Biosensors for chloride ions

Abstract

This invention provides biosensors, cell models, and methods of their use for monitoring chloride ion, where the biosensors can include targeting domains, sensing domains and reporting domains. Biosensors can be introduced into cells reprogrammed to represent experimental or pathologic cells of interest, including as detectors of chloride ions, as TempoChloro accomplishes.

Claims

1. A chloride ion sensor peptide construct comprising: a chloride ion sensing domain having a sequence at least 95% identical to SEQ ID NO: 1; and, a fluorescent reporter domain, wherein the fluorescent reporter domain is adapted to change fluorescent emissions characteristics in response to chloride ion concentration; and wherein the fluorescent reporter domain has a fluorescent emission range of 500 nm to 700 nm and a fluorescent excitation range of 400 nm to 700 nm.

2. The chloride ion sensor peptide construct of claim 1 wherein the fluorescent reporter comprises a sequence at least 95% identical to SEQ ID NO: 3.

3. The chloride ion sensor peptide construct of claim 1 wherein the chloride ion sensing domain binds to chloride ion.

4. The chloride ion sensor peptide construct of claim 1 further including one or more targeting peptide segments with affinity for a cellular target.

5. The chloride ion sensor peptide construct of claim 4 wherein the targeting peptide segments are one or more of: an NLS tag, an ER tag, an endosome tag, a human endosome sequence tag, a ciliary tag, a human synapsin tag, and a human synaptic vesicle tag.

6. A chloride ion sensor peptide construct comprising: a chloride ion sensing domain having a sequence at least 95% identical to SEQ ID NO: 1; and, a fluorescent reporter domain; wherein the fluorescent reporter domain is adapted to change fluorescent emissions characteristics in response to chloride ion concentration; and wherein the fluorescent reporter domain has a fluorescent emission range of 500 nm to 700 nm and a fluorescent excitation range of 400 nm to 700 nm.

7. The chloride ion sensor peptide construct of claim 6 wherein the fluorescent reporter comprises a sequence at least 95% identical to SEQ ID NO: 3.

8. The chloride ion sensor peptide construct of claim 6 wherein the chloride ion sensing domain binds to chloride ion.

9. The chloride ion sensor peptide construct of claim 6 further including one or more targeting peptide segments with affinity for a cellular target.

10. The chloride ion sensor peptide construct of claim 6 wherein the targeting peptide segments are one or more of: an NLS tag, an ER tag, an endosome tag, a human endosome sequence tag, a ciliary tag, a human synapsin tag, and a human synaptic vesicle tag.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

(2) FIG. 1 shows sample identification with the TempoChloro biosensor, which is chloride ion sensitive and picrotoxin sensitive, expressed in transformed neural human IPS progenitor cells, where detection is based on relative fluorescence.

(3) FIG. 2 shows sample identification with the TempoChloro biosensor, expressed in transformed neural human IPS progenitor cells, based on relative fluorescence.

(4) FIG. 3 shows sample identification with the TempoChloro biosensor, expressed in human IPS derived astrocytes, based on relative fluorescence.

DETAILED DESCRIPTION

Definitions

(5) As used in this specification and the appended claims, the singular forms a, an and the include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a surface includes a combination of two or more surfaces; reference to DNA peptide protein compound includes, respectively, multiple individual molecules, and the like.

(6) iPSC refers to inducible pluripotent stem cells; iN refers to inducible neurons; iG refers to inducible glial cells (including astrocytes); and iNPCs inducible neural progenitor cells.

(7) Near-Infrared refers to light wavelengths ranging from about 600 nm to about 1400 nm.

(8) The term conservative variant includes modifications of given sequences that result in conserved function. For example, in the context of nucleic acids, owing to the degeneracy of the genetic code, silent substitutions (i.e., substitutions in a nucleic acid sequence which do not result in an alteration in an encoded polypeptide) are an implied feature of every nucleic acid sequence which encodes an amino acid.

(9) Similarly, conservative variants in the context of peptide sequences can be expected to retain function. For example, Guo, Protein Tolerance to Random Amino Acid Change, (PNAS 101:9205-10; 2004), demonstrates that one of skill can modify peptides successfully even without detailed knowledge of the ways in which a protein's structure relates to its functional usefulness . . . Guo finds only 25% of random mutations lead to substantial loss of activity. Guo extensively discusses how one of skill can take into consideration active site location, alpha helices, beta sheets, hydrophobic interactions, turns and loops, conserved sites and the like to intelligently avoid loss of activity, e.g., by substitution avoidance at key positions or with conservative amino acid substitutions. Further, Guo states that his database can be a valuable resource for predicting the effects of mutations on protein function . . . Substitutions to known structures are predictable and in possession the of those having the structural information. Therefore, conservative amino acid substitutions, in which one or a few amino acids in an amino acid sequence are substituted with different amino acids with highly similar properties, are also readily identified as being highly similar to a disclosed constructs, and expected to retain function. One of skill will recognize that individual substitutions, deletions, or additions which alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 4%, 2% or 1%) in an encoded sequence are conservatively modified variations where the alterations result in the deletion of an amino acid, addition of an amino acid, or substitution of an amino acid with a chemically similar amino acid. Thus, conservative variations of a listed polypeptide sequence of the present invention include substitutions of a small percentage, typically less than 5%, more typically less than 2% or 1%, of the amino acids of the polypeptide sequence, with a conservatively selected amino acid of the same conservative substitution group.

(10) TABLE-US-00005 TABLE 1 Conservative Substitution Groups 1 Alanine (A) Serine (S) Threonine (T) 2 Aspartic Glutamic acid (D) acid (E) 3 Asparagine (N) Glutamine (Q) 4 Arginine (R) Lysine (K) 5 Isoleucine (I) Leucine (L) Methionine (M) Valine (V) 6 Phenylalanine (F) Tyrosine (Y) Trytophan (W)

(11) The present invention includes biosensor constructs, preparing cells useful in receiving the constructs, cells transformed with the constructs, and assay methods employing the biosensors, e.g., to detect the presence of active agents or signals in living cells.

(12) The biosensors generally include various complementary domain structures working together to sense a voltage or ion at a particular intracellular location, and to provide a distinct signal correlated to changes in the parameter. In a particularly useful embodiment, the biosensors are expressed in cells representative of a particular species (e.g., human), cell type of interest, or in a cell expressing a pathology of interest. Cells expressing the biosensors can be exposed to conditions or agents and monitored for signals indicating cell responses.

(13) Biosensors Generally.

(14) Biosensors of the present invention are generally engineered to include components specialized in providing, e.g., location, sensing, and reporting. In expression of a sensor nucleic acid construct, the peptide product can be positioned within the cell, e.g., by chaperone or transit sequences, hydrophobic affinities, or ligand/receptor interactions. The sensing domains typically change conformation in response to a changed intracellular condition or binding of a signal ion or molecule. Reporting domains display a detectable signal in response to conformational changes in the associated sensing domain.

(15) The intracellular location of a biosensor can optionally be controlled by a targeting domain or tag. In some biosensors, intracellular location is generalized or passively determined. For example, the sensor may be generally dispersed throughout the nucleus and/or cytoplasm. In other cases, the function or specificity of the biosensor signal may depend on localization at a particular intracellular membrane or organelle. Localizing biosensor tags can be done using hydrophobic peptides which interact with membranes, ionic peptides which disperse in cytoplasm, and/or other chaperone sequences directing the biosensor to a particular compartment including ligands directed to receptors. Tags useful in the study of intracellular signaling and in agent screening assays can include, e.g., NLS tags, lipid membrane tag, endoplasmic reticulum (ER) tags, golgi tags, endosome tags, ciliary tags, and others.

(16) Sensing domains in the biosensors change conformation in response to a changed physical condition, binding of a ligand (peptide or nucleic acid), or a change in an ionic environment, caused, e.g., by interaction with a protease, interaction with a phosphatase, changes in pH, changes in ionic strength, or changes in a voltage potential. The conformational changes can, e.g., cause conformational changes in an associated reporter domain, or reposition the reporter domain to enhance or diminish the signal.

(17) Reporter domains of the biosensors can be of any appropriate type known in the art. However, in preferred embodiments, the reporters comprise one or more peptide domains, e.g., so they can be readily employed in in vivo systems. Typically the peptide reporter domain provides a specific fluorescent emission in response to a specific interrogating excitation wavelength of light. In the context of sensor domain conformational changes, FRET strategies can be effective, e.g., wherein the biosensor construct comprises paired donor/acceptor peptide pairs. In certain embodiments, the reporter domain is adapted to provide an emission in the red to near-infrared spectrum, to allow the signal to pass and be detected in three dimensions through cells or tissue layers. For example, relatively penetrating reporter emission signals can range from 500 nm to 1400 nm, 550 nm to 900 nm, 600 nm to 850 nm, or 500 nm to 800 nm. In the 500 nm-800 nm range, the fluorescent properties can be easily changed to a point within that range, e.g. 625 nm emission.

(18) In an exemplary embodiment, the reporter domain includes fluorescence enhancing amino acids, such as tryptophan, tyrosine, phenylalanine, which contribute to their intrinsic fluorescence of the domain. Optionally, the reporter domain can include regions naturally modified post-translationally to provide the desired long wavelength emissions. For example, a domain that emits in the red regions can be a modified green fluorescent protein where the fluorophore originates from an internal serine-tyrosine-glycine sequence which is modified to a 4-(p-hydroxybenzylidene)-imidazolidin-5-one structure.

(19) Biosensor constructs can include, e.g., two, three, or four of the above described domain types, in any of a variety of functional configurations. For example, a sensor domain and reporter domain in any order can constitute a biosensor. Often, the biosensors of the invention include at least three domains, e.g., a targeting domain, a sensor domain, and a reporting domain. The three domains can be in any order, but typically the targeting domain is on one end of the construct, the sensor in the middle and the reporter on the second end. In certain configurations, the biosensor construct can be in the order (C-terminal/N-terminal or N-terminal/C-terminal) of: sensor/reporter; targeting/sensor/reporter; targeting/reporter/sensor; targeting/reporter1/sensor/reporter2 (e.g., FRET); reporter1/sensor/reporter2; sensor/reporter1/reporter2; and targeting/sensor/reporter1/reporter2.

(20) Typically, the domains are linked together in a commonly translated construct of a single linear peptide. Optionally, the constructs can include one or more domains not in the same peptide chain as another domain. For example, separate domains may be associated in a non-covalent interaction, such as a hydrophobic interaction, a chelation, a ligand/receptor interaction, an antibody/antigen interaction, and/or the like.

(21) In some cases, a single domain may have more than one function. For example, a sensor domain may also have a structure functioning as a targeting domain. In one embodiment, a domain may have a series of transmembrane domains, acting as both as a sensor (e.g., ligand responsive ion channel) and a membrane specific targeting domain. In another aspect, a sensor domain could also include a reporter function, e.g., acting as a donor or quencher member of a FRET pair with a separate reporter domain.

(22) Chloride Ion Sensing Biosensors.

(23) Chloride ion sensor constructs of the invention include many of the aspects described above for biosensors generally, but the sensor domain is responsive to changes in chloride ion levels. For example, the chloride ion sensor will typically bind Cl.sup. with a certain affinity and change conformation to some degree depending on the local Cl.sup. concentration. Cl.sup. can vary dramatically depending on cell type, condition, and the influence of induced signals. In living cells, Cl.sup. levels often vary with intracellular locations. The present chloride ion sensors can include targeting domains directing the sensors to any intracellular compartment of membrane, such as, e.g., a vacuole, the nucleus, cytoplasm, synapse, endoplasmic reticulum, and/or elsewhere.

(24) Chloride ion sensor domains are typically peptides homologous to portions of one or more chloride ion binding proteins. All that is necessary for the sensor role is that the peptide, or peptide fragment, changes conformation with changes in chloride ion concentration. The change in conformation will translocate the reporter group and typically change the emission profile or intensity. Modern protein engineering techniques can be used to engineer enhancements causing the translocation of the reporter to be enhanced or quenched, e.g., by induced contact or induced conformational changes in the reporter itself.

(25) A preferred chloride ion biosensor construct includes a combination of a tandem array of chloride ion binding domains and a fluorescent peptide. In an exemplary embodiment, the chloride ion sensor includes a modified chloride ion binding domain derived from a known chloride ion binding domain, and a fluorescent protein modified to provide red emissions. The domains of the constructs are typically configured from a combination of bioinformatics/database sequences as modified evolutionary mutagenesis.

(26) SEQ ID NO: 1 is a chloride ion sensing domain. Conservative variations of SEQ ID NO: 1 would be expected to retain substantial sensing function. The present chloride ion binding domains include peptides comprising sequences at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 1. SEQ ID NO: 3 is a particular reporter domain for the chloride ion sensor peptide. Conservative variations of SEQ ID NO: 3 would be expected to retain substantial useful function. The present fluorescent reporter sensor domains include peptides comprising sequences at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 3.

(27) Biosensor Constructs in Human or Mammalian Cells.

(28) The biosensors described herein can be incorporated into mammalian cells to monitor chloride ion conditions within the cells. For example, nucleic acid constructs encoding the biosensor peptide domains can be transformed or transfected into eukaryotic cells for expression, e.g., using appropriate promoters, as is known in the art. The cells can be mammalian, preferably human, providing the benefit of a reliable host cell model, e.g., for study of human signal transduction and disease states. The mammalian cells can be immortalized, but are preferably stem cells or cells induced to be pluripotent, or cells induced or otherwise differentiated to become a particular cell type of interest.

(29) The nucleic acid construct can encode any biosensor described herein. For example, the construct can encode a combination of a chloride ion sensor domain, a targeting domain, and/or a reporter domain. In one embodiment, the nucleic acid construct includes transient expression vector components directing expression of peptide chains comprising a combination of domains providing a functional biosensor. For example, the nucleic acid can express a single peptide chain comprising a combination of chloride ion binding domain, troponin domain, and reporter domain. In another example, the nucleic acid construct can encode a peptide comprising at least a combination of a transmembrane domain, a chloride ion sensing domain, and reporter domain.

(30) In other embodiments of the nucleic acid expression constructs, an expression vector includes sequences encoding peptides of any of SEQ ID NOs: 1, 3, or 5. In certain embodiments functioning as chloride ion biosensors, the vector can include a combination of nucleic acid sequences encoding the peptides of SEQ ID NOs: 1 and 3, e.g., along with a sequence encoding a transmembrane domain, such as an ion channel domain. Such a construct can encode functional peptide sequences at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the sequence of SEQ ID No: 1 and/or SEQ ID NO: 3. The cells transformed with TempoChloro used in the experiments shown in FIGS. 1 to 3, were transformed with nucleic acid sequences encoding (C to N terminus) the peptides of SEQ ID NO: 1 and SEQ ID NO: 3, i.e., these cells were transformed using vectors including SEQ ID NOs: 2 and 4.

(31) Useful nucleic acid constructs of the above expression vectors can include additional sequences encoding targeting tags, such as, e.g., a NLS tag, a lipid membrane tag, an ER tag, a golgi tag, an endosome tag, a mitochondrial tag, and/or a ciliary tag. For example, the targeting tag can include a sequence at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identical to a sequence encoding the peptide tag sequence of SEQ ID NO: 5.

(32) The present inventions include cells comprising the biosensors discussed herein. For example, nucleic acid constructs coding the biosensor peptides can be transduced or transfected into eukaryotic cells of choice. In preferred embodiments, the cells originate from a mammal, preferably from a human. In many embodiments, the cell is an immortalized stem cell, or a cell fully or partially differentiated from a stem cell.

(33) In the context of the present biosensors, cells can be initially derived from patient samples. The advantage of such cells is that they can provide very representative responses to active agents and changed conditions for that patient. Thus, such cell models are more likely to provide information on the modes of action or efficacy of a candidate therapeutic for that patient. For example, such cell models can aid in the identification of custom tailored treatment for patients with certain disease states, such as autoimmune diseases, neurological diseases, cancer, diabetes, or pathologies from genetic flaws.

(34) Alternately, the models can employ cells harboring a particular non-endogenous gene of interest, introduced by genetic engineering techniques. For example, the gene of interest can encode a receptor molecule, a G-protein coupled receptor, or an ion channel of interest. Typically, the host cell is a cell most representative of the cell type of interest in the research. Such model cells can be useful in providing more representative results, e.g., in monitoring a signal transduction or in screening prospective agents active in the modulation of the gene of interest.

(35) Cellular models can be derived from inducible cells available from primary culture of cells from living animals. For example, fibroblasts or undifferentiated cells from circulating blood can be induced to provide pluripotent cells. It is notable that epigenetic processes can play a key role in directing the status of a cell to stem cell, progenitor cell, or mature cell. In Lister (Nature 471 (7336): 68-73, 2011), aberrant epigenomic programming was found capable of inducing a variety of different pluripotent stem cells (iPSCs). Female lung fibroblasts, adipose cells, and foreskin fibroblasts were reprogrammed into induced pluripotent state using OCT4, SOX2, KLF4, and MYC genes. The iPSCs were found to be similar to embryonic stem cells in many characteristics, including DNA methylation patterns. Such concepts can be used to reprogram cells, e.g., in combination with further circadian synchronization techniques, described below.

(36) In addition to inducement by action of immortalizing genes, cell signaling was found to influence epigenetic processes governing differentiation. In the research of Baylin (e.g., Nature Biotechnology 28 (10): 1033-8, 2010), several signaling pathways were suggested as important in the induction and maintenance of embryonic stem cells and in their differentiation. For example, signaling pathways of growth factors can play a role in epigenetic regulation of cellular differentiation. These growth factors include, e.g., transforming growth factors (TGFs), fibroblast growth factors (FGFs), and bone morphogenetic proteins. Another important factor in induction and differentiation can be the Wnt signaling pathway.

(37) Circadian rhythm influences on cellular synchronization can be employed in inducement of pluoripotential cells. For example, cells can be reprogrammed into inducible pluripotent stem cells using human clock gene and human Bmal1/2/3/4 genes and their E-box promoters. In one aspect, fibroblast iPSCs can be generated by such reprogramming, and further directed to provide inducible neurons (iN), glial cells, or inducible neural progenitor cells (iNPCs), as desired. The reprogramming factor for each cell type is typically a transcriptional regulator that is specific for the cellular lineage. Each factor can be modified to be controlled by a circadian regulatory element (such as, E-box promoters or an artificial E-box-like promoter sequence tag). Such promoter sequences can be added to each transcriptional regulator, thus forming a novel transcriptional element for control regulated by human Clock and Bmal genes.

(38) To complement the biosensor systems described herein, induced cells can be cultured together in a tissue structure, e.g., in three dimensions. In this way, the cell to cell contacts of interest can be studied, e.g., using penetrating imaging available in the form of near infrared reporter signals. For example, co-cultures of iN and iG can be prepared to create a 3D model of a neuronal structure. The structure can be further controlled, e.g., using an appropriately structured scaffold, e.g., using materials not opaque to reporter signals, e.g., for confocal microscopic review.

(39) Screening and Assay Methods Using Biosensors in Model Cells

(40) The biosensor constructs, e.g., engineered into model cells of interest, can more predictably provide assay and screening results relevant to life science research and study of clinical pathologies of interest. That is, the present targeted biosensors in appropriate cell types, differentiated to a particular phenotype, can provide models more likely to anticipate a normal response in the modeled organism than, e.g., old art xenotypic models.

(41) The initial steps in preparing a model system can be to identify the cell type of interest and the signal to be detected. For example, to study certain therapeutic agents, one may choose to target a chloride ion sensor. Typically the biosensor constructs are transiently expressed in the cells using, e.g., a CMV constitutive promoter or a cell-type-specific promoter.

(42) Once the model system is established, the biosensor can be monitored in a single cell, or across an array of cells. For example, a cell can be exposed to a signaling agent to see if the cell type responds to that agent, e.g., a cytokine or candidate small molecule bioactive agent. Optionally, the cell can be co-transfected with a second expression construct of a peptide of interest (e.g., a tumor-associated antigen or oncogene) to monitor any influence of the external gene in a signaling pathway.

(43) In other embodiments, the cells can be segregated into arrays complementary to low, medium, or high throughput assay formats. For example, cells can be dispensed into 96-well plates, onto a micro-well array, or a FACS sorter, for separate exposure to library members of putative candidate agents. Such arrays can be reviewed suing standard fluorescent detection equipment. Optionally, the arrays can be reviewed photographically with digital CCD based cameras. Changes in a signal, e.g., as compared to a positive or negative reference, can be flagged for additional characterization.

(44) Targeting Tags

(45) In many cases, it is advantageous to direct the chloride ion sensor to a particular intracellular membrane or compartment. The biosensors of the invention can include peptide segments adapted to have an affinity for a cellular target.

(46) Exemplary peptide sequences useful in targeting biosensors to a desired intracellular location include, e.g.:

(47) TABLE-US-00006 NLStag(proteinsequence): (SEQIDNO:5) DPKKKRKV. ERtag: (SEQIDNO:6) KDEL Endosometag: (SEQIDNO:7) NPTY--DXXLL--YXXoo(proteinsequence;tandem motifs;oo= tworesidueswithhydrophobicside groups; Ciliarytag: SEQIDNO:8) VxPx-RVxP-KVHPSST-AxEGG(proteinsequence;tandem motifs- Humanendosomesequencetag (SEQIDNO:9) MTSRKKVLLKVIILGDSGVGKTSLMHRYVND SEQIDNO:10humansynapsintag: CCTGCAGGGCCCACTAGTATCTGCAGAGGGCCCTGCGTATGAG TGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGC CTACCTGACGACCGACCCCGACCCACTGGACAAGCACCCAACC CCCATTCCCCAAATTGCGCATCCCCTATCAGAGAGGGGGAGGG GAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGC ACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCAC CGCCGCCTCAGCACTGAAGGCGCGCTGACGTCACTCGCCGGTC CCCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCCGCGCC GCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAG GGGGGCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACTC AGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCG TGCCTGAGAGCGCAGCTGTGCTCCTGGGCACCGCGCAGTCCGC CCCCGCGGCTCCTGGCCAGACCACCCCTAGGACCCCCTGCCCCA AGTCGCAGCC SEQIDNO:11Humansynapticvesicletag: MDCLCIVTTKKYRYQDEDTPPLEHSPAHLPNQANSPPVIV NTDTLEAPGYELQVNGTEGEMEYEEITLERGNSGLGFSIA GGTDNPHIGDDPSIFITKIIPGGAAAQDGRLRVNDSILFV NEVDVREVTHSAAVEALKEAGSIVRLYVMRRKPPAEKVME IKLIKGPKGLGFSIAGGVGNSTSLEITASM

EXAMPLES

(48) FIG. 1 shows the TempoChloro biosensor (expressed in transformed neural human IPS progenitor cells) successfully identifying samples with chloride ion or picrotoxin, based on relative fluorescence.

(49) Picrotoxin, also known as cocculin, is a poisonous crystalline plant compound which has been shown to inhibit neurotransmitter GABA-ergic ion channels; acting, in vivo, as a plant-derived stimulant and convulsant. See, Newland, C F; Cull-Candy, S G (February 1992). On the mechanism of action of picrotoxin on GABA receptor channels in dissociated sympathetic neurones of the rat. The Journal of Physiology. 447: 191-213. doi:10.1113/jphysiol.1992.sp018998. PMC 1176032 Freely accessible. PMID 1317428.

(50) The controls for the assay in FIG. 1 were: HEPES Buffer (pH 7.4 and osmolarity at standard mOsm=300 (+/15)); 0.1 mM NaCl (1HBSS buffer pH7.4); and, 10 microMolar Picrotoxin (1HBSS buffer pH7.4). As a supplemental control against the HBSS buffer in the latter two controls, HBSS buffer pH 3.0 was used. The fluorescence detection parameters were: 517 nm Excitation/535 nm Emission (+/2).

(51) FIG. 2 shows the TempoChloro biosensor (expressed in transformed neural human IPS progenitor cells) successfully identifying samples with chloride ion or picrotoxin, based on relative fluorescence, as determined by kinetic readouts (X axis shows: seconds per scan). The controls were: HEPES Buffer (pH 7.4 and osmolarity at standard mOsm=300 (+/15)); 0.1 mM NaCl (1HBSS buffer pH7.4); and, 10 microMolar Picrotoxin (1HBSS buffer pH7.4). As a supplemental control against the HBSS buffer in the latter two controls, HBSS buffer pH 3.0 was used. The fluorescence detection parameters were: 517 nm Excitation/535 nm Emission (+/2).

(52) FIG. 3 shows the TempoChloro biosensor (expressed in transformed human iPS-derived astrocytes, which are glial cells) successfully identifying samples with chloride ion, based on relative fluorescence. The controls were: HEPES Buffer (pH 7.4 and osmolarity at standard mOsm=300 (+/15)); 0.1 mM NaCl (1HBSS buffer pH7.4); and, a supplemental control against the HBSS buffer in the latter control: HBSS buffer pH 3.0. The fluorescence detection parameters were: 517 nm Excitation/535 nm Emission (+/2).

(53) It can be seen, therefore, that the TempoChloro biosensor system is effective in detecting chloride ions in divergent cell types, including neurons and glial cells, and would be effective in other mammalian or human cell types, including, immortalized, native or induced: pluripotent stem cells, neurons, glial cells, and neural progenitor cells.

(54) The specific methods, procedures and apparatus described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and that they are not necessarily restricted to the orders of steps indicated herein or in the claims. Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants.

(55) The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.