A device and method for real-time detection of single nanoparticle protein crowns

Abstract

This invention discloses a device and method for real-time detection of single-nanoparticle protein crowns. The device includes a surface plasmon resonance imaging (SPRM) device comprising an optical module, a sample reaction unit, and a signal acquisition and analysis module. The SPRM device includes a laser emission module for heating single nanoparticles. This invention integrates the SPRM device with a laser emission module capable of heating single nanoparticles, achieving precise temperature control of single-particle protein crowns and enabling real-time, label-free, high spatiotemporal resolution detection of the protein crown formation process. Furthermore, this invention provides a high-precision experimental platform for quantitatively studying the thermodynamic processes of protein-nano interfaces and provides important theoretical and technical support for the design of nanomedicine carriers and biointerface engineering.

Description

Technical Field

[0001] This invention relates to an apparatus and method for detecting single nanoparticle protein corona, and more particularly to an apparatus and method for real-time detection of single nanoparticle protein corona. Background Technology

[0002] When nanoparticles enter a biological solution, they rapidly adsorb proteins from the solution, forming a "protein corona" on the particle surface. The formation of the protein corona typically involves two stages: first, the "hard protein corona," formed by the tight adsorption of high-affinity proteins onto the nanoparticle surface. This formation occurs in a short period, usually within seconds to minutes, and the structure is stable and difficult to displace. Then comes the "soft protein corona," gradually formed by the dynamic adsorption and desorption of low-affinity proteins on the outer layer of the hard protein corona. This formation takes tens of minutes to several hours, and its composition changes continuously with time and environmental conditions.

[0003] Measuring the formation and thickness of protein coronas is of significant scientific importance in nanobiointerface research. By monitoring the formation and evolution of protein coronas, the stability and potential toxicity of different nanomaterials in biological environments can be assessed, improving the enrichment and release efficiency of drugs in target tissues. Simultaneously, observing the dynamic impact of changes in the solution environment on protein corona thickness can serve as a sensitive indicator for identifying changes in pathological environments (such as pH or temperature), expanding its application potential in early disease diagnosis and intelligent response systems.

[0004] In recent years, surface plasmon resonance (SPR) imaging technology has been widely used for the detection of single nanoparticles due to its advantages such as label-free operation and high temporal resolution signal detection. For example, CN111551278A discloses a precise and rapid temperature measurement system and method for a single nanoparticle. However, this method lacks precise control over the local temperature of a single particle. Although SPR technology has been applied to the detection of protein crowns, it requires the detection of composition and thickness changes through mass spectrometry, TEM, DLS, etc., which limits its support for real-time, dynamic, and environmentally controllable observation. In addition, temperature is an important factor affecting the thermodynamics of protein adsorption, but existing technologies lack real-time control and quantitative detection methods for local temperature changes of single particles. Traditional photothermal heating methods usually achieve temperature increases by irradiating the entire solution with infrared lasers, which cannot achieve precise local temperature control of a single particle and make it difficult to reveal the quantitative relationship between temperature and protein adsorption thermodynamics. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a device capable of locally heating single nanoparticles to induce the formation of protein crowns and to detect it in real time. Another purpose of this invention is to provide a method for using this device to detect the evolution of single nanoparticle protein crowns in real time.

[0006] Technical solution: The device for real-time detection of single nanoparticle protein crowns of the present invention includes a surface plasmon resonance imaging device composed of an optical module, a sample reaction unit, and a signal acquisition and analysis module. The surface plasmon resonance imaging device has a laser emission module for temperature control of single nanoparticles.

[0007] Furthermore, the laser emitting module uses a near-infrared laser with a wavelength of 780~2500nm, preferably a near-infrared laser with a wavelength of 1064nm, and achieves temperature control of single nanoparticles by focusing through an objective lens.

[0008] Furthermore, the optical module includes a low-power red light source as the detection beam, an objective lens, a P-polarizer, a beam splitter, and a filter; the sample reaction unit includes a sample cell and an excitation substrate; and the signal acquisition and analysis module includes an image sensor.

[0009] The method for real-time detection of single nanoparticle protein corona by the device of the present invention includes the following steps:

[0010] (1) A functionalized layer is modified on the excitation substrate surface of the sample reaction unit so that the nanoparticles are adsorbed on the substrate surface, and then a protein solution is injected into the sample reaction unit.

[0011] (2) Excite surface plasmons on the substrate surface using a low-power red light source, collect the reflected signals using an objective lens, and record the dynamic image sequence of the intensity of single nanoparticle plasmons changing over time using an image sensor.

[0012] (3) The laser emission module is focused on a single nanoparticle through the aforementioned objective lens. The temperature of the single nanoparticle is controlled and a protein crown is induced to form on the surface of the single nanoparticle. During the heating process, the plasmon signal intensity and temperature calibration relationship are measured, the plasmon signal changes are recorded in real time, and the local temperature changes caused by heating are calculated. The thickness evolution and formation rate of the protein crown of the single nanoparticle are analyzed.

[0013] In step (1), the excitation substrate of the sample reaction unit is a glass slide with a gold thin film coated on its surface, which provides the clearest effect in surface plasmon resonance (SPR) imaging. The glass slide with the gold thin film coated on its surface is located at the bottom of the sample cell made of polydimethylsiloxane (PDMS).

[0014] Preferably, in step (1), the glass slide is plasma cleaned before being coated with a gold film to ensure that it is clean enough to prevent dust particles from interfering with the SPR imaging results and affecting the observation and detection of single nanoparticle protein crowns.

[0015] Preferably, in step (1), the thickness of the gold film is 45-50 nm, within which the SPR imaging effect is clearest.

[0016] In step (1), the functionalized layer is a carboxyl-functionalized layer. The functionalization modification is used to fix the nanoparticles on the excitation substrate surface of the surface plasmon resonance imaging. Among them, the carboxyl groups have a high affinity for the gold film surface, can firmly bind to the gold film surface, and prevent the protein from reacting with the gold film.

[0017] Preferably, the carboxyl functionalized layer is formed by the self-assembly of 11-mercaptoundecanoic acid (11-MUA) molecules.

[0018] In step (1), the nanoparticles are at least one of hard nanoparticles or soft nanoparticles. The nanoparticles are dispersed in phosphate (PBS) buffer at pH 7.4 and adsorbed onto the surface of the excitation substrate by Brownian motion, thereby achieving the fixation of spatially independent single nanoparticles.

[0019] Furthermore, the hard nanoparticles include at least one of metal nanoparticles, oxide ceramic nanoparticles, and polymer nanoparticles, and the soft nanoparticles include at least one of liposome nanoparticles and micelle nanoparticles. The nanoparticles are used to provide a substrate for protein adsorption and growth of protein crowns.

[0020] Further preferably, the metal nanoparticles are gold nanoparticles (AuNPs).

[0021] Preferably, in step (1), the protein solution is prepared using phosphate-buffered saline (PBS) buffer with a pH of 4-8. The protein can be selected from bovine serum albumin (BSA), hemoglobin (Hb), etc. When the protein is adsorbed onto the surface of the nanoparticles, it causes a local refractive index change. The brightness change ΔI in the plasmon image is proportional to the thickness of the protein crown. By recording ΔI(t) and performing time fitting, the protein adsorption kinetic curve can be obtained. By adjusting the pH of the PBS buffer, protein solutions under different acidic and alkaline conditions can be prepared to study the effect of pH on protein adsorption kinetics.

[0022] In step (2), the wavelength of the low-power red light source is 680 nm, at which wavelength the SPR excitation effect on the gold thin film is best. The low-power red light source is incident on the substrate surface coated with the gold thin film through an objective lens to excite surface plasmon resonance; the incident angle of the low-power red light source can be adjusted to be close to the critical angle to obtain the maximum resonance response.

[0023] In step (2), the objective lens is a 60x objective lens with a numerical aperture (NA) ≥ 1.2, which can provide a wider range of incident angles and collect reflected light from the substrate surface coated with a gold film. When light shines on the gold film surface through a high numerical aperture (NA) objective lens, a portion of the light can be incident at a large angle close to the interface, "brushing along the surface." Only under such a large incident angle condition can the momentum of the light match the free electron oscillations on the gold film surface, thereby exciting surface plasmon resonance (SPR) and forming an evanescent field on the gold film surface. If the NA of the objective lens is small, the range of incident angles it can provide is limited, and the light cannot reach the critical angle required to excite SPR, thus failing to effectively generate surface plasmon resonance and an evanescent field. Therefore, the higher the numerical aperture of the objective lens, the clearer the image resolution.

[0024] Preferably, in step (2), the objective lens is an oil immersion objective lens, which typically has a large NA value and can provide a sufficiently large incident angle to excite SPR.

[0025] In step (2), the image sensor is a digital camera. The digital camera uses a charge-coupled device to convert the optical image acquired by the objective lens into a digital signal in real time, forming a high-contrast plasmon interference image.

[0026] Preferably, in step (2), the digital camera is a CCD camera or a CMOS camera.

[0027] In step (3), the laser of the laser emitting module adopts one of the following modes: square wave modulation mode, sine wave modulation mode, triangular wave modulation mode or pulse modulation mode, to achieve local intermittent heating and precisely adjust the heating power.

[0028] Preferably, in step (3), the laser of the laser emitting module adopts a square wave modulation mode, which makes the operation more convenient.

[0029] Furthermore, in step (3), by measuring the calibration relationship between the change in plasmon signal intensity (ΔI) and the change in temperature (ΔT), the local temperature change caused by heating is calculated in real time, so that the temperature rise range of the system is controlled within 15K±5K, which can achieve stable local temperature regulation, ensure the integrity of protein conformation, and further reveal the protein adsorption behavior driven by heat.

[0030] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) It integrates the surface plasmon resonance imaging device (SPRM) with a laser emission module that can heat single nanoparticles to achieve precise temperature control of single-particle protein crowns and realize real-time, label-free, high spatiotemporal resolution detection of the protein crown formation process; (2) It provides a high-precision experimental platform for quantitative research on the thermodynamic process of protein-nano interface and provides important theoretical and technical support for the design of nanomedicine carriers and biointerface engineering. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the structure of the present invention;

[0032] Figure 2 This is a schematic diagram of the temperature correction of the present invention;

[0033] Figure 3 The curves showing the change in plasmonic signal intensity (ΔI) versus the change in temperature (ΔT) are shown in the present invention.

[0034] Figure 4 The SPR signal is generated during the growth of the heated bovine serum albumin (BSA) protein crown on the surface of gold nanoparticles according to the present invention.

[0035] Figure 5 Plasmon images before and after laser heating of bovine serum albumin (BSA) protein crown growth;

[0036] Figure 6 The variation of bovine serum albumin (BSA) plasmon signal in the x-direction after different laser heating times according to the present invention;

[0037] Figure 7 The 2D line profile of bovine serum albumin (BSA) plasmon signal changes over time after different laser heating times according to the present invention.

[0038] Figure 8 These are plasmon images of hemoglobin (Hb) protein corona before and after laser heating, as presented in this invention. Detailed Implementation

[0039] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0040] Example 1: Verification of Device Temperature Calibration and Control Accuracy

[0041] A clean glass slide was plasma-cleaned and then coated with a gold film approximately 45-50 nm thick to serve as the excitation substrate for surface plasmon resonance imaging (SPRM). A carboxyl-functionalized layer formed by 11-mercaptoundecanoic acid (11-MUA) molecules was then modified onto the gold film surface using a self-assembly method. The substrate was placed at the bottom of a sample cell made of polydimethylsiloxane (PDMS) to form the sample reaction unit. Commercially available 150 nm gold nanoparticles (AuNPs) were dispersed in phosphate-buffered saline (PBS) at pH 7.4, and the AuNPs were adsorbed and immobilized onto the functionalized substrate surface through Brownian motion. Hot water was added to the sample cell, and the overall temperature change and corresponding SPR signal intensity changes were measured simultaneously. Temperature and SPR intensity changes during the cooling process were recorded by thermocouples and SPRM, respectively (see details). Figure 2 The time-delayed SPR images were recorded by a CCD camera, and the curves of plasmon signal intensity change (ΔI) versus temperature change (ΔT) were obtained by fitting the images (see details). Figure 3 ),Depend on Figure 3 It can be seen that the linear correlation coefficient R of the system response 2 A value greater than 0.98 demonstrates that the system can achieve stable and controllable local heating without causing conformational changes in proteins. The aforementioned curves show that for every 1°C decrease in temperature, the plasmon signal intensity decreases by 60 IU. By calculating the local temperature change caused by heating in real time using the plasmon signal intensity, the system's temperature rise range is controlled within 15K ± 5K, preventing protein denaturation caused by overheating.

[0042] Example 2: Thermodynamically Controlled Adsorption Kinetics of Bovine Serum Albumin Coronavirus

[0043] (1) A clean glass slide is plasma-cleaned and coated with a gold film of approximately 45-50 nm thickness as the excitation substrate for surface plasmon resonance imaging (SPRM). A carboxyl functionalized layer formed by 11-mercaptoundecanoic acid (11-MUA) molecules is then modified onto the gold film surface using a self-assembly method. The substrate is placed at the bottom of a sample cell made of polydimethylsiloxane (PDMS) to form a sample reaction unit. Commercially available 150 nm gold nanoparticles (AuNPs) are dispersed in deionized water and dropped onto the 11-MUA-modified gold film surface to form a sparsely distributed array of single particles. Subsequently, phosphate-buffered saline (PBS) containing 5 mg / mL bovine serum albumin (BSA) is injected into the sample chamber. The carboxyl functionalized layer can also be other functionalized layers conventionally used by those skilled in the art.

[0044] (2) A low-power red light source with a wavelength of 680 nm was incident on the surface of the gold-coated substrate through an objective lens to excite surface plasmon resonance. The incident angle of the low-power red light source could be adjusted to be close to the critical angle to obtain the maximum resonance response. An oil immersion objective lens with a numerical aperture (NA) of 1.49 was used to collect the reflected signal, and a CCD camera was used to record a dynamic image sequence of the change in the intensity of single nanoparticle plasmons over time.

[0045] (3) A near-infrared laser with a wavelength of 1064 nm was focused onto a single nanoparticle using a lens system. Square wave modulation was used to heat the single nanoparticle and induce the formation of a protein crown on its surface. During the heating process, changes in plasmon signals were recorded in real time. The thickness evolution and formation rate of the protein crown on the single nanoparticle were analyzed. Furthermore, by measuring the calibration relationship between plasmon signal intensity and temperature, the local temperature changes caused by heating were calculated in real time. Figure 4 As shown in the figure. Experimental results indicate that temperature changes after laser heating induce the growth of protein crowns, manifested as an increase in the thickness of the hard layer on the particle surface. The SPR signal intensity on the particle surface significantly increases after heating, indicating the formation of a new hard protein crown layer on the surface, as shown in the figure. Figure 5 As shown. The SPR intensity distribution and time-lapse 2D line profile of a single BSA-AuNP complex along the x-axis reveal that the growth of the surface protein crown increases with the duration of laser operation, as shown. Figure 6 and Figure 7 As shown. The modulation of the near-infrared laser can also be replaced with a sine wave modulation mode, a triangular wave modulation mode, or a pulse modulation mode, all of which can achieve precise temperature control of single nanoparticles.

[0046] Example 3: Thermodynamically Controlled Adsorption Kinetics of Hemoglobin Corona

[0047] (1) A clean glass slide is plasma-cleaned and coated with a gold film of approximately 45-50 nm thickness as the excitation substrate for surface plasmon resonance imaging (SPRM). A carboxyl functionalized layer formed by 11-mercaptoundecanoic acid (11-MUA) molecules is then modified onto the gold film surface using a self-assembly method. The substrate is placed at the bottom of a sample cell made of polydimethylsiloxane (PDMS) to form a sample reaction unit. Commercially available 150 nm gold nanoparticles (AuNPs) are dispersed in deionized water and dropped onto the 11-MUA-modified gold film surface to form a sparsely distributed array of single particles. Subsequently, phosphate-buffered saline (PBS) containing 5 mg / mL hemoglobin (Hb) is injected into the sample chamber. The carboxyl functionalized layer can also be other functionalized layers conventionally used by those skilled in the art.

[0048] (2) A low-power red light source with a wavelength of 680 nm was incident on the surface of the gold-coated substrate through an objective lens to excite surface plasmon resonance. The incident angle of the low-power red light source could be adjusted to be close to the critical angle to obtain the maximum resonance response. An oil immersion objective lens with a numerical aperture (NA) of 1.49 was used to collect the reflected signal, and a CCD camera was used to record a dynamic image sequence of the change in the intensity of single nanoparticle plasmons over time.

[0049] (3) A near-infrared laser with a wavelength of 1064 nm was focused onto a single nanoparticle using a lens system. Square wave modulation was used to heat the single nanoparticle and induce the formation of protein crowns on its surface. During the heating process, changes in plasmon signals were recorded in real time. The thickness evolution and formation rate of the protein crowns on the single nanoparticles were analyzed. The local temperature changes caused by heating were calculated in real time by determining the calibration relationship between plasmon signal intensity and temperature. Experimental results show that temperature changes after laser heating induce the growth of protein crowns, manifested as an increase in the thickness of the hard layer on the particle surface. The protein crowns formed by Hb after heating exhibit obvious non-uniform spatial distribution characteristics. The density decreases in the central region due to the steric hindrance of prior adsorption, while molecular enrichment occurs at the edges, such as... Figure 8 As shown.

[0050] In addition, the gold nanoparticles in Examples 2 and 3 can be replaced with hard nanoparticles and / or soft nanoparticles, as well as other nanoparticles well known to those skilled in the art. The hard nanoparticles include at least one of metal nanoparticles, oxide ceramic nanoparticles, and polymer nanoparticles, and the soft nanoparticles include at least one of liposome nanoparticles and micelle nanoparticles. The nanoparticles are used to provide a substrate for protein adsorption and growth of protein crowns.

[0051] In summary, the detection system and method of this invention not only enable real-time, quantitative, and label-free observation of the formation process of single-particle protein crowns, but also reveal the synergistic regulatory mechanism of pH and temperature on protein crown structure and molecular fluctuations by analyzing the changes in adsorption kinetic parameters and thermodynamic potential energy through local photothermal regulation.

Claims

1. A device for real-time detection of single nanoparticle protein coronas, comprising a surface plasmon resonance imaging device consisting of an optical module, a sample reaction unit, and a signal acquisition and analysis module, characterized in that, The surface plasmon resonance imaging device has a laser emission module that allows for temperature control of single nanoparticles.

2. The device for real-time detection of single nanoparticle protein crowns according to claim 1, characterized in that, The laser emitting module uses near-infrared laser.

3. A method for real-time detection of single nanoparticle protein corona using the device of claim 1, characterized in that, Includes the following steps: (1) A functionalized layer is modified on the excitation substrate surface of the sample reaction unit so that the nanoparticles are adsorbed on the substrate surface, and then a protein solution is injected into the sample reaction unit. (2) Excite surface plasmons on the substrate surface using a low-power red light source, collect the reflected signals using an objective lens, and record the dynamic image sequence of the intensity of single nanoparticle plasmons changing over time using an image sensor. (3) The laser emission module is focused on a single nanoparticle through the aforementioned objective lens. The temperature of the single nanoparticle is controlled and a protein crown is induced to form on the surface of the single nanoparticle. During the heating process, the plasmon signal intensity and temperature calibration relationship are measured, the plasmon signal changes are recorded in real time, and the local temperature changes caused by heating are calculated. The thickness evolution and formation rate of the protein crown of the single nanoparticle are analyzed.

4. The method for real-time detection of single nanoparticle protein corona according to claim 3, characterized in that, In step (1), the excitation substrate of the sample reaction unit is a glass plate with a gold film coated on its surface.

5. The method for real-time detection of single nanoparticle protein coronas according to claim 3, characterized in that, In step (1), the functionalized layer is a carboxyl functionalized layer.

6. The method for real-time detection of single nanoparticle protein coronas according to claim 3, characterized in that, In step (2), the wavelength of the low-power red light source is 680nm.

7. The method for real-time detection of single nanoparticle protein coronas according to claim 3, characterized in that, In step (2), the objective lens is a 60x objective lens with a numerical aperture ≥ 1.

2.

8. The method for real-time detection of single nanoparticle protein corona according to claim 3 or 7, characterized in that, In step (2), the objective lens is an oil immersion objective lens.

9. The method for real-time detection of single nanoparticle protein corona according to claim 3, characterized in that, In step (2), the image sensor is a digital camera.

10. The method for real-time detection of single nanoparticle protein corona according to claim 3, characterized in that, In step (3), the laser emitted by the laser emitting module adopts one of the following modes: square wave modulation mode, sine wave modulation mode, triangular wave modulation mode, or pulse modulation mode.

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