Cholesterol Bio Sensors: getter better fast


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These slides use concepts from my (Jeff Funk) course entitled analyzing hi-tech opportunities to analyze the increasing economic feasibility of bio-sensors for measuring cholesterol in humans. Bio-sensors detect the level of cholesterol (and other biological materials) using enzymes, matrices, and transducers. The enzymes, which are held in a matrix, react with the cholesterol and an electric signal is produced from an amperometric transducer. Improvements in sensitivity, response time, shelf life, detection limit, and reusability have been achieved through creating more appropriate biological materials for the enzymes, matrices, and transducers.

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Cholesterol Bio Sensors: getter better fast

  1. 1. CHOLESTEROL BIO-SENSOR • NIHA Agarwalla • NAUSHAD Rahman • KARTHIKA Gogulakrishnan • Sun Chenxi MT-5009: ANALYZING HI-TECHNOLOGY OPPORTUNITIES For information on other technologies, please see Jeff Funk’s slide share account ( or his book with Chris Magee: Exponential Change: What drives it? What does it tell us about the future? ebook/dp/B00HPSAYEM/ref=sr_1_1?ie=UTF8&qid=1398325920&sr=8-1&keywords=exponential+change
  2. 2. CONTENT • What is Biosensor? • Why Cholesterol Biosensor? • Conventional Techniques V/s Biosensor • Current Trend of CB • Basic Parameters of CB • Different Materials of Biosensor • Commercial Biosensor • Future CB Biosensor • Market Analysis
  3. 3. BIOSENSOR • Biosensor is a compact analytical devices or sensor that integrates a biological element with a physiochemical transducer to produce an electronic signal proportional to a single analyte which is then conveyed to a detector. • Biosensor = Analyte + biorecognation element + transducer . • A biosensor is an analytical device that detects the level of glucose, cholesterol, urea or any other chemicals in our body by using the blood ,urine ,saliva or skin as a sample. Niha
  4. 4. Niha
  5. 5. WORKFLOW OF BIOSENSOR Transducer Electrochemical Optical Piezoelectric pH Change Matrix Electrode Array chip Metal Surface Immobilization of enzyme Entrapment Covalent Crosslinking Adsorption Bio receptor /Biomolecules Enzymes Antibodies Reagent Other molecules Analyte Blood Urea Saliva Skin Niha
  6. 6. CONTD.. Detector Niha
  8. 8. CONTD… Niha
  9. 9. EXPLANATION • Analyte:- It is a sample of Blood ,Urine, Saliva ,Skin • Analyte is reacted with bio receptor. • Bio receptors are enzymes, antigens. • E.g.:- For Glucose detection in blood an enzyme called glucose oxidase is used. • For Cholesterol detection in blood an enzyme called cholesterol oxidase or cholesterol esterase is used. • Matrix is a solid support which is used to holds the bio receptor. • Examples of matrixes are electrodes, array chip or any metal surface. Niha
  10. 10. EXPLANATION CONTD… • The only necessary property for matrix that it should not erode or get affected by pH, temperature or outside environment. • It is difficult to attach bio receptor to the matrix • So different immobilization techniques are used for the attachment • Immobilization techniques are entrapment, adsorption ,cross linking, covalent bonding etc. • The bio receptor comes in contact with the analyte and generates different kinds of signal. • Signal can be either movement of electron, change in color ,mass change etc. which is detected by transducer and covert this bio signals to electrical signals. • The electrical signal are amplified and is displayed in a monitor. Niha
  11. 11.  Food Analysis  Study of biomolecules and their interaction  Drug Development  Crime detection  Medical diagnosis (both clinical and laboratory use)  Environmental field monitoring  Quality control  Industrial Process Control  Detection systems for biological warfare agents  Manufacturing of pharmaceuticals and replacement organs APPLICATION OF BIOSENSOR Niha
  12. 12. BIOSENSOR FOR MEDICAL • Biosensor are used for applications ranging from screening for disease to allow early intervention, through to the management of chronic disease and the monitoring of health and wellbeing. • Biosensors are an essential tool in the detection and monitoring of a wide range of medical conditions from cancer to Parkinson’s disease. Niha
  13. 13. CHOLESTEROL BIOSENSOR • Cardiovascular diseases and cardiac arrest are number one cause of death globally. One of the most important reasons is hypercholesterolemia i.e. increased concentration of cholesterol in blood. • An estimated 17.3 million people died from CVDs in 2008, representing 30% of all global deaths . • The number of people who die from CVDs, mainly from heart disease and stroke, will increase to reach 23.3. million by 2030 .CVDs are projected to remain the single leading cause of death . • Estimation of cholesterol level in blood hence is the most important and challenging task for medical industry. • Development and Improvement of existing Cholesterol biosensor has got a worldwide attention. Niha
  14. 14. CONVENTIONAL TECHNIQUES TO DETERMINE CHOLESTEROL 1.Lieberman-Burchard Test Lieberman–Burchard is a reagent used in a colorimetric test to detect cholesterol, which gives a deep green color. This test uses acetic anhydride and sulfuric acid as reagents. Niha
  15. 15. ADVANTAGE OF BIOSENSOR OVER L-B TEST L-B Test Biosensor Requires lot of reagents Do not require any regent Time for detection is more than 20 min. Its response time is as less as 5 sec. It uses acetic acid and sulfuric acid as reagent which can cause severe burns so require specific care. It do not use any such reagent so handling is easy. For L-B test sample should be extracted from plasma and this extraction step constitutes a cumbersome extra step in the assay Sample can be taken from any part of body. It simple blood. Requires pretreatment of sample to avoid optical interference because of hemoglobin in blood No pretreatment of sample is required. Expensive and can only be used by trained people Less expensive and can be used by any people. Niha
  16. 16. CONTD.. L-B Test device :Huge apparatus Bio-sensor : Small and portable . Niha
  17. 17. NON BIOSENSOR TECHNIQUES TO DETERMINE CHOLESTEROL 2 .High-Performance liquid Chromatography(HPLC) HPLC used to separate the components in a mixture, to identify each component, and to quantify each component HPLC uses mass transfer process involving adsorption technique to separate different components. Niha
  18. 18. ADVANTAGE OF BIOSENSOR OVER L-B TEST HPLC Biosensor Irreversibly adsorbed compounds not detected Do not require any regent Requires pretreatment of sample to avoid optical interference because of hemoglobin in blood. No pretreatment of sample is required. Complex setup and can only be used by trained people Easy to handler it Bulky and Costly Smaller and Cheaper NIha
  19. 19. CONTD…. HPLC : Bulk Setup Bio-sensor : Small setup NIha
  20. 20. HISTORY OF CHOLESTEROL BIOSENSOR • First cholesterol biosensor was developed in 1993. Components Used Matrix Ppy/Pt covered with polycarbonate membrane Bio receptor Cholesterol Oxidase Immobilization technique Entrapment Transducer Amperometric Niha
  21. 21. PUBLICATIONS TREND IN CB 0 5 10 15 20 25 30 1993 1995 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 No of Publication No of Publication Niha
  22. 22. VARIOUS TYPES OF TRANSDUCER USED IN CHOLESTEROL BIOSENSOR Electrochemical Potentiometric Amperometric Cyclic Voltammetry Optical Fluorescence Absorption Reflection Niha
  23. 23. ELECTROCHEMICAL BASED CB. • Electrochemical biosensors are normally based on enzymatic catalysis of a reaction that produces or consumes electrons and generates signal, which are captured by transducer. • The signal is proportional to concentration of the analysed substance. • Electrochemical are considered to be the most important cholesterol biosensor. • Electrochemical sensor may be divided into conductometric, potentiometric, and amperometric biosensors depending upon the electrochemical property to be measured by detector system. Naushad
  24. 24. OPTICAL BASED CB • An optical fiber-based biosensor is a biosensor that employs an optical fiber, as a platform for the biological recognition element, and as a conduit for excitation light and/or the resultant signal. • The optical measurement method is critical to the sensitivity and detection limit of the sensor. Optical transducers use different types of measurement such as fluorescence, absorbance ,and chemiluminescence. Naushad
  25. 25. ADVANTAGES OF ELECTROCHEMICAL OVER OPTICAL Electrochemical Optical The color of the sample will not interfere with the redox reaction in electrochemical method and hence do not cause any change in the electrical signal. Color of the sample interfere with the wavelengths when using optical method which result in inaccurate data. The life time of the reagents used during reaction do not decrease. The life time of the reagents can be short under incident light. Its response time and sensitivity is very high. Because of the diffusion of analytes, it may cause slow response time. No specific reagent is required for electrochemical biosensor Fiber Optic Biosensor only works for specific reagent. Low cost and low power requirement High cost and high power requirement Naushad
  26. 26. ELECTROCHEMICAL V/S OPTICAL 0.00E+00 5.00E-01 1.00E+00 1.50E+00 2.00E+00 2.50E+00 3.00E+00 3.50E+00 4.00E+00 4.50E+00 5.00E+00 2001 2002 2003 2004 2005 2006 2007 2008 DetectionLevel Year Optical Biosensor Electrochemical Acrylamine glass beads + ChOx, ChEt, HRP+ Alkylamine glass beads+ Chox, Chet, HRP+ Cross- linking via Glutaraldehyde PANI/ITO+ ChOx+ Covalent via EDC/NHS TEOS (sol–gel)ITO+ ChOx, ChEt+ Entrapment Ferrocene monocarboxylica cid-PPy/Pt/Pt+ ChOx+ PPy/Pt+ ChOx+ Entrapment FeMC (physisorbed)- P(NMPY)-PTS/ITO+ ChOx+ Physical adso Naushad 𝜇𝑔/𝑚𝑙
  27. 27. 1. Linearity 2. Sensitivity 3. Response time 4. Reusability 5. Shelf life 6. Detection Limit Basic Parameters to Measure Efficiency of CB Naushad
  28. 28. SENSITIVITY 0 0.5 1 1.5 2 2.5 1994 1996 1998 2000 2002 2004 2006 2008 Sensitivity(nAM-1) Year Improvment In Sensitivity In CB Sensitivity 1 2 3 4 5 6 7 8 9 Naushad
  29. 29. CONTD…. Material No Tetramethoxy silane sol–gel/poly(1,2-diaminobenzene)/Pt + ChOx+ Entrapment followed by treatment with Glutaraldehyde vapors+ Ampero. at 0.6 V vs. Ag/AgCl 1 PPy/Pt/Pt+ ChOx+ Entrapment+ Ampero. at 0.5 V vs. Ag/AgCl 2 PPy-p(HEMA)-TEGDA/Pt+ ChOx+ Entrapment+ Ampero. at 0.7 V vs. Ag/AgCl 3 Ferrocene monocarboxylicacid-PPy/Pt/Pt+ ChOx+ Entrapment+ Ampero. at 0.375 V vs. Ag/AgCl 4 PPy/ITO+ ChOx, ChEt+ Entrapment+ Ampero. at 0.5 V vs. Pt wire 5 (a) BSA/dilauroylphosphatidylcholine (DLPC)/rhodium–graphite; (b) Agarose gel-Riboflavin- DLPC/rhodium–graphite + (a) RfP450scc; (b) Cytochrome P450scc+ (a) Cross-linking via Glutaraldehyde; (b)+ Electro. (CV) vs. Ag/AgCl 6 3-aminopropyl-modified controlled-pore glass (APCEG)/rotating disk + Chox, ChEt, HRP+ Cross-linking via Glutaraldehyde+ Ampero. at −0.15 V vs. Ag/AgCl) withTBC 7 Streptavidin/biotin/thioctic acid (SAM)/Au nanowires + ChOx, ChEt+ Covalent via Biotin-avidin+ Voltammetric (SWV) vs. Ag/AgCl 8 FeMC (physisorbed)-P(NMPY)-PTS/ITO+ ChOx+ Physical adsorption+ Electro. (CV) vs. Ag/AgCl 9 Naushad
  30. 30. EXPLANATION OF GRAPH • Sensitivity of an sensor indicates the capacity of the sensor to respond truly to the change in the output, corresponding to the change in the input. • Its depends on various factor like electrode , Enzymes and Immobilization technique. Naushad Rahman
  31. 31. RESPONSE TIME 0 0.5 1 1.5 2 2.5 3 3.5 1996 1998 2000 2002 2004 2006 2008 ResponseTime(sec) Year Response time in sec Response time in sec 1 2 3 4 5 6 7 8 9 12 11 10 13 14 15 Naushad
  32. 32. TABLE 1 PPy/Pt covered with polycarbonate membrane +ChOx + Entrapment+Ampero. at 0.7 V vs. SCE 8 PPy/Pt+ ChOx+ Entrapment+ Ampero. at 0.7 V vs. Ag/AgCl 2 2-aminoethanethiolate/Au +ChOx, ChEt for TC, ChOx for FC +Cross-linking via Glutaraldehyde +Amperometric at 0 V vs. SCE with thionin as electron mediator 9 ITO glass+ Cytochrome P450SCC (5 g in 40 LB)+ LB films+ Electro. (CV) vs. Ag/AgCl 3 Hexadecyl mercaptan (SAM)/Au+ Molecular imprints+ Template+ Electro. (CV) vs. Ag/AgCl 10 BSA/dilauroylphosphatidylcholine (DLPC)/rhodium– graphite; (b) Agarose gel-Riboflavin-DLPC/rhodium– graphite + (a) RfP450scc; (b) Cytochrome P450scc+ (a) Cross-linking via Glutaraldehyde; (b)+ Electro. (CV) vs. Ag/AgCl 4 TEOS derived solgel+ ChOx, HRP+ Entrapment+ Ampero. at 0.75 V vs. Ag/AgCl 11 Sol–gel/CNT-Pt/graphite electrode+ ChOx+ Entrapment+ Electro. (CV) and Ampero. −0.2 V vs. 5 Poly(ethylene imine)(PEI)/poly(styrene sulfonate)/ITO + ChOx+ LBL deposition of PEI and ChOx + Ampero. at 0.6 V vs. Ag/AgCl 12 1-hexadecanethiol (SAM)/Au+ Molecular imprinted layer+ Templete + Voltammetry (CV) vs. Ag/AgCl 6 PPy- doecylbenzene sulfonate/ITO+ ChOx+ Physical adsorption+ Electro. (CV) vs. Ag/AgCl 13 MWCN/Screen Printed Carbon Electrode+ Chox, Chet, HRP, K4 Fe(CN)6+ Physical adsorption+ Ampero. at 0.3 V vs. Ag/AgCl 7 (a) Octadecylsilica (ODS)/TEOS sol–gel for cholesterol in aqueous micelle solution; (b) ODS/HECMC/PVA gel for cholesterol in hydrophobic organic solvent + ChOx+ Entrapment+ Optical oxygen transducer 14 Multilayer of Pt-MWCNT-CHIT and poly(sodium-p - styrenesulfonate) onto Au + ChOx+ Cross-linking via Glutaraldehyde+ Ampero. at 0.1 V vs. SCE 15 ZnO/Au+ ChOx+ Physical adsorption+ Electro. (CV) vs. Ag/AgCl Naushad Rahman
  33. 33. EXPLANATION OF GRAPH • Response Time:- Time taken by a sensor to detect the cholesterol in a sample. • Lower the response time higher is the efficiency of the biosensor. NIha
  34. 34. REUSABILITY 0 20 40 60 80 100 120 140 160 180 200 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 Reusability(Times) Year Reusability RE 6 1 2 3 4 5
  35. 35. TABLE Component Component 1 PANI/Pt+ ChOx+ Electro. doping+ Ampero. at 0.6 V vs. SCE and 1% tritonX-100 5 Poly(dialyldimethylammonium chloride) (PDDA)/MWCNTs/Au + ChOx covered by o-PDD+ LBL deposition of PDDA and ChOx+ Ampero. at 0.7 V vs. SCE 2 Acrylamine glass beads+ ChOx, ChEt, HRP+ Covalent via diazotization+ Spectro. at 520 nm using APZ+ phenol 6 PANI/ITO+ ChOx+ Covalent via EDC/NHS+ Spectro. at 500 nm using o-dianisidine 3 Silisic sol–gel/PB/GC+ ChOx+ Entrapment+ Electro. (CV) at −0.05 V vs. Ag/AgCl 4 Polyacrylonitrile fiber+ ChOx+ Cross-linking via Glutaraldehyde+ Spectro. at 520 nm using APZ+ phenol Naushad Rahman
  36. 36. EXPLANATION OF GRAPH • Reusability means number times sensor can be use before losing its precision level . • It depends upon the electrode quality . • As sensor electrode will erode by interfacing element and reduce its surface area to volume ration will decrease. Naushad Rahman
  37. 37. SHELF LIFE Naushad 0 20 40 60 80 100 120 2000 2001 2002 2003 2004 2005 2006 2007 2008 ShelfLife(Days) Year Improvment in Shelf Life 1 2 3 4 5 6 7
  38. 38. TABLE 1 Tributylmethyl phosphonium chloride polymer membrane/pyrolitic graphite electrode + ChOx, HRP+ Entrapment+ Ampero. at −0.28 V vs. Ag/AgCl 4 PPy/ITO+ ChOx, ChEt+ Entrapment+ Ampero. at 0.5 V vs. Pt wire 2 Acrylamine glass beads+ ChOx, ChEt, HRP+ Covalent via diazotization+ Spectro. at 520 nm using APZ+ phenol 5 Sol–gel/CNT-Pt/graphite electrode+ ChOx+ Entrapment+ Electro. (CV) and Ampero. −0.2 V vs. 3 Silisic sol–gel/PB/GC+ ChOx+ Entrapment+ Electro. (CV) at −0.05 V vs. Ag/AgCl 6 BSA/TEOS/ITO+ ChOx, HRP+ Covalent+ Ampero. at 0.75 V vs. Ag/AgCl 7 BSA/Polycarbonate/Oxygen electrode+ ChOx, ChEt+ Cross-linking via Glutaraldehyde+ Polarographic at −0.7 V vs. Ag/AgCl Naushad Rahman
  39. 39. EXPLANATION OF GRAPH • Shelf Life:- Duration till which one can use electrode to detect cholesterol. • Higher the shelf life higher is the efficiency of the biosensor. Naushad
  40. 40. DETECTION LIMIT Naushad 0 10 20 30 40 50 60 70 1992 1994 1996 1998 2000 2002 2004 2006 2008 DetectionLimit(mgdl) Year Detection Limit DL 2 1 3 4 5 6 7 8 9 1310 11 12
  41. 41. TABLE Naushad Rahman Component Component 1 PPy/Pt covered with polycarbonate membrane +ChOx + Entrapment+Ampero. at 0.7 V vs. SCE 7 Poly(o-phenylenediamine)/PPy/Pt/Pt+ ChOx+ Entrapment in PPy+ Ampero. at 0.5 V vs. Ag/AgCl 2 HRP-hydroxymethyl ferrocene-carbon paste electrode +ChOx ChEt+In solution +Chronoamperometry at 0 V vs. SCE 8 Acrylamine glass beads+ ChOx ChEt HRP+ Covalent via diazotization+ Spectro. at 520 nm using APZ+ phenol 3 Laponite clay nanoparticls-poly((12-pyrrol-1- yldodecy)triethylammonium tetrafluoroborate)/Pt disk electrode + ChOx ChEt for TC ChOx for FC +Entrapment +Ampero. at 0.53 V vs. Ag/AgCl 9 PPy/Pt+ ChOx+ Entrapment+ Ampero. at 0.7 V vs. Ag/AgCl 4 Glassy carbon+ ChOx+ In solution + Electro. at 0 V vs. SCE using thionin as mediator 10 ITO glass+ Cytochrome P450SCC (5 g in 40 LB)+ LB films+ Electro. (CV) vs. Ag/AgCl 5 Tetramethoxy silane sol–gel/poly(12- diaminobenzene)/Pt + ChOx+ Entrapment followed by treatment with Glutaraldehyde vapors+ Ampero. at 0.6 V vs. Ag/AgCl 11 Silica sol–gel- chitosan (CHIT)-MWCNT/PB/GC + ChOx+ Entrapment+ Ampero. at −0.05 V vs. Ag/AgCl 6 Hexadecyl mercaptan (SAM)/Au+ Molecular imprints+ Template+ Electro. (CV) vs. Ag/AgCl 12 CHIT/NiNPs/histidine/MWCNT/GC electrode + ChOx+ Cross-linking via Glutaraldehyde+ Ampero. −0.2 V vs. SCE 13 TEOS (sol–gel)ITO+ ChOx ChEt+ Entrapment+ Spectro. at 500 nm using 4-APP + phenol
  42. 42. EXPLANATION OF GRAPH • DL is the lowest quantity of a substance that can be distinguished from the absence of that substance within a stated confidence limit. • Lower the DL greater is the selectivity of the CB. Naushd
  43. 43. DIFFERENT MATERIALS FOR AMPEROMETRIC BIOSENSORS Metallic Materials Thermoplastic Polymeric Material Thermosetting Polymeric Material Applications flow through reusable sensors Moderate temperature with no strong solvents, used as working electrode Used in combination with polar solvents, used as working electrode Advantages lower electronic noise low detection limits, high sensitivities, lower applied potential, reduction of background, efficient electron transfer Disadvantages Moderate performance commercial production of pure and defect- free polymers is difficult and costly Examples Gold, Ion, Silver Carbon, conductive polymer, sol-gel polymer, functional polymer Chenxi
  44. 44. METAL Chitosan-gold  High electron transfer rate (Electron transfer rate constant was estimated to be 15.6 s−1)  Enhanced stability  Moderate bioactivity  magnetic Fe3O4/chitosan  Fast response to H2O2 and excellent linear relationships  Excellent bioactivity  Long-time stability and good reproducibility Chenxi
  45. 45. CARBON NANOTUBES • Accuracy, efficiency and detection limit of a biosensor can be increased with the use of carbon nanotubes immobilized on bare electrodes • High electron transfer, high surface area, minimization of the surface fouling, high stability, excellent adsorptive and biocompatibility. Chenxi
  46. 46. SOL-GEL POLYMERIC MATERIALS Sol-gel materials provide a versatile way for immobilization due to the presence of inorganic M–O or M–OH–M bridges forming a continuous network containing a liquid phase which can then be dried out to form a solid, porous polymeric matrix Excellent sensitivity Good reproducibility Remarkable stability Rapid response Chenxi
  47. 47. CONDUCTIVE POLYMERIC MATERIALS Superior reusability Excellent thermal stability Large surface to volume ratio High conductivity, sensitivity Good biocompatibility Wide linear range Graphene/chitosan Chenxi
  48. 48. FUNCTIONAL POLYMERIC MATERIAL These are polymeric materials which possess different functional groups, for example, thiols, amines, carboxylic acids and others Low detection limit, wide detection range Fast response Good stability. Anti-interference ability Chenxi
  50. 50. INVASIVE CB • Invasive biosensors are the one in which blood sample is required and is obtained from the finger tip by pricking. • These sample is placed in the test strip and the test strip is inserted into the biosensor device and amount of cholesterol is detected. NIHA
  51. 51. NON INVASIVE BIOSENSOR. • Non Invasive biosensor are the one in which no pricking is required it uses skin cholesterol to measure the cholesterol level in human body. • Skin contains over 11% of the body cholesterol and ages in parallel with vascular connective tissue. As arterial walls accumulate cholesterol, so do the skin tissues. • A high skin cholesterol level is a reliable predictor of higher cholesterol accumulation in the arteries and, accordingly, can be used in combination with other risk factors to assess risk of coronary artery disease. NIHA
  52. 52. CONTD.. • The simple test is conducted by placing a drop of digitonin, which binds selectively to the cholesterol in the skin, on the palm of the hand. This liquid also contains an enzyme linked to the digitonin by a copolymer. After a one-minute incubation period, the area is blotted dry to remove any unbound digitonin solution. A second drop of liquid is then added, containing a substrate for the horseradish peroxidase enzyme. When combined, a blue color change occurs in direct proportion to the amount of digitonin that is bound to skin cholesterol. After two minutes, a hand-held spectrophotometer (color reader) is placed over the drop to measure the precise blue color, which indicates the skin cholesterol value. PREVU* Skin Cholesterol Test NIha
  53. 53. INVASIVE V/S NON INVASIVE Invasive Non Invasive It requires sample so finger pricking is done which is very painful No pricking is required so painless cholesterol determination It require patient preparation such as fasting before cholesterol determination No prior patient preparation is required. More sensitive and more accurate Less Sensitive and less accurate Latest cholesterol biosensor can detect total cholesterol, LDL,HDL and triglycerides separately. It can detect only total cholesterol It is the present. It can be the future as high research is done in the non invasive biosensor. NIha
  54. 54. Commercial Cholesterol biosensor Electrochemical techniques are simple, relatively cheap, and rapid as compared to other methods; but also have a potential for further improvement. Other methods. colorimetric, high performance liquid chromatography (HPLC) using gel electrophoretic chip, capillary electrophoresis, spectrophotometric and fluorometric 1st generation of Commercial cholesterol biosensors use cholesterol esterase or cholesterol oxidase .They depend on the presence of oxygen for the enzyme – catalysed reaction and the production and measurement of hydrogen peroxide. •2nd Generation use electron mediators which help to transfer electrons from the enzyme to electrode surface .Ferro/ferricyanide, ferrocene, conducting organic salts and redox dyes were used as mediators. Karthika
  55. 55. Commercial Cholesterol biosensor 3rd generation of cholesterol biosensors: Use direct electron transfer between enzymes and electrodes in the absence of mediator. The distance between immobilised enzyme and electrode should be as small as possible. Such biosensors are more sensitive. A wide range of cholesterol biosensors are available in market today using a range of matrix ,immobilisation and transducer technologies. Karthika
  56. 56. COMMERCIAL CB • Cholesterol Bio sensors used in Clinical analysis are usually more accurate than home kits. • Cholesterol biosensors used in Labs devices like Autoanalyzer which is capable of doing dozen of analyses simultaneously by a single machine from a small amount of serum. Karthika
  57. 57. COMMERCIAL CHOLESTROL BIOSENSOR cholesterol oxidase for assay of total and free cholesterol in serum by continuous-flow analysis. Desktop version Hitachi 7070 124017974- 103336093/Auto_Analyzer_HITACHI_7070_91 1_.html Evolution of Commercial biosensors Standalone Huge devices Portable Home kits Karthika
  58. 58. DESKTOP VERSION OF CB Karthika
  59. 59. CONTD.. Karthika
  60. 60. HOME KITS Detects as little as 200 nM (80 ng/ml) cholesterol Helps accurately measure cholesterol content of 0.01 µl of human serum Detects both free cholesterol and cholesteryl esters. High sensitivity and excellent linearity of the assay at low levels of cholesterol (0–0.015 µg⁄ml). Karthika
  61. 61. AMPLEX® RED ASSAYS DETECTION LIMIT Different product use different reagent (polyethylene glycol cholesteryl ether , Cholesterol oxidase , horseradish peroxidase ) and electrode combinations. Karthika high sensitivity and excellent linearity of the assay at low levels of cholesterol (0–0.015 µg⁄ml) 𝜇𝑔/𝑚𝐿 Fluorescence
  62. 62. COMMON TECHNOLOGY IN COMMERCIAL CB Most common technique in Home Kits Cholesterol Kit uses cholesterol oxidase to produce hydrogen peroxide, which is then detected by the reagent in the presence of horseradish peroxidase (HRP). Enzyme coupled technology Measure by reflectance of light(photometric). An electrochemical reaction which generates an electrical current proportional to the amount of Cholesterol- Electrochemical biosensors. Karthika
  63. 63. CONTD.. Karthika
  64. 64. FEATURES ON WHICH SUCCESS OF CB DEPENDS • Be stable under normal storage conditions and show good stability over a large number of assays (i.e. much greater than 100) • The reaction should be as independent of physical parameters as pH and temperature • The response should be accurate, precise, reproducible and linear over the useful analytical range. It should also be free from electrical noise. • Should be cheap , small, portable • Capable of being used by unskilled person. • Screen Printing played an important role in biosensors commercialization. • Variation of enzymes and electrode materials had improved performance in sensitivity storage and shelf life. Karthika
  65. 65. COMMERCIAL CB CONTD.. Karthika
  66. 66. CONTD.. Karthika
  67. 67. CONTD… Karthika
  68. 68. CONTD.. Karthika
  69. 69. FUTURE CHOLESTEROL BIOSENSOR Non invasive Biosensors and Wearable Biosensors are the future of CB. Wearable Biosensor • These are the devices that will monitor continuously the physiological signals. • They rely on wireless sensor ,the data are recorded and is used to monitor patients health condition. • They are very helpful to athletes, handicapped, old aged and to professional people. NIha
  70. 70. WEARABLE BIOSENSOR • Examples of wearable biosensor: Shoes Biosensor Shirt BiosensorRing Biosensor NIha
  71. 71. WEARABLE BIOSENSOR • Ring sensor Can measure the heart rate and oxygen saturation rate. • Shirt Sensor can measure body temperature, heart rate and respiration rate. • Their main applications are :-  Chronic Surveillance of abnormal heart failure  In cardio-vascular dieses to measure hyper tension. Wireless Monitoring for people in hazardous operations. NIha
  72. 72. WEARABLE BIOSENSOR Advantage:- Continuous Monitoring Easy to use Reduce Hospitalization Fee Disadvantage:- Initial Cost is high Limited number of physiological parameter is measured Niha
  73. 73. EFFECT OF TECHNOLOGY IN DEVELOPMENT OF CB Two most important technology that will play a significant role for the development of CB is Nanotechnology Microsystem Technology. Various kind of nanomaterial are applied to cholesterol biosensor such as gold nanoparticles, carbon nanotubes, Nanowires, Graphene and Quantum dots. Niha
  74. 74. WHY NANO TECHNOLOGY?? • These materials are generally used because of their unique physical, chemical, mechanical, magnetic and optical properties, • They markedly enhance the sensitivity and specificity of detection. • They have great potential in the detection of DNA, RNA, proteins, glucose ,pesticides and other small molecules from clinical samples, food industrial samples, as well as environmental monitoring. Niha
  75. 75. MULTI ANALYTE DETECTION They also help in Multi analyte Detection. • Development of sensors capable of determining several analyte simultaneously can represent an interesting tool in clinical industry. • This will help to reduce the cost and will also lessen the diagnostic time. NIha
  76. 76. MINIATURIZATION They also led to Miniaturization of biosensor. • Miniaturization allows the handling of low-volume samples, a reduction in reagent consumption and waste generation, and increases sample throughput . • Miniaturization can benefit Biosensor by making it inexpensive and easy-to-handle analytical devices. • Nanowire are smaller than the red blood cell whose diameter is 6.8 micro meter. • Use of Nanowire Biosensor can help to develop implantable biosensor too. NIha
  77. 77. EFFECT OF NANOPARTICLE ON SENSITIVITY 0 5 10 15 20 25 30 35 40 45 50 Sensitivity(mV) Sensitivity(mV) NIha
  78. 78. EFFECT OF NANOPARTICLE ON DETECTION LIMIT 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Detection Limit(microM) Detection Limit(microM) NIha
  79. 79. EFFECT OF MWCNT ON DETECTION LIMIT 0 0.1 0.2 0.3 0.4 0.5 0.6 2000 2002 2004 2006 2008 2010 2012 2014 Detection Limit(micromol) Detection Limit(micromol) NIha
  80. 80. EFFECT OF GRAPHENE ON DL AND SENSITIVITY NIHA 0 0.5 1 1.5 2 2.5 3 0 5 10 15 20 25 30 35 40 2009.5 2010 2010.5 2011 2011.5 2012 2012.5 2013 2013.5 2014 2014.5 LimirOFDetection(μM) Sensitivity(μAmM1 cm-2) YEAR Sensitivity LOD (μM)
  81. 81. TOTAL MARKET OF BIOSENSOR • The biosensor market is dominated by only a few products • For medical diagnostics, approximately 90% of biosensors are glucose monitors, blood gas monitors, and electrolyte or metabolite analyzers • Half of all biosensors produced worldwide are glucose monitors Sales are projected at $1.28 billion in the US in 2012 • The majority of the remaining market includes biosensors directed at environmental control, fermentation monitoring, alcohol testing, and food control NIha
  83. 83. MARKET ANALYSIS OF BIOSENSOR • According to a new market report published by Transparency Market Research “Biosensors Market - Global Industry Analysis, Size, Share, Growth, Trends and Forecast, 2012 - 2018,” in 2011, the global biosensors market was valued at USD 9.9 billion and it is expected to grow at a CAGR of 9.6% from 2012 to 2018 to reach a market of USD 18.9 billion by 2018. • In 2011, the biosensors market was valued to be USD 9,973.5 million and is expected to grow at a CAGR of 9.6% from 2012 to 2018. NIha
  84. 84. FURTHER ANALYSIS • The development of new biosensors and devices based on this technology is a highly capital intensive exercise. • Although miniaturization allows for economies of scale to be achieved in the actual manufacturing of biosensors, huge capital investment is required for research and development. • Though the U.S. remains the largest market in the world, Asian countries namely India and China are witnessing fast growth and are predicted to emerge as dominating markets in the near future. NIha
  85. 85. FURTHER TREND NIha
  86. 86. Thank You