Surface plasma imaging method and device

Abstract

The invention provides a surface plasma imaging method and a surface plasma imaging device, which are used for imaging surface plasma signals caused by interaction of biomolecules. More specifically, the invention provides a method and a device capable of imaging a surface plasmon resonance mode in a frequency domain through tiny change by comparing the light intensity in two sections of surface plasmon frequency domains.

Description

Technical Field

[0001] The present invention relates to the measurement of surface plasmon resonance signals, and more particularly to a method and device for imaging minute changes of surface plasmon resonance modes in the frequency domain. Background Art

[0002] Surface plasmon sensing systems are commonly used to detect target molecules. Target biomolecules interact with receptor molecules on surface plasmon sensors, changing the surface refractive index. These changes can be analyzed by measuring the angle or frequency (wavelength) of the surface plasmon resonance mode, typically the position of the trough in light intensity relative to the angle and the trough in light intensity relative to the wavelength. By monitoring this signal in real time, a sensing graph can be generated to analyze the signal's temporal changes and further analyze the affinity between the target molecule and the receptor. Because of its high sensitivity and calibration-free nature, surface plasmon sensing has become a popular method in the field of bioassays.

[0003] However, with the rapid development of the biological and pharmaceutical fields, the demand for high-throughput detection, such as biomarker discovery, clinical testing, and ligand display, has rapidly increased. However, the development of high-throughput surface plasmon systems has been hindered by the difficulty of integrating imaging systems with angle- or wavelength-resolving devices. For example, frequency-domain-sensitive detection uses a broadband light beam to illuminate a surface plasmon sensor. The reflected or transmitted light is split by a prism or grating, and a one- or two-dimensional photodetector analyzes the intensity of each frequency domain. The surface plasmon signal response is determined by tracking the troughs or peaks in the spectrum. Angle-sensitive detection uses a single-frequency light source with different wavelengths to illuminate the surface plasmon sensor. A one- or two-dimensional photodetector analyzes the intensity at each angle and detects the surface plasmon signal response by tracking the troughs or peaks in the light intensity at different angles. These methods can only measure the signal at a single point on the surface plasmon sensor. Measuring multiple points requires the addition of a position scanning device. This not only increases the complexity of the entire surface plasmon system, but the scanning time also reduces the temporal resolution of the detection. Therefore, a system and method for surface plasmon imaging are highly anticipated.

[0004] The first system combining surface plasmon sensing and imaging was invented by Yeatman in 1987. A line-scanning focused beam was used to excite surface plasmon waves, and by measuring the intensity of the reflected light in the scanned area, a 2.5-nanometer-thick dielectric film was imaged through a camera. Demonstrated a scanning-free surface plasmon imaging microscopy technique, where an expanded laser beam is used to excite surface plasmon waves, which are then imaged directly by a camera. Changes in the refractive index are reflected in the intensity of the image. Many devices are based on this approach, such as GWC Technologies' Products, including devices granted U.S. Patent No. 6,862,094B2 and Chinese Patent No. CN104730038B. Another approach to surface plasmon imaging involves adding a scanning device to the system. Bengt Ivarsson et al. proposed a method for measuring thin films on a surface plasmon sensor (see U.S. Patent No. 6,493,097B1), using two detectors to detect surface plasmon signals from different regions. Lutz Hoppe et al. used a self-made photomask in a surface plasmon system (see U.S. Patent No. 6,570,657B1). The incident light passed through the photomask and projected onto different locations on a prismatic surface plasmon sensor, providing multi-point surface plasmon sensing. Carsten Thirstrup proposed a complete surface plasmon imaging system in U.S. Patent No. 6,738,141B1. In this patent, a reflective diffractive optical component was used to generate a line of light that was focused onto different regions on the surface plasmon sensor, thereby forming a surface plasmon image. Stephen C. Weibel proposed a surface plasmon imaging device (see US Pat. No. 7,576,863 B2). This device uses a single-frequency light source to illuminate a surface plasmon sensor and employs dual lenses to scan the light angle, producing surface plasmon images based on angle-dependent surface plasmon signals. In addition to angle and frequency methods, phase difference methods can also be used to image surface plasmon signals. In US Pat. No. 2003 / 0219809 A1, Chen Xianzhen et al. proposed a surface plasmon imaging device based on Mach-Zehnder interferometry. In this patent, s-waves are used as reference signals, and p-waves are used to detect the refractive index. By combining s-waves and p-waves, an image with a spatial frequency can be produced.

[0005] Existing surface plasmon imaging technologies can be categorized into three types: wavelength / angle / position scanning, single-frequency light, and interferometry. Scanning methods, due to the additional time required for scanning, result in poor temporal resolution, making them unsuitable for detecting high-affinity molecular reactions. Single-frequency light methods, which utilize light intensity differences as a detection mechanism, have lower detection limits than angle or wavelength methods because light intensity is more susceptible to various noise interferences, including thermal disturbances and vibrations. Interferometry offers the best detection limits, but the optical system is more complex, requiring a very stable optical environment, and has a narrow detection dynamic range, limiting the size of target molecules that can be measured. Summary of the Invention

[0006] This paper proposes a method and related device for surface plasmon imaging that does not require a dispersion component. This method is based on contrast spectroscopy and can be used on any surface plasmon sensor, such as anomalous transmission type, prism type, and localized surface plasmon resonance type sensors. The images of the sensor in two different spectral bands are used to present the surface plasmon signal image. Its main advantages are as follows: (1) It can be used to measure anomalous transmission type, prism type, and localized surface plasmon resonance type sensors and provide surface plasmon signal images; (2) The dispersion component and scanning mechanism are unnecessary devices, so the system complexity will be simpler and the time resolution of the data acquisition will be improved; (3) By comparing the images of the two spectral bands, the surface plasmon signal has a wide dynamic range, which is suitable for biomolecule sensing; (4) By comparing the images of the two spectral bands, the light intensity noise will be reduced, so the detection limit will be improved; (5) It can be built on various imaging systems, such as scanners, microscopes, and smartphones.

[0007] The present invention provides a surface plasmon imaging device comprising a surface plasmon resonance sensing chip, an imaging module, and an image processing unit. The surface plasmon resonance sensing chip has a surface plasmon resonance wavelength. The imaging module is configured to receive a first-band plasmon resonance beam and a second-band plasmon resonance beam, and generate a first-band surface plasmon image signal and a second-band surface plasmon image signal, respectively. The image processing unit is coupled to the imaging module and receives the first-band surface plasmon image signal and the second-band surface plasmon image signal to generate a surface plasmon signal image. The surface plasmon resonance wavelength is less than the minimum wavelength of the first-band plasmon resonance beam and greater than the maximum wavelength of the second-band plasmon resonance beam, the difference between the minimum wavelength of the first-band plasmon resonance beam and the surface plasmon resonance wavelength is within 10 nanometers, and the difference between the maximum wavelength of the second-band plasmon resonance beam and the surface plasmon resonance wavelength is within 10 nanometers.

[0008] In one embodiment, the first wavelength band plasmon resonance beam and the second wavelength band plasmon resonance beam are both reflected beams or transmitted beams.

[0009] In one embodiment, the surface plasmon resonance wavelength of the surface plasmon resonance sensing chip varies with the refractive index of the surface environment.

[0010] In one embodiment, the surface plasmon resonance sensing chip has a periodic metal nanostructure.

[0011] In one embodiment, the surface plasmon resonance sensing chip is a transmission surface plasmon resonance sensing chip or a reflection surface plasmon resonance sensing chip.

[0012] In one embodiment, the surface plasmon signal image is calculated by the image processing unit using the following formula:

[0013]

[0014] In one embodiment, the surface plasmon imaging apparatus further includes a light source module that emits a first wavelength incident light beam and a second wavelength incident light beam, and a surface plasmon resonance sensing chip configured to receive the first wavelength incident light beam and the second wavelength incident light beam, and generate a first wavelength plasmon resonance beam and a second wavelength plasmon resonance beam, respectively.

[0015] In one embodiment, a light source module includes: a first-band light-emitting unit, a second-band light-emitting unit, and a switching unit. The first-band light-emitting unit emits a first-band incident light beam; the second-band light-emitting unit emits a second-band incident light beam; and the switching unit couples the first-band light-emitting unit and the second-band light-emitting unit to switch between the first-band light-emitting unit and the second-band light-emitting unit.

[0016] In one embodiment, the surface plasmon imaging apparatus further includes a spectrum modulation module comprising a first wavelength filter and a second wavelength filter. The spectrum modulation module receives a broadband light beam to generate a first wavelength incident light beam and a second wavelength incident light beam. The surface plasmon resonance sensing chip is configured to receive the first wavelength incident light beam and the second wavelength incident light beam and generate a first wavelength plasmon resonance light beam and a second wavelength plasmon resonance light beam, respectively.

[0017] In one embodiment, the spectrum modulation module further includes a switching unit coupled to the first band filter and the second band filter, such that the switching unit switches between the first band filter and the second band filter in the incident path from the broadband light source to the surface plasmon resonance sensing chip.

[0018] In one embodiment, the broadband light beam is generated by a broadband light source of a white light source.

[0019] In one embodiment, the white light source is selected from the group consisting of a white light LED, a halogen lamp, a tungsten lamp, and a xenon lamp.

[0020] In one embodiment, the imaging module includes an imaging unit and a lens. The lens is disposed between the surface plasmon resonance sensing chip and the imaging unit. The first wavelength band plasmon resonance beam and the second wavelength band plasmon resonance beam are projected onto the imaging unit through the lens.

[0021] In one embodiment, a surface plasmon resonance sensing chip is configured to receive a broadband light beam to generate a plasmon resonance beam. The surface plasmon imaging apparatus further includes a spectrum modulation module comprising a first wavelength filter and a second wavelength filter, the spectrum modulation module being configured to receive the plasmon resonance beam to generate the first wavelength plasmon resonance beam and the second wavelength plasmon resonance beam. The surface plasmon resonance wavelength of the surface plasmon resonance sensing chip is less than a minimum cutoff wavelength of the first wavelength filter and greater than a maximum cutoff wavelength of the second wavelength filter. The difference between the minimum cutoff wavelength of the first wavelength filter and the surface plasmon resonance wavelength is within 10 nanometers, and the difference between the maximum cutoff wavelength of the second wavelength filter and the surface plasmon resonance wavelength is within 10 nanometers.

[0022] In one embodiment, the spectrum modulation module further includes a switching unit coupled to the first band filter and the second band filter, such that the switching unit switches between the first band filter and the second band filter in the optical path between the surface plasmon resonance sensing chip and the imaging module.

[0023] In one embodiment, the spectrum modulation module further includes: a spectroscopic module configured to receive the plasma resonance beam and generate a first spectroscopic beam and a second spectroscopic beam, and a first band filter configured to receive the first spectroscopic beam and generate a first band plasma resonance beam, and a second band filter configured to receive the second spectroscopic beam and generate a second band plasma resonance beam.

[0024] In one embodiment, the light splitting module includes a light splitter configured to generate a first split light beam and a second split light beam.

[0025] In one embodiment, the light splitting module includes a light splitter and a reflector, and the reflector is disposed between the light splitter and the first wavelength band filter or the second wavelength band filter.

[0026] In one embodiment, the imaging module includes an imaging unit and a lens. The lens is disposed between the first and second wavelength filters and the imaging unit. The first and second wavelength plasma resonance beams are projected onto the imaging unit through the lens.

[0027] In one embodiment, the imaging module includes a first imaging submodule and a second imaging submodule. The first imaging submodule includes a first lens and a first imaging unit, the first lens being disposed between a first wavelength filter and the first imaging unit, and projecting a first wavelength band plasma resonance beam onto the first imaging unit through the first lens. The second imaging submodule includes a second lens and a second imaging unit, the second lens being disposed between a second wavelength filter and the second imaging unit, and projecting a second wavelength band plasma resonance beam onto the second imaging unit through the second lens.

[0028] In one embodiment, the plasmon resonance beam is a reflected beam or a transmitted beam.

[0029] The present invention also provides a surface plasmon imaging method, comprising the following steps: (1) an imaging module receives a first-band plasmon resonance beam and a second-band plasmon resonance beam from a surface plasmon resonance sensing chip to generate a first-band surface plasmon imaging signal and a second-band surface plasmon imaging signal, respectively. The surface plasmon resonance sensing chip has a surface plasmon resonance wavelength, and the surface plasmon resonance wavelength is less than the minimum wavelength of the first-band plasmon resonance beam and greater than the maximum wavelength of the second-band plasmon resonance beam. The difference between the minimum wavelength of the first-band plasmon resonance beam and the surface plasmon resonance wavelength is within 10 nanometers, and the difference between the maximum wavelength of the second-band plasmon resonance beam and the surface plasmon resonance wavelength is within 10 nanometers. (2) an image processing unit receives the first-band surface plasmon imaging signal and the second-band surface plasmon imaging signal to generate a surface plasmon signal image. The image processing unit is coupled to the imaging module.

[0030] In one embodiment, the surface plasmon signal image is calculated by the image processing unit using the following formula:

[0031]

[0032] In one embodiment, the difference between the minimum wavelength of the first wavelength band plasmon resonance beam and the surface plasmon resonance wavelength is within 10 nanometers.

[0033] In one embodiment, the difference between the maximum wavelength of the second-band plasmon resonance beam and the surface plasmon resonance wavelength is within 10 nanometers. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the surface plasmon signal imaging method.

[0035] Figure 2 Figure 3. Schematic diagram of the spectral working principle of the surface plasmon signal imaging method. The transmission spectra of the short-wavelength filter and the long-wavelength filter are represented by dashed and solid lines, respectively. The reflection and transmission spectra of the surface plasmon sensor are represented by red circular (reflection) and blue square (transmission) segments, respectively.

[0036] Figure 3 Schematic diagrams of theoretical verification of the dynamic range. (a) An example of a blue-shifted surface plasmon resonance wavelength and the relationship between (b) the signal value and the wavelength shift; (c) An example of a red-shifted surface plasmon resonance wavelength and the relationship between (d) the signal value and the wavelength shift.

[0037] Figure 4AThe figure is a schematic diagram of an embodiment of a surface plasma imaging device of the present invention.

[0038] Figure 4B FIG. 4 is a schematic diagram of another embodiment of a surface plasma imaging device of the present invention.

[0039] Figure 5A FIG. 1 is a schematic diagram of an embodiment of a dual-camera surface plasmon imaging device using an anomalous transmission type surface plasmon sensor according to the present invention.

[0040] Figure 5B FIG2 is a schematic diagram of an embodiment of a dual-camera surface plasmon imaging device using a prism-type surface plasmon sensor according to the present invention.

[0041] Figure 6A FIG1 is a schematic diagram of an embodiment of a single-camera surface plasmon imaging device using an anomalous transmission type surface plasmon sensor according to the present invention.

[0042] Figure 6B FIG2 is a schematic diagram of an embodiment of a single-camera surface plasmon imaging device using a prism-type surface plasmon sensor according to the present invention.

[0043] Figure 7A FIG. 1 is a schematic diagram of an embodiment of a surface plasmon imaging device using a prism-type surface plasmon sensor according to the present invention.

[0044] Figure 7B FIG. 4 is a schematic diagram of another embodiment of a surface plasmon imaging device using a prism-type surface plasmon sensor according to the present invention.

[0045] Figure 8 (a) is a schematic diagram of an embodiment of a scanner device of the present invention and an image of a nanostructured plasmonic biochip. Figure 8 (b) and Figure 8 (c) Schematic diagram illustrating the red, green, and blue color segment analysis method applied to a transmission-type plasma chip with narrow resonance peaks and narrow resonance troughs.

[0046] Figure 9 Surface plasmon signal images (top row: before introduction of high-refractive-index solution, bottom row: after introduction of high-refractive-index solution). Spectral-A image represents long-wavelength surface plasmon image, spectral-B image represents long-wavelength surface plasmon image, and gamma image represents long-wavelength surface plasmon signal image.

[0047] Figure 10Comparison of the detection limit of the method and device of the present invention with the standard wavelength shift analysis method. (Top left) Sensing curves obtained using the standard wavelength shift analysis method for measuring sugar solutions with different refractive indices. (Top right) Sensing curves obtained using the device and method of this invention for measuring sugar solutions with different refractive indices, where n0 = 1.3290, n1 = 1.3295, n2 = 1.3300, n3 = 1.3305, n4 = 1.3320, and n5 = 1.3355, in refractive index units (RIU). (Bottom left) Black dots: Changes in surface plasmon signals measured using the standard wavelength shift analysis method. Red line: Linear regression analysis. (Bottom right) Black dots: Changes in surface plasmon signals measured using the device and method of the present invention. Red line: Linear regression analysis.

[0048] Figure 11 Results of measuring protein-antigen interactions using the real-time surface plasmon imaging technology of the present invention. (Left) Surface plasmon signal images measured at the end of the experiment. (Right) Sensing curves extracted from the exp, ref, and non regions, respectively.

[0049] Figure 12 (a) A commercial transmission scanner and a double-layer nano-aluminum slit chip (left) were used to test different ratios of glycerol and water. The right side of the image shows the transmission images of the plasma sub-chip recorded by the scanner at different ratios of glycerol and water. The refractive index of glycerol and water ranged from 1.3290 to 1.346. (b) Relationship between the ambient refractive index and the transmission intensity using a custom formula for the structure in the red, green, and red-green color segments. (c) Relationship between the ambient refractive index and the change in transmission intensity using a custom formula for the structure in the red, green, and red-green color segments. (d) Transmission spectra of a double-layer nano-aluminum slit with a period of 430nm under different refractive index environments. (e) Relationship between the resonance peak wavelength and the ambient refractive index.

[0050] Figure 13A FIG2 is a schematic diagram of an embodiment of a surface plasmon imaging device using dual light sources and a prism-type surface plasmon sensor according to the present invention.

[0051] Figure 13B FIG. 1 is a schematic diagram of an embodiment of a surface plasmon imaging device using dual light sources and an anomalous transmission type surface plasmon sensor according to the present invention. DETAILED DESCRIPTION

[0052] The present invention provides a new method and related apparatus for surface plasmon imaging. Figure 1 The working principle of the method of the present invention is introduced. A metal surface with surface plasmon resonance has a resonance peak or trough in its transmission or reflection spectrum, and the center position of the peak or trough is λSPR (refer to Figure 2 The surface plasmon sensor is illuminated by a beam of light, which passes through two color filters designed for transmission wavelengths. The two color filters divide the surface plasmon sensor transmission peak into a short wavelength portion and a long wavelength portion. To determine the two-dimensional surface plasmon signal, the image formed by the light beam passing through the long-wavelength filter is called a long-wavelength surface plasmon image (Image A). ij ), i and j are the corresponding pixels in the x and y directions respectively. The image formed by the light beam passing through the short-wavelength filter is called the short-wavelength surface plasmon image (image B ij ), the image after calculation is the surface plasma signal image. Through the custom calculation formula: (A ij -B ij ) / (A ij +B ij ), we can get a self-referenced image, A in the formula ij With B ij where represents the light intensity at image pixel ij. This self-referencing calculation is typically used with a dual or quad position sensor to sense the position of a beam. If the light is noisy, the equation can be calculated and simplified:

[0053]

[0054] Because A~B>>dI, the impact of noise on the signal is greatly reduced.

[0055] This paper uses numerical methods to evaluate the dynamic range of this invention. Figure 3 (a) to Figure 3 (d) Demonstrates the sensing process in which the transmission peak position shifts with changes in the refractive index. Figure 3 (a) shows the blue-shifted surface plasmon spectroscopy signal, which represents the molecular dissociation event detected by the surface plasmon sensor. For comparison; Figure 3 (c) shows the red-shifted surface plasmon spectroscopy signal, representing the detection of a molecular binding event by the surface plasmon sensor. The contrast signal is defined as γ = ((A - B)) / ((A + B)). This is compared with the wavelength-shifted signal, as the wavelength-shifted method is well known for its wide dynamic range. Figure 3 (b) and Figure 3 (d) shows the correlation curve between the contrast signal and wavelength shift. Both the red-shift and blue-shift examples show a linear relationship with the wavelength shift range from -5 nm to +5 nm. This dynamic range corresponds to a refractive index range of -0.0106 to +0.0106. In other words, this method is suitable for most biomolecular interactions.

[0056] like Figure 4A As shown, to implement this method, an optical setup including an illumination system 1, two narrow-band filters 22, 24, a sensing unit 3 (including a surface plasmon resonance sensing chip) and a monochrome imaging device 42 is demonstrated (as shown in FIG. Figure 4A As shown). The above-mentioned sensing unit 3 is composed of a fluid device 33 and a surface plasmon sensor 35. The above-mentioned surface plasmon sensor 35 may be an abnormal transmission type surface plasmon sensor or a localized surface plasmon resonance type surface plasmon sensor. However, this embodiment is described by taking the surface plasmon sensor 35 as an example of an abnormal transmission type surface plasmon sensor. That is, although the present embodiment uses an abnormal transmission type surface plasmon sensor in combination with the measurement of transmitted light as a demonstration, the purpose can still be achieved by measuring reflected light, and its structure will be described later. The above-mentioned fluid device 33 is used to inject the object to be tested onto the surface plasmon sensor 35. The above-mentioned abnormal transmission type surface plasmon sensor 35 is composed of a nano-groove matrix covered with gold. The above-mentioned two narrow-band filters 22 and 24 both have narrow bandwidths (<40 nanometers), and the long-wavelength band boundary of one of the bandwidth filters is close to the surface plasmon resonance wavelength (λ long-boundary -λ SPR ≦3~5nm), the short-wave band boundary of the other bandwidth filter is close to the surface plasmon resonance wavelength (λ SPR -λ short-boundary ≤3-5nm). A drive device 26 is used to switch the positions of these bandwidth filters. The illumination system 1 is a halogen bulb, a light-emitting diode, or a white light source with a continuous wavelength band. The imaging module 4 is a monochrome imaging device 42. In the image captured by the imaging device 42, the image formed by the light beam passing through the long-wavelength filter 22 is called a long-wavelength surface plasmon image, and the image formed by the light beam passing through the short-wavelength filter 24 is called a short-wavelength surface plasmon image. The image calculated by the following formula is the surface plasmon signal image:

[0057]

[0058] Figure 4B exhibit Figure 4A A variation of the architecture shown. Figure 4A The architecture shown is roughly the same as Figure 4A The architecture shown differs in that Figure 4B The spectrum modulation module 2 (including the linear polarization plate 28 , the long-wavelength filter 22 , and the short-wavelength filter 24 ) is placed between the surface plasmon sensor 35 and the imaging device (camera) 42 .

[0059] Figure 5A An alternative architecture for implementing the present invention is shown. Figure 4A The architecture shown is roughly the same as Figure 4A The architecture shown differs in that, in Figure 5A In the illustrated optical setup, long-wavelength and short-wavelength surface plasmon images are captured by two monochrome imaging devices (cameras) 42 and 42', respectively. To implement this method, a 50:50 beam splitter (21000, Chroma) is used to split the beam, and two bandwidth filters 22 and 24 (ET640 / 20m and ZET660 / 20x, Chroma) are placed in front of the two imaging devices (cameras) 42 and 42', respectively. This setup allows for simultaneous capture of long-wavelength and short-wavelength surface plasmon images without requiring additional time to switch between the two bandwidth filters, resulting in improved temporal resolution.

[0060] Figure 5B exhibit Figure 5A A variation of the architecture shown. Figure 5A The architecture shown is roughly the same as Figure 5A The architecture shown differs in that Figure 5B The prism type surface plasma sensor 35' is used to replace the original Figure 5A Abnormal transmission type surface plasmon sensor 35.

[0061] Figure 6A Another alternative architecture for implementing the present invention is shown. Figure 5A The architecture shown is roughly the same as Figure 5A The architecture shown differs in that, in Figure 5A In the illustrated embodiment, long-wavelength and short-wavelength surface plasmon images are captured separately by the same imaging device (camera) 42. To implement this method, an image segmentation device 5 (A12801, Hamamatsu) with a built-in 50:50 beam splitter (21000, Chroma) and two bandwidth filters 22 and 24 (ET640 / 20m and ZET660 / 20x, Chroma) is used to segment the images, and the images are then captured by the same imaging device (camera 42). With this architecture, long-wavelength and short-wavelength surface plasmon images can be captured simultaneously by the same camera in different pixel areas without requiring additional time to switch between the two bandwidth filters 22 and 24, thereby improving temporal resolution.

[0062] Figure 6B exhibit Figure 6A A variation of the architecture shown. Figure 6A The architecture shown is roughly the same as Figure 6A The architecture shown differs in that Figure 6B The prism type surface plasma sensor 35' is used to replace the original Figure 6AAbnormal transmission type surface plasmon sensor 35.

[0063] Figure 7A The present invention is implemented in a prism-type surface plasmon sensor 35'. In this embodiment, light enters the prism-type surface plasmon sensor 35', and the reflected light is then imaged on an imaging device (camera) 42. A spectrum modulation module 2 (including a linear polarization plate 28, a long-wavelength filter 22, and a short-wavelength filter 24) is positioned between the broadband light source 13 and the prism-type surface plasmon sensor 35'. A driver 26 within the spectrum modulation module 2 allows light to pass through the long-wavelength filter 22, capturing a long-wavelength surface plasmon image. The driver 26 within the spectrum modulation module 2 then allows light to pass through the short-wavelength filter 24, capturing a short-wavelength surface plasmon image. Finally, a surface plasmon signal image is calculated.

[0064] However, the spectrum modulation module 2 (including the linear polarization plate 28, the long-wavelength filter 22 and the short-wavelength filter 24) is not limited to being placed in the following manner: Figure 7A As shown in FIG, it can also be placed between the surface plasmon sensor 35 and the imaging device (camera) 42. Figure 7B As shown, it is Figure 7A A variation of the architecture shown. Figure 7A The architecture shown is roughly the same as Figure 7A The architecture shown differs in that Figure 7B The spectrum modulation module 2 (including the linear polarization plate 28 , the long-wavelength filter 22 , and the short-wavelength filter 24 ) is placed between the prism-type surface plasmon sensor 35 ′ and the imaging device (camera) 42 .

[0065] In addition, the present invention also provides a surface plasmon imaging device, as shown in FIG13, comprising a light source module 6, a sensing unit 3 (including a surface plasmon resonance sensing chip), an imaging module 4, and an image processing unit. Figure 4A The structure shown in FIG13 is the same as that shown in FIG14 , except that a light source module having two incident light beams of different wavelengths is used instead of Figure 4AThe broadband light source and spectrum modulation module 2 are shown in FIG13 . The light source module includes a first-band light-emitting submodule unit 61 and a second-band light-emitting submodule unit 62. The first-band light-emitting submodule unit 61 includes a first-band light-emitting unit 611, a first lens 612, and a first linear polarizer 613. The first-band light-emitting unit 611 emits a first-band incident light beam, which sequentially passes through the first lens 612 and the first linear polarizer 613 before being incident on the surface plasmon sensor (surface plasmon resonance sensing chip) 35. Similarly, the second-band light-emitting submodule unit 62 includes a second-band light-emitting unit 621, a second lens 622, and a second linear polarizer 623. The second-band light-emitting unit 621 emits a second-band incident light beam, which sequentially passes through the second lens 622 and the second linear polarizer 623 before being incident on the surface plasmon resonance sensing chip. However, this embodiment uses the example of an anomalous transmission-type surface plasmon sensor as an example. That is, although this embodiment uses an anomalous transmission type surface plasmon sensor in conjunction with the measurement of transmitted light as an example, the purpose can also be achieved by measuring reflected light, and its structure will be described below. The aforementioned fluid device 33 is used to inject the object to be measured onto the surface plasmon sensor 35. The aforementioned anomalous transmission type surface plasmon sensor 35 is composed of a nano-groove matrix covered with gold. The two aforementioned incident light beams (the first wavelength incident light beam and the second wavelength incident light beam) both have narrow bandwidths (<40 nanometers), and the long wavelength band boundary of the second wavelength incident light beam is close to the resonant wavelength (λ) of the surface plasmon sensor. long-boundary -λ SPR ≤3-5nm), and the short-band boundary of the first-band incident light beam approaches the surface plasmon resonance wavelength of the surface plasmon sensor (λSPR - λshort-boundary ≤3-5nm). A drive device 26 is used to switch the positions of the first-band light-emitting sub-module unit 61 and the second-band light-emitting sub-module unit 62. The above-mentioned imaging system is a monochrome imaging device 42. Among the images captured by the imaging device 42, those with the long-band light beam (first-band incident light beam) are called long-band surface plasmon images, and those with the short-band light beam (second-band incident light beam) are called short-band surface plasmon images. The image calculated by the following formula is the surface plasmon signal image:

[0066]

[0067] Figure 13B exhibit Figure 13A A variation of the architecture shown. Figure 13A The architecture shown is roughly the same as Figure 13A The architecture shown differs in that Figure 13B The prism type surface plasma sensor 35' is used to replace the original Figure 13AAbnormal transmission type surface plasmon sensor 35.

[0068] Figure 8 (a) Schematic diagram of one of the scanner devices based on this invention and an image of a nanostructured plasmonic biochip. Figure 8 (b) and Figure 8 (c) is a schematic diagram illustrating the red, green and blue color segment analysis method applied to a transmission-type plasma chip with a narrow resonance peak and a narrow resonance trough. The white light LED light source of the transmission-type scanner can be divided into three color segments: red, green and blue, and the resonance peak or trough of the metal nanostructure can be adjusted and appear in the overlapping area of ​​the red and green or blue and green color segments. For the resonance peak, when the peak is red-shifted due to the increase in the refractive index of the environment, this movement will cause an increase in the intensity of the transmitted light in the red segment (AR) and a decrease in the intensity of the transmitted light in the green segment (BG). Conversely, for the resonance trough, the movement of the trough will cause a decrease in the transmitted light in the red segment and an increase in the intensity of the transmitted light in the green segment. The custom calculation formulas for the resonance peak and resonance trough in the red and green color segments can be expressed as γ=(A G -B R ) / (A G +B R ) and γ=(A R -B G ) / (A R +B G ), which can remove common noise and enhance sensing capabilities.

[0069] Example 1

[0070] Use as Figure 4A 、 Figure 4B In this setup, images of different spectra can be captured by the same camera, and surface plasmon signal images can also be calculated. In this architecture, a stable-intensity halogen lamp is used to generate a broadband light source, and two bandwidth filters (ET640 / 20m and ZET660 / 20x, Chroma) are mounted in an electrically controlled rotary mount for lightwave switching. A metal-coated nanogroove matrix structure is fabricated as a surface plasmon sensor. The period of this nanogroove matrix is ​​designed to be 470 nanometers, aligning the wavelength of surface plasmon resonance in water to 650 nanometers. This surface plasmon sensor is placed on the microscope's observation stage for imaging. A complementary monochrome metal-oxide semiconductor camera (C11440, Hamamatsu) is used to capture images of the surface plasmon sensor. Figure 9 The upper and lower images represent the transmission images of the surface plasmon sensor before and after injection of the high-refractive-index solution, respectively. Comparing the intensity differences between the long-wavelength and short-wavelength surface plasmon images before and after injection, no significant change is observed. However, the surface plasmon signal image clearly shows a strong signal in the flow channel region.

[0071] Example 2

[0072] The detection limit is an important indicator of surface plasmon sensors. In this example, the detection limit is Figure 4A 、 Figure 4B The detection limit of this invention was tested using a device and compared with a standard wavelength shift analysis method. In the test using the device and method of this invention, a structure consisting of a gold-coated nanogroove matrix was used as a surface plasmon sensor. The nanogroove matrix structure in the surface plasmon sensor was designed with a period of 470 nanometers. The surface plasmon sensor was placed on the observation platform of a microscope for imaging. A complementary monochrome metal oxide semiconductor (CMOS) camera (C11440, Hamamatsu) was used to capture the surface plasmon sensor image. Sugar solutions with different ratios and known refractive indices were sequentially introduced through a microfluidic device. By analyzing the change and stability of the sensing curve, the sensitivity and detection limit were determined. The standard wavelength shift analysis method uses a single-point measurement method, illuminating a single focused spot of light onto a surface plasmon sensor with a nanogroove matrix structure designed with a period of 470 nanometers. A microfluidic device sequentially introduces sugar solutions of varying proportions and known refractive indices, identical to those used in the aforementioned device and method tests. The transmitted light is collected by an optical fiber and transmitted to a spectrometer to read the spectral signal. The sensitivity and detection limit are analyzed by observing the wavelength shift of the spectral peak. Sensitivity is defined as the change in the surface plasmon signal per unit change in refractive index, while the detection limit is defined as the minimum refractive index change that the system can resolve. Figure 10 The upper left figure shows the sensing curve measured by the standard wavelength shift analysis device and method. Figure 10 The upper right figure shows the sensing curves measured by the present device and method. Both figures clearly show that as the refractive index of the object increases from n0 = 1.3290 to n5 = 1.3355, the wavelength and surface plasmon change signals also increase. When the refractive index returns from n5 = 1.3355 to n0 = 1.3290, the wavelength and surface plasmon change signals also return to the baseline. Comparing the two signals, it can be seen that in addition to forming a surface plasmon resonance image, the present device can also reduce noise effects due to its self-referenced signal, resulting in a better signal-to-noise ratio than the commonly used resonance wave peak change. The sensing curves were further analyzed using the linear regression method, as shown in Figure 2. Figure 10The lower left figure shows the relationship between peak wavelength and refractive index: λ = 184.64082 + 346.42185 × RIU (refractive index unit), where 346.42185 represents the detection sensitivity of the standard wavelength shift analysis method. System stability can be determined by analyzing the peak wavelength signal under the same refractive index sugar water over a period of time. In this example, the standard deviation of the peak wavelength signal obtained from the analysis of the n0 measurement interval is 0.0058 nm. Therefore, the detection limit of this method is 1.6743 × 10 -5 RIU; On the other hand, the relationship between the surface plasmon signal change and the refractive index is γ = -14.4292 + 10.8558 × RIU, where 10.8558 is the detection sensitivity of this case. In this embodiment, the surface plasmon signal standard deviation obtained by analyzing the n0 measurement interval is 1.12774 × 10 -4 Therefore, the detection limit of this method can be obtained as 1.0394×10 -5 RIU. This value clearly demonstrates that this method, by reducing the influence of self-reference signal noise, can achieve a lower detection limit than the standard wavelength-shift analysis method. More importantly, the standard wavelength-shift analysis method is only suitable for single-point measurement, while the method and device proposed in this case are suitable for full-field imaging and can simultaneously analyze multiple-point signals, significantly improving measurement throughput.

[0073] Example 3

[0074] The temporal variation of the surface plasmon signal is called a sensing curve. It is an important application in surface plasmon detection. The sensing curve can provide a considerable amount of information, such as affinity, the dissociation and association rates between the calibrant and the analyte, the concentration of the target molecule, and the specificity of the interacting molecules. Figure 11 This paper introduces an embodiment of the invention that can measure sensing curves with high throughput. In this embodiment, long-wavelength surface plasmon images and short-wavelength surface plasmon images are recorded sequentially, and time-series surface plasmon signal images are also calculated simultaneously. Because all surface plasmon signal images are recorded at all times, users can extract sensing images in specific areas. Figure 11 The right figure includes three sensing curves, labeled exp, ref, and non, corresponding to the surface plasmon signals extracted from regions exp, ref, and non in the left figure. Region exp is located within the flow channel and on the metal-coated nanogroove matrix structure, so it exhibits a surface plasmon signal. In contrast, region ref is not located on the nanogroove matrix structure. Region non is located outside the flow channel and exhibits no surface plasmon signal.

[0075] Example 4

[0076] To further confirm the feasibility of applying scanners and red, green, and blue color segment analysis methods to plasmon sensing chips. Figure 12 (a) shows an experiment using a commercial transmission scanner and a double-layer nano-aluminum slit chip (left) to measure glycerol-water mixtures at different ratios. The right side of the image shows transmission images of the plasmonic chip recorded using the scanner at different glycerol-water ratios. The refractive index of glycerol-water ranged from 1.3290 to 1.346. To ensure that the resonance peaks generated by the structure appear in the red and green color ranges, the period length of the double-layer nano-aluminum slit chip was adjusted to 430 nm.

[0077] First, pure water is dropped on the chip, and then a cover glass is placed on it to record the chip transmission image. Afterwards, the chip is blown dry and the above steps are repeated with different proportions of glycerol water. The image scanning results are as follows: Figure 12 (a) Shown on the right. Figure 12 (b) is the analysis of the image in red, green and red and green segments, and the transmission intensity of the structure at different refractive indices. As the refractive index of the environment increases, the resonance peak at a wavelength of 573nm produces a red shift, such as Figure 12 (d) As shown. Since the refractive index has a linear relationship with the peak position, its refractive index sensitivity is 427nm / RIU (see Figure 12 (e)). This value is close to the theoretical calculated value, and its refractive index sensitivity is determined by the period length, that is, S λ ~P nm / RIU, RIU is the unit of refractive index. Because the resonance peak moves toward the red segment as the refractive index increases, the transmission intensity of the structure in the red segment gradually increases. Conversely, because the resonance peak gradually moves away from the green segment as the refractive index increases, the transmission intensity of the structure in the green segment gradually decreases. Figure 12 As shown in (c), the change in transmitted intensity for both bands exhibits a linear relationship with the ambient refractive index, with slopes of 201% / RIU and 56% / RIU for the red and green bands, respectively. The intensity change for the custom calculation formula for both color bands also exhibits a linear relationship with the ambient refractive index, with an intensity sensitivity of 1241% / RIU. This sensitivity is superior to other two-color analysis methods. Therefore, these results demonstrate the feasibility of applying the scanner and red, green, and blue three-color analysis method to plasmon sensing chips.

Claims

1. A surface plasmon imaging device comprising: A surface plasmon resonance sensing chip having a surface plasmon resonance wavelength; an imaging module configured to receive the first-wavelength-band plasma resonance beam and the second-wavelength-band plasma resonance beam, and to generate a first-wavelength-band surface plasma image signal and a second-wavelength-band surface plasma image signal respectively; and an image processing unit coupled to the imaging module and receiving the surface plasma image signal of the first wavelength band and the surface plasma image signal of the second wavelength band to generate a surface plasma signal image; The surface plasmon resonance wavelength is smaller than the minimum wavelength of the first-band plasmon resonance beam and larger than the maximum wavelength of the second-band plasmon resonance beam. The difference between the minimum wavelength of the first-band plasmon resonance beam and the surface plasmon resonance wavelength is within 10 nanometers, and the difference between the maximum wavelength of the second-band plasmon resonance beam and the surface plasmon resonance wavelength is within 10 nanometers.

2. The device according to claim 1, wherein the first wavelength band plasmon resonance beam and the second wavelength band plasmon resonance beam are both reflected beams or transmitted beams. 3 . The device according to claim 1 , wherein the surface plasmon resonance wavelength of the surface plasmon resonance sensing chip varies with a surface environment refractive index. The device according to claim 1 , wherein the surface plasmon resonance sensing chip has a periodic metal nanostructure. 5 . The device according to claim 1 , wherein the surface plasmon resonance sensing chip is a transmission surface plasmon resonance sensing chip or a reflection surface plasmon resonance sensing chip.

6. The apparatus according to claim 1 , wherein the surface plasmon signal image is calculated by the image processing unit using the following formula:

7. The apparatus according to claim 1, further comprising: The light source module emits a first wavelength band incident light beam and a second wavelength band incident light beam. The surface plasmon resonance sensing chip is configured to receive the first wavelength band incident light beam and the second wavelength band incident light beam, and generate the first wavelength band plasmon resonance light beam and the second wavelength band plasmon resonance light beam respectively.

8. The device according to claim 7, wherein the light source module comprises: A first wavelength band light emitting unit, emitting an incident light beam in the first wavelength band; A second wavelength band light emitting unit, emitting an incident light beam in the second wavelength band; as well as The switching unit is coupled to the first-band light emitting unit and the second-band light emitting unit, thereby switching the first-band light emitting unit and the second-band light emitting unit.

9. The apparatus according to claim 1, further comprising: The spectrum modulation module includes a first-band filter and a second-band filter. The spectrum modulation module receives a broadband light beam to generate a first-band incident light beam and a second-band incident light beam. The surface plasmon resonance sensing chip is configured to receive the first-band incident light beam and the second-band incident light beam and generate the first-band plasmon resonance light beam and the second-band plasmon resonance light beam, respectively.

10. The device according to claim 9, wherein the spectrum modulation module further comprises: The switching unit is coupled to the first band filter and the second band filter, so that the switching unit switches the first band filter and the second band filter in an incident path from the broadband light source to the surface plasmon resonance sensing chip. The device according to claim 9 , wherein the broadband light beam is generated by a broadband light source of a white light source.

12. The device of claim 11, wherein the white light source is selected from the group consisting of a white light LED, a halogen lamp, a tungsten lamp, and a xenon lamp.

13. The device according to claim 1, wherein the imaging module comprises an imaging unit and a lens, the lens being disposed between the surface plasmon resonance sensing chip and the imaging unit, and the first-band plasmon resonance beam and the second-band plasmon resonance beam being projected onto the imaging unit through the lens.

14. The device according to claim 1, wherein the surface plasmon resonance sensing chip is configured to receive a broadband light beam to generate a plasmon resonance light beam; the device further comprising: a spectrum modulation module comprising a first wavelength band filter and a second wavelength band filter, wherein the spectrum modulation module is configured to receive the plasma resonance beam and generate the first wavelength band plasma resonance beam and the second wavelength band plasma resonance beam; The surface plasmon resonance wavelength of the surface plasmon resonance sensing chip is smaller than the minimum cutoff wavelength of the first band filter and larger than the maximum cutoff wavelength of the second band filter. The difference between the minimum cutoff wavelength of the first band filter and the surface plasmon resonance wavelength is within 10 nanometers, and the difference between the maximum cutoff wavelength of the second band filter and the surface plasmon resonance wavelength is within 10 nanometers.

15. The apparatus according to claim 14, wherein the spectrum modulation module further comprises: The switching unit is coupled to the first band filter and the second band filter, so that the switching unit switches the first band filter and the second band filter in the optical path between the surface plasmon resonance sensing chip and the imaging module.

16. The apparatus according to claim 14, wherein the spectrum modulation module further comprises: The spectroscopic module is configured to receive the plasma resonance beam and generate a first spectroscopic beam and a second spectroscopic beam, and the first band filter is configured to receive the first spectroscopic beam and generate a first band plasma resonance beam, and the second band filter is configured to receive the second spectroscopic beam and generate a second band plasma resonance beam. 17 . The device according to claim 16 , wherein the light splitting module comprises a light splitter configured to generate the first split light beam and the second split light beam. 18 . The device according to claim 16 , wherein the light splitting module comprises a light splitter and a reflector, and the reflector is disposed between the light splitter and the first wavelength band filter or the second wavelength band filter.

19. The device according to claim 16, wherein the imaging module comprises an imaging unit and a lens, the lens being disposed between the first and second wavelength band filters and the imaging unit, and the first and second wavelength band plasmon resonance beams being projected onto the imaging unit through the lens.

20. The apparatus of claim 14, wherein the imaging module comprises: a first imaging sub-module comprising a first lens and a first imaging unit, wherein the first lens is disposed between the first wavelength filter and the first imaging unit, and the first wavelength plasma resonance beam is projected onto the first imaging unit via the first lens; and The second imaging submodule includes a second lens and a second imaging unit. The second lens is disposed between the second wavelength filter and the second imaging unit. The second wavelength plasma resonance beam is projected onto the second imaging unit through the second lens.

21. The device according to claim 14, wherein the plasmon resonance beam is a reflected beam or a transmitted beam.

22. A method for surface plasmon imaging, comprising the following steps: An imaging module receives a first-wavelength plasmon resonance beam and a second-wavelength plasmon resonance beam from a surface plasmon resonance sensing chip to generate a first-wavelength surface plasmon image signal and a second-wavelength surface plasmon image signal, respectively. The surface plasmon resonance sensing chip has a surface plasmon resonance wavelength that is less than a minimum wavelength of the first-wavelength plasmon resonance beam and greater than a maximum wavelength of the second-wavelength plasmon resonance beam. The difference between the minimum wavelength of the first-wavelength plasmon resonance beam and the surface plasmon resonance wavelength is within 10 nanometers, and the difference between the maximum wavelength of the second-wavelength plasmon resonance beam and the surface plasmon resonance wavelength is within 10 nanometers. An image processing unit receives the first wavelength band surface plasma image signal and the second wavelength band surface plasma image signal to generate a surface plasma signal image, wherein the image processing unit is coupled to the imaging module.

23. The method according to claim 22, wherein the surface plasmon signal image is calculated by the image processing unit using the following formula:

24. The method of claim 22, wherein the difference between the minimum wavelength of the first wavelength band plasmon resonance beam or the maximum wavelength of the second wavelength band plasmon resonance beam and the surface plasmon resonance wavelength is within 10 nanometers.

Images