BIOSENSORS AND BIOELECTRONICS ASSIGNMENT

Assignment Submitted to:Dr. Rachana Sahney Amity Institute of Biotechnology, Amity University, Noida

Assignment Submitted by:RONEET GHOSH Roll no-BTBM/09/4004 Enrolment no- A0523109032 Semester-7, 4th Year, B.tech+M.tech (dual) Biotechnology Batch- 2009-2014 Amity Institute of Biotechnology

Question 1: What are the three important features that must be present in a biosensor?

Answer:

The optimum design of any biosensor is dictated by several physical properties of measuring system. Some of the most pertinent properties and characteristic behaviour of ideal biosensor characteristics are as follows:-

1. SENSITIVITY- The sensitivity is usually defined as the final steady state change in the magnitude of biosensor output signal with respect to change in concentration of a specific chemical species (Change in S/ Change in C). More often the change in concentration of a co-product or a co-reactant of a chemical reaction taking place within the biosensor are measured. The sensitivity of a biosensor with respect to the chemical substrate of interest ( the analyte) must then be related to the directly detected chemical species through the appropriate stoichiometry of the chemical reaction. In other cases, some physical property has been altered by the biological element,which is then measured by the transducer.

For some biosensor typs measurements are based upon the dynamic response of the biosensor. Sensitivity may be defined in this situation as the change in the signal with time for a given change in concentration, or some other relationship that depend on time. Time integration, frequency analysis or other data processing of the time varying signals may also be of vaue in relating them to the concentration of the analyte. There are many factors that determine the effective sensititvity of a given biosensor design to a target analyte These include the physical size of the sensor, the thickness of the membrabes and the resulting mass transport of chemical species of the sample to the sensing region, and various processes that deactivate the biosensor or otherwise impair its operation over time. Ideally the sensitivity of a given biosensor should remain constant during its lifetime and should be sufficiently high to allow convenient measurement of the transducer output signal with electronic instrumentation

2. CALIBRATION- An ideal biosensor should be easily calibrated by simply exposing it to a prepared standard solutions or gases containing different known concentrations of the target analyte. Calibration curves need not require many data points to obtain the sensitivity especially if the operational behaviour of the biosensor is known. Calibration points should

bracket the range of values that will be measured, to avoid possibly unreliable extrapolations outside the expected range. Ideally, it should be necessary to perform a calibration procedure only one time to determine the sensitivity of the biosensor for subsequent measurements. In practical terms, however, it is usually necessary to make periodic calibrations at regular intervals to characterise changes in the sensitivity with time.

3. DYNAMIC RESPONSE- The physical properties and relative size of a biosensor probe determine how quickly it will respond to change in concentration of the analyte molecule it measures. The principle mechanism is usually simple diffusion of the chemical species from sample to the active surface of the transducer. The mass flux of the target analyte and/ or the reactant that is being detected is proportional to the concentration differences, the effective diffusion coefficients Deff for each species moving through the different elements of the sensor (membranes, electrolytes and other structures), and the thickness of each element. This type of response is often referred to as being dependent on a diffusion-limited process.

Question 2: What are ISE? Discuss the principle and operationof glass pH electrode as H+ ion selective electrode?

Answer: An ideal I.S.E. consists of a thin membrane across which only the intended ion can be transported. The transport of ions from a high conc. to a low one through a selective binding with some sites within the membrane creates a potential difference.

Advantages: 1. Linear response: over 4 to 6 orders of magnitude of A. 2. Non-destructive: no consumption of analyte. 3. Non-contaminating. 4. Short response time: in sec. or min. useful in indust. applications. 5. Unaffected by color or turbidity. Limitations: 1. Precision is rarely better than 1%. 2. Electrodes can be fouled by proteins or other organic solutes. 3. Interference by other ions. 4. Electrodes are fragile and have limited shelf life. 5. Electrodes respond to the activity of uncomplexed ion. So ligands must be absent or masked. m must be kept constant.

Most of the metal cations (e.g. Na+) in the hydrated gel layer diffuse out of theglass membrane and into the solution. Concomitantly, H+ from solution diffuse into the membrane. There are two different types of Electrical Conductivity: 1) In Metals the electric current is carried by Electrons. 2) In Liquids the electric current is carried by Ions Every Electrochemical Process (Galvanic Cell, Electrolysis, Electro-Analysis) involves both these types of conductivity. The junctions where they meet and transfer the electrical charge are referred to as Metal-Liquid Interfaces. These interfaces were originally called Electrodes, but now this term is also used for various other devices such as welding electrodes or electro-cardiogram electrodes. At the Metal-Liquid interface there is an exchange of Electrons in one or other direction (details can be found in standard chemistry text books, in sections on Galvanic or Electrolytic Cells. (NB: Galvanic [Voltaic] Cells generate electricity; Electrolytic Cells consume electricity). For example, in a Copper-Silver Galvanic Cell, on one electrode an Oxidation reaction takes place: Cu (metallic) Cu 2+ (ionic, in solution) +2 eon the other electrode a Reduction reaction takes place: Ag+ (ionic, from solution) + e- Ag (metallic - deposited on electrode surface) This explains how the electric current in the wire (Electrons) becomes a current in the liquid (Ions). he Electrochemical Circuit for an Ion Selective Electrode measurement. An ISE (with its own internal reference electrode - more details later) is immersed in an aqueous solution containing the ions to be measured, together with a separate, external reference electrode. (NB: this external reference can be completely separate or incorporated in the body of the ISE

to form a Combination Electrode.) The electrochemical circuit is completed by connecting the electrodes to a sensitive milli-volt meter using special lownoise cables and connectors. A potential difference is developed across the ISE membrane when the target ions diffuse through from the high concentration side to the lower concentration side (a detailed description follows later).

General principle of ISE analysis At equilibrium, the membrane potential is mainly dependent on the concentration of the target ion outside the membrane and is described by the Nernst equation (see Glossary at www.nico2000.net). Briefly, the measured voltage is proportional to the Logarithm of the concentration, and the sensitivity of the electrode is expressed as the electrode Slope - in millivolts per decade of concentration. Thus the electrodes can be calibrated by measuring the voltage in solutions containing, for example, 10ppm and 100ppm of the target ion, and the Slope will be the slope of the (straight) calibration line drawn on a graph of mV versus Log concentration. i.e. S = [ mV(100ppm) mV(10ppm) ] / [Log100 Log10]

Thus the slope simply equals the difference in the voltages - since Log100Log10 = 1.

Unknown samples can then be determined by measuring the voltage and plotting the result on the calibration graph.

The exact value of the slope can be used as an indication of the proper functioning of an ISE and the following are typical values:

Monovalent: Cations +55 5, Anions -55 5. Anions -26 3.

Divalent: Cations +26 3,

The

Function

of

the

Reference

Electrode

The membrane potential cannot be measured directly. It needs a Metal-Liquid interface (or a metal-solid solution interface in modern "all-solid-state" ISEs) on both sides of the membrane. Theoretically these could just be metal wires immersed in the solutions. But the electrical potential on many simple metalliquid junctions is not stable; thus the need for a so-called reference system on both sides of the ISE membrane, with a particular metal-liquid interface which is known to have a stable potential. The magnitude of this potential need not be known because it is the same for all measurements of standards and samples and is thus eliminated during the calibration process. Nevertheless, it must be noted that this potential, plus any others that may be generated at any or all of the metal-liquid or liquid-liquid junctions in the circuit, is the value which is seen when the electrodes are immersed in pure water or any other solution which does not contain the target ion. This explains why the measured voltage is not expected to be zero when no target ion is present and also why it is not necessarily always positive when the target ion is present - it all depends on the difference between the ISE voltage and the sum of all the other voltages in the circuit. For example, for a

monovalent positive ion, the voltage could be -25 mV in 10ppm and +30mV in 100ppm (or even -60 mV in 10ppm and -5mV in 100ppm) but this still gives a slope of +55mV per decade of concentration and indicates that the ISE is functioning correctly. Reversing the charges above would describe the situation for a monovalent negative ion. It should be noted here that immersion in pure water should be avoided because it tends to leach out the target ion from the ISE membrane. This, together with the inherent instability of the liquid junction potential of the reference electrode, will cause an unstable voltage to be measured in pure water and require the ISE membrane to be re-equilibrated in a high concentration "pre-conditioning" solution before it will give stable readings again. In practice, the most common reference system is a silver wire coated with solid silver chloride and immersed in a concentrated solution (known as the "filling solution") of potassium chloride saturated with silver chloride. The reference electrode is a half-cell that provides a constant potential which is dependent only on the concentration of chloride ions in the filling solution. The reversible Redox reaction involves the chloride atoms in the solid silver chloride (plated on the silver wire) receiving an electron and the chloride ion going into solution, and vice versa. This electrode will give a constant potential of +205 mV (relative to the Standard Hydrogen Electrode) with a saturated KCl/AgCl solution at 25C.

Electrochemical Processes in the Membrane of an ISE There are various different charge-transfer processes, both outside and inside the membrane for the various different membrane types, and many of these are highly complex and poorly understood in detail. For example, even the apparently simple glass membrane of a pH electrode, which has traditionally been thought of as involving the passage of Hydrogen (H+), or

possibly hydroxonium (H3O+) ions, has recently been shown, by radioactive tagging experiments, to involve only the movement of Sodium (Na+) ions ! The following descriptions of the Calcium and Fluoride ISEs are typical examples of the basic principles of ion-selective membrane processes. Nevertheless, it must be noted that these processes may be far more complex than those described and may involve several layers of ions at each phase junction.

The potential difference across an ion-sensitive membrane is: E = K + (2.303RT/nF)log(a) where K is a constant to account for all other potentials, R is the gas constant, T is temperature, n is the charge of the ion (including the sign), F is Faraday's constant, and a is the activity of the analyte ion. A plot of measured potential versus log(a) will therefore give a straight line. ISEs are susceptible to several interferences. Samples and standards are therefore diluted 1:1 with total ionic strength adjuster and buffer (TISAB). The TISAB consists of 1 M NaCl to adjust the ionic strength, acetic acid/acetate buffer to control pH, and a metal complexing agent.

Question 3: What is BIO-MEMS and MEMS? How lithography can be used to pattern surface?

Answer: Microelectromechanical systems (MEMS) (also written as microelectro-mechanical, MicroElectroMechanical or microelectronic and

microelectromechanical systems) is the technology of very small devices; it merges at the nano-scale into nanoelectromechanical systems (NEMS)

and nanotechnology. MEMS are also referred to as micromachines (in Japan), or micro systems technology MST (in Europe). MEMS are separate and distinct from the hypothetical vision of molecular nanotechnology or molecular electronics. MEMS are made up of components between 1 to 100 micrometres in size (i.e. 0.001 to 0.1 mm), and MEMS devices generally range in size from 20 micrometres (20 millionths of a metre) to a millimetre (i.e. 0.02 to 1.0 mm). They usually consist of a central unit that processes data (the microprocessor) and several components that interact with the outside such as microsensors. At these size scales, the standard constructs of classical physics are not always useful. Because of the large surface area to volume ratio of MEMS, surface effects such

as electrostatics and wettingdominate over volume effects such as inertia or thermal mass.

The potential of very small machines was appreciated before the technology existed that could make themsee, for example, Richard Feynman's famous 1959 lecture There's Plenty of Room at the Bottom. MEMS became practical once they could be fabricated using modified semiconductor device fabrication technologies, normally used to makeelectronics. These include molding and plating, wet etching (KOH, TMAH) and dry etching (RIE and DRIE), electro discharge machining (EDM), and other technologies capable

of manufacturing small devices. An early example of a MEMS device is the resonistor an electromechanical monolithic resonator. Bio-MEMS is an abbreviation of biological microelectromechanical

systems and refers to a special class of microelectromechanical systems (MEMS) where biological matter is manipulated to analyze and measure its activity under any class of scientific study. This class of devices belongs to one of the areas of development based on microtechnology. Among the applications based in Bio-MEMS are: biological and biomedical analysis and measurements and micro total analysis systems (TAS). One of the more popular approaches to Bio-MEMS as of late has been through biomimetics.

Lithography in the MEMS context is typically the transfer of a pattern to a photosensitive material by selective exposure to a radiation source such as light. A photosensitive material is a material that experiences a change in its physical properties when exposed to a radiation source. If we selectively expose a photosensitive material to radiation (e.g. by masking some of the radiation) the pattern of the radiation on the material is transferred to the material exposed, as the properties of the exposed and unexposed regions differs

This discussion will focus on optical lithography, which is simply lithography using a radiation source with wavelength(s) in the visible spectrum. In lithography for micromachining, the photosensitive material used is typically a photoresist (also called resist, other photosensitive polymers are also used). When resist is exposed to a radiation source of a specific a wavelength, the chemical resistance of the resist to developer solution changes. If the resist is placed in a developer solution after selective exposure to a light source, it will etch away one of the two regions (exposed or unexposed). If the exposed material is etched away by the developer and the unexposed region is resilient, the material is considered to be a positive resist (shown in figure 2a). If the exposed material is resilient to the developer and the unexposed region is etched away, it is considered to be a negative resist

Once the pattern has been transferred to another layer, the resist is usually stripped. This is often necessary as the resist may be incompatible with further micromachining steps. It also makes the topography more dramatic, which may hamper further lithography steps.

Question 4: What is electronic transition in a molecule? What are the phenomenon of fluorescence and phosphorescence?

Answer: Photoluminescence in the ultraviolet-visible comprises two similar phenomena: fluorescence and phosphorescence. Molecules have energy levels determined by the molecular orbitals that hold the molecule bound together. In the case of atoms it is the atomic orbitals what determines the energy levels of the electrons. In this section we will concern ourselves with molecular photoluminescence. When discussing the nature of electronic states, it is important to distinguish between the terms electronic state and electronic orbital. An orbital is dened as the volume element in which there is a high probability (99.9%) of nding an electron. It is calculated from a one-electron wave function and is assumed to be independent of all other electrons in the molecule. Electronic states, on the other hand, are concerned with the properties of all the electrons in all the orbitals. In other words, the wave function of an electronic state is a combination of the wave functions of each of the electrons in each of the orbitals of the molecule. Another important distinction is that between exited electronic states and the transition state. Generally a transition state corresponds to a vibrationally exited ground state (i:e: ground state in a strained conguration), where as exited electronic states may contain no excess vibrational energy, but are still much higher in energy than the ground state. In fact a molecule in an exited state is best regarded as a completely new entity, only remotely related to the same molecule in the ground state. An exited state will have a completely dierent electron distribution from the ground state, a dierent geometry, and more than likely will undergo chemical reaction quite dierent from those of the ground state.

Electronic states of organic molecules can be grouped into two broad categories, singlet states and triplet states. A singlet state is one in which all of the electrons in the molecule have their spins paired. Triplet states are those in which one set of electron spin have become unpaired. As will be seen later, triplet states and singlet states significantly in there properties

Absorption of an ultraviolet or visible photon promotes a valence electron from its ground state to an excited state with conservation of the electrons spin. For example, a pair of electrons occupying the same electronic ground state have opposite spins and are said to be in a singlet spin state. Absorbing a photon promotes one of the electrons to a singlet excited state . This phenomenon is called excitation The excited states are not stable and will not stay indefinitely. If we observe a molecule in the excited state, at some random moment it will spontaneously return to the ground state. This return process is called decay, deactivation or relaxation. Under some special conditions, the energy absorbed during the excitation process is released during the relaxation in the form of a photon.

This type of relaxation is called emission. Emission of a photon from a singlet excited state to a singlet ground state, or between any two energy levels with the same spin, is called fluorescence. The probability of a fluorescent transition is very high, and the average lifetime of the electron in the excited state is only 105108 s. Fluorescence, therefore, decays rapidly after the excitation source is removed. In some cases an electron in a singlet excited state is transformed to a triplet excited state in which its spin isno longer paired with that of the ground state. Emission between a triplet excited state and a singlet ground state, or between any two energy levels that differ in their respective spin states, is called phosphorescence. Because the average lifetime for phosphorescence ranges from 104 to 104 s, phosphorescence may continue for some time after removing the excitation source.

History: The use of molecular fluorescence for qualitative analysis and

semiquantitative analysis can be traced to the early to mid-1800s, with more accurate quantitative fluorescence spectroscopy using filters and monochromators for wavelength selection appeared in, respectively, the 1930s and 1950s. Although the discovery of phosphorescenc Microelectromechanical systems (MEMS) (also written as micro-electro-mechanical) methods appearing in the 1920s. Instrumentation for

Question 5: What are the commercial aspects of developing a biosensor?

Answer: Biosensors have been under development for over 35 years and research in this field has become very popular for 15 years. Electrochemical biosensors are the oldest of the breed, yet sensors for only one analyte (glucose) have achieved widespread commercial success at the retail level. This perspective provides some cautions related to expectations for biosensors, the funding of science, and the wide gap between academic and commercial achievements for sensor research. The goal of this commentary is not to arrive at any particular truth, but rather to stimulate lively discussion. The purpose of a concentration sensor is to obtain a number that can be relied on to make a decision. Why are so many people working on developing sensors for analytes where no sensor is needed or desired? Quite often there are alternative methodologies that are much cheaper per data point, much more reliable, much more specific, and already commercially available in stable form at very low prices relative to the high data rate, automation and analyte flexibility available. Examples include chromatography, mass spectrometry, and immunoassays. Some have even gone to Mars, but would not come back. As a cynic, I think it fair to conclude that much biosensor work is done because it can be done and not because it needs to be done. We have a large differentiation between the nonprofit academic world and the tax paying commercial world in attitude about labor versus equipment. In academics, we view labor as cheap and equipment as expensive. In the commercial world, we view labor as expensive and equipment as cheap. The widely mistaken view is that this has something to do with relative salaries in these different worlds. This is not the case except to a very small degree. This perception primarily comes from the fact that labor and physical overhead (buildings, electricity, vacations, medical expense, retirement plans,

etc.) in academia are frequently paid for by taxpayers, students, endowments, and grants, whereas labor in industry must pay taxes. It also derives from the concept of depreciation expense, which chemistry Professors and

government scientists do not always worry about. They view an instrument as costing say $120,000 in one month, whereas a business person sees it as less that $1000 per month spent over 10 years or so. An enormous amount of talented human capital is perhaps wasted by the way grants are awarded, unless we consider academic research only as a means to train students. This leads to a focus on what looks like exceptionally low cost science, such as biosensors, which in reality can be very high cost science because much of it is not needed at all. This is slowly being corrected as we move more in the direction of academic center grants, collaborations and general cost sharing of major instruments. It is, of course, human nature to follow the money to some extent. There is, after all, profit in nonprofit institutions for the indivuals involved. Biosensors are very attractive for academic research. They do, in fact, require relatively little investment in equipment. They are an excellent way for students to become familiar with enzymes, antibodies, polymer films, kinetics, electrochemistry, fiber optics, biological selectivity, data acquisition, and materials science. Academic research involves the three-cornered stool of peer review of science, funding agencies, and politics. This is an a mixture that is inefficient and characterized by various conflicts of interest. While I make observations about it, I do not know a better system and this one has resulted in enormous scientific advances over the last 50 years. Nevertheless, it is often committees of academics who determine the priorities of funding agencies. They mix with politicians who seek dramatic justification for the funding they approve. The most recent rationale is the war on terror, chemical and biological. If a subject can be made to appear flashy and trendy, it has a better chance for success. Biosensors have a

certain cachet. They have been skillfully sold as a priority (most notably in Europe and Japan). This could be viewed as a situation where something is made a priority without careful consideration of the commercial realities. In my view, this is a mistake. Sensors are intended to be practical devices to be used. They employ basic science, but can hardly be justified as curiosity driven research. I suspect that the prefix bio is the blessing and the curse. It sells well to politicians who think it will do something for human health or the environment (and it might). A global problem is the ignorance of the general public for scientific subjects. There is little recognition that funding solid-state physics has aided brain surgery or that funding mathematics has aided medical imaging. ThatMRIis good andNMRis bad provides a nice example of bowing to public ignorance rather than working to correct it. Scientific progress is very inefficient and opportunities from basic science are largely unforeseen. We are now asked to rationalize our work in practical terms. This has merit, but too often it is overdone. Biosensor research is a clear example. There have been great successes (glucose, SPR) (Newman et al., 2002) among a much larger number of attempts.I fully recognize the restraints that faculty must deal with to obtain funding and publish results quickly. We must deal with the reality of the system as it exists today. At the same time, let us recognize the deficiencies and explore ways the process of funding science can be improved.

1. Do enough people want or need to have a sensor for the analyte of interest?

2. Are there existing sensors for the same analyte? Does the new sensor present clearly defined advantages over the older technology, or is it simply a small variation on a well established theme? Unless this new sensor is so

innovative that it is interesting for its conception alone, the following five issues should be addressed. 3. Has the chemical stability of each component used to prepare the sensor been considered? 4. Has the stability of the sensor itself been thoroughly tested both in use and in storage? At what temperatures? Sixmonths stability of a prepared sensor is perhaps the absolute minimum for any practical commercial application. 5. If the sensor is intended for biological samples, has it actually been tested with biological samples and not just aqueous buffers? 6. If a sensor is intended to be used in tissue, have biocompatible components been chosen? Has tissue reaction to the foreign implant been considered both for its effect on the sensor and on the organism? Has a means of sterilization and sterile packaging been considered? 7. Has the dynamic range of the sensor been tested appropriate to the anticipated analyte concentration in real samples?