FIELD OF THE INVENTION
[001] The present disclosure relates to the field of nanosensor, and more
particularly relates to fluorophores based pH- nanosensors.

BACKGROUND OF THE INVENTION
[002] Measurements of pH in acidic cellular compartments of mammalian cells
is important for our understanding of cell metabolism, and organelle acidification
is an essential event in living cells especially in the endosomal-lysosomal pathway
where pH is critical for cellular sorting of internalized material. Intracellular pH
can be measured by the use of fluorescence ratio imaging microscopy (FRIM),
however, available methods for pH measurements in living cells are not optimal.
Nanoparticle based optical sensor technology for quantification of metabolites in
living cells has been developed over the last two decades. However, even though
these sensor systems have proven themselves as superior to conventional
methods, there are still questions about the use of these sensors that need to be
addressed, especially regarding sensor design and calibration.
[003] There are two major design considerations for fabricating nanosensors.
Firstly, the sensor must have suitable physicochemical characteristics to be taken
up through the endocytic pathway with minimal impact on natural function. This
is mainly determined by the properties of the nanoparticle matrix. Secondly, the
sensor must have suitable optical characteristics. In this respect, an ideal sensor
would be ratiometric, show rapid response, have high signal to noise and be
sensitive to the entire pH range of the endocytic pathway. This is mainly
determined by the sensing elements of the sensor, which are the fluorophores.
[004] In the present invention relates to overcome the problem /obstacles, the of
prior art and relates to ratiometric polyacrylamide pH nanosensors to be utilized to
probe fundamental aspects of intracellular trafficking with the view of developing
biological insights to aid the rational design of nanomedicines.

[005] The information disclosed in this background of the disclosure section is
only for enhancement of understanding of the general background of the invention
and should not be taken as an acknowledgement or any form of suggestion that
this information forms the prior art already known to a person skilled in the art.

OBJECTS OF THE INVENTION

[006] A principal object of the present disclosure is to provide fluorophores
based pH- nanosensors.
[007] These and other objects and advantages of the present subject matter will
be apparent to a person skilled in the art after consideration of the following
detailed description taken into consideration with accompanying drawings in
which preferred embodiments of the present subject matter are illustrated.

SUMMARY OF THE INVENTION

[008] In an embodiment the present disclosure relates to fluorophores based pHnanosensors.
[009] In another embodiment ratiometric polyacrylamide pH nanosensors are
utilized to probe fundamental aspects of intracellular trafficking with the view of
developing biological insights to aid the rational design of nanomedicines.
[0010] In another preferred embodiment nanosensors are fabricated with a
dynamic range covering the entire range of the endocytic pathway (4.0 – 7.5),
with sizes between 50 and 100 nm.
[0011] In yet another embodiment endocytic uptake of nanosensors iss induced in
four different cell types (HeLa, 3T3, MRC-5 and JAWSII) by increasing the surface charge on the nanosensor.

[0012] The foregoing summary is illustrative only and is not intended to be in any
way limiting. In addition to the illustrative aspects, embodiments, and features
described above, further aspects, embodiments, and features will become apparent
by reference to the drawings and the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS
[0013] It is to be noted, however, that the appended drawings illustrate only typical embodiments of the present subject matter and are therefore not to be considered for limiting of its scope, for the invention may admit to other equally effective embodiments. The detailed description is described with reference to the accompanying figures. In the figures, the left-most digit(s) of a reference number identifies the figure in which the reference number first appears. The same numbers are used throughout the figures to reference like features and components. Some embodiments of system or methods in accordance with embodiments of the present subject matter are now described, by way of example, and with reference to the accompanying figures, in which:
[0014] FIG. 1 illustrates SEM micrographs of nanotips, in accordance with the embodiment of the present invention;
[0015] FIG. 2 illustrates the schematic representation of the fluorescence excitation and measurement procedure used for Cy5 detection, in accordance with the embodiment of the present invention; and
[0016] FIG. 3 illustrates the results of the characterization study, which involved detection of Cy5 labeled anti-cytochrome c immobilized on the nanotips, in accordance with the embodiment of the present invention.
[0017] The figures depict embodiments of the present subject matter for the purposes of illustration only. A person skilled in the art will easily recognize from the following description that alternative embodiments of the structures and methods illustrated herein may be employed without departing from the principles of the disclosure described herein.

DETAILED DESCRIPTION OF THE INVENTION
[0018] While the embodiments of the disclosure are subject to various modifications and alternative forms, specific embodiment thereof have been shown by way of example in the figures and will be described below. It should be understood, however, that it is not intended to limit the disclosure to the particular forms disclosed, but on the contrary, the disclosure is to cover all modifications, equivalents, and alternative falling within the scope of the disclosure.
[0019] The terms “comprise”, “comprising”, or any other variations thereof used in the disclosure, are intended to cover a non-exclusive inclusion, such that a device, system, assembly that comprises a list of components does not include only those components but may include other components not expressly listed or inherent to such system, or assembly, or device. In other words, one or more elements in a system or device proceeded by “comprises… a” does not, without more constraints, preclude the existence of other elements or additional elements in the system or device.
[0020] The present invention relates to fluorophores based pH- nanosensors
[0021] Reference may be made to Figure 1 illustrating the SEM micrographs of nanotips, in accordance with the embodiment of the present invention;
[0022] Reference may be made to Figure 2 illustrating the schematic representation of the fluorescence excitation and measurement procedure used for Cy5 detection, in accordance with the embodiment of the present invention; and
[0023] Reference may be made to Figure 3 showing results of the characterization study, which involved detection of Cy5 labeled anti-cytochrome c immobilized on the nanotips, in accordance with the embodiment of the present invention
[0024] In recent years, many details of the biochemical mechanisms involved in the normal cell cycle have been discovered. Scientists have found that cell signaling pathways are regulated by highly complex interactions between proteins in biochemical pathways. To understand cellular function, most scientists study parts of specific biochemical pathways, such as the cell cycle, that involve individual molecules, cells, groups of cells, and whole organisms. The ultimate goal is to be ablet o put all the parts together to understand normal cellular activities and how they function in normal cellular activities or how they malfunction in diseases. These studies are usually performed using bulk cell assays that are blind to heterogeneity of cells and the fact that cells in a population behave asynchronously to external stimuli. A good example is the apoptosis pathway. The differences between cells in their ability to activate caspases involved in apoptosis pathway contribute to their responsiveness of any given cell within a population to apoptotic stimuli. Therefore, to study and understand molecular mechanisms that underlie such differences it is necessary to measure caspase activity in intact individual living cells [4-6]. By measuring caspase activity in single living cells, it should be possible to test hypotheses and determine the molecular mechanisms that underlie cell-to-cell variability in response to poptotic stimuli. In this context, it is important to understand the dynamic relationships between chemicals and molecular events in living cells in the context of a cell as a system rather than a collection of organelles and individual processes. The information obtained from such studies can be used to accurately map biological behavior and function and in the process be used to effectively pursue drug discovery. It is also important to analysis cellular components such as proteins in their native form and environment. Protein analysis technique soften involve exposing proteins to pre-treatment conditions such as solubilization, denaturation and reduction. Exposing proteins to such conditions can cause modification that can end up causing artifacts in the results. In addition, it is important to have the ability to monitor slight differences in the amounts of protein and other biomolecules within the smallest possible detection volumes down to the single cell level for diagnostic and technological purposes. This can be invaluable in the sense that it would permit studies that would be otherwise difficult to perform, such as, performing experiments in primary human cells from surgical specimens that are only available in very small numbers. Accurate cellular studies can be performed if we did not have to study parts or components of a cell and then piece all the parts together like a jigsaw puzzle in order to study cell-signaling pathways. This means that we require technologies capable of performing sensitive and specific biochemical analysis within single living cells. Technologies for performing sensitive and specific biochemical analyses at the single cell level are mainly important for understanding intracellular signaling pathways that control cell function and behavior. Such technologies should have the capability of penetrating cell membranes without disrupting or terminating cellular reactions. Disruption of the cell membrane can lead to significant changes in the parameter of interest which can occur during the course of cell lysis, resulting in an in accurate view of the actual physiologic state of a cell. Cell lysis leads to disruption of the plasma membrane, which can result in order of magnitude changes in the concentrations of the analyte or proteins of interest. In addition, compartmentalized enzymes can be released and activated leading to a high background that can potentially obscure the detection of the target analyte or protein. If we could “go easier” on the cells, this information might become available to researchers.
[0025] This work focuses on the development and application of optical nanosensor technology for possibly determining all of the many molecular interactions, their components, and the order in which they occur during a biochemical-signaling pathway. The main aim of this work is to identify and possibly catalogue components in the apoptosis pathway. The hope is to yield information of the processes of apoptosis that can potentially aid researchers seeking to study or treat diseases in which cellular processes go awry and are only armed with bulk cell assay information that is not representative of what is happening at the single cell level. This information can also be used by scientists seeking to learn more about the ways single cells respond to external signals, which are often conveyed when molecules bind to special receptors in cell membranes or when molecules traverse the cell membrane. The fundamental knowledge developed through single cell analysis studies can lead to new ways to diagnose, treat, cure, or even prevent diseases such as Alzheimer’s Disease (AD), which involves neuronal apoptosis, or cancer, which involves a defect in the apoptosis machinery of a cell. Single cell analysis involves monitoring the activities of single cells, preferably live, in their physiological conditions. These types of studies are important due to the enhanced sensitivity of single living cells to their environment. The results obtained from such studies are important because they can give an additional dimension to standard cellular assays that rely on population-averaged studies that are blind to heterogeneity and the presumed existence of subpopulations. Studies on single cells are crucial to determine the activities and functional relationships of signaling molecules within the complex cellular networks that comprise biological systems. However, to accurately measure the complex interrelationships of the elements in the biological systems, measurements must be performed on individual cells with their signaling pathways intact. Therefore, continued progress in understanding cellular physiology requires technologies capable of biochemical measurements in solitary living cells with spatio-temporal control over the regions of cells interrogated, techniques that do not compromise the integrity of the cell membrane or cause physiological damage with biochemical consequences. The disruption of the cell membrane can lead to significant changes in the parameter of interest, resulting in an inaccurate view of the actual physiologic state of the cell. This means that the accurate measurement of many cellular properties will require a technique that is minimally invasive and does not disrupt the cell, and perhaps no recent technology has greater potential for this application the optical nanosensors.
[0026] Optical pH sensor offers an alternative approach. As compared to pH electrodes, optical pH sensors have several advantages. They can be miniaturized down to sub micrometer or even nanometer dimensions. pH optical fiber sensors and pH nanosensors have been extensively studied and applied for monitoring intracellular pH.
[0027] The main aim of this research is to try answering the fundamental question, “Can we probe the workings of cells without causing physiological of biological damage that may have resultant biochemical consequences?”. As we have gained a better understanding the overall nature of bulk cell assays over time, and achieved a greater understanding of many macroscopic biological processes, we have been led to ponder the purpose of an increasingly smaller important entity, the basic unit of life, the single cell. The cell is the fundamental unit of life, a very small compartment with the capability to replicate itself. It is full of proteins, which support its life, replication, and interactions with other cells as well as its environment. Proteins are molecules made up of amino acids arranged in a specific order determined by the genetic code and are essential for all life processes. Since the mid-1940's, biomedical researchers have made enormous progress in identifying and understanding proteins and how they interact in many cellular processes. Much of this research was “basic”, scientific research that sought to discover how systems work and develop a base of knowledge that other scientists can use in order to achieve practical goals, such as treatments or cures for diseases. In the 1950's, scientists started to develop the current picture of the cell as a complex and highly organized entity. They found that a typical cell is like a miniature body containing tiny "organs," called organelles. One organelle is the command center, others provide the cell with energy, while still others manufacture proteins and additional molecules that the cell needs to survive and to communicate with its environment. The cell components are enclosed in a plasma membrane that not only keeps the cell intact, but it also provides channels that allow communication between the cell and its external environment
[0028] Measurements of pH in acidic cellular compartments of mammalian cells is important for our understanding of cell metabolism, and organelle acidification is an essential event in living cells especially in the endosomal-lysosomal pathway where pH is critical for cellular sorting of internalized material. Intracellular pH can be measured by the use of fluorescence ratio imaging microscopy (FRIM), however, available methods for pH measurements in living cells are not optimal. Nanoparticle based optical sensor technology for quantification of metabolites in living cells has been developed over the last two decades. However, even though these sensor systems have proven themselves as superior to conventional methods, there are still questions about the use of these sensors that need to be addressed, especially regarding sensor design and calibration.
[0029] Surface-enhanced Raman scattering (SERS) offers high specificity in molecular identification and is a promising technique for making biological and chemical sensors. However, a major weakness of this technique is the lack of reliable SERS substrates. Currently available substrates usually contain irregular active sites that suffer from strong spatial and temporal fluctuation in Raman intensity and thus do not produce stable Raman output. On the other hand, because of their well-defined structure, periodic metallic arrays excite more controllable local fields and can be exploited for achieving highly sensitive SERS. The SERS obtained from these periodic structures exhibit strong spectral and angular dependences that are associated with the generation and decay processes of the various electromagnetic resonance modes.
[0030] Further, a pH nanosensor with high specificity and sensitivity is developed based on surface-enhanced Raman scattering by encapsulating 4-mercaptobezonic acid functionalized silver nanoparticles in a protonpermeable silica shell. The performance of silica protected nanosensor against aggregation and biomolecular interference is investigated. The nanosensors are introduced to report intracellular pH in living macrophages.
[0031] This work explores and focuses on the development and application of novel optical nanosensors for single living cell analysis. In this context, single cell analysis involves the application of optical nanosensor technology to observe and possibly map molecular events inside single living cells. Previous studies have focused on the bulk response of cells and this largely increases the probability of missing critical underlying mechanisms specific to the single cell. The ability to perform single cell analysis can dramatically improve our understanding of basic cellular processes e.g., signal transduction as well as improving our knowledge of the intracellular transport and the fate of therapeutic agents at the single cell level. This is important not only because of the capability to perform minimally invasive analysis, but also to overcome the problem of ensemble averaging. This capability to overcome ensemble averaging has the potential to yield new information that is not available from population averaged cellular measurements.
[0032] This research also involves the development and application of optical nanosensors for specific and sensitive chemical and protein analysis within single living cells. The ability of these sensors to successfully perform chemical and protein analysis at the single cell level, lay in their design specifications, size, specificity, sensitivity and eliminating interferences. With regard to their specifications, their size was in the nanometer regime, which is relative to the scale of a single mammalian cell (~ 10 µm)to allow non-invasive-to-minimally-invasive measurements in single living cells. In addition, they incorporated biological recognition molecules to achieve specificity and finally, near-field evanescent wave excitation and detection to achieve high sensitivity. High specificity and sensitivity allowed for precise and accurate identification of physicochemically detectable substances in complex matrices to eliminate any potential interference.
[0033] The field of nanomedicine has progressed to a stage where a diverse set of materials are available for controlling how a drug is delivered in the body. Although these materials can be engineered to overcome many of the obstacles associated with drug delivery, the complexity of cellular trafficking mechanisms means controlling intracellular delivery remains a major challenge. The primary portal for the cellular internalization of nanomedicines is endocytosis, which involves transport through a network of highly complex intracellular compartments undergoing a dynamic process of acidification. As a result, nanoparticle-based pH sensors offer a new perspective from which to investigate this process.
[0034] In this study, ratio metric polyacrylamide pH nanosensors were utilized to probe fundamental aspects of intracellular trafficking with the view of developing biological insights to aid the rational design of nanomedicines. Nanosensors were fabricated with a dynamic range covering the entire range of the endocytic pathway (4.0 – 7.5), with sizes between 50 and 100 nm. Endocytic uptake of nanosensors was induced in four different cell types (HeLa, 3T3, MRC-5 and JAWSII) by increasing the surface charge on the nanosensor. Dynamic pH measurements were found to be highly sensitive to experimental methodology for performing ratio metric measurements, particularly image analysis. Consequently, an optimized procedure for performing ratio metric measurements was developed, and subsequently validated by correlating pH measurements with intracellular location using 3D structured illumination microscopy (3D-SIM).
[0035] We further explore Application of pH nanosensors in studies investigating fundamental aspects of intracellular trafficking resulted in three key findings: 1) HeLa, 3T3 and JAWS II cells process material in different ways with respect to the extent and rate of acidification in endocytic organelles, 2) surface charge does not affect the final intracellular location of polyacrylamide nanoparticles internalized by endocytosis, and 3) lipid-mediated transfection of siRNA is associated with a greater degree of lysosomal disruption compared to cationic polymer-mediated transfection, with the former observed to show increased toxicity. These findings represent biological insights, which can be utilized to provide a rational basis for tailoring the response of pH-sensitive nanomedicines to a specific cell type, tuning the physicochemical properties of a material for more efficient intra cellular trafficking and optimizing siRNA formulations for endo-lysosomal release.
[0036] There are two major design considerations for fabricating nanosensors. Firstly, the sensor must have suitable physicochemical characteristics to be taken up through the endocytic pathway with minimal impact on natural function. This is mainly determined by the properties of the nanoparticle matrix. Secondly, the sensor must have suitable optical characteristics. In this respect, an ideal sensor would be ratio metric, show rapid response, have high signal to noise and be sensitive to the entire pH range of the endocytic pathway. This is mainly determined by the sensing elements of the sensor, which are the fluorophores. These two critical design considerations are addressed in in our research work.
[0037] Synthesis of silver nanoparticles: Silver nanoparticles were synthesized using ascorbic acid (Spectrum Chemicals MFG., Gardena, CA) as a reducing reagent. Briefly, for a 10mL solution, 1 mM silver nitrate (Spectrum Chemicals MFG., Gardena, CA) was reduced by 1 mM of ascorbic acid in the presence of 1 mM sodium citrate (Spectrum Chemicals MFG., Gardena, CA) in a five-dram glass vial. The reaction took place at room temperature overnight. The obtained nanoparticles were centrifuged with distilled water for three times to remove the excess reagents. The nanoparticles were concentrated to 1mL in distilled water and the concentration is estimated to be approximately 1 mg/mL, assuming all the silver nitrate was completely reduced. To prepare Ag-MBA, 100 µL 4-MBA in ethanol (3 mM) was also added to 1 mL silver nanoparticles (1 mg/mL).
[0038] Synthesis and characterization of Ag-MBA@SiO2 nanoparticles:Ag-MBA@SiO2 were synthesized via reverse microemulsion method according to literature with some modifications.[61-62] For a typical reaction, 1.5 mL Triton X-100 (Alfa Aesar, Ward Hill, MA), 7.5 mL cyclohexane (Alfa Aesar, Ward Hill, MA) and 1.6 mL hexyl alcohol(Sigma Aldrich, St. Louis, MO) were added to a 100 mL flask to form a transparent oil phase under stirring, then silver nitrate (1 mM), sodium citrate (1 mM), ascorbic acid (1mM) and 100 µL 4-MBA (Sigma Aldrich, St. Louis, MO) in ethanol (3 mM) were added to the oil phase sequentially. 5 min later, 40 µL of tetraethyl orthosilicate (TEOS) (TCI America, Portland, OR) was added and then 60 µL of ammonium hydroxide (BDH, West Chester, PA) was added to initialize the silica coating process. The flask was sealed and stirred at room temperature for 24 h. After the reaction was completed, 10 mL ethanol was added to precipitate the nanoparticles and then the nanoparticles were washed and centrifuged with both ethanol and water for three times to remove excess reagents. The nanoparticles were also concentrated to 1 mL, dispersed in water and stored at room temperature. No settling was observed by eye for up to one month in water, while aggregation forms slowly in buffer solutions. The size and morphology of the nanoparticles were characterized with both SEM and TEM. One drop of either type of nanoparticle solution was added to TEM grids and both the SEM and TEM images were taken with a HD-2000 (200 kV, Hitachi) with a magnification of 110 k. A Zetasizer Nano dynamic light scattering (DLS) instrument (Malvern, Westborough, MA) was also used to determine hydrodynamic size distribution of nanoparticles dispersed in water. UV absorbance spectra of both kinds of nanoparticles dispersed in water were taken with aUV-2101pc spectrometer (Shimadzu, Torrance, CA).
[0039] pH calibration curves and interference studies: To calibrate the SERS sensors, a 100µL solution of either Ag-MBA or Ag-MBA@SiO2 was added to a small glass vial, then400 µL different buffers were added and 300 µL distilled water was added afterwards. The pH values of all the samples were verified with a pH meter (Fisher Scientific, Pittsburgh, PA). The samples were left still overnight to let the functionalized nanoparticles precipitate to the bottom before further experiments. To study to effect of BSA on the functionality of the functionalized silver nanoparticles, 20 µL BSA (2mg/mL) (Thermo Scientific, Rockford, IL) was added to Ag-MBA with a pH of 7. Both20 µL BSA (2 mg/mL) and 100 µL of BSA (500 mg/mL) was added to Ag-MBA@SiO2with a pH of 5, respectively. SERS spectra were obtained both before and after addition of BSA. To study the effect of NaCl on the nanosensor performance, NaCl with a final concentration of 50 mM was added to either Ag-MBA in a pH 7 buffer, or Ag-MBA@SiO2 in a pH 5 buffer. SERS spectra were also obtained before and after adding NaCl.
[0040] Settling effect on SERS intensity: To study the influence of nanoparticle settling on SERS signal and pH functionality, SERS spectra were kinetically obtained for Ag-MBA at pH 7 and Ag-MBA@SiO2 at pH 5. The samples were first dispersed by sonication and SERS spectra were kinetically obtained while the particles were settling down. For Ag-MBA, SERS measurements were obtained every 2s for 2.78 h with an integration time of3s, while for Ag-MBA@SiO2, SERS measurements were obtained every 27 s for 12 h with an integration time of 3 s. By the end of acquisition, both types of nanoparticles had settled to the bottom of glass vials.
[0041] SERS acquisition setup: The following setup was introduced for all the SERS signal acquisition. Samples were placed on the stage of an inverted microscope ((DMI 5000,eica Microsystems, Germany). A 5 mW 632.8 nm He-Ne laser (Thorlabs, Newton, NJ),filtered with a 633 nm laser line excitation filter (Chroma Technology Corp, Bellows Falls, VT), was directed to a 10x or 50x objective lens and focused to the bottom of the glass vial. The scattered light was collected with the same objective and the Raman scattered light was passed through a 646 nm long pass emission filter (Chroma Technology Corp, Bellows Falls, VT) before being focused to a spectrometer (DNS 300,DeltaNu, Laramie, WY), equipped with a cooled CCD camera (iDUS-420BV, Andor, South Windsor, CT). SERS spectra were obtained with an integration time of 5 s unless otherwise indicated. The spectra displayed were recorded with the corresponding software without further normalization. For calculating the peak height, the SERS spectra were first smoothed with a second order Savitzky-Golay filter with a frame size of 19pixels (28.3 cm-1). The peak heights were calculated by deducting the minimum value near the peak from maximum peak intensity.
[0042] Intracellular pH measurement: The mouse macrophage cell line (J774A.1) was purchased from American Type Culture Collections. The cells were cultured in high glucose Dulbecco’s modified Eagle medium (DMEM, Media tech, Manassas, VA)supplemented with 10% heat inactivated fetal bovine serum (Thermo Scientific, Logan, UT), penicillin (100 units/mL) and streptomycin (100 µg/mL) The cells were cultured on coverslips with diameter of 22 mm in 33 mm diameter-petri dishes at 37 °C in a humidified incubator with 5% CO2. After being incubated overnight, 50 µL of Ag-MBA@SiO2 was added to the culture medium and incubated for 6 h, as to the control plate, 50 µL of 1x PBS was added. The cell plates were rinsed with 1x PBS three times before SERS measurements. The coverslip was mounted to a small incubator in microscope stage, and 1 mL of 1x PBS was added to it. The temperature was set to 37 oC to mimic the culturing conditions for the cells. Before acquiring SERS spectra with a 50x objective lens, different regions of the coverslips were imaged with the microscope under both bright and dark fields.
Experiment-1
[0043] 600 µm plastic clad silica (PCS) optical fiber was purchased from Fiber guide Industries, Stirling, New Jersey. The benefits o PCS optical fiber include, cost effective, high numerical aperture (NA) for efficient light collection from extended sources, radiation resistant, laser damage resistant, dielectric nonmagnetic construction, broad operating wavelength range from 220nm to 700n, excellent laser beam delivery. PCS optical fibers were cut into 8 inch pieces and each end of the fiber was polished using Aluminium oxide lapping paper. The fibers were drawn to various tip diameters using Sutter P-2000 micropipette puller. The P-2000 works well with small diameter glasses such as PCS optical fibers. The programmable parameters laser power level, scan width, trip velocity, delay/ laser on time, and hard pull strength, were modified before each pulling to obtain nanotips of different diameters. After pulling the optical fiber to nanotips, they were coated with 99.999 % silver metal in silver thermal deposition chamber. After coating the nanotips with silver, scanning electron microscope (SEM) images of the nanotips were acquired with Hitachi S-4700 SEM at the High Temperature Materials Laboratory, ORNL.
[0044] Table below shows the programmable parameters include; heat, trip velocity, delay/ laser on time, and hard pull strength (heat, filament, velocity, delay and pull) that were adjusted to draw nanotips of different tip diameters. This table shows the change in each parameter and the results obtained. FIG. 1 show SEM micrographs of the nanotips acquired with Hitachi S-4700 SEM. From the results, it can be concluded that increasing the pull strength, decreases the tip diameters obtained. These results were used as guide in the production of uniform optical nanosensors to enhance the reproducibility factor between single cell measurements.
Sutter P-2000 Input Parameters
Program Heat (Degree Celcius) Velocity Delay Pull Size (nm)
40 850 20 450 200 50
10 850 60 126 130 1000
3 850 60 126 160 500
9 850 40 126 170 400
22 850 40 126 180 300
19 850 20 126 190 200
18 850 20 126 200 100

[0045] The greatest challenge of developing nanoscale materials and structures lies in being able to look at what has been created and to determine the presence or absence of elements and their distribution. I devised a simple but straightforward method to characterize the optical nanosensor and in the process confirm protein immobilization chemistry. The optical nanosensors were characterized by immobilizing Cy-5 labeled antibody onto functionalized nanotips and detecting the presence or absence of Cy-5labeled antibody. Cy-5 was used because of its intense fluorescence, low hydrophobicity, and their high photostability which renders them useful for single molecule fluorescence detection. In this study, anti-cytochrome c was labeled according to the following protocol and immobilized on nanotips (antibody was labeled in a column and collected). 1mg of protein was labeled to a final average molar dye/ protein ratio of 8 (this assumes an average protein molecular weight of 155 KDa). Cytochrome c antibody was labeled according to the following protocol and immobilized on nanotips. Protein (1mg/ml in PBS) + Coupling buffer + ReactiveCy-5 dye. Mixture is incubated at room temperature for 30 minutes. Separation of labeled protein from non-conjugated (free) dye using a gel filtration column.
[0046] Collection tubes used to collect labeled protein. One batch of functionalized nanotips were incubated in the labeled protein solution while the others were incubated in unlabeled protein solution. Interaction between the Cy-5 labeled and unlabeled antibody, with the nanotip was investigated. Interaction between the Cy-5 labeled antibody and the nanotip was investigated. Fluorescence signal produced when the antibody-antigen complex is excited; is converted to a measurable electrical signal.
Experiment-2
[0047] Analytical grade materials were used as long as they were commercial yavailable. Cy-5 labeling kit containing, buffers, Cy-5 monofunctional dye as well asPD-10 columns were purchased from Amer sham Pharmacia Biotech. Cytochrome c antibody was purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA).Labeling of Cytochrome c with Cy5In the standard procedure, the contents of 1 vial (to label 1 mg of protein) ofCy-5 monofunctional dye was dissolved in 50 µL of DMSO. Cytochrome c antibody was dissolved in buffer provided, typically at 1 mg/mL protein concentration. 10 µL of dye/DMSO mixture was pipetted into 200 µL cytochrome c solution under slow vortexing. After 30 min incubation at 25°C in the dark the reaction was terminated by freezing at –4oC. For separation of unbound dye, 300 µL of 100 mM NaH2PO4 (to suppress further labeling after thawing) was added to a frozen sample and the sample was incubated in a 25°C water bath until thawed. The sample was loaded on a PD-10column (10 mL bed of Sephadex G-25M), which had been pre-equilibrated in buffer. After washing the column with buffer A (2 *1 mL) the labeled protein was eluted by adding 2 mL of buffer to the column top. Another 10 mL of buffer was added to elute all unbound dye, and the column was regenerated with 20 mL of buffer. Higher Cy-5dye concentrations can be achieved by dissolving 2 vials of Cy-5 dye in 50 µL of DMSO.
Immobilization of Labeled Anti-Cytochrome c
[0048] Preparation of anti-cytochrome c optical nanosensors involved the construction of nanofibers and functionalizing the nanotips to facilitate antibody immobilization. PCS optical fibers were pulled using the laser based fiber puller, from 600 µm diameter to 50 nm diameters. The pulled fibers were coated with silver metal in silver thermal deposition chamber. After coating the nanotips with silver, the nanotips were functionalized as described previously to allow the immobilization of Cy5 labeled anti-cytochrome c. The measurements were performed using labeled antibody for the experimental and unlabeled antibody for the control.
[0049] FIG. 2 is a schematic representation of the fluorescence excitation and measurement procedure used for Cy5 detection during the characterization study of optical nanosensor. FIG. 3 shows the results of the characterization study, which involved the detection of Cy5 labeled anti-cytochrome c immobilized on the nanotips. This study involved two treatment groups, optical nanosensor with Cy5 labeled anti cytochrome c and without Cy5 labeled anti-cytochrome c. These results show a difference of an order of magnitude in the fluorescent intensity measurements. This difference is significant enough to determine the successful immobilization of Cy5labeled anti-cytochrome c.
Characterization of Optical Nanosensor: -AFM measurements
[0050] In addition, we attempted to determine the distribution of antibody using a recently acquired an atomic force microscope (AFM). AFM is a method of measuring surface topography on a scale from angstroms to 100 microns. The technique involves imaging a sample through the use of a probe, or tip, with a radius of 20 nm.
[0051] The tip is held several nanometers above the surface using a feedback mechanism that measures surface–tip interactions on the scale of nanoNewtons. Variations in tip height are recorded while the tip is scanned repeatedly across the sample, producing a topographic image of the surface. The AFM has the ability to image at atomic resolution (sub-angstrom level) and can achieve a resolution of 10 pm, and unlike electron microscopes, can image samples in air and under liquids. The AFM’s resolution combined with its ability to image a wide variety of samples under a wide variety of conditions such as air and in fluids. Images have appeared in the literature showing DNA, single proteins, structures such as gap junctions, and living cells. AFM operates by measuring attractive or repulsive forces between a tip and the sample. In its repulsive "contact" mode, the instrument lightly touches a tip at the end of a leaf spring or "cantilever" to the sample. As a raster-scan drags the tip over the sample, some sort of detection apparatus measures the vertical deflection of the cantilever, which indicates the local sample height. Thus, in contact mode the AFM measures hard-sphere repulsion forces between the tip and sample. In noncontact mode, the AFM derives topographic images from measurements of attractive forces; the tip does not touch the sample.
Experiment-3
[0052] PCS optical fibers were pulled using the laser based fiber puller, from 600 µm diameter to 50 nm diameters. The pulled fibers were coated with 99.999 % silver metal in silver thermal deposition chamber. After coating the nanotips with silver, the nanotips were functionalized to allow the immobilization of Cy-5 labeled cytochrome c antibody. After the construction of antibody-modified nanotips, we imaged the nanotips using contact mode to locate the distribution of the immobilized cytochrome c antibody. Contact mode is the most common method of operation of the AFM and as the name suggests the tip and sample remain in close contact as the scanning proceeds.
[0053] The results obtained revealed that we were unable to achieve sub-angstrom resolution and therefore unable to determine the distribution of cytochrome c antibody immobilized on the nanotips. However, we were able to obtain, with the acquired images, the dimensions of the nanotips. Idealistically, if this worked and we were able to determine the location and distribution of cytochrome c antibody, it would essentially allow us to study the biophysics of molecular interactions and its role in important processes such as signal transduction.
[0054] Experiment-4
[0055] PCS optical fibers were pulled using the laser based fiber puller, from 600 µm diameter to 50 nm diameter. The pulled fibers were coated with 99.999 % silver metal in silver thermal deposition chamber. After coating the nanotips with silver, the nanotips were functionalized to facilitate the immobilization of antibody to fluorescein for the experimental group, and without antibody to fluorescein for the control group. Anti-fluorescein solution is prepared by dissolving the lyophilizate in 1ml double distilled water results in a concentration of 0.1 mg antibody/ml. The antibody is suitable for the detection of fluorescein and fluorescein-labeled compounds. The detection of bound antibody can be carried out directly in one step using an antibody to fluorescein. Both groups of optical nanosensors were incubated in fluorescein (10-4M) prepared in PBS. Both were incubated for 5 minutes in fluorescein and the evaluation of optical nanosensors study involved two groups, experimental: antibody to fluorescein was immobilized on functionalized nanotips, and, control: without antibody on functionalized nanotips.
[0056] The various actions, acts, blocks, steps, or the like in the flow chart may be performed in the order presented, in a different order or simultaneously. Further, in some embodiments, some of the actions, acts, blocks, steps, or the like may be omitted, added, modified, skipped, or the like without departing from the scope of the invention.
[0057] The embodiments disclosed herein can be implemented using at least one software program running on at least one hardware device and performing network management functions to control the elements.
[0058] The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.

We Claim:

1. A system for studying intracellular trafficking of cells, comprising: a substrate; and a nanosensor interacting with the linker.
2. The system for studying intracellular trafficking of cells as claimed in claim 1, wherein Nanosensors cover the entire range of the endocytic pathway (4.0 – 7.5), with sizes between 50 and 100 nm.
3. The system for studying intracellular trafficking of cells as claimed in claim 1, wherein the Endocytic uptake is induced in four different cell types (HeLa, 3T3, MRC-5 and JAWSII) by increasing the surface charge on the nanosensor.
4. The system for studying intracellular trafficking of cells as claimed in claim 1, wherein the nanosensors aid in the rational design of nanomedicines.
5. The system for studying intracellular trafficking of cells as claimed in claim 1, wherein the nanosensors are validated by correlating pH measurements with intracellular location using 3D structured illumination microscopy (3D-SIM).