Patent Application
20180045675
None.
The present invention relates to the field of nanopore scale devices and sensors, in particular for pH sensing of fluids and solutions within a single cell.
Presented below is background information on certain aspects of the present invention as they may relate to technical features referred to in the detailed description, but not necessarily described in detail. That is, individual compositions or methods used in the present invention may be described in greater detail in the publications and patents discussed below, which may provide further guidance to those skilled in the art for making or using certain aspects of the present invention as claimed. The discussion below should not be construed as an admission as to the relevance or the prior art effect of the patents or publications described.
Personalized medicine holds great potential, especially in treating cancer, which remains a major medical challenge due to both intrinsic and acquired resistance to conventional chemotherapeutics1-3. In the last decade, advances have been made in the development of personalized cancer therapeutics to increase the efficacy of chemotherapy4. Despite every effort to tailor drugs to the individual, results vary5. This fact has been correlated with the presence of genetically distinct cells within an individual tumor6. In recent studies genome sequencing technology has been employed to identify these genetic alterations in a large population of cells7-9. While genetic aspects of cancer cell heterogeneity and the relationship between mutations and drug resistance have been studied extensively, development of pre-screening technologies to detect heterogeneity, that is, to find cancer cells that differ in their cellular metabolism and physiology within large cell populations, is under-investigated.
Evaluation of cell heterogeneity can be performed through the measurement of cytoplasmic ions and molecules. Accumulation of metal ions10, changes in reactive oxygen (ROS) and nitrogen species (RNS) levels11, and protein expression12 are important markers of cancerous cells within cell populations. Although less recognized, pH is also a distinctive factor of cancer cells. pH is one of the most intriguing features in initiating and regulating a myriad of cellular events, such as multi-drug resistance in tumors13, protein processing14, endocytosis15 and apoptosis16. Due to its vital importance, the pH of the intracellular environment is strictly regulated through various ion channels and intracellular weak acids and bases, such as alkali cation-H+ exchangers, bicarbonate and acid loading transporters. In mammalian cells, subcellular compartments have different pH values in order to sustain optimum operational conditions for certain metabolic functions17. In normal physiological conditions, the resting intracellular pH of mammalian cells is maintained between 6.8 and 7.318. On the other hand, extracellular pH values are slightly alkaline with the range of 7.2 to 7.4. A dysregulation of intracellular pH is often associated with altered cell functions, proliferation and drug resistance, and is observed in cancerous tumors19. Moreover pH has a great effect on tumor growth and cancer cell migration and therefore the potential for metastases20,21.
Carcinogenic tumors are heterogeneous and widely assumed to be acidic due to the high metabolic rate of cancer cells coupled with poor blood supply. This regional high metabolism and lack of perfusion triggers an anaerobic metabolism which leads extracellular pH levels to decrease to ˜6.022. Additionally, aerobic metabolism can increase the intracellular concentration of carbon dioxide (CO2), which results in a decrease of local pH levels. These two mechanisms of acidosis are commonly accepted in cancer research. Little is known, however, about whether intracellular pH levels contribute to intratumoral heterogeneity, and if it is an indicator of preexisting metabolic heterogeneity in cancer cells in a large cell population. Greater granularity of pH data will be of great importance not only for the development of new anti-cancer drugs and carriers, as most new drug delivery systems propose to use pH sensitive polymers or pH sensitive polymeric nanoparticles, but also to ascertain how effectively anti-cancer drugs work over the course of treatment. Therefore, real-time quantitative measurement of intracellular pH may be crucial to link intratumoral cell heterogeneity, drug resistance and drug delivery systems for effective treatment.
pH can be used as a marker for the identification of variants of cancer cells in a tumor tissue. Once identified, these cells can be tagged and followed over the course of drug treatment. Then samples can be collected from the tagged cells to sequence their RNA and DNA to illuminate what makes these cells drug-resistant.
Detecting pH at the cellular level is not only important to investigate single cancer cells and cell heterogeneity in a tumor environment but also to understand neurodegeneration and aging. Neurodegenerative diseases, such as Parkinson's and Alzheimer's diseases, create heterogeneous physico-chemical environments due to mitochondrial oxidative phosphorylation, and therefore it is important to measure pH and understand its effect on neural recovery at the damaged site of brain23. Additionally, cerebral pH has been found to be one of the major markers of metabolic disturbance and lethality after brain injury24. Many of these studies have suffered from the lack of an appropriate analytical tool.
Commonly utilized analytical techniques to measure intracellular pH values include nuclear magnetic resonance (NMR)25, electrochemistry26,27, confocal microscopy28, and absorbance and fluorescence spectroscopy29,31. Of these, fluorescence spectroscopy and imaging are the most widely used techniques. However, fluorescence intensity is hard to quantify directly and suffers from experimental factors such as dye localization, photobleaching, excitation wavelength and cellular uptake and release rate. Additionally, fluorescence intensity can be affected by autofluorescence. Moreover, fluorescence probes do not allow continuous and site-specific detection of intracellular pH levels.
Thus, intracellular pH is both an indicator of cell metabolism and also plays an important role in the initiation and regulation of a myriad of cellular functions such as multi-drug resistance, protein processing and apoptosis. Even within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences of intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially as we move toward more personalized medicine. Therefore, the detection of intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurement of intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies.
Functionalized Nanopipette Biosensor, Karhanek et al. in US Patent Application Publication 2010/0072080, published on Mar. 25, 2010, disclose methods and devices for biomolecular detection, comprising a nanopipette, exemplified as a hollow inert, non-biological structure with a conical tip opening of nanoscale dimensions, suitable for holding an electrolyte solution which may contain an analyte such as a protein biomolecule to be detected as it is passed through the tip opening.
Nanopore Device for Reversible Ion and Molecule Sensing or Migration, Pourmand et al. in US Patent Application Publication 2012/0222958, published on Sep. 6, 2012, disclose methods and devices for detection of ion migration and binding, utilizing a nanopipette adapted for use in an electrochemical sensing circuit. Chitosan is used on a PAA (polyacrylic acid) layer attached first to the nanopipette, and for measuring binding of ions such as copper.
Actis et al. in “Functionalized nanopipettes: toward label-free, single cell biosensors,” Bioanalytical Reviews 1:177-185 (2010) disclose a nanopipette as a label-free biosensor capable of identifying DNA and proteins.
Umehara et al. in “Label-free biosensing with functionalized nanopipette probes,” Proc. Nat Acad. Sci. 106(12): 4611-4616 (2009) disclose a label-free, real-time protein assay using functionalized nanopipette electrodes. The proteins interact with the nanopipette tip coated with probe molecules. It is shown that electrostatic, biotin-streptavidin, and antibody-antigen interactions on the nanopipette tip surface affect ionic current flowing through a 50-nm pore.
The following brief summary is not intended to include all features and aspects of the present invention, nor does it imply that the invention must include all features and aspects discussed in this summary.
The present invention comprises, in certain embodiments, a device for measuring pH inside a single cell, comprising (a) a nanopipette structure that (i) is operatively connectable to a micromanipulator and sensing device for piercing a cell on a support, (ii) contains a working electrode therein, said (iii) contains a polymer coating that selectively absorbs hydrogen ions; (b) said nanopipette structure further connected to an amplifier circuit constructed to apply different voltages between the working electrode and a reference electrode in a solution and further constructed to measure an ionic current between the working electrode and the reference electrode under different voltages; and (c) logic means for correlating different ionic currents measured by said amplifier circuit with pH values within a cell outside the nanopipette structure.
In certain embodiments, the present invention comprises a device wherein the micromanipulator and sensing device comprises an SICM (scanning ion conductance microscope) and xyz controller controlling the nanopipette for movement to and into a single cell. In certain embodiments, the present invention comprises a device wherein the amplifying circuit comprises a detection circuit with gain controls and with a low pass filter for detecting ionic currents. In some embodiments, the present invention comprises a device comprising an array of nanopipette structures connected to a single logic means, as shown, e.g. in
In some embodiments, the present invention comprises a device wherein the polymer coating is selected from the group consisting of sulfonated tetrafluorethylene copolymer (Nafion®), poly-1-lysine, and alginate. In certain embodiments, the present invention comprises a device wherein the amplifier circuit comprises a potentiostat connected to the reference electrode and responsive to input from an amplifier having an input from the working electrode. In further embodiments, the present invention comprises a device wherein the potentiostat is connected to a counter electrode that is also connected to the potentiostat's reference electrode. In certain embodiments, the present invention comprises a device wherein the working electrode and the counter electrode are Ag/AgCl.
In certain embodiments, the present invention comprises a device for measuring pH inside a single cell, comprising (a) a nanopipette electrically connected to a circuit that measures ionic current versus potential at various potentials and is attached to an insertion device for inserting the nanopipette into a single cell; (b) logic means for correlating a rectification value with known pH values, wherein a rectification value obtained in a cell can be correlated with a known rectification value, thereby providing an output identifying a measured pH value; (c) said nanopipette having a layer of chitosan material directly bound to the surface of the nanopipette and porous to hydrogen ions; and (d) a circuit comprising a reference electrode that also functions as an auxiliary electrode and is connected to a potentiostat.
In further embodiments, the present invention comprises a device wherein the logic means is programmed for scanning the potential of the working electrode at a given potential range with respect to the reference electrode by measuring the current at an auxiliary electrode. The device may comprise an i/V amplifier that is bridged by a filter selection and a sensitivity selection circuit, wherein the components are adjusted to adjust the detectable current range based on the current passing through the electrolyte solution.
In certain embodiments, the present invention comprises a method for making a device for measuring pH inside a single cell, comprising (a) preparing a nanopipette structure that (i) is operatively connectable to a micromanipulator and sensing device for piercing a cell on a support, (ii) contains a working electrode therein, and (iii) contains a polymer coating that selectively absorbs hydrogen ions; (b) connecting said nanopipette structure to an amplifier circuit constructed to apply different voltages between the working electrode and a reference electrode in a solution and further constructed to measure an ionic current between the working electrode and the reference electrode under different voltages; (c) connecting said nanopipette structure to logic means for correlating different ionic currents measured by said amplifier circuit with pH values within a cell outside the nanopipette structure.
In further embodiments, the present invention comprises a method as described above wherein said polymer coating is applied by binding a chitosan material layer to the nanopipette; further comprising connecting said working electrode to an amplifier that conducts and measures an I-V curve for ionic current through the nanopipette.
In certain embodiments, the present invention comprises a method of measuring pH in a cell, comprising (a) providing a nanopipette structure, having an interior layer responsive to pH ions, and being electrically connected by a working electrode to a circuit comprising a potentiostat configured to measure ionic current through said nanopipette structure versus potential at various potentials in a electrochemical cell containing said nanopipette structure and a reference electrode; (b) inserting said nanopipette structure into a cell in said electrochemical cell; and (c) using said circuit to measure said ionic current, wherein said current is correlated to a known pH.
In some embodiments, the present invention comprises a method as described above wherein said inserting said nanopipette comprises using an SICM and an x-y-z controller. In certain embodiments, the present invention comprises a method wherein said circuit further comprises an amplifying circuit comprising a detection circuit with gain controls and with a low pass filter for detecting ionic currents. In certain aspects and embodiments, the present invention comprises a method wherein said interior layer comprises a layer of chitosan material having an average pore size between 50 nm and 150 nm diameter. The chitosan may have a monomer number between about 30,000 and 60,000 units, and may comprise a hemeprotein attached thereto.
In certain embodiments, the present invention comprises a method as described above wherein the interior layer comprises a polymer coating that is selected from the group consisting of sulfonated tetrafluorethylene copolymer (Nafion®), poly-1-lysine, and alginate.
In certain embodiments, the present invention comprises a method as described above wherein the circuit comprises a potentiostat connected to the reference electrode and responsive to input from an amplifier in turn having an input from the working electrode. In further embodiments, the present invention comprises a method wherein the potentiostat is connected to a counter electrode connected to the reference electrode. The working electrode and the counter electrode may be Ag/AgCl.
In some embodiments, the present invention comprises a method as described above wherein the voltage is between 0.5V and 0.7V. In futher embodiments, the present invention comprises a method wherein a variety of voltages is set on the potentiostat.
In certain embodiments, the present invention comprises a method as described above wherein the pH value is taken on a cancerous cell and compared to a pH on a noncancerous cell.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. Generally, nomenclatures utilized in connection with, and techniques of, cell and molecular biology and chemistry are those well-known and commonly used in the art. Certain experimental techniques, not specifically defined, are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. For purposes of clarity, the following terms are defined below.
Ranges: For conciseness, any range set forth is intended to include any sub-range within the stated range, unless otherwise stated. As a non-limiting example, a range of 120 to 250 is intended to include a range of 120-121, 120-130, 200-225, 121-250 etc. The term “about” has its ordinary meaning of approximately and may be determined in context by experimental variability. In case of doubt, the term “about” means plus or minus 5% of a stated numerical value.
The term “nanopipette” means a hollow self-supporting, inert, non-biological structure with a conical tip opening of nanoscale, i.e., a nanopore, having a tip opening of 0.05 nm to about 500 nm, preferably about (+ or −20%) 50 nm or about 80 nm or about 100 nm. The hollow structure may be e.g. glass or quartz, and is suitable for holding inside of it a fluid which is passed through the tip opening. The interior of the nanopipette is selected or modified to minimize nonspecific binding of analyte. The interior of a nanopipette typically is in the form of an elongated cone, with a uniform wall thickness of a single layer of quartz or other biologically inert material, and is sized to allow insertion of an electrode that contacts solution in the nanopipette. The nanopipettes used herein typically have a single bore, but nanopipettes with multiple concentric bores can be prepared by pulling dual bore capillary tubes. The outer diameter is typically less than about 1 μm in the tip region.
The term “nanopore” means a small hole in an electrically insulating membrane, preferably the tip of a nanopipette, as described. The nanopore will be in a tip region, which is the last few mm of the nanopipette bore, adjacent the nanopore. The nanopore, as described below, is sized so that small molecular complexes will affect movement of ions and molecules through the nanopore. The nanopore is designed to function in a device that monitors an ionic current passing through the nanopore as a voltage is applied across the membrane. The nanopore will have a channel region formed by the nanopipette body, and, preferably, will be of a tapered, e.g. frusto-conical configuration. By pulling a quartz capillary as described below, a reproducible and defined nanopore shape may be obtained.
As described below, the term “nano-pH probe” refers to a device comprising a nanopipette containing an electrode inside and a functionalized interior portion, further comprising circuitry connected to the electrode to sense small changes in ionic current in the nanopipette, indicative of a pH in a material.
The term “quartz” means a nanopipette media is a fused silica or amorphous quartz, which is less expensive than crystalline quartz. Crystalline quartz may, however, be utilized. Ceramics and glass ceramics and borosilicate glasses may also be utilized but accuracy is not as good as quartz. The term “quartz” is intended and defined to encompass that special material as well as applicable ceramics, glass ceramics or borosilicate glasses. It should be noted that various types of glass or quartz may be used in the present nanopipette fabrication. A primary consideration is the ability of the material to be drawn to a narrow diameter opening. The preferred nanopipette material consists essentially of silicon dioxide, as included in the form of various types of glass and quartz. Fused quartz and fused silica are types of glass containing primarily silica in amorphous (non-crystalline) form.
The term “chitosan” is used herein in its conventional sense, to refer to a linear polysaccharide composed of randomly distributed β-(1-4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). The amino group in chitosan has a pKa value of ˜6.5, which leads to a protonation in acidic to neutral solution with a charge density dependent on pH and the % DD (degree of deacetylation) value. This makes chitosan water soluble and a bioadhesive which readily binds to negatively charged surfaces such as mucosal membranes. Chitosan enhances the transport of polar drugs across epithelial surfaces, and is biocompatible and biodegradable. Chitosan is produced commercially by deacetylation of chitin, which is the structural element in the exoskeleton of crustaceans (crabs, shrimp, etc.) and cell walls of fungi. The degree of deacetylation (% DD) can be determined by NMR spectroscopy, and the % DD in commercial chitosans is in the range of 60-100%. On average, the molecular weight of commercially produced chitosan is between 3800 to 20,000 daltons.
The term “chitosan material” means the naturally occurring chitosan polysaccharide described above, and various allotropes and derivatives, described, e.g. in Rinaudo, “Chitin and chitosan: Properties and application,” Prog. Polym. Sci 31:603-632 (2006). As described there, chitosan can have a variety of degrees of solubility, acetylation or molecular weight. As described, the chitosan material or native chitosan may be formed in a layer that is thin and dilute so as to result in nanoscopic or microscopic pores that receive ions within the layer.
The term “highly hydroxylated” is used in connection with quartz materials (SiO2) used in the present nanopore bearing hydroxyl groups. For example, α-quartz (0001) can be hydroxylated as described in Yang et al. “Water adsorption on hydroxylated α-quartz (0001) surfaces,” Phys. Rev. b 73:035406 (2006). See e.g. Konecny et al. “Reactivity of free radicals on hydroxylated quartz surface and its implications for pathogenicity experimental and quantum mechanical study,” J. Environ Pathol Toxicol Oncol. 2001; 20 Suppl 1:119-32.
The term “hemeprotein” refers to a metalloprotein containing a heme prosthetic group—an organic compound that allows a protein to carry out several functions that it cannot do alone. The heme contains a reduced iron atom, Fe2+ in the center of a highly hydrophobic, planar, porphyrin ring. Hemeproteins include hemoglobin, myoglobin, neuroglobin, cytoglobin and leghemoglobin.
The term pH has the commonly accepted definition, i.e., a measure of acidity or alkalinity of water soluble substances (pH stands for ‘potential of Hydrogen’). A pH value is a number from 1 to 14, with 7 as the middle (neutral) point. Values below 7 indicate acidity which increases as the number decreases, 1 being the most acidic. Values above 7 indicate alkalinity, which increases as the number increases, 14 being the most alkaline. This scale, however, is not a linear scale like a centimeter or inch scale (in which two adjacent values have the same difference).
The term “logic means” means a logical circuit that is programmable or is programmed to convert a series of electronic signals to one or more tangible measurable value(s). For example, U.S. Pat. No. 4,124,899 to Birkner, et al shows a programmable logic circuit which is referred to as a programmable array logic, or PAL, circuit. The present logic means produces a pH value based on a given change in ionic current though the described probe (containing a nanopipette sensitive and responsive to hydrogen ions and containing an electrode) relative to a reference probe. As will be appreciated, any required computer program may be loaded onto a computer, including without limitation a general purpose computer or special purpose computer, or other programmable processing apparatus to produce a machine, such that the computer program instructions which execute on the computer or other programmable processing apparatus create means for implementing the functions. Accordingly, appropriate logic means as used here may be software provided for use by a user on an extrinsic computer programmed to sense and control the present device.
The present invention provides a means and device that can measure pH within a single cell, as well as changes in pH in the cell, without the necessity of any exogenous materials. The measurement is in real time, and can track changes in pH while the nanopipette is inserted into the cell and the sensitive circuit measures ionic current at the nanopore opening of the nanopipette, which is in the cell, e.g. in the cytoplasm, nucleus, mitochondrion, etc. The detection circuit provides a high degree of sensitivity on the order of 0.1-0.01 pH units, with a described example showing detection of 0.09 pH units. Furthermore, the system has a large dynamic range, between about pH 2-11.
The present invention further comprises a method and device for measuring a current that varies in response to pH changes in a solution in a cell. In one method, the device is calibrated using different standard pH solutions. A calibration curve reflects current vs. pH and is calculated and used to measure pH in the sample. A preferred voltage setting for a current measurement is 0.6V, or within a range of 0.5V-0.7V. The measured current (measured with the potentiostat) increases as the pH in the sample decreases. The potentiostat reports the current and is swept across a voltage range to determine various responses and/or to determine an optimal operating voltage. Typically the applied voltage is swept from about 0.2 to 0.6V. In a presently preferred method of use, the potentiostat applies a chosen voltage to the system and records the current, that is correlated to the pH.
In one aspect, the invention comprises the use of a controlled concentration of highly porous chitosan material that forms a molecular sponge to trap ions including H+ to increase ionic rectification, as shown in the traces of
Ionic current rectification, as is known in the art, is characterized by an increase of the ion conduction for one voltage polarity but a decrease of it for the same voltage magnitude with opposite polarity, producing an asymmetric I-V curve. A positive and negative voltage is applied to the electrodes; the difference between the ionic current response is indicative of the pH in the pore, and, as a result, in the cell.
The highly porous chitosan material may be prepared by using a relatively low concentration of chitosan material in coating the nanopipette interior pore. In some aspects, the chitosan material is applied in a concentration of between 0.25% to 1% chitosan material. The chitosan material is directly bonded to hydroxyl groups on the quartz material of the nanopipette, in the vicinity of the interior of the nanopore. Preferably, short chain chitosan material is used, having a monomer number of about 30,000 to 60,000. Bonding may be enhanced by reacting the quartz with chemicals to increase surface functionality, such as sulfuric acid, hydrogen hydroxide, ammonium hydroxide, etc. This will serve to reduce contaminants and hydroxylate the quartz.
In another aspect, the present invention comprises modification of the chitosan material layer so as to contain a material sensitive to the redox potential in the cell. The redox potential of a cell is used in the conventional sense, to refer to a measure used to infer the direction and free energy cost of reactions involving electron transfer. The redox potential, or more accurately the reduction potential, of a compound refers to its tendency to acquire electrons and thereby to be reduced.
For example, one may use the redox potential to connect these two molecular protagonists, and estimate an upper bound on the number of ATP molecules that can be produced from the oxidation of NADH (produced for example in the TCA cycle). The redox potential of a cell may be perturbed by various diseases.
In another aspect, the present invention comprises a sensitive electronic device and arrangement of the working and reference electrodes used between the bulk solution and the interior of the nanopipette. The reference electrode also functions as an auxiliary electrode and is connected to a potentiostat. The system functions by scanning the potential of the working electrode at a given potential range with respect to the reference electrode by measuring the current at an auxiliary electrode. An i/V amplifier is bridged by a filter selection and a sensitivity selection circuit. These are used to adjust the detectable current range based on the current passing through the electrolyte solution.
Referring now to
As described below, the nanopipette (nano-pH probe) is operatively connected to a micromanipulator (not shown) such as a scanning ion conductance microscope that detects current feedback for positioning the nanopiette and inserting it into a selected cell.
As shown schematically in
The two circuits, shown in
The system functions by scanning the potential of the working electrode at a given potential range with respect to the reference electrode by measuring the current at an auxiliary electrode. The potentiostat is connected to a gain selector 154 used to control the frequency at which the signal amplification is done. The working electrode (in the nanopipette) is connected to the input of an i/V (current to voltage) amplifier 158 that outputs to a digital attenuator 156 and from there back—as described above—to the reference electrode to create a feedback circuit. The i/V amplifier 158 further is bridged by a filter selection 162 and a sensitivity selection circuit 164. These are used to adjust the detectable current range based on the current passing through the electrolyte solution.
The amplifier 158 outputs to a low pass filter 146 and the output connection 148 (circle) shown connected to the low pass filter. This and the potentiostat provide input terminals (circles) to a monitor that can measure and record ionic current through the nanopipette. The monitor may comprise a computer programmed to monitor and control signals produced by the above components.
The computer will contain logic means that will convert a detected current, from the potentiostat circuit, to a pH value, based on a calibration established during use, or, alternatively built into the device.
The single cell in which the nanopipette is inserted may be a cell in culture in liquid or immobilized on a substrate. The single cell may be part of a tissue. It is identified microscopically and the nanopipette is controlled by an x-y-z controller to be inserted into the cell. Scanning ion-conductance microscopy (SICM) may be used for this purpose.
The present nano-pH probe can be used as an analytical tool to illuminate the relationship between pH and a variety of diseases. The present nano-pH probe may utilize scanning ion conductance microscopy (SICM) principles32. Nanopipettes are electrical devices that can measure the differences in ionic current at a nanopore. Their small size enables direct, real-time in vitro measurements with high spatial resolution and reduced invasiveness, allowing the monitoring of intracellular changes of an individual cell over the course of drug treatment. Recently nanopipettes have gained importance as novel sensing tools and have been investigated for the detection of proteins33,34, metal cations32,35, DNA36 and carbohydrates37. Quartz nanopipettes can be functionalized with various recognition materials. In this work, chitosan material, a biopolymer, is used as a pH-sensitive surface coating of the internal surface of nanopipettes. Chitosan is biocompatible and has low-toxicity which makes it ideal for biological purposes. It possesses unique film-forming ability, high adherence to surfaces and remarkable mechanical strength. In addition, chitosan has been shown as a selective coating for biosensor fabrication38-40.
It is demonstrated here the development and characterization of chitosan-modified quartz nanopipettes for pH measurements in physiological buffers and cell media. The chitosan-modified nanopipettes were then used for the direct measurement of intracellular pH in four different cells types, including human fibroblast, HeLa, MCF-7 and MDA-MB-231. As described, in vitro specificity of chitosan-modified nano-pH probes using a chloride channel blocker can be achieved. The nano-pH probe is a powerful candidate not only to investigate cell heterogeneity in a variety of pathologic states, including cancerous tumors, but also neurodegenerative states and aging.
The present device has been shown to overcome the limitations of intracellular pH measurement at the single-cell level. Direct measurement of intracellular pH has been demonstrated in a new way via simple physisorption of a chitosan material into a quartz nanopipette. This approach takes advantage of a pH-responsive chitosan polymeric layer and the small size of a nanopipette for intracellular pH measurement at the single-cell level. Described here is nano-pH probe prepared through physisorption of chitosan, a biocompatible pH-responsive polymer, onto highly hydroxylated quartz nanopipettes with extremely small pore size (˜97 nm). Changes of pH alter the surface charge of chitosan which can be measured as a change in ionic current at the nanopore. The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 pH units. Leveraging a scanning ion conductance microscope customized for single-cell navigation, we were able to insert nano-pH probes into individual cells. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the nano-pH probe. In vitro results showed that chitosan-functionalized nanopipettes measure intracellular pH selectively with high temporal resolution. The average intracellular pH levels were 7.37±0.29, 6.75±0.27, 6.91±0.20 and 6.85±0.11 for human fibroblast, HeLa, MCF-7 and MDA-MB-231, respectively. These results show good separation between fibroblast and cancerous cells, which have a more acidic cytoplasmic environment than non-cancerous cells. Additionally, our findings reveal that individual cells within a population may differ in their intracellular pH. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing cellular pH response to pharmaceutical manipulations. An NPPB exposure experiment demonstrates that the nano-pH probe enables real-time, continuous interrogation of a single cell upon biochemically induced changes in intracellular pH.
Our data show that chitosan-modified nanopipette sensing technology is a powerful approach for interrogating single-cell pH levels with high spatial and temporal resolution with high selectivity and sensitivity. Further application of this nano-pH probe technology may provide a deeper understanding of cell heterogeneity and drug resistance. To achieve this aim, we are working on the development of a fully automated system for high-throughput screening of cell populations over the course of drug treatment. Additionally, we will use nano-pH probes to investigate pH changes and differences in tumorous microenvironments (e.g. tumor tissues).
General Method and Materials
Reagents and materials. Chitosan (low molecular weight), 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), sodium phosphate dibasic and monobasic were purchased from Sigma Aldrich. Sodium chloride (ACS grade), hydrochloric acid, sodium hydroxide and hydrogen peroxide were obtained from Fisher Scientific. Acetic acid (glacial) was supplied from Riedel-de-Haen. 2-propanol was obtained from Spectrum Chemicals. 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) was bought from Invitrogen. Dimethyl sulfoxide (anhydrous) was supplied from Fluka. Minimum essential medium eagle (MEM), Dulbecco's modified eagle medium (DMEM) and trypsin were purchased from CellGro while fetal bovine serum (FBS) and penicillin-streptomycin from Gibco. All aqueous solutions are prepared in distilled, deionized water (Millipore, Synthesis System) with a resistivity of 18.2 Ωcm.
Preparation of nano-pH probe. Nanopipettes were fabricated from quartz capillaries with filament (QF100-70.7.5, Sutter Instrument). Prior to pulling, capillaries were treated with piranha solution (sulfuric acid:hydrogen peroxide, 3:1 v/v) (Caution: piranha solution′ reacts violently with organic materials and may become extremely hot when prepared.) and rinsed thoroughly with distilled water and 2-propanol. Treated capillaries were kept in 2-propanol until use to prevent contamination. Capillaries were pulled using a P-2000 laser puller (Sutter Instrument) with a two-line program with following parameters; Line 1: Heat 700, Fil 4, Vel 20, Del 170, Pull 0 and Line 2: Heat 680, Fil 4, Vel 40, Del 170, Pull 200. The resulting nanopipettes had a pore diameter of ˜97 nm detected by a FEI Quanta 3D field emission microscope. Nanopipettes were stored in a sealed box until modification. Nanopipettes were functionalized by backfilling 10 μl of 0.25% chitosan solution and centrifuged at 4000 rpm to assure the coverage of the nanopipette tip with chitosan matrix. After centrifugation, excess chitosan was aspirated and nanopipettes were left to air-dry overnight. Dried nanopipettes were backfilled with 10 mM phosphate buffer saline (PBS) solution at pH 7.0, then centrifuged to remove residual air bubbles trapped at the tip of nanopipettes. Once filled all nanopipettes were kept in 10 mM PBS (pH 7.0) until pH measurements to prevent clogging of the nanopore.
Sensing setup. To carry out analytical characterization experiments of chitosan-modified nanopipette sensors, a two-electrode setup connected to a potentiostat (1030C, CH Instruments Inc.) was used for sensing. A 125 μm platinum wire (Goodfellow Corporation) placed into nanopipettes filled with electrolyte served as the working electrode while a pseudo Ag/AgCl electrode placed in bulk solution (PBS or cell media) served as the reference electrode. Linear sweep voltammetry was utilized for all in vitro measurements with a scan rate of 0.1 V/sec.
Intracellular measurements were performed by combining the potentiostat and scanning ion conductance microscope (SICM) with a low-noise mechanical switch. The SICM setup consisted of an Axopatch 200B amplifier (Molecular Devices) for current feedback measurements, a MP-285 motorized micromanipulator (Sutter Instrument) for coarse positioning of the nano-pH probe, a piezo stage (NanoCube, Physik Instrumente) for fine positioning and insertion of the nano-pH probe sensors, and a programmable interface for hardware control of the setup. This system is run by custom software written in LabVIEW (National Instruments). All experiments with cells were conducted on an inverted fluorescence microscope (Olympus IX 70) equipped with an eyepiece camera (Dino-Eye, Big C).
Cell culture. HeLa, MCF-7, MDA-MB-231 and human fibroblast cells were cultured in a conditioned environment with 5% CO2 and 90% humidity at 37° C. HeLa, MCF-7 and MDA-MB-231 cells were cultured in 1×MEM, while human fibroblasts in 1×DMEM. All media were supplemented with 10% FBS and 1% Penicillin-Streptomycin.
Fluorescence microscopy. MDA-MB-231 cell cultures were exposed to a pH-sensitive fluorescent indicator, BCECF-AM. The working solution was prepared to a concentration of 1 μM in Hank's Buffered Salt Solution (HBSS) and incubated at 37° C. for 15 min before fluorescent imaging. Cells were washed with Dulbecco's phosphate-buffered saline (DPBS) before loading of 1 μM BCECF-AM solution. After incubation, excess fluorescent dye was rinsed off the cells with HBSS was loaded on the culture for imaging.
For intracellular pH buffer calibration, cell cultures were exposed to complete pH calibration buffer prepared according to the protocol supplied with the Intracellular pH Calibration Buffer Kit (Life Technologies, P35379), and were incubated at 37° C. for 10 min before imaging. Intracellular pH calibration was done in three replicates. All fluorescence microscopy analyses were carried out with a Leica SP5 confocal microscope using the Leica Application Suite Advance Fluorescence (LAS AF 3) software. Further image analyses were performed with Fiji-ImageJ software.
The measurement principle of nanopipettes is based on the ionic current at the tip. This ionic current is highly dependent on the pore size and surface charge of the nanopipette34. The surface charge of a quartz nanopipette is negative due to dissociation of silanol groups at the glass-liquid interface. Quartz undergoes protonation at extremely acidic pH values41. These surface properties of quartz reduce pH sensing capabilities, making bare nanopipettes inappropriate for measuring very small pH changes. Limitations associated with the low sensitivity of bare quartz surfaces can be overcome through the incorporation of pH responsive polymeric entities onto nanopipette surfaces. Here, we employed chitosan as the pH sensitive surface coating. Chitosan, with a strong positive charge at acidic pH, is attracted to hydroxyl moieties on the negatively charged quartz surface through electrostatic interactions. In addition to alterations of the surface charge, the thickness of the chitosan layer has been shown to change with pH which may enhance the sensitivity of the nanopipette42,43. To evaluate the presence and impact of the chitosan layer on the nanopipette surface, we monitored the changes in current responses as a result of surface modification.
Once the presence of the chitosan layer was confirmed with SEM and electrochemistry, analytical characterization of the functionalized nanopipettes was conducted using linear sweep voltammetry. The potential range spanned from −0.5 to 0.5 V with a scan rate of 0.1 V/sec. The modulation of pH was achieved by a conventional acid-base titration approach. Calibration of chitosan-modified nanopipettes was performed by consecutive additions of 20 μl of first 1 M NaOH and second HCl into 0.1 M PBS (pH 7.0). Current rectification of modified nanopipettes at +/−0.5 V changed in response to the changing pH of the buffer solution, as expected with the alteration of charge on the chitosan layer. Chitosan contains a glucosamine residue on its polysaccharide backbone (pKa˜6.5) making chitosan pH-responsive38. pH values below the pKa protonate the chitosan layer making the nanopipette surface positively charged, whereas basic conditions deprotonate chitosan's amine functional group, increasing the net negative charge at the surface (
Our motivation for developing a solid nanopore pH probe is to measure intracellular pH at the single-cell level and to identify cancer cells with their distinctive metabolic characters. To perform intracellular pH measurements, chitosan-modified nanopipettes were further calibrated in cell culture media, MEM and DMEM. As cell media contain various amino acids, vitamins and other ingredients, optimum working parameters were different from those determined for PBS. The scanned potential range was from −0.2 to 0.6 V with a scan rate of 0.1 V/sec. The sensitivity of chitosan-modified nanopipettes for pH changes was the highest at 0.6 V.
Direct measurement of intracellular pH is challenging due to the small size of cells and the complexity of the physiological matrix. While the physiological pH level is marginally alkaline, the intracellular pH level of individual cells in a large population and subcellular compartments is unknown. Conventionally, fluorescence dyes (e.g. BCECF-AM, oregon green) are utilized for indirect detection of pH in cells30. Although these pH indicators reveal an approximation of pH over a large cell population, there are several disadvantages of using fluorescence dyes: i) low sensitivity due to short pH range, ii) fast photobleaching, iii) cytotoxicity. Additionally, accumulation of these dyes in certain organelles and their rate of leakage can result in incorrect interpretations. Our studies using a conventional pH indicator, BCECF-AM, to measure intracellular pH of MDA-MB-231 cells have proven the drawbacks of using fluorescence for accurate and sensitive evaluation of intracellular events. In these studies, in which fluorimetric intracellular pH measurements were made, cells were exposed to BCECF-AM and incubated for 15 min. Then, cells were washed and exposed to nigericine containing cellular pH calibration buffer (pH 7.5, 6.5, 5.5 and 4.5) for 10 min. BCECF-AM has dual excitation wavelengths; therefore, images were taken at 458 and 488 nm. Bright field and fluorescence micrographs were obtained for the two excitation wavelengths of each pH value. A ratiometric calibration curve was obtained using fluorescence intensities of 16 to 23 individual cells (data not shown). One group of cells served as negative control (without BCECF-AM) to evaluate the presence of intracellular autofluorescence. In the absence of the pH dye, there was no observable fluorescence for MDA-MB-231. Cells exposed to BCECF-AM were used to estimate the intracellular pH values of individual cells. The average intracellular pH value obtained from 10 individual cells was calculated to be 6.78 (±0.83). However, the micrographs taken after BCECF-AM exposure revealed that fluorescence intensity over the cell body varies (data not shown). Fluorescence intensity was higher where cells were thicker. Additionally, any two regions in close proximity to one another in an individual cell were found to have large variation in pH values. These variations can be attributed to (i) unequal distribution or accumulation of the fluorescence dye; (ii) cross-reactivity of the fluorescence dye with another molecule. Another drawback of fluorescence measurements is the sample preparation step that requires the frequent change of media, which can stress cells and alter the basal intracellular levels. Moreover, the use of fluorescence dyes does not allow continuous interrogation of a single cell over the course of treatment, such as in drug testing, or toxicity measurements, because the presence of these dyes along with the compound of interest may cause false experimental conclusions by changing the physiology of a cell or by cross-reacting with the compound to be tested. In other words, continuous interrogation of a single cell over the course of time for evaluating the cellular impact of therapeutics, channel activators, or toxins cannot be carried out with conventional fluorescence probes.
In order to directly and accurately measure intracellular pH, chitosan-modified nanopipettes were inserted in the cytoplasm of the cells in culture. We used this sensing technology, for the first time, for the direct monitoring of intracellular pH of human cancerous and non-cancerous cell lines, including human fibroblast, HeLa, MCF-7 and MDA-MB-231. Human fibroblast cells are selected as a non-cancerous model to investigate intracellular pH levels at normal cytoplasmic conditions. HeLa cell lines are the most commonly used human cancer type due to their rapid and continuous growth in cell culture. Additionally, because of reports of contamination and heterogeneity of HeLa cells, determination of the intracellular pH levels of these cells may allow us to evaluate the cell heterogeneity44. MCF-7 and MDA-MB-231 are distinct breast cancer cell lines. MCF-7 is a hormone-responsive cell line and its growth is stimulated with estrogen; MDA-MB-231 derives from an invasive breast cancer which was found to be highly metastatic45. We chose to interrogate these two breast cancer cell lines because they exhibit different drug sensitivities and we sought to determine whether this could be correlated with differences in the intracellular pH levels.
Chitosan-modified nanopipettes were inserted to individual cells using a customized scanning ion conductance microscope which detects current feedback for positioning the nanopipettes. Recently we have demonstrated that this custom-built platform can perform nanobiopsies at the single-cell level for genomic investigations46.
From voltammetric current responses at 0.6 V versus Ag/AgCl, the calculated intracellular pH levels of individual cells and average pH values for all cell lines are shown in
We also used the nano-pH probes to investigate the metabolic differences between non-cancerous and cancerous cells. As cancer cells have a faster metabolic rate compared to non-cancerous cells, production of acidic species and CO2 in cancer cells is higher as well. Using the chitosan-modified nano-pH probe in 14 individual HeLa cells for intracellular pH measurements, we found the average pH for HeLa cells to be 6.75±0.27 (
To compare whether a similarly acidic intracellular environment is present in other cancer cell lines, we performed pH measurements on breast cancer lines. Using the nano-pH probe, was observed an average intracellular pH level for 14 individual MCF-7 cells of 6.91±0.20 (
To more fully deploy the pH nano-probe described herein, one builds a fully-automated high-throughput robotic system that will allow us to interrogate hundreds of cells in a range of minutes. Cells having lower or higher pH values compared to the general population of cells will be identified and then tagged with a molecular marker to nanobiopsy for DNA and RNA sequencing.
The present nano-pH probe can be used to monitor intracellular pH changes during drug therapy. To this end, the present nano-pH probe was arranged for continuous monitoring at a single cell during the addition of a known chloride channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB). NPPB has been shown previously to block chloride channels in renal epithelial and macrophage cells, with a resulting increase in acidity of the intracellular environment. Conventionally, the change in pH has been measured indirectly by introduction of fluorescent dye (BCECF-AM)47,48. Thus, this pharmaceutical manipulation test not only serves to demonstrate the capability of real-time measurement of nano-pH probes but also the specificity towards pH detection. To obtain a baseline, nano-pH probes were inserted in MDA-MB-231 cells and consecutive pH measurements were performed for every 21 second for 7 min. This real-time pH monitoring in MDA-MB-231 cells showed minimal drift around a value 7 over the course of measurement (
The device described above can be further modified with a layer attached to the chitosan layer on the nanopipette that is responsive to oxidation or reduction of components in the cell.
The above-described chitosan-modified quartz nanopipettes can be modified with immobilized proteins such as hemeproteins and enzymes. This immobilization to chitosan can be realized through either a peptide bond formation mechanism or catalytic reactions as chitosan possesses carboxylic groups and randomly distributed glucosamine residues on its polymeric backbone. Immobilization of redox active small proteins onto the chitosan layer makes the so-functionalized nanopipette sensitive to highly reactive radicals such as reactive oxygen (ROS) and nitrogen species (RNS), and hydrogen peroxide. For details on ROS, see Salehi, et al., “Hemeproteins including hemoglobin, myoglobin, neuroglobin, cytoglobin and leghemoglobin,” J. Photochemistry and Photobiology B: Biology 133 11-178 (2014).
These radical (e.g. reactive oxygen species) are known to contribute to many disease states, such as cancer, aging, stroke, Parkinson's and Alzheimer's diseases. Therefore the measurement of physiological levels of ROS and RNS is of great importance.
In the presence of ROS or RNS, redox sensitive surface functionalities inside the nanopipette undergo either reduction or oxidation depending upon the oxidation states. Such changes in the oxidation state result in a change of the surface charge. The change in surface charge is in correlation with the amount of ROS or RNS present in the aqueous environment. The detection of reactive species is done by measuring the fluctuations of ionic current at the nanopore when a potential difference is applied across the quartz nanopore.
The device described above can be further constructed in a multiplexed array of nano-pH probes. In addition, a number of surface recognition materials can be added to the interior of various nanopipettes used in the array. The number nanopipette structures can be varied, and not all of them may contain the chitosan pH sensing coating.
One possible method for making conical nanopipette structures for use in this array is described in Meyyappan, U.S. Pat. No. 9,182,394. This patent describes an array of nanopipette channels, formed and controlled in a metal-like material that supports anodization. As described there, a thin substrate of anodizable metal such as Al, Mg, Zn, Ti, Ta and/or Nb is anodized at temperature T=20-200° C. in a chemical bath of pH=4-6 and electrical potential 1-300 Volts, to produce an array of anodized nanopipette channels, having diameters 10-50 nm, with oxidized channel surfaces of thickness 5-20 nm. A portion of exposed non-oxidized anodizable metal between adjacent nanopipette channels, of length 1-5 μm, is etched away, exposing inner and outer surfaces of a nanopipette channel.
In addition, magnetic structures 168a, 168b are provided to provide a removable attachment between the support structure 163 and the insulating layer 166. This provides access to nanopipettes in the array and allows modification of pipettes, as well as filling them with the supporting electrolyte.
The modification is done prior to insertion of the electrodes (161) by casting the inner surface of pipette structures with polymers or recognition molecules. The surface coating process can be performed for the entire inner surface but not necessarily since the ionic current changes are dominated by the first 0.1 to 5 μm of the nanopore.
These surface recognition materials can be polymers including Nafion®, phenylenediamine, poly-1-lysine, poly-acrylic acid and polypyrrole; enzymes including oxidoreductase and dehydrogenase families; proteins including avidin and prion; and antigens, RNA fragments and aptamers. These substances can be utilized alone or in combination for the functionalization of individual or an array of nanoprobes for targeted sensing purposes. The surface modification protocols must be optimized for each recognition material including surface chemistry for immobilization, concentration, incubation time and temperature. The nanopipette filling solution's properties such as pH, electrolyte type and concentration for each sensing array should be evaluated for the highest detection sensitivity.
After necessary surface modifications are completed and filling electrolyte is introduced, a customized printed circuit board (PCB) with built-in sensing electrodes is placed on top of the nanopipette array by aligning the electrodes to perforations. Sensing electrodes are metallic including silver, platinum, gold; or redox-based (Silver-silver(I)chloride) or non-metals including glassy carbon, graphite and boron-doped diamond. When electronics are inserted into the nanopipette array, inner components of the nanopipette array are completely sealed. Magnetic structures 168 made of neodymium ensure both electronics and the nanopipette array are locked to each other. The electronic structure of this invention contains all the necessary circuitry for individual channels and is able to perform synchronized or customized sensing.
The above specific description is meant to exemplify and illustrate the invention and should not be seen as limiting the scope of the invention, which is defined by the literal and equivalent scope of the appended claims. Any patents or publications mentioned in this specification are intended to convey details of methods and materials useful in carrying out certain aspects of the invention which may not be explicitly set out but which would be understood by workers in the field. Such patents or publications are hereby incorporated by reference to the same extent as if each was specifically and individually incorporated by reference and contained herein, as needed for the purpose of describing and enabling the method or material referred to.
This application claims priority from U.S. Provisional Patent Application No. 62/120,624 filed on Feb. 25, 2015, which is hereby incorporated by reference in its entirety.
This invention was made with Government support under Contract Number U54CA143803 awarded by the National Cancer Institute, Contract Number P01-35HG000205 awarded by the National Institutes of Health, and Contract Number R21NS082927 awarded by the National Institute of Neurological Disorders and Stroke. The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2016/019333 | 2/24/2016 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62120624 | Feb 2015 | US |
