Wavelength modulation SPR imaging sensing system and method

Abstract

The invention relates to the technical field of optical sensing imaging, in particular to a wavelength modulation SPR (Surface Plasmon Resonance) imaging sensing system and method, and the system comprises an image collection unit which comprises an incident light path assembly, a sensing assembly and a reflected light path assembly; the temperature control unit comprises a heating element positioned on one side, deviating from the coupling prism, of the sensing chip; and the data processing unit is used for fitting the sensing surface image acquired by the surface array detector and outputting the refractive index of the liquid to be detected. By adopting the low-cost monochromatic LED array, the system cost is greatly reduced, the stability is improved, and high-precision measurement in a wide dynamic range is realized through a self-adaptive spectrum fitting algorithm of the data processing unit; the temperature control unit is used for accurately controlling the temperature field of the liquid drops and manipulating the internal flowing mode of the liquid drops, so that directional enrichment of target molecules in a preset area is realized, substrate modification or exogenous additives are not needed, and the problem of probe saturation of the traditional coffee ring effect is effectively solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of optical sensing imaging technology, and in particular to a wavelength modulation SPR imaging sensing system and method. BACKGROUND

[0002] Surface plasmon resonance (SPR) sensing technology has become an important tool for biochemical analysis due to its advantages such as label-free and real-time monitoring. However, the existing SPR technology faces two major bottlenecks: one is the limited penetration depth of evanescent field, which can only detect molecules within a hundred nanometers of the sensor surface; the other is the slow diffusion of low-concentration biomarkers, which seriously restricts the detection sensitivity and speed. Although researchers have developed various signal enhancement strategies, traditional external field driving methods require complex equipment, while droplet evaporation enrichment strategies have problems such as probe saturation, complex process, and interference with biological activity. There is a limitation that low-concentration biomolecules are difficult to identify.

[0003] To improve the sensitivity of SPR sensing technology, researchers have proposed various enhancement strategies, which can be summarized as passive enhancement and active enhancement. Among the four main SPR modulation methods (intensity, angle, phase, and wavelength), wavelength-modulated SPR sensing can flexibly optimize the excitation wavelength of different samples, making it the most suitable modality for imaging applications. Since the pioneering research by Yuk, wavelength-modulated SPR imaging (WSPRi) technology has made significant progress. Although WSPRi technology can achieve high consistency and throughput, it usually requires expensive spectral components (such as liquid crystal tunable filters, monochromators, or acousto-optic tunable filters), which not only increase the cost of the system but also affect its stability. More importantly, ensuring a wide dynamic range requires a large range of spectral scanning, and the response time of spectral devices will limit the time resolution of the method. Traditional WSPRi measures the resonance wavelength shift caused by the interaction between the target and the probe to quantify the concentration of target molecules, but during the evaporation enrichment process of the test liquid, the increase in the concentration of non-specific substances will also change the resonance wavelength signal, so this sensing method requires high time resolution to accurately obtain the SPR signal before the liquid evaporates, thereby minimizing the detection error.

[0004] Therefore, the prior art still needs to be improved and developed. SUMMARY

[0005] In view of the deficiencies of the prior art described above, the purpose of the present application is to provide a wavelength modulation SPR imaging sensing system and method, which aims to solve the problem that the existing SPR imaging sensing system cannot achieve high-precision measurement in a wide dynamic range.

[0006] The technical solution of the present application is as follows: A wavelength modulation SPR imaging sensing system, comprising: An image acquisition unit, comprising an incident light path assembly, a sensing assembly, and a reflected light path assembly; the incident light path assembly comprises an LED array composed of six single-wavelength LED lamp beads; the sensing assembly comprises a coupling prism for receiving incident light and a sensing chip arranged on the top of the coupling prism; the reflected light path assembly comprises a surface array detector for receiving reflected light; A temperature control unit, comprising a heating element arranged on the side of the sensing chip away from the coupling prism, for heating the to-be-tested liquid on the sensing chip; A data processing unit for fitting the sensing surface image collected by the surface array detector and outputting the refractive index of the to-be-tested liquid.

[0007] The wavelength modulation SPR imaging sensing system, wherein the incident light path assembly comprises, in sequence along the light propagation direction, an integral uniform light element, a collimating lens, and a first polarizer; the light emitted by the LED array is uniformly lighted by the integral uniform light element and then enters the collimating lens through a multi-mode optical fiber; the reflected light path assembly comprises, in sequence along the light propagation direction, a second polarizer and an imaging lens group; the sensing assembly is located between the first polarizer and the second polarizer, and the surface array detector is located on the side of the imaging lens group away from the second polarizer.

[0008] The wavelength modulation SPR imaging sensing system, wherein the wavelength of the LED lamp bead is between 730 nm and 805 nm, and the wavelengths of the six single-wavelength LED lamp beads are different.

[0009] The wavelength modulation SPR imaging sensing system, wherein the data processing unit comprises: A data processing module for measuring a reflectivity value data set corresponding to the wavelengths of the six LED lamp beads based on the sensing surface image; A data fitting module for identifying the wavelength point corresponding to the minimum reflectivity in the reflectivity value data set, and performing four-order polynomial fitting on the five largest or five smallest wavelength points of the six wavelengths based on the wavelength point to obtain a spectral curve; A resonance wavelength extraction module for deriving the resonance wavelength according to the fitting function of the spectral curve; A refractive index output module for outputting the refractive index according to the resonance wavelength.

[0010] The wavelength modulation SPR imaging sensing system, wherein the heating element is in contact with the to-be-tested liquid and is arranged spaced apart from the sensing chip; the surface of the sensing chip is modified with probe molecules.

[0011] A wavelength modulation SPR imaging sensing method based on a wavelength modulation SPR imaging sensing system, comprising the steps of: The test liquid is dropped onto the surface of the sensor chip, and the test liquid is heated using a heating element; Six single-wavelength LED beads are lit in turn in sequence. The emitted light is incident on the coupling prism and then received by the surface array detector after reflection, so as to obtain the sensing surface image at different wavelengths. The dataset of reflectance values ​​corresponding to the wavelengths of the six LED beads is measured based on the sensor surface image. Identify the wavelength point corresponding to the minimum reflectance in the reflectance value dataset, and perform fourth-order polynomial fitting based on the wavelength point, taking the five largest or five smallest wavelength points out of six wavelengths, to obtain the spectral curve. The resonance wavelength is obtained by differentiating the fitting function of the spectral curve. The refractive index is output based on the resonant wavelength.

[0012] In the wavelength modulation SPR imaging sensing method, the wavelengths of the six LED beads are 730nm, 740nm, 756nm, 777nm, 780nm and 805nm, respectively.

[0013] The wavelength modulation SPR imaging sensing method described above, wherein the reflectance value dataset is used... It means that, among them, Indicates the wavelength of different LED beads. Indicates wavelength as The corresponding reflectivity value; and the wavelength value satisfies < < < < < ; The wavelength point corresponding to minimum reflectivity In i When ≤3, take , , , , A fourth-order polynomial fitting was performed to obtain the spectral curve; The wavelength point corresponding to minimum reflectivity In i When >3, take , , , , A fourth-order polynomial fitting was performed to obtain the spectral curve.

[0014] The wavelength modulation SPR imaging sensing method, wherein the step of deriving according to the fitting function of the spectrum curve is specifically: the extreme point of the first derivative of the fitting function being zero and the second derivative being positive.

[0015] The wavelength modulation SPR imaging sensing method, wherein the switching response time of the LED lamp bead is 1-2 ms, and the exposure time of the area array detector is 18-20 ms.

[0016] Beneficial effects: the wavelength modulation SPR imaging sensing system and method provided by the application, the wavelength modulation SPR imaging sensing system comprises: an image acquisition unit, which comprises an incident light path assembly, a sensing assembly and a reflected light path assembly; the incident light path assembly comprises an LED array composed of six single-wavelength LED lamp beads; the sensing assembly comprises a coupling prism for receiving incident light and a sensing chip arranged on the top of the coupling prism; the reflected light path assembly comprises an area array detector for receiving reflected light; a temperature control unit, which comprises a heating element located on the side of the sensing chip away from the coupling prism; the heating element is used for heating a to-be-tested liquid on the sensing chip; and a data processing unit, which is used for fitting the sensing surface image collected by the area array detector and outputting the refractive index of the to-be-tested liquid. The low-cost single-color LED array adopted by the application not only greatly reduces the system cost and improves the stability, but also realizes high-precision measurement in a wide dynamic range through the adaptive spectrum fitting algorithm of the data processing unit; and the temperature control unit is used for accurately controlling the temperature field of the liquid drop, manipulating the internal flow pattern of the liquid drop and realizing directional enrichment of target molecules in a preset area. The method does not need substrate modification or external additives, effectively overcomes the probe saturation problem of the traditional coffee ring effect; meanwhile, by combining the spatial resolution of the imaging SPR with the active temperature control enrichment strategy brought by the temperature control unit, the imaging SPR technology plays a unique advantage in full-field detection, and provides a complete solution for low-concentration biomarker analysis. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 FIG. 1 is a partial structure schematic diagram of the wavelength modulation SPR imaging sensing system of the application; Figure 2 FIG. 2 is a flowchart of the wavelength modulation SPR imaging sensing method of the application; Figure 3 FIG. 3 is a fitting result diagram of different fitting orders on different bandwidth spectra; Figure 4 FIG. 4 is a flowchart of the adaptive sampling algorithm; Figure 5 FIG. 5 is a diagram of actual RW values and RW calculation results based on the adaptive sampling algorithm; Figure 6A schematic diagram of the principle and verification of the dual-mechanism enrichment strategy; Figure 7 A schematic diagram of the standard refractive index sample test for the embodiment; Figure 8 A schematic diagram of the antigen-antibody specific binding test for Example 2; Figure 9 A result of the Tau protein detection for Example 3; BRIEF DESCRIPTION OF DRAWINGS: incident light path assembly 10, LED array 11, integrating light homogenizing element 12, collimating lens 13, first polarizer 14, multi-mode optical fiber 15, sensing assembly 20, coupling prism 21, sensing chip 22, reflected light path assembly 30, area array detector 31, second polarizer 32, imaging lens group 33, first imaging lens 331, second imaging lens 332. DETAILED DESCRIPTION

[0018] The present application provides a wavelength modulation SPR imaging sensing system and method. To make the purpose, technical solution and effect of the present application more clear and explicit, the present application is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application.

[0019] Those skilled in the art can understand that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs. It should also be understood that terms such as those defined in general dictionaries should be understood as having meanings consistent with those in the prior art, and should not be interpreted in an idealized or overly formal sense unless specifically defined as such.

[0020] To improve the sensitivity of SPR sensing technology, researchers have proposed various enhancement strategies, which can be summarized as passive enhancement and active enhancement. Passive enhancement refers to a label-free SPR sensing strategy that relies only on diffusion without applying an external field to change the motion of the analyte, which usually enhances the SPR response through various sensing substrates. Active enhancement aims to break through the diffusion limit barrier, thereby improving sensitivity, shortening detection time and reducing detection limit. Traditional active enhancement methods usually use external fields (light field, electric field or magnetic field) to drive target molecules to move or enrich in a specific location to increase the contact probability of target molecules with probe molecules on the surface of the sensing chip, thereby reducing the detection limit of SPR technology. However, these methods often require complex equipment (such as lasers, alternators, magnetic field generators) and precise control of field strength, frequency and other parameters.

[0021] Marangoni convection and capillary-driven molecular enrichment (such as the coffee ring effect) during droplet evaporation is another active sensitization strategy. Compared to externally driven methods, this strategy has the significant advantages of simple operation and no need for complex equipment. However, due to the limited number of probe molecules on the sensor chip surface, when analytes are enriched at the droplet edge, probe binding in the edge region may become saturated, causing some analyte molecules to become undetectable. Therefore, evaporation conditions need to be optimized when using this sensitization method. Surface wettability patterning techniques such as photolithography and plasma treatment can be used to construct alternating hydrophilic and hydrophobic substrates, achieving multi-regional molecular enrichment while alleviating probe oversaturation on the chip surface; however, these methods have complex and demanding fabrication processes. Furthermore, adding surfactants to regulate droplet surface tension can also affect molecular enrichment, but this triggers complex surface rheological properties and flow field changes, which is a passive and unpredictable mechanism, and exogenous reagents may interfere with the activity of biomolecules.

[0022] Meanwhile, traditional evaporation-driven enrichment methods have inherent limitations: (1) Difficulty in optimizing droplet volume: Although the coffee ring effect promotes the enrichment of molecules at the edge of the droplet, too small a volume will exacerbate the edge pinning effect, causing non-specific adsorbed molecules to be retained and interfering with the SPR signal; while too large a volume will dilute the enrichment effect.

[0023] (2) Probe binding site saturation: Although molecules tend to accumulate in the edge region of the coffee ring, the probe density in this region is limited. When the target molecule occupies all available binding sites, the detection signal will reach saturation, thus limiting the dynamic range and accuracy of the detection.

[0024] Based on this, such as Figure 1 As shown, the present invention provides a wavelength modulation SPR imaging sensing system, comprising: The image acquisition unit includes an incident light path assembly, a sensing assembly, and a reflected light path assembly; the incident light path assembly 10 includes an LED array 11 composed of six single-wavelength LED beads; the sensing assembly 20 includes a coupling prism 21 for receiving incident light and a sensing chip 22 disposed on top of the coupling prism 21; the reflected light path assembly 30 includes a surface array detector 31 for receiving reflected light. The temperature control unit includes a heating element located on the side of the sensing chip 22 away from the coupling prism 21, for heating the liquid to be tested on the sensing chip; The data processing unit is used to fit the sensing surface image acquired by the surface array detector and output the refractive index of the liquid to be tested.

[0025] In this embodiment, the low-cost monochrome LED array not only greatly reduces the system cost and improves the stability, but also realizes high-precision measurement in a wide dynamic range through the adaptive spectral fitting algorithm of the data processing unit. The temperature control unit is used to control the droplet temperature field accurately, manipulate the internal flow pattern, and realize the directional enrichment of target molecules in the preset area. This method does not require substrate modification or external additives, effectively overcoming the saturation problem of traditional coffee ring effect. At the same time, by combining the spatial resolution of imaging SPR with the active temperature control enrichment strategy brought by the temperature control unit, the unique advantages of SPR imaging technology in full-field detection are played, and a complete solution is provided for low-concentration biomarker analysis.

[0026] Specifically, the temperature control unit of the present application can make the wavelength modulation SPR imaging sensing system realize a double-mechanism enrichment strategy, which is limited by two factors, including edge enrichment (evaporation driven) and center enrichment (temperature gradient driven). The edge enrichment is caused by the higher evaporation rate at the edge of the droplet (due to larger specific surface area), which leads to solvent replenishment flow from the center to the edge, forming a radial outward flow, which pushes the molecules to the edge and deposits. The center enrichment is due to the establishment of a radial temperature gradient (high in the center and low at the edge) by the heating element (higher than the ambient temperature), which drives the Marangoni flow from the cooler edge to the center with higher temperature, which brings the molecules back to the center area and deposits. Therefore, by using the active temperature control enrichment strategy of the temperature control unit, the internal flow pattern is manipulated by accurately controlling the droplet temperature field, and the directional enrichment of target molecules in the preset area is realized. Combined with the adaptive algorithm of the data processing unit, the sensing surface image collected by the area array detector is fitted, and finally the refractive index of the test liquid is output. This can reduce the conditions of hardware equipment and reduce the cost.

[0027] In contrast, active temperature control provides a more robust and adjustable strategy for regulating the coffee ring effect. By establishing a non-uniform temperature field to control the internal flow pattern, directional dispersion and enrichment of molecules can be achieved without the need for substrate modification or external additives.

[0028] In some embodiments, the incident light path assembly 10 comprises, sequentially arranged along the light propagation direction, an integrating light homogenizing element 12, a collimating lens 13, and a first polarizer 14; the light emitted by the LED array 11 is collected and homogenized by the integrating light homogenizing element 12, and then enters the collimating lens 13 through a multi-mode optical fiber 15; the reflected light path assembly 30 comprises, sequentially arranged along the light propagation direction, a second polarizer 32 and an imaging lens group 33; the sensing assembly 20 is located between the first polarizer 14 and the second polarizer 32, and the area array detector 31 is located on the side of the imaging lens group 33 away from the second polarizer 32. The light emitted by the LED array 11 is collected and homogenized by the integrating light homogenizing element 12, and then enters the collimating lens 13 through the multi-mode optical fiber 15. The collimated parallel light enters the sensing assembly 20 through the first polarizer 14, and the reflected light from the sensing assembly 20 passes through the second polarizer 32 and the imaging lens group 33 and is finally accepted by the area array detector (CMOS). The sensing assembly is composed of a coupling prism and a sensing chip, and the sensing chip is located above the prism. The air between the two is exhausted by refractive index matching oil.

[0029] Specifically, when the wavelength modulation SPR imaging sensing system is working, six single-wavelength LED lamp beads of the LED array are sequentially and alternately lit, and the CMOS synchronously collects the sensing surface images to record the gray scale information at a specific wavelength. After completing the full-band scanning, the data processing unit automatically calculates the SPR spectrum of the region of interest (ROI) and obtains the resonance wavelength (RW) of the position.

[0030] In a preferred embodiment, the LED switching response time is 1 ms, and the CMOS exposure time is about 20 ms. Therefore, the time required for completing one full-spectrum scan is (1+20)×6=126 ms.

[0031] In some embodiments, the wavelength of the LED lamp bead is between 730 nm and 805 nm, and the wavelengths of the six single-wavelength LED lamp beads are different.

[0032] In a preferred embodiment, the wavelengths of the six single-wavelength LED lamp beads are 730 nm, 740 nm, 756 nm, 777 nm, 780 nm, and 805 nm, respectively.

[0033] In some embodiments, the data processing unit comprises: a data processing module, configured to measure a reflectivity value data set corresponding to the wavelengths of the six LED lamp beads based on the sensing surface images; a data fitting module, configured to identify a wavelength point corresponding to a minimum reflectivity in the reflectivity value dataset, and perform a fourth-order polynomial fitting on five largest or five smallest wavelength points based on the wavelength point to obtain a spectrum curve; a resonance wavelength extraction module, configured to derive a resonance wavelength based on a derivative of a fitting function of the spectrum curve; a refractive index output module, configured to output a refractive index based on the resonance wavelength.

[0034] Specifically, the data processing unit intelligently captures key features (especially a resonance valley) of a surface plasmon resonance (SPR) spectrum curve by using a limited number of carefully selected sampling points, and reconstructs the spectrum profile with high accuracy in a local range by using a low-order polynomial fitting, so as to obtain a resonance wavelength and further obtain a refractive index.

[0035] In some embodiments, the heating element is in contact with the liquid to be measured and is spaced apart from the sensing chip; and the surface of the sensing chip is modified with probe molecules. By arranging the heating element, a double-mechanism enrichment strategy of the system can be realized, the internal flow pattern can be manipulated by accurately controlling the temperature field of the droplet, directional enrichment of target molecules in a preset area can be realized, and a complete solution is provided for low-concentration biomarker analysis.

[0036] In some embodiments, the heating element is a heating probe, which has a high heating rate and can better realize the active temperature control enrichment strategy.

[0037] In some embodiments, the imaging lens group 33 includes a first imaging lens 331 and a second imaging lens 332.

[0038] In addition, as shown in Figure 2 The application further provides a wavelength modulation SPR imaging sensing method based on the wavelength modulation SPR imaging sensing system, including the following steps: Step S10: adding a liquid droplet to be measured on the surface of a sensing chip, and heating the liquid to be measured by using a heating element; Step S20: sequentially lighting six single-wavelength LED lamp beads, and emitting light to a coupling prism and being reflected by the coupling prism to be received by a surface array detector to obtain sensing surface images under different wavelengths; Step S30: measuring a reflectivity value dataset corresponding to the wavelengths of the six LED lamp beads based on the sensing surface images; Step S40: identifying a wavelength point corresponding to a minimum reflectivity in the reflectivity value dataset, and performing a fourth-order polynomial fitting on five largest or five smallest wavelength points based on the wavelength point to obtain a spectrum curve; Step S50: Derivation according to the fitting function of the spectrum curve to obtain the resonance wavelength; Step S60: Output the refractive index according to the resonance wavelength.

[0039] In this embodiment, the wavelength modulation SPR imaging sensing method is realized based on an adaptive sampling algorithm, which intelligently captures the key features (especially the resonance valley) of the surface plasmon resonance (SPR) spectrum curve through limited and carefully selected sampling points, and reconstructs the spectrum profile with high precision in a local range by using a low-order polynomial fitting, so as to extract the resonance wavelength and further obtain the refractive index.

[0040] In some embodiments, the wavelengths of the six LED lamp beads are 730 nm, 740 nm, 756 nm, 777 nm, 780 nm and 805 nm respectively.

[0041] As shown in Figure 3 , a typical surface plasmon resonance (SPR) spectrum curve presents obvious nonlinear characteristics. Generally speaking, the greater the spectral width of the SPR spectrum, the greater the corresponding detection dynamic range, which is beneficial to improve the sensitivity and application range of the measurement. However, the increase of the spectral width also brings higher function complexity, which requires a higher-order polynomial to be used for fitting, thereby putting forward higher requirements for the number of spectral sampling points. For example, for a spectral width of 75 nm, a sixth-order polynomial is theoretically required to achieve accurate fitting, but limited by the hardware conditions, the system is difficult to meet the corresponding dense sampling requirements, so the adaptive sampling algorithm (steps S30-S60) is used to complete the fitting of the spectrum curve and the solution of the resonance wavelength and the refractive index, and the flow chart is as shown in Figure 4 .

[0042] In some embodiments, in step S30, specifically comprising: the algorithm first measures the corresponding reflectivity values =730nm, =740nm, =756nm, =777nm, =780nm, =805nm at six pre-set discrete excitation wavelength points , through this step, the continuous spectrum function is discretized into a set of six data points . The selection of these six wavelengths is based on the prior knowledge of the typical SPR spectrum characteristics, aiming to effectively cover the entire 75nm spectral width of the potential resonance interval with the least number of points, and to sensitively reflect the approximate position of the resonance valley.

[0043] In some embodiments, the reflectivity value data set is used represents the wavelength of different LED lamp beads, represents the wavelength corresponding to the minimum reflectivity value; and the value of the wavelength satisfies ; the wavelength point corresponding to the minimum reflectivity i ≤3, take , , , , perform four-order polynomial fitting to obtain the spectral curve; the wavelength point corresponding to the minimum reflectivity i >3, take , , , , perform four-order polynomial fitting to obtain the spectral curve.

[0044] Specifically, the step S40 is the decision-making core of the entire algorithm, which dynamically determines the optimal fitting strategy according to the preliminary data, including: 1) Identify the minimum reflectivity wavelength: among the six sampling points obtained in step S30, find and determine the wavelength point with the minimum reflectivity value min , which is a preliminary and rough estimate of the SPR resonance valley position, because it represents the position with the strongest energy attenuation in the current sampling set.

[0045] 2) Conditional judgment and fitting interval selection: based on the position of , a conditional judgment branch is executed to select the most suitable local spectral interval for fine fitting; when ≤3 (i.e., the minimum reflectivity point appears on the shorter wavelength side, such as , , ), which indicates that the resonance peak is likely to be located in the first half of the spectrum (730nm-780nm interval). Therefore, the fitting range is locked between 730nm and 780nm, which covers the identified resonance valley and its left rising edge and right partial falling edge, ensuring that the fitting curve can completely contain the characteristics of the resonance valley. When >3 (i.e., the minimum reflectivity point appears on the longer wavelength side, such as​​​​​​​​​​ , , This indicates that the resonance peak is likely located in the latter half of the spectrum (740nm-805nm range). Accordingly, the algorithm adjusts the fitting range to 740nm to 805nm.

[0046] 3) Perform fourth-order polynomial fitting: Within the selected local interval (730-780nm or 740-805nm), using all available sampling point data within this interval (usually 4 to 5 points), a fourth-order polynomial is applied. Least square fitting is performed. The fourth-order polynomial is chosen based on a trade-off between the nonlinearity of the SPR curve in this local interval. It can well describe the curvature of the curve, avoid the overfitting problem that may be caused by higher-order fitting, and has a much lower requirement for sampling points than the full-spectrum sixth-order fitting.

[0047] In some embodiments, step S50, the step of differentiating the fitting function of the spectral curve, specifically involves finding the extreme point where the first derivative of the fitting function is zero and the second derivative is positive, which is the resonance wavelength. After completing the fourth-order polynomial fitting, an analytical fitting function can be obtained. Resonant wavelength Theoretically, this corresponds to the point of minimum reflectivity, i.e., the extreme point where the first derivative of the fitted function is zero and the second derivative is positive. Mathematically, this is achieved by solving the first derivative equation of the fitted polynomial. Find the root within the fitted interval and verify its second derivative. This allows for the precise calculation of the wavelength value corresponding to the minimum point. This calculation yields... This is the highly accurate estimated resonance wavelength that is ultimately output by the algorithm.

[0048] This method dynamically selects key wavelength regions, effectively covering a spectral range of up to 75 nm using only six sampling points, significantly reducing dependence on sampling resources while maintaining accuracy. To verify the accuracy of the algorithm, the calculated resonance wavelength is compared with the actual experimental value (e.g., Figure 5 (As shown). The results show that the two agree well within a broad spectral range of 80 nm, exhibiting excellent consistency. Further quantitative analysis reveals that the slope of the fitted resonance wavelength variation curve is k=4845.71, with a relative deviation of only 2.7% compared to the experimental reference value (k=4858.93). This result fully demonstrates the robustness and practical value of the adaptive sampling algorithm in broadband SPR measurements.

[0049] In some embodiments, the switching response time of the LED beads is 1-2ms, and the exposure time of the area array detector is 18-20ms.

[0050] In some implementations, a schematic diagram illustrating the principle of using a heating probe to achieve molecular enrichment through heating and evaporation is shown below. Figure 6 As shown in (a) in 6; specifically, this embodiment uses PBS solution to verify the performance of the proposed enrichment strategy. This dual-mechanism enrichment strategy is implemented through a simple heating unit, as shown in (b) in 6. The heating probe is inserted into the top of the droplet, and the experimental environment is maintained at 20°C and 45% relative humidity. According to the characteristics of protein molecular reactions (such as IgG or Tau protein), the optimal temperature is set to 37°C. This temperature can maximize the stability and activity of the protein and improve the antigen-antibody binding efficiency.

[0051] Given that excessively large droplet volumes would suppress the coffee ring effect, while excessively small volumes might cause pinning effects due to rapid evaporation—leading to the immobilization of non-specifically adsorbed molecules and affecting detection accuracy—4 μL was ultimately selected as the optimal droplet volume. After adding 4 μL of PBS to the chip surface, a heating probe was immediately inserted, and the sample reached 37°C within 10 seconds of probe insertion. Under these conditions, evaporation altered the spatial distribution of molecules within the droplet, thereby causing changes in the SPR signal. After 5 minutes, the resonant wavelength shift distribution on the droplet sensing surface exhibited the following characteristics: Figure 6 The pattern shown in (c) illustrates a regional variation in the resonant wavelength signal within the droplet, with the offset being significantly higher in the edge and center regions (within the white circle) than in other regions. This non-uniform distribution originates from the directional enrichment of salt molecules towards the center and edges under specific conditions, while the changes in resonant wavelength in other regions mainly stem from the overall concentration increase caused by droplet evaporation.

[0052] To more clearly illustrate the enrichment behavior at the edges and centers, representative data rows (such as...) are selected. Figure 6 The analysis was performed on (c) marked by the dashed line, and the results are as follows. Figure 6 As shown in (d) above, the data clearly show that the resonant wavelength shifts at the edges and center are significantly greater than in other regions, confirming that molecules preferentially accumulate in these two specific regions within the droplet under heating conditions.

[0053] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention.

[0054] Example 1 This embodiment performs standard refractive index solution testing based on the optimal scheme of the wavelength modulation SPR imaging sensing system, as detailed below: To verify the imaging capability of the SPRi system, NaCl solution was used for testing. NaCl solutions of different concentrations (0%, 2.5%, 5%, 7.5%) were dropped onto the chip surface, and the obtained resonant wavelength imaging results are shown below. Figure 7 As shown in (a) above, the results indicate that the resonance wavelength increases with increasing NaCl solution concentration, demonstrating that the system has good imaging capabilities.

[0055] Dynamic range and refractive index resolution are important performance parameters of a sensing system. First, the dynamic range of the system was tested: NaCl solutions with concentrations of 0%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, and 7.5% were prepared before the experiment. By monitoring the changes in resonance wavelength caused by different samples (each sample was tested three times), the relationship between the refractive index of the NaCl solution and the resonance wavelength shift was obtained as follows: Figure 7 As shown in (b) above, the resonance wavelength exhibits a good linear relationship with the refractive index of the NaCl solution (R0). 2 =0.994). In this experiment, the refractive index of the sample varied from 1.3333 to 1.3460 RIU, therefore the dynamic range of the system is 0.013 RIU. According to Figure 7 The fitting result in (b) shows that the refractive index resolution of this system is 1.96 × 10⁻⁶. -6 RIU.

[0056] Example 2 Based on Example 1, an antigen-antibody specific binding test was performed, as follows: During detection, 4 μL of goat anti-human IgG droplets are added to the chip surface, at which point the molecules are randomly spatially distributed. Figure 8 (a1)). Immediately afterwards, a heating probe was inserted to heat the droplet. Under the combined effects of evaporation and the temperature gradient, molecules aggregated towards the edge and center regions. Figure 8 (a2)). To ensure sufficient antigen-antibody binding (usually takes about 5 minutes) and to avoid the pinning effect caused by the drying of the droplet edges (about 9 minutes), PBS washing is initiated 7 minutes after sample addition. This time setting ensures that molecular binding is completed before evaporation interference occurs. 10 μL of PBS is introduced to allow unbound target molecules to diffuse into the PBS solution, while bound target molecules remain on the chip surface (a2). Figure 8 (a3) in the middle.

[0057] Figure 8Images (b1), (b2), and (b3) show the differences in resonance wavelengths before and after the reaction, corresponding to the interactions of goat anti-human IgG with immobilized human IgG, PBS with rabbit IgG, and goat anti-human IgG with immobilized rabbit IgG, respectively. Image (b1) shows the difference in resonance wavelengths before and after the interaction of 7.5 ng / mL goat anti-human IgG with immobilized human IgG, with significant signal shifts observed in both the edge and central regions. The detection specificity was repeatedly validated through control experiments (interaction of PBS with immobilized human IgG and interaction of 7.5 ng / mL goat anti-human IgG with immobilized rabbit IgG probe). Figure 8 (b2) and (b3) in the text). Since there was no specific binding, no significant difference in resonance wavelength was observed under either condition. Furthermore, the relationship between goat anti-human IgG concentration and mean resonance wavelength shift under different heating and non-heating modes is shown in the curves below. Figure 8 As shown in (c) in the figure.

[0058] Example 3 Based on Example 1, a Tau protein test was performed, as follows: Based on the above detection protocol, this method was applied to Tau protein analysis. For example... Figure 9 As shown in (a), the average resonant wavelength shift exhibits an excellent linear relationship with Tau protein concentration in the range of 1 pg / mL to 10 ng / mL (R0). 2 =0.9997), confirming the detection capability at clinically relevant concentrations. The detection limit for Tau protein was calculated to be 429 fg / mL (7.8 fM) according to Equation 2. In the specificity test, high-abundance serum albumin and human IgG were selected as negative controls. Figure 9 As shown in (b), the resonance wavelength shifts caused by human IgG and albumin were 0.0336 nm and 0.05 nm, respectively. Compared with the signal shift generated by 5 pg / mTau protein, the magnitudes of these nonspecific responses are almost negligible. This significant differential response confirms the high specificity of this method. In summary, these results demonstrate that this system provides a reliable technical solution for achieving highly sensitive and specific detection of Tau protein.

[0059] In summary, the present application provides a wavelength modulation SPR imaging sensing system and method, the wavelength modulation SPR imaging sensing system comprising: an image acquisition unit comprising an incident light path assembly, a sensing assembly, and a reflected light path assembly; the incident light path assembly comprising an LED array composed of six single-wavelength LED lamp beads; the sensing assembly comprising a coupling prism for receiving incident light and a sensing chip arranged on the top of the coupling prism; the reflected light path assembly comprising a surface array detector for receiving reflected light; a temperature control unit comprising a heating element located on the side of the sensing chip away from the coupling prism; for heating the to-be-measured liquid on the sensing chip; a data processing unit for fitting the sensing surface image collected by the surface array detector and outputting the refractive index of the to-be-measured liquid. The present application uses a low-cost single-color LED array, which not only greatly reduces the system cost and improves the stability, but also realizes high-precision measurement in a wide dynamic range through the adaptive spectral fitting algorithm of the data processing unit; and by using the temperature control unit to accurately control the droplet temperature field and manipulate the internal flow pattern of the droplet, the directional enrichment of target molecules in the preset area is realized, which does not require substrate modification or exogenous additives, effectively overcoming the probe saturation problem of the traditional coffee ring effect; at the same time, by combining the spatial resolution capability of the imaging SPR with the active temperature control enrichment strategy brought by the temperature control unit, the unique advantages of the SPR imaging technology in full-field detection are played, and a complete solution is provided for low-concentration biomarker analysis.

[0060] It should be understood that the application of the present application is not limited to the above examples, and those of ordinary skill in the art can make improvements or changes according to the above description, and all these improvements and changes shall belong to the protection scope of the appended claims of the present application.

Claims

1. A wavelength modulated SPR imaging sensor system, characterized by, The application relates to a refractive index detection device and a detection method thereof. The device comprises an image acquisition unit, a temperature control unit and a data processing unit. The image acquisition unit comprises an incident light path assembly, a sensing assembly and a reflected light path assembly. The incident light path assembly comprises an LED array composed of six single-wavelength LED lamp beads.

2. The wavelength-modulated SPR imaging sensor system of claim 1, wherein, The sensing assembly comprises a coupling prism for receiving incident light and a sensing chip arranged on the top of the coupling prism.

3. The wavelength-modulated SPR imaging sensor system of claim 1, wherein, The reflected light path assembly comprises a surface array detector for receiving reflected light.

4. The wavelength-modulated SPR imaging sensor system of claim 1, wherein, The temperature control unit comprises a heating element arranged on the side of the sensing chip away from the coupling prism, which is used for heating the to-be-detected liquid on the sensing chip. The data processing unit is used for fitting the sensing surface images collected by the surface array detector and outputting the refractive index of the to-be-detected liquid. The incident light path assembly comprises, sequentially arranged along the light propagation direction, an integral uniform light element, a collimating lens and a first polarizer. The light emitted by the LED array is uniformly lighted by the integral uniform light element and then enters the collimating lens through a multimode optical fiber. The reflected light path assembly comprises, sequentially arranged along the light propagation direction, a second polarizer and an imaging lens group.

5. The wavelength-modulated SPR imaging sensor system of claim 1, wherein, The sensing assembly is arranged between the first polarizer and the second polarizer, and the surface array detector is arranged on the side of the imaging lens group away from the second polarizer.

6. A wavelength modulation SPR imaging sensing method based on the wavelength modulation SPR imaging sensing system according to any one of claims 1-5, characterized in that, The wavelength of the LED lamp bead is between 730 nm and 805 nm, and the wavelengths of the six single-wavelength LED lamp beads are different. The data processing unit comprises: a data processing module for measuring a reflectivity value dataset corresponding to the wavelengths of the six LED lamp beads based on the sensing surface images; a data fitting module for identifying the wavelength point corresponding to the minimum reflectivity in the reflectivity value dataset and performing four-order polynomial fitting on the maximum five wavelength points or the minimum five wavelength points in the six wavelength points based on the wavelength point to obtain a spectrum curve; a resonance wavelength extraction module for deriving the fitting function of the spectrum curve to obtain a resonance wavelength; a refractive index output module for outputting the refractive index according to the resonance wavelength. The heating element is in contact with the to-be-detected liquid and is arranged at intervals from the sensing chip. 7.The wavelength-modulated SPR imaging sensing method of claim 6, wherein, The surface of the sensing chip is modified with probe molecules. The application further discloses a detection method of the device. The method comprises the following steps: dropping the to-be-detected liquid on the surface of the sensing chip and heating the to-be-detected liquid by using the heating element; sequentially lighting the six single-wavelength LED lamp beads, and the emitted light is incident to the coupling prism and is received by the surface array detector after reflection to obtain sensing surface images under different wavelengths; measuring a reflectivity value dataset corresponding to the wavelengths of the six LED lamp beads based on the sensing surface images; identifying the wavelength point corresponding to the minimum reflectivity in the reflectivity value dataset and performing four-order polynomial fitting on the maximum five wavelength points or the minimum five wavelength points in the six wavelength points based on the wavelength point to obtain a spectrum curve; deriving the fitting function of the spectrum curve to obtain a resonance wavelength; outputting the refractive index according to the resonance wavelength. The wavelengths of the six LED lamp beads are 730 nm, 740 nm, 756 nm, 777 nm, 780 nm and 805 nm, respectively. 8.The wavelength-modulated SPR imaging sensing method of claim 6, wherein, The reflectance value dataset is represented as wherein, represents the wavelength of different LED lamp beads, represents the reflectance value corresponding to the wavelength ; and the value of the wavelength satisfies ;​​​​​ Wavelength point corresponding to minimum reflectivity In i When ≤3, take , , , , Perform fourth-order polynomial fitting to obtain the spectral curve; Wavelength point corresponding to minimum reflectivity In the case of i > 3, take , , , , A fourth-order polynomial fitting is performed to obtain the spectral curve. 9.The wavelength-modulated SPR imaging sensing method of claim 6, wherein, The step of derivation according to the fitting function of the spectrum curve is specifically: the extreme point of the first derivative of the fitting function is zero and the second derivative is positive. 10.The wavelength-modulated SPR imaging sensing method of claim 6, wherein, The switching response time of the LED lamp bead is 1-2 ms, and the exposure time of the area array detector is 18-20 ms.

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