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WO2024057329A1 - Fluorometric sensory arrays for the detection of urinary bladder cancer-related vocs - Google Patents

Fluorometric sensory arrays for the detection of urinary bladder cancer-related vocs Download PDF

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Publication number
WO2024057329A1
WO2024057329A1 PCT/IN2022/050828 IN2022050828W WO2024057329A1 WO 2024057329 A1 WO2024057329 A1 WO 2024057329A1 IN 2022050828 W IN2022050828 W IN 2022050828W WO 2024057329 A1 WO2024057329 A1 WO 2024057329A1
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sensor
bladder cancer
voc
urinary bladder
ethyl benzene
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Inventor
Neha BHATTACHARYYA
Soumendra Singh SINGH
Dipanjan MUKHERJEE
Gulam NABI
Asim Kumar Mallick
Samir Kumar Pal
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    • G01N33/57557—
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/493—Physical analysis of biological material of liquid biological material urine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N2021/7769—Measurement method of reaction-produced change in sensor
    • G01N2021/7786—Fluorescence

Definitions

  • the present invention provides for a point of care non-invasive screening system for urinary bladder cancer comprising: fluorometric sensor array including array of select fluorescent sensor dyes comprising: cellulosic substrate supported photosensitizer fluorescent sensor dye selected from Nile Red (NR), Eosin Y (EY) and Rose Bengal (RB) having specific selectivity for absorption of select VOC, ethyl benzene, present in urinary bladder cancer, urine sample holder adapted for supporting said fluorometric sensor array that absorbs/traps said VOC, ethyl benzene, present in urinary bladder cancer generated from heated urine sample onto said photosensitizer fluorescent sensor dye; and a configured colorimetric unit for detecting spectroscopic change including fluorescence change induced in said fluorometric sensor array having said fluorescent sensor dyes upon interaction with said trapped VOC, ethyl benzene present in urinary bladder cancer subjects.
  • fluorometric sensor array including array of select fluorescent sensor dyes comprising: cellulosic substrate supported photos
  • Urinary bladder cancer is the most common malignancy of the genitourinary tract ranking as the tenth most common malignancy worldwide [H. Sung, J. Ferlay, R. L. Siegel, M. Laversanne, I. Soerjomataram, A. Jemal, et al., "Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries," CA: a cancer journal for clinicians, vol. 71, pp. 209-249, 2021].
  • Bladder tumours represent a heterogeneous range of cancer. At one end of the spectrum is the low-grade non-invasive Ta stage, which doesn't constitute a threat to the patient population and requires initial endoscopic treatment and surveillance [A.
  • the disease recurrence rate of superficial bladder cancer even after Transurethral resection range from 50-70% which may progress to invasive cancers [ E. J. Small, S. Halabi, G. Dalbagni, R. Pruthi, G. Phillips, M. Edelman, et al., "Overview of bladder cancer trials in the Cancer and Leukemia Group B," Cancer: Interdisciplinary International Journal of the American Cancer Society, vol. 97, pp. 2090-2098, 2003].
  • urinary biomarkers miss a substantial population of patients with urinary bladder cancer and are subjected to false positive results. Additionally, the accuracy of diagnosis is also poor in low stage and low-grade tumours [R. Chou, J. L. Gore, D. Buckley, R. Fu, K. Gustafson, J. C. Griffin, et al., "Urinary biomarkers for diagnosis of bladder cancer: a systematic review and meta-analysis," Annals of internal medicine, vol. 163, pp. 922-931, 2015]. On the contrary, diagnosis of urinary bladder cancer by the presence of volatile organic compounds (VOC) in the urine has been employed for the past decade using standard analytical methods such as GC or GC-MS [F. C.-Y. Wang, K. Qian, and L. A. Green, "GCx MS of diesel: a two-dimensional separation approach,” Analytical Chemistry, vol. 77, pp. 2777-2785, 2005.]
  • VOC volatile organic compounds
  • a Point of care non-invasive screening system for urinary bladder cancer comprising: fluorometric sensor array including array of fluorescent sensor dyes comprising cellulosic substrate supported photosensitizer fluorescent sensor dye selected from Nile Red (NR), Eosin Y (EY) and Rose Bengal (RB) having specific selectivity for absorption of select VOC, ethyl benzene, present in urinary bladder cancer; an urine sample holder adapted for supporting said fluorometric sensor array with said photosensitizer fluorescent sensor dye such as to absorb/trap said VOC, ethyl benzene, present in urinary bladder cancer generated from heated urine sample onto said photosensitizer fluorescent sensor dye; colorimetric unit for detecting spectroscopic change including fluorescence change induced in said fluorometric sensor array having said fluorescent sensor dyes upon interaction with said trapped VOC, ethyl benzene present in urinary bladder cancer.
  • fluorometric sensor array including array of fluorescent sensor dyes comprising cellulosic substrate supported photosensit
  • said point of care non-invasive screening system wherein said fluorometric sensor array comprises flurometric sensor array strip including regions of said photosensitizer fluorescent sensor dye cast upon cellulosic substrate and adapted for detachable operative fixing with respect to said (a) urine sample holder top such as to face the urine sample in said urine sample holder and (b) sensor holder of said colorimetric unit, said colorimetric unit comprises: multi-wavelength spectroscopic device having customised light/optical excitation source and power supply module and including set up to hold said detachable flurometric sensor array strip to examine said flurometric sensor array strip involving diffused light signals passing therethrough; and micro-controller and CCD based detector for triggering of light from said light/optical excitation source for passing through said flurometric sensor array strip and collecting related fluorescent signals for detection of the said VOC, ethyl benzene trapped in said fluorometric sensor array present in urinary bladder cancer by way of optical chemical sensing.
  • said colorimetric unit comprises: multi-wavelength
  • said point of care non-invasive screening system comprises graphical user interface (GUI) based acquisition and analytics for acquiring data from micro-spectrometer including means for operative connection with said microcontroller for tuning LED based optical power into ON/OFF mode, storage means and display means; spectroscopic converter for converting said acquired light signal based spectroscopic inputs into quadrant based and/or linear distribution analytic based indications on presence of carcinoma of urinary bladder for ready and user friendly detection indicative of both early and advanced stages of urinary bladder cancer including postoperative surveillance based on VOC generation having sensitivity at even 10 ppm for bladder cancer VOC ethyl benzene.
  • GUI graphical user interface
  • the point of care non-invasive screening system of the present invention comprises microcontroller for tuning average power of required pulse signal by involving PWM (Pulse width modulation) and intensity control of LED for said strategic tuning of said LED.
  • PWM Pulse width modulation
  • said sensor holder is adapted to hold fluorometric sensor array strip, and cuvette like sample holder to hold ethanol washings of VOC ethyl benzene from said urine sample holder top
  • said colorimetric unit comprising multi-wavelength spectroscopic device for both differential optical chemical sensing based on array of differential fluorescence change induced in said sensor array upon exposure to and interaction with bladder cancer VOC ethyl benzene as biomarker present in urine for operative interaction with said spectroscopic converter for conversion to quadrant based qualitative indication of bladder cancer carcinoma, and, means for recordal of concentration dependent absorbance of VOC ethyl benzene at select wavelength for spectroscopic superposition/fit on concentration vs. absorbance based linear distribution of VOC ethyl benzene for quantitative mass screening of urinary bladder cancer respectively with minimal cost.
  • said customized light/optical excitation source of said multiwavelength spectroscopic device includes UV LEDs of wavelength 395 nm each that cooperates with micro-controller to trigger said sensor array in a chronological order for collection and detection of fluorescence based optical signal in said CCD based detector for said detection of trapped VOC, ethyl benzene including said sensor array thereby favouring for easy colorimetric detection of bladder cancer VOCs with minimized need of extensive signal transduction hardware.
  • a point of care non-invasive screening system wherein said cellulose based flurometric sensor array strip material include selective hydrophilicity/porosity of Pore size: 20-25 pm (Particle retention), thickness: 205 pm, ash: ⁇ 0.06% and basic weight: 92 g/m 2 for select absorption of VOC ethyl benzene onto said casted including drop casted sensor dye based array adapted for retention of up to at least 2 hours without any change of fluorescence intensity.
  • said point of care non-invasive screening system comprising optical chemical sensing colorimetry means cooperative with said fluorometric sensor array for sensing fluorescence changes based on select three hydrophobic photo sensitizer molecules of said Nile Red (NR), Eosin Y (EY), and Rose Bengal (RB) due to solvation effect of Nile Red and the radical generation behaviour of Eosin Y along with Rose Bengal upon interaction with ethyl benzene VOCs based de-coloration of the flurophore with indicative increment of fluorescence intensity and related spectral shift of the fluorophores, means for tracking, recording, analytics and display as said quadrant based qualitative indication for determining both early and advanced stages of urinary bladder cancer including postoperative surveillance.
  • optical chemical sensing colorimetry means cooperative with said fluorometric sensor array for sensing fluorescence changes based on select three hydrophobic photo sensitizer molecules of said Nile Red (NR), Eosin Y (EY), and Rose Bengal (RB) due to solvation effect of Nile
  • said point of care non-invasive screening system wherein said colorimetric unit comprising multi-wavelength spectroscopic device enabling quantitative screening of urinary bladder cancer includes colorimetric unit based micro-controller triggered acquisition of spectral data including UV-Vis absorbance spectral data from cuvette comprising ethyl benzene VOC in ethanol at various concentrations via UV-LED based excitation and absorption by said VOC in the UVC region of 150 to 280 nm and selectively at ⁇ 268 nm for clear linear distribution/fit at ⁇ 268 nm against varying concentration of ethyl benzene VOC in storage means as calibration curve and/ or calibrating the system; micro-controller based acquisition of real time absorbance data of ethyl benzene VOC in ethanol from cuvette generated by urinary bladder cancer for cooperative ready feed and analytics based on sequence of defined instructions allowing fitting said real time data with said calibration curve to yield absorbance dependent quantitative real time concentration of
  • the point of care non-invasive screening system is provided wherein said fluorometric sensor array based fluorescence change enabling said qualitative quadrant-based indication/screening including said colorimetric unit based sensor array analytics generation based on: significant but regular increase of EY fluorescent intensity after exposure of the EY sensor towards the urine VOCs compared to that of healthy control; manifold increase of RB fluorescent intensity after exposure of the VOC towards RB sensor as compared to healthy control; significant blue shift of the NR sensor intensity with negligible increment of the intensity in presence of urine VOCs as compared to healthy control; system storage records, as instrumental index and analytic means for clusters towards generation of quadrant-based indication selectively including means for tracking differential positive variation of fluorescent intensity amplitude of EY and RB, differential positive variation of fluorescent intensity amplitude of NR followed by differential negative intensity amplitude of NR throughout the wavelength range of 400-700nm as compared to negligible variations of healthy control enabling normalized photon counts; means for analytics by plotting for "E-R test" to
  • a method for point of care non-invasive screening for urinary bladder cancer comprising the steps of providing urine sample from urinary bladder cancer subject in a urine sample holder having a releasable fitting lid; providing a fluorometric sensor array as sensor strip including array of fluorescent sensor dyes comprising cellulosic substrate supported photosensitizer fluorescent sensor dye selected from Nile Red (NR), Eosin Y (EY) and Rose Bengal (RB) having specific selectivity for absorption of select urinary bladder cancer VOC ethyl benzene; incubating by heating said urine sample holder covered with the lid at temperatures of 35-degree Centigrade with said releasable fitted lid of urine sample holder detachably attaching said sensor array on said cellulosic substrate and facing said urine sample for absorbing/trapping select bladder cancer VOC ethyl benzene on said fluorometric sensor array; removing said sensor strip from said releasable fitted lid of urine sample holder after
  • said urine sample holder top lid having condensed VOC ethyl benzene is washed in ethanol for collection in cuvette like sample holder for placing in sensor holder of said colorimetric unit comprising spectroscopic device for detecting UV-vis absorbance intensity based spectroscopic change due to concentration of said VOC ethyl benzene for assisting non-invasive quantitative screening for urinary bladder cancer.
  • said step of detection of fluorescence change induced in said fluorometric sensor array upon interaction with said trapped urinary bladder cancer VOC ethyl benzene is carried out based on the following steps examining the fluorometric sensor array based sensor strip by said colorimetric unit comprising multi-wavelength spectroscopic device including four UV LEDs of wavelength 395 nm each for illuminating four positions of the sensor strip having said three select sensor dyes of Nile Red (NR), Eosin Y (EY) and Rose Bengal (RB) and one blank position; collecting diffused light signals from said sensor holder including collection of diffused light signals from three sensor dye positions and one blank position of said sensor strip for sending said collected diffused light signals to CCD based spectrometer detector of said colorimetric unit; and performing spectroscopic conversion of thus collected/ acquired light signals into quadrant based indications on presence of carcinoma of urinary bladder for ready and user friendly detection of both early and advanced stages
  • said step of screening for urinary bladder cancer includes collecting diffused light signals from sensor holder including collecting light signals from sensor array strip, and/or, cuvette so as to obtain quadrant based and/or linear distribution fit based indications of urinary bladder cancer by carrying out said colorimetric unit based sensor array analytics generation based on monitoring: significant but regular increase of EY fluorescent intensity after exposure of the EY sensor towards the urine VOCs compared to that of healthy control; manifold increase of RB fluorescent intensity after exposure of the VOC towards RB sensor as compared to healthy control; significant blue shift of the NR sensor intensity with negligible increment of the intensity in presence of urine VOCs as compared to healthy control; correlating with system storage records, as instrumental index and analytic means for cluster location towards generation of quadrant-based indication selectively including tracking differential positive variation of fluorescent intensity amplitude of EY and RB, differential positive variation of fluorescent intensity amplitude of NR followed by differential negative intensity amplitude of NR throughout the wavelength range
  • said collection of diffused light signals for screening of urinary bladder cancer is done in the absence and presence of said sensor strip on the sensor holder not exposed to urine sample for generating baseline and reference UV-vis absorbance spectra for recordal, followed by collecting diffused fluorescent light signals by placing the sensor strip with said sensor array on sensor holder exposed to said urine sample VOC ethyl benzene for converting said light signals into fluorescence spectral data and collating the same with reference spectral data for each sensor of said sensor array for spectroscopic conversion and display as said quadrant based indications/screening of urinary bladder carcinoma.
  • FIG. 1 (a) Schematic of the technique used in our 'NABIL' device to collect the volatile organic compounds (VOCs) from the urine sample.
  • the red arrows are the guide to the eyes to depict the deposition of the VOCs through condensation in the cold surface of the lid of the urine chamber,
  • the absorption spectra of the VOCs of the cancer patient as well as healthy control is shown in the same micrograph,
  • EBZ ethyl benzene
  • VOC of the EBZ at different concentrations has been collected by dissolving the EBZs at different concentrations in water and finally the whole mixtures is subjected towards gentle heating (at heat chamber) followed by condensation.
  • Black arrows signify the different peak positions of the UV-Visible absorption of the VOC of EBZ in water
  • Inset depicts the steady-state emission of the VOCs of EBZ by dissolving the condensed VOCs into the lid of the urine chamber in water;
  • Figure 2 (a)
  • the strip has been developed on cellulose paper from Whatman.
  • Three different colour holes with a white hole within the black strip signify the location of the sensors and the corresponding reference of the measurement in strip.
  • Fluorophore molecules with variable solvatochromisms Rose Bengal (RB), Eosin Y (EY) and Nile Red (NR)
  • Sensor strips for healthy control and cancer patients are shown to depict the transformation after interacting of the strip with the condensed VOCs of urinary bladder cancer patients.
  • EY Eosin Y
  • RB Rose Bengal
  • NR Nile Red
  • Scheme 1 Schematic of the collection of the deposited VOCs into the lid of the Urine chamber through condensation through Step 1 (condensation of VOCs); Step 2 (dissolving the VOCs in ethanol); Step 3 (collection of the mixture);
  • Scheme 2 Schematic of the possible excited state reaction mechanism through radical pathway of EY (a) and RB (b) in presence of VOCs of EBZ;
  • Scheme 3 Schematic of the excited state charge transfer reaction of NR in presence of EBZ VOC
  • Figure 3 Intensity variation of the EY, RB and NR in our indigeneously developed sensor strip after exposing the strip to the condensed VOCs of cancer patients as well as healthy control are shown in (a), (b) and (c) respectively.
  • Reference signifies the intensity of the EY, RB and NR in sensor strip before exposing the strip to the VOCs.
  • (d) Differential intensity variation of the EY, RB and NR in cancer patients as well as healthy control. Differential spectra have been calculated by subtracting the intensity spectra of the reference from the intensity after exposing the strip to the condensed VOCs;
  • Figure 4 depict the typical NABILOGY E-R and N-R reports respectively, (c), (d), (e), and (f) show the probability of occurrence of urinary bladder cancer in a control subject, a diseased subject, a subject undergoing treatment and a subject suffering from prostate cancer respectively;
  • Figure 5 depict the NABILOGY reports of in-vitro test results. Intensity variation of the EY, RB and NR in sensor strip in our in-vitro and in- vivo study recorded in our "NABIL" device.
  • the solid line represents the intensity of the particular sensor when the strip is exposed towards urine VOCs.
  • the dotted line represents the intensity of the same sensor when the strip is exposed towards the artificially created VOC by dissolving same concentration of ethyl benzene in water;
  • Figure 6 illustrates overall advantages of our techniques of urinary bladder cancer detection over other existing in the market.
  • the present invention provides for a care non- invasive screening system for urinary bladder cancer
  • fluorometric sensor array including array of fluorescent sensor dyes comprising cellulosic substrate supported photosensitizer fluorescent sensor dye selected from Nile Red (NR), Eosin Y (EY) and Rose Bengal (RB) having specific selectivity for absorption of select VOC, ethyl benzene, present in urinary bladder cancer
  • an urine sample holder adapted for supporting said fluorometric sensor array with said photosensitizer fluorescent sensor dye such as to absorb/trap said VOC, ethyl benzene, present in urinary bladder cancer generated from heated urine sample onto said photosensitizer fluorescent sensor dye
  • colorimetric unit for detecting spectroscopic change including fluorescence change induced in said fluorometric sensor array having said fluorescent sensor dyes upon interaction with said trapped VOC, ethyl benzene present in urinary bladder cancer.
  • Three select photo sensitizer molecules (Nile Red, Eosin Y, and Rose Bengal) allows detection of a specific VOC, ethyl benzene which is specifically present in the urine of patients with urinary bladder cancer.
  • NR Nile Red
  • EY Eosin Y
  • RB Rose Bengal
  • the sensor having onto it trapped/ absorbed VOC ethyl benzene allowing optical chemical sensing by the system thus has been utilized to detect the fluorescence change induced in an array of three fluorescent dyes namely Eosin Y (EY), Rose Bengal (RB) and Nile Red (NR) upon interaction with the odiform carcinogenic compounds preferably and selectively ethyl benzene present in urine.
  • EY Eosin Y
  • RB Rose Bengal
  • NR Nile Red
  • spectroscopic change including fluorescence change induced in an array of three fluorescent dyes namely Eosin Y (EY), Rose Bengal (RB) and Nile Red (NR) upon interaction with ethyl benzene VOC, fed into the present system and processed by system means comprising graphical user interface (GUI) based acquisition and analytics for acquiring select data from micro-spectrometer including means for operative connection with said microcontroller for tuning LED based optical power into ON/OFF mode, storage means and display means, together with, spectroscopic converter for converting said acquired light signal based spectroscopic inputs into quadrant based and/or linear distribution analytic based indications on presence of carcinoma of urinary bladder, thus allows ready and user friendly detection indicative of both early and advanced stages of urinary bladder cancer including postoperative surveillance based on VOC generation having sensitivity at even 10 ppm for bladder cancer VOC ethyl benzene.
  • GUI graphical user interface
  • the choice of the sensor paper with defined hydrophilicity/porosity (Pore size: 20-25 pm (Particle retention), thickness: 205 pm, ash: ⁇ 0.06% and basic weight: 92 g/m 2 ) and presence of cellulose component is the key to stop the detachment of the sensitive materials upon VOC exposure. It was found that the sensor paper facilitates the absorption of VOC and its retention up to two hours without any change of the fluorescence of the sensitive materials on it.
  • the indigenously developed sensor strip is attached on the top lid of urine sample holder. Then the sample holder is closed with the lid and is placed in the warm (37° C) incubation chamber. After 15 minutes, the sensor strip from the sample holder is taken from the bath and placed on to the sensor holder.
  • the developed instrument/system coined as includes as system means a multi-wavelength spectroscopic device (STS-VIS, manufactured by Ocean Optics, Florida), and a customized power supply module. It consists of a set-up to examine the sensor strip by its exposure to a set of four UV LEDs (3W, 700LUX) of wavelength 395 nm each. The diffused light signals collected from the four sensors of the strip are sent to the CCD based spectrometer respectively. The detector and the associated electronics are controlled by a microcontroller. The microcontroller also would control triggering of the LEDs in a chronological order and the detector (spectrometer) to collect the fluorescence signal. Development of an Intuitive technique/Algorithm for Data Acquisition and Real-Time Analysis:
  • the device/system is fed with an indigenously developed Graphical User Interface (GUI) based acquisition and analytical tool.
  • GUI Graphical User Interface
  • the on-board computer uses the microcontroller to acquire data from the micro-spectrometer and sequentially and strategically turning LED lights in the OFF/ON mode.
  • the intensity of LED based optical power source were programmatically controlled by tuning average power of the pulse signal using PWM mechanism.
  • the acquired data were analysed by an intuitive algorithm based technique which converts the spectroscopic information into a quadrant-based indication of presence of carcinoma thus making easier for the medical personnel to decipher.
  • the analytical tool is entirely developed in LabVIEW platform for automated data acquisition and visualization of spectra as well as clinical condition of the patient. After getting powered up, health check-up and initialization of the instrument takes place. If there is any discrepancy, the device auto-corrects different conditions and restarts automatically, followed by a pop-up window asking for patient details including name, age, sex, medical conditions etc. to be saved along with data in individual folders.
  • the analytical tool next guides to store 'baseline' and 'reference' spectra one time for a particular ambience.
  • the baseline spectrum is collected under the absence of sensor strip on the sensor holder, then the reference spectrum is collected by placing the strip which has not been exposed to any urine sample.
  • the integration time of the spectrometer is kept fixed at 60 seconds and the boxcar width (Smoothening factor) to be 2 in this entire study for maintaining proper signal to noise ratio (S/N) of the spectra.
  • the main data collection window opens and after clicking the 'start' button along with placing the sensor strip exposed to the urine sample on the holder, the software starts analysing the spectra.
  • the collected fluorescence signals are processed using an indigenously developed analytics and compared with an in-built library in the device in order to get probability of urinary bladder cancer in the urine sample under test.
  • the machine learning strategy included in the developed software automatically train the device for the revision of in-built library using the data from the samples under test.
  • the self-developed analytical tool/ analytics uploads all data to cloud for future big data analysis and implementation of further machine learning algorithm.
  • the device is loT and telemedicine enabled for quicker medical intervention if required.
  • the experimental protocol consisted of the data collection using the system/device coined as (NABIL) of the present invention, parameters being calculated from data, and the intuitive algorithm being trained using the parameters, to create a model that could estimate the probability of urinary bladder cancer in the urine sample under test.
  • Ethyl Benzene, Rose Bengal, Eosin Y and Nile Red are purchased from Sigma Aldrich (Saint Louis, USA). These compounds are declared as highest purified grade and used without any further purification. Ethanol (Merck, India) and Water (Mili-Q) are used as solvents. Black strip and the glass paper for the sensor strip preparation has been purchased from local super market.
  • 2.5 cm x 2.5 cm sensor blocks are generated by simple paper cutting.
  • the 5 mm each perforation within the sensor strip is created by using a puncher machine.
  • the four perforations within the sensor strip are blocked by fabricating Whatman 4 filter paper with the size slightly greater than 5 mm.
  • the other side of the sensor block which remains unexposed towards the urine VOCs is covered by a glass paper for further protection from any contamination.
  • the sample size was estimated using the Everald's equation for power calculation in diagnostics tests. Assuming the prevalence of urinary bladder cancer to be 70% the minimal sample size required was found to be 52.
  • NABIL and NRSMH were responsible for uploading the NABIL readings to the database.
  • the histological parameters were measured by expert doctors of the Department of Urology, NRSMH.
  • the TURBT reports with proper identification numbers of the selected subjects were uploaded by another laboratory technician hired for the study purpose.
  • the readings of both the methods were matched based on the identification number by one research staff, to ensure complete blindness of the study. Complete blinding was maintained to keep the two sets of readings separate.
  • FIG. la displays the schematic of the collection of the VOCs from urine samples for the detection of the urinary bladder cancers in indigenously developed present system coined as "NABIL" device through a sensor strip. Details of the collection of the VOCs from several urine subjects of bladder cancer patients as well as healthy control has been mentioned in the experimental section of the manuscript.
  • the UV-Visible absorption spectra of the collected VOCs have been shown in Figure lb. As observed from the figure, VOCs of the bladder cancer patients produce multiple absorption maxima in UVC region ( ⁇ 150-280 nm) of the spectral wavelength with a characteristic absorption peak at ⁇ 268 nm, whereas, in contrast, no such absorption peak has been observed for the VOCs collected from healthy control at this spectral range.
  • VOC markers were selected to investigate the crude UV-Visible absorption of individual VOCs by maintaining the similar ppm level of concentrations to that of urine (namely, ethyl benzene, hexanal and nonyl chloride) since the study of Jain et. al. has further explored that the concentrations of these 3 particular VOC biomarkers are increased to a significant extent compare to that of other biomarkers.
  • the marker solvents are dissolved in water and were collected as VOCs by following similar technique as mentioned earlier for the collection of VOCs present in urine (Schematic 1).
  • Figure 1c displays the UV-Visible absorption of crude ethylbenzene as VOC marker in different concentrations ranging from ⁇ 25 ppm to ⁇ 200 ppm.
  • VOC level is increased due to the various metabolic and nutritional activities of cells in various physiological conditions which being solely a demographic phenomenon, depends on the food habits as well as quality of the daily life of the common people.
  • the observation regarding the prevalence of nonyl chloride is inconclusive mainly due to the very limited clinical trial, performed by Nabi et. al.
  • the absorption intensities at the select peak of ⁇ 268 nm have been plotted against the concentration of ethyl benzene in Figure Id which surprisingly shows a very good linear fit.
  • the purpose of such construction of the present invention is to generate a calibration curve for the quantitative determination of concentration of ethyl benzene in the urine of various degree of bladder cancers which is useful for the sensitivity determination of the newly developed system coined as 'NABIL' device.
  • Sensor strip ( Figure 2a) is based on optical fluorescence sensors array and highly sensitive towards environmental changes like pH, redox as well as solvatochromisms.
  • the select fluorophores [Eosin Y (EY), Rose Bengal (RB) and Nile Red (NR)] are selectively chosen as hydrophobic to reduce the possibility of the interference of water during excited state photochemical reactions.
  • Figure 2a depicts the de-coloration of the fluorophore which is visually distinguishable after getting the sufficient exposure of the bladder cancer VOCs compare to that of healthy control.
  • the grade of cancer has also been corroborated by the histopathological examination of the cancer lesions following TURBT.
  • the tissue shows papillary morphology for both the low-grade and high-grade cancer tissues.
  • patches of skip lesions were present corroborating the high-grade cancer.
  • EY is one of the xanthene group of dyes that has enormous utility in cosmetics industry because of its intense orange colour.
  • ethyl benzene is modelled as VOC by taking into account previous observation in accordance with the invention that UV-Visible absorption of VOCs of urine of the patients suffering from bladder cancer is comparable to that of crude ethyl benzene as only VOC marker.
  • EY by absorbing photon (Aex ⁇ 375 nm), EY generates EY* (singlet), which undergoes immediately to a triplet excited state through an intersystem crossing (ISC) by which Br atom of the aromatic ring carbon-Br bond of EY* becomes less stable and causes bromination of the ethyl benzene through a radical chain reaction.
  • ISC intersystem crossing
  • Certain increment of fluorescence intensity of RB is observed after reacting with ethyl benzene VOC biomarker ( Figure 2b).
  • RB acts as a photosensitizer, participates in photoexcitation in its excited state of ethyl benzene at benzylic positions by forming a radical ion pair with superoxide anion of other VOCs like trace amount of H 2 O 2 as a consequence of sensitization (Scheme 2c).
  • steady state fluorescence spectra of NR in presence of ethyl benzene VOC are characterized by a remarkable blue shift with significant fluorescence quenching.
  • the blue shifted emission of NR ( Figure 2d) originates from the nonplanar LE state of NR which is entrapped by the non-polar VOC environment where the probe is significantly screened from the exposure of the bulk water vapour.
  • This differential variation of the intensity of the sensors after getting exposure of the VOCs of the urinary bladder cancer patients is further involved as an instrumental index for assisting the analytics of the present system towards generation of the diagnostic report. In order to generate a score from the present method based on the system, cluster analysis have been performed, where the normalized photon counts from the sensitive materials in the sensor strip are plotted in the 2 graphs.
  • E-R test The plot depicting normalized photon numbers from sensitive material RB versus that from EY is called "E-R test” as shown in Figure 4a.
  • the entire data set has been categorized into four quadrants after analysing the results obtained from the limited clinical trial and comparing the same with reports generated from our "NABIL" device.
  • E-R test all normal subjects are essentially confined in the third quadrant of the plot. Normalized deviation for a particular subject, can be considered as the probability of the occurrence of urinary cancer.
  • N-R test Another projection revealing normalized photon number from RB versus that from NR is called “N-R test” is considered and is depicted in Figure 4b.
  • NABILOGY report which is the outcome of NABIL device.
  • the NABILOGY test report of a control subject is shown in the Figure 4c, where both E-R and N-R tests reveal the cancer occurrence probability of 0% (both in quadrant 3). Similarly, that of a urinary bladder cancer patient showed the occurrence probability to be 89% and 88% respectively (in quadrant 1 and 4).
  • the Line of Significance (15%), obtained comparing the data of the control and diseased samples generated from both the histopathological report and the NABIL device, is also shown in the test report.
  • the medical history of the subject indicates that the patient underwent Transurethral Resection of Bladder Tumor (TURBT) recently and the biopsy report stated that the grade of cancer to be T1HG, which means that the tumor has spread to the connective tissue and that it is of high grade in nature.
  • TURBT Transurethral Resection of Bladder Tumor
  • the concentration of the EBZ in urinary bladder cancer patients has been determined by observing 268 nm UV-Visible absorption peak of the collected VOC from urine and taking into account molar extinction coefficient of pure EBZ at 268 nm.
  • Intensity of each sensor (EY, RB and NR) is significantly higher ( ⁇ 2-3 times) after getting exposure to the urine VOC compared to that of pure ethyl benzene at same concentration in the present in-vitro study. This may be due to fact that the ethyl benzene is not the only biomarker, but there are several other organic volatile compounds in trace amounts that interacts with the sensor fluorophores as well.
  • VOCs bladder cancer volatile organic compounds
  • the sensitivity and the specificity of the instrument as opposed to the gold standard method of cystoscopy was estimated.
  • the instrument/ system of the present invention is fund to have a specificity of 86%, which evaluates the potential of the device to detect proportions of true negatives in a population, who are also tested as negative by the instrument.
  • the sensitivity of the instrument which evaluates the proportions of true positives which are also tested as positive by the instrument is 87 %.
  • the instrument can specifically screen urinary bladder patients from the control population by the help of the VOC marker with a sensitivity of 87 %.
  • urinary bladder cancer is one of the most common cancers having extensively high possibility of recurrence and mortality rate across the globe, current clinical diagnostic approaches are either invasive or inaccurate.
  • a cost effective, system/ device based on select activation of select environmental sensitive fluorophores namely Nile Red (NR), Eosin Y (EY) and Rose Bengal (RB) as sensor materials for detecting bladder cancer VOCs from urine samples as a biomarker has been devised.
  • NR Nile Red
  • EY Eosin Y
  • RB Rose Bengal
  • the present invention contributes a novel, non-invasive, easy-to-use, inexpensive, real-time, accurate method for selectively urinary bladder cancer diagnosis, which is useful for personalized care/diagnosis and postoperative surveillance, resulting in saving more lives.

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Abstract

A system/"NABIL" device based on an array of environmentally sensitive f luorophores, selected from Nile Red (NR), Eosin Y (EY) and Rose Bengal (RB) as sensor materials for detecting bladder cancer VOCs from urine samples. Large increment of fluorescence intensity with a spectral shift of fluorophores could be recorded by the calorimetric unit upon the interaction of the fluorescent sensor dyes with trapped VOC, ethyl benzene present in urinary bladder cancer.

Description

FLUOROMETRIC SENSORY ARRAYS FOR THE DETECTION OF URINARY BLADDER CANCER-RELATED VOCS
FIELD OF INVENTION:
The present invention provides for a point of care non-invasive screening system for urinary bladder cancer comprising: fluorometric sensor array including array of select fluorescent sensor dyes comprising: cellulosic substrate supported photosensitizer fluorescent sensor dye selected from Nile Red (NR), Eosin Y (EY) and Rose Bengal (RB) having specific selectivity for absorption of select VOC, ethyl benzene, present in urinary bladder cancer, urine sample holder adapted for supporting said fluorometric sensor array that absorbs/traps said VOC, ethyl benzene, present in urinary bladder cancer generated from heated urine sample onto said photosensitizer fluorescent sensor dye; and a configured colorimetric unit for detecting spectroscopic change including fluorescence change induced in said fluorometric sensor array having said fluorescent sensor dyes upon interaction with said trapped VOC, ethyl benzene present in urinary bladder cancer subjects.
Also provided is a method for point of care non-invasive screening for urinary bladder cancer for ready and user friendly detection indicative of both early and advanced stages of urinary bladder cancer including postoperative surveillance based on VOC generation having sensitivity at even 10 ppm for bladder cancer VOC ethyl benzene. Keywords: Diagnosis, Bladder cancer, Volatile Organic Compounds, Non-invasive detection.
BACKGROUND ART:
Urinary bladder cancer is the most common malignancy of the genitourinary tract ranking as the tenth most common malignancy worldwide [H. Sung, J. Ferlay, R. L. Siegel, M. Laversanne, I. Soerjomataram, A. Jemal, et al., "Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries," CA: a cancer journal for clinicians, vol. 71, pp. 209-249, 2021]. Bladder tumours represent a heterogeneous range of cancer. At one end of the spectrum is the low-grade non-invasive Ta stage, which doesn't constitute a threat to the patient population and requires initial endoscopic treatment and surveillance [A. Lopez-Beltran, "Bladder cancer: clinical and pathological profile," Scandinavian journal of urology and nephrology, vol. 42, pp. 95-109, 2008;J. T. Matulay, M. Soloway, J. A. Witjes, R. Buckley, R. Persad, D. L. Lamm, et al., "Risk-adapted management of low-grade bladder tumours: Recommendations from the international bladder cancer group (IBCG)," BJU international, vol. 125, pp. 497-505, 2020]. On the other end of the spectrum, is the invasive high-grade tumours which possess a high malignant potential and is associated with significant cancer progression and cancer death rates [A. Lopez-Beltran, "Bladder cancer: clinical and pathological profile," Scandinavian journal of urology and nephrology, vol. 42, pp. 95-109, 2008;J. T. Matulay, M. Soloway, J. A. Witjes, R. Buckley, R. Persad, D. L. Lamm, et al., "Risk-adapted management of low-grade bladder tumours: Recommendations from the international bladder cancer group (IBCG)," BJU international, vol. 125, pp. 497-505, 2020]. In addition, a high rate of recurrence is also associated with the urinary bladder cancer. The disease recurrence rate of superficial bladder cancer even after Transurethral resection range from 50-70% which may progress to invasive cancers [ E. J. Small, S. Halabi, G. Dalbagni, R. Pruthi, G. Phillips, M. Edelman, et al., "Overview of bladder cancer trials in the Cancer and Leukemia Group B," Cancer: Interdisciplinary International Journal of the American Cancer Society, vol. 97, pp. 2090-2098, 2003].
The most common diagnosis of urinary bladder cancer of patients presented with hematuria include urine cytology, hematuria dipstick, tumour biopsy and cystoscopy [H. B. Grossman, L. Gomella, Y. Fradet, A. Morales, J. Presti, C. Ritenour, et al., "A phase III, multicenter comparison of hexaminolevulinate fluorescence cystoscopy and white light cystoscopy for the detection of superficial papillary lesions in patients with bladder cancer," The Journal of urology, vol. 178, pp. 62-67, 2007; D. Schrag, L. J. Hsieh, F. Rabbani, P. B. Bach, H. Herr, and C. B. Begg, "Adherence to surveillance among patients with superficial bladder cancer," Journal of the National Cancer Institute, vol. 95, pp. 588-597, 2003; C. L. Pashos, M. F. Botteman, B. L. Laskin, and A. Redaelli, "Bladder cancer: epidemiology, diagnosis, and management," Cancer practice, vol. 10, pp. 311-322, 2002; M. Babjuk, M. Burger, R. Zigeuner, S. F. Shariat, B. W. Van Rhijn, E. Comperat, et al., "EAU guidelines on non-muscle- invasive urothelial carcinoma of the bladder: update 2013," European urology, vol. 64, pp. 639-653, 2013; M. C. Hall, S. S. Chang, G. Dalbagni, R. S. Pruthi, J. D. Seigne, E. C. Skinner, et al., "Guideline for the management of nonmuscle invasive bladder cancer (stages Ta, Tl, and Tis): 2007 update," The Journal of urology, vol. 178, pp. 2314-2330, 2007; T. Dudderidge, J. Stockley, G. Nabi, J. Mom, N. Umez-Eronini, D. Hrouda, et al., "A novel, non- invasive test enabling bladder cancer detection in urine sediment of patients presenting with haematuria— a prospective multicentre performance evaluation of ADXBLADDER," European Urology Oncology, vol. 3, pp. 42-46, 2020; F. J. P. van Valenberg, A. M. Hiar, E. Wallace, J. A. Bridge, D. J. Mayne, S. Beqaj, et al., "Validation of an mRNA-based urine test for the detection of bladder cancer in patients with haematuria," European Urology Oncology, vol.
4, pp. 93-101, 2021] As cystoscopy is an invasive technique and costly, non- invasive diagnosis of urinary bladder cancer is sought after. For this purpose, several studies have reported the presence of urine-based biomarkers as potential alternatives for the initial diagnosis of bladder cancer [ M. Herman, R. Svatek, Y. Lotan, P. Karakiewizc, and S. Shariat, "Urine-based biomarkers for the early detection and surveillance of non-muscle invasive bladder cancer," Minerva urologica e nefrologica= The Italian journal of urology and nephrology, vol. 60, pp. 217-235, 2008; S. D. Smith, M. A. Wheeler, J. Plescia, J. W. Colberg, R. M. Weiss, and D. C. Altieri, "Urine detection of survivin and diagnosis of bladder cancer," Jama, vol. 285, pp. 324-328, 2001; E. J. Pietzak, A. Bagrodia, E. K. Cha, E. N. Drill, G. Iyer, S. Isharwal, et al., "Next-generation sequencing of nonmuscle invasive bladder cancer reveals potential biomarkers and rational therapeutic targets," European urology, vol. 72, pp. 952-959, 2017; S. K. Arya and P. Estrela, "Electrochemical ELISA- based platform for bladder cancer protein biomarker detection in urine," Biosensors and Bioelectronics, vol. 117, pp. 620-627, 2018; M. MacGregor, H.
5. Shirazi, K. M. Chan, K. Ostrikov, K. McNicholas, A. Jay, et al., "Cancer cell detection device for the diagnosis of bladder cancer from urine," Biosensors and Bioelectronics, vol. 171, p. 112699, 2021; K. W. Kim, Y. Shin, A. P. Perera, Q. Liu, J. S. Kee, K. Han, et al., "Label-free, PCR-free chip-based detection of telomerase activity in bladder cancer cells," Biosensors and Bioelectronics, vol. 45, pp. 152-157, 2013]. However, urinary biomarkers miss a substantial population of patients with urinary bladder cancer and are subjected to false positive results. Additionally, the accuracy of diagnosis is also poor in low stage and low-grade tumours [R. Chou, J. L. Gore, D. Buckley, R. Fu, K. Gustafson, J. C. Griffin, et al., "Urinary biomarkers for diagnosis of bladder cancer: a systematic review and meta-analysis," Annals of internal medicine, vol. 163, pp. 922-931, 2015]. On the contrary, diagnosis of urinary bladder cancer by the presence of volatile organic compounds (VOC) in the urine has been employed for the past decade using standard analytical methods such as GC or GC-MS [F. C.-Y. Wang, K. Qian, and L. A. Green, "GCx MS of diesel: a two-dimensional separation approach," Analytical Chemistry, vol. 77, pp. 2777-2785, 2005.]
Although, several strategies involving mobile sensors like acoustic wave, conductive polymers, quartz crystal microbalance, etc. continue to be reported to detect the VOCs [ T. Kida, M.-H. Seo, S. Kishi, Y. Kanmura, N. Yamazoe, and K. Shimanoe, "Application of a solid electrolyte CO2 sensor for the analysis of standard volatile organic compound gases," Analytical chemistry, vol. 82, pp. 3315-3319, 2010; T. Kida, H. Harano, T. Minami, S. Kishi, N. Morinaga, N. Yamazoe, et al., "Control of electrode reactions in a mixed-potential-type gas sensor based on a BiCuVOx solid electrolyte," The Journal of Physical Chemistry C, vol. 114, pp. 15141-15148, 2010; C. Tucker, N. Chen, J. Engel, Y. Yang, S. Pandya, and C. Liu, "High-sensitivity bidirectional flow sensor based on biological inspiration of animal haircell sensors," in SENSORS, 2006 IEEE, 2006, pp. 1440-1442; X. Zhong, D. Li, W. Du, M. Yan, Y. Wang, D. Huo, et al., "Rapid recognition of volatile organic compounds with colorimetric sensor arrays for lung cancer screening," Analytical and bioanalytical chemistry, vol. 410, pp. 3671-3681, 2018] their performance suffer from several drawbacks like low sensitivity, low selectivity, lack of reproducibility, interference from humidity, and limited stability have confined their applications [A. Hierlemann and R. Gutierrez-Osuna, "Higher- order chemical sensing," Chemical reviews, vol. 108, pp. 563-613, 2008; A.- C. Romain and J. Nicolas, "Long term stability of metal oxide-based gas sensors for e-nose environmental applications: An overview," Sensors and Actuators B: Chemical, vol. 146, pp. 502-506, 2010; H. Lin, M. Jang, and K. S. Suslick, "Preoxidation for colorimetric sensor array detection of VOCs," Journal of the American Chemical Society, vol. 133, pp. 16786-16789, 2011]. Hence there is a need in the art to provide for point of care non-invasive screening system based on select fluorometric sensor dye array of select dye molecules that would enable tracking of select bladder cancer VOC based on system means and analytics to advantageously allow bedside use of said system at point of care setting that would be of practical importance for non- invasive mass diagnosis or screening of urinary cancer with minimal cost at a point of care.
OBJECTS OF THE INVENTION:
It is thus the basic object of the present invention to provide for point of care non-invasive screening system based on select fluorometric sensor dye array of select dye molecules that would enable tracking of select bladder cancer VOC based on system configuration for acquiring spectral data from said sensor array and select analytics.
It is another object of the present invention to provide for said system that would advantageously allow bedside use of said system at point of care setting to be of practical importance for non-invasive mass diagnosis or screening of urinary bladder cancer with minimal cost at a point of care.
It is yet another object of the present invention to provide for said system and a method thereof that would allow ready and user friendly detection indicative of both early and advanced stages of urinary bladder cancer including postoperative surveillance based on VOC generation having sensitivity at the levels of even 10 ppm of urinary bladder cancer VOC ethyl benzene.
It is still another object of the present invention to provide for said system that would allow both qualitative followed by quantitative screening of urinary bladder cancer VOCs of subjects. SUMMARY OF THE INVENTION:
Thus according to the basic aspect of the present invention there is provided a Point of care non-invasive screening system for urinary bladder cancer comprising: fluorometric sensor array including array of fluorescent sensor dyes comprising cellulosic substrate supported photosensitizer fluorescent sensor dye selected from Nile Red (NR), Eosin Y (EY) and Rose Bengal (RB) having specific selectivity for absorption of select VOC, ethyl benzene, present in urinary bladder cancer; an urine sample holder adapted for supporting said fluorometric sensor array with said photosensitizer fluorescent sensor dye such as to absorb/trap said VOC, ethyl benzene, present in urinary bladder cancer generated from heated urine sample onto said photosensitizer fluorescent sensor dye; colorimetric unit for detecting spectroscopic change including fluorescence change induced in said fluorometric sensor array having said fluorescent sensor dyes upon interaction with said trapped VOC, ethyl benzene present in urinary bladder cancer.
Preferably said point of care non-invasive screening system wherein said fluorometric sensor array comprises flurometric sensor array strip including regions of said photosensitizer fluorescent sensor dye cast upon cellulosic substrate and adapted for detachable operative fixing with respect to said (a) urine sample holder top such as to face the urine sample in said urine sample holder and (b) sensor holder of said colorimetric unit, said colorimetric unit comprises: multi-wavelength spectroscopic device having customised light/optical excitation source and power supply module and including set up to hold said detachable flurometric sensor array strip to examine said flurometric sensor array strip involving diffused light signals passing therethrough; and micro-controller and CCD based detector for triggering of light from said light/optical excitation source for passing through said flurometric sensor array strip and collecting related fluorescent signals for detection of the said VOC, ethyl benzene trapped in said fluorometric sensor array present in urinary bladder cancer by way of optical chemical sensing.
More preferably said point of care non-invasive screening system comprises graphical user interface (GUI) based acquisition and analytics for acquiring data from micro-spectrometer including means for operative connection with said microcontroller for tuning LED based optical power into ON/OFF mode, storage means and display means; spectroscopic converter for converting said acquired light signal based spectroscopic inputs into quadrant based and/or linear distribution analytic based indications on presence of carcinoma of urinary bladder for ready and user friendly detection indicative of both early and advanced stages of urinary bladder cancer including postoperative surveillance based on VOC generation having sensitivity at even 10 ppm for bladder cancer VOC ethyl benzene.
According to another preferred aspect the point of care non-invasive screening system of the present invention comprises microcontroller for tuning average power of required pulse signal by involving PWM (Pulse width modulation) and intensity control of LED for said strategic tuning of said LED.
According to yet another aspect of the present invention there is provided point of care non-invasive screening system wherein said sensor holder is adapted to hold fluorometric sensor array strip, and cuvette like sample holder to hold ethanol washings of VOC ethyl benzene from said urine sample holder top, said colorimetric unit comprising multi-wavelength spectroscopic device for both differential optical chemical sensing based on array of differential fluorescence change induced in said sensor array upon exposure to and interaction with bladder cancer VOC ethyl benzene as biomarker present in urine for operative interaction with said spectroscopic converter for conversion to quadrant based qualitative indication of bladder cancer carcinoma, and, means for recordal of concentration dependent absorbance of VOC ethyl benzene at select wavelength for spectroscopic superposition/fit on concentration vs. absorbance based linear distribution of VOC ethyl benzene for quantitative mass screening of urinary bladder cancer respectively with minimal cost.
Preferably said point of care non-invasive screening system is provided wherein said customized light/optical excitation source of said multiwavelength spectroscopic device includes UV LEDs of wavelength 395 nm each that cooperates with micro-controller to trigger said sensor array in a chronological order for collection and detection of fluorescence based optical signal in said CCD based detector for said detection of trapped VOC, ethyl benzene including said sensor array thereby favouring for easy colorimetric detection of bladder cancer VOCs with minimized need of extensive signal transduction hardware.
According to another preferred aspect of the present invention there is provided a point of care non-invasive screening system wherein said cellulose based flurometric sensor array strip material include selective hydrophilicity/porosity of Pore size: 20-25 pm (Particle retention), thickness: 205 pm, ash: <0.06% and basic weight: 92 g/m2 for select absorption of VOC ethyl benzene onto said casted including drop casted sensor dye based array adapted for retention of up to at least 2 hours without any change of fluorescence intensity.
According to yet another preferred aspect of the present invention there is provided said point of care non-invasive screening system comprising optical chemical sensing colorimetry means cooperative with said fluorometric sensor array for sensing fluorescence changes based on select three hydrophobic photo sensitizer molecules of said Nile Red (NR), Eosin Y (EY), and Rose Bengal (RB) due to solvation effect of Nile Red and the radical generation behaviour of Eosin Y along with Rose Bengal upon interaction with ethyl benzene VOCs based de-coloration of the flurophore with indicative increment of fluorescence intensity and related spectral shift of the fluorophores, means for tracking, recording, analytics and display as said quadrant based qualitative indication for determining both early and advanced stages of urinary bladder cancer including postoperative surveillance.
According to another preferred aspect of the present invention there is provided said point of care non-invasive screening system wherein said colorimetric unit comprising multi-wavelength spectroscopic device enabling quantitative screening of urinary bladder cancer includes colorimetric unit based micro-controller triggered acquisition of spectral data including UV-Vis absorbance spectral data from cuvette comprising ethyl benzene VOC in ethanol at various concentrations via UV-LED based excitation and absorption by said VOC in the UVC region of 150 to 280 nm and selectively at ~ 268 nm for clear linear distribution/fit at ~ 268 nm against varying concentration of ethyl benzene VOC in storage means as calibration curve and/ or calibrating the system; micro-controller based acquisition of real time absorbance data of ethyl benzene VOC in ethanol from cuvette generated by urinary bladder cancer for cooperative ready feed and analytics based on sequence of defined instructions allowing fitting said real time data with said calibration curve to yield absorbance dependent quantitative real time concentration of said ethyl benzene VOC to thereby ascertain the degree of bladder cancers.
Preferably the point of care non-invasive screening system is provided wherein said fluorometric sensor array based fluorescence change enabling said qualitative quadrant-based indication/screening including said colorimetric unit based sensor array analytics generation based on: significant but regular increase of EY fluorescent intensity after exposure of the EY sensor towards the urine VOCs compared to that of healthy control; manifold increase of RB fluorescent intensity after exposure of the VOC towards RB sensor as compared to healthy control; significant blue shift of the NR sensor intensity with negligible increment of the intensity in presence of urine VOCs as compared to healthy control; system storage records, as instrumental index and analytic means for clusters towards generation of quadrant-based indication selectively including means for tracking differential positive variation of fluorescent intensity amplitude of EY and RB, differential positive variation of fluorescent intensity amplitude of NR followed by differential negative intensity amplitude of NR throughout the wavelength range of 400-700nm as compared to negligible variations of healthy control enabling normalized photon counts; means for analytics by plotting for "E-R test" to depict the normalized photon count for RB sensor vs. EY sensor; and/or by plotting for "N-R test" to depict the normalized photon count for said RB sensor vs. NR sensor wherein, normalized deviation for a particular subject is considered as the probability of the occurrence of urinary bladder cancer with normal subjects of 0% cancer occurrence probability being essentially confined to the third quadrant for both ER and NR tests.
According to another aspect of the present invention there is provided a method for point of care non-invasive screening for urinary bladder cancer comprising the steps of providing urine sample from urinary bladder cancer subject in a urine sample holder having a releasable fitting lid; providing a fluorometric sensor array as sensor strip including array of fluorescent sensor dyes comprising cellulosic substrate supported photosensitizer fluorescent sensor dye selected from Nile Red (NR), Eosin Y (EY) and Rose Bengal (RB) having specific selectivity for absorption of select urinary bladder cancer VOC ethyl benzene; incubating by heating said urine sample holder covered with the lid at temperatures of 35-degree Centigrade with said releasable fitted lid of urine sample holder detachably attaching said sensor array on said cellulosic substrate and facing said urine sample for absorbing/trapping select bladder cancer VOC ethyl benzene on said fluorometric sensor array; removing said sensor strip from said releasable fitted lid of urine sample holder after absorbing/trapping select bladder cancer VOC ethyl benzene; placing said sensor strip in sensor holder of colorimetric unit comprising a spectroscopic device for detecting spectroscopic change including fluorescence change induced in said fluorometric sensor array having said fluorescent sensor dyes upon interaction with said trapped urinary bladder cancer VOC ethyl benzene and involving optical chemical sensing of select dyes to thereby enable point of care non-invasive screening for urinary bladder cancer.
Preferably in said method wherein said urine sample holder top lid having condensed VOC ethyl benzene is washed in ethanol for collection in cuvette like sample holder for placing in sensor holder of said colorimetric unit comprising spectroscopic device for detecting UV-vis absorbance intensity based spectroscopic change due to concentration of said VOC ethyl benzene for assisting non-invasive quantitative screening for urinary bladder cancer.
According to another preferred aspect of the present method for point of care non-invasive screening for urinary bladder cancer said step of detection of fluorescence change induced in said fluorometric sensor array upon interaction with said trapped urinary bladder cancer VOC ethyl benzene is carried out based on the following steps examining the fluorometric sensor array based sensor strip by said colorimetric unit comprising multi-wavelength spectroscopic device including four UV LEDs of wavelength 395 nm each for illuminating four positions of the sensor strip having said three select sensor dyes of Nile Red (NR), Eosin Y (EY) and Rose Bengal (RB) and one blank position; collecting diffused light signals from said sensor holder including collection of diffused light signals from three sensor dye positions and one blank position of said sensor strip for sending said collected diffused light signals to CCD based spectrometer detector of said colorimetric unit; and performing spectroscopic conversion of thus collected/ acquired light signals into quadrant based indications on presence of carcinoma of urinary bladder for ready and user friendly detection of both early and advanced stages of urinary bladder cancer including postoperative surveillance with detection sensitivity levels of about even 10 ppm of bladder cancer VOC ethyl benzene coupled with minimized influence of confounding factors based on method steps related to VOC collection from urine and related analysis.
Preferably in said method for point of care non-invasive screening for urinary bladder cancer said step of screening for urinary bladder cancer includes collecting diffused light signals from sensor holder including collecting light signals from sensor array strip, and/or, cuvette so as to obtain quadrant based and/or linear distribution fit based indications of urinary bladder cancer by carrying out said colorimetric unit based sensor array analytics generation based on monitoring: significant but regular increase of EY fluorescent intensity after exposure of the EY sensor towards the urine VOCs compared to that of healthy control; manifold increase of RB fluorescent intensity after exposure of the VOC towards RB sensor as compared to healthy control; significant blue shift of the NR sensor intensity with negligible increment of the intensity in presence of urine VOCs as compared to healthy control; correlating with system storage records, as instrumental index and analytic means for cluster location towards generation of quadrant-based indication selectively including tracking differential positive variation of fluorescent intensity amplitude of EY and RB, differential positive variation of fluorescent intensity amplitude of NR followed by differential negative intensity amplitude of NR throughout the wavelength range of 400-700nm as compared to negligible variations of healthy control to enable normalized photon counts; carrying out analytics by plotting for "E-R test" to depict the normalized photon count for RB sensor vs. EY sensor; and/or by plotting for "N-R test" to depict the normalized photon count for said RB sensor vs. NR sensor wherein, normalized deviation for a particular subject is considered as the probability of the occurrence of urinary bladder cancer with normal subjects of 0% cancer occurrence probability is essentially confined to the third quadrant for both ER and NR tests for a quadrant based selectivity and sensitivity report generation of tests.
Preferably in said method for point of care non-invasive screening for urinary bladder cancer said collection of diffused light signals for screening of urinary bladder cancer is done in the absence and presence of said sensor strip on the sensor holder not exposed to urine sample for generating baseline and reference UV-vis absorbance spectra for recordal, followed by collecting diffused fluorescent light signals by placing the sensor strip with said sensor array on sensor holder exposed to said urine sample VOC ethyl benzene for converting said light signals into fluorescence spectral data and collating the same with reference spectral data for each sensor of said sensor array for spectroscopic conversion and display as said quadrant based indications/screening of urinary bladder carcinoma.
BRIEF DESCRIPTION OF FIGURES:
Figure 1: (a) Schematic of the technique used in our 'NABIL' device to collect the volatile organic compounds (VOCs) from the urine sample. The red arrows are the guide to the eyes to depict the deposition of the VOCs through condensation in the cold surface of the lid of the urine chamber, (b) UV- Visible absorption of the collected VOCs in the lid of the chamber through condensation by dissolving the VOCs in 300 pL ethanol and finally 30 pL of the whole mixture in 1970 pL in water. The absorption spectra of the VOCs of the cancer patient as well as healthy control is shown in the same micrograph, (c) UV-Visible absorption spectra of the VOC of ethyl benzene (EBZ) in water. VOC of the EBZ at different concentrations has been collected by dissolving the EBZs at different concentrations in water and finally the whole mixtures is subjected towards gentle heating (at heat chamber) followed by condensation. Black arrows signify the different peak positions of the UV-Visible absorption of the VOC of EBZ in water, (d) Linear distribution of the UV-Visible absorption of the condensed VOCs with the concentration of dissolved EBZs in water. Inset depicts the steady-state emission of the VOCs of EBZ by dissolving the condensed VOCs into the lid of the urine chamber in water;
Figure 2: (a) Indigenously developed urine strip for the detection of presence of urinary bladder cancer for our 'NABIL' device. The strip has been developed on cellulose paper from Whatman. Three different colour holes with a white hole within the black strip signify the location of the sensors and the corresponding reference of the measurement in strip. Fluorophore molecules with variable solvatochromisms (Rose Bengal (RB), Eosin Y (EY) and Nile Red (NR)), act as the sensors. Sensor strips for healthy control and cancer patients are shown to depict the transformation after interacting of the strip with the condensed VOCs of urinary bladder cancer patients. Variation of the intensity of Eosin Y (EY), Rose Bengal (RB) and Nile Red (NR) of the sensor strip in our 'NABIL' device after interacting with the condensed VOCs of EBZ at two different concentrations are shown in (b), (c) and (d) respectively. 'Initial' corresponds the fluorescence intensity of the sensor before interacting with the EBZ VOCs in our invitro experiment;
Scheme 1 : Schematic of the collection of the deposited VOCs into the lid of the Urine chamber through condensation through Step 1 (condensation of VOCs); Step 2 (dissolving the VOCs in ethanol); Step 3 (collection of the mixture);
Scheme 2: Schematic of the possible excited state reaction mechanism through radical pathway of EY (a) and RB (b) in presence of VOCs of EBZ;
Scheme 3: Schematic of the excited state charge transfer reaction of NR in presence of EBZ VOC;
Figure 3: Intensity variation of the EY, RB and NR in our indigeneously developed sensor strip after exposing the strip to the condensed VOCs of cancer patients as well as healthy control are shown in (a), (b) and (c) respectively. Reference signifies the intensity of the EY, RB and NR in sensor strip before exposing the strip to the VOCs. (d) Differential intensity variation of the EY, RB and NR in cancer patients as well as healthy control. Differential spectra have been calculated by subtracting the intensity spectra of the reference from the intensity after exposing the strip to the condensed VOCs;
Figure 4: (a) and (b) depict the typical NABILOGY E-R and N-R reports respectively, (c), (d), (e), and (f) show the probability of occurrence of urinary bladder cancer in a control subject, a diseased subject, a subject undergoing treatment and a subject suffering from prostate cancer respectively;
Figure 5: (a) and (b) depict the NABILOGY reports of in-vitro test results. Intensity variation of the EY, RB and NR in sensor strip in our in-vitro and in- vivo study recorded in our "NABIL" device. In (c), (d) and (e), the solid line represents the intensity of the particular sensor when the strip is exposed towards urine VOCs. The dotted line represents the intensity of the same sensor when the strip is exposed towards the artificially created VOC by dissolving same concentration of ethyl benzene in water;
Figure 6: illustrates overall advantages of our techniques of urinary bladder cancer detection over other existing in the market.
DETAILED DESCRIPTION OF THE INVENTION:
As discussed hereinbefore, the present invention provides for a care non- invasive screening system for urinary bladder cancer comprising: fluorometric sensor array including array of fluorescent sensor dyes comprising cellulosic substrate supported photosensitizer fluorescent sensor dye selected from Nile Red (NR), Eosin Y (EY) and Rose Bengal (RB) having specific selectivity for absorption of select VOC, ethyl benzene, present in urinary bladder cancer; an urine sample holder adapted for supporting said fluorometric sensor array with said photosensitizer fluorescent sensor dye such as to absorb/trap said VOC, ethyl benzene, present in urinary bladder cancer generated from heated urine sample onto said photosensitizer fluorescent sensor dye; colorimetric unit for detecting spectroscopic change including fluorescence change induced in said fluorometric sensor array having said fluorescent sensor dyes upon interaction with said trapped VOC, ethyl benzene present in urinary bladder cancer.
Three select photo sensitizer molecules (Nile Red, Eosin Y, and Rose Bengal) allows detection of a specific VOC, ethyl benzene which is specifically present in the urine of patients with urinary bladder cancer. The system of the present invention and the sensor strip therein based on select three photo sensitizer molecules configured to a colorimetric unit comprising a multi-wavelength spectroscopic device advantageously capturing by way of optical chemical sensing the solvation effect of Nile Red (NR) and the radical generation behaviour of Eosin Y (EY) along with Rose Bengal (RB) upon interaction with select VOC ethyl benzene was found to favour an easy colorimetric technique/method that minimizes the need for extensive signal transduction hardware. The sensor having onto it trapped/ absorbed VOC ethyl benzene allowing optical chemical sensing by the system thus has been utilized to detect the fluorescence change induced in an array of three fluorescent dyes namely Eosin Y (EY), Rose Bengal (RB) and Nile Red (NR) upon interaction with the odiform carcinogenic compounds preferably and selectively ethyl benzene present in urine.
The thus captured spectroscopic change including fluorescence change induced in an array of three fluorescent dyes namely Eosin Y (EY), Rose Bengal (RB) and Nile Red (NR) upon interaction with ethyl benzene VOC, fed into the present system and processed by system means comprising graphical user interface (GUI) based acquisition and analytics for acquiring select data from micro-spectrometer including means for operative connection with said microcontroller for tuning LED based optical power into ON/OFF mode, storage means and display means, together with, spectroscopic converter for converting said acquired light signal based spectroscopic inputs into quadrant based and/or linear distribution analytic based indications on presence of carcinoma of urinary bladder, thus allows ready and user friendly detection indicative of both early and advanced stages of urinary bladder cancer including postoperative surveillance based on VOC generation having sensitivity at even 10 ppm for bladder cancer VOC ethyl benzene.
Such a system and method is not workable unless optical chemical sensing based data acquisition of VOC ethyl benzene interaction with select three dyes are acquiesced by the system for processing by analytics of said system.
The in-vitro studies and the photo-physics behind the VOC interaction indicates a sensitivity of 10 ppm with the present developed sensor, which together with UV LED, and micro spectrometer as an excitation source and detector respectively enabled a system as bedside point of care non-invasive screening system of practical importance for non-invasive mass diagnosis or screening of urinary cancer with minimal cost at a point of care setting.
EXAMPLES:
MATERIALS AND METHODS:
Experimental Setup:
The choice of the sensor paper with defined hydrophilicity/porosity (Pore size: 20-25 pm (Particle retention), thickness: 205 pm, ash: <0.06% and basic weight: 92 g/m2) and presence of cellulose component is the key to stop the detachment of the sensitive materials upon VOC exposure. It was found that the sensor paper facilitates the absorption of VOC and its retention up to two hours without any change of the fluorescence of the sensitive materials on it. The indigenously developed sensor strip is attached on the top lid of urine sample holder. Then the sample holder is closed with the lid and is placed in the warm (37° C) incubation chamber. After 15 minutes, the sensor strip from the sample holder is taken from the bath and placed on to the sensor holder.
The developed instrument/system coined as (NABIL) includes as system means a multi-wavelength spectroscopic device (STS-VIS, manufactured by Ocean Optics, Florida), and a customized power supply module. It consists of a set-up to examine the sensor strip by its exposure to a set of four UV LEDs (3W, 700LUX) of wavelength 395 nm each. The diffused light signals collected from the four sensors of the strip are sent to the CCD based spectrometer respectively. The detector and the associated electronics are controlled by a microcontroller. The microcontroller also would control triggering of the LEDs in a chronological order and the detector (spectrometer) to collect the fluorescence signal. Development of an Intuitive technique/Algorithm for Data Acquisition and Real-Time Analysis:
The device/system is fed with an indigenously developed Graphical User Interface (GUI) based acquisition and analytical tool. The on-board computer uses the microcontroller to acquire data from the micro-spectrometer and sequentially and strategically turning LED lights in the OFF/ON mode. The intensity of LED based optical power source were programmatically controlled by tuning average power of the pulse signal using PWM mechanism. The acquired data were analysed by an intuitive algorithm based technique which converts the spectroscopic information into a quadrant-based indication of presence of carcinoma thus making easier for the medical personnel to decipher.
The analytical tool is entirely developed in LabVIEW platform for automated data acquisition and visualization of spectra as well as clinical condition of the patient. After getting powered up, health check-up and initialization of the instrument takes place. If there is any discrepancy, the device auto-corrects different conditions and restarts automatically, followed by a pop-up window asking for patient details including name, age, sex, medical conditions etc. to be saved along with data in individual folders. The analytical tool next guides to store 'baseline' and 'reference' spectra one time for a particular ambience. The baseline spectrum is collected under the absence of sensor strip on the sensor holder, then the reference spectrum is collected by placing the strip which has not been exposed to any urine sample. The integration time of the spectrometer is kept fixed at 60 seconds and the boxcar width (Smoothening factor) to be 2 in this entire study for maintaining proper signal to noise ratio (S/N) of the spectra. Finally, the main data collection window opens and after clicking the 'start' button along with placing the sensor strip exposed to the urine sample on the holder, the software starts analysing the spectra. The collected fluorescence signals are processed using an indigenously developed analytics and compared with an in-built library in the device in order to get probability of urinary bladder cancer in the urine sample under test. The machine learning strategy included in the developed software, automatically train the device for the revision of in-built library using the data from the samples under test. The self-developed analytical tool/ analytics uploads all data to cloud for future big data analysis and implementation of further machine learning algorithm. Thus, the device is loT and telemedicine enabled for quicker medical intervention if required.
Experimental Protocol:
The experimental protocol consisted of the data collection using the system/device coined as (NABIL) of the present invention, parameters being calculated from data, and the intuitive algorithm being trained using the parameters, to create a model that could estimate the probability of urinary bladder cancer in the urine sample under test.
Chemicals:
Ethyl Benzene, Rose Bengal, Eosin Y and Nile Red are purchased from Sigma Aldrich (Saint Louis, USA). These compounds are declared as highest purified grade and used without any further purification. Ethanol (Merck, India) and Water (Mili-Q) are used as solvents. Black strip and the glass paper for the sensor strip preparation has been purchased from local super market.
Preparation of the sensor strip:
2.5 cm x 2.5 cm sensor blocks are generated by simple paper cutting. The 5 mm each perforation within the sensor strip is created by using a puncher machine. The four perforations within the sensor strip are blocked by fabricating Whatman 4 filter paper with the size slightly greater than 5 mm. The other side of the sensor block which remains unexposed towards the urine VOCs is covered by a glass paper for further protection from any contamination.
Preparation of the fluorescence dye solution (sensors):
For the preparation of the stock solution, requisite number of fluorophores are dissolved in ethanol and corresponding concentrations of the individual fluorophores has been estimated from the UV-Visible absorbance and corresponding molar extinction coefficients. The ~1 mM stock dye solution is further diluted to ~ 50 pM in each three cases and 2pL of the diluted solution is drop casted over the Whatman paper of the sensor strip. The rest one is kept as it is and further used for recording blank or instrumental index before performing the real sample. Characterization technique:
Steady state absorption and emission spectra are recorded with a Shimadzu UV2600 spectrophotometer and Jovin Yvon Fluorolog fluorimeter included in the present system. Microscopy images of the histopathological slides are captured in Leica DMI8A microscope through DFC550 camera equipped with the microscope. All the sensing experiments through the sensor strip after exposing with the urine VOCs has been performed in "NABIL" device. Details of the discuss part has been discussed in the particular section.
Study Settings:
Prospective observational study conducted over 5 months starting from January 2022 at the Department of Urology, Nil Ratan Sircar Medical College and Hospitals (NRSMH, a Govt, aided tertiary hospital), Kolkata, India.
Sample Size Estimation:
The sample size was estimated using the Everald's equation for power calculation in diagnostics tests. Assuming the prevalence of urinary bladder cancer to be 70% the minimal sample size required was found to be 52.
Study Design and Subjects:
A small-scale clinical trial on 85 subjects were performed among which, 52 patients were reported to have confirmed urinary bladder cancer, 7 subject revealed to have fresh haematuria and 5 subjects were reported to have other types of cancer. Urine samples from 21 normal volunteers were also collected for the control study. Comprehensive details of the subjects are provided in Table 1.
It is worth mentioning that the recruitment of patients was not consecutive as not all physicians practicing in the department were involved in the study. The cancer patients getting treatment under the physicians associated with the study were inducted.
Quality Assurance in Data Collection:
Care was taken that a similar clinical protocol i.e., study, reference, and sample collection methods, and patient enrolment strategies were prospectively maintained throughout the experimental period. To avoid bias in measurements, particular care was taken to keep the technicians, clinicians, investigators, and data analysts at data collection sites blinded to the NABIL and the histopathological findings. Data of each subject on pre-defined variables like the date, identification number, sex, tobacco consumption habits, occupation, habitat, carcinogenic history, whether having any risk factors, treatment details, etc. was collected from clinical charts on a tablet having required database with the in-built proforma by one laboratory technician hired for the study purpose. Urine collection and measurements by system of the present invention coined as NABIL were performed by trained nurses of the Department of Urology, NRSMH. They were responsible for uploading the NABIL readings to the database. The histological parameters were measured by expert doctors of the Department of Urology, NRSMH. The TURBT reports with proper identification numbers of the selected subjects were uploaded by another laboratory technician hired for the study purpose. The readings of both the methods (NABIL and the conventional) were matched based on the identification number by one research staff, to ensure complete blindness of the study. Complete blinding was maintained to keep the two sets of readings separate.
Ethical Considerations:
For the present work, all necessary ethical permissions were taken from the Institutional Medical Ethics Committee, NRSMH, Kolkata (Ref. No. : NRSMC/IEC/41/2022, dated May 20, 2022). All studies involving human subjects were performed following the Declaration of Helsinki (46) and guidelines provided by the Indian Council for Medical Research (ICMR), Govt, of India. Written informed consent was obtained from the subjects who agreed to participate in the study after understanding the relevant details and the consequences. All data and information about the subjects were kept under anonymous head for maintaining confidentiality and used only for this study.
Results and Discussions:
Identification of VOC marker in urine:
Figure la displays the schematic of the collection of the VOCs from urine samples for the detection of the urinary bladder cancers in indigenously developed present system coined as "NABIL" device through a sensor strip. Details of the collection of the VOCs from several urine subjects of bladder cancer patients as well as healthy control has been mentioned in the experimental section of the manuscript. The UV-Visible absorption spectra of the collected VOCs have been shown in Figure lb. As observed from the figure, VOCs of the bladder cancer patients produce multiple absorption maxima in UVC region (~150-280 nm) of the spectral wavelength with a characteristic absorption peak at ~ 268 nm, whereas, in contrast, no such absorption peak has been observed for the VOCs collected from healthy control at this spectral range. The appearance of multiple absorption peaks in the spectrum of VOCs of the bladder cancer patients compared to that of healthy control, indicates the presence of several organic compounds. Jobu. et. al. have earlier applied GC-MS analysis to urine samples of bladder cancer patients as well as healthy controls, observed the appearance of several characteristic peaks within GC-MS signals of bladder cancer patients whereas such signal is completely absent in healthy controls [K. Jobu, C. Sun, S. Yoshioka, J. Yokota, M. Onogawa, C. Kawada, et al., "Metabolomics study on the biochemical profiles of odor elements in urine of human with bladder cancer," Biological and Pharmaceutical Bulletin, vol. 35, pp. 639-642, 2012; S. Zhu, S. Corsetti, Q. Wang, C. Li, Z. Huang, and G. Nabi, "Optical sensory arrays for the detection of urinary bladder cancer-related volatile organic compounds," Journal of biophotonics, vol. 12, p. e201800165, 2019].
Those peaks are identified by them as ethylbenzene, nonanoyl chloride, dodecanal, 5-dimethyl-3(2H)-isoxazolone etc [ K. Jobu, C. Sun, S. Yoshioka, J. Yokota, M. Onogawa, C. Kawada, et al., "Metabolomics study on the biochemical profiles of odor elements in urine of human with bladder cancer," Biological and Pharmaceutical Bulletin, vol. 35, pp. 639-642, 2012]. Furthermore, Jian et. al. has reported 11 different VOC markers associated with urinary bladder cancer where the level of each VOC increases or decreases upon development of cancer into the urinary bladder wall through transition cells, compared to that of healthy control, detected by their own developed chemi-resistive sensor of artificial pattern recognition [ Y. Jian, N. Zhang, T. Liu, Y. Zhu, D. Wang, H. Dong, et al., "Artificially Intelligent Olfaction for Fast and Noninvasive Diagnosis of Bladder Cancer from Urine," ACS sensors, 2022]. The sensing response of such chemi-resistive sensor lies in between 25 to 200 ppm for various VOCs, depending on the gradation of the bladder cancer during their clinical trial [Y. Jian, N. Zhang, T. Liu, Y. Zhu, D. Wang, H. Dong, et al., "Artificially Intelligent Olfaction for Fast and Noninvasive Diagnosis of Bladder Cancer from Urine," ACS sensors, 2022]. In our further study, the investigation of the abundance of a particular VOC biomarker or group of biomarkers in the urine of bladder cancer patients have been emphasized which may be useful to understand the cancer prognosis as well as treatment direction in early stages. In this direction, 3 particular VOC markers were selected to investigate the crude UV-Visible absorption of individual VOCs by maintaining the similar ppm level of concentrations to that of urine (namely, ethyl benzene, hexanal and nonyl chloride) since the study of Jain et. al. has further explored that the concentrations of these 3 particular VOC biomarkers are increased to a significant extent compare to that of other biomarkers. Typically, the marker solvents are dissolved in water and were collected as VOCs by following similar technique as mentioned earlier for the collection of VOCs present in urine (Schematic 1). Figure 1c displays the UV-Visible absorption of crude ethylbenzene as VOC marker in different concentrations ranging from ~25 ppm to ~200 ppm. Surprisingly, it was found that the absorption spectra of crude ethylbenzene as VOC are almost identical to that of mixture of VOCs in urine for the bladder cancer patients with comparable peak positions (Figure lb), indicating significant generation of ethylbenzene as a result of liposomal peroxidation induced by loss of balance of reactive oxygen species (ROS) in cancerous cells [S. Zhu, Z. Huang, and G. Nabi, "Fluorometric optical sensor arrays for the detection of urinary bladder cancer specific volatile organic compounds in the urine of patients with frank hematuria: a prospective case-control study," Biomedical Optics Express, vol. 11, pp. 1175-1185, 2020; D. F. G. Rodrigues, "Volatile Organic Compounds for the Detection of Bladder Cancer: an In Vitro Metabolomic Approach," 2016; H. Piri-Moghadam, A. Miller, D. Pronger, F. Vicente, J. Charrow, S. Haymond, et al., "A rapid LC-MS/MS assay for detection and monitoring of underivatized branched-chain amino acids in maple syrup urine disease," Journal of Mass Spectrometry and Advances in the Clinical Lab, vol. 24, pp. 107-117, 2022]. On the contrary, in case of nonyl chloride and hexanal, no such identical spectral characteristics of UV-Visible absorption of crude urine could be obtained.
In this regard, it is important to mention that apparently the observation by virtue of the present invention is quite contradictory to that of observation of Nabi et. al. where prevalence of nonyl chloride over all other VOCs in the urine of bladder cancer patients is confirmed from the optical sensors array as well as GC-MS in their UK based clinical trial.
Here we rationalize the fact in two different prospects. Firstly, VOC level is increased due to the various metabolic and nutritional activities of cells in various physiological conditions which being solely a demographic phenomenon, depends on the food habits as well as quality of the daily life of the common people. Secondly, the observation regarding the prevalence of nonyl chloride is inconclusive mainly due to the very limited clinical trial, performed by Nabi et. al.
However, the absorption intensities at the select peak of ~268 nm have been plotted against the concentration of ethyl benzene in Figure Id which surprisingly shows a very good linear fit. The purpose of such construction of the present invention is to generate a calibration curve for the quantitative determination of concentration of ethyl benzene in the urine of various degree of bladder cancers which is useful for the sensitivity determination of the newly developed system coined as 'NABIL' device.
Interaction of the photo-sensitizer with Ethyl benzene
In the next part, sensors strips for the determination of urinary bladder cancer VOCs as the marker of bladder cancers was developed. Details of the development of the sensors is described in experimental section. Sensor strip (Figure 2a) is based on optical fluorescence sensors array and highly sensitive towards environmental changes like pH, redox as well as solvatochromisms. Herein the select fluorophores [Eosin Y (EY), Rose Bengal (RB) and Nile Red (NR)] are selectively chosen as hydrophobic to reduce the possibility of the interference of water during excited state photochemical reactions. Figure 2a depicts the de-coloration of the fluorophore which is visually distinguishable after getting the sufficient exposure of the bladder cancer VOCs compare to that of healthy control. The grade of cancer has also been corroborated by the histopathological examination of the cancer lesions following TURBT. The tissue shows papillary morphology for both the low-grade and high-grade cancer tissues. In addition, patches of skip lesions were present corroborating the high-grade cancer. EY is one of the xanthene group of dyes that has enormous utility in cosmetics industry because of its intense orange colour. In in-vitro model of the present invention, ethyl benzene is modelled as VOC by taking into account previous observation in accordance with the invention that UV-Visible absorption of VOCs of urine of the patients suffering from bladder cancer is comparable to that of crude ethyl benzene as only VOC marker.
The fluorescence spectra of EY after interacting with ethyl benzene VOC, a large blue shift of the peak is observed with significant enhancement of emission intensity relative to that of reference (before interacting with VOC) (Figure 2b). The remarkably blue-shifted emission of EY in presence of ethyl benzene VOC mainly originates from the non-planar locally excited state conformer of EY, stabilized by the ethyl benzene VOC. In the present invention, EY acts as a photoredox catalysts which catalyzes the bromination process of ethyl benzene through a radical mechanism pathway. Details of the plausible mechanistic pathway has been described in Scheme 2a. In brief, by absorbing photon (Aex~375 nm), EY generates EY* (singlet), which undergoes immediately to a triplet excited state through an intersystem crossing (ISC) by which Br atom of the aromatic ring carbon-Br bond of EY* becomes less stable and causes bromination of the ethyl benzene through a radical chain reaction. Certain increment of fluorescence intensity of RB is observed after reacting with ethyl benzene VOC biomarker (Figure 2b). RB acts as a photosensitizer, participates in photoexcitation in its excited state of ethyl benzene at benzylic positions by forming a radical ion pair with superoxide anion of other VOCs like trace amount of H2O2 as a consequence of sensitization (Scheme 2c). Furthermore, steady state fluorescence spectra of NR in presence of ethyl benzene VOC are characterized by a remarkable blue shift with significant fluorescence quenching. The blue shifted emission of NR (Figure 2d) originates from the nonplanar LE state of NR which is entrapped by the non-polar VOC environment where the probe is significantly screened from the exposure of the bulk water vapour. On the other hand, quenching of the NR fluorescence (Figure 2d) likely originates from the inability of the some of the NR molecules to incorporate within the VOC environment which manifests significant exposure of the NR towards bulk environment, favours the transformation of LE to CT through a non-radiative deactivation channel (Scheme 3).
Diagnosis of cancer from urinary cancer patients
In order to validate the observation of the present invention as per the in- vitro study, total 85 subjects, including 52 bladder cancer patients were selected having different degrees of cancer and among rest of the 33 patients, most of the patients are suffering from various disorders other than cancer and some were taken as control. In order to develop an idea of the demographic scenario of the urinary bladder cancer, the urine samples from the villages far apart from the central town (Kolkata) were collected. As depicted in Figure 3a, EY fluorescent intensity increases significantly after exposure of the sensor strip towards the urine VOCs compare to that of healthy control. Furthermore, careful observation suggests this increment of EY fluorescence intensity is quite regular, depending on the degree and recurrences of the bladder cancer. After getting exposure of the VOC towards RB of the sensor strip, RB fluorescence increases many folds, similar to that of our invitro experiment (Figure 3b). The intensity spectrum of NR significantly blue shifted with a negligible increment of the intensity in presence of urine VOCs of the patients suffering from bladder cancer, similar to that of ethyl benzene VOC invitro study, confirms the significant population of ethyl benzene in the VOCs of bladder cancer compare to that of healthy control. Figure 3d shows the differential spectrum of fluorescence sensors on sensor strip as the responds to the VOCs vapours in the present "NABIL" device. This has been typically characterized by the positive variation of the intensity amplitude in case of EY and RB but NR is characterized by the positive followed by negative amplitude of the intensity variation throughout the wavelength of our interest for the urinary bladder cancer patients. On the other hand, this variation is negligible small in case of healthy control. This differential variation of the intensity of the sensors after getting exposure of the VOCs of the urinary bladder cancer patients is further involved as an instrumental index for assisting the analytics of the present system towards generation of the diagnostic report. In order to generate a score from the present method based on the system, cluster analysis have been performed, where the normalized photon counts from the sensitive materials in the sensor strip are plotted in the 2 graphs. The plot depicting normalized photon numbers from sensitive material RB versus that from EY is called "E-R test" as shown in Figure 4a. The entire data set has been categorized into four quadrants after analysing the results obtained from the limited clinical trial and comparing the same with reports generated from our "NABIL" device. In the E-R test, all normal subjects are essentially confined in the third quadrant of the plot. Normalized deviation for a particular subject, can be considered as the probability of the occurrence of urinary cancer. Another projection revealing normalized photon number from RB versus that from NR is called "N-R test" is considered and is depicted in Figure 4b. Similarly, in the N-R test, while all the normal subjects are confined in the third quadrant, the normalized deviation depicts occurrence of cancer according to NABILOGY analysis report as coined. The coupled scoring tests together are called NABILOGY report, which is the outcome of NABIL device.
The NABILOGY test report of a control subject is shown in the Figure 4c, where both E-R and N-R tests reveal the cancer occurrence probability of 0% (both in quadrant 3). Similarly, that of a urinary bladder cancer patient showed the occurrence probability to be 89% and 88% respectively (in quadrant 1 and 4). The Line of Significance (15%), obtained comparing the data of the control and diseased samples generated from both the histopathological report and the NABIL device, is also shown in the test report. The medical history of the subject indicates that the patient underwent Transurethral Resection of Bladder Tumor (TURBT) recently and the biopsy report stated that the grade of cancer to be T1HG, which means that the tumor has spread to the connective tissue and that it is of high grade in nature. Moreover, the sample from the above patient was again studied after he was treated with 6 cycles of BCG and our results show significant fall in the probability of occurrence to 16% and 17% respectively (in quadrant 1 and 4). Additionally, the experiment with the urine sample of a patient suffering from Prostate cancer was performed, and the test report suggested that the probability of occurrence of Urinary Bladder cancer is below the Line of Significance (both 0%). Thus, the results generated out of the system of the present invention are consistent with the medical findings that the subject is suffering from high grade tumor cancer.
Thereafter, the difference of the sensitivity of the sensor strip of the present invention in the present "NABIL" device between the artificially created ethyl benzene VOC by dissolving it into water and the VOC originates from the urine sample of the patient suffering from urinary bladder cancer was tested. Hence, the intensity of EY, RB and NR was monitored by exposing the strip towards the VOC where the concentration of the EBZ is more or less same to that of VOC of urinary bladder cancer patients (Figure 5). In this context, it is important to mention that the concentration of the EBZ in urinary bladder cancer patients has been determined by observing 268 nm UV-Visible absorption peak of the collected VOC from urine and taking into account molar extinction coefficient of pure EBZ at 268 nm. Intensity of each sensor (EY, RB and NR) is significantly higher (~ 2-3 times) after getting exposure to the urine VOC compared to that of pure ethyl benzene at same concentration in the present in-vitro study. This may be due to fact that the ethyl benzene is not the only biomarker, but there are several other organic volatile compounds in trace amounts that interacts with the sensor fluorophores as well. This observation clearly supports the observation of Morgan et. al. where they have identified 200 bladder cancer volatile organic compounds (VOCs) biomarkers in human urine by using gas-chromatography and massspectroscopy including aldehydes, ketones, alcohols, aromatics, and several hydrocarbons etc [M. Cauchi, C. Weber, B. Bolt, P. Spratt, C. Bessant, D. Turner, et al., "Evaluation of gas chromatography mass spectrometry and pattern recognition for the identification of bladder cancer from urine headspace," Analytical Methods, vol. 8, pp. 4037-4046, 2016].
Sensitivity and Specificity of the instrument:
In order to evaluate the accuracy of the present system/instrument to diagnose urinary cancer from the control population, the sensitivity and the specificity of the instrument as opposed to the gold standard method of cystoscopy was estimated. The instrument/ system of the present invention is fund to have a specificity of 86%, which evaluates the potential of the device to detect proportions of true negatives in a population, who are also tested as negative by the instrument. Additionally, the sensitivity of the instrument, which evaluates the proportions of true positives which are also tested as positive by the instrument is 87 %. Thus, the instrument can specifically screen urinary bladder patients from the control population by the help of the VOC marker with a sensitivity of 87 %.
Table 1
Figure imgf000031_0002
Figure imgf000031_0001
It is thus possible by way of the present advancement to provide for a system/ platform and method thereof for the detection of urinary bladder cancer from select bladder cancer VOC ethyl benzene by involving an array of select three sensitive fluorophores, namely EY, RB and NR as sensor materials with said system allowing to monitor, record and analyze array of spectroscopic changes in said sensor materials based on system analytics due to optical chemical sensing by said array of sensor materials upon interaction with said VOC, ethyl benzene when trapped/absorbed in the sensor material, thereby enabling both qualitative and quantitative mass screening of urinary bladder cancer respectively with minimal cost. Thus the indigenously developed present system coined as "NABIL" device with very high sensitivity and specificity could be provided. The experiments have shown that the fluorophores sporting on a sensor strip can participate in a remarkable change in spectral property in terms of fluorescence increment or spectral shift after getting exposure towards select VOC ethyl benzene of the urine of the urinary bladder cancer patients. In addition to that, during the clinical trial in several cases it is found that the sensor strip of the system named as "NABIL' device could successfully identify post operative recurrences with similarly high performance while exhibiting negligible influences by confounding factors. Overall, the present system enables a precise sensitive method for point of care non-invasive screening for urinary bladder cancer based on cost-effective sensitive sensors suiting rapid detection by the device with great potential applicability in rapid and non-invasive diagnosis of urinary bladder cancer. It is envisioned that the strategy of select VOC based urinary bladder cancer detection enabled by a select system having specific system features is extremely helpful in clinical application and opens a new window in offering a painless early detection and screening method of urinary bladder cancer based on said system.
While urinary bladder cancer (UBLC) is one of the most common cancers having extensively high possibility of recurrence and mortality rate across the globe, current clinical diagnostic approaches are either invasive or inaccurate. Herein, a cost effective, system/ device based on select activation of select environmental sensitive fluorophores, namely Nile Red (NR), Eosin Y (EY) and Rose Bengal (RB) as sensor materials for detecting bladder cancer VOCs from urine samples as a biomarker has been devised. A large increment of fluorescence intensity with spectral shift of the fluorophores, could be recorded in the present system coined as "NABIL" device that originates from the excited state photophysical changes upon exposure of the fluorophores with the bladder cancer VOCs. On the contrary, spectral shape of the fluorophores remains unaltered for healthy control. In clinical trial, 21 healthy controls and 52 UBLC patients are assessed by the sensors and our indigenously developed device. With the assistance of our analysis technique based on LabVIEW platform, very high sensitivity and accuracy from healthy controls are achieved, exceeding those obtained by the currently available commercial techniques that are in practice. In addition, the recurrences of both early and advanced stages are diagnosed well, with the effect of confounding factors on the performance of the collection of the VOCs from urine and have a negligible influence on the diagnostic performance. Overall, the present invention contributes a novel, non-invasive, easy-to-use, inexpensive, real-time, accurate method for selectively urinary bladder cancer diagnosis, which is useful for personalized care/diagnosis and postoperative surveillance, resulting in saving more lives.

Claims

Claims:
1. Point of care non-invasive screening system for urinary bladder cancer comprising: fluorometric sensor array including array of fluorescent sensor dyes comprising cellulosic substrate supported photosensitizer fluorescent sensor dye selected from Nile Red (NR), Eosin Y (EY) and Rose Bengal (RB) having specific selectivity for absorption of select VOC, ethyl benzene, present in urinary bladder cancer; an urine sample holder adapted for supporting said fluorometric sensor array with said photosensitizer fluorescent sensor dye such as to absorb/trap said VOC, ethyl benzene, present in urinary bladder cancer generated from heated urine sample onto said photosensitizer fluorescent sensor dye; colorimetric unit for detecting spectroscopic change including fluorescence change induced in said fluorometric sensor array having said fluorescent sensor dyes upon interaction with said trapped VOC, ethyl benzene present in urinary bladder cancer.
2. The point of care non-invasive screening system as claimed in claim 1 wherein said fluorometric sensor array comprises flurometric sensor array strip including regions of said photosensitizer fluorescent sensor dye cast upon cellulosic substrate and adapted for detachable operative fixing with respect to said (a) urine sample holder top such as to face the urine sample in said urine sample holder and (b) sensor holder of said colorimetric unit, said colorimetric unit comprises: multi-wavelength spectroscopic device having customised light/optical excitation source and power supply module and including set up to hold said detachable flurometric sensor array strip to examine said flurometric sensor array strip involving diffused light signals passing therethrough; and micro-controller and CCD based detector for triggering of light from said light/optical excitation source for passing through said flurometric sensor array strip and collecting related fluorescent signals for detection of the said VOC, ethyl benzene trapped in said fluorometric sensor array present in urinary bladder cancer by way of optical chemical sensing.
3. The point of care non-invasive screening system as claimed in anyone of claims 1 or 2 comprising graphical user interface (GUI) based acquisition and analytics for acquiring data from micro-spectrometer including means for operative connection with said microcontroller for tuning LED based optical power into ON/OFF mode, storage means and display means; spectroscopic converter for converting said acquired light signal based spectroscopic inputs into quadrant based and/or linear distribution analytic based indications on presence of carcinoma of urinary bladder for ready and user friendly detection indicative of both early and advanced stages of urinary bladder cancer including postoperative surveillance based on VOC generation having sensitivity at even 10 ppm for bladder cancer VOC ethyl benzene.
4. The point of care non-invasive screening system as claimed in anyone of claims 1-3 comprising microcontroller for tuning average power of required pulse signal by involving PWM (Pulse width modulation) and intensity control of LED for said strategic tuning of said LED.
5. The point of care non-invasive screening system as claimed in anyone of claims 1-4 wherein said sensor holder is adapted to hold fluorometric sensor array strip, and cuvette like sample holder to hold ethanol washings of VOC ethyl benzene from said urine sample holder top, said colorimetric unit comprising multi-wavelength spectroscopic device for both differential optical chemical sensing based on array of differential fluorescence change induced in said sensor array upon exposure to and interaction with bladder cancer VOC ethyl benzene as biomarker present in urine for operative interaction with said spectroscopic converter for conversion to quadrant based qualitative indication of bladder cancer carcinoma, and, means for recordal of concentration dependent absorbance of VOC ethyl benzene at select wavelength for spectroscopic superposition/fit on concentration vs. absorbance based linear distribution of VOC ethyl benzene for quantitative mass screening of urinary bladder cancer respectively with minimal cost.
6. The point of care non-invasive screening system as claimed in anyone of claims 1-5 wherein said customized light/optical excitation source of said multi-wavelength spectroscopic device includes UV LEDs of wavelength 395 nm each that cooperates with micro-controller to trigger said sensor array in a chronological order for collection and detection of fluorescence based optical signal in said CCD based detector for said detection of trapped VOC, ethyl benzene including said sensor array thereby favouring for easy colorimetric detection of bladder cancer VOCs with minimized need of extensive signal transduction hardware.
7. The point of care non-invasive screening system as claimed in anyone of claims 1-6 wherein said cellulose based flurometric sensor array strip material include selective hydrophilicity/porosity (Pore size: 20-25 pm (Particle retention), thickness: 205 pm, ash: <0.06% and basic weight: 92 g/m2) for select absorption of VOC ethyl benzene onto said casted including drop casted sensor dye based array adapted for retention of up to at least 2 hours without any change of fluorescence intensity.
8. The point of care non-invasive screening system as claimed in anyone of claims 1-7 comprising optical chemical sensing colorimetry means cooperative with said fluorometric sensor array for sensing fluorescence changes based on select three hydrophobic photo sensitizer molecules of said Nile Red (NR), Eosin Y (EY), and Rose Bengal (RB) due to solvation effect of Nile Red and the radical generation behaviour of Eosin Y along with Rose Bengal upon interaction with ethyl benzene VOCs based de-coloration of the flurophore with indicative increment of fluorescence intensity and related spectral shift of the fluorophores, means for tracking, recording, analytics and display as said quadrant based qualitative indication for determining both early and advanced stages of urinary bladder cancer including postoperative surveillance.
9. The point of care non-invasive screening system as claimed in anyone of claims 1-8 wherein said colorimetric unit comprising multi-wavelength spectroscopic device enabling quantitative screening of urinary bladder cancer includes colorimetric unit based micro-controller triggered acquisition of spectral data including UV-Vis absorbance spectral data from cuvette comprising ethyl benzene VOC in ethanol at various concentrations via UV-LED based excitation and absorption by said VOC in the UVC region of 150 to 280 nm and selectively at ~ 268 nm for clear linear distribution/fit at ~ 268 nm against varying concentration of ethyl benzene VOC in storage means as calibration curve and/ or calibrating the system; micro-controller based acquisition of real time absorbance data of ethyl benzene VOC in ethanol from cuvette generated by urinary bladder cancer for cooperative ready feed and analytics based on sequence of defined instructions allowing fitting said real time data with said calibration curve to yield absorbance dependent quantitative real time concentration of said ethyl benzene VOC to thereby ascertain the degree of bladder cancers.
10. The point of care non-invasive screening system as claimed in anyone of claims 1-9 wherein said fluorometric sensor array based fluorescence change enabling said qualitative quadrant-based indication/screening including said colorimetric unit based sensor array analytics generation based on: significant but regular increase of EY fluorescent intensity after exposure of the EY sensor towards the urine VOCs compared to that of healthy control; manifold increase of RB fluorescent intensity after exposure of the VOC towards RB sensor as compared to healthy control; significant blue shift of the NR sensor intensity with negligible increment of the intensity in presence of urine VOCs as compared to healthy control; system storage records, as instrumental index and analytic means for clusters towards generation of quadrant-based indication selectively including means for tracking differential positive variation of fluorescent intensity amplitude of EY and RB, differential positive variation of fluorescent intensity amplitude of NR followed by differential negative intensity amplitude of NR throughout the wavelength range of 400-700nm as compared to negligible variations of healthy control enabling normalized photon counts; means for analytics by plotting for "E-R test" to depict the normalized photon count for RB sensor vs. EY sensor; and/or by plotting for "N-R test" to depict the normalized photon count for said RB sensor vs. NR sensor wherein, normalized deviation for a particular subject is considered as the probability of the occurrence of urinary bladder cancer with normal subjects of 0% cancer occurrence probability being essentially confined to the third quadrant for both ER and NR tests.
11. A method for point of care non-invasive screening for urinary bladder cancer comprising the steps of providing urine sample from urinary bladder cancer subject in a urine sample holder having a releasable fitting lid; providing a fluorometric sensor array as sensor strip including array of fluorescent sensor dyes comprising cellulosic substrate supported photosensitizer fluorescent sensor dye selected from Nile Red (NR), Eosin Y (EY) and Rose Bengal (RB) having specific selectivity for absorption of select urinary bladder cancer VOC ethyl benzene; incubating by heating said urine sample holder covered with the lid at temperatures of 35-degree Centigrade with said releasable fitted lid of urine sample holder detachably attaching said sensor array on said cellulosic substrate and facing said urine sample for absorbing/trapping select bladder cancer VOC ethyl benzene on said fluorometric sensor array; removing said sensor strip from said releasable fitted lid of urine sample holder after absorbing/trapping select bladder cancer VOC ethyl benzene; placing said sensor strip in sensor holder of colorimetric unit comprising a spectroscopic device for detecting spectroscopic change including fluorescence change induced in said fluorometric sensor array having said fluorescent sensor dyes upon interaction with said trapped urinary bladder cancer VOC ethyl benzene and involving optical chemical sensing to thereby enable point of care non-invasive screening for urinary bladder cancer.
12. The method for point of care non-invasive screening for urinary bladder cancer as claimed in claim 11 wherein said urine sample holder top lid having condensed VOC ethyl benzene is washed in ethanol for collection in cuvette like sample holder for placing in sensor holder of said colorimetric unit comprising spectroscopic device for detecting UV-vis absorbance intensity based spectroscopic change due to concentration of said VOC ethyl benzene for assisting non-invasive quantitative screening for urinary bladder cancer.
13. The method for point of care non-invasive screening for urinary bladder cancer as claimed in claim 11 wherein said step of detection of fluorescence change induced in said fluorometric sensor array upon interaction with said trapped urinary bladder cancer VOC ethyl benzene is carried out based on the following steps examining the fluorometric sensor array based sensor strip by said colorimetric unit comprising multi-wavelength spectroscopic device including four UV LEDs of wavelength 395 nm each for illuminating four positions of the sensor strip having said three select sensor dyes of Nile Red (NR), Eosin Y (EY) and Rose Bengal (RB) and one blank position; collecting diffused light signals from said sensor holder including collection of diffused light signals from three sensor dye positions and one blank position of said sensor strip for sending said collected diffused light signals to CCD based spectrometer detector of said colorimetric unit; and performing spectroscopic conversion of thus collected/ acquired light signals into quadrant based indications on presence of carcinoma of urinary bladder for ready and user friendly detection of both early and advanced stages of urinary bladder cancer including postoperative surveillance with detection sensitivity levels of about even 10 ppm of bladder cancer VOC ethyl benzene coupled with minimized influence of confounding factors based on method steps related to VOC collection from urine and related analysis.
14. The method for point of care non-invasive screening for urinary bladder cancer as claimed in claims 11-13 wherein said step of screening for urinary bladder cancer includes collecting diffused light signals from sensor holder including collecting light signals from sensor array strip, and/or, cuvette so as to obtain quadrant based and/or linear distribution fit based indications of urinary bladder cancer by carrying out said colorimetric unit based sensor array analytics generation based on monitoring: significant but regular increase of EY fluorescent intensity after exposure of the EY sensor towards the urine VOCs compared to that of healthy control; manifold increase of RB fluorescent intensity after exposure of the VOC towards RB sensor as compared to healthy control; significant blue shift of the NR sensor intensity with negligible increment of the intensity in presence of urine VOCs as compared to healthy control; correlating with system storage records, as instrumental index and analytic means for cluster location towards generation of quadrant-based indication selectively including tracking differential positive variation of fluorescent intensity amplitude of EY and RB, differential positive variation of fluorescent intensity amplitude of NR followed by differential negative intensity amplitude of NR throughout the wavelength range of 400-700nm as compared to negligible variations of healthy control to enable normalized photon counts; carrying out analytics by plotting for "E-R test" to depict the normalized photon count for RB sensor vs. EY sensor; and/or by plotting for "N-R test" to depict the normalized photon count for said RB sensor vs. NR sensor wherein, normalized deviation for a particular subject is considered as the probability of the occurrence of urinary bladder cancer with normal subjects of 0% cancer occurrence probability is essentially confined to the third quadrant for both ER and NR tests for a quadrant based selectivity and sensitivity report generation of tests.
15. The method for point of care non-invasive screening for urinary bladder cancer as claimed in claims 11-14 wherein said collection of diffused light signals for screening of urinary bladder cancer is done in the absence and presence of said sensor strip on the sensor holder not exposed to urine sample for generating baseline and reference UV-vis absorbance spectra for recordal, followed by collecting diffused fluorescent light signals by placing the sensor strip with said sensor array on sensor holder exposed to said urine sample VOC ethyl benzene for converting said light signals into fluorescence spectral data and collating the same with reference spectral data for each sensor of said sensor array for spectroscopic conversion and display as said quadrant based indications/screening of urinary bladder carcinoma.
PCT/IN2022/050828 2022-09-16 2022-09-16 Fluorometric sensory arrays for the detection of urinary bladder cancer-related vocs Ceased WO2024057329A1 (en)

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Citations (1)

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JP2021501321A (en) * 2017-10-26 2021-01-14 エッセンリックス コーポレーション Devices and methods for tissue and cell staining

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JP2021501321A (en) * 2017-10-26 2021-01-14 エッセンリックス コーポレーション Devices and methods for tissue and cell staining

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