CN115290616A - Polymer Optical Waveguide Evanescent Field Sensing Structure - Google Patents

Abstract

The invention discloses a polymer optical waveguide evanescent field sensing structure for exciting an evanescent field, which consists of an aspheric cylindrical lens and a thin-layer planar waveguide structure. The aspheric cylindrical lens is positioned at the front end of the structure and used for refractively coupling the parallel incident laser to the waveguide structure, and the thin-layer planar waveguide is positioned at the rear end of the structure and used for generating an evanescent field and serving as a carrier of biological reaction. Collimated incident light is coupled into the rear end thin-layer waveguide at a certain angle after passing through the front end aspheric coupling structure, and when light is transmitted in the thin-layer waveguide, an evanescent field with hundreds of nanometer magnitude can be generated on the upper surface of the waveguide, so that fluorescence excitation of biological substances on the upper surface of the waveguide is realized, and emitted fluorescence signals are filtered and finally captured and imaged by an optical camera.

Description

Polymer Optical Waveguide Evanescent Field Sensing Structure

technical field

The invention relates to a planar waveguide sensing structure, in particular to a sensing structure that excites a large-scale evanescent field illumination area, and its application fields include biological detection and microscopic imaging.

Background technique

Since the early 1960s, biosensors were first proposed and verified to be feasible, and this field has continued to grow and develop with the advancement of technologies such as biochemical technology and microelectronics. Among many biosensing technologies, fluorescent biosensors are widely used due to their high sensitivity, good selectivity, and strong real-time performance. Sensors often need to use complex optical systems to improve the signal-to-noise ratio. For this reason, evanescent field illumination is used in the field of fluorescent biosensing.

When light is incident from an optically denser medium to an optically rarer medium, the refracted light will gradually deviate from the normal line as the incident angle increases until the light propagates along the interface between the two media. At this time, the refraction angle is 90 degrees. The incident angle continues to increase, the refracted light will no longer appear in the optically sparse medium, and the incident light will be completely reflected back into the optically dense medium. The incident angle at which total reflection begins to occur is called the critical angle, and this phenomenon is called light of total internal reflection. The boundary continuity condition of the electromagnetic field shows that when full emission occurs at the interface of the medium, a non-uniform field with extremely fast attenuation and height limitation will be generated on the surface of the optically dense medium, which is the evanescent field. The evanescent field has the same wavelength as the incident light, and its intensity decays exponentially with the distance away from the waveguide surface. For the incident light in the visible band (380-780 nanometers), only a hundred nanometers of evanescent field illumination area will be formed on the surface of the waveguide. The penetration depth of the evanescent field is defined as the position where the energy decays to the initial energy 1/e, and the penetration depth of the evanescent field can be changed by changing the wavelength of the incident light, the angle of incidence, or the relative refractive index of the material.

Most of the excitation wavelengths of fluorescent markers used for biological detection are in the visible light band, and the depth of the evanescent field excited in this band is much smaller than the average diameter of cells, which means that only fluorescent markers close to the surface of the waveguide can be excited. The excitation method only selectively excites the target fluorescence, while greatly reducing the interference of incident light, scattered light, cell autofluorescence and background fluorescence, so that the signal-to-noise ratio of the detection system is greatly improved.

Biosensor devices based on the principle of evanescent field fluorescence have been applied in many fields such as cell imaging, environmental monitoring, drug development and in vitro diagnosis. In order to make such sensors obtain higher signal-to-noise ratio, detection range, uniformity and integration, And to achieve multi-wavelength detection, reduce the cost of production and use, the structure, material, supporting system and fluorescent labeling method of the sensor have been improved. Structurally, the typical structure of evanescent field sensing lighting includes optical fiber and planar waveguide. The optical fiber structure has the advantages of small size, high sensitivity, and strong anti-interference ability, but the detection area it can provide is small, and the operation and manufacturing requirements more complicated. Therefore, in practical applications, planar waveguides are preferred due to many advantages such as large sensing area, various coupling methods, and relatively simple manufacturing. In addition, common planar waveguide coupling methods include direct coupling, prism coupling, lens coupling, grating coupling, etc., where the lens coupling has a converging effect while deflecting the incident light. In contrast, using this method for incident light coupling , without the need for supporting complex alignment and angle adjustment devices. At the same time, the focusing effect makes the incident light undergo continuous total internal reflection at a certain distance in the waveguide, and a continuous large-scale illumination sensing area will be produced on the surface of the waveguide. In terms of materials, the early evanescent field sensing structure was developed based on glass slides. However, glass materials have great limitations in the processing of irregular structures. The research focus is to reduce the manufacturing difficulty and production cost while ensuring the sensing performance, making the large-scale use of such sensors possible.

At present, there is no evanescent field sensing structure/system that can be mass-produced, which can achieve large effective sensing area and fast multi-wavelength detection while achieving high sensing area uniformity and high integration. Therefore, in order to further To improve the performance of evanescent field fluorescence sensors, reduce production and use costs, and realize multi-scenario applications, it is necessary to develop a sensing structure/system based on polymer-integrated slab waveguides.

Contents of the invention

The invention discloses an improved evanescent field illumination sensing structure with wavelength robustness in the visible light band. The structure couples the input light into the thin-layer planar waveguide through the coupling structure, so as to realize the continuous and full generation of the incident light in the waveguide. Internal reflection, the specific structure is shown in Figure 1, including a coupling structure 110, a thin-layer waveguide structure 120 and a skirt structure 130, each structure is characterized by:

The coupling structure 110 is located at the front end of the illumination sensing structure and is integrated by a lens 111 and a polygonal prism 112 . The surface of the lens 111 is designed as an aspheric surface, thereby eliminating the spherical aberration caused by the lens structure, and minimizing the chromatic aberration in the 380-780nm visible light band, and the structure includes at least a quarter of a complete cylinder; The position of the short side of the polygonal prism 112 should not exceed the position of the second reflection point of the input light in the thin-layer waveguide, so as to avoid the mismatch between the coupling structure and the thin-layer waveguide, causing the incident light to directly pass through the coupling structure, The structure is also provided with a tangential inclined surface, which can play the role of installation limit.

The thin-layer waveguide structure 120 is located at the rear end of the illumination sensing structure. The length and width of the structure are the size of a standard slide glass, such as 75mm×25mm, and the thickness of the waveguide is between 0.05-0.8mm.

The skirt structure 130 is arranged on the lower surface of the thin-layer waveguide structure, and is an integrated structure with the thin-layer waveguide. This structure can reduce the Z-direction warping of the thin-layer waveguide due to processing stress, and the value should be less than 0.5mm; The structure also acts as a spacer to form a fluid cavity between the waveguide surface and the superstructure. A similar effect can also be achieved by setting the skirt structure on the upper surface of the thin-layer structure.

The present invention uses a high-precision mold to perform injection molding on the waveguide structure, and integrates the coupling structure 110, the thin-layer waveguide structure 120, and the skirt structure 130. Among them, the lens 111 and the polygonal prism 112 of the coupling structure 110 are molded into inserts. Design, as shown in Figure 2(c), for further optimization and improvement of the structure in the future. In addition, for polymer thin-layer waveguides with a thickness less than 0.4mm, splicing can be used for processing, and the coupling structure 110 is molded by an insert mold, as shown in Figure 2(d), the thin-layer waveguide structure 120 and the skirt The structure 130 is integrally injection-molded by another mold, and a refractive index matching liquid is used to realize bonding and splicing of the two processed structures.

The evanescent field illumination sensing structure involved in the present invention satisfies the following characteristics: the coupling structure 110, the thin-layer waveguide structure 120 and the skirt structure 130 are all processed with the same material, and the refractive index of the material is between 1.44-2.50, and for The incident light with a wavelength of 380-780nm is transparent, and the material can be selected from optical polymer or optical glass according to different processing methods. In the structure shown in Figure 3, the roughness of the upper surface 310, the lower surface 320 of the waveguide structure and the free-form surface 330 of the coupling structure is less than 10nm, the flatness of the upper plane 310 of the waveguide structure is less than 0.5mm, and the Z direction of the waveguide structure The warpage is less than 0.5mm, the thickness tolerance of the waveguide structure is controlled within ±0.05mm, and the surface roughness of the side surface 340 of the illumination sensing structure is less than 100nm; in order to improve the utilization efficiency of incident light, the free-form surface 330 can be coated to increase through treatment.

The invention also discloses an optical system for producing large-scale uniform evanescent field illumination area. After the input laser is collimated, it is incident on the beam shaping element, and then emits a light spot with uniform energy distribution at a certain angle. After passing through the cylindrical lens, a collimated and shaped light spot that does not diverge can be obtained. The collimated light spot is horizontally incident on the sensor structure. The coupling structure focuses the incident angle greater than the critical angle of total internal reflection onto the upper surface of the thin-layer waveguide structure. After the beam undergoes continuous total internal reflection in the thin-layer waveguide structure, the initial energy of the evanescent field on the surface of the thin-layer waveguide is finally tends to be stable, forming a large-scale, high-uniformity sensing area. The system is specifically shown in FIG. 4 , including a light source part 410 , an illumination sensing structure 420 , and a detection area 430 . The main characteristics of each part are as follows:

The light source part 410 is composed of a light source 411 , a collimating lens 412 , a shaping device 413 and a lenticular lens 414 . The light source adopts laser diode (LD) or light-emitting diode (LED); according to the divergence angle of the light source and the required spot size, the collimator lens collimates the divergent spot from the light source into a beam that propagates along the horizontal direction; the shaping device collimates the circular spot The light spot becomes linear, elliptical, rectangular or square, and the energy distribution of the light spot is improved at the same time. The present invention uses a diffractive optical element (DOE), a Powell lens (Powell Lens), a microlens array (MLA) or a cylindrical lens to realize shaping of the light spot.

The illumination sensing structure 420 has a front-end coupling structure that couples the reshaped incident light into the thin-layer waveguide structure for transmission. When the incident light is transmitted in the thin-layer waveguide, its energy attenuates, and its surface energy approaches the thin-layer waveguide structure. The end of the waveguide tends to be stable; due to the converging effect of the front-end coupling lens, the beam changes from continuous parallel total internal reflection to multi-mode continuous total internal reflection, and the mixing of different modes finally forms a continuous sheet at the end of the waveguide. Uniformly illuminated area, the optical path is shown in Figure 5.

The detection area 430 is set at the end of the illumination sensing structure 420, the size of this area is set to be no less than 10mm×10mm, and the uniformity of illumination in the area is required to be less than 10%, and this area is also set A certain number of sample fixed points, the substance to be detected marked with fluorescent substances are fixed on the surface of the thin-layer waveguide by these points, and the fluorescent biosensing can be realized after being excited by the evanescent field.

The present invention also provides a biological detection system for fluorescence analysis using a large-scale uniform evanescent field illumination area. The main structure of the system is shown in FIG. Displacement structure 620 , carrying structure 640 , filter system 650 and imaging system 660 . After the light beam emitted by the light source is shaped, it is coupled into the thin-layer waveguide by the coupling structure at an incident angle greater than the critical angle of total internal reflection. In the evanescent field illumination area, the fluorescently labeled substance to be detected on the surface of the waveguide emits a fluorescent signal of a specific wavelength after being excited by the evanescent field. The optical signal will only pass through the fluorescent signal of a specific band after passing through the optical filter system, and the fluorescent signal will pass through the imaging system. After the capture, the sensing result is finally obtained after image processing. The characteristics of each part of the system are as follows:

The light source part 610 is used to provide a light beam propagating in the horizontal direction, and complete collimation and shaping of the light beam, so that the output light beam is a shaped light spot with uniform energy distribution.

The displacement structure 620 can realize the displacement of the light source relative to the sensing waveguide in two degrees of freedom, horizontal and vertical, for adjusting the relative position between the light source and the sensing waveguide, wherein the displacement in the horizontal direction can change the input light incident to the coupling structure The horizontal position on the top can realize image stitching in the horizontal direction, and the displacement in the vertical direction can change the vertical position of the input light incident on the coupling structure, so as to realize the coupling of the incident light into the thin-layer waveguide structure at different incident angles. In one embodiment, the structure is capable of a displacement of 25 mm in the horizontal and vertical directions, respectively.

The illumination sensing waveguide 630 couples the incident light into the thin-layer waveguide structure for transmission through the front-end coupling structure, and then generates an evanescent field illumination area on the surface of the thin-layer waveguide; a certain number of fixed points of the sample to be tested are set at the end of the thin-layer waveguide surface , the fixed point adopts different spot sizes and arrangements according to the type and fixing method of the sample to be tested. The fixed point can be prefabricated on the surface of the waveguide, or it can be made on-site according to the needs of different application scenarios.

The carrying structure 640 supports and fixes the illumination sensing waveguide 630 through a clamping structure.

The filter system 650 is equipped with different filters according to the wavelength of the input light and the wavelength of the emitted fluorescence. In order to improve the filtering effect of the system, multiple filters can be selected to form a filter group, so as to filter the light from the detection area, so that only The fluorescence emitted by the fluorescent substance can pass through; the filter/group is installed in a lens tube and further installed into the imaging system.

The imaging system 660 captures the fluorescent signal through a sCMOS or CCD camera, and can be replaced by a sCMOS or CCD chip to simplify the system; the imaging camera or chip transmits the collected fluorescent image to the computer, and processes and analyzes the image, and then obtains the transmitted signal. feel the result.

Description of drawings

Fig. 1 is a schematic diagram of the polymer evanescent field optical waveguide sensing structure of the present invention

Fig. 2 is a schematic diagram of the processing scheme of the optical waveguide sensing structure of the present invention

Figure 3 is a schematic diagram illustrating the labeling of each surface of the optical waveguide sensing structure of the present invention

Fig. 4 is a schematic diagram of the optical system for producing a large-scale uniform evanescent field illumination area according to the present invention

Fig. 5 is a schematic diagram of the optical path of the optical waveguide sensing structure of the present invention and the previous prism coupling sensing structure

Fig. 6 is the flow chart and system diagram of fluorescence analysis biological detection of the present invention

Fig. 7 is a graph showing the test parameter results of the actual processing sample of the optical waveguide sensing structure of the present invention

Fig. 8 is the real object and result diagram of the optical coupling test of the optical waveguide sensing structure of the present invention

Fig. 9 is a curve diagram of local energy distribution on the upper surface of the optical waveguide sensing structure of the present invention under the condition of COC material

Figure 10 is a schematic diagram of the results of multi-wavelength detection regions of the optical waveguide sensing structure of the present invention

Detailed ways

In order to further illustrate the technical points of the present invention, the present invention will be described in detail below in conjunction with the accompanying drawings and examples.

The illumination sensing structure shown in Figure 1, the present invention provides an embodiment, wherein the surface type of the lens 111 of the coupling structure 110 adopts a second-order aspheric surface, wherein the conic constant k is -0.56, and the curvature radius c is 0.3, Then the surface equation is:

The length and width of the waveguide structure 120 are 75mm×25mm, and its thickness adopts two sizes of 0.8mm and 0.6mm. A skirt structure 333 with a width of 1mm is arranged at the lower end of the waveguide.

The processing of the lighting sensor structure adopts high-precision molds for injection molding, and four materials are used: polystyrene (PS), polymethyl carbonate (PC), cycloolefin copolymer (COC), and polymethyl methacrylate (PMMA). For integrated injection molding processing, the lighting sensing structure processed in the embodiment needs to be polished. The roughness of the structure was measured with a roughness measuring instrument, and the flatness, warpage, and length and width tolerances of the waveguide structure were tested with a height gauge and a CNC projector. The results are shown in Figure 7. Obviously, all parameters are consistent. The design requirements are met, and the introduction of the skirt structure 130 effectively controls the Z-direction warpage of the waveguide structure.

In another embodiment, the input light source is a laser diode with a wavelength of 520nm, and the laser beam passes through a collimating lens to a circular collimated spot with a diameter of 1mm, and the output light power of the spot after passing through a diaphragm with a diameter of 1mm is 5.24 mW, the collimated light beam is incident on the coupling structure of the illumination sensing structure along the horizontal direction, the illumination sensing structure is made of PS material, the refractive index at 520nm is 1.60, and the upper medium is air, then the critical angle of total internal reflection is 38.68°,

After the deflection and focusing of the coupling structure, the light beam is well coupled into the sensing waveguide structure for transmission. The energy distribution on the upper surface of the measured waveguide structure is shown in Figure 8. The results are averaged from five measurements. The front end of the curve is The flat part is to verify that the system is in a stable state. After that, it is the optical power distribution along the optical transmission direction on the upper surface of the waveguide, which begins to show energy attenuation. As the light beam transmits in the waveguide, the energy distribution tends to be stable, and a more uniform shape is formed near the end of the waveguide structure. Flat lighting area.

Based on the sensing illumination structure 100, the present invention further provides an optical system for generating a large-scale uniform evanescent field illumination area, as shown in FIG. 4 . When the system is working, the divergent light beam emitted by the light source 411 passes through the collimating lens 412 to obtain a beam of collimated light beams that exit in parallel. The circular spot is transformed into a bundle of oblate ellipse/square/rectangular/linear divergent spot, at this time, the energy distribution of the spot is uniform, and the shaped spot is collimated again by the cylindrical lens 414, and a beam of oblate ellipse/square with uniform energy distribution can be obtained /Rectangular/linear collimated light spot, the collimated and shaped light spot is coupled by the coupling structure at the front end of the illumination sensing structure 420 and focused to the upper surface of the waveguide structure, and total internal reflection occurs at the interface of the medium, after which the collimated and shaped light spot is in the Continuous total internal reflection occurs in the waveguide. With the increase of the transmission distance in the waveguide, a continuous piece of uniform evanescent field illumination area will be formed on the upper and lower surfaces of the waveguide. The present invention uses the variation coefficient CV (Coefficient of Variation) to measure this area The energy distribution uniformity of :

Wherein σ is the average value of the energy in the area, and μ represents the standard deviation of the energy distribution. The present invention adopts the evanescent field illumination area with CV<10% as the detection area 430, which can be prefabricated/on-site for a certain number of sample capture points. For fixed biological samples, biological samples with fluorescent markers will emit fluorescent signals of specific wavelengths under the excitation of highly uniform evanescent field illumination.

Figure 9 shows the local energy distribution curve on the upper surface of the illumination sensing structure of the COC material. The thickness of the waveguide structure is 0.8 mm. The end of the structure forms an illumination area with a uniformity lower than 10%, which is the detection area 430 set in the present invention.

FIG. 10 shows the size of the multi-wavelength detection area that can be formed on the upper surface of the sensor structure illuminated by the COC material under the condition of input light of three different wavelengths using the evanescent field illumination optical system 400 proposed by the present invention. The three wavelengths are commonly used fluorescence excitation wavelengths, respectively 488nm, 520nm and 646nm. The incident light is coupled through the coupling structure 110 at the front end. Due to the existence of chromatic aberration, different evanescent field illumination field distributions will be formed on the upper surface of the waveguide structure 120 , to calculate the overlapping area of the illumination field with CV<10% excited by the three wavelengths, which is wavelength robust and can be used for multicolor detection of samples.

Claims (24)

1. A sensing structure for generating an evanescent field illumination area, the structure comprising:

the coupling structure is positioned at the front end of the sensing structure, is used for changing the transmission direction angle of incident light and has a focusing function on the incident light;

and the thin-layer waveguide structure is positioned at the rear end of the sensing structure and used for guiding the light obliquely incident from the coupling structure, and the light excites an evanescent field on the surface of the waveguide structure.

2. A sensing structure according to claim 1, wherein the coupling structure of the front end is such that the complementary angle to the direction of deflection of the incident light is greater than the critical angle for total internal reflection at the upper interface.

3. A sensing structure according to claim 1, wherein the coupling structure of the front end is comprised of an aspheric lens and a polygonal prism structure.

4. The coupling structure of claims 1 and 3, wherein the aspheric lens and the polygonal prism structure are integrally formed by insert molding.

5. The coupling structure as claimed in claims 1 and 3, wherein the aspheric lens adopts a second, third or higher order profile to achieve coupling of incident light having a wavelength of 380nm to 780 nm.

6. A sensing structure according to claim 1, wherein the back end thin waveguide is a slab waveguide structure having a thickness of between 0.05-0.8mm using standard rectangular glass slide dimensions.

7. A sensing structure according to claim 1, wherein the lower surface of the thin layer waveguide structure is provided with a skirt structure to control the warpage of the waveguide.

8. A sensing structure according to claim 1, wherein the coupling structure and the thin waveguide structure are injection molded using a high precision mold to form an integrated structure, and the coupling structure and the thin waveguide structure are bonded using an index matching fluid after injection molding.

9. A sensing structure according to claims 1 to 8, wherein a high-transparency optical polymer such as cyclic olefin copolymer, polycarbonate, polystyrene, polymethylmethacrylate or the like is used for processing in the injection moulding.

10. A sensing structure according to claim 1, wherein the process material further comprises optical glass, tantalum pentoxide, silicon nitride, etc., depending on the process.

11. An optical system for generating an evanescent field illumination area, the system comprising:

a light source section providing a shaped beam of energy propagating in a horizontal direction with a uniform distribution;

the sensing structure is used for coupling incident light into the thin-layer waveguide structure through the front-end coupling structure for transmission, and the incident light generates an evanescent field on the surface of the thin-layer waveguide;

a detection region in which a substance to be detected labeled with a fluorescent substance is immobilized.

12. The optical system of claim 11, wherein the light source portion further comprises a laser diode/light emitting diode, collimating means, and beam shaping means, such that diverging light emitted from the laser diode/light emitting diode becomes a shaped, non-diverging spot having a uniform energy distribution.

13. Beam shaping device according to claim 12 for shaping an input spot, which can be realized by a Diffractive Optical Element (DOE), a Powell Lens (Powell Lens), a Micro Lens Array (MLA) or a cylindrical Lens.

14. The shaped light spot according to claims 11 to 13 can be arranged in a line, an ellipse, a rectangle or a square according to application requirements.

15. The optical system of claim 11, wherein the evanescent field strength excited by the sensing structure at the upper surface of the slab waveguide decays along the direction of beam propagation, forming a uniform illumination area at the end of the slab waveguide structure.

16. An optical system as claimed in claim 11, wherein the detection region is arranged at an end position of the upper surface of the thin-layer waveguide structure.

17. The detection zone of claims 11 and 16, wherein the uniformity of illumination intensity of the zone is less than 10% and the length and width of the zone are greater than 10mm.

18. The detection region of claims 11 and 16, wherein the region is wavelength robust, and the uniformity of the intensity of illumination incident on the region for light having a wavelength of 380-780nm is less than 10%.

19. An evanescent field fluorescence sensing system for biological detection, the system comprising:

a light source section providing a light beam traveling in a horizontal direction;

a displacement section for adjusting a relative position between the light source and the sensing waveguide;

the sensing structure is used for coupling incident light into the thin-layer waveguide structure through the front-end coupling structure for transmission, and the incident light generates an evanescent field on the surface of the thin-layer waveguide;

a clamping portion for securing the sensing structure;

a detection region in which a substance to be detected labeled with a fluorescent substance is immobilized;

a filter section that filters light from the detection area by a filter so that only emitted fluorescence can pass therethrough;

and the imaging part is composed of an sCMOS or CCD sensing chip and is used for capturing and imaging the fluorescence signal.

20. The optical system of claim 19, wherein the light source portion provides the light beam as a uniformly energy distributed non-diverging shaped spot.

21. The optical system of claim 19, wherein the displacement section is capable of effecting displacement of the light source relative to the sensing waveguide in both horizontal and vertical degrees of freedom, with a displacement stroke of 25mm being achievable in both degrees of freedom.

22. The displacement section according to claim 19 and 21, wherein the displacement in the horizontal direction can change the horizontal position of the input light incident on the coupling structure to achieve image stitching in the horizontal direction.

23. The displacement section according to claim 19 and 21, wherein the vertical displacement can change the vertical position of the input light incident on the coupling structure to realize the coupling and focusing of the incident light on the upper surface of the thin-layer waveguide at different incident angles.

24. The optical system of claim 19, wherein the detection zone is prefabricated/fabricated on-site as many capture sites for immobilizing the biological subject as desired.

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