WO2021210027A1 - Dispositif de détection et de pronostic d'une maladie chronique et son procédé de détection - Google Patents

Dispositif de détection et de pronostic d'une maladie chronique et son procédé de détection Download PDF

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WO2021210027A1
WO2021210027A1 PCT/IN2021/050385 IN2021050385W WO2021210027A1 WO 2021210027 A1 WO2021210027 A1 WO 2021210027A1 IN 2021050385 W IN2021050385 W IN 2021050385W WO 2021210027 A1 WO2021210027 A1 WO 2021210027A1
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biosensor
type
saliva
carbon
biomarkers
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PCT/IN2021/050385
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English (en)
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Yogeeswari Perumal
Sankalp KODUVAYUR GANESHAN
Srinivasan MADABHOOSHI PADMANABAN
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Yogee's Bioinnovations Private Limited
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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/72—Signal processing specially adapted for physiological signals or for diagnostic purposes
    • A61B5/7271—Specific aspects of physiological measurement analysis
    • A61B5/7275—Determining trends in physiological measurement data; Predicting development of a medical condition based on physiological measurements, e.g. determining a risk factor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96466—Cysteine endopeptidases (3.4.22)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders

Definitions

  • the present invention relates to a device for detection and prognosis of chronic disease and method of detection thereof and generally relates to the field of bioelectronics including biosensors. More specifically, the present invention relates to device that includes various biosensors for detecting various biomarkers including protein like cathepsins indicated in chronic diseases. The device detects different biomolecules including protein, mRNA, DNA etc. which helps in detection of various fatal human diseases like cancer, neurological disorders etc.
  • cancer biomarker including protein like cathepsins (D, S, L, K, etc) indicated in chronic diseases constitutes one of the most rapidly advancing fields in clinical diagnostics.
  • Conventional histopathology based on assessing morphology has remained the standard diagnostic method for many years.
  • Other techniques such as immunohistochemistry, in situ hybridization (FISH, CSH), PCR, RT-PCR (real time- PCR), flow cytometry, and microarray are used nowadays for diagnosis.
  • the International Publication WO2013172866 discloses sensor for detection of mesothelin, and thus is useful for screening patients for cancers that overexpress mesothelin.
  • the sensor comprises a solid support, carbon nanotube on a surface of the solid tube and antibody capable of binding mesothelin dispersed in the carbon nanotubes.
  • the method comprises contacting a sample from a subject (preferably serum) with the sensor, measuring the change in electrical property of the carbon nanotube and correlating the change in the electrical property with the concentration of mesothelin.
  • Methods of identifying gene and protein expression profiles associated with the likelihood of recurrence/metastasis of colorectal cancer in a patient tissue sample using assays comprising of an immunohistochemistry (IHC) test in which tissue samples, preferably arrayed in a tissue microarray (TMA) (W02009126543).
  • IHC immunohistochemistry
  • TMA tissue microarray
  • a kit for assessing methylation in a test sample to detect and treat colon cancer was reported (W02008100913).
  • Multigene diagnostic assay for malignant thyroid neoplasm using different reagents and protocols was reported (US20080280302A1).
  • the US Patent Publication No. 20170037450 discloses a method to detect a disease having a marker enzyme, comprising: collecting a biological sample using a support tool embedded with a magnetic nanoparticle biosensor; attaching a marker peptide coupled carboxy terminated magnetic bead to a gold sensor support using a specific method; reacting the biological sample and the marker peptide coupled carboxy terminated magnetic bead to form a conjugate; and measuring to detect an association or dissociation of the conjugate on the gold sensor support for presence and intensity of the disease, effect of treatment for the disease and absence of the disease in a mammal.
  • the US Publication No. 20130330747 discloses nanosubstrates as biosensors, wherein the nanosubstrates comprise a first substrate layer; a sacrificial layer deposited on the first substrate layer; a second substrate layer deposited on the sacrificial layer, the second substrate layer comprising a photoresist material; a conductive layer deposited on the second substrate layer; and an insulating layer deposited on the conductive layer, the insulating layer interrupted by one or more nanotrenches or nanowells having a width of at least about 20 nm.
  • the nanosubstrates have nanoparticles deposited in one or more nanotrenches or nanowells and nanoparticles are covalently bound to a different antibody or antigen-binding fragment thereof.
  • the Indian Patent No. 204308 discloses a biosensor comprising a polymer film and a binder layer, characterized in that the binder layer is ink-jet printed in a pattern onto the polymer film wherein the binder layer comprises an antibody that is specific for an analyte.
  • the polymer film comprises metal coating and the binder layer has been inactivated in a pattern by ink jet printing a substance capable of destroying its analyte-binding activity, wherein the substance is selected from a protease or a strongly anionic surfactant.
  • the present invention provides a simple device which is easy to develop and efficiently detects the chronic diseases by employing non-invasive samples.
  • the present invention also describes a method to diagnose and prognosis of multiple cancer with one test using single or combination of biomarker. This is disruptive with respect to using one to few drops of saliva on to a simple Nanobiosensor detecting electrochemical changes or piezoelectric variation to detect the saliva biomarkers in one go. Using the same protocol by varying the biomarker combination the method could also be extended to chronic diseases like diabetes, cardiovascular, neurological and other metabolic disease detection and prognosis.
  • the Nanobiosensor utilized in the invention is frugal and yields affordable sensors or chips that can facilitate mass screening as saliva collection do not require professional support. Another advantage of this invention is that the prognosis of chronic diseases especially cancer during and after treatment can boost the confidence and quality of life of survivors especially those of cancer, neurological and cardiovascular patients.
  • the main object of the present invention is to provide a device for detecting chronic diseases by employing simple, economical, and easily available components.
  • the device is portable and non-invasive device.
  • Another object of the invention is to provide a device for detecting chronic diseases which employs the subject sample obtained by non-invasive methods such as such as saliva testing, urine testing, and sweat analyses.
  • the primary intention of developing the biodevice for diagnosis and prognosis of human diseases like cancer is to detect the biomarker including protein like cathepsins indicated in chronic diseases non-invasively using saliva, urine, sweat etc.
  • Yet another object of the invention is to provide a device for detecting probability of cancer or neuro diseases and indicate the stage of the diseases.
  • Another object of the invention is to provide a device for detecting chronic diseases which is based on nanotechnology or Nanobiosensors including quartz crystal microbalance.
  • Another object of the present invention is to provide a device and technique which does not need any expertise to use and can be used without any technical or medical assistance unlike conventional technique using blood.
  • Another object of the invention is to provide a device for detecting chronic diseases wherein the results are transferred to a smart phone for regular health monitoring.
  • Another main object of the of the present invention is to provide biosensors to detect saliva biomarkers like cathepsin D, K, L, S and other protein markers involved in cancer, and metabolic diseases.
  • Another main object of the present invention is to provide biosensors and detection of biomarkers using said biosensors wherein samples such as saliva, urine, and sweat are used for detection. It is the object of the present invention to use saliva as a biomarker for cancer detection and other disease detection.
  • Another object of the present invention is to provide the method for detection and prognosis of the chronic diseases.
  • the present invention provides a device for detection of chronic diseases which is simple, economical and employs non-invasive samples of the subject.
  • a device for detection of chronic diseases comprising: a. a strip; b. a biosensor; c. a signal processor; and d. a communication unit, wherein the strip receives test sample selected from saliva, urine or sweat and propagates the sample to flow through the strip wherein the test sample encounters the biosensor provided in conjunction with the strip, which biosensor detects the one or more biomarkers present in the test sample of saliva, urine or sweat; wherein, the processor is a high-end micro-controller and it gives input signal and power to the biosensor to runs and function the whole detection device; wherein the communication unit is an interface between a user detection device and the processor providing information and test results or readings to be communicated to the user; wherein the biosensor of the device detects and quantify the saliva, urine or sweat biomarkers selected from cathepsin D, K, L, S and other protein markers or combination thereof, involved in cancer, and metabolic diseases.
  • the strip comprises of a microfluidic channel coated with super hydrophobic material, wherein test sample selected from saliva, urine or sweat flows through the said channel and encounters the biosensor; wherein the biosensor is inserted into the device to quantify for diagnosis and prognosis and detects the saliva biomarkers including cathepsin D, K, L, S and other protein markers involved in cancer, and metabolic diseases; and wherein, the signal processor is a high-end microcontroller and it gives input signal and power to the biosensor.
  • the biosensor detects the one or more biomarkers based on antibody conjugation to nanomaterials, wherein the said nanomaterial comprises group selected from gold, titanium, carbon, Molybdenum or a combination of two or more thereof.
  • the said nanomaterial is selected from group comprising of Molybdenum disulfide quantum dots, carbon nanotubes, titanium dioxide nanotubes or nanowires or gold nanoparticles on a metal surface through covalent conjugation.
  • the biosensor may be a type of biosensor which comprises: the carbon/Molybdenum disulfide quantum dots are coated with gold/titanium dioxide nanoparticles or nanotubes and conjugated with antibody and the sample; or
  • QCM Quantum Crystal Microbalance
  • the biosensor comprises a type of biosensor selected from Type I, Type II, Type III, Type IV and Type V biosensor or combination thereof, wherein
  • Type I biosensor comprises Biosensor made of Titanium dioxide nanostrips coated with quantum dots conjugated with specific antibodies;
  • Type II biosensor comprises Biosensor made of M0S 2 Paper Strips layered with quantum dots conjugated with specific antibodies;
  • Type III biosensor comprises Biosensor made of Carbon materials [Carbon(charcoal) pencil tips or carbon papers or whatman filter paper coated with carbon or graphene] layered with quantum dots conjugated with specific antibodies;
  • Type IV biosensor comprises Biosensor made of QCM (Quantum Crystal Microbalance) layered with gold nanoparticles conjugated with specific antibodies; and
  • Type V biosensor comprises Biosensor as a modification of type I, II, and III to have multiple biomarker detection using multi-channeled sensors using Molybdenum disulfide quantum dots conjugated with antibody on metal surfaces that can be Titanium dioxide nanotubes, or carbon based or MoSicoated paper-strips; wherein above said biosensors are capable of detection of the one or more biomarkers present in test sample of saliva, urine or sweat based on antibody conjugation to nanomaterials.
  • the device comprises a display unit which displays the information and test results or readings to be communicated to the user after detection and quantification of the biomarkers present in the test samples.
  • the communication unit is a Bluetooth module or Wi-Fi module, internet network and the user device is a smartphone.
  • the communication unit may send out data, information and test results obtained by the detection device to external database/memory device to store such data via the said communication unit of the device.
  • the device comprises memory to store one or more of codes, software, programmed instructions and/or algorithms which enable the processor to run the device and execute the various functions of the detection device including measuring, detecting, identifying, calculating, quantifying, displaying, sending, receiving etc.
  • the device is all in one non-invasive device to quantify salivary bio markers including cathepsins (D, K, L, S, etc) along with other protein biomarkers to detect multiple cancers and other metabolic chronic diseases.
  • salivary bio markers including cathepsins (D, K, L, S, etc) along with other protein biomarkers to detect multiple cancers and other metabolic chronic diseases.
  • the device is a portable diagnostic device to detect multiple cancer and chronic diseases using a simple non-invasive spit test (saliva) that is integrated to mobile phone to regularly monitor the disease progression and treatment.
  • a simple non-invasive spit test saliva
  • a portable non-invasive detection device which detection device comprises: a strip; biosensor; processor; and communication unit, wherein the strip receives test sample selected from saliva, urine or sweat and propagates the sample to flow through the strip wherein the test sample encounters the biosensor provided in conjunction with the strip, which biosensor detects the one or more biomarkers present in the test sample of saliva, urine or sweat; wherein, the processor is a high-end micro-controller and it gives input signal and power to the biosensor to runs and function the device; wherein the communication unit is an interface between a user detection device and the processor providing information and test results or readings to be communicated to the user; wherein the biosensor of the device detects and quantify the saliva, urine or sweat biomarkers selected from cathepsin D, K, L, S and other protein markers or combination thereof, involved in cancer, and metabolic diseases.
  • the strip comprises of a microfluidic channel coated with super hydrophobic material, wherein test sample selected from saliva, urine or sweat flows through the said channel and encounters the biosensor; wherein the biosensor is inserted into the device to quantify for diagnosis and prognosis and detects the saliva biomarkers including cathepsin D, K, L, S and other protein markers involved in cancer, and metabolic diseases; wherein, the signal processor is a high-end microcontroller and it gives input signal and power to the biosensor.
  • the biosensor detects the one or more biomarkers based on antibody conjugation to nanomaterials, wherein the said nanomaterial comprises group selected from gold, titanium, carbon, Molybdenum or a combination of two or more thereof; or wherein the said nanomaterial is selected from group comprising of Molybdenum disulfide quantum dots, carbon nanotubes, titanium dioxide nanotubes or nanowires or gold nanoparticles on a metal surface through covalent conjugation.
  • the said nanomaterial comprises group selected from gold, titanium, carbon, Molybdenum or a combination of two or more thereof; or wherein the said nanomaterial is selected from group comprising of Molybdenum disulfide quantum dots, carbon nanotubes, titanium dioxide nanotubes or nanowires or gold nanoparticles on a metal surface through covalent conjugation.
  • the biosensor comprises a type of biosensor selected from Type I, Type II, Type III, Type IV and Type V biosensor or combination thereof, wherein
  • Type I biosensor comprises Biosensor made of Titanium dioxide nanostrips coated with quantum dots conjugated with specific antibodies;
  • Type II biosensor comprises Biosensor made of M0S2 Paper Strips layered with quantum dots conjugated with specific antibodies;
  • Type III biosensor comprises Biosensor made of Carbon materials [Carbon(charcoal) pencil tips or carbon papers or Whatman filter paper coated with carbon or graphene] layered with quantum dots conjugated with specific antibodies;
  • Type IV biosensor comprises Biosensor made of QCM (Quantum Crystal Microbalance) layered with gold nanoparticles conjugated with specific antibodies; and
  • Type V biosensor comprises Biosensor as a modification of type I, II, and III to have multiple biomarker detection using multi-channeled sensors using Molybdenum disulfide quantum dots conjugated with antibody on metal surfaces that can be Titanium dioxide nanotubes, or carbon based or M0S2 coated paper-strips; wherein above said biosensors are capable of detection of the one or more biomarkers present in test sample of saliva, urine or sweat based on antibody conjugation to nanomaterials.
  • the device is all in one non-invasive device to quantify salivary biomarkers including cathepsins (D, K, L, S, etc) along with other protein biomarkers to detect multiple cancers and other metabolic chronic diseases; and wherein the device is a portable diagnostic device to detect multiple cancer and chronic diseases using a simple non-invasive spit test (saliva) that is integrated to mobile phone to regularly monitor the disease progression and treatment.
  • salivary biomarkers including cathepsins (D, K, L, S, etc) along with other protein biomarkers to detect multiple cancers and other metabolic chronic diseases
  • the device is a portable diagnostic device to detect multiple cancer and chronic diseases using a simple non-invasive spit test (saliva) that is integrated to mobile phone to regularly monitor the disease progression and treatment.
  • salivary biomarkers including cathepsins (D, K, L, S, etc) along with other protein biomarkers to detect multiple cancers and other metabolic chronic diseases
  • the device is a portable diagnostic device to detect multiple cancer and chronic diseases using a
  • Determining a stage of cancer by obtaining a saliva sample from a subject; and measuring a quantity of biomarkers including cathepsins D, L, K, S, and other biomarkers singly and in combination, the quantity of biomarkers or a combination thereof, above or below a pre-determined cut-off or reference level is indicative of the stage of cancer.
  • the said method using the said detection device comprises the steps of:
  • Step I Biosensor testing and biomarker standard graph
  • Step II Saliva testing wherein in Step-I, the biosensor types I, II, III and V sensors are electro conducting and hence the sensors when fixed with two electrodes which are part of the device circuit and at a constant voltage applied (1-5V) would develop resistance based on the material used in different types of sensors which are noted as the starting reading of current in nano to micro Ampere current; and different concentrations of the protein biomarkers of interest selected from Cathepsin D, S, 1, K etc or Bcl2, Bcl-x, CEA, CA 19-9, or combination thereof etc are prepared (2, 5, 10, 15, 20, 30, 50, 75, 100, 200 and 300 ng/ml); and 1-2 drops of protein biomarkers when dropped in the centre of the biosensors (Type I, II, III and V) causes increase in current under a constant voltage and was found to be a linear which means increasing concentration of biomarker increase the current; and wherein Step II comprises Saliva testing wherein one to two drops of saliva is dropped in the centre of the biosensors (types I, II, III and V
  • the said method using the said detection device comprises the steps of:
  • Step I Biosensor testing and biomarker standard graph
  • Step II Saliva testing wherein said biosensor is a type IV biosensor which is QCM based coated with gold nanoparticles conjugated with specific antibodies; wherein in Step-I, different concentrations of the protein biomarkers of interest like Cathepsin D, S, L, K etc or Bcl2, Bcl-x, CEA, CA 19-9, etc or combination thereof were prepared (2, 5, 10, 15, 20, 30, 50, 75, 100, 200 and 300 ng/ml); and 1-2 drops of protein biomarkers when dropped in the centre of the biosensors (Type IV) causes increase in frequency due to piezoelectric property directly correlated to concentration and was found to be a linear, which means increasing concentration of biomarker increase the weight on the QCM and hence increases resonant frequency; and wherein Step II Saliva testing comprises one to two drops of saliva are dropped in the centre of the QCM biosensor (type IV) fixed at the end of the device right that detects change in resonant frequency due to binding of the protein to the specific antibody coated on the biosensors, the frequency change
  • the said method using the said detection device comprises the steps of:
  • a method to detect human saliva, urine and sweat biomarkers including cathepsins (D, K, L, S, etc) along with other protein biomarkers indicating in cancer and chronic metabolic diseases including diabetes, neurological and cardiovascular comprising a nano biosensor or quartz crystal piezo electric biosensor, wherein said biosensor comprises a type of biosensor selected from Type I, Type II, Type III, Type IV and Type V biosensor or combination thereof, wherein
  • Type I biosensor comprises Biosensor made of Titanium dioxide nanostrips coated with quantum dots conjugated with specific antibodies;
  • Type II biosensor comprises Biosensor made of M0S2 Paper Strips layered with quantum dots conjugated with specific antibodies;
  • Type III biosensor comprises Biosensor made of Carbon materials [Carbon(charcoal) pencil tips or carbon papers or Whatman filter paper coated with carbon or graphene] layered with quantum dots conjugated with specific antibodies;
  • Type IV biosensor comprises Biosensor made of QCM (Quantum Crystal Microbalance) layered with gold nanoparticles conjugated with specific antibodies; and Type V biosensor comprises Biosensor as a modification of type I, II, and III to have multiple biomarker detection using multi-channeled sensors using Molybdenum disulfide quantum dots conjugated with antibody on metal surfaces that can be Titanium dioxide nanotubes, or carbon based or MoSicoated paper-strips. wherein above said biosensors are capable of detection of the one or more biomarkers present in test sample of saliva, urine or sweat based on antibody conjugation to nanomaterials.
  • biosensor to detect human saliva, urine and sweat biomarkers including cathepsins (D, K, L, S, etc) along with other protein biomarkers indicating in cancer and chronic metabolic diseases including diabetes, neurological and cardiovascular, wherein said biosensor is one selected from below types of biosensors I-V:
  • Type-I biosensor wherein said Type-I biosensor comprises Biosensor made of Titanium dioxide nanostrips coated with quantum dots conjugated with specific antibodies; or
  • Type II biosensor comprises Biosensor made of M0S2 Paper Strips layered with quantum dots conjugated with specific antibodies; or
  • Type III biosensor comprises Biosensor made of Carbon materials [Carbon(charcoal) pencil tips or carbon papers or Whatman filter paper coated with carbon or graphene] layered with quantum dots conjugated with specific antibodies; or
  • Type IV biosensor comprises Biosensor made of QCM (Quantum Crystal Microbalance) layered with gold nanoparticles conjugated with specific antibodies; or
  • Type V biosensor comprises Biosensor as a modification of type I, II, and III to have multiple biomarker detection using multi- channeled sensors using Molybdenum disulfide quantum dots conjugated with antibody on metal surfaces that can be Titanium dioxide nanotubes, or carbon based or MoS2Coated paper-strips.
  • the antibody includes Cathepsin Antibodies selected from D, K, S, L, or the like, antibodies against Bcl2, Bcl-x, CEA, CA 19-9, etc or combination thereof.
  • the device, method, biosensors and the biomarker are described in the description and are shown in figures 1 to 14.
  • biosensors are described in details in the description and are shown in figures 1 to 5.
  • the Device and method are also described in details in the description and are shown in figures 11 and 12, and figure 13-14 respectively.
  • Figure 1 Shows Type I biosensor of present invention.
  • Figure 3 Shows Type III biosensor of present invention.
  • Biosensor made of Carbon materials [Carbon (charcoal) pencil tips or carbon papers or Whatman filter paper coated with carbon or graphene ] layered with quantum dots conjugated with specific antibodies.
  • Figure 4 (A, B, C): Shows Type IV biosensor of present invention.
  • Figure 5 Shows Type V biosensor of present invention.
  • Biosensor as a modification of type I, II, and III to have multiple biomarker detection using multi -channeled sensors using Molybdenum disulfide quantum dots conjugated with antibody on metal surfaces that can be Titanium dioxide nanotubes, or carbon based or M0S 2 coated paper-strips.
  • Figure 6 Shows a diagram illustrating individual components of the device of the present invention (CIRCUIT- 1).
  • Figure 7 Shows a PCB that can be used as a hand-held device of present invention.
  • Figure 8 Circuit System 2 of the Device according to invention (CIRCUIT-2).
  • Figure 9 Grounded CE configuration of Amperometric sensor (CIRCUIT-3).
  • Figure 10 Grounded WE configuration of Amperometric sensor (CIRCUIT-3).
  • Figure 11A Shows one fabrication design of the device.
  • Figure 11B Shows the device of figure 11A in communication with the user device (mobile);
  • Figure 12 (A, B, C): Shows the different fabrication designs of the device.
  • Figure 13 Flow Chart showing the operational steps of DEVICE Type-1 wherein above said Type I, II, III and V biosensors work on the electrochemical or amperometric devices with CIRCUIT 1 of the present invention.
  • Figure 14 Flow Chart showing the operational steps of DEVICE Type-2 wherein above said Type IV biosensor works on the piezoelectric device with CIRCUIT 2 provided in the present invention to measure resonant frequency.
  • the present invention provides a device for detection of chronic diseases and a method thereof.
  • the primary intention of developing the biodevice for diagnosis and prognosis of human diseases like cancer is to detect the biomarker including protein like cathepsins (D, K, L, S) indicated in chronic diseases through samples obtained non-invasively such as using saliva, urine, sweat etc.
  • the naturally occurring molecule, gene, protein, RNA, DNA or a characteristic by which a particular condition including disease, pathological or psychological process, which are the direct feedback from the body have been utilized as biomarkers for developing the device for detection of chronic diseases.
  • concentration of different biomarker including protein like cathepsins (D, K, L, S) indicated in chronic diseases provides a vital information about the functioning of our body.
  • Electrochemical Biosensors are based on the electrons movement, i.e. electronic current determination as the molecules land on the surface. When a molecule lands having charge binds to surface some current flows through the electrode typically of nA -mA order.
  • the sensing molecules are either coated onto or covalently bonded to a probe surface.
  • a membrane holds the sensing molecules in place, excluding interfering species from the analyte solution.
  • the sensing molecules react specifically with compounds to be detected, sparking an electrical signal proportional to the concentration of the analyte.
  • the electrochemical biosensors can employ potentiometric, amperometric and impedimetric transducers converting the chemical information into a measurable amperometric signal.
  • Quartz is something we come across daily without even noticing the complexity it works with. Almost every electronic gadget has quartz oscillator which helps in keeping track of time with the help of its property to oscillate at high frequencies. Natural frequency of any quartz crystal is inversely proportional to its thickness. Any amount of deposition over quartz crystal tends to decrease its frequency and the change in frequency is directly proportional to the amount of mass deposited, hence the Quartz Crystal Microbalance.
  • a Quartz Crystal Microbalance is a device that measures a mass per unit area by measuring the change in frequency of a quartz crystal resonator.
  • disk configuration of quartz crystal with silver electrodes on either face and coated (electrically) one side with antibody solution which has a high affinity towards Cathepsin D (CAT D) has been used.
  • CAT D Cathepsin D
  • the biomarker can be accurately measured even in the low concentrations like 1 ng/ml.
  • the thickness increases; consequently, the frequency of oscillation decreases from the initial value. This frequency change can be quantified and correlated precisely to the mass change using Sauerbrey's equation.
  • the sensor device of the present invention includes a hybrid between electrochemical QCM or quantum dots and carbon or titanium nanotubes and also utilizing simple day-to-day material like carbon paper and carbon pencil to be utilized as sensor probes coated with nanomaterials.
  • the details of the test and results are then communicated to the smartphone via the said unit and this data is uploaded to the database maintained by the company server where with the help of the machine learning the best advice would be presented to the user.
  • multiple biomarker including protein like cathepsins (D, K, L, S) indicated in chronic diseases levels can be stored and added to database which would further help to diagnosis the disease stage accurately using artificial intelligence. This would lead to a device to be used as Doctor on chip.
  • the algorithm may suggest the user to take multiple tests at different time of day in order to confirm the doubtful cases. However to explicitly mention no kind of medicine or drugs are recommended by this interface.
  • the said device of the present invention is used for detection of chronic diseases by estimation of biomarkers which get elevated or identified in the said disease. Further, the device provides the estimation of biomarkers including protein like cathepsins (D, K, L, S) and screening of cancer patients. Also, the device according to the invention employs the subject sample obtained by non-invasive methods for detection of chronic diseases.
  • a device for detection and prognosis of chronic disease and method of detection thereof comprising: a. a strip, b. a biosensor, c. a signal processor and d. a communication unit.
  • the device is a non-invasive, simple, portable device that can be communicated with an external device such as a mobile or any similar device either by physical connection or wireless or mobile communication.
  • the portable diagnostic detection device is wirelessly connected with a smart phone device.
  • the strip consists of a microfluidic channel coated with super hydrophobic material.
  • the hydrophobic materials are known as non-polar materials with a low affinity to water, which makes them water repelling.
  • the strips may be coated. The coating is done to get rid of bubbles in the saliva.
  • the saliva flows through the channel of the strip, where it encounters the sensors.
  • the sensor head is coated with hydrophilic material.
  • the output of the sensors is transmitted through the wires which are connected to the signal processor when the sensor strip is inserted into it.
  • the sensors sense the biomarkers present in the sample materials sampled on the strip channel and provide output reading which is displayed in the display unit of the device.
  • the strip receives test sample selected from saliva, urine or sweat and propagates the sample to flow through the strip wherein the test sample encounters the biosensor provided in conjunction with the strip, which biosensor detects the one or more biomarkers present in the test sample of saliva, urine or sweat;
  • the sensor is the most important part of the device of the present invention.
  • the sensor comprises of biosensor which converts the biological response into an electrical signal and functions by coupling of a biological sensing element with a detector system.
  • the biosensor is a modification of presently available ion- sensitive field effect transistor, where the sensitivity has been increased significantly.
  • the cutting edge nanowire sensors are perfect fit for such biosensor but handling such tiny nanowire is one of the challenges.
  • the inventive feature of the present invention is in development of hybrid sensor or mechanism where the sensitivity of the order of nanowire has been achieved and also at the same time simplicity to manufacture the technique has been maintained.
  • Biosensors are placed to detect the saliva biomarkers like cathepsin D, K, L, S and other protein markers involved in cancer, and metabolic diseases.
  • the said detection is based on antibody conjugation to nanomaterials selected from the group of Molybdenum disulfide quantum dots (Molybdinium disulphide quantum dots), carbon nanotubes, titanium dioxide nanotubes or nanowires or gold nanoparticles on a metal surface through covalent conjugation.
  • Molybdenum disulfide quantum dots Molybdinium disulphide quantum dots
  • carbon nanotubes carbon nanotubes
  • titanium dioxide nanotubes or nanowires or gold nanoparticles on a metal surface through covalent conjugation.
  • the biosensors used in the device to converts the biological response into an electrical signal and functions by coupling of a biological sensing element with a detector system can be of following types:
  • Type I biosensor of present invention Biosensor made of Titanium dioxide nanostrips coated with quantum dots conjugated with specific antibodies.
  • Type II biosensor of present invention Biosensor made of M0S 2 Paper Strips layered with quantum dots conjugated with specific antibodies.
  • Type III biosensor of present invention Biosensor made of Carbon materials [Carbon (charcoal) pencil tips or carbon papers or Whatman filter paper coated with carbon or graphene] layered with quantum dots conjugated with specific antibodies).
  • Type IV biosensor of present invention Biosensor made of QCM ( Quantum Crystal Microbalance) layered with gold nanoparticles conjugated with specific antibodies).
  • Type V biosensor of present invention Biosensor as a modification of type I, II, and III to have multiple biomarker detection using multi -channeled sensors using Molybdenum disulfide quantum dots conjugated with antibody on metal surfaces that can be Titanium dioxide nanotubes, or carbon based or MoSicoated paper- strips).
  • Type I biosensor has structure which act as inbuilt nanowire.
  • the Figure 1 shows the carbon coated nanoparticles of gold-coated antibody and the sample. As shown in the figure the carbon/ Molybdenum disulfide quantum dots are coated with gold/titanium dioxide nanoparticles or nanotubes conjugated with antibody and the sample. TiC -Nanotube Layers and Nanobeads Carrying Quantum Dots (Molybdenum disulfide) are conjugated with antibody.
  • FIG 1(A) it shows the source (1), drain (2), gate (3) and oxide layer (4) of the FET as a biosensor.
  • the field-effect transistor (FET) is a type of transistor that has three terminals: source, gate, and drain and uses an electric field to control the flow of current.
  • Figure 1(B) shows an image of biosensor type-I.
  • the Biosensor is made of Titanium dioxide nanostrips coated with quantum dots conjugated with specific antibodies (monoclonal) for cathepsins (D, K, L, S etc) or proteins like Bcl2, Bcl-x, CEA, CA 19-9, etc.
  • Step 1 Preparation of Molybdenum disulfide quantum dots (M0S2QD) ⁇
  • Step 2 Preparation of antibody conjugated Molybdenum disulfide quantum dots (M0S2OD) ⁇
  • the M0S2QD (1000 pi) as obtained above were dissolved in phosphate buffer (pH 7.4) solution.
  • a solution of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-Hydroxysuccinimide in the ratio of 2:1 was made in phosphate buffer with pH 7.4 and was added to the M0S2QD solution.
  • a range of 5-20m1 of specific antibody (lOng-lOOng/ml, changes with different biomarker) was added to the above solution and stirred for l-2h at room temperature.
  • the resultant was purified by a Millipore and kept in refrigerator at 4°C. The conjugation is confirmed by SEM or TEM and by photoluminescence tests.
  • Step 3 Preparation of Titanium dioxide nanotubes (TiChNT).
  • a foil of Titanium foil measuring 1cm x 3cm is cleaned with sand paper and rinsed with acetone using ultrasonicator. Isopropanol for used to rinse by ultra- sonication and then washed with distilled water.
  • a black tape was used to stick the forming nanotubes to make then as a strip. This strip was then placed in electrolytic solution (ethylene glycol and ammonium fluoride in distilled water) and a 25V was applied with stirring at 300 rpm in a magnetic stirrer for 90 minutes.
  • electrolytic solution ethylene glycol and ammonium fluoride in distilled water
  • Titanium dioxide nanotubes were rinsed thoroughly with distilled water and annealing was done at 450°C for 2 hours with a ramp rate of 1.5°C/minute to reduce impurities and to obtain poly-crystalline nanotubes. Average size, composition, and morphology of nanotubes were examined using SEM and transmission electron microscope (TEM).
  • Step 4 Preparation of biosensor type-1: Titanium dioxide nanostrips coated with quantum dots conjugated with specific antibodies.
  • the T1O2NT strips were cut into smaller strips of size 1 cm x 3 cm (wxh) dimension and were coated with 200 pi of M0S2QD solution three times with a gap of 2-3h for drying using drop casting method.
  • the functionalized biosensors are kept for drying for 24h in cold environment in a clean room and stored in refrigerator.
  • the antibody-conjugated biosensors were analysed for successful coating and functional activity using SEM and electrical conductivity, respectively.
  • the Type II biosensors includes the Molybdenum disulfide quantum dots (Molybdinium disulphide quantum dots) conjugated with antibody coated on to metal surfaces like carbon either laser treated graphene or Whatman filter paper coated with carbon or Molybdenum disulfide powder.
  • Figure 2 illustrates the Type II biosensor.
  • the type II biosensor is made of M0S2 Paper Strips layered with quantum dots conjugated with specific antibodies (monoclonal) for cathepsins (D,L,K, etc) or proteins like Bcl2, Bcl-x, CEA, CA 19-9, etc
  • Step 1 Preparation of Molybdenum disulfide quantum dots (M0S2QD) ⁇
  • Step 2 Preparation of antibody conjugated Molybdenum disulfide quantum dots (M0S2OD) ⁇
  • the M0S2QD (1000 pi) as obtained above were dissolved in phosphate buffer (pH 7.4) solution.
  • a solution of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-Hydroxysuccinimide in the ratio of 2:1 was made in phosphate buffer with pH 7.4 and was added to the M0S2QD solution.
  • a range of 5-20m1 of specific antibody (lOng-lOOng/ml, changes with different biomarker) was added to the above solution and stirred for l-2h at room temperature.
  • the resultant was purified by a Millipore and kept in refrigerator at 4°C. The conjugation is confirmed by SEM or TEM and by photoluminescence tests.
  • Step 3 Preparation of Molybdenum disulfide coated Whatman filter paper (MoSrPaper Strips).
  • the Molybdenum disulfide powder obtained in step 1 was dissolved in distilled water, poured onto the Whatman filter paper in a Buchner funnel, and repeated till the whole paper was pitch black and was air-dried the filter paper thoroughly for 5h.
  • Step 4 Preparation of biosensor M0S 2 Paper Strips coated with quantum dots conjugated with specific antibodie s.
  • the M0S2 Paper Strips were cut into smaller strips of size 1 cm x 3 cm (wxh) dimension and was coated with 200 pi of M0S2QD solution three times with a gap of 2-3h for drying using drop casting method.
  • the functionalized biosensors are kept for drying for 24h in cold environment in a clean room or using nitrogen flow and stored in refrigerator.
  • the antibody-conjugated biosensors were analysed for successful coating and functional activity using SEM and electrical conductivity, respectively.
  • Type III biosensors Carbon pencils or papers coated with nanomaterial: FIG 3 (A, B, C, D)
  • the Type III sensor includes carbon pencils or carbon papers with nanomaterial- conjugated antibodies to sense biomarkers have been illustrated in Figure 3. Further, in one embodiment the type III sensors are the graphene treated with laser with nanomaterial-conjugated antibodies to sense biomarkers.
  • the Type III Biosensor is made of Carbon materials [Carbon(charcoal) pencil tips or carbon papers or Whatman filter paper coated with carbon or graphene] layered with quantum dots conjugated with specific antibodies (monoclonal) for cathepsins (D,L,K, etc) or proteins like Bcl2, Bcl-x, CEA, CA 19-9, etc
  • Step 1 Preparation of Molybdenum disulfide quantum dots (M0S2QD) ⁇
  • N-Hydroxysuccinimide in the ratio of 2:1 was made in phosphate buffer with pH 7.4 and was added to the M0S2QD solution. Then a range of 5-20m1 of specific antibody (lOng-lOOng/ml, changes with different biomarker) was added to the above solution and stirred for l-2h at room temperature. The resultant was purified by a Millipore and kept in refrigerator at 4°C. The conjugation is confirmed by SEM or TEM and by photoluminescence tests.
  • Step 3A Preparation of Carbon (charcoal) coated Whatman filter paper (Carbon
  • Step 3B Preparation of Laser treated polymer sheets to graphene.
  • LIG Laser-induced graphene
  • Step 4A Preparation of biosensor IIIA Carbon Paper Strips or Carbon sheets coated with quantum dots conjugated with specific antibodies.
  • the Carbon Paper Strips or commercially available carbon papers were cut into smaller strips of size 1 cm x 3 cm (wxh) dimension and was coated with 200 m ⁇ of M0S2QD solution three times with a gap of 2-3h for drying using drop casting method.
  • the functionalized biosensors are kept for drying for 24h in cold environment in a clean room or using nitrogen flow and stored in refrigerator.
  • the antibody-conjugated biosensors were analysed for successful coating and functional activity using SEM and electrical conductivity respectively.
  • Step 4B Preparation of biosensor IIIB Laser induced graphene coated with quantum dots conjugated with specific antibodies.
  • the Laser induced graphene with a size 1 cm x 3 cm (wxh) dimension was coated with 200 m ⁇ of M0S2QD solution three times with a gap of 2-3h for drying using drop casting method.
  • the functionalized biosensors are kept for drying for 24h in cold environment in a clean room or using nitrogen flow and stored in refrigerator.
  • the antibody-conjugated biosensors were analysed for successful coating and functional activity using SEM and electrical conductivity respectively.
  • Step 4C Preparation of biosensor IIIC Carbon Pencil tips coated with quantum dots conjugated with specific antibodies.
  • Carbon pencil tips obtained from commercial source were coated with 100 m ⁇ of M0S2QD solution two times with a gap of 2-3h for drying using dip and electrochemical coating procedures.
  • the functionalized biosensors are kept for drying for 24h in cold environment in a clean room or using nitrogen flow and stored in refrigerator.
  • the antibody conjugated biosensors were analysed for successful coating and functional activity using SEM and electrical conductivity of Carbon materials [Carbon(charcoal) pencil tips or carbon papers or Whatman filter paper coated with carbon or graphene] layered with quantum dots conjugated with specific antibodies (monoclonal) for cathepsins (D,L,K, etc) or proteins like Bcl2, Bcl-x, CEA, CA 19-9, etc
  • Type IV biosensors QCM sensors coated with nanomaterial [FIG 4 (A, B, C)]
  • the QCM sensors are coated with nanomaterial-conjugated sensors with the circuit schematic as illustrated in Figure 4.
  • Biosensor is made of QCM (Quantum Crystal Microbalance) layered with gold nanoparticles conjugated with specific antibodies (monoclonal) for cathepsins (D,L,K, etc) or proteins like Bcl2, Bcl-x, CEA, CA 19-9, etc
  • Step 1 Preparation of Gold nanoparticles (AuNP).
  • About 200 ml of 0.01 % Tetrachloroauric acid (HAuCU ⁇ 3H2O) solution was prepared by boiling the solution with stirring in a magnetic stirrer in phosphate buffer pH 7.4. Then 4.5 ml of 1 % trisodium citrate was added and stirred continuously for 15 min till the color of the solution changed gradually from light yellow to wine red.
  • the solution was cooled to room temperature and the obtained AUNPs were stored at 4 °C.
  • the size of the AuNPs was determined to be 10-200 nm.
  • Step 2 Preparation of GSH-capped AuNPs.
  • a stock solution of glutathione (10 mM) was prepared. Dilute HC1 solution (10 mM, 100 mL) was added to colloidal AuNP solution (3 mL) to lower the pH ( ⁇ 4). An aliquot of GSH solution (10 mM, 30 mL) was added to the acidic gold solution. The color of the solution changes from deep red to blue, indicating formation of aggregates of AuNPs.
  • Step 3 Preparation of antibody conjugated AuNPs.
  • the GSH-capped AuNPs was dissolved in phosphate buffer (pH 7.4) solution.
  • a range of 5-20pl of specific antibody (lOng-lOOng/ml, changes with different biomarker) was added to the above solution and stirred for 2.5h at room temperature. This mixture was then added to the GSH-capped AuNP solution and left to react for lh.
  • the resultant was purified by a Millipore and kept in refrigerator at 4°C. The conjugation is confirmed by SEM.
  • Step 4 Preparation of biosensor IV QCM (Quantum Crystal Microbalance) layered with gold nanoparticles conjugated with specific antibodies.
  • the QCMs were coated with 200 m ⁇ of M0S2QD solution using drop-casting method.
  • the functionalized biosensors are kept for drying for 24h in cold environment in a clean room or using nitrogen flow and stored in refrigerator.
  • the antibody-conjugated biosensors were analysed for successful coating and functional activity using piezoelectric method for measuring resonant frequency.
  • Type V biosensors :
  • the Type V biosensors are modified biosensors to have multiple biomarker detection using multi-channeled sensors further using Molybdenum disulfide quantum dots (Molybdinium disulphide quantum dots) conjugated with antibody on metal surfaces as illustrated in Figure 5.
  • Molybdenum disulfide quantum dots Molybdinium disulphide quantum dots conjugated with antibody on metal surfaces as illustrated in Figure 5.
  • the type V Biosensor sensors are a modification of type I, II, and III biosensor to have multiple biomarker detection using multi-channeled sensors using Molybdenum disulfide quantum dots conjugated with antibody on metal surfaces that can be Titanium dioxide nanotubes, or carbon based or MoSicoated Paper-strips.
  • the preparation of the sensor is as per the protocol given above but multiple channels are coated with different antibodies conjugated M0S2QDS.
  • Monoclonal antibodies for the biomarkers detected in saliva for various diseases including cancer, Alzheimer’s, diabetes, and metabolic disorders include Cathepsin Antibodies (D, K, S, L, etc), antibodies against Bcl2, Bcl-x, CEA, CA 19-9, etc.
  • biosensors are coated and fabricated.
  • the signal processor is a high-end microcontroller and it gives input signals and power to the biosensor. Accordingly, the output is read from the sensor by this unit.
  • the resistance and capacitance of the biosensor is measured.
  • the amount of CAT D present on the biosensor in nano-molar is estimated by the device.
  • Figure 6 shows circuit diagram of the device according to invention showing a microcontroller.
  • the microcontroller used is BLUE PILL STM32F103C6T8 microcontroller.
  • Communication unit is basically an interface between user device and the signal processor. Details of the test and results are communicated to the user device via this unit and this data is uploaded to the database where with the help of the machine learning the best advice is presented to the user as explained below in the disclosure.
  • the user device has an interface where the user can checkbox various other symptoms like fever, cold, etc. These are taken as the activity of CAT D which might be higher in case of fever.
  • CAT D which might be higher in case of fever.
  • biomarker including protein like cathepsins (D, K, L, S) indicated in chronic disease levels can be stored and added to database which would further help in diagnosis of the disease stage accurately using artificial intelligence. This would lead to a device to be used as Doctor on chip.
  • the algorithm may suggest the user to take multiple tests at different time of day in order to confirm the doubtful cases.
  • the Communication units includes Bluetooth modules and the results are displayed on the display unit provided in the detection device itself or in the display unit of the user device where the user device is a smartphone.
  • connection may be via physical connection by wire or may be by wireless connection such as Bluetooth, Wi-Fi or the like.
  • the measured resistance of the sensor and the biomarker varies.
  • a test voltage is applied, and the current flow in the circuit is measured.
  • the resistance offered by a circuit is the ratio of the voltage applied to the current flow through the circuit.
  • test voltage in the implemented circuit is 1V-5V.
  • the current flow through the biosensor (along with the sample) was found to possess a linear relation with the concentration of the biomarker present in the saliva sample.
  • test voltage used (1V-5V) needs to be highly accurate, to avoid any voltage swings and any unwanted effects of noise.
  • a precision reference voltage source of IV is used for this purpose.
  • the current range to be measured was found to be from a few nano amperes to a few hundred nano amperes.
  • the shunt resistor was implemented using a precision lkQ resistor with a very low tolerance value. This results in the burden voltage varying from a few microvolts to a few hundred microvolts. This voltage needs to be measured by an external ADC.
  • a 24-bit external ADC is used.
  • the total error in the voltage measured by the ADC is the sum of the errors associated with the stages preceding the ADC viz., ADC measurement error and the error associated with its reference voltage, amplifier output voltage error, the effect of temperature on the shunt resistance and the amplifier feedback resistance and the reference voltage of the applied test voltage of 1V-5V.
  • the overall error in the measured voltage was calculated to be a few hundred microvolts. Dividing this voltage error by the shunt resistor gives an accuracy of less than a nano ampere (a few hundred picoamperes). Since the current flow due to the test voltage and the salival biomarker concentration varies linearly, the corresponding concentration error was found to be 0.0608ng/mL.
  • FIG. 6 shows the individual component details of the device of the present invention.
  • the device comprises of multiple modules or components placed in the circuit.
  • the components include a BLUE PILL STM32F103C6T8 microcontroller.
  • the high-end microcontroller is the signal processor of the device. It further may include low pass filter (as mentioned in figure 8), current amplifier, analog to digital convertor (ADC), and reference voltage generator for ADC.
  • a sensor interface is connected in the circuit for detection and prognosis of the device.
  • the sensor interface comprises of a sensor reference generation circuit and sensor inputs.
  • a Bluetooth module is connected in the device as a communication unit between the detection device and the user device.
  • a power supply unit circuit is provided to supply power to the device for its operation along with an OLED display. There are five LEDs as an extra feature to the circuit to add indicators in the future depending on each protein biomarker.
  • the sensors are interfaced on a PCB board to be inserted rather than manually examined.
  • the PCB can be used as a hand-held device. Its dimension is 35mm x 92mm which has been also miniaturized to 15mm x 45mm to accommodate in the device DESIGN 1 (of Figure 11 A).
  • the sensor area provided is 25mm x 7.62mm and can be varied to accommodate smaller chips from 10mm x 2.5mm or a plug and check model like a cylindrical probe.
  • the sensor will have four holes on either breadth with an inter-hole gap of lmm-2.54mm.
  • the circuit system-2 for the device set up according to the invention QCM 1 and QCM 2 are uniformly pretreated with antibody.
  • the QCM 1 is submerged in a reference solution whose concentration is about 5 ng/ml.
  • the circuit gives an output of col - co2.
  • Q is >5ng/ml
  • the output frequency is function of concentration which can be identified through the set up according to invention.
  • the circuit also uses a XOR Gate.
  • the XOR gate is a digital logic gate that gives a true output when the number of true inputs is odd. Further it also implement a low pass filter to pass the signals with a frequency lower than a selected cutoff frequency and attenuates signals with frequencies higher than the cutoff frequency.
  • FIGS 9-10 show the circuit system 3 of the present invention.
  • Amperometric sensors are configured as either grounded CE or grounded WE configurations.
  • Figure 9 shows a Grounded Counter Electrode (CE) configuration of
  • Amperometric sensor (CIRCUIT-3). This is a grounded CE configuration. This is complex and vulnerable to component mismatch. In cases which shielding and screening of WE from external EMI are difficult to implement this configuration may improve current measurement. Transimpedance amplifier used to force a virtual ground at WE and at the same time, it generates an output voltage.
  • FIG. 10 shows a Grounded Working Electrode (WE) configuration of
  • WE Working Electrode
  • the output of this operational amplifier is a array of resistors in parallel with Counter Electrode (CE).
  • CE Counter Electrode
  • the measured signal is passed through low pass filter and connected to instrumentation amplifier (INA121).
  • INA121 instrumentation amplifier
  • the output of INA121 is given to anti-aliasing low pass filter then to ADC.
  • the device can be fabricated either by simple electrochemical deposition, coating, or photolithography technique or by electron beam lithography. After the device is designed, required doping of the electrodes is done using ion implantation due to its increased controllability as compared to other doping techniques. Later on, to protect the gate electrode from surrounding charge influence and to increase the binding of the antibody, an oxide layer is created using plasma oxidation. Now, the oxide layer is treated in order to equip it with thiol terminal groups. These groups help in binding the antibody onto the oxide layer with the help of a heterobifunctional crosslinker. This method of binding antibody to the device is very efficient.
  • Figure 11A show the device (10) in the thermometer shape.
  • the device as seen the figures shows the strip (11) which is mounted on the tip of the device.
  • the strip is covered with the plastic cap (12) to present contamination of the strip.
  • the signal -processing unit, the communication unit along with other components is mounted in the form of a chip (13) inside the tubular body of the device.
  • the device consists of an OLED display (14) to display the information on the device.
  • the display unit (14) displays the information and test results or readings to be communicated to the user after detection and quantification of the biomarkers present in the test samples.
  • Figure 11B illustrate the communication between the output device i.e. a smart phone (20) and the device (10) of the present invention to show the results of the Detection and Prognosis perform by the device as shown in Figure 11 A.
  • the user of the device such as a smart phone can communicate with the detection device of present invention.
  • the mobile phone can be installed with an app or mobile application designed specifically for the purpose.
  • the mobile phone (10) is installed with an app named “iCanO” specifically designed to communicate with the detection device of Figure 11 A.
  • the communication may be achieved via physical connection or wireless connection.
  • the detection device is connected with the mobile smart phone (10) via wireless connection or Wi-Fi.
  • the user needs to install the app in the smart phone and connected the device using the app to the smart phone device. With the interface as shown in the figure 11B, the user can gets the details of the analysis performed by the detection device.
  • the readings of the device are shown in smart phone (10) of the user.
  • the communication unit is a Bluetooth module or Wi-Fi module, internet network and the user device is a smartphone. In one embodiment the both the devices are in communication with each other using the wireless medium preferred via a Bluetooth connection.
  • the device also comprises memory (not shown) to store one or more of codes, software, programmed instructions and/or algorithms which enable the processor to run the device and execute the various functions of the detection device including measuring, detecting, identifying, calculating, quantifying, displaying, sending, receiving etc.
  • Figure 12A-12C illustrates the different fabrication designs of the said device.
  • the figures describe the various design on the device as a rectangular or square box.
  • the present invention discloses a method to detect human saliva biomarkers like cathepsins (D, K, L, S, etc) along with other protein biomarkers indicating in cancer and chronic metabolic diseases including diabetes, neurological and cardiovascular.
  • the method is performed using the said device as described above.
  • the method comprising a nano biosensors or quartz crystal piezo electric sensors.
  • the method quantify salivary biomarkers like cathepsins (D, K, L, S, etc) along with other protein biomarkers with a point of care device to diagnose and prognosis of chronic diseases including multiple cancers employing one saliva test for clinical use.
  • the method is also used for determining a stage of cancer.
  • the determination of the stage is by obtaining a saliva sample from a subject; and measuring a quantity of biomarkers like cathepsins D, L, K, S, and other biomarkers singly and in combination, the quantity of biomarkers, or a combination thereof, above or below a pre-determined cut-off or reference level is indicative of the stage of cancer.
  • the method for detection and prognosis of the chronic disease is performed by the following steps:
  • the Device Step wise operation of the device
  • the portable detection device of the present invention can be of two types viz. DEVICE- 1 and DEVICE-2 based on type of biosensors used.
  • DEVICE TYPE 1 DEVICE TYPE 1:
  • the detection device type-1 of the present invention uses Type I, II, III and V sensors which work on the electrochemical or amperometric devices with CIRCUIT 1 as described above.
  • step-wise working/operation of device- 1 of present invention are as below:
  • Step I Biosensor testing and biomarker standard graph
  • the biosensor types I, II, III and V sensors are electro conducting and hence the sensors when fixed with two electrodes which are part of the CIRCUIT 1 and at a constant voltage applied (1-5V) would develop resistance based on the material used in different types of sensors which are noted as the starting reading of current in nano to micro Ampere current.
  • Different concentrations of the protein biomarkers of interest like Cathepsin D, S, 1, K etc or Bcl2, Bcl-x, CEA, CA 19-9, or combination thereof etc were prepared (2, 5, 10, 15, 20, 30, 50, 75, 100, 200 and 300 ng/ml).
  • 1-2 drops of protein biomarkers when dropped in the centre of the biosensors causes increase in current under a constant voltage and was found to be a linear. That means increasing concentration of biomarker increase the current.
  • Step II Saliva testing
  • Standard graph can be developed for each biomarker of our interest (Cathepsin D, S, L, K etc or Bcl2, Bcl-x, CEA, CA 19-90) or combination thereof.
  • any unknown sample can be screened for a specific biomarker using specific antibody coated biosensors of types I, II, III and V.
  • saliva sample is collected usually early in the morning or any time in the day after rinsing the mouth with water or antiseptic gargles to avoid contagious disease.
  • One to two drops of saliva is dropped in the centre of the biosensors (types I, II, III and V) fixed at the end of the device right above the electrodes that detects change in current due to binding of the protein to the specific antibody coated on the biosensors.
  • the current change from the basic current before addition of saliva and after the drop of saliva is measured and correlated to the exact concentration as determined from the standard graph developed in step 1. This is done automatically by the device which is programmed.
  • Cathepsin D screened as a biomarker saliva level less than 10 ng/ml is considered normal and above 10 ng/ml can indicate the progression of cancers like colon, oral, liver, pancreas, kidney, or Alzheimer or other metabolic diseases.
  • the detection device type-2 of the present invention uses Type IV sensor which would work on the piezoelectric device with CIRCUIT 2 provided above to measure resonant frequency.
  • step-wise working/operation of device-2 of present invention are as below:
  • Step I Biosensor testing and biomarker standard graph
  • the biosensor type IV sensors are QCM based coated with gold nanoparticles conjugated with specific antibodies.
  • one or more antibody conjugated gold nanoparticles can be coated to make it single biomarker sensor or multiple biomarkers sensors.
  • Step II Saliva testing
  • any unknown sample can be screened for a specific biomarker using specific antibody coated biosensors of type IV.
  • saliva sample is collected usually early in the morning or any time in the day after rinsing the mouth with water or antiseptic gargles to avoid contagious disease.
  • One to two drops of saliva are dropped in the centre of the QCM biosensor (type IV) fixed at the end of the device right that detects change in resonant frequency due to binding of the protein to the specific antibody coated on the biosensors.
  • the frequency change before addition of saliva and after the drop of saliva is measured and correlated to the exact concentration as determined from the standard graph developed in step 1. This is done automatically by the device which is programmed.
  • Cathepsin D screened as a biomarker saliva level less than 10 ng/ml is considered normal and above 10 ng/ml can indicate the progression of cancers like colon, oral, liver, pancreas, kidney, or Alzheimer or other metabolic diseases.
  • Non-invasive technique attracts many to come forward for tests without fear and can help in the early diagnosis compared to the conventional blood and tissue sampling for diseases like cancer.
  • Mass screening is possible at a time and also self-testing at home at affordable cost.
  • the biosensor detects saliva biomarkers like cathepsin D, K, L, S and other protein markers involved in cancer, and metabolic diseases based on antibody conjugation to nanomaterials like Molybdenum disulfide quantum dots, carbon nanotubes, titanium dioxide nanotubes or nanowires or gold nanoparticles on a metal surface through covalent conjugation.
  • the antibody-nanoparticle conjugate as obtained above, wherein the nanoparticle comprises gold, titanium, carbon, Molybdenum or a combination of two or more thereof.
  • a method is provided to detect human saliva biomarkers like cathepsins (D, K, L, S, etc) along with other protein biomarkers indicating in cancer and chronic metabolic diseases including diabetes, neurological and cardiovascular using nanbiosensors or quartz crystal piezo electric sensors.
  • human saliva biomarkers like cathepsins (D, K, L, S, etc) along with other protein biomarkers indicating in cancer and chronic metabolic diseases including diabetes, neurological and cardiovascular using nanbiosensors or quartz crystal piezo electric sensors.
  • a method is provided to detect using a point of care device to diagnose and prognoses of multiple cancer using one saliva test for clinical use.
  • the detection device of present invention is all in one non-invasive device to quantify salivary biomarkers like cathepsins (D, K, L, S, etc) along with other protein biomarkers to detect multiple cancers and other metabolic chronic diseases.
  • the device is a portable diagnostic device to detect multiple cancer and chronic diseases using a simple non-invasive spit test (saliva) that is integrated to mobile phone to regularly monitor the disease progression and treatment.
  • the portable device as described above, wherein the biosensors of Type-I, II, III, IV or V are inserted into the device to quantify for diagnosis and prognosis.
  • the invention provides a method of determining a stage of cancer by obtaining a saliva sample from a subject; and measuring a quantity of biomarkers like cathepsins D, L, K, S, and other biomarkers singly and in combination.
  • the quantity of biomarkers, or a combination thereof, above or below a pre determined cut-off or reference level is indicative of the stage of cancer.
  • the present invention provides an all in one non-invasive device to quantify salivary biomarkers like cathepsins (D, K, L, S, etc) along with other protein biomarkers to detect multiple cancers and other metabolic chronic diseases. It further provides a portable diagnostic device to detect multiple cancer and chronic diseases using a simple non-invasive spit test (saliva) that is integrated to mobile phone to regularly monitor the disease progression and treatment.

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Abstract

La présente invention concerne un dispositif de détection et de pronostic d'une maladie chronique et un procédé de détection de celle-ci. La bandelette comprend un canal microfluidique revêtu d'un matériau superhydrophobe, de la salive s'écoule à travers ledit canal et rencontre les biocapteurs. Ledit biocapteur est inséré dans le dispositif pour quantifier le diagnostic et le pronostic et détecte les biomarqueurs de salive tels que la cathepsine D, K, L, S et d'autres marqueurs protéiques impliqués dans le cancer, et des maladies métaboliques. Ledit processeur de signal est un microcontrôleur haut de gamme et fournit des signaux d'entrée et de l'énergie au capteur et l'unité de communication est une interface entre un dispositif utilisateur et le processeur de signal. En particulier, le dispositif est utile dans le dépistage de patients pour un cancer.
PCT/IN2021/050385 2020-04-18 2021-04-19 Dispositif de détection et de pronostic d'une maladie chronique et son procédé de détection WO2021210027A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114177200A (zh) * 2021-12-02 2022-03-15 中南大学 一种habt-c纳米材料及其制备和应用
CN114839343A (zh) * 2022-07-04 2022-08-02 成都博瑞科传科技有限公司 一种便携式水质监测巡检仪装置及使用方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150056099A1 (en) * 2004-01-27 2015-02-26 Altivera, Llc Diagnostic radio frequency identification sensors and applications thereof
WO2017165800A2 (fr) * 2016-03-25 2017-09-28 The Board Of Regents Of The University Of Texas System Nanocapteurs et procédés pour la détection de marqueurs biologiques
US20180231533A1 (en) * 2006-03-24 2018-08-16 Theranos Ip Company, Llc Systems and Methods of Sample Processing and Fluid Control in a Fluidic System

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150056099A1 (en) * 2004-01-27 2015-02-26 Altivera, Llc Diagnostic radio frequency identification sensors and applications thereof
US20180231533A1 (en) * 2006-03-24 2018-08-16 Theranos Ip Company, Llc Systems and Methods of Sample Processing and Fluid Control in a Fluidic System
WO2017165800A2 (fr) * 2016-03-25 2017-09-28 The Board Of Regents Of The University Of Texas System Nanocapteurs et procédés pour la détection de marqueurs biologiques

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114177200A (zh) * 2021-12-02 2022-03-15 中南大学 一种habt-c纳米材料及其制备和应用
CN114839343A (zh) * 2022-07-04 2022-08-02 成都博瑞科传科技有限公司 一种便携式水质监测巡检仪装置及使用方法

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