General Introduction and Application in Blood- Glucose Level Montoring Biosensors General Introduction and Application in Blood- Glucose Level Montoring Pooja Tomar, HU Zain Hassep, HU

General introduction (Principle, Classification &Applications) Content 1 General introduction (Principle, Classification &Applications) Glucose biosensors (Importance, Principle & Classification) Validation of glucose biosensors. Calibration of glucose biosensors. Homogeneous & heterogeneous glucose biosensors. Reusable Biosensors

2 Definition The term “biosensor” was coined by Cammann, and its definition was introduced by IUPAC A chemical sensor is a device that transforms chemical information, ranging from the concentration of a specific sample component to total composition analysis, into an analytically useful signal. Chemical sensors usually contain two basic components connected in series: a chemical (molecular) recognition system (receptor) and a physicochemical transducer. Biosensors are chemical sensors in which the recognition system utilizes a biochemical mechanism. There are three main parts of a biosensor: (i) the biological recognition elements that differentiate the target molecules in the presence of various chemicals, (ii) a transducer that converts the biorecognition event into a measurable signal, and (iii) a signal processing system that converts the signal into a readable form [19-21]. (1) D. R. Thevenot, K. Toth, R. A. Durst, G. S. Wilson, Electrochemical biosensors: recommended definitions and classification, Pure Appl Chem, 1999, 71 , 2333–2348, (2) D. Grieshaber , R. MacKenzie , J. Vörös, E. Reimhult , Electrochemical Biosensors - Sensor Principles and Architectures, Sensors , 2008, 8, 1400-1458.

3 Properties Specificity: a biosensor should be specific to the analyte which it interact. Durability: it should withstand repeated usage. (iii) Independent nature: It should not be affected by variations in the environment like temperature, pH etc. Stability in results: the results produced by interaction should be corresponding to the concentration of analyte. (v) Ease of use and transport: it should be small in size so that it can be easily carried and used. The biocatalyst must be highly specific for the purpose of the analysis, be stable under normal storage conditions and show a low variation between assays. The reaction should be as independent as manageable of such physical parameters as stirring, pH and temperature. This will allow analysis of samples with minimal pre-treatment. If the reaction involves cofactors or coenzymes these should, preferably, also be co-immobilized with the enzyme. The response should be accurate, precise, reproducible and linear over the concentration range of interest, without dilution or concentration. It should also be free from electrical or other transducer induced noise. If the biosensor is to be used for invasive monitoring in clinical situations, the probe must be tiny and biocompatible, having no toxic or antigenic effects. Furthermore, the biosensor should not be prone to inactivation or proteolysis. For rapid measurements of analytes from human samples it is desirable that the biosensor can provide real-time analysis. The complete biosensor should be cheap, small, portable and capable of being used by semi-skilled

Biological Sensing Component 4 Classification Classification Biological Sensing Component Enzymatic Receptor Nuclear Transduction Process Electrochemical Amperometric Potentiometric Conductometric Magnetic Thermometric Optical Piezoelectric The molecular recognition elements include receptors, enzymes, antibodies, nucleic acids, microorganisms and lectins [22,23]. The five principal transducer classes are electrochemical, optical, thermometric, piezoelectric, and magnetic [24]

Applications 5 Health Care Industrial Process Control Military Applications Environmental Monitoring Pregnancy test Detects the hCG protein in urine. Glucometer Field tool kit Food quality analyis

Glucose Biosensors Glucose sensors 6 Plays a major role in our life “fuel molecule” Normal level (fasting) : 3.9 and 5.5 mmol/L (70 to 100 mg/dL). Glucose-related disease: Diabetes from 347 million people in 2005 to 700 million people in 2030. One person dies from diabetes every 10 seconds. Glucose sensors Point sample test Continuous glucose monitor(CGM) Most sensors are enzyme-based.

Glucose oxidase (GOD) is the standard enzyme for biosensors The earliest sensor 7 - lack of selectivity Glucose oxidase (GOD) is the standard enzyme for biosensors Easy to obtain, Cheap - Can withstand greater extremes of pH, ionic strength, and temperature. Other enzymes : Hexokinase and glucose-1-dehydrogenase (GDH). journal of food and drug anal ysi s 2 3 ( 2 0 1 5 ) 1 9 1 - 2 0 0

Principle 8 1st generation of Glucose Biosensors: Drawbacks . (high potential for selectivity, oxygen deficit, Deactivation of GOD by H2O2. “The fact that normal O2 concentrations are about 1 order of magnitude lower than the physiological level of glucose”

in vivo monitoring of blood glucose 9 2nd generation of Glucose Biosensors 3rd generation of Glucose Biosensors - Reagentless - ferrocene, ferricyanide, quinines,… - Organic conducting materials based on charge-transfer complexes. Drawbacks (Competition between O2 and Med., interference, Med. Leaching, toxicity of mediators) in vivo monitoring of blood glucose RSC Adv., 2013, 3, 4473–4491

How to use ?? 10

Continuous glucose monitoring(CGM) 11 Self testing is limited by the number of tests per a 24 h period.(neglect nighttime variations) Continuous ex vivo monitoring was proposed in 1974. 1st application for in vivo glucose monitoring was demonstrated in 1982. Requirements:- Proper attention to the issues of : Biocompatibility/biofouling - Miniaturization, - Long-term stability of the enzyme and transducer - Oxygen deficit - Baseline drift, - Short stabilization times, - In vivo calibration, - Safety, and convenience CGMs Invasive CGMs Subcutaneous & microdialysis Non-invasive CGMs Transdermal & Optical

Invasive CGMs 12 Subcutaneous CGM Microdialysis CGM

Non-invasive CGMs 13 Transdermal CGM Optical CGM

Glucose content in beverages and fruit juices 14

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Analytical Performance Validation of Glucose Biosensors 16 Suboptimal measurement quality can lead to significant inaccuracies and increased patient morbidity and mortality. The regulatory guidelines address: Precision (Repeatability& Intermediate precision) Accuracy - Linearity User performance: to demonstrate that users are able to operate the glucose biosensors, given only the instructions and training materials routinely provided with the system and obtain valid glucose results. At least 50 subjects, with varying demographics (age, gender, and education level), should be included for each lot. Sensors 2010, 10, 4558-4576

17 Interferences : Hematocrit, hypoxemia, hypotension, altitude, temperature, and humidity >>>> affect the reliability of the test. Electrochemical interferents >>> false high glucose readings. standard interferents developed by the FDA include: acetaminophen, salicylic acid, tetracycline, dopamine, ephedrine, ibuprofen, methy-DOPA, ascorbic acid. GDH based biosensors ### peritoneal dialysis using icodextrin

18 Calibration The sensitivity or slope of Pt/PCS+PEI/GOD glucose biosensor system has been found to be 0.72 ± 0.15 nA/µM. Fig: The amperometric response of the enzyme electrode to successive glucose additions Fig: Calibration curve of the enzyme electrode for glucose The response time depends upon the analyte, co-substrate and the product transport through different membranes. The thickness and permeability of the polymer matrix (membrane) are very critical in determining the response behaviour. They also depend upon the activity of the molecular reorganization system. The higher the activity of the system, the smaller will be the response time. The existence of this linear relationship between the current and concentration of glucose is important for the accurate determination of glucose levels in human blood which lie within a narrow range of 3.5 to 5.0 mM U. B. Trivedi, D. Lakshminarayana, I. L. Kothari, N. G. Patel, H. N. Kapse, P. B. Patel, C. J. Panchal , Amperometric Glucose Biosensor Based on Immobilization of Glucose Oxidase in Polyethylenemine and Poly (carbamolylsuphonate) Polymer Matrix , Sensors & Transducers, 2010, 119, 129-141.

Hetrogeneous v/s Homogeneous assay 19 Hetrogeneous v/s Homogeneous assay Heterogeneous assay Homogeneous assay Diffusion of the analyte molecules within the sample solution volume towards the sensor surface for signal generation. Rely on signal generation within the whole sample volume. Usually, the signal generating probes are mixed with the sample solution, and measurements are carried out on this complex mixture. Heterogeneous assay principles generally display high sensitivity and wide dynamic range, labor intensive sample preparation steps that usually comprise multiple washing and incubation steps are disadvantages that limit their applicability The three dimensional diffusion of both, analyte molecules and capture probes, leads to reduced total assay times compared to heterogeneous assay principles Include amperometric and conductometric type of sensors. Include fluorescence polarization, fluorescence correlation spectroscopy, single component noble metal nanoparticles and magnetic nanoparticles sensors. In the former case, the wavelength of the Nanoparticles, plasmon resonances is red-shifted on the adsorption of target molecules, while in the latter case, the Brownian relaxation time increases for nanoparticles with bound target molecules due to their increased hydrodynamic diameter

Example of homogeneous glucose biosensor- fluoroscent assay 20 Example of homogeneous glucose biosensor- fluoroscent assay The current approach employs two labels that absorb and fluoresce visible light, FITC-Dextran as a donor (fluorescer) and tetramethylrhodamine isothiocyanate labeled ConA (TRITCConA) as the acceptor (quencher). The competing reactions for the system are given in Eqns. 1 and 2. When FITC-Dextran reversibly binds to TRITCConA, the donor and acceptor chromophores are sufficiently dose together (less than approximately 50 A> for a significant portion of the fluorescein signal to be quenched by rhodamine. As glucose is added to the system, FITC-Dextran is liberated from the TRITC-ConA causing the fluorescein signal to increase. With this approach, the fluorescein signal is used to monitor glucose concentrations. D.L. Meadows and J.S. Schultz, Design, manufacture and characterization of an optical fiber glucose affinity sensor based on an homogeneous fluorescence energy transfer assay system , Analyricu Chimku /lcra, 280 (1993) 21-30

21 Reusable Biosensors

22 Reusable Biosensors In this work, a label-free minimally invasive rectangular meandered line (RML) resonator functioning at a microwave frequency of 9.20 GHz was analysed for the detection of the glucose level in human serum and compared with an aqueous solution of deionised (D) water glucose solution. The detection of the glucose levels is based on the variation of the dielectric property with the different concentrations of glucose present in the serum and the change in the electrical behaviour due to the skin effect of the meandered conductive line. The reusability of the biosensor was characterised by measuring the resonance frequency before and after the use of serum and the Dglucose solution. No device response deterioration was observed over the long iteration of the measurement process. As a result, the relative standard deviation (RSD) of less than 1% was observed for each concentration of serum samples and aqueous solution of D-glucose.

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