A microfluidic raman system and method for in-situ dynamic monitoring of plant root surface iron plaque

Abstract

The application discloses a microfluidic Raman system and method for in-situ dynamic monitoring of plant root surface iron film. The system comprises a plant incubator, a microfluidic chip and a Raman spectrometer, and the microfluidic chip has a microchannel for the plant roots to grow into. During monitoring, the plant incubator and the microfluidic chip are assembled, the plant to be detected is planted in the plant incubator, the roots of the plant to be detected grow into the microchannel through the pipeline, nutrient solution is added into the microchannel through the liquid inlet, the Raman spectrometer is used for in-situ root surface iron film detection of the plant roots in the microchannel, and the characteristic peak at 715 cm ‑1 is used as the characteristic peak of the in-situ root surface iron film for quantification. The application constructs a highly integrated and controllable research platform, realizes real in-situ and non-destructive monitoring, and integrates chemical recognition and quantitative analysis.

Description

Technical Field

[0001] This invention belongs to the field of environmental monitoring and plant physiological analysis technology, specifically relating to a microfluidic Raman system and method for in-situ dynamic monitoring of iron film on plant root surfaces. Background Technology

[0002] Iron films, commonly formed on the root surface of wetland plants, are iron (hydroxyl) oxide coverings. As a key biogeochemical barrier at the root-soil interface, they play an irreplaceable role in regulating nutrient absorption, pollutant migration and transformation, and carbon cycling. For example, invention application CN112136673A discloses a method for controlling arsenic pollution using iron films formed on the roots of wetland plant seedlings under hydroponic conditions. Invention application CN115231709A discloses the application of iron films in improving the remediation of chromium-contaminated water bodies by the hyperaccumulating plant *Rhizophora stylosa*. Therefore, accurately analyzing the formation kinetics, spatial structure, and content changes of iron films is crucial for understanding their environmental functions.

[0003] To overcome this limitation, existing technologies have developed several methods. For example, mild ultrasonic extraction is used to obtain intact iron films for in vitro characterization, or transparent root boxes based on mineral colorimetry are used for visualization evaluation, and iron film analogs are collected in situ using polyethylene films for in vitro X-ray fluorescence and near-edge absorption structure analysis.

[0004] While these methods offer valuable insights, they also have significant limitations: on the one hand, destructive sampling methods such as ultrasonic extraction completely disrupt the activity of the root system and the continuity of the rhizosphere microenvironment, making it impossible to capture the dynamic processes of iron film nucleation, growth, and phase transition on the surface of living roots, and lacking the necessary spatiotemporal resolution; on the other hand, while methods such as transparent root boxes or thin film collection have partially achieved in-situ or semi-in-situ observation, they usually cannot simultaneously perform precise, molecular-level chemical characterization, making it difficult to correlate the morphological changes of the iron film with its specific mineral phase composition and quantitative information in real time.

[0005] In other words, the current technological system lacks an integrated solution that can simultaneously achieve in-situ dynamic visualization of the iron film formation process, chemical identification of specific mineral phases, and quantitative monitoring of content changes without disturbing the living root system and rhizosphere microenvironment. This technological bottleneck limits a deeper understanding of the microscopic mechanisms of iron film formation and its environmental regulation mechanisms.

[0006] In addition, existing technologies disclose the use of microfluidic chips to study plant root systems. For example, invention application CN113655037A discloses a research device and method for the microdomain of rhizosphere soil in mature plants. The research device includes a microfluidic chip and a root box. The microfluidic chip includes a substrate and a cover plate. Microchannels are etched on the inner surface of the substrate. The microchannels and the inner surface of the cover plate enclose the inner cavity of the microfluidic chip. The microfluidic chip also has an inlet hole, an outlet hole, and a plant root inlet. The inlet hole, outlet hole, and plant root inlet are all connected to the inner cavity. A through hole is provided at the bottom of one side wall of the root box. The root box is connected to a pipe through the through hole. A connector is provided at the free end of the pipe. The pipe is connected to the plant root inlet through the connector. The pipe and the connector are filled with cotton and quartz sand. Summary of the Invention

[0007] To address the aforementioned shortcomings in existing technologies, this invention provides a microfluidic Raman system and method for in-situ dynamic monitoring of iron films on plant root surfaces. This system couples a microfluidic chip capable of simulating the day-night environment of the rhizosphere with a confocal Raman spectrometer to construct a "Rhizo-Raman" platform. This platform provides a growth chamber for living roots and allows for in-situ laser penetration detection, enabling in-situ, non-destructive observation of chemical reactions at the root surface interface.

[0008] This invention first provides a microfluidic Raman system for in-situ dynamic monitoring of iron films on plant root surfaces, comprising:

[0009] A plant culture box for growing plants to be tested, the plant culture box having an outlet for the plant roots to extend out;

[0010] The microfluidic chip has microchannels inside for plant roots to enter, and an inlet at one end of the microchannels for plant roots to enter. A pipe connects the outlet of the plant culture box to the inlet of the microfluidic chip. The microfluidic chip also has an inlet and an outlet that communicate with the microchannels.

[0011] A Raman spectrometer is used to detect the iron film on the root surface of plant roots located in the microchannel.

[0012] Preferably, the cross-sectional shape of the microchannel is square.

[0013] Preferably, the microfluidic chip includes an upper layer and a lower layer that overlap each other. The microchannel is processed on the top surface of the lower layer, the inlet is provided on the side of the lower layer, and the liquid inlet and liquid outlet are provided on the upper layer.

[0014] More preferably, the upper and lower layers are made of PMMA. PMMA has excellent light transmittance in the visible and near-infrared regions and is compatible with Raman detection.

[0015] This invention further provides a method for in-situ dynamic monitoring of iron film on plant root surfaces, using a microfluidic Raman system for in-situ dynamic monitoring of iron film on plant root surfaces. The method includes the following steps:

[0016] (1) After assembling the plant culture box and the microfluidic chip, the plant to be tested is planted in the plant culture box. The roots of the plant to be tested extend into the microchannel through the pipe. Nutrient solution is added into the microchannel through the liquid inlet.

[0017] (2) In situ detection of the iron film on the root surface of plant roots located in the microchannel was performed using a Raman spectrometer at a depth of 715 cm. -1 The characteristic peak at the location was used as the characteristic peak of the in-situ root surface iron film for quantitative analysis.

[0018] Preferably, in step (1), the nutrient solution is used for pre-culture first, and then a nutrient solution containing ferrous ions is used for induction culture to induce the formation of an iron film.

[0019] More preferably, in step (2), when using a nutrient solution containing ferrous ions for induction culture, the ferrous ions in the nutrient solution can be derived from FeSO4, FeCl2, ferrous citrate, and EDTA-Fe. 2+ .

[0020] More preferably, in step (2), the concentration of ferrous ions is 0.05~0.3 mM.

[0021] Preferably, in step (2), when using a Raman spectrometer for detection, samples are collected at a depth of 400~900 cm⁻¹. -1 The Raman spectra within the range were smoothed and baseline corrected, and the 715 cm⁻¹ value was calculated. -1 Differential intensity I at the point 差分 I 差分 =I 715 -I 400 .

[0022] By identifying the root surface iron film at 715 cm -1 The characteristic Raman peaks at the location were identified, and a linear quantitative relationship between their peak intensity and iron content was established. Based on this, time-series spectral acquisition can be performed on the same observation point to achieve dynamic, continuous, and quantitative monitoring of iron film formation.

[0023] In step (2), when using a Raman spectrometer for detection, a root observation point is selected, and Raman spectra are collected in situ at fixed time intervals. The intensity values ​​of characteristic peaks are extracted and calculated, and the curve of the intensity value changing with time is plotted to achieve quantitative tracking of the formation dynamics of the iron film at that point.

[0024] Alternatively, in step (2), when using a Raman spectrometer for detection, a target area on the root surface is selected, and a two-dimensional point scan is performed using the Raman spectrometer. The intensity of the characteristic peaks at each scan point is recorded to generate a two-dimensional chemical image reflecting the distribution of the iron film. Furthermore, the thickness information of the iron film layer can be obtained through line scan analysis perpendicular to the root surface.

[0025] Using the point-scan imaging function of confocal Raman spectroscopy, a two-dimensional scan of a specific region on the root surface was performed with a certain step size. This was achieved by extracting data from a 715 cm⁻¹ area. -1 The intensity information of characteristic peaks is used to generate a spatial distribution chemical image of the iron film and to measure its thickness variation, with a resolution down to the micrometer level and a temporal resolution down to several hours. The step size can be selected from 2 μm to 100 μm, preferably the smallest scale of 2 μm; the smaller the step size, the higher the resolution.

[0026] Beneficial effects of this invention:

[0027] (1) Realized in-situ and non-destructive monitoring: The system directly detects in the simulated environment of living root growth, without damaging the natural state of the root system and iron film, and can truly reflect the in-situ formation process of the iron film, solving the fundamental problem that destructive sampling cannot obtain dynamic information.

[0028] (2) It integrates chemical identification and quantitative analysis: by utilizing the molecular fingerprint characteristics of Raman spectroscopy, it is possible to identify the main mineral phase of the iron film in situ (such as ferrihydrite, with a characteristic peak at 715 cm⁻¹). -1 By establishing a quantitative relationship between characteristic peak intensity and iron content, a leap from "observing its presence" to "knowing its quantity" has been achieved, providing quantitative data that traditional visualization methods lack.

[0029] (3) Micrometer-level spatial resolution imaging: Through confocal Raman scanning imaging technology, a two-dimensional chemical distribution map of the iron film covering the root surface can be obtained with a spatial resolution of micrometers (about 1.3 μm), and the thickness of the iron film layer can be accurately measured. This makes it possible to observe the spatial heterogeneity of the iron film, local deposition hotspots, and thickness evolution.

[0030] (4) Provides a means of visualizing dynamic processes: Combining time-resolved quantitative spectroscopy with spatially resolved chemical imaging not only allows for tracking the accumulation rate of iron film content with high temporal resolution, but also intuitively displays the dynamic evolution of its spatial structure (such as from discrete point deposits to continuous layered coverage), revealing the microscopic formation mechanism that cannot be observed by traditional endpoint methods.

[0031] (5) A highly integrated and controllable research platform has been constructed: the environmental simulation and manipulation capabilities of microfluidic chips, in vivo biological culture and in-situ spectral detection technology have been deeply integrated to form a stable, reproducible and parameter-controllable standardized analysis platform (Rhizo-Raman platform), which provides a powerful general tool for in-depth research on other biogeochemical processes at the root-soil interface. Attached Figure Description

[0032] Figure 1 This is a system configuration and physical diagram of the Rhizo-Raman integrated platform described in this invention. Figure 1 In the diagram, 'a' represents the exploded structure of a microfluidic chip. Figure 1 b in the diagram represents the system architecture of the Rhizo-Raman integration platform. Figure 1 In the image, 'c' represents a physical diagram of the Rhizo-Raman integrated platform. Figure 1 In the diagram, d represents a physical image of the microfluidic chip and other components.

[0033] Figure 2 The image shows a comparison of the Raman spectra of the in-situ iron film, the in vitro extracted iron film, and the standard ferrophosphate obtained in Example 1, used to illustrate the identification of characteristic peaks.

[0034] Figure 3 The intensity of the characteristic Raman peak of the iron film established in Example 2 (I) 715 -I 400 Linear quantitative relationship between iron content and chemically extracted iron content.

[0035] Figure 4 This is a diagram showing the spatial distribution and thickness evolution of the iron film obtained by time-series Raman imaging in Example 3.

[0036] Figure 5 This is a graph showing the results of the root surface cover layer thickness detection in Example 3. Wherein, Figure 5 In this context, 'a' represents the result from scanning electron microscopy. Figure 5 In the image, 'b' represents the Raman scan result, 'C' represents carbon, 'O' represents oxygen, and 'Fe' represents iron. Detailed Implementation

[0037] Example 1: Construction of a root microfluidic coupled Raman system and verification of characteristic Raman peaks of iron film

[0038] The purpose of this embodiment is to verify the feasibility of the root microfluidic coupled Raman (Rhizo-Raman) platform and to determine the Raman characteristic fingerprint of the in-situ formed iron film.

[0039] (1) System setup: according to Figure 1As shown, the microfluidic chip is assembled. The upper and lower layers of the chip are CNC-machined PMMA boards, with a silicone rubber gasket in the middle, and are secured with screws. A confocal Raman spectrometer is used, equipped with a 532 nm laser, 10× and 50× objectives, and a motorized stage.

[0040] (2) Sample preparation and iron film induction: After surface sterilization and germination, rice was hydroponically cultured in a greenhouse. Uniformly growing seedlings were selected, and the roots were carefully passed through the M5 threaded holes in the lower layer of the chip (with the pagoda connector for sealing) and placed in the chip chamber. The seedlings were first pre-cultured in a half-strength Yoshida nutrient solution for 3 days, and then replaced with the same nutrient solution containing 0.1 mM FeSO4·7H2O to induce iron film formation for 3 days.

[0041] (3) Spectral acquisition and peak identification: Place the chip on the stage of the Raman system and focus on the area where the root surface appears reddish-brown. Use a 10× objective lens, set the laser power to 0.5 mW, and the spectral range to 0-2000 cm⁻¹. -1 Raman spectra of the iron film on the root surface were collected in situ. Raman spectra of standard ferrihydrite, goethite, lepidocrocite, and hematite were collected simultaneously as controls.

[0042] (4) Results and Analysis: such as Figure 2 As shown, the in-situ iron film at 715 cm -1 A distinct characteristic peak appears at this location. This peak's position is similar to the main characteristic peak of standard ferrohydrate (~710 cm⁻¹). -1 The peak position is most similar to that of goethite and significantly different from other minerals. This confirms that, under the conditions of this system, the main mineral composition of the iron film formed in situ on the rice root surface is ferrihydrite (or ferrihydrite analogues), and establishes the 715 cm peak position. -1 Peaks serve as chemical fingerprint markers for subsequent quantitative and imaging analyses.

[0043] Example 2: Establishment of a quantitative relationship between Raman signal intensity and iron content in iron film

[0044] The purpose of this embodiment is to establish a method for in-situ quantitative determination of iron content in iron films using Raman intensity.

[0045] (1) Gradient concentration induction experiment: Five groups of different Fe were prepared 2+ Yoshida nutrient solution at concentrations of (0.05, 0.1, 0.15, 0.2, 0.3 mM). Three parallel chips were set up in each group to culture rice seedlings (method as in Example 1) for 1 day.

[0046] (2) In-situ Raman measurement: After cultivation, a fixed point was selected on the surface of the middle part of the root maturation zone within each chip, and a 10× objective lens was used to collect data at a depth of 400-900 cm. -1Raman spectra within the range. After smoothing and baseline correction of the spectra, the 715 cm⁻¹ value was calculated. -1 The difference intensity at point I (difference) = I 715 -I 400 .

[0047] (3) Determination of iron content in vitro: After completing the in situ measurement, the root segment (about 2 cm) that was just measured was immediately cut off, and the Fe content of the iron film on its surface was determined by the standard chemical extraction method of dithionite-citrate-bicarbonate (DCB). The results were expressed as the mass of iron per unit dry weight of the root.

[0048] (4) Data analysis: The average I (difference) value of the three parallel samples in each group was correlated with the corresponding average DCB-Fe content.

[0049] (5) Results: such as Figure 3 As shown, with the Fe in the induction solution 2+ As the concentration increased from 0.05 mM to 0.3 mM, the DCB-Fe content in the root iron film gradually increased from approximately 2 g / kg to approximately 10 g / kg. Simultaneously, the differential intensity I (difference) of the Raman characteristic peak measured in situ also showed a synchronous, approximately linear increase. Linear regression analysis showed a highly significant positive correlation between the two (R0). 2 > 0.99), the formula is as follows: y =14.308 x +7.925.

[0050] in y For the Raman differential intensity of the iron film, x The content of Fe in the DCB-iron film is shown. 0.12 mM and 0.23 mM Fe were added respectively. 2+ After being added to the chip and cultured for one day, the Raman differential intensities were measured to be 68.3 and 95.7, respectively. Substituting these values ​​into the formula, the DCB-Fe content of the iron film was found to be 4.2 g / kg and 6.1 g / kg, respectively. This is basically consistent with the DCB-Fe content of the iron film measured by ICP-MS, which was 4.2 g / kg and 6.0 g / kg. These results confirm that using a 715 cm⁻¹ chip... -1 The intensity of the characteristic Raman peak can reliably invert the iron content of the in-situ iron film, providing a theoretical basis for the quantitative monitoring method of this invention.

[0051] Example 3: Micro-region dynamic imaging observation of the iron film formation process

[0052] The purpose of this embodiment is to demonstrate the ability of the system of the present invention to perform in-situ, dynamic imaging of iron film spatial structures.

[0053] (1) Time-series imaging experiment: A plant was selected under 0.1 mM Fe 2+Rice seedlings cultured under the same conditions as in Example 1 were timed starting from the time the induction solution was changed (0 hours).

[0054] (2) Raman imaging parameter settings: Use a 50× long working distance objective lens (NA=0.5). Set the imaging mode: Scan range 400-900 cm. -1 The central window is 715 cm. -1 For thickness measurement, the scanning area is set as a rectangular region with a width of 10 μm, starting from the root edge and extending 32 μm inwards. The scanning step size is set to 2 μm, the integration time per pixel is 10 seconds, and the scan is performed twice.

[0055] (3) Imaging time points and operation: At 0, 6, 12, 24 and 36 hours after the start of cultivation, the chip was placed on the stage and the above Raman scanning imaging was repeated at the same preset position in the root maturity area.

[0056] (4) Image generation and processing: Using the instrument's software, the dataset obtained from scanning at each time point was processed into images with each pixel corresponding to a 715 cm² value. -1 Peak I (differential) intensity values ​​are pseudo-color encoded to generate a two-dimensional chemical distribution image of the iron film. By extracting intensity profiles perpendicular to the root surface direction, the effective thickness of the iron film layer can be determined (the width of the region where the intensity value is higher than three standard deviations of the mean background noise is typically defined as the thickness).

[0057] (5) Results: such as Figure 4 As shown, the generated series of images clearly demonstrates the dynamic spatial process of iron film formation. In the 0-hour image, there is no significant Raman signal in the root surface region (shown as a blue background in the pseudo-color image). The 6-hour image shows sporadic, irregular dotted or small patchy high-signal areas (yellow / red) on the root surface, indicating spatial heterogeneity in the initial deposition of the iron film. From 12 to 24 hours, these high-signal areas gradually expand and connect with each other, forming a discontinuous sheet-like coverage on the root surface. By 36 hours, the high-signal areas have basically merged into one, forming a relatively continuous and uniform coverage layer. The thickness of this layer was measured to be approximately 10 micrometers through cross-sectional analysis. This thickness result is consistent with the root surface coverage layer thickness observed by scanning electron microscopy (SEM) after slicing parallel culture samples (…). Figure 5 The results are essentially the same. This embodiment demonstrates that the method of the present invention can reveal the complete spatial structural evolution process of the iron film from initial nucleation and local growth to the final formation of a continuous capping layer in situ and dynamically at micron-level resolution, which is impossible to achieve by any in vitro or destructive analysis method.

Claims

1. A method for in-situ dynamic monitoring of iron film on plant root surfaces, characterized in that, A microfluidic Raman system for in-situ dynamic monitoring of iron films on plant root surfaces, comprising: A plant culture box for growing plants to be tested, the plant culture box having an outlet for the plant roots to extend out; The microfluidic chip has microchannels inside for plant roots to enter, and an inlet at one end of the microchannels for plant roots to enter. A pipe connects the outlet of the plant culture box to the inlet of the microfluidic chip. The microfluidic chip also has an inlet and an outlet that communicate with the microchannels. Raman spectrometer is used to detect the iron film on the root surface of plant roots located in the microchannels in situ. The method includes the following steps: (1) After assembling the plant culture box and the microfluidic chip, the plant to be tested is planted in the plant culture box. The roots of the plant to be tested extend into the microchannel through the pipe. Nutrient solution is added into the microchannel through the liquid inlet. In step (1), the nutrient solution is used for pre-culture, and then a nutrient solution containing ferrous ions is used for induction culture to induce the formation of an iron film. (2) In situ detection of the iron film on the root surface of plant roots located in the microchannel was performed using a Raman spectrometer at a depth of 715 cm. -1 The characteristic peaks at the location were used as the characteristic peaks of the in-situ root surface iron film for quantitative analysis. In step (2), when using a Raman spectrometer for detection, samples were collected from 400 to 900 cm⁻¹. -1 The Raman spectra within the range were smoothed and baseline corrected, and the 715 cm⁻¹ value was calculated. -1 Differential intensity I at the point 差分 I 差分 =I 715 -I 400 ; In step (2), when using a Raman spectrometer for detection, a root observation point is selected, and Raman spectra are collected in situ at fixed time intervals. The intensity values ​​of characteristic peaks are extracted and calculated, and the curve of the intensity value changing with time is plotted to achieve quantitative tracking of the formation dynamics of the iron film at that point. Alternatively, in step (2), when using a Raman spectrometer for detection, the target area of ​​the root surface is selected, and a two-dimensional point scan is performed using a Raman spectrometer to record the intensity of the characteristic peak at each scan point, thereby generating a two-dimensional chemical image reflecting the distribution of the iron film.

2. The method for in-situ dynamic monitoring of iron film on plant root surfaces according to claim 1, characterized in that, In step (2), when using a nutrient solution containing ferrous ions for induction culture, the ferrous ions in the nutrient solution are sourced from FeSO4, FeCl2, Fe-citrate, or EDTA-Fe. 2+ .

3. The method for in-situ dynamic monitoring of iron film on plant root surfaces according to claim 1, characterized in that, In step (2), the concentration of ferrous ions is 0.05~0.3 mM.

4. The method for in-situ dynamic monitoring of iron film on plant root surfaces according to claim 1, characterized in that, The microchannel has a square cross-sectional shape.

5. The method for in-situ dynamic monitoring of iron film on plant root surfaces according to claim 1, characterized in that, The microfluidic chip includes an upper layer and a lower layer that overlap each other. The microchannel is processed on the top surface of the lower layer, and the inlet is provided on the side of the lower layer. The liquid inlet and liquid outlet are provided on the upper layer.

6. The method for in-situ dynamic monitoring of iron film on plant root surfaces according to claim 5, characterized in that, The upper and lower layers are made of PMMA material.

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