TWI472745B - Plasmonic biosensors and fabricating method thereof - Google Patents

Description

Surface plasma resonance biochemical sensor and forming method thereof

The invention relates to a surface plasma resonance biochemical sensor, in particular to a surface plasma resonance biochemical detector manufactured by direct pressure metal technology and a forming method thereof.

In terms of biological activity and behavior, biomolecular identification includes enzyme-substrate, ligand-receptor, deoxyribonucleic acid hybridization, ribonucleic acid translation, and even biosynthesis, which can be said to be the basis of biological behavior. The understanding of these behaviors is the basis for the development of pharmaceutical design, and also the focus of protein body research after genetic mapping. Therefore, biomolecular interaction analysis is very important in biotechnology research or medical clinical testing.

In general, a biosensor consists of two components: a specific molecular recognition component and a transducer that converts molecular recognition into a quantized signal. Among them, signal conversion can be achieved in many ways, such as: fluorescent marking, optical interference, heavy force measurement, and the like. As for the optical transduction method, due to the fluorescent-free marking, such as surface plasma resonance spectroscopy, grating coupled waveguide spectroscopy, ellipsometry and resonant mirror technology, because the device is simple and can observe unlabeled biomolecules. The interaction is greatly noticed.

As mentioned above, in the past ten years, surface plasma resonance sensors have been widely used as surface biochemical molecular detection tools by biological, chemical, medical and other research units. The advantages are: label-free, specific, quantitative and instant biomolecules. Kinetic analysis of interaction, high sensitivity, massive parallel screening, etc. Wait. The surface plasma resonance phenomenon of metallic materials is sensitive to the dielectric constant of the environment and is therefore often used as a biochemical sensor that does not require biometric calibration.

In the past, surface plasma waves were typically excited by a neon, highly focused laser beam, or a periodic metal structure. First, the Kretschmann architecture is usually used in the manner of erbium excitation. In a fairly mature way, the change in the refractive index of the environment can be known by observing the change in the resonant wavelength or angle of the surface plasma.

The sub-wavelength periodic metal structure is another option that can be used to excite surface plasma waves, such as metal hole arrays. Usually, the metal hole array is formed by electron beam lithography or focused ion beam. The metal hole array can also effectively excite the surface plasma wave and cause abnormal penetration. By observing the band in which the abnormal penetration occurs, it is possible to know the change in the refractive index of the environment. Metal hole arrays are widely used in biochemical sensors, whether they are non-biometric or immobile biochemical sensors, have been proposed in past studies.

On the other hand, the one-dimensional nanometal slit exhibits the SPP Bloch-Wave mode. Depending on the size of the slit, these one-dimensional nanostructures may inhibit or enhance the transmitted light intensity, and the ambient refractive index can also be known by observing the bands in the spectrum where the inhibition or enhancement of light penetration occurs. The change.

However, although the above various methods have good sensitivity, the measurement architecture and method are quite complicated and troublesome, and the cost increase and inconvenience are caused.

In view of this, in order to be able to quickly manufacture biochemical sensors, the present invention utilizes nano direct metal technology to produce biochemical sensors to provide biochemical sensing applications that are fast and inexpensive to use. Among them, an object of the present invention is to provide a surface plasma resonance biochemical sensor comprising at least a substrate and a sensing layer. The sensing layer covers the substrate and has a plurality of protrusions on its surface. Wherein, the material of the sensing layer is a metal material, and the selected one is selected from the group consisting of gold, silver, copper, platinum and aluminum.

In an embodiment of the invention, the substrate is a polymer material, and the material may be a polycarbonate material or a polyethylene terephthalate material.

In an embodiment of the invention, wherein the thickness of the sensing layer is 70 nm, and the pitch of the protrusions is 800 nm.

In an embodiment of the present invention, the sensing method of the surface plasma resonance biochemical sensor provided by the present invention comprises at least the following steps: first dropping different concentrations of the second biomolecule, and then measuring one of the sensing layers The amount of displacement of one of the surface plasma resonance wavelengths.

In an embodiment of the present invention, the step of dropping the second biomolecules at different concentrations further comprises the step of modifying the surfaces of the second biomolecules with a plurality of first modifying materials.

In an embodiment of the present invention, before the step of dropping the second biomolecules of different concentrations, the method further comprises the steps of: firstly, connecting one end of the plurality of first biomolecules to the sensing layer. Then, the other ends of the first biomolecules are respectively connected to a plurality of second modifying materials.

In an embodiment of the invention, the first biomolecule and the second biomolecule are both cysteine molecules.

In an embodiment of the invention, wherein the first modifying material is a gold nanoparticle and the second modifying material is a copper ion.

Another object of the present invention is to provide a method for forming a surface plasma resonance biochemical sensor, comprising at least the following steps: first providing a substrate, and then covering a sensing layer on the substrate, wherein the sensing layer is A metallic material selected from the group consisting of gold, silver, copper, platinum, and aluminum. Next, a mold is provided, one of which has a plurality of grooves on its surface. Then, the surface of the mold having the groove faces the sensing layer, and then the mold is applied to cause the grooves to press the sensing layer to thereby have a plurality of protrusions on one surface of the sensing layer.

In an embodiment of the invention, the step of applying the force to the mold to cause the trenches to squeeze the sensing layer and thereby have the protrusions on the surface of the sensing layer further comprises the steps of: heating the substrate To one of the glass transition temperatures.

Therefore, the advantages and spirit of the present invention can be further understood from the following detailed description of the invention and the accompanying drawings.

In view of the above, the present invention utilizes nano direct metal technology to produce a one-dimensional periodic wavy metal structure, which can effectively couple incident light into surface plasma waves, thereby causing abnormal penetration. phenomenon. In addition, since such a wavy metal structure has a sharp corner having a relatively small radius of curvature, the electric field is concentrated and enhanced at the sharp corners. This electric field enhancement affects the transmittance of the light wave, so by adjusting the curvature of the sharp corner, we can adjust the transmittance of this metal structure. In addition, such local electric field enhancement can also increase the sensitivity of the refractive index change of the detection environment. Worth mentioning Yes, the modality of the surface plasmon resonance of such a wavy metal structure is relatively simple compared to other metal structures, so it is easy to detect and detect changes in the signal. This wavy metal structure therefore has the potential to be a highly sensitive chemical sensor.

First, please refer to FIGS. 1A to 1D, and FIGS. 1A to 1D are cross-sectional views showing a method of forming a surface plasma resonance biochemical sensor according to a preferred embodiment of the present invention.

As shown in FIG. 1A, the method for forming a surface plasma resonance biochemical sensor according to the present invention first provides a substrate 10 first. Preferably, the substrate 10 is a polymer material, and it may be a polycarbonate material or a polyethylene terephthalate material. Basically, the present invention is directed to replacing a conventional hard substrate with a polymer material to reduce the manufacturing cost and to make the biochemical sensor flexible. Therefore, the material of the substrate 10 is selected as a preferred embodiment. The invention is not intended to be limited thereto.

Next, a sensing layer 20 is overlaid on the substrate 10. Preferably, the sensing layer 20 has a thickness of about 70 nm. In addition, the sensing layer 20 is a metal material selected from the group consisting of gold, silver, copper, platinum, and aluminum. Basically, the metal material of the sensing layer 20 needs to be a metal material having excellent surface plasma behavior, and the present invention is not intended to be limited to the above embodiments.

As shown in FIG. 1B, a mold 30 is further provided, and one surface of the mold 30 has a plurality of grooves 30a, and then the surface of the mold 30 faces the sensing layer 20.

Subsequently, the mold 30 is applied to cause the plurality of grooves 30a to press the sensing layer 20 as shown in FIG. 1C.

Finally, as shown in FIG. 1D, the mold 30 is taken out, at this time, the sensing layer 20 The surface has a plurality of projections 20a.

In a preferred embodiment, before applying the force to the mold 30 to cause the plurality of grooves 30a to press the sensing layer 20 and thereby the surface of the sensing layer 20 to have the plurality of protrusions 20a, The material 10 is heated to its glass transition temperature. Preferably, the substrate 10 is a plastic substrate and its glass transition temperature is 150 ° C, but the invention is not intended to be limited thereto.

In addition, when the mold 30 presses the sensing layer 20, the mold 30, the sensing layer 20, and the substrate 10 are also added together. After cooling, the mold 30 is removed again. At this time, since the plurality of grooves 30a on the mold 30 press the sensing layer 20, the surface of the sensing layer 20 is pressed to generate a plurality of protrusions 20a. . That is, the sensing layer 20 has a plurality of protrusions 20a on the other surface of the cover substrate 10, and the plurality of protrusions 20a form a periodically wavy metal structure with a spacing therebetween (ie, The peak of one protrusion to the peak of the other protrusion is preferably 800 nm. At this time, a surface plasma resonance biochemical sensor 100 is completed.

Compared with the traditional method of using optical lithography or electron beam lithography, it is simpler and more convenient to make a surface plasma resonance biochemical sensor by a single step of nano direct metal technology. In addition, since nanoimprint technology is suitable for continuous processes, there is potential for a large number of inexpensive disposable chemical sensors. It is further explained that the protrusions 20a of different curvatures can be controlled by adjusting the pressure at the time of imprinting, for example, a larger imprinting pressure can obtain the protrusions 20a having a larger curvature, and the protrusions 20a make the surface of the plasma waves Energy can be coupled more efficiently to the other side.

Please refer to FIG. 2, which shows a dispersion curve of the surface plasma resonance wave of the sensing layer according to a preferred embodiment of the present invention, wherein the vertical axis is a different wave. The long and horizontal axes are different incident angles, and the color depth represents different transmittances, and the white portion is where the transmitted light is more.

It is worth mentioning that another change in the refractive index of the environment will have an optical signal change that is the intensity of the abnormal penetration phenomenon, and the intensity of the abnormal penetration phenomenon depends on whether the energy of the surface plasma wave can be efficiently Coupled to another interface of the metal film. Therefore, when the environmental refractive indices of the upper and lower interfaces of the sensing layer 20 are equal, the coupling efficiency is the highest at this time. That is to say, this special index matching phenomenon, when the refractive index of the object to be tested is closer to the substrate, the abnormal penetration strength of the surface plasma wave will be higher.

In view of the above, since the surface plasma resonance biochemical sensor 100 has a certain thickness, the light transmittance is not good, but it can be understood from the figure that when the incident angle is 0 degrees, the pole has a wavelength of 800 to 830 nm. The good penetration rate is evidenced by the above description.

Please refer to FIGS. 3A-3B. FIG. 3A shows a calibration regression line of the resonance wavelength of the sensing layer and the environmental medium modal surface under different environmental refractive indexes in a preferred embodiment of the present invention, and FIG. 3B shows a first embodiment of the present invention. A preferred regression line of the plasma resonance strength of the sensing layer and the substrate modal surface at different ambient refractive indices in the preferred embodiment. As shown, the sensor 100 fabricated using the sensing layer 20 having the plurality of bumps 20a has a sensitivity of up to 800 nm/RIU, which is sufficient compared to the sensors described in the prior literature. Match or even higher sensitivity. In addition, the measurement using the intensity variation (Fig. 3B) can be performed without the need for a spectrometer, which indeed simplifies many complicated detection steps.

Please refer to FIGS. 4A-4D. FIG. 4A shows a sensing layer for measuring the penetration spectrum of alcohol at different concentrations in a preferred embodiment of the present invention, and FIG. 4B shows a sensing layer in a preferred embodiment of the present invention. Comparing with the refractive index of the ambient medium modal surface plasma wave resonance wavelength to measure the refractive index of the alcohol solution and the theoretical value, FIG. 4C shows the resonance intensity of the plasma using the sensing layer and the substrate modal surface in a preferred embodiment of the present invention. The comparison between the refractive index of the alcohol solution and the theoretical value is measured, and the 4D graph is a comparison of the average value of the measured values of the 4B and 4C graphs with the theoretical value. As shown in the figure, the present invention further senses the refractive indices of different concentrations of alcohol solution by two different sensing methods of wavelength shift and intensity variation, all of which are below 10 ^-4 RIU.

Next, as shown in FIG. 5, FIG. 5 shows an instant of surface plasma resonance wavelength (sensing layer and environmental medium mode) and intensity (sensing layer and substrate mode) in a preferred embodiment of the present invention. Change measurement. In the instant fluid test, the sensor 100 fabricated by the present invention exhibits its highly immediacy sensing properties both in the movement of the surface plasma resonance wavelength or in the variation of the abnormal penetration strength.

Please refer to FIGS. 6A to 6B. FIGS. 6A to 6B are cross-sectional views showing a sensing method of a surface plasma resonance biochemical sensor according to a preferred embodiment of the present invention. First, the first modifying material 50 is first modified on the first biomolecule 40 to be sensed. Preferably, the first modifying material 50 is a gold nanoparticle. Next, the second biomolecule 60 is first modified on the sensing layer 20, and the other end of the second biomolecule 60 is attached to the second modifying material 70. When the concentration of the first biomolecule 40 is to be measured, the first biomolecule can be dropped into the sensor 100, and finally the displacement of one of the surface resonance wavelengths of the sensing layer 20 is measured. A concentration of a biomolecule 40 or the like.

In a preferred embodiment, the first biomolecule 40 and the second biomolecule 60 may be the same or different. For example, in a preferred embodiment of the invention, both the first biomolecule 40 and the second biomolecule 60 are cysteine molecule.

Please refer to FIG. 7. FIG. 7 shows the relationship between the concentration of cysteine molecules at different concentrations and the resonance wavelength of the surface plasma in a preferred embodiment of the present invention. As shown in the figure, if the concentration of the first biomolecule (e.g., cysteine) to be tested is 10 ^-3 M, the surface plasma wave resonance wavelength shifts by 8 nm. On the other hand, when the concentration of the first biomolecule 40 to be tested is as low as 10 ^-6 M, the wavelength still shifts significantly by 2 nm, and the wavelength exhibits a linear shift at different concentrations. Therefore, the periodic wavy metal structure proposed by the present invention can indeed be developed into a highly sensitive biosensor.

In summary, the surface plasma resonance biochemical sensor provided by the invention and the manufacturing method thereof are manufactured by using the simple step nano imprint technology, and have the advantages of high yield. On the other hand, the use of a plastic substrate can greatly reduce the cost, and the sensor 20 fabricated on the plastic substrate has the advantage of being flexible and disposable. That is to say, the present invention has industrial applicability, and greatly reduces the cost required for developing a highly sensitive chemical sensor, and provides two sensing bases for surface plasma resonance band and intensity, and provides high Sensitive, highly accurate, highly immediacy chemical sensing.

The above is only the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention; all other equivalent changes or modifications which are not departing from the spirit of the present invention should be included in the following. Within the scope of the patent application.

100‧‧‧ surface plasma resonance biochemical sensor

10‧‧‧Substrate

20‧‧‧Sensor layer

20a‧‧‧ bump

30‧‧‧Mold

30a‧‧‧ trench

40‧‧‧First biomolecule

50‧‧‧First modified material

60‧‧‧Second biomolecule

70‧‧‧Second modification material

F‧‧‧力力

1A to 1D are cross-sectional views showing a method of fabricating a surface plasma resonance biochemical sensor according to a preferred embodiment of the present invention; and Fig. 2 is a view showing a surface plasma resonance wave of a sensing layer according to a preferred embodiment of the present invention. Dispersion curve; FIG. 3A is a view showing a calibration regression line of the resonance wavelengths of the sensing layer and the environmental medium modal surface in different environmental refractive indexes according to a preferred embodiment of the present invention; FIG. 3B is a view showing different environmental refractions in a preferred embodiment of the present invention; A calibration regression line for the resonance intensity of the sensing layer and the substrate modal surface; FIG. 4A shows a sensing layer for measuring the penetration spectrum of alcohol at different concentrations in a preferred embodiment; FIG. 4B shows In a preferred embodiment of the invention, the refractive index of the alcohol solution is measured by the resonance wavelength of the sensing layer and the environmental medium modal surface, and the theoretical value is compared; FIG. 4C shows the sensing using a sensing method according to a preferred embodiment of the present invention. Comparing the refractive index of the layer and the substrate modal surface to the theoretical value of the refractive index of the alcohol solution; the 4D is the comparison of the average value of the measured values of the 4B and 4C graphs with the theoretical value; The figure shows the instantaneous change measurement of the surface plasma resonance wavelength (sensing layer and environmental medium mode) and the intensity (sensing layer and substrate mode) in a preferred embodiment of the present invention; FIGS. 6A to 6B show the present Surface plasma resonance in a preferred embodiment of the invention A schematic cross-sectional view of a sensor sensing method; and Figure 7 shows the relationship between the concentration of cysteine molecules at different concentrations and the surface resonator resonance wavelength shift in a preferred embodiment of the invention.

10‧‧‧Substrate

20‧‧‧Sensor layer

30‧‧‧Mold

30a‧‧‧ trench

F‧‧‧力力

Claims (2)

A sensing method of a surface plasma resonance biochemical sensor, wherein the surface plasma resonance biochemical sensor has a substrate and a sensing layer, the sensing layer having a plurality of convex periodic wave structures, including at least The following steps: modifying a surface of one of the plurality of cysteine molecules with a plurality of gold nanoparticles; respectively connecting one end of the plurality of cysteine molecules to a sensing layer; and making the plurality of cysteine molecules different One end is respectively connected with a plurality of copper ions; a plurality of the cysteine molecules of different concentrations are dropped; and a displacement amount of one of the surface plasma resonance wavelengths of the sensing layer is measured. A method for forming a surface plasma resonance biochemical sensor, wherein the surface plasma resonance biochemical sensor has a substrate and a sensing layer, the sensing layer having a plurality of raised periodic wave-like structures, including at least the following Step: providing a substrate, wherein the substrate is selected from the group consisting of a polycarbonate material and a polyethylene terephthalate material; covering a sensing layer on the substrate, wherein the sensing layer is Selected from a group of gold, silver, copper, platinum and aluminum, the thickness of the sensing layer comprises 70 nanometers (nm); providing a mold having a plurality of grooves on one surface thereof; The surface faces the sensing layer; heating the substrate to a glass transition temperature of the substrate; and applying a force to the mold to cause the plurality of trenches to compress the sensing layer, Further, one surface of the sensing layer has a plurality of protrusions, and the plurality of protrusions form a periodic wave structure, and a pitch of the periodic wave structure comprises 800 nanometers (nm).