Claims:1. A spore selective fluorescent dye composition comprising 7,7-Bis(n-hexylamino)-8,8-dicyanoquinodimethane (BHADQ) compound in dimethylsulfoxide (DMSO) solvent.
2. The composition as claimed in claim 1 has 20 - 40 nmols of BHADQ.
3. The composition as claimed in claim 1, wherein the compound BHADQ is represented by the formula (C22H32N4):

4. The composition as claimed in claim 1, wherein the composition is used for staining and identifying spores.
5. The composition as claimed in claim 4, wherein the spores comprise of bacterial spores.
6. The composition as claimed in claim 4, wherein the spores comprise of dormant and cryptic spores.
7. The composition as claimed in claim 4, wherein the spores comprise of both endo and exo spores.
8. The composition as claimed in claim 1, wherein the composition is non-cytotoxic.
9. A process for staining and identifying spores comprising the steps of:
(a) Mixing the composition as claimed in claim 1, with the spores in an aqueous medium and incubating for 2-60 minutes under ambient conditions (24oC) to obtain stained spores;
(b) Centrifuging the stained spores of step (a) at 4°C to obtain deposited pellets of stained spores;
(c) Re-suspending the deposited pellets of step (b) in water (100 µL) to obtain a solution; and
(d) Using a drop of the solution of step (c) for imaging.
10. The process as claimed in claim 9, wherein the amount of composition mixed with the spores in in the range of 5-10 µL of 4 mM solution.
11. The process as claimed in claim 9, wherein the amount of the composition mixed with the spores is 24 nmols in 6 µL of 4 mM solution
12. The process as claimed in claim 9, wherein the process is carried out without prior heating or chemical treatment of the spores.
, Description:FIELD OF THE INVENTION

The present invention is related to a novel composition comprising molecules based on the diaminodicyanoquinodimethane (DADQ) framework. More particularly, the invention relates to a specific DADQ derivative, 7,7-bis(n-hexylamino)-8,8-dicyanoquinodimethane (BHADQ), in staining bacterial spores (both endo and exospores). The invention also provides a process for staining and identifying the spores.

BACKGROUND OF THE INVENTION

Spore formation is the ingenious mechanism, which allows bacteria to pass through adverse environmental conditions, and transfer their genetic information to ensuing generations. Bacterial spores are formed in response to adverse environmental conditions and help in the survival of the organisms. However, they do not play any role in the reproduction of these organisms. These dormant states (i.e., no metabolic activity) are highly resistant to debilitating effects such as heat, dehydration, radiation and chemicals. This is because of various parts of the spores such as the exosporium providing adherence and biocide resistance, keratin-based spore coat, cortex and spore wall made of peptidoglycan and dipicolinate, as well as low water content in the core. The increasing susceptibility of humans and animals to infections arising from the quickly developing antibiotic resistance of several bacterial strains is reaching alarming levels. With the antibiotics and sterilization techniques being ineffective against the spores, the food and medical industries are facing high risks. Additionally, these spores can also act as ominous biological weapons. Therefore, development of efficient, cost-effective and facile methods for the detection of spores, and assessment of their viability is an urgent and critical imperative.

Even though electronic absorption and fluorescence emission-based imaging are desirable, facile and relevant techniques, there are certain inherent challenges faced due to chemical resistance and permeability barriers posed by the protective coats of dormant spores. Additionally, the common non-fluorescence-based imaging methods utilize staining protocols such as Gram-stain, Ziehl-Neelsen, Dorner and Schaeffer-Fulton; however, these methods generally require lengthy preparatory steps involving extended heating in many cases, and decolorization using media such as acidified alcohol or acids. Preferable alternatives based on flow cytometry and fluorescence microscopy require efficient fluorescence probes, even though the former could utilize techniques such as mass spectrometry. The common fluorescence probes such as 4',6-Diamidino-2-Phenylindole (DAPI), acridine orange, Hoechst 34580 and SYTO9 are easy to use and suitable for clinical applications, however, they have crucial limitations. For example, as DAPI is not cell-permeable it is useful only for imaging dead spores. Similarly, the others are ineffective in differentiating spores from bacteria, as they stain both. The imaging process generally involves killing the spores by methods like heating, followed by treatment with nucleic acid stains. Lethal germination induced by dodecylamine leads to core dehydration and spore destruction. As dormant bacterial spores are highly resistant towards staining, lethal germination which leads to the termination of dormancy is necessitated for the incorporation of the fluorescence dye or stain. There are practically no effective fluorescence dyes at the moment, that can be used to image bacterial spores (both endo and exospores) in their dormant state.

US4659177A discloses nonlinear optical medium comprising a transparent solid substrate with a surface coating of at least one monomolecular layer of uniaxial aligned quinodimethane molecules such as 7,7-bis(hexadecylamino)-8,8-dicyanoquinodimethane.

201741017444 discloses staining and imaging of biological tissue by DADQ derivatives, particularly 7,7-bis(piperazinium)-8,8- dicyanoquinodimethane bis (p-toluene sulfonate). The derivatives can be used to selectively stain guard cells in plant tissue.

CN103175768B discloses fluorescent staining kit for rapid detection on biological cell viability. The kit comprises an anthocyanin dye and a propidium iodide dye. It uses dimethyl sulfoxide (DMSO) solvent for fluorescent dyes.

Gangopadhyay P. et al. discloses process of synthesis of 7,7-bis(n-alkylamino)-8,8-dicyanoquinodimethanes. It also discloses the structural study of the said molecule. Powder studies on 7,7-Bis(n-alkylamino)-8,8-dicyanoquinodimethanes indicate that moderate solid-state optical SHG is obtained when the alkyl chains are of length 4, 5, and 6, and no detectable second harmonic generation occurs when the chains are of length 0, 3, 7, 8, and 12.

N Senthilnathan et al. discloses general use of DADQs as bio-imaging dyes on a broader aspect. This is demonstrated through their efficient application in epidermal and stomatal imaging with selective staining of cell walls and nuclei.

CG Chandaluri et al. discloses 7,7-diamino-8,8-dicyanoquinodimethanes (DADQs) exhibiting strong fluorescence enhancements. The amorphous-to-crystalline transformation of a DADQ-based nanoparticle is accompanied by substantial fluorescence enhancement and switching of the emission color.

It would be highly desirable to provide an agent for fluorescence imaging of spores which would be cost-effective, easily synthesized and non-cytotoxic. It should also possess good chemical and photo-stability.

OBJECT OF THE INVENTION

An objective of the present invention is to provide a spore selective fluorescent dye composition (Sporotan) for identification and staining of spores.

Another objective of the present invention is to provide a composition for staining of endo and exospores.

Yet another objective of the present invention is to provide a process for the staining and identification of the spores.

Another object of the present invention is to provide an agent and process for fluorescence imaging of spores which is practical and efficient.

SUMMARY OF THE INVENTION

The present invention as embodied and broadly described herein discloses novel compositions comprising specific DADQ derivative, 7,7-bis(n-hexylamino)-8,8-dicyanoquinodimethane (BHADQ), in staining bacterial spores (both endo and exospores). A class of molecules based on the DADQ framework employed in the present invention have been explored in connection with a wide range nonlinear optical and fluorescence-based applications and are found to be ideally suited for effective and selective imaging of bacterial spores (both endo and exospores), especially in their dormant state. Accordingly, an aspect of the present invention is to provide an agent for fluorescence imaging of spores which is practical and efficient. It is cost-effective, easily synthesized and non-cytotoxic, possesses good chemical and photostability, with an optimal level of hydrophobicity necessary for selective staining of the spores.

In one embodiment of the invention, the spore selective fluorescent dye composition comprises 20-40 nmols of 7,7-Bis(n-hexylamino)-8,8-dicyanoquinodimethane (BHADQ) compound in DMSO solvent.

In another embodiment of the invention, the compound BHADQ is represented by the formula (C22H32N4).

In another embodiment of the invention, the composition is used for staining and identifying spores.

In one aspect of the present invention, the spores comprise bacterial spores.

In another aspect of the present invention, the spores comprise dormant and cryptic spores.

In another aspect of the present invention, the spores comprise both endo and exospores.

In another aspect of the present invention, it is provided that the composition is non-cytotoxic.

One preferred embodiment of the invention, provides a process for staining and identifying spores comprising the steps of:
(a) Mixing the composition with the spores in an aqueous medium and incubating for 2-60 minutes under ambient conditions (24°C) to obtain stained spores;
(b) Centrifuging the stained spores of step (a) at 4°C to obtain deposited pellets of stained spores;
(c) Re-suspending the deposited pellets of step (b) in 100 µL water to obtain a solution; and
(d) Using a drop of the solution of step (c) for imaging.

In another embodiment, the amount of the composition mixed with the spores is 5 - 10 µL of 4 mM solution.
In another embodiment of the invention, it is provided that the process for staining and identifying the spores is carried out without prior heating or chemical treatment of the spores.

To further clarify the advantages and features of the present invention, a more particular description of the invention will be rendered by reference to specific embodiments thereof, which is illustrated in the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope. The invention will be described and explained with additional specificity and detail with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying drawings, wherein:

Figure 1 illustrates molecular structure of the composition (7,7-Bis(n-hexylamino)-8,8-dicyanoquinodimethane, BHADQ).

Figure 2 illustrates confocal laser scanning microscope (CLSM) images of the endospores of Halobacillus dabanensis stained using 4 mM solution of BHADQ in DMSO. In this, (a) scale = 20 µm and (b) scale = 5 µm; excitation wavelength = 405 nm, emission range = 410-485 nm and (c) 3-D images viewed at different angles.

Figure 3 illustrates CLSM images of (a) E.coli (Gram-stain-negative bacteria) and (b) Halobacillus dabanensis (Gram-stain-positive bacteria) (scales = 5 µm), and endospores together with bacteria of Halobacillus dabanensis (c) scale = 5 µm and (d) scale = 20 µm, treated with 4 mM solution of BHADQ in DMSO; excitation wavelength = 405 nm, emission range = 410-485 nm.

Figure 4 illustrates CLSM images of endospores of (a) Bacillus subtilis (b) Bacillus cereus and (c) Bacillus paralicheniformis stained using 4 mM solution of BHADQ in DMSO respectively; excitation wavelength = 405 nm, emission range = 410-485 nm and scale = 5 µm.

Figure 5 illustrates CLSM images of Bacillus subtilis bacteria and its endospores stained using 4 mM solution of BHADQ in DMSO; excitation wavelength = 405 nm, emission range = 410-485 nm and scale: 10 µm.

Figure 6 illustrates the photostability of the BHADQ in (a) solution and (b) solid state.

Figure 7 illustrates Raman spectra of the endospores of Halobacillus dabanensis, untreated (Control) and treated with 4 mM solution of BHADQ in DMSO (Stained).

Figure 8 illustrates Phase contrast microscopy images of the Halobacillus dabanensis endospores, (a) unstained, and stained using (b) 4 mM of BHADQ in DMSO. Scale = 10 µm.

Figure 9 illustrates CLSM images of the exospores of Planctobacteria stained using 4 mM BHADQ in DMSO; excitation wavelength = 405 nm, emission range = 410-485 nm. (a - c) fluorescence, bright field and overlay images (scale: 10 µm), and (d) overlay image at higher magnification (scale: 10 µm). So = exospore; Sp = sporangium.

Figure 10 illustrates the flowchart of the process for identifying and staining the spores.

DESCRIPTION OF THE INVENTION

It should be understood at the outset that although illustrative implementations of the embodiments of the present disclosure are provided below, the present invention may be implemented using any number of techniques, whether currently known or in existence. The present disclosure should in no way be limited to the illustrative implementations, drawings, and techniques illustrated below, including the exemplary design and implementation illustrated and described herein, but may be modified within the scope of the appended claims along with their full scope of equivalents. Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps of the process, features of the product, referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.

The terminology and structure employed herein is for describing, teaching and illuminating some embodiments and their specific features and elements and does not limit, restrict or reduce the scope of the claims or their equivalents. For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are collected here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. More specifically, any terms used herein such as but not limited to “includes,” “comprises,” “has,” “consists,” and grammatical variants thereof do NOT specify an exact limitation or restriction and certainly do NOT exclude the possible addition of one or more features or elements, unless otherwise stated, and furthermore must NOT be taken to exclude the possible removal of one or more of the listed features and elements, unless otherwise stated with the limiting language “MUST comprise” or “NEEDS TO include.”

Reference is made herein to some “embodiments.” It should be understood that an embodiment is an example of a possible implementation of any features and/or elements presented in the attached claims. Some embodiments have been described for the purpose of illuminating one or more of the potential ways in which the specific features and/or elements of the attached claims fulfill the requirements of uniqueness, utility, and non-obviousness.

While the invention is susceptible to various modifications and alternative forms, specific embodiments thereof are described in detail in Examples section below. It should be understood, however that it is not intended to limit the invention to the particular forms disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternatives falling within the scope of the invention as defined by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.
The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally equivalent products and methods are clearly within the scope of the disclosure, as described herein.

In accordance with an embodiment of the present invention, a spore selective fluorescent dye composition and the process of staining and identifying bacterial spores thereof is disclosed.

A wide array of DADQs have been synthesized and demonstrated for enhanced florescence emission in various states of these molecular materials. These have also been shown to be efficient probes for imaging stomatal cells. The utility of a specific DADQ derivative, 7,7-bis(n-hexylamino)-8,8-dicyanoquinodimethane has been demonstrated, in staining bacterial spores (both endo and exospores). This molecule is cheap to synthesize, photo and thermally stable, easy to store and handle in solid form and non-cytotoxic. The staining procedure is simple and requires no prior heating or chemical treatment. Dormant spores (both endo and exospores) could be stained by BHADQ, and subsequently germinated in a nutrient medium suggesting the non-invasive nature of the stains. BHADQ is represented by the formula given in Figure 1.

Figure 1 illustrates the structure of a DADQ molecule (C22H32N4). These are highly dipolar molecules with a zwitterionic structure arising from the push-pull character of the structure because of the amino and cyano substituents.

According to an aspect of the present invention, the spore selective fluorescent dye composition comprises 20-40 nmols of 7,7-Bis(n-hexylamino)-8,8-dicyanoquinodimethane (BHADQ) compound in DMSO solvent. This composition is used for staining and identifying spores. The spores may comprise bacterial spores, but not limited to and may include any other types of spores. The spores comprise dormant and cryptic endospores and exospores.

As provided in the present invention, several DADQs with a variety of substituents including aromatic and aliphatic cycles, without and with H-bonding or basic groups, as well as alkyl chains were explored. The alkyl chain derivatives were found to be relevant in the context of spore (both endo and exospores) staining. Butylamine and pentylamine derivatives were relatively less efficient. Interestingly, the hexylamine derivative, BHADQ showed very efficient staining. This was mainly due to the reason of aqueous solubility issues because of which higher alkyl chain derivatives could not be used effectively.

In accordance with an embodiment of the present invention, the staining protocol disclosed by the present invention comprises of the following general steps: (a) Mixing the composition with the spores in an aqueous medium and incubating for 2-60 minutes under ambient conditions (24°C) to obtain stained spores; (b) Centrifuging the stained spores of step (a) at 4°C to obtain deposited pellets of stained spores; (c) Re-suspending the deposited pellets of step (b) in 100 µL water to obtain a solution; and (d) Using a drop of the solution of step (c) for imaging. This process is illustrated by the flowchart illustrated in Figure 10.

In accordance with another aspect of the present invention, the amount of the composition mixed with the spores is 5 - 10 µL of 4 mM solution.

In accordance with another aspect of the present invention, the amount of the composition mixed with the spores is 6 µL of 4 mM solution.

In accordance with one aspect of the present invention, the process for staining and identifying the spores is carried out without prior heating or chemical treatment of the spores.

In accordance with another aspect of the present invention, the time used to incubate the composition mixed with the spores in an aqueous medium is approximately 5 minutes under ambient conditions (24°C) to obtain stained spores.

Figure 2 illustrates the images obtained for endospores of Halobacillus dabanensis stained with BHADQ. The 3-D images (see Figure 2c) reveal that the cell coat and the core of the endospores are stained successfully. Most significantly, BHADQ stains the endospores selectively, with absolutely no staining of Gram-stain-negative bacteria and very weak and rare staining of Gram-stain-positive bacteria.

Figure 3 illustrates the staining of Gram-stain-negative bacteria and Gram-stain-positive bacteria. Figure 3c and Figure 3d show the selective staining of endospores when they are present together with the bacteria.

Figure 4 illustrates the experiments with other bacterial endospores such as those of Bacillus subtilis, Bacillus cereus and Bacillus paralicheniformis which indicate that BHADQ is an efficient dye for imaging them as well.

Figure 5 illustrates the case of Bacillus subtilis, in which bacterium was present along with the endospores, and the latter were selectively stained. The photostability and cytotoxicity are of critical importance in evaluating the practical utility of fluorescent dyes for imaging applications.

Figure 6 illustrates that by monitoring the fluorescence emission under continuous excitation (405 nm), BHADQ is found to be stable for extended periods of time in the molecular state in solution, and to a reasonable extent in the bulk solid state. Cytotoxicity studies using murine fibroblast cells (L929) and Human cancer cell line/ (HeLa) cell lines showed that the lethal dose 50% (IC50) value for BHADQ is > 500 ?g mL-1, as the viability remained > 65% even at this concentration. The concentrations of BHADQ used in our imaging experiments (84 ?g mL-1) is well within safe limits.

A series of experiments including controls, involving propidium iodide staining, clearly rule out the possibility of BHADQ destroying the spore cell coats or inducing lethal germination of the endospores. The endospores which were stained with these dye molecules, do not germinate automatically. These experiments demonstrate that the BHADQ can be used to image endospores in their dormant state, and possibly monitor them through their germination under favorable conditions subsequently.

Figure 7 illustrates Raman spectral studies and Figure 8 illustrates phase contrast microscopy of untreated endospores and stained ones which indicate that the staining process does not alter their chemical structure in any definitive way. Figure 7 shows that the crucial peak due to the symmetric ring breathing mode of calcium dipicolinate at 1018 cm-1 is retained in the sample treated with BHADQ; if dead or germinated, loss of calcium dipicolinate would cause this peak to diminish or disappear (see Figure 7 for former result).

Figure 9 illustrates that the exospores of Planctobacteria are stained successfully with BHADQ. These experiments reveal that BHADQ stains both endospores and exospores of bacterial spores.

EXAMPLES

In accordance with the present invention, the technique has been tested on 6 endo and exospores producing bacteria and found to be very useful in identifying cryptic spores, which are otherwise thought to be poly-ß-hydroxyalkanoate granules. Table 1 illustrates this. The fluorophore developed is very useful in identifying spores (both endo and exospores) producing taxa of Firmicutes and non-Firmicutes like Proteobacteria. This helps investigators to extend sporogenesis in members of other phyla, which was otherwise thought to be confined to the phylum Firmicutes.

Sr. No. Dyes used for imaging the spores Spores (both endo and exospores) Bacteria cells Spore
dormancy
1 Hoechst
Live spores Live bacterial cells No
2 SYTO
Live spores Bacterial cells Maybe
3 DAPI
Dead spores Bacterial cells No
4 Acridine orange
Dead spores Bacterial cells No
5 PI
Dead spores Dead bacterial cells No
6 BHADQ
(Present invention) Live spores No stain on the bacteria Yes

Therefore, it is seen that the BHADQ can selectively stain the spores only. It does not stain the bacteria.

While specific language has been used to describe the disclosure, any limitations arising on account of the same are not intended. As would be apparent to a person in the art, various working modifications may be made to the method in order to implement the inventive concept as taught herein. The scope of the embodiments is by no means limited by these specific examples. The scope of the embodiments is at least as broad as given by the following claims.