CN106918578B

CN106918578B - Sensing chip

Info

Publication number: CN106918578B
Application number: CN201510981843.9A
Authority: CN
Inventors: 林鼎晸; 高丰生; 施亭宇; 陈品诚; 朱仁佑
Original assignee: Industrial Technology Research Institute ITRI
Current assignee: Industrial Technology Research Institute ITRI
Priority date: 2015-12-24
Filing date: 2015-12-24
Publication date: 2020-06-09
Anticipated expiration: 2035-12-24
Also published as: CN106918578A

Abstract

The invention discloses a sensing chip which comprises a substrate, a plurality of metal nano structures, a first surface modification layer and a second surface modification layer. The metal nanostructures are located on the substrate. The first surface modification layer is located on the surface of the metal nanostructure, wherein the first surface modification layer comprises a plurality of molecules having thiol groups. A second surface modifying layer is on the surface of the substrate, the second surface modifying layer comprising a plurality of molecules having silane groups. The sensing chip of the present invention has high sensitivity and high linearity with respect to the concentration of the molecule to be detected.

Description

Sensing chip

Technical Field

The present invention relates to a chip, and more particularly, to a sensing chip.

Background

Localized Surface Plasmon Resonance (LSPR) is a fluorescence-free calibration detection method. Besides, the detection process and time can be shortened, and the problem that secondary antibody (containing fluorescent molecules) grafting is difficult due to steric hindrance can be avoided. However, the sensitivity of the current LSPR chip is lower than that of the traditional ELISA method. The main reason is that besides the wider plasma resonance spectrum, it is also a major key point for whether the molecule to be measured can be close to the position of the sensing hot spot (hot spot) on the metal nanostructure to generate effective spectral shift. Therefore, how to effectively graft the analyte onto the metal nanostructure is a problem that researchers are trying to solve.

Disclosure of Invention

It is an object of the present invention to provide a sensing chip having a high sensitivity and a high linearity with respect to the concentration of a molecule to be detected.

To achieve the above objective, the present invention provides a sensing chip, which includes a substrate, a plurality of metal nanostructures, a first surface modification layer, and a second surface modification layer. The metal nanostructures are located on the substrate. The first surface modification layer is located on the surface of the metal nanostructure, wherein the first surface modification layer comprises a plurality of molecules having thiol groups. A second surface modifying layer is on the surface of the substrate, the second surface modifying layer comprising a plurality of molecules having silane groups.

Based on the above, since the metal nanostructure of the sensing chip of the invention is away from the substrate by a distance, the sensing hot spot is increased from the substrate and exposed, thereby having higher sensitivity. In addition, the sensing chip of the invention is subjected to two-stage surface modification, so that the probability of grafting the object to be detected to the effective sensing area can be increased, the noise interference caused by sticking the object to be detected to the non-sensing area is reduced, the linear relation between the object to be detected and signals under different concentrations of molecules to be detected is further improved, and the sensing sensitivity is improved.

In order to make the aforementioned and other features and advantages of the invention more comprehensible, embodiments accompanied with figures are described in detail below.

Drawings

Fig. 1A to fig. 1F are schematic cross-sectional views illustrating a manufacturing process of a sensing chip according to a first embodiment of the invention;

fig. 2A to fig. 2C are schematic cross-sectional views illustrating a manufacturing process of a sensing chip according to a second embodiment of the invention;

FIG. 3 is a schematic diagram of characteristic spectra of the sensor chip of embodiment 1 in air and water;

FIG. 4 is a schematic diagram of characteristic spectra of the sensor chip of comparative example 1 in air and water;

FIG. 5A is a schematic representation of FDTD simulation results of the energy hot spot profile of example 1;

FIG. 5B is a graph showing FDTD simulation results of the energy hot spot profile of comparative example 1;

FIG. 6 is a graph comparing the results of sensitivity experiments with FDTD simulations;

FIG. 7 is a graph of the relationship between the concentration of the molecule to be measured and the characteristic red shift of the spectrum in example 2;

FIG. 8 is a graph of the relationship between the concentration of the molecule to be measured and the red shift of the characteristic spectrum in comparative example 2;

FIG. 9A is a schematic representation of an atomic force micrograph of example 2;

fig. 9B is a schematic diagram of an atomic force micrograph of comparative example 2.

Description of the symbols

10. 20: sensing chip

100. 100a, 300, 400: substrate

110: photoresist layer

111: patterned photoresist layer

120: patterned mask layer

122. 144, and (3) 144: opening of the container

130. 132: stacking structure

140: layer of metallic material

142. 302, 402: metal nanostructures

142 a: upper surface of

142 b: lower surface

142 c: corner area

150. 160, 250, 260: surface finishing layer

152. 252: molecule having thiol group

162. 262: molecule having silane group

202: support structure

204: contact surface

304. 404: sensing hot spots

L1: size of the Metal nanostructures

L2: width of contact surface

H1: height of metal nanostructures

H2: height of the supporting structure

P: period of time

Detailed Description

Fig. 1A to fig. 1F are schematic cross-sectional views illustrating a manufacturing process of a sensing chip according to a first embodiment of the invention. First, a substrate 100 is provided, and the substrate 100 is, for example, a glass substrate. A photoresist layer 110 and a patterned mask layer 120 on the photoresist layer 110 have been formed on the substrate 100. In the present embodiment, the photoresist layer 110 is an organic photoresist layer, and the patterned mask layer 120 is a patterned inorganic photoresist layer. The patterned mask layer 120 is formed, for example, by forming an inorganic photoresist layer on the photoresist layer 110, and then patterning the inorganic photoresist layer by phase change of the inorganic photoresist through a blue laser thermal lithography (blue laser thermal lithography) process. The patterned mask layer 120 has a plurality of openings 122, and the openings 122 expose portions of the photoresist layer 110. In the present embodiment, the exposed region of the opening 122 is a predetermined region for defining the metal nanostructure.

Next, referring to fig. 1B, an etching process is performed using the patterned mask layer 120 as an etching mask to remove a portion of the photoresist layer 110 exposed by the opening 122, so as to form a plurality of spaced-apart stacked structures 130. The etching process may be an isotropic dry etching process or an anisotropic dry etching process. The isotropic dry Etching process is, for example, Reactive Ion Etching (RIE). The anisotropic dry etching process is, for example, Inductively Coupled Plasma etching (ICP) stack 130 including a patterned photoresist layer 111 and a patterned mask layer 120 thereon. In this step, the pattern of the patterned mask layer 120 is transferred to the patterned photoresist layer 111, that is, the pattern position of the patterned photoresist layer 111 approximately corresponds to the patterned mask layer 120.

Then, referring to fig. 1C, a metal material layer 140 is formed on the substrate 100 between the adjacent stacked structures 130 and on each stacked structure 130, wherein the stacked structures 130 and the metal material layer 140 thereon form stacked structures 132. The metal material layer 140 is formed by, for example, electron beam evaporation (e-beam evaporation). The material of the metal material layer 140 is, for example, silver, gold, platinum, copper, aluminum, or a combination thereof, but the present invention is not limited thereto. The thickness of the metal material layer 140 is, for example, 10nm to 100 nm. According to the present embodiment, since the photoresist layer 111 and the mask layer 120 have a height (<140nm), the metal material layer 140 deposited on the substrate 100 is separated from the metal material layer 140 deposited on the mask layer 120 when the metal material layer 140 is formed.

Thereafter, each stacked structure 132 on the substrate 100 is removed. The method of removing the stacked structure 132 includes performing a wet stripping process, a dry stripping process, or a combination thereof. More specifically, the mask layer 120 and the metal material layer 140 on the photoresist layer 111 may be simultaneously stripped while the photoresist layer 111 is removed by wet etching or dry etching. Next, as shown in fig. 1D, a thermal annealing process is performed to form a plurality of metal nanostructures 142 on the metal material layer 140 on the substrate 100. According to the present embodiment, there are openings 144 between adjacent metal nanostructures 142, and the openings 144 expose a portion of the substrate 100. The size L1 of the metal nanostructures 142 is, for example, between 10nm and 900 nm. The height H1 of the metal nanostructures 142 is, for example, between 10nm and 100 nm.

In the present embodiment, as shown in fig. 1C and fig. 1D, the openings 122 of the patterned mask layer 120 are distributed in a periodic and regular manner. Thus, the position of each opening 122 corresponds to each metal nanostructure 142. That is, the metal nanostructures 142 are periodically and regularly arranged on the substrate 100, and the arrangement position pattern thereof corresponds to the opening 122. The period P of the metal nanostructures 142 is, for example, between 15nm and 1000nm, where P > L1. The periodically arranged structure has the advantage of high uniformity, and does not influence the signal strength due to the change of the measurement position. In addition, in the present embodiment, the metal nanostructure 142 has a hemispherical shape (as shown in fig. 1D). In another embodiment, the metal nanostructure may be cylindrical, disc-shaped, moth-eye-shaped, triangular prism-shaped, or a combination thereof, but the invention is not limited thereto, and those skilled in the art can change the shape of the metal nanostructure according to their needs.

Then, referring to fig. 1E, a surface modification layer 150 is formed on the surface of the metal nano structure 142 for capturing an antibody (antibody) or an aptamer (aptamer) corresponding to a molecule to be detected (e.g., a virus, an antigen, or a protein). Surface modification layer 150 includes molecules 152 having a plurality of thiol groups (-SH). The molecule 152 having a thiol group is, for example, 11-mercaptoundecanoic acid (11-mercaptodectanoic acid,11-MUA), 11-mercaptoundecanamine (11-Amino-1-uncacetanol, 11-AUT), Cysteamine (cysteine), 4-Aminothiophenol (4-Aminothiophenol), 4-methylthiothiophenol (4-methylthiothiophenol), thiolated aptamer (thiolated aptamer), or a combination thereof. The surface modification layer 150 can be formed by, for example, immersing the entire structure shown in fig. 1D in a solution of the above-mentioned compounds. During the soaking, one end of the molecule 152 having a thiol group reacts with the metal of the surface of the metal nanostructure 142 to form a covalent bond.

Although fig. 1E shows that the surface modification layer 150 is in contact with a portion of the substrate 100, fig. 1E is only an example, and in this embodiment, the molecule 152 having a thiol group only reacts with the metal and does not react with the substrate 100, so that the surface modification layer 150 is formed only on the surface of the metal nanostructure 142 and the surface modification layer 150 is not formed on the surface of the substrate 100. Fig. 1E illustrates the surface modification layer 150, and no reaction occurs between the surface modification layer 150 and the substrate 100, so that the molecules of the surface modification layer 150 are not actually formed on the surface of the substrate 100.

In addition, in the embodiment, the antibody or aptamer corresponding to the molecule to be detected can be effectively grafted (coupling) with the surface modification layer 150, so that the probability that the molecule to be detected is immobilized (immobilized) in the effective sensing region can be increased, and the sensitivity of the chip sensing can be further improved.

Thereafter, referring to fig. 1F, a surface modification layer 160 is formed on the surface of the substrate 100 to resist adhesion of the molecules to be detected. The surface modification layer 160 includes a molecule 162 having a plurality of silane groups. Molecules 162 having silane groups such as diethanol silane (poly (ethylene glycol) -silane, PEG-silane), Polyvinylpyrrolidone silane (PVP-silane), polyoxyethylene silane (PEO-silane), or combinations thereof. The surface modification layer 160 can be formed by, for example, immersing the entire structure shown in fig. 1E in a solution of the above-mentioned compounds. During the soaking, the silane groups of the molecules 162 having the silane groups react with silicon dioxide of the surface of the substrate 100 to form covalent bonds. Thus, the fabrication of the sensing chip 10 is completed.

In the present embodiment, since the molecules 162 having silane groups only react with the substrate 100 and do not react with the metal nanostructures 142, the surface modification layer 160 is formed only on the surface of the substrate 100, and the surface modification layer 160 is not formed on the surface of the metal nanostructures 142. Similarly, the surface modification layer 160 shown in fig. 1F is a schematic view, and the surface modification layer 160 and the metal nanostructure 142 do not react with each other, so that the molecules of the surface modification layer 160 are not actually formed on the surface of the metal nanostructure 142.

In the embodiment, the surface modification layer 160 can effectively inhibit the molecules to be detected (e.g., antigens or proteins) from adhering to the surface of the substrate 100, so as to reduce noise interference caused by non-specific binding of the molecules to be detected, and increase the probability of the molecules to be detected being fixed in the effective sensing region, thereby improving the sensitivity and accuracy of sensing.

Fig. 2A to fig. 2C are schematic cross-sectional views illustrating a manufacturing process of a sensing chip according to a second embodiment of the invention. It should be noted that, in the following embodiments, a part of the manufacturing process of the foregoing embodiments is followed, and a subsequent manufacturing process is performed after fig. 1D. Therefore, the following embodiments will follow the reference numerals and parts of the contents of the foregoing embodiments, wherein the same reference numerals are used to designate the same or similar elements, and the description of the same technical contents is omitted. For the description of the omitted parts, reference may be made to the foregoing embodiments, and the following embodiments will not be repeated.

Referring to fig. 2A, after the metal nano-structure 142 shown in fig. 1D is formed, a wet etching process (for example, using an HF or KOH aqueous solution) is performed on the substrate 100 using the metal nano-structure 142 as an etching mask to form a substrate 100a and a plurality of supporting structures 202 thereon, wherein the supporting structures 202 are located between the substrate 100a and the metal nano-structure 142, so that the metal nano-structure 142 and the substrate 100a are spaced apart from each other. In the present embodiment, the depth of the wet etching is substantially the same as the height H2 of the support structure 202, and the height of the support structure can be controlled by controlling the etching time. The height H2 of the support structure 202 is, for example, between 10nm and 100 nm.

In the present embodiment, the metal nanostructure 142 has an upper surface 142a and a lower surface 142 b. In the embodiment, the upper surface 142a is a circular arc surface, but the invention is not limited thereto. The lower surface 142b has a corner region 142 c. The lower surface 142b has an interface 204 with the support structure 202. Since the wet etching method is an isotropic etching, in the process of performing the wet etching method, in addition to etching the substrate 100 exposed by the partial opening 144, the substrate 100 under the partial metal nanostructure 142 is also laterally etched, and thus the width L2 of the contact surface 204 is smaller than the width L1 of the metal nanostructure 142.

Next, referring to fig. 2B, a surface modification layer 250 is formed on the surface of the metal nanostructure 142 for capturing an antibody or aptamer corresponding to the molecule to be detected. Surface modification layer 250 includes a plurality of molecules 252 having thiol groups (-SH). The molecule 252 having a thiol group is, for example, 11-mercaptoundecanoic acid (11-mercaptodectanoic acid,11-MUA), 11-mercaptoundecanamine (11-Amino-1-uncacetanol, 11-AUT), Cysteamine (cysteine), 4-Aminothiophenol (4-Aminothiophenol), 4-methylthiothiophenol (4-methylthiothiophenol), thiolated aptamer (thiolated aptamer), or a combination thereof. The surface modification layer 250 can be formed by, for example, immersing the entire structure shown in fig. 2A in a solution of the above-mentioned compounds. During the soaking process, one end of the molecule 252 having a thiol group reacts with the metal of the upper surface 142a, the lower surface 142b and the corner region 142c of the metal nanostructure 142 to form a covalent bond.

Although it can be seen in fig. 2B that the surface modification layer 250 is in contact with a portion of the supporting structure 202, fig. 2B is only an example, and in this embodiment, since the molecule 252 having a thiol group only reacts with the metal and does not react with the supporting structure 202 and the substrate 100a, the surface modification layer 250 is formed only on the upper surface 142a, the lower surface 142B and the corner region 142c of the metal nanostructure 142, and the surface modification layer 250 is not formed on the supporting structure 202 and the substrate 100 a.

In addition, in the present embodiment, an antibody or aptamer corresponding to a molecule to be detected (e.g., an antigen or a protein) can be effectively grafted to the surface modification layer 250, so that the probability of the molecule to be detected being immobilized in the effective sensing region of the chip can be increased, and the sensing sensitivity can be further improved.

In the embodiment, since the supporting structure 202 is located between the substrate 100a and the metal nanostructure 142, so that a distance is formed between the metal nanostructure 142 and the substrate 100a, the sensing hot spot located at the junction between the corner region 142c of the metal nanostructure 142 and the substrate 100a can be upwardly adjusted away from the substrate 100a, which is beneficial for the object to be measured to approach from all directions, and reduces the influence of the space obstacle during fixing. In addition, the lateral etching effect of the wet etching process can empty the substrate below the sensing hot spot, so that the sensing hot spot is exposed in the surrounding environment, and the sensing sensitivity is further improved.

Then, referring to fig. 2C, a surface modification layer 260 is formed on the surface of the substrate 100a and the surface of the supporting structure 202 to resist adhesion of the molecules to be measured. The surface modification layer 260 includes a plurality of molecules 262 having silane groups. Molecules 262 having silane groups such as diethanol silane (poly (ethylene glycol) -silane, PEG-silane), Polyvinylpyrrolidone silane (PVP-silane), polyoxyethylene silane (PEO-silane), or combinations thereof. The surface modification layer 260 may be formed by, for example, immersing the entire structure shown in fig. 2B in a solution of the above-mentioned compounds. During the soaking, the silane groups of the molecules 262 having silane groups react with silicon dioxide of the surface of the substrate 100a and the support structure 202 to form covalent bonds. At this point, the fabrication of the sensing chip 20 is completed.

In the embodiment, the surface modification layer 260 can effectively prevent molecules to be detected (e.g., antigens or proteins) from being adhered to the substrate 100a and the surface of the supporting structure 202, so as to reduce noise interference caused by non-specific binding of the molecules to be detected, increase the probability of the molecules to be detected being fixed in the effective sensing region, and further improve the sensitivity and accuracy of sensing.

The present invention will be described more specifically below with reference to examples of the present invention. However, the materials, methods of use, and the like shown in the following examples may be appropriately modified without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited to the examples shown below.

[ Structure design experiment of LSPR sensing chip ]

In this experiment, the simulated structures 1-3 were analyzed using the Finite-Difference-Time-Domain (FDTD) method. The dummy structure 1 has a structure as shown in fig. 2A, in which the material of the metal nanostructure is gold; the dummy structure 2 has a structure as shown in fig. 1D, in which the metal nanostructure is gold; the structure of the pseudo structure 3 is similar to that of the pseudo structure 2, and the difference is that the nano structure of the pseudo structure 3 is a multi-layer sandwich metal nano structure composed of gold-alumina-gold.

Table 1 shows the sensitivity and the yield of the manufacturing process of the simulated structure 1-3 after the FDTD analysis. As shown in Table 1, the sensitivities of mock structure 1, mock structure 2, and mock structure 3 were 331 nm/RIU, 230nm/RIU, and 280nm/RIU, respectively. From the above results, the dummy structure 1 has the highest sensitivity and high manufacturing yield, and thus the dummy structure 1 is suitable for designing as a structure of an LSPR chip.

TABLE 1

	Sensitivity (nm/RIU)	Yield of the manufacturing process
			Simulation structure
1	331	Height of
			Simulation architecture 2	230	Height of
Simulation architecture 3	280	Is low in

[ sensitivity test of LSPR sensing chip ]

Example 1

Example 1 is a sensing chip having the structure shown in fig. 2A (i.e., a substrate is subjected to a wet etching process).

The sensing chip of example 1 was placed in air (refractive index of 1.0) and water (refractive index of 1.33), respectively, and the sensitivity thereof was estimated by measuring the change in its LSPR characteristic spectrum (characteristic spectrum). Fig. 3 is a characteristic spectrum of the sensing chip of embodiment 1 in air and water. As shown in FIG. 3, the sensitivity of the sensor chip of example 1 is 351 nm/RIU, which is calculated by moving the air with a lower refractive index into the water with a higher refractive index and red-shifting the characteristic spectrum by 117nm due to the increase of the refractive index of the medium.

Comparative example 1

Comparative example 1 is a sensor chip having the structure shown in fig. 1D (i.e., the substrate is not subjected to the wet etching process).

The sensing chip of comparative example 1 was placed in air (refractive index of 1.0) and water (refractive index of 1.33), respectively, and the sensitivity thereof was estimated by measuring the change in the LSPR characteristic spectrum thereof. FIG. 4 shows the characteristic spectra of the sensor chip of comparative example 1 in air and water. As shown in FIG. 4, the sensitivity of the sensor chip of comparative example 1 was calculated to be 189nm/RIU by shifting the characteristic spectrum of the sensor chip from air having a low refractive index to water having a high refractive index to red shift 63nm as the refractive index of the medium increases.

From the above results, it is understood that example 1 has higher sensitivity than comparative example 1.

Fig. 5A is the result of FDTD simulation of the energy hot spot distribution of example 1. Fig. 5B is the FDTD simulation result of the energy hot spot distribution of comparative example 1. Compared to the sensing hot spot 404 of the comparative example 1 located at the intersection of the substrate 400 and the metal nanostructure 402, the sensing hot spot 304 of the embodiment 1 is exposed due to the distance between the metal nanostructure 302 of the embodiment 1 and the substrate 300, so that the embodiment 1 is more beneficial for the object to be measured to approach the sensing hot spot from all directions.

[ Effect of etching depth on sensitivity ]

Since the etching time can correspond to the etching depth, in order to further test the sensitivity of the sensing chips with different etching depths, the wet etching process with different etching time conditions can be performed on the sensing chip shown in fig. 1D, and the sensitivity of the sensing chip manufactured with different etching time conditions can be measured. In addition, the above measured results are superimposed with the corresponding FDTD simulation results. Fig. 6 is a graph comparing the results of the sensitivity experiment with FDTD simulation. As shown in fig. 6, the results of the sensitivity experiment were consistent with the trend of the FDTD simulation results. The deeper the etching depth, the higher the sensitivity of the manufactured sensing chip. This phenomenon can be explained by the position of the sensing hot spot of the sensing chip, when the etching depth is zero (i.e. the structure shown in fig. 1D), the sensing hot spot is located at the boundary between the metal nanostructure and the substrate, so most of the energy is buried in the substrate, and the sensitivity of the sensing chip is low. However, as the etching time is longer (i.e. the etching depth and the lateral etching are increased), the distance between the sensing hot spot and the substrate is increased, the sensing hot spot is more exposed to the surrounding environment, and thus the sensitivity of the sensing chip is higher.

[ biotin-avidin System test ]

In order to test the actual test result of the LSPR sensing chip manufactured by the present invention on the biological sample, in this embodiment, a biotin-avidin system (biotin-avidin) with specificity and high affinity is used for the experiment, wherein the biotin is NH2-PEG4-biotin and the avidin is NeutrAvidin. NeutrAvidin is modified avidin with avidin deglycosylated, and is less sticky.

Example 2 (two-stage surface modification)

First, the sensing chip shown in FIG. 1D was dipped in a 0.1mM 11-amino-1-undecanethiol (11-AUT) solution for 24 hours to perform the first-stage surface modification. Then, the sensor chip was immersed in a 6mM 2- [ methoxy (polyoxyethylene) propyl ] trimethoxysilane (2- [ methoxy (polyoxyethylene) propyl ] trimethyoxysilane, m-PEG silane) solution and heated at 60 ℃ under nitrogen atmosphere for 24 hours to perform the second stage surface modification. Then, the sensing chip was dipped in a 0.25% Glutaraldehyde (GTA) solution. In this process, the aldehyde group at one end of glutaraldehyde will form a covalent bond with the amine group of 11-AUT. Then, 1mM NH2-PEG4-biotin solution was dropped on the sensing chip and shaken for half an hour, during which the aldehyde group at the other end of glutaraldehyde would form a covalent bond with the amine group of NH2-PEG 4-biotin. Then, the sensing chip is washed with a phosphoric acid buffer solution. Thereafter, NeutrAvidin solutions of different concentrations (0.5. mu.g/mL, 5. mu.g/mL, 50. mu.g/mL, 500. mu.g/mL) were added dropwise to the sensor chip and shaken for half an hour. In the process, NH2-PEG4-biotin specifically binds to NeutrAvidin.

Comparative example 2 (only one-stage surface modification)

A similar method as in example 2 was used, with the only difference that the sensing chip of comparative example 2 was subjected to only the first stage (11-AUT) surface modification, and was not subjected to the second stage (m-PEG silane) surface modification.

FIG. 7 is a graph of the relationship between the concentration of the molecule to be measured and the characteristic red shift of the spectrum in example 2. FIG. 8 is a graph of the relationship between the concentration of the molecule to be measured and the characteristic red shift of the spectrum in comparative example 2.

Referring to fig. 7, in the concentration range of 0.5-50 μ g/mL, as the concentration of the molecule to be detected (i.e., NeutrAvidin) increases, the characteristic spectrum red shift also increases, that is, the concentration of the molecule to be detected and the characteristic spectrum red shift (which may correspond to signal intensity) are in a linear relationship, and the linear relationship leads to a gradual spectrum change due to saturation of the molecule to be detected when the concentration of the molecule to be detected exceeds 50 μ g/mL because the molecule to be detected is fully spread on the surface of the chip.

Referring to fig. 8, as the concentration of the molecules to be detected increases, the characteristic spectrum red shift does not increase, that is, the concentration of the molecules to be detected and the characteristic spectrum red shift are not in a linear relationship.

FIG. 9A is an Atomic Force Microscope (AFM) photograph of example 2. Fig. 9B is an atomic force micrograph of comparative example 2. Referring to fig. 9A and 9B, it can be seen from AFM images of the chips grafted with the analyte (i.e., NeutrAvidin), that many analytes are adsorbed on the glass substrate to form aggregates at the position of the glass substrate without two-stage modification (i.e., comparative example 2), and the surface roughness (Rq) is 6.7nm, so that it can be seen that a large portion of the analytes are lost, and the linear relationship of concentration measurement is also affected. In contrast, in the chip modified in two stages (i.e., embodiment 2), the adhesion of the glass substrate is much less, the surface roughness (Rq) is 2.3nm, and theoretically, there is no number loss, so that most of the objects to be measured can be grafted only with the metal nanostructure, and the stability and reproducibility of the system measurement can be improved.

From the above results, the sensing chip of the present invention performs two-stage surface modification, so that the adhesion of the molecules to be tested to the non-sensing region can be avoided, the probability of grafting the molecules to be tested to the sensing region can be increased, and the linear relationship between the concentration of the molecules to be tested and the signal intensity can be further improved.

In summary, the metal nanostructure of the sensing chip of the present invention is spaced apart from the substrate by a distance, so that the sensing hot spot is far away from the substrate and exposed, thereby having higher sensitivity. In addition, the sensing chip of the invention is subjected to two-stage surface modification, so that the probability of grafting the object to be detected to the effective sensing area can be increased, the noise interference caused by sticking the object to be detected to the non-sensing area is reduced, the linear relation between the object to be detected and signals under different concentrations of molecules to be detected is further improved, and the sensing sensitivity is improved.

Although the present invention has been described with reference to the above embodiments, it should be understood that the invention is not limited thereto, and that various changes and modifications can be made by those skilled in the art without departing from the spirit and scope of the invention.

Claims

1. A sensing chip, comprising:

a substrate;

a plurality of metal nanostructures on the substrate;

a plurality of support structures positioned between the substrate and each of the metal nanostructures to keep a distance between each of the metal nanostructures and the substrate,

a first surface modification layer on a surface of the metal nanostructure and not on a surface of the substrate, wherein the first surface modification layer comprises a plurality of molecules having thiol groups; and

a second surface modification layer on the surface of each support structure and the substrate and not on the metal nanostructures, the second surface modification layer comprising a plurality of molecules with silane groups,

wherein each metal nanostructure has an upper surface and a lower surface, the lower surface has a corner region, and the molecules of the first surface modification layer having thiol groups are fixed on the upper surface, the lower surface and the corner region of the metal nanostructure.

2. The sensing chip of claim 1, wherein the height of the support structure is 10nm to 100 nm.

3. The sensing chip of claim 1, wherein:

a contact surface is arranged between the lower surface and the support structure, wherein the width of the contact surface is smaller than that of each metal nano structure.

4. The sensor chip of claim 1, wherein the material of the support structure is the same as the material of the substrate.

5. The sensing chip of claim 1, wherein the metal nanostructures are between 10nm and 900nm in size.

6. The sensing chip of claim 1, wherein the height of the metal nanostructures is between 10nm and 100 nm.

7. The sensing chip of claim 1, wherein the metal nanostructures are periodically and regularly arranged on the substrate, and the period of the metal nanostructures is between 15nm and 1000 nm.

8. The sensing chip of claim 1, wherein the shape of the metal nanostructure comprises a cylinder shape, a hemisphere shape, a dish shape, a moth-eye shape, a triangular prism shape, or a combination thereof.

9. The sensing chip of claim 1, wherein the material of the metal nanostructure comprises silver, gold, platinum, copper, aluminum, or a combination thereof.

10. The sensing chip of claim 1, wherein the molecule having a thiol group comprises 11-mercaptoundecanoic acid, 11-mercaptoundecanamine, cysteamine, 4-aminothiophenol, 4-methylphenylthiol, a thiolated nucleic acid aptamer, or a combination thereof.

11. The sensing chip of claim 1, wherein the molecules having silane groups comprise polydiethanolisilane, polyvinylpyrrolidone silane, polyethylene oxide silane, or a combination thereof.