Photoacoustic enhanced microcavity misfolded protein detection system and method

Abstract

The invention provides a photoacoustic enhanced microcavity misfolded protein detection system and method.The photoacoustic enhanced microcavity misfolded protein detection system comprises a micro laser cavity unit, the micro laser cavity unit is a liquid crystal micro laser cavity doped with a quantum dot material with the fluorescence lifetime larger than or equal to 10 ns, and the liquid crystal micro laser cavity takes a cationic surface active agent solution as a water phase; a liquid crystal material is mixed with a quantum dot material to serve as an oil phase, the surface of the oil phase is modified with an aptamer for specifically capturing misfolded protein, and the oil phase is used for generating WGM resonance light; a pulse laser excitation unit; the photoacoustic material unit is used for responding to photoacoustic excitation to generate photoinduced ultrasonic waves; the photoacoustic excitation unit is used for exciting the photoacoustic material unit, generating controlled photoinduced ultrasonic waves, acting on the liquid crystal micro laser cavity and generating a modulation effect on WGM resonant wavelength drift caused by misfolded protein binding; and the spectrum detection unit is used for collecting a spectrum signal of the WGM resonance light drifted and modulated by the photoacoustic excitation unit.

Description

Technical Field

[0001] This invention relates to an optical whispering-gallery microcavity, a technique for detecting misfolded proteins using whispering-gallery mode and photo-ultrasound, and belongs to the fields of laser technology and biophotonics. Background Technology

[0002] Misfolded proteins are proteins that fail to form or maintain their normal three-dimensional spatial structure. They are a core pathological marker of many neurodegenerative diseases, such as Alzheimer's and Parkinson's. In the progression of these diseases, severe imbalances occur in protein homeostasis within neurons, leading to the abnormal aggregation of proteins with misfolded spatial conformations. These aggregates are not only cytotoxic themselves but can also spread between neurons, inducing a chain reaction of conformational changes in normal proteins. This process disrupts intracellular homeostasis, triggering oxidative stress and neuroinflammation, ultimately leading to progressive neuronal death and cognitive impairment. Currently, elucidating the mechanisms of misfolded protein aggregation and propagation, and exploring effective strategies for regulating protein homeostasis, has become a scientific challenge for developing early intervention and treatment methods.

[0003] Whispering Gallery Mode (WGM) is a special optical mode defined as a continuous total internal reflection that occurs when light propagates along the inner edge of a rotationally symmetric structure, confining the light to the edge of the resonant cavity. When the optical path travels through one revolution and is an integer multiple of the wavelength, interference enhancement occurs, i.e., resonance. This rotationally symmetric structure is called a whispering gallery mode optical microcavity. Whispering gallery mode microlaser cavities possess high quality factor, small mode volume, low excitation threshold, and narrow linewidth, thus showing broad application prospects in biological, chemical, and magnetic field detection fields. Summary of the Invention

[0004] Based on the above background, the present invention aims to propose a photoacoustic enhanced microcavity misfolded protein detection system and method, and the specific technical solution of the present invention is as follows: A photoacoustic enhanced microcavity misfolded protein detection system, characterized in that it comprises: The micro-laser cavity unit is a liquid crystal micro-laser cavity doped with quantum dot material with a fluorescence lifetime ≥10 ns. The liquid crystal micro-laser cavity is made with a cationic surfactant solution as the aqueous phase and a mixture of liquid crystal material and quantum dot material as the oil phase. Its surface is modified with aptamers for specifically capturing misfolded proteins and for generating WGM resonant light. A pulsed laser excitation unit is used to pump the micro-laser cavity unit with a first high-frequency pulsed laser to excite the quantum dot material to emit light and form WGM resonant light. The photoacoustic material unit is a photoacoustic thin film that can absorb the light emitted by the second high-frequency pulsed laser and is used to generate photo-induced ultrasound in response to photoacoustic excitation. The photoacoustic excitation unit is used to excite the photoacoustic material unit to generate controlled photo-induced ultrasound and act on the liquid crystal microlaser cavity, thereby modulating the WGM resonant wavelength drift caused by misfolded protein binding. The spectral detection unit is used to acquire the spectral signal of the WGM resonant light after it has been drifted and modulated by the photoacoustic excitation unit.

[0005] Furthermore, the photoacoustic excitation unit includes a second high-frequency pulsed laser, a second optical attenuator, and a reflector. The laser generated by the second high-frequency pulsed laser 7 has its intensity modulated by the second optical attenuator, and the reflector controls the direction of light propagation to irradiate the surface of the photoacoustic film, generating photoinduced ultrasound. The generated ultrasound propagates to the surface of the liquid crystal micro-laser cavity.

[0006] Furthermore, the laser generated by the second high-frequency pulsed laser is a 532 nm laser; the WGM resonant light, after drifting and modulation by the photoacoustic excitation unit, passes through a long-pass filter before entering the spectral detection unit, and the cutoff wavelength of the long-pass filter is 505 nm.

[0007] Furthermore, the photoacoustic film is a PDMS-rGO film formed by a two-step spin coating method.

[0008] Furthermore, the pulsed laser excitation unit includes the first high-frequency pulsed laser, the first optical attenuator, the dichroic mirror, and the objective lens. The laser emitted by the first high-frequency pulsed laser is modulated and attenuated by the first optical attenuator and modulated in direction by the dichroic mirror before being collimated and incident into the objective lens 3 to achieve beam focusing. The laser beam is then incident on the side of the liquid crystal micro-laser cavity to achieve fluorescence excitation of the quantum dot material and generate the WGM effect through the liquid crystal micro-laser cavity.

[0009] Furthermore, the laser emitted by the first high-frequency pulsed laser is a 473 nm laser; the dichroic mirror parameters are: cutoff wavelength 493 nm.

[0010] Furthermore, the doped quantum dot material with a fluorescence lifetime ≥10 ns is a CdSe / ZnS quantum material; the fabrication method of the liquid crystal microlaser cavity is as follows: CdSe / ZnS quantum material is added to the liquid crystal material to form a uniform quantum dot-doped liquid crystal; using microfluidic technology, poly-L-lysine PLL is used as the aqueous phase and quantum dot-doped liquid crystal is used as the oil phase to fabricate a liquid crystal microlaser cavity with PLL embedded on the surface.

[0011] Furthermore, the misfolded protein is Aβ, a biomarker for neurodegenerative diseases. 1-42The corresponding aptamer is the specific aptamer Aptamer 15-1; the method for modifying the surface of the liquid crystal microlaser cavity for the specific capture of misfolded proteins is as follows: Aptamer 15-1 is added to the liquid crystal microlaser cavity solution in which the surface is embedded in the PLL; bovine serum albumin (BSA) is added and allowed to stand to bind to sites on the surface of the liquid crystal microlaser cavity that have not reacted with the aptamer; unbound Aptamer 15-1 molecules and BSA molecules are removed by washing.

[0012] The present invention also provides a detection method using the aforementioned microcavity misfolded protein detection system, comprising the following steps: Place a fluorescently labeled misfolded protein solution on the surface of a glass slide; Add the fabricated liquid crystal microlaser cavity; Place the photoacoustic film at another location on the glass slide, at a distance of ≤1 cm from the fluorescently labeled misfolded protein solution; The laser emitted by the first high-frequency pulsed laser is modulated and attenuated by the first optical attenuator and its direction is modulated by the dichroic mirror. It is then collimated and incident into the objective lens to achieve beam focusing. The laser beam is then incident on the side of the liquid crystal micro-laser cavity to achieve fluorescence excitation of the quantum dot material and generate WGM resonant light through the liquid crystal micro-laser cavity. After the surface aptamer of the liquid crystal microlaser cavity binds to the misfolded protein, it leads to the recombination behavior of the ionic interfacial surfactant modified on the surface of the liquid crystal microlaser cavity, which in turn changes the liquid crystal orientation in the liquid crystal microlaser cavity and modulates the WGM resonant light. The laser generated by the second high-frequency pulsed laser is modulated in intensity by the second optical attenuator. The reflector controls the direction of light propagation and irradiates the surface of the photoacoustic material, exciting the photothermoelastic effect and generating photoinduced ultrasound. The generated ultrasound propagates to the surface of the liquid crystal micro-laser cavity and produces spectral drift under the action of ultrasound. The objective lens receives the signal light emitted by the liquid crystal microlaser cavity, and passes the light through a dichroic mirror and a long-pass filter to filter out noise from the incident light signal. The signal light then enters the imaging spectrometer.

[0013] Furthermore, a delay time is set for the imaging spectrometer. The delay time of the imaging spectrometer is set according to the fluorescence lifetime of the doped quantum dot material, so that the delay time is shorter than its fluorescence lifetime, in order to eliminate noise signals.

[0014] This invention achieves enhanced optical detection of misfolded proteins by constructing a detection system that synergistically combines signal generation and physical enhancement, and has the following technical effects: 1. Dynamic capture and signal generation Unlike traditional liquid crystal microlaser cavities that use short-lived fluorescent dyes (<5 ns), this invention utilizes quantum dot materials (which have at least a timescale difference of more than one time with short-lived fluorescent dyes) and combines them with a high-Q WGM microcavity to enhance the intrinsic time-resolved detection window, thereby improving the detection effect from a temporal perspective.

[0015] 2. Physical enhancement and signal amplification By utilizing the photothermoelastic effect to introduce non-contact, controllable photoinduced ultrasound, controlled transient perturbations are applied to the boundary conditions of the liquid crystal microlaser cavity. This specifically amplifies the resonant mode redistribution caused by energy transfer, thereby amplifying the microcavity resonant wavelength drift. The weak biochemical binding signal is transformed into an easily detectable and significantly enhanced optical wavelength displacement signal. Furthermore, the non-contact nature effectively avoids contamination and damage to misfolded proteins caused by close-range human manipulation. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the structure of a liquid crystal microlaser cavity; Figure 2 This is a schematic diagram of the experimental apparatus of the present invention; Figure 3 This is a schematic diagram of the spectral response of the WGM effect excited by a liquid crystal microlaser cavity; Figure 4 This is a schematic diagram showing the wavelength shift of the liquid crystal microlaser cavity when ultrasound is excited. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and the accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0018] Example 1 In this invention, a method for fabricating liquid crystal microlaser cavities, functionalizing them, modifying misfolded proteins with fluorescence, and creating photoacoustic thin films based on the fluorescence energy resonance transfer (FRET) effect is proposed. The FRET effect is only one specific implementation of misfolded protein sensing; it can also be achieved through other modulation methods.

[0019] The materials used in this invention include, but are not limited to: CdSe / ZnS quantum dot material; 5CB liquid crystal; InP / ZnSeS quantum dot dye; poly-L-lysine (PLL) as the cationic surfactant for modification; and Aβ, a neurodegenerative disease marker, as the misfolded protein to be tested. 1-42The corresponding specific aptamer is Aptamer 15-1; the photoacoustic thin film material is a mixture of polydimethylsiloxane-reduced graphene oxide (PDMS-rGO). The emission spectrum (excitation range: 510 nm~570 nm) of the CdSe / ZnS quantum dot material significantly overlaps with the absorption spectrum (absorption range: 550 nm~580 nm; excitation range: 650 nm~680 nm) of the InP / ZnSeS quantum dot material, satisfying the FRET effect conditions.

[0020] Fabrication of the liquid crystal microlaser cavity: 1.1 mg of CdSe / ZnS quantum material was added to 200 μL of 5 CB solution. After ultrasonic bath for 30 min, a uniform quantum dot-doped liquid crystal (QDs-LC) was formed. Using a microfluidic system, 1 ml of 0.01% poly-L-lysine (PLL) was used as the aqueous phase, and QDs-LC was used as the oil phase to fabricate a liquid crystal microlaser cavity with a diameter of 20 μm and PLL embedded in its surface. The structural schematic diagram of the fabricated liquid crystal microlaser cavity is shown below. Figure 1 As shown.

[0021] Surface functionalization of liquid crystal microlaser cavities: 1 mL of 200 µg / ml Aptamer 15-1 was added to the liquid crystal microlaser cavity solution for 1 h. Due to the positive charge of PLL, electrostatic interactions caused negatively charged Aptamer 15-1 molecules to adsorb onto the surface of the liquid crystal microlaser cavity. Subsequently, 10 mL of 1 mg / ml bovine serum albumin (BSA) was added to the liquid crystal microlaser cavity and allowed to stand for 30 min to fill the blank binding sites on the microcavity surface. The microcavity was then slowly washed with 1 mg / ml sodium dodecyl sulfate (SDS) solution to remove unbound Aptamer 15-1 and BSA molecules from the surrounding solution.

[0022] Preparation of fluorescently labeled misfolded proteins: Water-soluble InP / ZnSeS quantum dots with carboxyl groups modified on their surface were selected as fluorescent dyes, and the quantum dots were labeled with Aβ... 1-42 Both were placed together in physiological buffer solution at pH 7.2–7.4 and incubated with gentle shaking at 37 °C for 1 h. Due to Aβ... 1-42 The surface is rich in amino groups, and the carboxyl groups on the quantum dot surface can react with Aβ. 1-42 The amino groups on the surface form electrostatic interactions, achieving specific direct binding and completing fluorescent labeling. Subsequently, gel filtration chromatography is used to remove unbound free quantum dots, ultimately yielding specifically fluorescently labeled Aβ. 1-42 Store at 4 ℃ in the dark for later use.

[0023] PDMS-rGO film fabrication: First, PDMS main agent and curing agent were mixed at a mass ratio of 10:1, and 5.0% (by mass) of reduced graphene oxide (rGO) powder was added and initially stirred. Then, the mixture was stirred at 2000 rpm for 60 minutes using a centrifugal mixer to achieve uniform powder dispersion, and air bubbles were removed using a vacuum pump. Next, the mixed gel was added to a clean substrate and shaped using a two-step spin-coating method: first, spin-coating at 500 rpm for 15-20 seconds to allow the slurry to spread, and then spin-coating at 1500-2000 rpm for 60 seconds to control the thickness. Finally, the film was cured at 80 °C for 4 hours, cooled, and peeled off to obtain a PDMS-rGO film with a uniform thickness of 300 μm.

[0024] Example 2 In this invention, a method for detecting misfolded proteins based on the FRET effect is proposed.

[0025] Figure 2 The diagram shows a microcavity misfolded protein detection system and method based on photoacoustic enhancement. The system includes: a high-frequency pulsed laser 1, an optical attenuator 2, an objective lens 3, a dichroic mirror 4, a long-pass filter 5, an imaging spectrometer 6, a high-frequency pulsed laser 7, an optical attenuator 8, a reflector 9, and a computer 10. The parameters of the high-frequency pulsed laser 1 are: output wavelength 473 nm, pulse width 5 ns, and frequency 500 Hz; the parameters of the objective lens 3 are: magnification 20 X, numerical aperture (NA) 0.4; the parameters of the dichroic mirror 4 are: cutoff wavelength 493 nm; the parameters of the long-pass filter 5 are: cutoff wavelength 505 nm; and the parameters of the high-frequency pulsed laser 7 are: output wavelength 532 nm, pulse width 5 ns, and frequency 500 Hz.

[0026] A fluorescently labeled misfolded protein solution was placed on a glass slide, and the liquid crystal microlaser cavity solution prepared in the example was dropped into it. Then, a PDMS-rGO film was placed on the other side of the glass slide, at a distance ≤1 cm from the solution. The laser emitted by the high-frequency pulsed laser 1 was first modulated and attenuated by the optical attenuator 2 and oriented by the dichroic mirror 4, before being collimated and focused into the objective lens 3. The beam was then incident on the side of the liquid crystal microlaser cavity, achieving fluorescence excitation of the quantum dot material and generating the WGM effect through the liquid crystal microlaser cavity. The spectrum is as follows: Figure 3As shown, as the aptamers on the surface of the liquid crystal microlaser cavity continuously bind to the misfolded protein, the FRET effect is excited. CdSe / ZnS transfers some energy to the InP / ZnSeS bound to the misfolded protein via the FRET effect, causing it to emit fluorescence. Subsequently, the signal light emitted by the liquid crystal microlaser cavity is received by objective lens 3, and the light is filtered out by dichroic mirror 4 and long-pass filter 5 to remove noise from the incident light signal. The signal light then enters the imaging spectrometer 6. A delay time of 5 ns is set for the imaging spectrometer 6. By delaying the spectral acquisition time window, the long fluorescence lifetime of the material is utilized to suppress short-lifetime background fluorescence and scattering signals. Within this time window, the WGM resonant wavelength change induced by the binding of the misfolded protein can be resolved with a high signal-to-noise ratio to eliminate environmental noise and receive the signal light.

[0027] Simultaneously, the 532 nm laser generated by the high-frequency pulsed laser 7 is modulated in intensity by the optical attenuator 8, and the reflector 9 controls the direction of light propagation to perpendicularly irradiate the PDMS-rGO thin film surface, exciting the photothermoelastic effect and generating photoinduced ultrasound. The generated ultrasound propagates along the stage to the surface of the liquid crystal microlaser cavity, and the spectral shift caused by the ultrasound is as follows: Figure 4 As shown. Computer 10 is responsible for controlling the instruments and equipment in the system, including the switching on and off of the imaging spectrometer 6 and the setting of the delay time; setting the excitation time of the high-frequency pulsed laser 7 so that it is triggered approximately 2 μs earlier than the high-frequency pulsed laser 1, to ensure that the ultrasound can act for the entire duration of the FRET effect. Figure 4 This is a comparison of wavelength shift results when ultrasound is applied.

Claims

1. A photoacoustic enhanced microcavity misfolded protein detection system, characterized in that, include: The micro-laser cavity unit is a liquid crystal micro-laser cavity doped with quantum dot material with a fluorescence lifetime ≥10 ns. The liquid crystal micro-laser cavity is made with a cationic surfactant solution as the aqueous phase and a mixture of liquid crystal material and quantum dot material as the oil phase. Its surface is modified with aptamers for specifically capturing misfolded proteins and for generating WGM resonant light. A pulsed laser excitation unit is used to pump the micro-laser cavity unit with a first high-frequency pulsed laser to excite the quantum dot material to emit light and form WGM resonant light. The photoacoustic material unit is a photoacoustic thin film that can absorb the light emitted by the second high-frequency pulsed laser and is used to generate photo-induced ultrasound in response to photoacoustic excitation. The photoacoustic excitation unit is used to excite the photoacoustic material unit to generate controlled photo-induced ultrasound and act on the liquid crystal microlaser cavity, thereby modulating the WGM resonant wavelength drift caused by the binding of misfolded proteins. The spectral detection unit is used to acquire the spectral signal of the WGM resonant light after it has been drifted and modulated by the photoacoustic excitation unit.

2. The photoacoustic-enhanced microcavity misfolded protein detection system according to claim 1, characterized in that, The photoacoustic excitation unit includes a second high-frequency pulsed laser, a second optical attenuator, and a reflector. The laser generated by the second high-frequency pulsed laser 7 has its intensity modulated by the second optical attenuator, and the reflector controls the direction of light propagation to irradiate the surface of the photoacoustic film, generating photoinduced ultrasound. The generated ultrasound propagates to the surface of the liquid crystal micro-laser cavity.

3. The photoacoustic enhanced microcavity misfolded protein detection system according to claim 2, characterized in that, The laser generated by the second high-frequency pulsed laser is a 532 nm laser; the WGM resonant light, after drifting and modulation by the photoacoustic excitation unit, passes through a long-pass filter before entering the spectral detection unit, with a cutoff wavelength of 505 nm.

4. The photoacoustic-enhanced microcavity misfolded protein detection system according to claim 1, characterized in that, The photoacoustic film is a PDMS-rGO film formed by a two-step spin coating method.

5. The photoacoustic-enhanced microcavity misfolded protein detection system according to claim 1, characterized in that, The pulsed laser excitation unit includes a first high-frequency pulsed laser, a first optical attenuator, a dichroic mirror, and an objective lens. The laser emitted by the first high-frequency pulsed laser is modulated and attenuated by the first optical attenuator and modulated in direction by the dichroic mirror before being collimated and incident into the objective lens 3 to achieve beam focusing. The laser beam is then incident on the side of the liquid crystal micro-laser cavity to achieve fluorescence excitation of the quantum dot material and generate the WGM effect through the liquid crystal micro-laser cavity.

6. The photoacoustic-enhanced microcavity misfolded protein detection system according to claim 5, characterized in that, The laser emitted by the first high-frequency pulsed laser is 473 nm; the dichroic mirror parameters are: cutoff wavelength 493 nm.

7. The photoacoustic enhanced microcavity misfolded protein detection system according to claim 1, characterized in that, The doped quantum dot material with a fluorescence lifetime ≥10 ns is a CdSe / ZnS quantum material; the fabrication method of the liquid crystal microlaser cavity is as follows: CdSe / ZnS quantum material is added into the liquid crystal material to form a uniform quantum dot-doped liquid crystal; using microfluidic technology, poly-L-lysine PLL is used as the aqueous phase and quantum dot-doped liquid crystal is used as the oil phase to fabricate a liquid crystal microlaser cavity with PLL embedded on the surface.

8. The photoacoustic-enhanced microcavity misfolded protein detection system according to claim 1, characterized in that, The misfolded protein is Aβ, a biomarker for neurodegenerative diseases. 1-42 The corresponding aptamer is the specific aptamer Aptamer 15-1; the method for modifying the surface of the liquid crystal microlaser cavity for the specific capture of misfolded proteins is as follows: Aptamer 15-1 is added to the liquid crystal microlaser cavity solution in which the surface is embedded in the PLL; bovine serum albumin (BSA) is added and allowed to stand to bind to sites on the surface of the liquid crystal microlaser cavity that have not reacted with the aptamer; unbound Aptamer 15-1 molecules and BSA molecules are removed by washing.

9. A detection method using the microcavity misfolded protein detection system according to any one of claims 1-8, characterized in that, Includes the following steps: Place a fluorescently labeled misfolded protein solution on the surface of a glass slide; Add the fabricated liquid crystal microlaser cavity; Place the photoacoustic film at another location on the glass slide, at a distance of ≤1cm from the fluorescently labeled misfolded protein solution; The laser emitted by the first high-frequency pulsed laser is modulated and attenuated by the first optical attenuator and its direction is modulated by the dichroic mirror. It is then collimated and incident into the objective lens to achieve beam focusing. The laser beam is then incident on the side of the liquid crystal micro-laser cavity to achieve fluorescence excitation of the quantum dot material and generate WGM resonant light through the liquid crystal micro-laser cavity. After the surface aptamer of the liquid crystal microlaser cavity binds to the misfolded protein, it leads to the recombination behavior of the ionic interfacial surfactant modified on the surface of the liquid crystal microlaser cavity, which in turn changes the liquid crystal orientation in the liquid crystal microlaser cavity and modulates the WGM resonant light. The laser generated by the second high-frequency pulsed laser is modulated in intensity by the second optical attenuator. The reflector controls the direction of light propagation and irradiates the surface of the photoacoustic material, exciting the photothermoelastic effect and generating photoinduced ultrasound. The generated ultrasound propagates to the surface of the liquid crystal micro-laser cavity and produces spectral drift under the action of ultrasound. The objective lens receives the signal light emitted by the liquid crystal microlaser cavity, and passes the light through a dichroic mirror and a long-pass filter to filter out noise from the incident light signal. The signal light then enters the imaging spectrometer.

10. The detection method according to claim 9, characterized in that, A delay time is set for the imaging spectrometer. The delay time of the imaging spectrometer is set according to the fluorescence lifetime of the doped quantum dot material, so that the delay time is shorter than its fluorescence lifetime, in order to eliminate noise signals.

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