biosensors and bioelectronics assignment

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

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Page 1: Biosensors and Bioelectronics Assignment

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

Page 2: Biosensors and Bioelectronics Assignment

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.

Page 3: Biosensors and Bioelectronics Assignment

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

Page 4: Biosensors and Bioelectronics Assignment

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.

Page 5: Biosensors and Bioelectronics Assignment

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.

Page 6: Biosensors and Bioelectronics Assignment

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.

Page 7: Biosensors and Bioelectronics Assignment

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) à Cu 2+ (ionic, in solution) +2 e-

on the other electrode a Reduction reaction takes place:

Ag+ (ionic, from solution) + e- à 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

Page 8: Biosensors and Bioelectronics Assignment

to form a Combination Electrode.) The electrochemical circuit is completed by

connecting the electrodes to a sensitive milli-volt meter using special low-

noise 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]

Page 9: Biosensors and Bioelectronics Assignment

Thus the slope simply equals the difference in the voltages - since Log100-

Log10 = 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.      Divalent: Cations +26 ± 3,

Anions -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 metal-

liquid 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

Page 10: Biosensors and Bioelectronics Assignment

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 25°C.

 

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

Page 11: Biosensors and Bioelectronics Assignment

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.

Page 12: Biosensors and Bioelectronics Assignment

Question 3: What is BIO-MEMS and MEMS? How lithography can be

used to pattern surface?

Answer: Microelectromechanical systems (MEMS) (also written as micro-

electro-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).

Page 13: Biosensors and Bioelectronics Assignment

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 them—see, 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.

Page 14: Biosensors and Bioelectronics Assignment

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 

Page 15: Biosensors and Bioelectronics Assignment

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

Page 16: Biosensors and Bioelectronics Assignment

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.

Page 17: Biosensors and Bioelectronics Assignment

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 distin-

guish between the terms electronic state and electronic orbital. An orbital is

de¯ned 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 elec-

trons 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 vibra-

tionally exited ground state (i:e: ground state in a strained con¯guration),

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 mole-

cule 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 di®erent electron distribution from the ground state,

a di®erent geometry, and more than likely will undergo chemical reaction

quite di®erent from those of the ground state.

Page 18: Biosensors and Bioelectronics Assignment

Electronic states of organic molecules can be grouped into two broad cate-

gories, 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 electron’s

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.

Page 19: Biosensors and Bioelectronics Assignment

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 10–5–10–8 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 10–4 to 104 s, phosphorescence may continue for some time

after removing the excitation source.

Page 20: Biosensors and Bioelectronics Assignment

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 methods appearing in the 1920s. Instrumentation for

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)

Page 21: Biosensors and Bioelectronics Assignment

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,

Page 22: Biosensors and Bioelectronics Assignment

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

Page 23: Biosensors and Bioelectronics Assignment

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

Page 24: Biosensors and Bioelectronics Assignment

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?