Description:FIELD OF THE INVENTION:
The present invention relates to diagnosis of cancer. More specifically, the present invention is directed to develop a spectroscopic system for instant point of care non-invasive, non-contact detection of cancer including malignant cells, small ulcer-like precancerous lesions including malignantoral lesions, cysts or other neoplasmson tongue and floor of the mouth.

BACKGROUND OF THE INVENTION:
Oral cancer is the 11th most common cancer in the world [Ref: R. Sankaranarayanan, K. Ramadas, H. Amarasinghe, S. Subramanian, and N. Johnson, "Oral cancer: prevention, early detection, and treatment," DISEASE CONTROL PRIORITIES, THIRD EDITION, p. 85, 2015] and has secured its place in the top 3 common cancers in India[Ref: K. R. Coelho, "Challenges of the oral cancer burden in India," Journal of cancer epidemiology, vol. 2012, 2012].
Oral cancer or oral cavity cancer falls in the category of head and neck cancer which includes the malignancy of any tissues in the mouth, mucosa of the lip, floor of the mouth, plate, the gingiva, the inner walls of the cheek also including the salivary glands, tonsils or other lymphoid tissues [Ref: E. E. Vokes, R. R. Weichselbaum, S. M. Lippman, and W. K. Hong, "Head and neck cancer," New England Journal of Medicine, vol. 328, pp. 184-194, 1993]. But the most common locations are that of the epithelia of the lining of the mouth and the tongue [Ref: C. Moore and D. Catlin, "Anatomic origins and locations of oral cancer," The American Journal of Surgery, vol. 114, pp. 510-513, 1967]. These types of epithelia present aresquamous; hence termed assquamous cell carcinoma.
Oral cancer, often referred as “poor man’s disease” [Ref: H. D. Sgan-Cohen and J. Mann, "Health, oral health and poverty," The Journal of the American Dental Association, vol. 138, pp. 1437-1442, 2007], is caused by usage of tobacco in various forms (through smoking and chewing) both in young and old. Also heavy alcohol drinking [Ref: W. J. Blot, J. K. McLaughlin, D. M. Winn, D. F. Austin, R. S. Greenberg, S. Preston-Martin, et al., "Smoking and drinking in relation to oral and pharyngeal cancer," Cancer research, vol. 48, pp. 3282-3287, 1988], poor oral hygiene, malnutrition and genetics [Ref: N. W. Johnson, S. Warnakulasuriya, P. Gupta, E. Dimba, M. Chindia, E. Otoh, et al., "Global oral health inequalities in incidence and outcomes for oral cancer: causes and solutions," Advances in dental research, vol. 23, pp. 237-246, 2011] are also responsible for its occurrence. Apart from the confirmed cases of malignancy in the forms of lesions, cysts and neoplasms there are also cases of localized small ulcer-like precancerous lesions on the tongue and the floor of the mouth (homogenous and non-homogeneous types), plaque like leukoplakia or erythroplakia. If these can be detected through careful visual screening in the early stage where these oral cancers haven’t propagated to the lymph node yet, the patient can be treated with an 80% survival rate.
It is thus there has been a need for developing an accurate, fast and effective technique for detecting localized small ulcer-like precancerous lesions on the tongue and the floor of the mouth in the early stage where the oral cancers haven’t propagated to the lymph node yet to ensure survival of the patient.

OBJECT OF THE INVENTION:
It is thus the basic object of the present invention is to develop a system for accurate, fast and effective detection of malignant cells, small ulcer-like precancerous lesions.

Another object of the present invention is to develop a non-invasive, non-contact point of care spectroscopic system for instant detection of localized small ulcer-like precancerous lesions on the tongue and the floor of the mouth in the early stage where the oral cancers haven’t propagated to the lymph node yet to ensure survival of the patient.
Yet another object of the present invention is to develop a non-invasive, non-contact spectroscopic technique for quantitative detection of oral cancer.

Yet another object of the present invention is to develop a staining agent for the diagnosis of oral cancer through non-invasive, non-contact spectroscopic technique.

SUMMARY OF THE INVENTION:
Thus according to the basic aspect of the present invention there is provided a non-invasive and non-contact reflection spectrometry based cancer detection system comprising
a light source,
reflectance probe to transmit light form said light source and accept diffuse optical signal from sample for testing stained with staining dye capable of interacting with nucleic acid at physiological pH;
spectrometric means to receive the diffuse optical signal for generating graph components indicative of spectra obtained from the nucleic acid of malignant lesions in the sample interacting with said staining dye having atleast one distinctly different pattern than the nucleic acid of normal cells interacting with said staining dye.

In a preferred embodiment of the present non-invasive and non-contact reflection spectrometry based cancer detection system for oral cancer detection comprising said spectrometric means generating graph components indicative of spectra obtained from the nucleic acid of the malignant lesions in the sample interacting with cationic staining dye Toluidine Blue (TB) having atleast one distinctly different pattern than the nucleic acid of normal cells in the sample interacting with said cationic staining dye Toluidine Blue TB.

In a preferred embodiment of the present non-invasive and non-contact reflection spectrometry based cancer detection system, the spectrometric means comprises
a spectrometer to generate absorption spectra from the received optical signal; and
a microcontroller for computing spectral deconvolution of the absorption spectra and plotting the same to enable determination of the precancerous or malignant cell in the sample based on presence of the spectral line in said plotted spectral deconvolution corresponding to interaction of the staining dye Toluidine Blue TB with only the precancerous or malignant cells.

In a preferred embodiment, the present non-invasive and non-contact reflection spectrometry based cancer detection system comprises GUI based microcontroller to acquire the signals and plot the spectral deconvolution in real time for ready detection and screening of patients.

In a preferred embodiment of the present non-invasive and non-contact reflection spectrometry based cancer detection system, the microcontroller is configured to include reference and dark values selectively including preloaded in interfacing computer for facilitating the detection based thereon.

In a preferred embodiment of the present non-invasive and non-contact reflection spectrometry based cancer detection system, the reflectance probe comprise means for light incident (400-900nm) on tissue of sample/oral cavity and illuminate the lesion under test from the sample/oral cavity and retro-reflected to spectrograph for non-contact and non-invasive detection.

In a preferred embodiment of the present non-invasive and non-contact reflection spectrometry based cancer detection system, the spectrometer enables generating distinctly different pattern of TB stain as a potential marker for increase in nuclear material characteristic of malignant cells.

In a preferred embodiment of the present non-invasive and non-contact reflection spectrometry based cancer detection system, the spectrometer cooperate with arrangement of spectroscopic investigation and microscopic imaging means for generating deconvoluted TB stain based absorption spectra indicative of Monomeric TB form (610 nm) only present in the malignant/cancer cell lines in contrast to its normal counterpart.

In a preferred embodiment of the present non-invasive and non-contact reflection spectrometry based cancer detection system, the Toluidine Blue (TB) is configured to interact with the cells of the sample at physiological pH value of 7.4.

In a preferred embodiment of the present non-invasive and non-contact reflection spectrometry based cancer detection system, the precancerous or malignant cells of the tissue stained with the TB includes the TB in monomeric form;
wherein the TB in monomeric form provides the specific spectral line in the spectral deconvolution of the optical signal from the TB stained tissues indicating presence of the precancerous or malignant cells of the tissue.

In a preferred embodiment of the present non-invasive and non-contact reflection spectrometry based cancer detection system, the light source includes 3-watt LED light to illuminate the TB stained tissues with light beam having wave length 400-900 nm.

In a preferred embodiment of the present non-invasive and non-contact reflection spectrometry based cancer detection system, the reflectance probe includes multiple illumination optical fibers and one acquisition optical fiber;
said illumination optical fibers are disposed surrounding the acquisition optical fiber having open ends of all the optical fibers coplanar with respect to tip of the probe;
each of said illumination optical fibers at other end is operatively connected to the LED light to enables transmitting of light to the tissue; and
said acquisition optical fiber at other end is operatively connected to the spectrometer to enables collection of the light reflected from the tissue and send to the spectrometer for the spectral analysis.

In a preferred embodiment of the present non-invasive and non-contact reflection spectrometry based cancer detection system, the microcontroller is operatively connected with the computer for plotting the absorption spectra for the TB stained tissue in real time, whereby absence of the spectral line at wavelength 610 nm which corresponds to the monomeric form of TB indicates non-malignant lesion in the TB stained tissue and presence of the spectral line at wavelength 610 nm indicates malignant lesion in the TB stained tissue.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS:
Figure 1 shows schematic diagram of non-invasive, non-contact system for spectroscopic detection of the oral cancer in accordance with an embodiment of the present invention.

Figure 2 shows work flow of the developed system for spectroscopic detection of the malignant cancer cells in accordance with the present invention.

Figure 3 shows (a) absorption spectrum of staining agent Toluidine Blue (TB) in water and in human serum albumin (HSA protein at pH 7); (b) spectral deconvolution of the TB in water; (c) minor but district difference of TB deconvoluted spectrum in HSA at pH 7 compared to that in water; (d) absorption spectral characteristics of TB in HSA solution at various pH conditions; (e) deconvoluted spectrum of TB-HSA in pH 2; and (f) deconvoluted spectrum of TB-HSA in pH 7 in accordance with the present invention.

Figure 4 shows (a) absorption spectrum of Toluidine Blue (TB) in Genomic DNA (from Calf Thymus) and in water; (b) deconvoluted spectrum of TB-DNA at pH=7.4 in accordance with the present invention.

Figure 5 shows förster Resonance Energy Transfer (FRET) from ethidium bromide (EB) to toluidine blue (TB) is genomic DNA where (a) shows spectral overlap of TB absorption with the emission spectrum of EB and the steady state and picosecond resolved emission quenching events of EB-DNA in presence of TB are shown in (b) and (c) respectively in accordance with the present invention.

Figure 6 shows (a) bright field microscopic image of lung cancer cell lines (A549) wherein fluorescence images of the cell lines with Ethidium bromide (Etbr) labelling in absence and presence of toluidine blue (TB) are shown in (b) and (c); a quantitative estimation from red component in the nucleus images from RGB analysis is shown in (d).

Figure 7 shows (a) photographic representative image of 96 well plates containing either normal embryonic kidney (HEK) cell or lung cancer (A549) cell lines with TB staining of different concentrations; (b) Deconvoluted TB absorption spectra for HEK cells and (c) Deconvoluted TB absorption spectra for A549 cells.

Figure 8 shows (a) absorption spectra acquired through the present system in a clinical trial on malignant and non-malignant human subjects; (b) spectral deconvolution for non-malignant human subjects; (c) spectral deconvolution for malignant human subjects; and (d) corresponding confirmatory biopsy (histopathological slide) image of the TB stained tissue from the malignant subject.

Figure 9 shows (a) absorption spectra acquired through the present system in a clinical trial on malignant and over-stained non-malignant human oral lesion; (b) corresponding deconvoluted spectra for non-malignant case; and (c) corresponding deconvoluted spectra for malignant.

Figure 10 shows absorption spectra acquired through the present system in a clinical trial on an under-stained non-malignant human oral lesion.

DETAILED DESCRIPTION OF THE INVENTION WITH REFERENCE TO THE ACCOMPANYING DRAWINGS:
As stated hereinbefore, the present invention discloses reflection spectroscopy based system for malignant cell detection. The present invention basically discloses a probing or staining element to interact with sample tissues at physiological pH value and spectrometer means to carry out spectrometric arrangement on such sample tissues interacted with the staining element for spectrometry based determination of presence of malignant cell in the sample tissues.
In the present invention, Toluidine Blue (TB) is used as a staining element or staining agent for the diagnosis of cancer cell or malignant cell. The staining element or staining agent TB can interact with Human Serum Albumin (HSA), a model protein at very high pH conditions which is very hard to achieve physiologically. On the other hand, TB significantly interacts with the nucleic acid such as DNA of tissue cells at physiological pH value of 7.4. The molecular studies strengthen the understanding of the Toluidine Blue staining of cancer cells, where the relative ratio of the nucleic acids is higher than the normal intracellular content.
The present invention also discloses a non-invasive, non-contact spectroscopic system for quantitatively detecting the cancer cell/ malignant cell/ precancerous cell including instant detection of oral cancer by exploiting the interaction of the TB with the cell DNA. The system embodiment of the present invention for the detection of the Oral cancer is named as “Oral-o-scope”.
The Toluidine Blue/Toluidine Blue O/ Basic Blue 17/ Tolonium chloride/ Blutene chloride (Molecular Formula: C15H16ClN3S) is a phenothiazine cationic basic, hence acidophilic dye. By exploiting its metachromatic properties it is expected to bind to acidic components of the tissues like sulfates, carboxylates, and phosphates. It interacts with macromolecules and provides a unique yet simple visual molecular recognition technique by staining nucleic acid and polysaccharides. This compound (?654.572 g/mol) fulfills the criteria of being a suitable a charged stain as it cannot cross the plasma membrane of the cells, thus finding its application in in-vivo staining. It found its first application in in situ detection of cervical cancer in 1963 [Ref: D. P. Shedd, P. B. Hukill, and S. Bahn, "In vivo staining properties of oral cancer," The American Journal of Surgery, vol. 110, pp. 631-634, 1965]. Similarly for the patients, with oral lesions, cysts or other neoplasms or for the patients with potential malignancy in their aerodigestive tracts, a 1-2% rinse of the Toluidine Blue (TB) dye or an aqueous based or a weak acid based application will ensure that the dye only retains in those malignant or the potentially malignant tissues (and also the non-specific retention of the dye in dead tissues such as on the upper surface of the tongue) in a qualitative manner. The relative staining of the tissues which provides almost a gradient of colors in the bluish-purple range in the localized malignancies are indicative of the intensity of the malignancy and a give us a qualitative or a visual idea of the stages of the carcinoma. Since TB is a basic and a cationic dye it will have an affinity towards the negatively charged components of the tissues such as DNA and RNA. The malignant epithelia of the mouth contain more amounts of nucleic acids along with wider intracellular channels which help in an increased penetration and retention of the dye than the non-malignant tissues. Thus, after rinsing off the buccal cavity, only the malignant areas would retain the stain and the non-malignant parts would eject out the bulky stain which won’t be able to cross its plasma membranes and interact with the macromolecules [Ref: G. Sridharan and A. A. Shankar, "Toluidine blue: A review of its chemistry and clinical utility," Journal of oral and maxillofacial pathology: JOMFP, vol. 16, p. 251, 2012; J. Wang, Y. Guo, B. Liu, C. Cheng, Z. Wang, G. Han, et al., "Spectroscopic analyses on interaction of bovine serum albumin (BSA) with toluidine blue (TB) and its sonodynamic damage under ultrasonic irradiation," Journal of Luminescence, vol. 131, pp. 231-237, 2011].
In the present invention, the basic photochemistry behind the application mentioned above and possibility of quantitatively detecting cancer cell including detection of the oral cancer via spectroscopic methods exploiting the interaction of TB with cell DNA have been explored.
In a recent study it is shown that absorption spectrum of TB can fitted in to different bands of six different aggregation species simultaneously present in the sample under investigation [Ref: R. Matassa, C. Sadun, L. D'Ilario, A. Martinelli, and R. Caminiti, "Supramolecular organization of toluidine blue dye in solid amorphous phases," The Journal of Physical Chemistry B, vol. 111, pp. 1994-1999, 2007. And L. D'Ilario and A. Martinelli, "Toluidine blue: aggregation properties and structural aspects," Modelling and Simulation in Materials Science and Engineering, vol. 14, p. 581, 2006.]. The overall TB spectrum may be mainly attributed to the H-type aggregation, although some of the species also show the J type bands with distinct spectrum band. The interest is to recheck the evidences of interaction between the dye and the macromolecules such as DNA and protein. Though it has already been reported in the literature with stress that the dye must interact with the nucleic material of the virtue of its anionic and acidic nature [Ref: J. Wang, Y. Guo, B. Liu, C. Cheng, Z. Wang, G. Han, et al., "Spectroscopic analyses on interaction of bovine serum albumin (BSA) with toluidine blue (TB) and its sonodynamic damage under ultrasonic irradiation," Journal of Luminescence, vol. 131, pp. 231-237, 2011.], the strong confirmation of whether this dye interacts with protein at different pH is still unrevealed and this area is focused in the present invention by exploiting the spectroscopic or absorption properties of the chosen model protein and the dye. The understanding of pH dependent interaction of TB will enable to exploit its character in a direction where one can use it for cancer detection.
Materials, System and Methods:
The stock solution of HSA is prepared in 100 mM of Phosphate Buffer Solution (PBS) of physiological pH=7. The stock solution of TB is made in simple distilled water. Milli Q (from Millipore) water is used throughout the experiment. The concentrations of the HSA, DNA and TB stock are spectrophotometrically measured by reported procedures. To understand the interaction of TB with protein and DNA, absorption spectroscopy is carried out. For TB Absorption measurements are made by Shimadzu UV-2450 UV-Visible Spectrophotometer. Using the Lambert-Beer’s Law the concentration of the stock solution is found to be 42 mM. A quartz cuvette of path length 1 cm is used to take the absorption measurements. This solution is later diluted to 100X and 200 X for further use. The experiment is carried out at the room temperature of 25 degrees C. The stock solution is prepared freshly before use. To compare the absorption spectra of HSA along with TB the measurements are made by Shimadzu UV-2450 UV-Visible Spectrophotometer in the same procedure as above to obtain the spectra of equal concentrations (1:1 of 1uM each) of HSA along with added TB in buffer. The experiments are carried out at the room temperature of 25oC. For carrying out Absorption spectral measurements of equal concentrations of TB and HSA between a range of pH, 22 Eppendorftubes are prepared having PBS and divided into two sets. Set A had 11 of the tubes had a constant concentration of TB with a range of pH from 2-11. Set B had 11 of the tubes had a 1:1 ratio of 5uM each [HSA]/[TB] with a range of pH from 2-11. Different amounts of HCl and NaOH are added into the respective sets of buffers by estimation of the eye and are repeatedly checked on the pH paper to get the approximate desired pH value. Absorption measurements are made in the same procedure as above. The stock solution is prepared freshly before use. A similar comparison study is conducted for DNA interaction with TB. A peak at 260 nm for DNA is obtained and a shift of 10 nm is observed.
Commercially available time-correlated single-photon counting (TCSPC) setup with MCP-PMT from Edinburgh instrument, U.K (instrument response function (IRF) of ~90 ps) using a 409 nm excitation laser source is used for measuring all picosecond resolved fluorescence transients. A controller from Julabo (Model:F32) is used to maintain the sample temperature. In the present work, methodology for estimating the FRET efficiency of energy donor (D) to various acceptors (A) has been used. To briefly explain the phenomenon, the equation r6=[R06 (1-E)]/E], can be used to calculate the D-A distance (r), the E in the equation denotes the energy transfer efficiency between donor and acceptor, and R0 is Förster distance.
Human lung carcinoma (A549) cell, human embryonic kidney (HEK293) are cultured in DMEM media (pH 7.4) supplemented with 10% FBS and antibiotic-antimycotic solution 100? (containing 10,000 units penicillin, 10 mg streptomycin, and 25 µg amphotericin B per mL in 0.9% normal saline). The cell lines are maintained at 37°C in an air-jacketed 5% CO2 incubator and are routinely passaged.

All the cells are separately plated at a density of ~100 cells in a 96-well plate. After 24 h incubation, Toluidine blue dye is treated at specified concentrations (write the concentrations) for 30 mins at 37°C. Then the cells are washed with 1X Phosphate Saline Buffer and OD is taken at (check it from data). The same procedure is repeated after the 2nd wash with 1X PBS.
A549 cells and Hek cells are seeded on the cover slip and grown in DMEM media. Cells are fixed for 30 min with 4% paraformaldehyde and subjected to confocal microscopic studies. Cells are then incubated with 2?M Toluidine blue for 15 m, washed twice with 1x PBS, and incubated with EtBr for 15 m. After washing in the same procedure, cells are mounted with anti-quenching agent n-propyl gallate for microscopic slide preparation and detected by confocal fluorescence microscope.
Reflection spectrometry is used for the development of system capable for non-invasive and non-contact detection of malignant cell including oral cancer cells. This system of the present invention consists of the staining element i.e. the staining agent Toluidine Blue (TB), a light source, a cooperative reflectance probe, a spectrograph or spectrometer and microcontroller. The schematic of the developed system for detection of the oral cancer cells is shown in Figure 1.
As shown in the figure 1, the present system can operates with staining element i.e. TB strained sample tissues. In the present case it is TB strained oral cavity wherein the TB interacts with the oral cavity tissues at physiological pH value.
The present system includes a light source (1), a reflectance probe (2), a spectrometer (3) and a microcontroller (4). The light source (1) preferably comprises a 3-watt white LED light to illuminate the TB stained tissues with light beam having wavelength 400-900 nm. The reflectance probe (2) is operatively connected to the light source (1) and adapted to be held proximal to the oral cavity/sample tissues. This enables transmission of the light from the light source (1) to the TB stained tissues/oral cavity.
The reflectance probe (2) is also operatively connected with the spectrometer (3). The reflectance probe (2) accepts diffuse optical signal from the TB stained tissues/oral cavity and send the optical signal to the spectrometer (3). The spectrometer (3) upon receiving the optical signal generates absorption spectra corresponding to the received optical signal. The microcontroller (4) is disposed in operative connection to the spectrometer (3) to receive the absorption spectra and computes spectral deconvolution thereof. The microcontroller (4) is also configured to plot the spectral deconvolution in a cooperative computer or user interface (5). This potting of the spectral deconvolution enables determination of the malignant cell in the tissues/oral cavity based on presence of spectral line in the plotted spectral deconvolution corresponding to interaction of the staining element with only the malignant cells.
The present system also includes a power supply preferably a 5 Volt AC-DC adapter for driving all the system elements. In a preferred embodiment, the microcontroller is based on Arduino Uno platform and embodies software for acquiring necessary information and produces the real time plot in the connected computer. The GUI based software is specifically developed to acquire signals and plot them in real time and quickly arrive at the decision of screening the patient. The computer connects to the developed system via a USB and listens to the interface for incoming data. The acquired data will be an array of the intensity values at various wavelength after necessary calibration. The software developed in LabVIEW platform, is simple, intuitive and needs no trained manpower to operate.
The reflectance probe as used in the present system is basically a lab grade diffuse reflectance probe. The diffuse reflectance probe includes multiple (preferably six) illumination fibers (a) around one acquisition fiber (b). As shown in the figure 1, the illumination optical fibers (a) are disposed surrounding the acquisition optical fiber (b) keeping their open ends coplanar with respect to tip of the probe. The other end each illumination optical fibers is operatively connected to the LED light to enables transmitting of light to the tissues/oral cavity. The acquisition optical fiber is also operatively connected to the spectrometer at its other end to enables collection of the light reflected from the tissues/oral cavity and sending to the spectrometer for the spectral analysis.
Particularly for the case of detection of the malignant cells/precancerous cells in the oral cavity by involving the present system, oral debris from the oral cavity is removed by rinsing with water for 30 sec. This is followed by a 1% acetic acid rinse for 20 sec. Finally, the oral cavity is rinsed with (1%W/W) TB for full 30 sec. The mechanically retained TB stain is then eliminated using 1% acetic acid rinse for 20 sec.
The working principle of the developed system “Oral-o-scope” has been demonstrated in Figure 2. After initiation of the GUI, it performs its “homework” about the availability and health of the connected equipment. After successful establishment of the link to the instrument, the software asks the user to perform at reference and dark value determination procedure. This is a very important step to arrive at the Optical density guided by Beer Lambert’s law.
The reference and dark values can also be a preloaded set of value stored on the hard drive as text files or can be instantaneously provided. The users are then provided with the live intensity (pixel counts) window, stored values for reference intensity and the Optical density window. The reflectance probe is held close to the oral cavity of the patient. The light incident (400-900nm) on the tissue of the oral cavity illuminates the lesion under test and the retro-reflected light is collected by the center fiber and is taken to spectrograph. All obtained graphs are fitted with multiple Gausian peaks to evaluate the individual contribution of each component in the plot. The process of fitting was followed according to one of earlier work [Ref: A. Halder, M. Banerjee, S. Singh, A. Adhikari, P. K. Sarkar, A. M. Bhattacharya, et al., "A Novel Whole Spectrum-based Non-invasive Screening Device for Neonatal Hyperbilirubinemia," IEEE journal of biomedical and health informatics, 2019.]. Each component of the graph corresponds to certain form of molecular TB or some tissue spectroscopic signature. It is observed that the spectra obtained from malignant lesions showed a distinctly different spectral pattern than the normal cells. Multiple sets of data are acquired and averaged to produce a steady trace free from errors that may arise from hand or subject shaking during the time of data acquisition. The software automatically decides to acquire the valid coming from the muscle and ignore any spurious signal.

Results and Discussions:
The figure 3 (a) shows absorption spectrum of Toluidine Blue (TB) in water and in human serum albumin (HSA protein at pH 7). Spectral deconvolution of TB in water is shown in the panel (b); presence of monomer (610 nm) along with some dimer (580 nm and 640 nm) are evident. Panel (c) shows minor but district difference of TB deconvoluted spectrum in HSA at pH 7 compared to that in water. The absorption spectral characteristics of TB in HSA solution at various pH conditions are shown in panel (d). While deconvoluted spectrum of TB-HSA in pH 2 (e) is consistent with that of pH 7 (panel c), the spectrum at pH 11 (f) is distinctly different as the protein undergoes structural denaturation revealing unprecedented absence of dimeric form (580 nm and 640 nm) rather indicate the presence of trimeric species (550 nm). As high pH conditions are rare in biological systems and it is almost impossible to achieve in oral cavities this study gives us the basis for using the dye as an oral cancer detection probe. As the pH condition in cancer cells are deregulated and lowered, TB will not be able to bind with protein in cancer cells in normal circumstances. However, denatured protein is shown have binding affinity, which may have some physiological significance. For example, it was observed that filiformpapillae, when exposed to the toluidine blue, always retain the dye. Although the mechanism was not clear, it might be related to a high protein-synthesis rate. In a recent study it is also reported the presence of denatured sensory proteins in the filiformpapillae.
Figure 4 (a) shows absorption spectrum of Toluidine Blue (TB) in Genomic DNA (from Calf Thymus). The spectrum of TB in water is also shown for comparison. An apparent shift in the TB-DNA spectrum compared to that in water is evident. Panel (b) shows deconvoluted spectrum of TB-DNA, where the presence of monomeric form of TB (610 nm) along with dimeric form are evident (580 nm, 640 nm). This establishes that DNA interacts with the dye in a specific manner consistent with reported literature (Biochim. Biophys. Acts, 145 (1967) 436 445) and offers opportunity to exploit the property to identify malignant cells using the spectroscopic technique. As malignant cells have more amount of nucleic acid as it is highly proliferating, therefore, the cells are expected to be strongly stained with TB. In order to study the specific molecular recognition of TB by the genomic DNA, the FRET studies have been performed to investigate the energy transfer from EtBr (a known intercalary dye for DNA) to TB in the condensate.
Figure 5 (a) shows the emission spectra of EtBr overlaps with the absorption spectrum of TB. The fluorescence transient of EtBr is quenched in presence of TB as shown in Figure 5 (b). The efficiency of energy transfer in the above system is found to be 45% from the temporal fluorescence decay (Figure 5 (c)). The distance between the donor and acceptor was calculated to be 36 Angstrom. Thus, the experimental finding indicates that EtBr and TB can be intercalated simultaneously maintaining a distance of about 10 base pairs. These results help indicate that TB and EtBr both binds the DNA. Hence, TB stain can be a potential marker for increase in nuclear materials which is the characteristic of malignant cells.
After conducting the biophysical study on the specific interaction of TB with DNA, the effect of TB on cell lineshas been investigated. For experiment propose, a normal HEK cell line and A549 lung cancer cell lines have been selected as shown in Figure 6. Bright field microscopic image of lung cancer cell lines (A549) is shown in the panel (a). The fluorescence images of the cell lines with Ethidium bromide (EtBr) labelling in absence and presence of toluidine blue (TB) are shown in the panels (b) and (c). The fluorescence quenching of the nucleus in presence of TB is evident from the insets of the panels. A quantitative estimation from red component in the nucleus images from RGB analysis is shown in the panel (d). A marked decrease in the red fluorescence of EtBr is observed which indicates increased localization of TB in malignant cells and thus quenching of EtBr emission. This further strengthens the present proposition of TB being a predominant staining agent particularly for malignant cells.
Figure 7 (a) depicts photographic image of 96 well plate containing either normal embryonic kidney (HEK) cell or lung cancer (A549) cell lines with TB staining of different concentrations. The arrangement of spectroscopic investigation and microscopic image of TB stained A549 cell lines is also shown. Deconvoluted TB absorption spectra for HEK and A549 are shown in (b) and (c) panels. Note that Monomeric TB form (610 nm) is only present in the cancer cell lines in contrast to its normal counterpart. The experimental observation clearly depicts that presence of 610 nm spectral line may be accompanied with dimeric or trimeric TB species is indicative of malignant cell.
Figure 8 (a) shows absorption spectra acquired through the developed “Oral-o-scope” in a clinical trial on malignant and non-malignant human subjects. It is found that the instrument is able to produce repeatable data under the clinical setting. The spectral deconvolution as shown in panels (b) and (c) clearly reveals absence of monomeric form of TB (610 nm) in non-malignant lesion (b) compared to that in malignant lesion (c). The presence of monomeric TB associated with tetrameric forms (520 nm, 580 nm and 670) is distinctly evident in the malignant tissue. Corresponding confirmatory biopsy (histopathological slide) image of the TB stained tissue from the malignant subject is shown in panel (d). The provisional diagnosis suggested solitary bit of soft tissue from the left buccal mucosa. Histopathological evaluation revealed hyperplastic squamous epithelium with intact basement membrane. However, at one place an invasion of islands of dysplastic epithelium with cellular changes and nuclear hyper-chromatism were observed along with few keratin pearl formations and 1 or 2 abnormal mitoses. High power field of microscope shows highly cellular and vascular tissue stroma as shown in Figure 8d. This is suggestive of ‘moderately differentiated squamous cell carcinoma.
The possibility of detection of over staining of oral lesion, which often led “false-positive” in the visual interpretation of TB-staining has also been explored in the present invention. Figure 9a shows absorption spectra acquired through “Oral-o-scope” in a clinical trial on malignant and over-stained non-malignant human oral lesion. The corresponding deconvoluted spectra are shown in the panels (b) and (c). The remarkable absence of monomeric form of TB in the non-malignant over stained lesion has been noted. The understating of oral tissue is found to be detected by the developed “Oral-o-scope”. Figure 10 shows absorption spectra acquired through “Oral-o-scope” in a clinical trial on an under-stained non-malignant human oral lesion. The absence of monomeric TB form (610 nm) and presence of tissue information represented by 546 nm and 576 nm has been noted which are the signature of oxygenated hemoglobin. Table 1 summarizes the individual contributions of all the deconvoluted peaks in percentage for all the acquired data in this work.
Table 1
Sample
(TB in) Spectral Contributions (%) Forms of TB
520 nm 546 nm 550 nm 576
nm 580
nm 610 nm 640 nm 670 nm
Water - - - 32.64 33.05 34.3 - Monomer+ Dimer
Protein (HSA), pH=7 - - - 15.3 49.67 35.01 - Monomer+ Dimer
Protein (HSA), pH=2 - - - 33.6 39.4 26.96 - Monomer+ Dimer
Protein (HSA), pH=11 - 66.79 - 33.2 - - Monomer+ Trimer
TB-DNA
pH 7.4 - - - 26.54 43.51 30.13 - Monomer+ Dimer
Normal cell (HEK) - - - 41.6 0 58.44 - Dimer
Lung cancer cell (A549) - - - 66.33 13.15 20.50 - Monomer+ Dimer
Non-malignant oral lesion 36.16 - - 21.91 0 - 42.95 Tetramer only
Malignant oral lesion 19.39 - - 0.78 49.9 - 29.87 Tetramer+ Monomer
Overstained malignant oral lesion 7.06 - - 00.24 58.54 - 34.14 Tetramer+ Monomer
Under-stained non-malignant oral lesion - 8.18 - 2.79 48.17 - - 40.84 Oxygenated Hemoglobin+Tetramer

Table 1: Weightage of the deconvoluted spectra of TB sample in various conditions. The individual contribution of monomeric form (610 nm) of TB is substantially high in cells with malignancy and hence can be used as a suitable marker for oral cancer detection.

Claims:WE CLAIM:

1. A non-invasive and non-contact reflection spectrometry based cancer detection system comprising
a light source,
reflectance probe to transmit light form said light source and accept diffuse optical signal from sample for testing stained with staining dye capable of interacting with nucleic acid at physiological pH;
spectrometric means to receive the diffuse optical signal for generating graph components indicative of spectra obtained from the nucleic acid of malignant lesions in the sample interacting with said staining dye having atleast one distinctly different pattern than the nucleic acid of normal cells interacting with said staining dye.

2. The non-invasive and non-contact reflection spectrometry based cancer detection system as claimed in claim 1, for oral cancer detection comprising said spectrometric means generating graph components indicative of spectra obtained from the nucleic acid of the malignant lesions in the sample interacting with cationic staining dye Toluidine Blue (TB) having atleast one distinctly different pattern than the nucleic acid of normal cells in the sample interacting with said cationic staining dye Toluidine Blue TB.

3. The non-invasive and non-contact reflection spectrometry based cancer detection systemas claimed in claim 1 or 2, wherein the spectrometric means comprises
a spectrometer to generate absorption spectra from the received optical signal; and
a microcontroller for computing spectral deconvolution of the absorption spectra and plotting the same to enable determination of the precancerous or malignant cell in the sample based on presence of the spectral line in said plotted spectral deconvolution corresponding to interaction of the staining dye Toluidine Blue TB with only the precancerous or malignant cells.

4. The non-invasive and non-contact reflection spectrometry based cancer detection systemas claimed in anyone of claims 1 to 3, comprises GUI based microcontroller to acquire the signals and plot the spectral deconvolution in real time for ready detection and screening of patients.

5. The non-invasive and non-contact reflection spectrometry based cancer detection system as claimed in anyone of claims 1 to 4, wherein the microcontroller means is configured to include reference and dark values selectively including preloaded in interfacing computer for facilitating the detection based thereon.

6. The non-invasive and non-contact reflection spectrometry based cancer detection system as claimed in anyone of claims 1 to 5, wherein the reflectance probe comprise means for light incident (400-900nm) on tissue of sample/oral cavity and illuminate the lesion under test from the sample/oral cavity and retro-reflected to spectrograph for non-contact and non-invasive detection.

7. The non-invasive and non-contact reflection spectrometry based cancer detection system as claimed in anyone of claims 1 to 6, wherein the spectrometer enables generating distinctly different pattern of TB stain as a potential marker for increase in nuclear material characteristic of malignant cells.

8. The non-invasive and non-contact reflection spectrometry based cancer detection system as claimed in anyone of claims 1 to 7, wherein the spectrometer cooperate with arrangement of spectroscopic investigation and microscopic imaging means for generating deconvoluted TB stain based absorption spectra indicative of Monomeric TB form (610 nm) only present in the malignant/cancer cell lines in contrast to its normal counter-part.

9. The non-invasive and non-contact reflection spectrometry based cancer detection system as claimed in anyone of claims 1 to 8, wherein the Toluidine Blue (TB) is configured to interact with the cells of the sample at physiological pH value of 7.4.

10. The non-invasive and non-contact reflection spectrometry based cancer detection system as claimed in anyone of claims 1 to 9, wherein the precancerous or malignant cells of the tissue stained with the TB includes the TB in monomeric form;
wherein the TB in monomeric form provides the specific spectral line in the spectral deconvolution of the optical signal from the TB stained tissues indicating presence of the precancerous or malignant cells of the tissue.

11. The non-invasive and non-contact reflection spectrometry based cancer detection system as claimed in anyone of claims 1 to 10, wherein the light source includes 3-watt LED light to illuminate the TB stained tissues with light beam having wave length 400-900 nm.

12. The non-invasive and non-contact reflection spectrometry based cancer detection system as claimed in anyone of claims 1 to 11, wherein the reflectance probe includes multiple illumination optical fibers and one acquisition optical fiber;
said illumination optical fibers are disposed surrounding the acquisition optical fiber having open ends of all the optical fibers coplanar with respect to tip of the probe;
each of said illumination optical fibers at other end is operatively connected to the LED light to enables transmitting of light to the tissue; and
said acquisition optical fiber at other end is operatively connected to the spectrometer to enables collection of the light reflected from the tissue and send to the spectrometer for the spectral analysis.

13. The non-invasive and non-contact reflection spectrometry based cancer detection system as claimed in anyone of claims 1 to 12, wherein the microcontroller is operatively connected with the computer for plotting the absorption spectra for the TB stained tissue in real time, whereby absence of the spectral line at wavelength 610 nm which corresponds to the monomeric form of TB indicates non-malignant lesion in the TB stained tissue and presence of the spectral line at wavelength 610 nm indicates malignant lesion in the TB stained tissue.