Patent Application
20240319101
Current sensors suffer from numerous limitations, such as in the multiplexed sensing of different analytes and environmental conditions. Various embodiments of the present disclosure seek to address these limitations.
In some embodiments, the present disclosure pertains to a composition that includes: silica nanofibers; and one or more detection particles associated with the silica nanofibers. In some embodiments, at least one of the one or more detection particles are operational to exhibit a change in a property upon interaction with one or more analytes, upon detection of one or more environmental conditions, or combinations thereof. In some embodiments, the change in property includes, without limitation, a change in optical intensity, a change in emission wavelength peak, a change in emission wavelength phase, fluorescence resonance energy transfer (FRET), a shift in localized surface plasmonic resonances (LSPR), and combinations thereof.
In some embodiments, the compositions of the present disclosure are a component of a sensor. In some embodiments, the sensor includes: a surface associated with the silica nanofibers; a light source positioned near the silica nanofibers and operational to transmit light to the detection particles associated with the silica nanofibers; and a measuring device operational to measure light emitted from the detection particles associated with the silica nanofibers. In some embodiments, the sensor also includes an optical fiber that is connected to the measuring device and positioned near the silica nanofibers.
Further embodiments of the present disclosure pertain to methods of sensing one or more analytes and/or one or more environmental conditions from a sample. In some embodiments, the methods of the present disclosure include the following steps: associating the sample with a composition of the present disclosure, where the composition includes silica nanofibers and one or more detection particles associated with the silica nanofibers; detecting a change in a property of the detection particles; and correlating the change in the property of the detection particles to a presence or absence of one or more analytes, one or more environmental conditions, or combinations thereof.
Additional embodiments of the present disclosure pertain to methods of making the compositions of the present disclosure. In some embodiments, the methods of the present disclosure include: growing silica nanofibers from at least one precursor material; and associating one or more detection particles with the silica nanofibers.
It is to be understood that both the foregoing general description and the following detailed description are illustrative and explanatory, and are not restrictive of the subject matter, as claimed. In this application, the use of the singular includes the plural, the word “a” or “an” means “at least one”, and the use of “or” means “and/or”, unless specifically stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements or components comprising one unit and elements or components that include more than one unit unless specifically stated otherwise.
The section headings used herein are for organizational purposes and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.
Current sensors, such as those utilized in localized surface plasmon resonance (LSPR) and fluorescence, suffer from numerous disadvantages. For instance, real-time pH measurements are of great importance in chemical reactions, environmental monitoring, and physiological processes. Measuring pH values, especially for that in small volumes, is difficult but necessary for a variety of scientific applications.
In particular, it is well known that the local microenvironment pH has great influence on immunization, cell metabolism, and enzyme activity. Commercially available glass electrode pH sensors are widely used in laboratories for pH measurements. However, the glass electrode pH sensors have the disadvantages for limited accessibility of low volume detection, show slow drift, and are unsuitable for long-term pH monitoring. Furthermore, the glass-electrode-based pH probes have an inherent measurement error on the order of 0.1 pH unit, which limits their uses in precision measurements.
Optical pH sensors have proved to be an ideal alternative because of its advantages including rapid response, low sample volume, and the possibility of contactless measurement. Many optical transducers such as organic molecules, have been widely used to monitor local pH values. These organic molecules' fluorescence intensity changes when the environmental pH varies. Examples of such organic molecules include fluorophores like Fluorescein, Rhodamine, Oregon Green 488, and Coumarin. However, the accuracy of the pH measurement based on the single fluorescence emission intensity change are susceptible to both extrinsic and intrinsic factors, such as the stability of the excitation light, the concentration of the organic molecules, and the photobleaching of the organic molecules.
Therefore, efforts have been made to enhance the repeatability of the fluorescence pH sensors using ratiometric detections, which can eliminate or alleviate the environmental factors' influence on the accuracy of the measurements. The ratiometric sensors have fluorescence emissions with peaks of two different wavelengths. These emission peaks can be from different dyes or from the same dye that has two different emission wavelengths. To achieve ratiometric measurements, one of the fluorescence emission peak intensities should be sensitive to the measurand, while the other emission peak intensity should be insensitive to the measurand and be used as the reference signal.
By normalizing the measurand-sensitive peak intensity change with the measurand-insensitive peak intensity, the ratiometric measurements provide self-calibrated results with much better reliability. Specifically for pH sensing, the combination of the pH responsive dye and the reference dye can be carefully designed so that they can be excited by a single light source and the emission peak wavelengths are separated as far as possible to avoid signal distortions.
Silica is an intrinsically non-toxic, environmentally friendly material possessing great mechanical strength. Silica is widely used in sensing applications due to the solid encapsulating molecules by virtue of the rigidity of the inorganic cage. The use of silica-based material for pH probes embedding and immobilization has been demonstrated. In most studies, pH-probes are embedded in silica nanoparticles due to the unique properties, such as large specific surface area and thermal stability. However, most of these nanosensors are used for imaging or intracellular pH monitoring.
As such, a need exists for more effective systems and methods for sensing analytes and environmental conditions. Various embodiments of the present disclosure address the aforementioned need.
In some embodiments, the present disclosure pertains to a composition that includes silica nanofibers and one or more detection particles associated with the silica nanofibers. In some embodiments illustrated in
The compositions of the present disclosure can include various types of silica nanofibers. For instance, in some embodiments, the silica nanofibers of the present disclosure are in the form of a matrix. In some embodiments, the silica nanofibers are in the form of an extracellular matrix (ECM). In some embodiments, the ECM is a multifunctional ECM. In some embodiments, the silica nanofibers of the present disclosure are composed of silicon dioxide. In some embodiments, the silica nanofibers of the present disclosure have a nanoscale structure.
The silica nanofibers of the present disclosure can also include various sizes and geometries. For instance, in some embodiments, the silica nanofibers of the present disclosure include a length of at least about 500 nm and a diameter of at least about 50 nm. In some embodiments, the silica nanofibers of the present disclosure include a length of at least about 1 m and a diameter of at least about 100 nm. In some embodiments, the silica nanofibers of the present disclosure include a length of at least about 1 m and a diameter of at least about 400 nm. In some embodiments, the silica nanofibers of the present disclosure include a length of at least about 1 m and a diameter of about 410 nm.
In some embodiments, the silica nanofibers of the present disclosure have a length to width aspect ratio of at least about 10. In some embodiments, the silica nanofibers of the present disclosure have a length to width aspect ratio of at least about 20. In some embodiments, the silica nanofibers of the present disclosure have a length to width aspect ratio of at least about 30. In some embodiments, the silica nanofibers of the present disclosure have a length to width aspect ratio ranging from about 10 to about 30. In some embodiments, the silica nanofibers of the present disclosure include a hollow cavity.
Detection particles generally refer to particles that are operational to exhibit a change in a property upon interaction with one or more analytes, upon detection of one or more environmental conditions, or combinations thereof. In some embodiments, the change in property includes, without limitation, a change in optical intensity, a change in emission wavelength peak, a change in emission wavelength phase, fluorescence resonance energy transfer (FRET), a shift in localized surface plasmonic resonances (LSPR), and combinations thereof.
The detection particles of the present disclosure may be associated with silica nanofibers in various manners. For instance, in some embodiments, the detection particles are on an outer surface of the silica nanofibers (e.g., detection particles 14 illustrated in
The silica nanofibers of the present disclosure may be associated with various types of detection particles. For instance, in some embodiments, the detection particles include, without limitation, chromophores, fluorophores, plasmonic nanoparticles, Ru(2,2′-bipyridine)3 (Ru(bpy)3), fluorescein isothiocyanate (FITC), quantum dots, and combinations thereof. In some embodiments, the detection particles include FITC. In some embodiments, the detection particles include Ru(bpy)3. In some embodiments, the detection particles include FITC and Ru(bpy)3.
In some embodiments, the detection particles include one or more plasmonic nanoparticles. In some embodiments, the plasmonic nanoparticles can include, without limitation, metal nanoparticles, magnetic nanoparticles, functionalized nanoparticles, functionalized magnetic nanoparticles, and combinations thereof.
In some embodiments, the detection particles include a plurality of plasmonic nanoparticles. In some embodiments, the plurality of plasmonic nanoparticles include plasmonic nanoparticles with different sizes. In some embodiments, the plasmonic nanoparticles are gold nanoparticles. In some embodiments, the plasmonic nanoparticles are arranged on the silica nanofibers in such a manner that the arranged plasmonic nanoparticles can enhance the electromagnetic field of the incident light at the vicinity of the surface of the plasmonic nanoparticles.
In some embodiments, the detection particles of the present disclosure may also include one or more analyte binding agents. In some embodiments, the analyte binding agent can include, without limitation, antibodies, aptamers, nucleic acids, small molecules, and combinations thereof.
The detection particles of the present disclosure may be associated with the silica nanofibers in various manners. For instance, in some embodiments, the detection particles of the present disclosure are non-randomly arranged on the silica nanofibers. In some embodiments, the detection particles are patterned on the silica nanofibers. In some embodiments, the detection particles are separated from one another at predetermined distances. In some embodiments, the predetermined distances have a spatial resolution as high as 6 nm. In some embodiments, the detection particles are within a hollow cavity of the silica nanofibers.
In some embodiments, the detection particles include a first particle and a second particle. In some embodiments, the first particle and the second particle exhibit different changes in a property upon interaction with one or more analytes, upon detection of one or more environmental conditions, or combinations thereof. In some embodiments, the first particle and the second particle are associated with the silica nanofibers in such a manner that the first particle is able to transfer energy to the second particle when the first particle is in an electronic excited state.
In some embodiments, the first particle is on an outer surface of the silica nanofibers, and the second particle is within a hollow cavity of the silica nanofibers. In some embodiments, the first particle includes fluorescein isothiocyanate (FITC) and the second particle includes Ru(2,2′-bipyridine)3 (Ru(bpy)3).
The compositions of the present disclosure may be incorporated as components of various devices. For instance, in some embodiments, the compositions of the present disclosure are a component of a sensor. As such, in some embodiments, the present disclosure pertains to sensors that include the compositions of the present disclosure.
In some embodiments, the sensors of the present disclosure include a surface that is associated with the silica nanofibers of the present disclosure, a light source positioned near the silica nanofibers and operational to transmit light to the one or more detection particles associated with the silica nanofibers, and a measuring device operational to measure light emitted from the detection particles associated with the silica nanofibers.
In some embodiments, the light source includes a laser. In some embodiments, the laser includes, without limitation, a gas laser, a solid-state laser, a fiber laser, a liquid laser, a semiconductor laser, and combinations thereof.
In some embodiments, the measuring device measures an emission wavelength peak from the light emitted from the detection particles associated with the silica nanofibers. In some embodiments, the measuring device includes a spectrometer.
In some embodiments, the sensors of the present disclosure also include an optical fiber. In some embodiments, the optical fiber is connected to the measuring device and positioned near the silica nanofibers.
The sensors of the present disclosure can include various types of surfaces. For instance, in some embodiments, the surfaces can include polymers. In some embodiments, the polymers are plasma treated. In some embodiments, the polymers include polystyrene. In some embodiments, the polystyrene is a plasma-treated polystyrene. In some embodiments, plasma treatment of a polymer surface modifies the surface, thereby resulting in a specifically targeted behavior and hydrophilicity, which allows the silica nanofibers to be bonded to the polymer (e.g., polystyrene) surface through hydrogen bonds.
In some embodiments illustrated in
Sensing Analytes and/or Environmental Conditions
Additional embodiments of the present disclosure pertain to methods of sensing one or more analytes and/or one or more environmental conditions from a sample by utilizing the compositions and sensors of the present disclosure. In some embodiments illustrated in
The methods of the present disclosure can sense various analytes and environmental conditions from various types of samples. For instance, in some embodiments, the sample can include, without limitation, a liquid, a gas, and combinations thereof. In some embodiments, the sample includes a liquid. In some embodiments, the sample includes an actual environment.
Associating a Sample with a Composition
Various methods may also be utilized to associate a sample with the compositions of the present disclosure. For instance, in some embodiments, the associating includes flowing the sample through the composition. In some embodiments, the associating includes incubating the sample with the composition. In some embodiments, the associating includes exposing the sample to the composition.
Various changes in properties of the detection particles of the present disclosure can occur to identify the presence or absence of one or more analytes, one or more environmental conditions, or combinations thereof. For instance, in some embodiments, the detected change in the property of the one or more detection particles includes, without limitation, a change in optical intensity, a change in emission wavelength peak, a change in emission wavelength phase, fluorescence resonance energy transfer (FRET), a shift in localized surface plasmonic resonances (LSPR), and combinations thereof.
Various methods may be utilized to detect a change in a property of the one or more detection particles. For instance, in some embodiments, the detecting occurs by a method that includes, without limitation, visualization, microscopy, dark field microscopy, spectrometry, spectroscopy, colorimetric analysis, localized surface plasmon resonance (LSPR), nuclear magnetic resonance (NMR), surface plasmon resonance, electrochemistry, and combinations thereof. In some embodiments, the detecting includes visualizing a color or image change of the detection particles.
In some embodiments, the detecting includes: exposing the composition to a light source, where the exposing transmits light to the detection particles associated with the silica nanofibers, and utilizing a measuring device to measure light emitted from the detection particles associated with the silica nanofibers. In some embodiments, the measured light emitted includes a measured emission wavelength peak.
Various methods may also be utilized to correlate the change in the property of the detection particles to a presence or absence of one or more analytes, one or more environmental conditions, or combinations thereof. For instance, in some embodiments, the correlating includes comparing the change in the property of the particles with known properties in a database.
In some embodiments, the correlating includes correlating the change in the property of the detection particles to the presence of a concentration of the one or more analytes, the one or more environmental conditions, or combinations thereof. As such, in some embodiments, the methods of the present disclosure may be utilized to quantify the presence of one or more analytes, one or more environmental conditions, or combinations thereof.
In some embodiments, the correlating includes utilization of a machine-learning algorithm. In some embodiments, the correlating includes comparing the change in the property of the one or more detection particles with results from other existing detection particles.
In some embodiments, the detection particles of the silica nanofibers include a first particle and a second particle. In some embodiments, the first particle and the second particle exhibit different changes in a property upon interaction with the one or more analytes, upon detection of the one or more environmental conditions, or combinations thereof. In some embodiments, the changes in the property include changes in emission wavelength peaks of the first particle and the second particle. In some embodiments, the ratio of the change in the emission wavelength peak of the first particle relative to the change in the emission wavelength peak of the second particle is utilized to correlate the change in the property of the detection particles to the presence or absence of the one or more analytes, the one or more environmental conditions, or combinations thereof.
In some embodiments, the first particle and the second particle are associated with the silica nanofibers in such a manner that the first particle is able to transfer energy to the second particle when the first particle is in an electronic excited state. In some embodiments, the transfer of energy from the first particle to the second particle is utilized to correlate the change in the property of the detection particles to the presence or absence of the one or more analytes, the one or more environmental conditions, or combinations thereof.
In some embodiments, the first particle is on an outer surface of the silica nanofibers. In some embodiments, the second particle is within a hollow cavity of the silica nanofibers.
The methods of the present disclosure may be utilized to sense various analytes and environmental conditions. In some embodiments, the methods of the present disclosure are utilized to sense one or more analytes from a sample. In some embodiments, the methods of the present disclosure are utilized to sense one or more environmental conditions from a sample. In some embodiments, the methods of the present disclosure are utilized to sense one or more analytes and one or more environmental conditions from a sample.
In some embodiments, the one or more analytes include, without limitation, environmental analytes, pollutants, biomolecules, cellular analytes, nucleic acids, DNA, single-stranded DNAs, double-stranded DNAs, RNAs, messenger RNAs (mRNA), proteins, antibodies, hormones, enzymes, antigens, cells, and combinations thereof. In some embodiments, the one or more analytes include a plurality of analytes.
In some embodiments, the one or more environmental conditions include, without limitation, cellular metabolism level, pH, temperature, cellular force, force, changes in biochemical environment near cells in the sample, and combinations thereof. In some embodiments, the one or more environmental conditions include pH of the environment. In some embodiments, the one or more environmental conditions include a plurality of environmental conditions.
The methods of the present disclosure can detect or monitor a sample for various analytes and environmental conditions in various manners. For instance, in some embodiments, the detecting and/or correlating steps are conducted in real-time. In some embodiments, the detecting and/or correlating steps include continuous monitoring of a sample.
In some embodiments, the methods of the present disclosure can be repeated with various samples. In some embodiments, the methods of the present disclosure can be continually repeated. In some embodiments, the methods of the present disclosure can provide for real-time monitoring of the one or more analytes, one or more environmental conditions, or combinations thereof.
In some embodiments, the sensors of the present disclosure may be utilized to sense various analytes and environmental conditions in accordance with the method of the present disclosure. For instance, in some embodiments, sensor 20 shown in
As a result, detection particles 14 and 16 emit lights 22 and 24. The emitted lights are transmitted by tip 28 of optical fiber 26 to measuring device 38 after filtration through emission filter 40. Thereafter, measuring device 38 detects the emitted lights. The emitted lights may then be correlated to the presence or absence of one or more analytes, one or more environmental conditions, or combinations thereof.
Additional embodiments of the present disclosure pertain to methods of making the compositions of the present disclosure. In some embodiments illustrated in
The methods of the present disclosure may utilize various steps to grow silica nanofibers. For instance, in some embodiments, the growth occurs by hydrolysis of the at least one precursor material. In some embodiments, the at least one precursor material includes tetraethyl orthosilicate (TEOS).
Association of Detection Particles with Silica Nanofibers
Detection particles may be associated with silica nanofibers at various stage of growth. For instance, in some embodiments, the associating occurs during the growing of the silica nanofibers. In some embodiments, the associating occurs after the growing of the silica nanofibers. In some embodiments, the associating occurs during and after the growing of the silica nanofibers.
In some embodiments, the associating occurs by a method that includes, without limitation, coating, doping, covalent coupling, or combinations thereof. In some embodiments, the associating occurs by covalent coupling. In some embodiments, the covalent coupling includes the utilization of an alkyl alkoxysilane that couples the detection particles to the silica nanofibers.
The methods of the present disclosure can utilize various alkyl alkoxysilanes for coupling detection particles to silica nanofibers. For instance, in some embodiments, the alkyl alkoxysilane includes at least one amino group and at least one silanol group. In some embodiments, the amino group covalently bonds to the one or more detection particles through amide bonds while the silanol group couples to the silica nanofibers through a co-condensation reaction. In some embodiments, the alkyl alkoxysilane includes 3-aminopropyl)triethoxysilane (APTES).
Various detection particles may be associated with silica nanofibers. Suitable detection particles were described previously in this patent application. In some embodiments, the detection particles include a first particle and a second particle. In some embodiments, the first particle and the second particle exhibit different changes in a property upon interaction with one or more analytes, upon detection of one or more environmental conditions, or combinations thereof.
In some embodiments, the first particle becomes associated on an outer surface of the silica nanofibers. In some embodiments, the second particle becomes associated within a hollow cavity of the silica nanofibers. In some embodiments, the first particle becomes associated on an outer surface of the silica nanofibers after the growing step. In some embodiments, the second particle becomes associated within a hollow cavity of the silica nanofibers during the growing step. In some embodiments, the first particle includes fluorescein isothiocyanate (FITC) and the second particle includes Ru(2,2′-bipyridine)3 (Ru(bpy)3).
The present disclosure can have various advantages. For instance, in some embodiments, the compositions and sensors of the present disclosure can allow real-time and continuous monitoring of various analytes and environmental conditions. In some embodiments, the compositions and sensors of the present disclosure have multiplex sensing capability, thereby allowing them to detect different environmental conditions and analytes simultaneously.
As such, the compositions and sensors of the present disclosure can have numerous applications. For instance, in some embodiments, the compositions and sensors of the present disclosure can be utilized for real-time, continuous monitoring of various analytes and pH values of various environments.
Reference will now be made to more specific embodiments of the present disclosure and experimental results that provide support for such embodiments. However, Applicants note that the disclosure below is for illustrative purposes only and is not intended to limit the scope of the claimed subject matter in any way.
In this Example, applicant reports a sensitive and robust pH sensor based on dual fluorescence doped hollow silica nanofibers (hSNFs) for long-term pH monitoring. Fluorescein isothiocyanate (FITC) and Tris(2,2-bipyridyl)dichlororuthenium(II) hexahydrate (Ru(BPY)3) were chosen as pH sensitive dye and reference dye, respectively. Two-step synthesized hSNFs from a reverse micelle system had an average length of 6.20 μm and an average diameter of 410 nm. The intensity ratio of FITC/Ru(BPY)3 is used to calibrate with pH changes. Applicant has also developed an optical-fiber-based fluorescent detection system that enables feasible and highly efficient near-field fluorescent detection. The system has a fully automated fluorescent detection procedure, where components including the light source, detector, and data acquisition are all controlled by the computer. The results show that pH sensor works in a linear range of pH 4.6-pH 8.5 with a fast response time of less than 10 seconds. In addition, the as-prepared hSNFs-based pH sensors also have optimal long-term durability.
Fluorescein isothiocyanate (FITC), Tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate (Ru(BPY)3), 3-aminopropyl triethyloxylsilane (APTES), 1-Pentanol, tetraethyl orthosilicate (TEOS), trisodium citrate dihydrate, polyvinylpyrrolidone (PVP, 40 kDa), ammonium hydroxide (25 wt % aq.) and ethanol (200-proof) were purchased from Sigma-Aldrich. Water used was from a Milli-Q water ultrapure water purification system. All chemicals were used as received without any further purification.
A handheld high frequency generator (Electro-Technic Products, BD-20) was used for plasma treatment of the polymer surface. Transmission electron microscopy (TEM) was performed on a Tecnai F20ST field emission gun (FEG) transmission electron microscope operating at an accelerating voltage of 200 kV. Scanning electron microscopy (SEM) was performed on the Thermo Scientific Helios 5cx Dual Beam scanning electron microscope. The energy-dispersive X-ray spectroscopy (EDS) measurements were performed with the spectrometer attached on the Tecnai F20ST FEG electron microscope. Fluorescent images were taken by Olympus BX51 microscope.
The Ru(BPY)3 doped hSNFs were prepared following reported methods with modifications. In a typical reaction, 5 mL of 1-pentanal and PVP solution were first added in a 15 mL glass scintillation vial and mixed at 50° C. for 30 minutes. Then, 475 mL of ethanol, 100 mL of DI water and 40 mL of Ru(BPY)3 solution (30 mM in water) were added to the mixed solution, and the mixture was vortexed for 1 min. Next 50 μL of 0.18 M aqueous trisodium citrate dihydrate was injected and vortexed for 1 min, followed by the addition of 100 μL of ammonia hydroxide (25 wt % aq.) with vortex mixing for 1 min. Lastly, 50 μL of TEOS was injected and then immediately vortexed for 20 s. The mixtures reacted without stirring at 70° C. overnight for 12 h. After the reaction, the solution was poured into a 15 mL centrifuge tube, and 1 mL of acetone was added to break the microemulsion. hSNFs were isolated by centrifuging (1500 rpm, 15 min), and washed with water and ethanol (1000 rpm, 5 min) for 3 times (ultrasonic cleaning, 5 min each time) to remove the surface impurities.
FITC was coated on the surfaces of hSNFs using a fluorescence coating method. 20 mg Ru(BPY)3 doped hSNFs fabricated in the last step were dispersed in 2 mL ethanol in a 15 mL centrifuge tube. Next, 100 μL of ammonia hydroxide (25 wt % aq.), 100 μL of DI water and 20 μL of TEOS were added sequentially and vortexed for 20 s. 10 μL of FITC solution (15 mg FITC and 150 μL APTES reacted for 24 h in 1 mL of ethanol at room temperature) was then added. The above mixture was left without stirring at 40° C. for 8 h. The hSNFs doped with both FITC and Ru(BPY)3 were isolated by centrifuging (700 rpm, 15 min), and washed 2 times with DI water (ultrasonic cleaning, 5 min each time) and 2 times with ethanol (ultrasonic cleaning, 5 min each time).
20 mg of the fabricated dual fluorescence hSNFs were dispersed in 2 mL ethanol. Petri dish (Ø60×H15 mm, polystyrene) was first plasma treated using the handheld high frequency generator for 1 min to induce a hydrophilic bottom surface by adding hydroxyl groups into hydrocarbon chains. Right after the plasma treatment, 50 μL of the dual fluorescence hSNFs solution were dispensed onto the center of petri dish, followed by spin-coating process (200 rpm for 5 s, and 400 rpm for 15 s).
To easily read out the fluorescent spectrum of the hSNFs, Applicant has developed a customized fluorescent detection system based on optical fibers, as shown in
The emission filter blocked light lower than 500 nm, which includes the 488 nm excitation laser. This prevented the spectrometer to be overloaded by the strong scattered excitation light and the distortion of the fluorescent spectrum. Compared to fluorescent pH sensors based on microscope imaging, in which the fluorescent signal is read out in the form of pixel intensities, Applicant's fiber-based system can read out the whole spectrum of the fluorescent signal in real time. As will be detailed in this Example, real-time pH measurements are based on this acquired fluorescent spectrum.
The synthesis of the dual fluorescence hSNFs is illustrated in
In Applicant's experiments, hydrolyzed TEOS molecules (silica oligomers) formed on the droplet surface at high ammonia content. They were then deposited immediately as solid silica before reaching the droplet center, so that hSNFs were synthesized instead of solid silica nanorods.
The synthesis process can be summarized as follows: Firstly, Ru(BPY)3 was dissolved in the water droplets in microemulsion and encapsulated inside the hollow center of hSNFs. Then, FITC-APTES conjugate was grafted onto silica matrix while additional TEOS was added to grow a thin silica film simultaneously. The hollow structure allows having most Ru(BPY)3 inside SNFs, which prevented its interaction from the environment and made it a stable reference signal in the ratiometric measurement. Coating FITC outside allows it to respond to environmental pH variation.
The average length of hSNFs before coating FITC was (6.20±1.59) μm and the average diameter was (238±51) nm. By adjusting reaction parameters (i.e., concentration of chemical reagents, temperature, and reaction time), Applicant achieved a relative long aspect ratio of 26.5.
The shell thickness of hSNFs was about 41 nm (
To further confirm that FITC and Ru(BPY)3 fluorophores have been successfully doped to the hSNFs, and to identify the fluorescent peak wavelengths of the dual fluorescence hSNFs, Applicant conducted four emission spectrum analysis using the microplate reader (BioTek).
Applicant notes that the peak fluorescent wavelengths of both FITC and Ru(BPY)3 have shifted after being coated to the SNFs. These shifts were due to the coupling of the transition dipole moment of the fluorophores with the silica nanofibers and the change of the fluorophores' molecular conformation. Emission spectrum of the dual fluorescence hSNFs was used to identify the fluorescence peaks of FITC and Ru(BPY)3, as shown in
Applicant has experimentally characterized the pH responses of the dual fluorescence hSNFs using the optical characterization setup shown in
In the experiments, the environmental pH of the dual fluorescence hSNFs coated petri dish was changed from alkaline (pH=7.76) to acidy (pH=5.31) by adding 0.01M HCl solution to a diluted NaOH solution. Fluorescent spectra of the dual fluorescence hSNFs at seven different pH values were characterized. At each pH value, 10 spectra were recorded with an exposure time of 0.5 seconds. The averaged spectra at each pH value were plotted in
Since such photobleaching also acted on the FITC, Applicant used a ratiometric characterization method to decouple the pH-induced and photobleaching-induced FITC fluorescent intensity decrease. In this method, the environmental pH change was characterized by the ratio of FITC over Ru(BPY)3 intensity.
A linear regression model was used to characterize the relationship between the IFITC/IRu(BPY)3 ratio and the pH values. The fitting results show that the IFITC/IRu(BPY)3 ratio has a linear relationship with the pH value and a pH sensitivity of 0.34 [pH−1]. The strength of the linear fitting was characterized using the R2 value, which was calculated to be 0.95. It is noted that for pH in the range of 5.5 to 7.5, the IFITC/IRu(BPY)3 ratio had a much higher linearity of R2>0.99 with respect to the pH values.
In many pH sensing applications, it is desirable that the sensor has good reversibility. Here, Applicant has experimentally characterized the dual fluorescence hSNFs' response to fluctuating pH values. In the experiment, the hSNFs were first deposited onto a petri dish using the method discussed above. Then 0.01M HCl was added into the petri dish to achieve the initial acidic environment of pH=3.44. The pH value of the solution was then abruptly changed between 3.44 and 10.88 in cycles by adding the concentrated HCl and NaOH solution intermittently, while the fluorescent light emitted by the hSNFs was recorded every 10 seconds with an exposure time of 0.5 seconds, as shown in
The spectra in
To differentiate the spectra measured at different pH states, and to differentiate the overlapping spectra at the same pH state, different colors were assigned to the spectra in
To better characterize the pH responsiveness and reversibility of the dual fluorescence hSNFs, the IFITC/IRu(BPY)3 ratio variations in
This IFITC/IRu(BPY)3 ratio plot showed that during the acidic-alkaline cycles, the IFITC/IRu(BPY)3 ratio of the hSNFs maintained a highly reversible response even when the environmental pH was abruptly fluctuating. The fluctuation of the IFITC/IRu(BPY)3 ratio in
In all the cases, the response times were less than 30 seconds, and in some cases, the response times were less than 10 seconds. As mentioned above, the ratiometric characterization method based on the FITC and Ru(BPY)3 fluorescent intensity ratio can effectively decouple the pH induced FITC fluorescent intensity variation from that caused by the photobleaching. Such decoupling is particularly beneficial in long-term measurements, where the accumulated exposure time is often long enough to induce photobleaching of the fluorophores.
Here, Applicant experimentally demonstrated the effectiveness of the ratiometric characterization method in long-term pH sensing applications. In the experiment, dual fluorescence hSNFs coated petri dish was filled with pH=7 buffer. The fiber-based fluorescent detection system was used to read out the fluorescent spectrum every 20 minutes for over more than 6 hours. As shown by the recorded spectra in
However, as shown in
In summary, Applicant demonstrated in this Example a novel ratiometric fluorescence pH sensor based on hSNFs doped with FITC and Ru(BPY)3 fluorophores. Using water-in-oil droplets created in microemulsion system, the dual fluorophore doped hSNFs with emission peaks at around 515 and 595 nm were synthesized in a straightforward fashion. The pH response of the dual fluorescence hSNFs was characterized using a customized optical fiber-based fluorescence detection system. The characterization results show that the dual fluorescence hSNFs have a pH sensitivity of 0.34 [pH−1] and a response time of less than 10 seconds. Thanks to the ratiometric measurement approach based on the FITC and Ru(BPY)3 fluorescent intensity ratio, the developed fluorescence pH sensor was able to achieve long term pH measurements with only minimum influence from photobleaching. Applicant have experimentally demonstrated that the sensor has a linear pH response between pH 4.6 to pH 8.5 and a long-term stability over more than 6 hours.
The photobleaching independent pH measurements of Applicant's hSNFs pH sensors made it ideal for applications that require long-term pH monitoring, such as the long-term cell metabolic profiling. Beyond the demonstrated pH sensing application, the developed rational synthesis method of hSNFs can potentially enable other sensing applications by rationally design the fluorophores doped to the hSNFs.
Without further elaboration, it is believed that one skilled in the art can, using the description herein, utilize the present disclosure to its fullest extent. The embodiments described herein are to be construed as illustrative and not as constraining the remainder of the disclosure in any way whatsoever. While the embodiments have been shown and described, many variations and modifications thereof can be made by one skilled in the art without departing from the spirit and teachings of the invention. Accordingly, the scope of protection is not limited by the description set out above, but is only limited by the claims, including all equivalents of the subject matter of the claims. The disclosures of all patents, patent applications and publications cited herein are hereby incorporated herein by reference, to the extent that they provide procedural or other details consistent with and supplementary to those set forth herein.
This application claims priority to U.S. Provisional Pat. App. No. 63/220,594, filed on Jul. 12, 2021. The entirety of the aforementioned application is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/033156 | 6/11/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63220594 | Jul 2021 | US |
