Microbial sers detection method and kit based on super-hydrophobic evaporation concentration and magnetic guiding enrichment

Abstract

This invention discloses a method and kit for detecting microbial SERS based on superhydrophobic evaporation concentration and magnetically guided enrichment, belonging to the field of in vitro diagnostics and analytical chemistry. The method includes: mixing and incubating the sample to be tested with a magnetic SERS probe modified with targeted recognition molecules to form a complex; adding the mixed droplet to a superhydrophobic surface for evaporation concentration; applying a magnetic field below the detection site during droplet evaporation, using the magnetic gradient force to directionally enrich and pin the complex to the detection site, resulting in a dense solid enrichment spot in situ after drying, creating nanoscale gaps between the metal layers of adjacent probes and exciting SERS hotspots; irradiating the spot with a laser and acquiring the SERS signal to achieve microbial detection. This invention also provides a kit for implementing this method, including a magnetic SERS probe, a superhydrophobic detection card, and a positioning base with an embedded magnet. This invention eliminates the dependence on traditional solid-phase SERS substrates, overcomes the defect of easy droplet rolling on superhydrophobic surfaces through magnetic pinning, achieves self-alignment and high-sensitivity detection, and has advantages such as strong anti-interference, low cost, and ease of automation.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostics and analytical chemistry. More specifically, this invention relates to a method and kit for detecting microorganisms using surface-enhanced Raman scattering (SERS) based on superhydrophobic magnetic trapping microspheres, without dependence on traditional solid-phase or colloidal substrates. Particularly, it relates to a technique and supporting apparatus for achieving highly sensitive detection of trace microorganisms in liquid samples by utilizing the evaporation and concentration effect of superhydrophobic interfaces and the synergistic effect of magnetic nanoprobes. Background Technology

[0002] Surface-enhanced Raman scattering (SERS) technology has shown great application potential in in vitro diagnostics, food safety monitoring, and environmental pathogen detection due to its advantages such as ultra-high detection sensitivity, non-destructive analysis, and the ability to provide molecular "fingerprint" spectra. When detecting trace microorganisms in liquid samples (such as pathogens in blood, urine, or environmental water samples), effectively capturing and introducing them into the SERS "hotspot" region is crucial to determining the detection limits and stability. However, existing SERS microbial detection technologies still face the following technical challenges in their practical clinical application and commercialization:

[0003] (1) Severe base dependence and susceptibility to contamination Traditional SERS assays typically rely on solid-phase substrates with periodic or dense micro / nano structures (e.g., gold / silver nanoarrays prepared by photolithography, etching, or physical vapor deposition). These substrates are not only complex to prepare and costly to produce, but they can also exhibit non-specific protein adsorption (the "protein halo effect") when exposed to real, complex biological samples (such as serum or culture media containing high levels of protein). This contamination rapidly masks highly active sites on the substrate surface, hindering effective contact between the target microorganism and the SERS substrate, significantly reducing the enhancement effect and even leading to false negative results.

[0004] (2) Contact problem of superhydrophobic interface To address the concentration issue of trace samples, recent studies have introduced superhydrophobic surfaces to generate an evaporation and concentration effect. However, when performing detection on existing superhydrophobic SERS substrates, compatibility issues arise: due to the typical Cassie-Baxter wetting state of superhydrophobic surfaces, droplets are primarily supported by the top of the solid-phase micro / nanostructure, with a large amount of trapped air at the bottom. As the droplets evaporate and concentrate, the analytes inside often remain suspended and aggregated at the top of the micro / nanostructure, making it difficult for them to truly penetrate or fall into the extremely narrow electromagnetic field enhancement region within the structure. This results in a situation of "concentration without signal," limiting the application.

[0005] (3) Difficulty in positioning during the evaporation and concentration process The core characteristic of superhydrophobic surfaces lies in their extremely low water adhesion; the roll-off angle of droplets on such surfaces is typically less than 10°. During the evaporation and shrinkage process of large sample droplets (tens of microliters) over a period of tens of minutes, even minute airflow disturbances in the external environment, slight vibrations of the substrate, and even Marangoni convection within the droplet can easily disrupt the force equilibrium of the droplet. This causes the shrinking droplets to deviate from their initial predetermined positions, resulting in unpredictable random rolling or sliding. This makes the final position of the concentrated microspheres formed after drying highly random. Current confocal Raman spectrometers or automated detection equipment struggle to simultaneously achieve accurate and rapid focusing and high-throughput acquisition, severely hindering the automation and instrumentation of this technology. Summary of the Invention

[0006] To address the shortcomings of existing surface-enhanced Raman scattering (SERS) microbial detection technologies, such as strong substrate dependence, susceptibility to complex matrix contamination, the "contact problem" of superhydrophobic surfaces, and the "positioning difficulty" during droplet evaporation and concentration, this invention aims to provide a substrate-independent SERS microbial detection method and kit based on superhydrophobic magnetically trapped microspheres. This invention utilizes a synergistic strategy of "hybrid system combining trapping, suspension concentration, and magnetic field pinning" to eliminate dependence on traditional expensive SERS substrates, achieving highly sensitive, interference-resistant, and easily automated qualitative and quantitative detection of trace microorganisms in complex liquid samples.

[0007] Technical solution.

[0008] To achieve the above-mentioned objectives, this invention provides a substrate-independent SERS microbial detection method based on superhydrophobic magnetic trapping microsphere concentration, characterized by comprising the following core steps:

[0009] (1) Liquid-phase capture: The sample to be tested is mixed with a functionalized magnetic SERS probe. A larger volume of the sample to be tested is mixed with a functionalized magnetic SERS probe. The probe surface is modified with a targeting recognition molecule (such as 4-mercaptophenylboronic acid, 4-MPBA), which can specifically recognize and covalently bind to the microbial cell wall structure, forming a preliminary "probe-microbe-probe" aggregate in the liquid phase before the droplet dries.

[0010] (2) Superhydrophobic suspension concentration: The above-mentioned mixed liquid is added to the surface of a superhydrophobic substrate with a water contact angle greater than 150° for evaporation. Due to the high contact angle characteristics of the superhydrophobic substrate surface, the droplets always maintain a spherical shrinkage during the evaporation process, physically concentrating a large volume of sample of tens of microliters to the micrometer level, thereby achieving a significant increase in the concentration of analytes.

[0011] (3) Magnetic pinning to overcome disturbances (self-alignment): During the droplet evaporation and shrinkage stage, a point-like micro-magnetic field is applied below the substrate detection site. The magnetic field gradient force overcomes the defect that droplets on superhydrophobic surfaces are easily disturbed by airflow and roll, forcing the shrinking droplet to be positioned directly above the magnetic pole at the drying endpoint, thus achieving a self-alignment effect.

[0012] (4) Raman hotspot construction and acquisition: As the water evaporates completely, the magnetic force tightly attracts the probe-microbe complex to the substrate surface, forming dense solid microspheres (diameter <200 μm). Under this extremely compressed state, a large number of nanoscale gaps are formed between the metal shells of adjacent magnetic SERS probes, which excites a strong localized surface plasmon resonance (LSPR) coupling signal (i.e., SERS hotspot). The signal is then acquired by directly irradiating the microsphere spot with a laser.

[0013] The present invention also provides a matching kit for implementing the above method, comprising: a magnetic SERS probe in the form of lyophilized powder; a disposable detection card with a superhydrophobic nanocoating on its surface; and a matching base with a micro-magnetic column array embedded at the corresponding detection position.

[0014] Beneficial effects

[0015] Compared with the prior art, the present invention has the following significant advantages:

[0016] Improved resistance to matrix interference and elimination of dependence on solid substrates: This invention eliminates the dependence on solid-phase SERS substrates, with SERS characteristic signals originating from magnetic nanoprobes that actively bind to targets in the liquid phase. This mechanism helps reduce the risk of surface passivation and signal attenuation caused by non-specific adsorption of proteins or other interfering components in complex biological samples on traditional solid-phase reinforced surfaces.

[0017] Significantly reduces consumable manufacturing costs and possesses high mass production capability: Existing technologies often rely on expensive and inefficient micro-nano fabrication processes such as photolithography and electron beam etching to prepare SERS substrates. The superhydrophobic detection card used in this invention can be fabricated using only conventional surface spraying or chemical modification processes. Its extremely low cost per detection consumable facilitates the large-scale fabrication and mass application of the technology.

[0018] Overcoming the inherent limitations of random rolling, this invention achieves high-precision spatial positioning and automated adaptation: Addressing the inherent limitations of superhydrophobic surface droplets with small roll angles (<10°) and susceptibility to random sliding due to airflow or vibration disturbances during evaporation, this invention introduces a magnetic field-guided in-situ anchoring (magnetic pinning) mechanism. This mechanism forces concentrated microspheres to achieve "self-alignment" above the magnetic poles through magnetic gradient force, effectively solving the engineering challenge of focusing in spectrometers and laying the foundation for high-throughput, array-based automated detection in clinical settings.

[0019] This invention improves solid-liquid phase contact efficiency, enhances detection sensitivity, and simplifies the operation process: The target capture process is transferred to the liquid phase system, effectively avoiding the physical limitation of analytes easily suspending at the Cassie-Baxter gas-solid interface in traditional superhydrophobic SERS detection. The liquid probe can efficiently encapsulate the target and form a high-density SERS hotspot under evaporation-driven high physical compression. Furthermore, the entire detection process is highly integrated into droplet injection, evaporation concentration and shaping, and in-situ optical acquisition, eliminating the need for complex sample pretreatment steps and meeting the rapid diagnostic needs of point-of-care testing (POCT). Detailed Implementation

[0021] Testing process

[0022] Example 1

[0023] Step 1: Preparation of functionalized magnetic SERS probe (Fe3O4@Ag-MPBA) like Figure 1As shown, 10 mL of an aqueous dispersion of magnetic Fe3O4 nanoparticles with a concentration of 2 mg / mL and an average particle size of approximately 150 nm was prepared. 0.5 g of polyvinylpyrrolidone (PVP) was added, and the mixture was ultrasonically dispersed for 30 min to improve particle dispersibility and system stability during subsequent silver deposition. Subsequently, 2 mL of a 0.1 M AgNO3 aqueous solution was slowly added while continuously stirring, and stirring was continued for 15 min. Then, 5 mL of a 0.05 M ascorbic acid aqueous solution was slowly added as a reducing agent, and the reaction was continued at room temperature (approximately 25 °C) for 2 h. After the reaction was completed, the particles were separated by an external magnetic field and washed three times with pure water to obtain Fe3O4@Ag magnetic particles with a silver-coated surface. Characterization revealed a rough silver-coated structure on the surface of the Fe3O4 particles, with an equivalent thickness of approximately 20 nm in some areas. The Fe3O4@Ag magnetic particles were resuspended in 10 mL of anhydrous ethanol, and 1 mL of 0.1 mM 4-mercaptophenylboronic acid (4-MPBA) aqueous solution was added. The mixture was incubated at 25 °C in the dark for 4 h with shaking, allowing 4-MPBA to self-assemble onto the silver surface via Ag-S bonds. The 4-MPBA serves as a surface functional molecule, enhancing the probe's affinity recognition of microbial surface components and providing corresponding characteristic Raman signals. After incubation, the particles were magnetically separated and washed, then redispersed in PBS buffer and incubated at room temperature with shaking for 5 min to reduce non-specific adsorption and particle self-aggregation caused by unmodified sites. The particles were then magnetically separated and washed again, and the resulting functionalized magnetic SERS probe was resuspended in 10 mL of PBS buffer (0.01 M, pH 7.4) and stored at 4 °C for later use.

[0024] Step Two: Preparation of Testing Consumables (Superhydrophobic Test Card and Positioning Base) A clean glass slide is uniformly coated with a nano-silica suspension containing a low surface energy modifier (such as perfluorodecyltriethoxysilane). After curing, a superhydrophobic testing card with a surface water contact angle >150° and a roll-off angle <10° is obtained. A matching plastic base is also prepared. Figure 3 The device structure shown has an array of cylindrical neodymium iron boron micro-magnets with a diameter of about 1 mm embedded directly below the detection point on the base, so that when the detection card is placed on the base, the magnets can generate a local strong magnetic field on the superhydrophobic surface.

[0025] Step 3: Liquid-phase mixing and specific capture refer to Figure 2The method flowchart is as follows: Take a sterile centrifuge tube, add 50 μL of E. coli test suspension, and then add 10 μL of the magnetic SERS probe solution prepared in step one. Gently pipette and mix several times, then incubate at room temperature for 15 min. During incubation, the 4-MPBA on the probe surface can interact with recognizable components on the bacterial surface, thereby promoting the formation of the "probe-bacterium" complex; in this embodiment, a "probe-bacterium-probe" aggregate structure can be further formed. (e.g.) Figure 3 (As shown in the floating state on the left)

[0026] Step 4: Superhydrophobic suspension concentration of large-volume samples Place the superhydrophobic test card flat on the magnetic positioning base. Use a pipette to draw 20 μL of the bacterial mixture incubated in step three and drop it onto the test card directly above the micromagnet at the bottom. Due to the superhydrophobic properties of the surface, the mixed droplet exhibits a full, near-spherical shape and begins to evaporate moisture at room temperature (or with the aid of gentle infrared heating), causing the droplet volume to continuously shrink.

[0027] Step 5: Magnetic guided deposition (magnetic pinning) During the evaporation and contraction of the droplet, the magnetic gradient force generated by the micromagnet on the base acts on the magnetic complex inside the droplet. For example... Figure 3 As shown in the force-concentration process on the right, the magnetic field effectively overcomes the random rolling tendency of droplets on the superhydrophobic surface caused by airflow disturbance or microfluidic convection, guiding the complex to settle and aggregate directly above the magnetic pole. After the solvent has completely evaporated, all the complexes are pinned to the substrate surface, forming dense, metallic-luster solid spots with a diameter of less than 500 μm.

[0028] Step Six: In-situ SERS Signal Acquisition and Analysis The solid spots were directly irradiated using a 785 nm laser from a portable Raman spectrometer. Due to the concentrated enrichment of the magnetic complex at the end of evaporation, the spacing between adjacent probes decreased, which facilitated the formation of more SERS enhancement sites, thereby increasing the Raman detection signal intensity. Highly sensitive detection of trace bacteria can be achieved by reading the characteristic spectral peak intensity of 4-MPBA, without relying on traditional, expensive solid-phase SERS substrates.

[0029] Optional Implementation Scope and Equivalent Replacements Although the above specific embodiments use the Fe3O4@Ag-MPBA probe to detect Escherichia coli as an example, those skilled in the art should understand that the scope of protection of the present invention is not limited thereto.

[0030] (1) The Raman-active metal layer of the magnetic SERS probe is not limited to a silver layer, but can also be gold (Au), gold-silver alloy or other noble metal nanostructures with surface plasmon resonance effect.

[0031] (2) The targeted recognition molecule is not limited to small chemical molecules such as 4-mercaptophenylboronic acid (4-MPBA), but can also be selected from macromolecular ligands such as antibodies, nucleic acid aptamers, antimicrobial peptides or vancomycin that can specifically recognize target microorganisms.

[0032] (3) The method and kit described in this invention have broad applicability. The detection targets are not limited to Escherichia coli, but also include other Gram-negative bacteria, Gram-positive bacteria, fungi, viruses and other microorganisms in the environment that contain characteristic surface antigens or chemical structures. Attached Figure Description

[0034] Figure 1 This is a schematic diagram illustrating the preparation process and surface modification mechanism of the functionalized magnetic SERS probe (Fe3O4@Ag-MPBA) in Example 1 of the present invention.

[0035] Figure 2 This is a schematic diagram of the overall operation process of the substrate-independent SERS microbial detection method based on superhydrophobic magnetic trapping microspheres concentration of the present invention.

[0036] Figure 3 This is a cross-sectional schematic diagram illustrating the principle of droplet evaporation and concentration on the surface of a superhydrophobic detection card and "magnetic pinning" by a micromagnet, demonstrating the physical process of magnetic field gradient force constraining droplets and guiding the self-alignment of the complex.

[0037] reagent kit composition

[0038] Core reagent: Broad-spectrum / specific magnetic SERS probe in lyophilized powder form.

[0039] Test card: A glass slide or plastic sheet with a superhydrophobic nano-coating on its surface.

[0040] Matching positioning base: A matching base with a miniature magnet (such as a neodymium iron boron magnet) embedded in the corresponding detection position.

Claims

1. A method for detecting microorganisms using surface-enhanced Raman scattering, characterized in that, Includes the following steps: S1, the sample to be tested is mixed with a magnetic SERS probe whose surface is modified with targeted recognition molecules and incubated, so that the magnetic SERS probe binds to the microorganisms in the sample to form a complex. S2, the mixture containing the complex is dropped onto the superhydrophobic surface, and the droplet is evaporated and shrunken on the superhydrophobic surface to concentrate the complex; S3, during the droplet evaporation and shrinkage process, a magnetic field is applied below the detection site on the superhydrophobic surface. The magnetic field gradient force drives the composite to gather towards the detection site, and after the droplet dries, solid enrichment spots are formed in situ at the detection site. This causes the surface-enhanced Raman active metal layers of adjacent magnetic SERS probes to be tightly squeezed together, forming nanoscale gaps and exciting local surface plasmon resonance (LSPR) coupling hotspots. S4, the solid enrichment spots (i.e. the coupled hot spot region) are irradiated with laser and surface-enhanced Raman scattering signals are collected to achieve microbial detection.

2. The method according to claim 1, characterized in that, The superhydrophobic surface has a static water contact angle greater than 150° and a roll-off angle less than 10°.

3. The method according to claim 1 or 2, characterized in that, The superhydrophobic surface is prepared by spraying a modified nanoparticle suspension, dipping in a fluorinated silane solution, or imprinting a micro / nano structure template.

4. The method according to claim 1, characterized in that, The magnetic field is generated by a micromagnet positioned on the back side of the superhydrophobic surface and directly below the detection site.

5. The method according to claim 1 or 4, characterized in that, The magnetic field is applied for all or part of the period from when the droplet is applied to the superhydrophobic surface until the droplet is completely dry; preferably, it is applied from when the droplet volume shrinks to 10% of its initial volume until it is completely dry.

6. The method according to claim 1, characterized in that, The magnetic SERS probe comprises a magnetic core and a surface-enhanced Raman-active metal layer, the surface of which is modified with targeted recognition molecules for recognizing microbial surface components. In some embodiments, the targeted recognition molecules are molecules capable of binding to or interacting with characteristic components of the microbial surface.

7. The method according to claim 6, characterized in that, The magnetic core is iron(III) oxide (Fe3O4) nanoparticles, the surface-enhanced Raman active metal layer is a silver layer, and the targeted recognition molecule is 4-mercaptophenylboronic acid (4-MPBA).

8. The method according to claim 1, characterized in that, The mixture in step S1 also includes an anti-agglomeration agent, which is used to reduce the non-specific aggregation of the magnetic SERS probe during the concentration process.

9. The method according to claim 8, characterized in that, The anti-agglomeration agent is polyethylene glycol or bovine serum albumin.

10. The method according to claim 1, characterized in that, The method causes the target component in the sample to be tested to accumulate at the detection site and form solid enrichment spots with a diameter of less than 500 μm.

11. A microbial SERS detection kit for implementing the method according to any one of claims 1-10, characterized in that, include: Magnetic SERS probe; Test cards with superhydrophobic surfaces; The device also includes a positioning base for use with the detection card. The positioning base has a support structure for supporting the detection card, and a magnet for generating a local magnetic field is embedded directly below the detection site on the superhydrophobic surface. The magnetic gradient force generated by the local magnetic field is used to penetrate the detection card, pinning the droplets on the superhydrophobic surface in an evaporating and contracting state in situ and self-aligning them with the detection site.

12. The kit according to claim 11, characterized in that, The magnetic SERS probe reagent is in individually packaged lyophilized powder form.

13. The kit according to claim 11, characterized in that, The test card is a disposable test card, and its surface is uniformly coated with a superhydrophobic nano-coating.

14. The kit according to claim 11, characterized in that, The magnet is a cylindrical array of neodymium iron boron miniature strong magnets, each with a diameter of approximately 1 mm.

15. The kit according to claim 11, characterized in that, The substrate material of the test card is a glass slide or injection-molded plastic sheet.

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