedical sensing device of the present invention increases greatly.

The biomedical sensing device of the present invention increases the refractive index and reflectance of the light source by forming a sandwich immune structure in the inverse opal photonic crystal structure and the surface plasmon resonance of the nanometer particles, thereby improving the biomedical sensing device. Detection performance (for example: increasing the fluorescence intensity and / or the amount of red shift, etc.). Therefore, the biomedical sensing device of the present invention can detect low-concentration antigens, and the change in antigen concentration can also be judged from the color change of the biomedical sensing device. This biomedical sensing device takes average samples and can monitor the changes in antigen concentration over time.

Although the present invention has been disclosed as above with several embodiments, it is not It is used to limit the present invention. Any person with ordinary knowledge in the technical field to which the present invention pertains can make various modifications and retouches without departing from the spirit and scope of the present invention. The ones defined in the scope of patent application shall prevail.

Claims (10)

A biomedical sensing device includes: a substrate, wherein the substrate includes a first region and a second region connected to each other, and the second region is located on one side of the first region; a first A polymer layer is provided in the first region, wherein a plurality of complex antibodies are distributed in the first polymer layer, and each of the complex antibodies includes a labeled molecule and a first antibody connected to each other; and a first Two polymer layers are provided in the second region, wherein the second polymer layer has an inverse opal photonic crystal structure, the inverse opal photonic crystal structure includes a plurality of holes, and a hole wall of each of the holes A plurality of gold nanoparticles and a plurality of second antibodies are provided, and the first antibody and the second antibodies recognize the same antigen. The biomedical sensing device according to item 1 of the patent application scope, wherein the second area is located above the first area. According to the biomedical sensing device described in item 1 of the scope of the patent application, wherein the first region and the second region sequentially concentrically center one of the substrates from outside to inside. The biomedical sensing device according to item 3 of the scope of the patent application, wherein one of the bottom masks of the first region has a recessed profile, and the recessed profile has a first depth; The symmetrical U-shaped depression profile has a second depth, and the second depth is greater than the first depth. The biomedical sensing device according to item 1 of the scope of the patent application, wherein the inverse opal photonic crystal structure is an inverse structure of a face-centered cubic stack of nano-beads, and the nano-beads have a thickness of 100 nm to 1000 nm A particle size, and the nano-particles are distributed on a surface of the nano-beads. The biomedical sensing device according to item 1 of the scope of the patent application, wherein the labeling molecule includes a fluorescent molecule, and the gold nano particles have a particle diameter of 5 nanometers to 80 nanometers. The biomedical sensing device described in item 3 of the scope of the patent application, further includes a third polymer layer disposed above the first polymer layer. The biomedical sensing device according to item 3 of the patent application scope, wherein the biomedical sensing device is a contact lens, the center of the substrate is an optical area of the contact lens, and the first area and the first area are The two areas are distributed in a non-optical area of the contact lens. At least the second polymer layer is exposed from one surface of the contact lens, and the surface is an opposite surface of a direct contact surface of the eyeball. A method for detecting an antigen content, comprising: providing a biomedical sensing device according to any one of claims 1 to 8, wherein a substrate of the biomedical sensing device includes a first Area and a second area, and the second area is located on one side of the first area; and a biological liquid sample containing an antigen flows from the second area of the biomedical sensing device to the first area, Releasing at least one of the plurality of complex antibodies, and allowing the at least one of the complex antibodies, the antigen, and at least one of the plurality of second antibodies to react for a time, so as to form a complex in the second region ; Detecting an optical property of the complex in the second region of the biomedical sensing device with a light source of a specific wavelength, wherein the optical property includes a fluorescent intensity or a coloration; and obtaining the antigen according to the optical property Content. The method for detecting an antigen content according to item 9 in the scope of the patent application, wherein the specific wavelength is 200 nm to 700 nm, and the inverse opal photonic crystal structure of one of the second regions is increased by at least 2 times the fluorescence intensity. .