BIOSENSORS
Applications and Detection
Created by the faculty at
Morehouse College
© 2006-7
ACKNOWLEDGMENTS
The authors would like to thank the following people for their advice and support in the
development of this module:
2
FOREWORD
3
TABLE OF CONTENTS
Acknowledgments
Foreword
Introduction
What is a Biosensor?
2
Types of Biosensors
3
Brief History of Biosensors
Trends of the Past 4 Decades
6
7
Scientific Principles
The Components of a Biosensor
Manufacturing of Biosensors
“Not-So-Common” Applications of Biosensors
References
Resources
Teacher Demonstrations
Demonstration Materials & Equipment Grid
Demonstration #1 Chemiluminescence (Luminol Reaction)
Demonstration #2 Testing Know Concentrations of Peroxide
Lab Activities
Laboratory Materials Equipment Grid
Was it really a poppy seed bagel?
Is it a boy or a girl?
Low carbs or no carbs?
Bioluminescence
Experiment #5 (TBD)
Experiment #6 (TBD)
Design Project
Build-Your-Own Electrochemical Glucose Monitor
Biosensor Quiz
Assessments
Glossary
4
INTRODUCTION
Module Objective:
The objective of this module is to identify and characterize the fundamental properties of
biosensors and to recognize the impact that biosensors have on human societies today.
Key Concepts Covered:





Types of sensors and their associated technologies
The history of biosensors
Basic understanding of signal transduction
The fundamental elements of biosensor devices and their design
Technical definitions including, but not limited to: calibration, selectivity,
sensitivity, reproducibility, detection limits, response time, immobilization
Prerequisites:
A familiarity with the following concepts would be helpful in understanding the information in
contained within this module.




Fundamental knowledge of organic chemistry
An understanding of interparticle forces (e.g. van der Waals)
The nature of the chemical bond (both ionic and covalent bonds)
Basic laboratory technique / laboratory safety precautions
Placement in Curriculum:
This module, as presented, could be used at the college level in either an introductory or
advanced chemistry course, as well as any general physical science course.
5
WHAT IS A BIOSENSOR?
Put simply, a biosensor is an analytical tool. It consists of a biologically active material
used in close conjunction with a device that will convert a biochemical signal into a quantifiable
electrical signal. Compared to other types of sensors, biosensors have many advantages. For
example, biosensors are hailed for both simple and low-cost instrumentation, fast response times,
minimum sample pretreatment, and high sample throughput. Despite these inherent advantages,
relatively few biosensors have been commercialized, though market demands are bringing
biosensors closer to both field testing and commercialization in the United States, Europe, and
Japan. To maintain the current push towards broader implementation of biosensors devices in
the marketplace, research in this area continues to demand the development of both new and
novel materials, new and better analytical techniques, and new and improved biosensors.7-11
In general, a biosensor has two key components: a receptor and a detector. The receptor
is responsible for the selectivity of the sensor. Examples of common receptors include enzymes,
antibodies, nucleic acids, and lipids. The detector, which plays the role of the transducer,
translates the physical or chemical change occurring at the receptor by recognizing the analyte
and relaying it through an electrical signal.
As a rule, then, the detector component of a
Potential Applications of Biosensors: 1-6








Agricultural, horticultural and veterinary analysis
Pollution, water, and microbial contamination analysis
Clinical diagnosis and biomedical applications
Fermentation analysis and control
Industrial gases and liquids
Mining and toxic gases
Explosives and military arena
Flavors, essences and pheromones
6
Figure 1. A biosensor configuration that allows measurement
of a target analyte without using reagents.
biosensor is not selective. Examples transducer elements range from pH and oxygen electrodes
to piezoelectric crystals.
Figure 1 describes a typical biosensor configuration that allows
measurement of a target analyte without using reagents. The device incorporates a biologicalsensing element with a traditional transducer.
The biological-sensing element selectively recognizes a particular biological molecule
through a reaction, specific adsorption, or other physical or chemical process, and the transducer
converts the result of this recognition into a usable signal, which can be quantified. Common
transduction systems are optical, electro-optical, or electrochemical; this variety offers many
opportunities to tailor biosensors for specific applications.1-6 For example, the glucose
concentration in a blood sample can be measured directly by a biosensor (which is made
specifically for glucose measurement) by simply dipping the sensor into the sample.
7
TYPES OF BIOSENSORS
In this section we will discuss the various types of possible biosensors. We will analyze
the working mechanism of each one of them.
Resonant Biosensors
In this type of biosensors an acoustic wave transducer is coupled with antibody (bioelement). When the analyte molecules (antigen) get attached to the membrane, the membrane
mass changes, resulting in a subsequent change in the resonant frequency of the transducer. This
frequency change is measured out [19].
Optical-Detection Biosensors
The output transduced signal that is measured is light signal for this type of biosensors.
The biosensors can be made based on optical diffraction or electrochemilluminence. In optical
diffraction based devices, a silicon wafer is coated with a protein via covalent bonds. The wafer
is exposed to UV light through a photomask and the antibodies made inactivated in the exposed
regions. The diced wafer chips when incubated in analyte antigen-antibody binding is formed in
active regions, thus creating diffraction grating. This grating produces diffraction signal when
illuminated with a light source such as laser. This signal can be measured or can be further
amplified before measuring for improving sensitivity [19].
8
Thermal-Detection Biosensors
This type of biosensors are constructed combining enzymes with temperature sensors.
When the analyte comes in contact with the enzyme, the heat reaction of the enzyme is measured
and is calibrated against the analyte concentration [19].
Ion-Sensitive Biosensors
These are basically semiconductor FETs having ion-sensitive surface [7, 19]. The surface
electrical potential changes when the ions and the semiconductor interact. This potential change
can be measured. The Ion Sensitive Field Effect Transistor (ISFET) can be constructed by
covering the sensor electrode with a polymer layer. This polymer layer is selectively permeable
to analyte ions. The ions diffuse through the polymer layer and in turn cause a change in the FET
surface potential. Fig. 11 shows an ISFET having enzyme enzyme layer Figure 10: Specificity of
Enzymes, Source : [5] placed on it; also called ENFET (Enzyme Field Effect Transistor) [7].
This type of biosensor are primarily used for pH detection.
Electrochemical Biosensors
Electrochemical biosensors are mainly used for detection of hybridised DNA, DNAbinding drugs, glucose concentration, etc. The underlying principle for this class of biosensors is
that many chemical reactions produce or consume ions or electrons which in turn cause some
change in the electrical properties of the solution which can be sensed out and used as measuring
parameter [1, 19]. The electrochemical biosensors can be classified based on the measuring
electrical parameters as : (1) conductimetric, (2) amperometric and (3) potentiometric [1]. A
comparative discussion of these:
9
Conductimetric Biosensors
The measured parameter is the electrical conductance/resistance of the solution. When
ectrochemical reactions produce ions or electrons the overall conductivity/resistivity of the
solution changes. This change is measured and calibrated to a proper scale. Conductance
measurement has relatively low sensitivity. The electric field generated using sinusoidal voltage
(AC) which helps in minimizing undesirable effects such as Faradaic process, double layer
charging and concentration polarization [1].
Amperometric Biosensors
This high sensitivity biosensor cab detect electroactive species present in biological test
samples. Since the biological test samples may not be intrinsically electro-active, enzymes
needed to catalyze production of radio-active species. In this case, the measured parameter is
current [1].
Potentiometric biosensors
In this type of sensors the measured parameter is oxidation/reduction potential (of an
electrochemical reaction). The working principle of that when a ramp voltage is applied to an
electrode in solution the current flow occurs because of electrochemical reaction. The voltage at
which these reactions occurs indicate a particular reaction and particular species [1].

Piezoelectric Sensors
In this mode, sensing molecules are attached to a piezoelectric surface – a mass to
frequency transducer – in which interactions between the analyte and the sensing
molecules set up mechanical vibrations that can be translated into an electrical
signal proportional to the amount of the analyte (Figure 3). Example of such a
sensor is quartz crystal micro or nano balance.
10
Potentiometric biosensors (Continued)

Optical Sensors
In optical biosensors, the optical fibers allow detection of analytes on the basis of
absorption, fluorescence or light scattering. Since they are non-electrical, optical
biosensors have the advantages of lending themselves to in vivo applications and
allowing multiple analytes to be detected by using different monitoring
wavelengths. The versatility of fiber optics probes is due to their capacity to
transmit signals that reports on changes in wavelength, wave propagation, time,
intensity, distribution of the spectrum, or polarity of the light. In general,
acquisition of the signal from these devices is accomplished through flexible
cables, which can transmit light to the biological component. Optrodes use fiber
optics for performing optical measurement away from the measuring locations
(e.g., intra–arterial determination using FIA systems). A powerful and sensitive
analytical methodology has been constructed based on the luciferin/luciferase
bioluminescence reaction.

Electrochemical Sensors
In this configuration, sensing molecules are either coated onto or covalently
bonded to a probe surface. A membrane holds the sensing molecules in place,
excluding interfering species from the analyte solution. The sensing molecules
react specifically with compounds to be detected, sparking an electrical signal
proportional to the concentration of the analyte. The bio-molecules may also
respond to an entire class of compounds such as opiates and their metabolites. The
most common detection method for electrochemical biosensors involves
measurement of current, voltage, conductance, capacitance and impedance.
11
A BRIEF HISTORY OF BIOSENSORS
His invention of the oxygen electrode, and its
subsequent modification with enzymes, allow us to
clearly identify Professor Leland C. Clark, Jr. as the
father of the biosensor concept.
In 1956, Clark
published his definitive paper on the oxygen
Photo Credit Needed
electrode. Based on this experience and addressing
Figure 2. Professor Leland C. Clark
his desire to expand the range of analytes that could
be measured in the body, he made a landmark address in 1962 at a New York Academy of
Sciences symposium.
In this he described "how to make electrochemical sensors (pH,
polarographic, potentiometric or conductometric) more intelligent" by adding "enzyme
transducers as membrane enclosed sandwiches." The concept was illustrated by an experiment in
which glucose oxidase was entrapped at a Clark oxygen electrode using a dialysis membrane.
The
decrease
in
measured
oxygen
concentration was proportional to glucose
concentration. In the published paper, Clark
and
Lyons
coined
the
term
enzyme
electrode, which many reviewers have
mistakenly attributed to Updike and Hicks,
who expanded on the experimental detail
necessary to build functional enzyme
electrodes for glucose.
Figure 3. Taken from Professor Clark’s notebook
12
The biosensor, essentially invented by Clark, was the basis of numerous variations on the
basic design and many other (oxidase) enzymes were immobilised by various workers as a result.
Indeed, Clark's basic design was so successful that many research biosensors and at least one
commercial biosensor are still produced using the original concept of oxygen measurement.
However, nowadays the preferred alternative is to measure hydrogen peroxide. Most notable of
the commercially-available biosensors today is probably the range of biosensors sold by the
Yellow Springs Instrument Company (YSI, Ohio, USA). Their glucose biosensor was
successfully launched commercially in 1975 and was based on the amperometric detection of
hydrogen peroxide. This was the first of many biosensor-based laboratory analysers to be built by
companies around the world.
Guilbault and Montalvo were the first to detail a potentiometric enzyme electrode. They
described a urea sensor based on urease immobilised at an ammonium-selective liquid membrane
electrode. The use of thermal transducers for biosensors was proposed in 1974 and the new
devices were christened thermal enzyme probes and enzyme thermistors, respectively. Lubbers
and Opitz coined the term optode in 1975 to describe a fibre-optic sensor with immobilised
indicator to measure carbon dioxide or oxygen. They extended the concept to make an optical
biosensor for alcohol by immobilising alcohol oxidase on the end of a fibre-optic oxygen sensor.
Commercial optodes are now showing excellent performance for in vivo measurement of pH,
pCO2 and pO2, but enzyme optodes are not yet widely available.
The biosensor took a further fresh evolutionary route in 1975, when Divie suggested that
bacteria could be harnessed as the biological element in microbial electrodes for the
measurement of alcohol. This paper marked the beginning of a major research effort in Japan and
elsewhere into biotechnological and environmental applications of biosensors.
13
In 1976, Clemens et al. incorporated an electrochemical glucose biosensor in a bedside
artificial pancreas and this was later marketed by Miles (Elkhart) as the Biostator. Although the
Biostator is no longer commercially available, a new semi-continuous catheter-based blood
glucose analyser has recently been introduced by VIA Medical (San Diego). In the same year, La
Roche (Switzerland) introduced the Lactate Analyser LA 640 in which the soluble mediator,
hexacyanoferrate, was used to shuttle electrons from lactate dehydrogenase to an electrode.
Although this was not a commercial success at the time, it turned out in retrospect to be an
important forerunner of a new generation of mediated biosensors and of lactate analysers for
sports and clinical applications. A major advance in the in vivo application of glucose biosensors
was reported by Shichiri et al. who described the first needle-type enzyme electrode for
subcutaneous implantation in 1982. Companies are still pursuing this possibility, but no device
for general use is available as yet.
The idea of building direct immunosensors by fixing antibodies to a piezoelectric or
potentiometric transducer had been explored since the early seventies, but it was a paper by
Liedberg et al. that was to pave the way for commercial success. They described the use of
surface plasmon resonance to monitor affinity reactions in real time. The BIAcore (Pharmacia,
Sweden) launched in 1990 is based on this technology.
It was during the 1980s, however, that large-scale commercial success was first achieved.
YSI had built up a steady and thriving business, but it was not in the same league as the success
which had been predicted and, indeed, widely expected. The basic problem lay largely with the
cost of producing the biosensors of the time, which made them uncompetitive with the other
technologies widely used in the massive rapid testing sector. It was within this sector that the
hopes were pinned, since it was (and still is) a huge market.
14
In 1984, a much cited paper on the use of ferrocene and its derivatives as an immobilised
mediator for use with oxidoreductases was published. These were crucial components in the
construction of inexpensive enzyme electrodes, and formed the basis for the screen-printed
enzyme electrodes launched by MediSense (Cambridge, USA) in 1987 with a pen-sized meter for
home blood-glucose monitoring. The electronics have since been redesigned into popular creditcard and computer-mouse style formats, and MediSense's sales showed exponential growth,
reaching $175 million per annum by 1996, when they were purchased by Abbott. Boehringer
Mannheim (now Roche Diagnostics), Bayer and LifeScan now have competing mediated
biosensors and the combined sales of the four companies dominate the world market for
biosensors and are rapidly displacing conventional reflectance photometry technology for home
diagnostics.
Academic journals now contain descriptions of a wide variety of devices exploiting
enzymes, nucleic acids, cell receptors, antibodies and intact cells, in combination with
electrochemical, optical, piezoelectric and thermometric transducers. Within each permutation
lies a myriad of alternative transduction strategies and each approach can be applied to numerous
analytical problems in healthcare, food and drink, the process industries, environmental
monitoring and defence and security.
Some defining events in the history of biosensor development show that the 1980s was a
very inventive decade, with commercialization being the theme of the 1990s. There appears to be
no sign of the latter theme changing as we enter the new millennium. This demonstrates that, in
some areas, biosensors have become a mature technology. It must be remembered, however, that
this commercial success and maturity is limited to a small number of applications and that it
15
came as a result of a great deal of research and development and has only really taken place
where market size/share justified significant financial investment.
16
TRENDS OVER THE PAST FOUR DECADES (TIMELINE?)
17
SCIENTIFIC PRINCIPLES
In very nonspecific terms, a sensor is any device that can be used to measure the
presence or absence of an object. For example, a sensor can be used to detect the presence of
an object as large as a person walking into a supermarket equipped with automatic doorways.
A sensor can also be used to detect the presence of objects as small as molecules. In this
way, a carbon monoxide detector can be used to denote the presence, at levels of harm to
humans, of the gas, carbon monoxide (Molecular formula CO). For our purposes here, a
biosensor is a device that combines a biological component (i.e. enzyme, antibody, DNA)
with a physicochemical detector component, the combination of which can be used
specifically for the detection of any object, large or small. Despite its narrow definition, the
biosensor is becoming increasingly important as a tool in everyday life, but especially in the
field of medicine. In fact, the most widespread example of a commercial biosensor is the
blood glucose monitor, which uses an enzyme to break blood glucose down. In doing so it
transfers an electron to an electrode and this is converted into a measure of blood glucose
concentration. The high market demand for such sensors has fueled development of
associated sensor technologies. Recently, arrays of many different detector molecules have
been applied in so called electronic nose devices, where the pattern of response from the
detectors is used to fingerprint a substance. A canary in a cage, as used by miners to warn of
gas could be considered a biosensor. Many of today's biosensor applications are similar, in
that they use organisms which respond to toxic substances at a much lower level than us to
warn us of their presence. Such devices can be used both in environmental monitoring and in
water treatment facilities. Below we learn some of the scientific principles involved in the
preparation and utilization of biosensors.
The Components of a Biosensor
A basic biosensor consists of at least three (3) parts:



A sensitive biological element
A transducer
A detector element
To gain a greater appreciation/understanding of each of these three components, let’s explore
each of them in some detail.
Biorecognition Systems
Biocatalysis-based biosensors for environmental applications depend universally on the
use of enzymes. These enzyme-based biosensors primarily rely on two operational mechanisms.
The first mechanism involves the catalytic transformation of a pollutant (typically from a nondetectable form to a detectable form). The second mechanism involves the detection of pollutants
that inhibit or mediate the enzyme's activity.
For environmental applications, the first mechanism involving catalytic transformation of
the pollutant, shows certain advantages and limitations. They are simple in design and operation.
Examples of this format include the use of tyrosinase for the detection of phenols11-12 and the use
of organophosphate hydrolase for the detection of organophosphorus pesticides.13 These
biosensors can be configured to operate continuously and reversibly. They can also be configured
such that the only required reagent is the analyte of interest
Inherent limitations for this type of biosensor are primarily those imposed by the nature of
the enzyme itself and include the limited number of environmental pollutants which happen to be
substrates for the enzyme and the relatively high detection limits (compared to those required by
many environmental monitoring applications) for environmental pollutants. The detection limits
19
for these sensors are determined by the enzyme's catalytic properties and are defined by Km and
Vmax values. Biosensor formats have been devised which substantially reduce these inherent
limitations. For example, in the case of the tyrosinase enzyme electrode, catalytic cycling of the
enzyme intermediate between the quinone and catachol oxidation states can significantly amplify
the biosensor response. This results in lower detection limits for phenols than expected from Km
and Vmax values.12 Mechanisms for the tyrosinase biosensors involve the detection of phenols
either through the electrochemical reduction of quinone intermediates or through oxygen
consumption (O2 is a co-substrate) using a Clark electrode.14
In another example, a biosensor was designed to increase both sensitivity and the range of
substrates typically measured using the tyrosinase enzyme electrode. With this biosensor, a wide
range of chlorinated phenols are detected as oxidation-reduction mediators in a glucose oxidase
electrode system.15 This is accomplished by chemically oxidizing the chlorophenols which then
cycle between the quinone and hydroquinone oxidation states. In this case, the quinone acts as an
electron shuttle for the glucose oxidase-catalyzed oxidation of glucose. This configuration results
in low nM detection limits for a number of chlorinated phenols.
The other primary mechanism used in biocatalytic biosensors for environmental
applications is inhibition of the enzyme by the pollutant. Inherent advantages for these formats
involve the larger number of environmental pollutants, usually of a particular chemical class, that
inhibit the enzyme and the low concentrations needed to affect the enzyme activity.
Detection limits for biosensors based on irreversible inhibitors are usually within the
range required for a variety of environmental applications.16 For example, detection limits for
cholinesterase biosensors are reported to be in the µg/l to ng/l range for compounds such as
aldicarb, carbaryl, carbofuran, and dichlovos.17
20
There are several inherent limitations for biosensors based on enzyme inhibition. In
addition to the analyte of interest, these assay formats require the use of substrates and in some
cases, cofactors and mediators. Further, for a number of these assays, pollutants must be
chemically oxidized to metabolic intermediates to show maximum sensitivities. The irreversible
nature of many analyte-enzyme interactions that result in increased sensitivity also renders the
biosensor inactive after a single measurement. This may not be a problem, however, for those
systems that can be reactivated or that employ disposable sensing elements. Another potential
limitation to this type of biosensor mechanism involves the sometimes diverse classes of
pollutants that inhibit a specific enzyme. In most circumstances, this would not be expected to
present problems. In some cases e.g., the co-contamination of environmental samples with
organics and heavy metals, such interferences may lead to unexpected results.
The interface of these devices to the contaminated matrix is critical to the successful
application of biocatalytic-based biosensors, particularly for in situ applications. These matrices
may range from pristine drinking water to highly contaminated industrial sludges. Although a
considerable amount of work has been done with respect to use of biosensors in biological
matrices (such as blood and fermentation media), little work has focused on the development of
membrane barriers for the direct sampling of groundwater or pore water in sludges and saturated
sediments.
Bioaffinity-based biosensors for environmental applications primarily depend on the use
of antibodies because of the availability of monoclonal and polyclonal antibodies directed toward
a wide range of environmental pollutants as well as the relative affinity and selectivity of these
recognition proteins for a specific compound or closely related groups of compounds.18-19 In
addition to the wide range of antibodies directed toward different environmental pollutants, a
21
range of assay formats has also been demonstrated with virtually every type of reported signal
transducer.
Because most small molecular weight organic pollutants in the environment have few
distinguishing optical or electrochemical characteristics, the detection of stoichiometric binding
of these compounds to antibodies is typically accomplished using competitive binding assay
formats. Competitive immunosensor formats rely on the use of an antigen-tracer which competes
with the analyte for a limited number of antibody binding sites. For affinity-based biosensors,
this is typically accomplished in one of several ways. In one type of format, antigen-tracer
competes with analyte for immobilized antibody binding sites (Figure 2). This format is often
used in fluorescence-based systems. In another commonly used format, the antigen is
immobilized to the signal transducer (operationally becoming the analyte-tracer) while free
binding sites on the antibody, which has been previously exposed to the analyte, bind to the
surface-immobilized antigen (Figure 2). Because the antibody is a relatively large molecule, its
binding to the surface can be detected by signal transduction methods such as surface plasmon
resonance, acoustic systems, and optical systems that measure changes in refractive index and
thus, do not require an optical tag. The third commonly used format requires an indirect
competitive assay and relies on the use of an enzyme-labeled antigen-tracer (Figure 2). In this
format, the assay is completed in two steps. First, the enzyme-tracer competes with analyte for
immobilized antibody binding sites. Then, after removal of the unbound tracer (by means of a
washing step), a non-detectable substrate is catalytically converted to an electrochemically or
optically detectable product. This assay format is used almost universally with electrochemical
signal transduction.
22
Immunosensors are becoming the most frequently reported type of biosensor for
environmental applications.19 Rather than expanding the envelope of fundamental understanding,
however, immunosensors (and biosensors in general) for the most part represent technological
advances for existing bioanalytical assays. It is important to address the issue of whether or not a
biosensor shows the potential to improve the characteristics of a particular assay with respect to
known or anticipated applications. Because of the wide variety of scientifically established and
commercially available immunoassays (test kits), this is particularly relevant in the area of
immunosensors.
Although there are a variety of ways to group immunosensors based on signal transducers
or format considerations, one functionally useful discriminator involves classification based on
reusable/regenerable or disposable format configurations. Because immunosensors (particularly
those using disposable formats) are most closely related to immunoassay test kit technology,
issues which become important for this comparison are more practical in nature and involve the
potential for multi-analyte capability, format versatility, assay time, assay sensitivity, system cost,
assay cost, shelf life, reproducibility, ruggedness, etc.
In contrast to the disposable formats, the multi-use immunosensors, which can be
recharged or regenerated, offer certain advantages, particularly for use as detectors for
chromatographic and flow injection analysis systems.20 For example, continuous flow and fiber
optic immunosensors that have been reported for detection of explosive residues in ground water
use multi-assay formats and perform comparably to chemical and immunoassay test kit methods.
Initial cost estimates suggest that for a limited number of assays (e.g., < 400), the test kits are
more cost-effective. For long-term monitoring projects, however, the biosensor methods which
23
require an initial instrument investment but offer lower cost per assay would be more costeffective.21
Nucleic acid-based affinity biosensors which may potentially be developed for
environmental applications have recently been reported. Application areas for these biosensors
include the detection of chemically induced DNA damage22 and the detection of microorganisms
through the hybridization of species-specific sequences of DNA.23 Although results from these
reports are still preliminary, they appear to offer promising avenues for further investigation.
Microorganism-based biosensors tend to use one of three primary mechanisms. For the
first mechanism, the pollutant is a respiratory substrate. Biosensors that detect biodegradable
organic compounds measured as biological oxygen demand (BOD) are the most widely reported
of the microorganism-based biosensors using this mechanism. Several of these devices are
commercially available from vendors including: Nisshin Electric Co. Ltd., Tokyo; Autoteam,
GmbH, Berlin; Prufgeratewrk, Medingen GmbH, Dresden; and Dr. Lange, GmbH, Berlin. The
use of these devices has been incorporated into industrial standard methods in Japan.24-25
Biological oxygen demand is widely used as an indicator of the amount of biodegradable
organic compounds found in industrial waste water. The standard procedure (termed BOD5)
involves a 5 day incubation of the environmental or industrial water sample with an inoculum of
microorganisms typically present in the waste treatment system to yield an endpoint
measurement for oxygen consumption.25 The use of indicator microorganisms interfaced to
signal transducers allows the measurement of the rate of organic compound metabolism rather
than an endpoint; thus, data can be acquired in a significantly shorter time frame (e.g., 15 min to
1 hr), rendering this technology highly advantageous for process control applications. Although
these biosensors appear to work well for in situ monitoring of industrial waste waters that result
24
in high BOD values, they currently require improvements in several areas. The primary
limitations for these methods involve the variability encountered in calibration of the biosensor
response to BOD5 values. This arises from the fact that specific microbial species (used in
biosensors) have characteristic substrate spectra which may or may not correspond well with the
spectrum of compounds present in the sample. Additional variability results from the presence of
polymers (such as protein, starch, and lipid) which must be broken down to monomers before
they can be metabolized; this changes the linearity of the response over time and can make
interpretation of the result problematic.
Current progress on these technologies involves several areas. These areas include: the
acid-induced breakdown of biological polymers prior to biosensor analysis, the selection of
microorganisms with broad substrate spectra, and the introduction of novel transduction
techniques. In addition, a recent report exploring the feasibility of disposable BOD sensors
suggests considerable promise for advancement in this area.26
Another mechanism used for microorganism-based biosensors involves the inhibition of
respiration by the analyte of interest. Microbial respiration and its inhibition by various
environmental pollutants have been measured both optically27-28 and electrochemically.29
Inherent advantages of these techniques apply primarily to the use of microorganisms as
compared to isolated enzymes.24 Microorganism-based biosensors are relatively inexpensive to
construct and can operate over a wide range of pH and temperature. General limitations involve
the long assay times including the initial response and return to baseline. These characteristics
are primarily determined by the cellular diffusion characteristics that can be modified by using
genetically engineered microorganisms. The broad specificity of these biosensors to
environmental toxins may be an advantage or disadvantage depending on the intended
25
application. In this respect, these devices might be most applicable for general toxicity screening
or in situations where the toxic compounds are well defined, or where there is a desire to measure
total toxicity through a common mode of action.
Biosensors have also been developed using genetically engineered microorganisms
(GEMs) that recognize and report the presence of specific environmental pollutants. The
microorganisms used in these biosensors are typically produced with a constructed plasmid in
which genes that code for luciferase or -galactosidase are placed under the control of a promoter
that recognizes the analyte of interest. Because the organism's biological recognition system is
linked to the reporting system, the presence of the analyte results in the synthesis of inducible
enzymes which then catalyze reactions resulting in the production of detectable products. With
respect to environmental applications, the primary disadvantage for this type of biosensor is the
limited number of GEMs which have been constructed to respond to specific environmental
pollutants. Nevertheless, reported advances include the development of GEMs involving a
variety of bioremediation pathways and mechanisms. GEMs that could report both the metabolic
condition of the relevant microorganisms as well as the rates of pollutant breakdown could be
very useful.
Physico-chemical transducers
Biosense Detectors
26
Manufacturing of Biosensors
Biosensor
•Specific•Robust
•Cheap
•Portable
•Simple
•Easy to use
1) Low incentive to use (effects are long term)
2) Cost high
3) What do you do with the information?
4) Learnt nothing from the Glucose industries development.
Integration
•Sensor systems
•Integration of several steps
•Multiple analytes
•Expensive
•Lab environment
•Trained users
Miniaturisation
•Making integrated systems smaller
•Mass production
•Cheaper components
27
Common Applications of Biosensors
The driving force behind advances in sensor technology came from health care area, where it is
now generally recognized that measurements of blood gases, ions and metabolites are often
essential and allow a better estimation of the metabolic state of a patient. In intensive care units
for example, patients frequently show rapid variations in biochemical levels that require an
urgent remedial action. Also, in less severe patient handling, more successful treatment can be
achieved by obtaining instant assays. At present, the list of the most commonly required instant
analyses is not extensive. In practice, these assays are performed by analytical laboratories,
where discrete samples are analyzed, frequently using the more traditional analytical techniques.
Many of the technologies first made useful in the hospital setting have now been commercialized
into units for home use. A few examples of such devices follows:
Blood Glucose Monitors
ClearBlue Home Pregnancy Test
Cholesterol monitoring
28
“Not-So-Common” Applications of Biosensors
Remote Sensing Of Airborne Bacteria
Universal Detection Technology (UDTT) has licensed a spore detection technology from
NASA's Jet Propulsion Laboratory (JPL) and has developed a first of its kind real-time
continuous detection device capable of identifying abnormal levels of bacterial spores in the air,
an indication of a possible anthrax attack.
Other “Not-So-Common” Uses for Biosensors






Remote detection of pathogens
Determining levels of toxic substances before and after bioremediation
Detection and determining of organophosphate
Routine analytical measurement of folic acid, biotin, vitamin B12 and
pantothenic acid as an alternative to microbiological assay
Determination of drug residues in food, such as antibiotics and growth
promoters, particularly meat and honey
Drug discovery and evaluation of biological activity of new compounds
29
Activity 4(JW)
Bioluminescence
Background
Bioluminescence (bī´´ōl´´mĭnĕs´ns), the production of light by living organisms. Organisms
that are bioluminescent include certain fungi and bacteria that emit light continuously. The
dinoflagellates, a group of marine algae, produce light only when disturbed. Bioluminescent
animals include such organisms as ctenophores, annelid worms, mollusks, insects such as
fireflies, and fish. The production of light in bioluminescent organisms results from the
conversion of chemical energy to light energy. In fireflies, one type of a group of substances
known collectively as luciferin combines with oxygen to form an oxyluciferin in an excited state,
which quickly decays, emitting light as it does. The reaction is mediated by an enzyme,
luciferase, which is normally bound to ATP (see adenosine triphosphate) in an inactive form.
When the signal for the specialized bioluminescent cells to flash is receive, the luciferase is
liberated from the ATP, causes the luciferin to oxidize, and then somehow recombines with ATP.
Different organisms produce different bioluminescent substances. Bioluminescent fish are
common in ocean depths; the light probably aids in species recognition in the darkness. Other
animals seem to use luminescence in courtship and mating and to divert predators or attract prey.
Objectives
Upon completion of this exercise, students should
• Know aseptic techniques.
• Understand some of the biochemistry of bioluminescence.
• Be familiar with some of the natural history of Vibrio fischeri.
Time Requirements
This kit can be completed in two or three consecutive class periods.
Materials
Materials for this kit are sufficient for a class of 30 students working in 6
groups of 5. The materials have been supplied for the sole use of this lab
exercise. Carolina Biological Supply Company disclaims all responsibility
for any other use of these materials.
Included in the kit
Vibrio fischeri culture tube
6 metal inoculating loops
7 bottles photobacterium agar
40 sterile disposable petri dishes
autoclave disposal bag
Needed, but not provided
30
marking pen
a disinfectant such as a 10% bleach solution or 70% alcohol
Bunsen burner or alcohol lamp
water bath or pan of boiling water
Discussion
A few species of bacteria exhibit bioluminescence, the extraordinary ability of living organisms
to produce light. Many of these bacteria live in marine habitats, some as parasites of saltwater
fish. Most bioluminescent bacteria give off a blue-green light, while some give off a yellow light.
None of these species are harmful. Vibrio fischeri is a bioluminescent marine bacterium that
commonly inhabits fish. This bacterium has a Gram-negative cell wall, is motile by means of
flagella, and the cell shape is a curved rod. It requires some salt in the medium in order to grow,
and it is cultured on photobacterium agar. Photobacterium agar provides the nutrients necessary
for good growth and bioluminescence by Vibrio fischeri. Various species of fish, insects, fungi,
and microbes can express bioluminescence. Bacteria of the Vibrio genus have been found in
association with squid, nematodes, microscopic organisms, and with insects that feed on
nematodes. Some gather in the pockets of fish in a symbiotic relationship. The fish are luminous
because of the photobacteria. The purpose, if any, of the many various vibrio pigments is still a
mystery. However, researchers do know that the occurrence of luminescence is due to the
bacterial cells’ electron transport system, involving an aldehyde, an enzyme, oxygen, and an
altered form of riboflavin. Though Vibrio isolates
are facultative anaerobes, they are bioluminescent only when O2 is present. Several components
are needed for bacterial bioluminescence: the enzyme luciferase, a long-chain aliphatic aldehyde,
flavin mononucleotide (FMN), and O2. The primary electron donor is NADH, and the electrons
pass through FMN to the luciferase. The reaction can be expressed as FMNH2 + O2 + RCHO
➝FMN + RCOOH + H2O + light (Luciferase)
The light-generating system constitutes a bypass route for shunting electrons from MNH2 to O2,
without involving other electron carriers such as quinones and cytochromes. The enzyme
luciferase shows a unique kind of regulatory synthesis called autoinduction. The bioluminous
bacteria produce a specific substance, the autoinducer, which accumulates in the culture medium
during growth, and when the amount of this substance has reached a critical level, induction of
the enzyme occurs. The autoinducer in V. fischeri has been identified as N-β ketocaproylhomoserine lactone. Thus cultures of bioluminous bacteria at low cell density are not
bioluminous, but become bioluminous when growth reaches a sufficiently high density so that
the autoinducer can accumulate and function. Because of the autoinduction phenomenon, it is
obvious that the free-living bioluminescent bacteria in seawater will not be bioluminescent
because the autoinducer could not accumulate, and bioluminescence only develops when
conditions are favorable for the development of high population densities. Although it is not
clear why bioluminescence is density dependent in free-living bacteria, in symbiotic strains of
bioluminescent bacteria, the rationale for density-dependent bioluminescence is clear:
bioluminescence only develops when sufficiently high population densities are reached in the
light organ of the fish to allow a visible flash of light.
Much new information about bioluminescence has emerged from studies of the genetics of this
process. Several lux operons have been identified in bioluminescent Vibrio species, and the key
structural genes cloned and sequenced. The luxA and the luxB genes code for the a and b
31
subunits, respectively, of bacterial luciferase. The luxC, luxD, and the luxE genes code for
polypeptides that function in the bioluminescence reaction and in
the generation and activation of fatty acids for the bioluminescence system. Light-emitting strains
of Escherichia coli have been constructed by inserting lux genes cloned from a Vibrio species
into cells of E. coli and then placing their expression under the control of specific and easily
manipulable E. coli promoters. This has served to accelerate the pace of research on the
regulatory aspects of bioluminescence because many E.
coli genetic tools can be employed to probe aspects of lux genes’ control. In addition, cloned lux
genes have stimulated biotechnological exploitation of bioluminescence in clinical diagnostics
and other biomedical fields.
Procedure
Note: The culture of Vibrio fischeri you received will probably have passed its prime of
bioluminescence. Therefore, it is necessary to transfer the bacteria upon receipt of this kit to
successfully demonstrate their bioluminescent ability. However, the culture itself will remain
viable for at least 2 weeks at room temperature storage.
Part 1. Medium Preparation
1. Slightly loosen the bottle caps and set the bottles in a boiling water bath (or pan of boiling
water) to melt the agar. Make sure the water level is even with the agar level. Using an oven mitt,
swirl the bottles gently to be sure that all of the agar has melted.
2. Cool the agar to 45°C (the bottle should feel comfortably hot to the touch) by cooling the
water bath to that temperature or by letting the bottles sit for several minutes at room
temperature.
3. Wipe down the work surfaces with a disinfectant such as 70% alcohol or 10% bleach solution.
Wash your hands.
4. Swirl the bottle of agar, remove the cap, flame the mouth over a Bunsen burner for a few
seconds, and begin pouring five petri dishes:
• Lift the lid of a dish just enough to pour in the molten agar.
• Pour just enough agar to cover the bottom of the petri dish.
• Replace the lid immediately to prevent contamination.
5. Repeat step 4 with the other photobacterium agar bottles, using one bottle for five dishes.
6. Allow about 2 hours for the agar to solidify. The agar dishes may be stored for up to 1 week at
room temperature or can be stored in the refrigerator for up to 3 weeks.
Part 2. Preparing Vibrio fischeri Stock Cultures
32
1. Set aside 6 (1 for each student group) of the photobacterium agar dishes for inoculation with
the original Vibrio fischeri tube culture and store the other 34 of the 40 dishes for the students to
use for their transfers.
2. Before transferring Vibrio fischeri, observe the culture tube in total darkness. Allow at least 5
minutes for your eyes to adjust to the dark in a room with no light leaks. (Note: A room with the
lights out and shades drawn does not provide enough darkness to observe
bioluminescence). You may or may not observe any growth depending on the bioluminescent
phase the bacteria has reached and the time that has elapsed since the initial transfer. Locate an
area in the classroom where you and the students can incubate the cultures in an undisturbed,
totally dark area (e.g., a cleaned, disinfected cabinet or covered box at room temperature.
3. Transfer from the bioluminescent areas, if any were apparent. To transfer bacteria, place the
stock tube in the palm of one hand. With the other hand, flame an inoculating loop until it turns
red (about 1 min). Remove the cap with the “loop” hand and flame the mouth of the tube. Insert
the cooled loop into the stock tube and pick up a loopful of the
culture. Flame the mouth of the tube, replace the cap, and set the tube aside. Gently raise the
cover of the petri dish. Touch the inoculating loop to the top of the dish and streak from side to
side all the way to the bottom edge. Lower the cover and flame the loop between each transfer.
Flame the mouth of the tube and replace the cap.
4. Repeat for the remaining dishes (each of the six plates of transferred culture needs to provide
enough growth for 5 students to obtain a sample for their secret message).
5. After inoculating the six plates, incubate cultures at room temperature for 18 to 24 hours in the
dark.
6. When checking the cultures for bioluminescence, again allow at least 5 minutes for your eyes
to adjust to the dark in a room with no light leaks. Note: A room with the lights out and shades
drawn does not provide enough darkness to observe bioluminescence. Bioluminescence is most
vivid 18 to 24 hours after subculture. Thereafter, the amount of light emitted decreases inversely
with time elapsed.
7. To retain a bioluminescent culture, subculture from bioluminescent areas every 3 to 4 days. If
this is not practical, a new subculture should be made 18 to 24 hours before any observations are
to be made.
Part 3. Student Activity
1. Divide into six groups of five people. Each group needs the following materials: a Vibrio
fischeri culture, five photobacterium agar plates, a metal inoculating loop, and a gas (Bunsen)
burner or alcohol lamp.
33
2. Disinfect the work surface with a disinfectant, such as 70% ethanol or 10% sodium
hypochlorite (see above for preparation). Wash your hands.
3. Each student should label the underside of a petri dish with initials and the date.
4. Before transferring Vibrio fischeri, each group should observe the culture plate in total
darkness. Allow at least 5 minutes for your eyes to adjust to the dark in a room with no light
leaks. Note: A room with the lights out and shades drawn does not provide enough darkness to
observe bioluminescence. Bioluminescence is most vivid 18 to 24 hours after subculture.
Thereafter, the amount of light emitted decreases inversely with time elapsed.
5. Light the burner as directed by your instructor.
6. Begin the transfer procedure one at a time. Use aseptic technique, and flame-sterilize the wire
end of the metal inoculating loop (heat to redness).
7. After the inoculating loop has cooled, use aseptic technique and obtain a small portion of the
bacteria from the prepared culture dish and quickly replace its cover.
8. Inoculate a dish by printing a secret message with the inoculating loop across the surface of the
medium (do not penetrate or stab the surface of the agar, or the message may become
unreadable). Replace the cover.
9. Flame-sterilize the inoculating loop to remove any remaining bacteria. The next student should
begin at step 6 and continue until all transfers are complete.
10. Invert all petri dishes and incubate at room temperature (20–26° C) for 18 to 24 hours in total
darkness.
11. After 18–24 hours, locate your petri dish and observe the secret message. Observe the culture
in total darkness. Allow at least 5 minutes for your eyes to adjust to the dark.
12. Bioluminescence is most vivid at 18–24 hours and will then begin to decrease in light
intensity. At or around hour 96 (the third day), there may be little or no bioluminescence. Try to
observe your culture as many times as possible to observe this phenomenon.
13. Observe the message in a lighted area and note the difference.
14. After all observations have been made, dispose of the culture dishes in
a designated autoclave disposal bag.
(More Background if needed)
Bioluminescence is simply light produced by a chemical reaction which originates in an
organism. It can be expected anytime and in any region or depth in the sea. Its most common
occurrence to the sailor is in the often brilliantly luminescent bow wave or wake of a surface
ship. In these instances the causal organisms are almost always dinoflagellates, single-cell algae,
often numbering many hundreds per liter. They are mechanically excited to produce light by the
34
ship's passage or even by the movement of porpoises and smaller fish. The deep-sea fish
Aristostomias has more than one light organ.
Bioluminescence is a primarily marine phenomenon. It is the predominant source of light in the
largest fraction of the habitable volume of the earth, the deep ocean. In contrast, bioluminescence
is essentially absent (with a few exceptions) in fresh water, even in Lake Baikal. On land it is
most commonly seen as glowing fungus on wood (called foxfire), or in the few families of
luminous insects.
Bioluminescence has evolved many times in the sea as evidenced by the several distinct chemical
mechanisms by which light is emitted and the large number of only distantly related taxonomic
groups that have many bioluminescent members.
Bioluminescent bacteria occur nearly everywhere, and probably most spectacularly as the rare
"milky sea" phenomenon, particularly in the Indian Ocean where mariners report steaming for
hours through a sea glowing with a soft white light as far as the eye can see.
Spectrum
In the sea, bioluminescent light is concentrated in the blue window of greatest optical
transparency of seawater. Most organisms emit between 440 nm and 479 nm. Some cnidarians
have green fluorescent proteins that absorb an initially blue emission and emit
it shifted towards the green (~505 nm). One remarkable fish has a similar mechanism to shift the
initial emission into the red for use in viewing prey in the near infrared with its red-sensitive
eyes. Measurements in situ at various depths confirm emission clustering in the blue to green
region of the spectrum.
Intensity
The luminescence of a single dinoflagellate is readily visible to the dark adapted human eye, as
the demonstration will show. Most dinoflagellates emit about 6e8 photons in a flash lasting only
about 0.1 second. Much larger organisms such as jellyfish emit about 2e11 photons per second
for sometimes tens of seconds. The intensity of luminescence by
photosynthetic dinoflagellates is strongly influenced by the intensity of sunlight the previous day.
The brighter the sunlight the brighter the flash.
Kinetics
Some organisms emit light continuously, but most emit flashes of durations ranging from about
0.1 s to 10 s. Some dinoflagellates can respond repetitively to excitation over a short period. In
most multicellular species luminescence is neurally controlled. Thus in some fish the sympathetic
nervous system controls luminescence by way of the neurotransmitter nor-adrenaline. In fireflies
the transmitter is glutamate. In most marine invertebrates the transmitters are unknown. In such
forms the "trigger" to luminescence is some detected behaviorally significant event. In single cell
organisms like dinoflagellates or radiolarians luminescence is triggered by deformation of the
35
cell surface by minute forces(1 dyne per square cm). Mechanical deformation causes an action
potential sweeping over the vacuole membrane and this is thought to induce light emission by
admitting protons from the acidic vacuole into contact with the cellular elements that contain the
light emission chemistry. In a some instances in marine invertebrates with eyes or other light
receptors, light emission can be induced by photic excitation, even by another luminescing
organism. Called "empathetic" luminescence, this phenomenon has as yet undemonstrated
potential to enhance the luminescence generated by a moving source by photic transfer from the
luminescent organisms mechanically triggered by the moving source.
36
Activity 5(JW)
Are We Expecting??
The search for hCG using biosensors
Background
HUMAN CHRIONIC GONADOTROPIN (hCG)
The term “pregnancy test” is actually is a misnomer since most pregnancy tests do not actually
determine pregnancy but rather the level of hCG. The pregnancy test detects only the unique
betasubunit of hCG. Since hCG is only
produced in the developing embryo (except in
rare cases of secretion of hCG by hydatidiform
moles or choriocarcinoma), and since it is
detectable within a few days of implantation,
it is a very specific and early test for
pregnancy.
HCG is a glycoprotein hormone composed of
two dissimilar units, a and b, joined
noncovalently. There are multiple forms of
hCG in serum and urine samples in early
HCG polypeptide structure
pregnancy, including intact hCG (molecular
weight- 36700) and its free subunits free
a(molecular weight- 14500) and free b (molecular weight- 10000) free b Unit is degraded by
macrophage enzymes and in the kidney to make a b subunit core fragment is the principal hCGrelated molecule present in urine during pregnancy(1) intact hCG comprises approximately 70%
amino acids and 30% sugar residues.
The human chorionic gonadotropin (hCG) test is done to measure the amount of the hormone
hCG in blood or urine to see whether a woman is pregnant. HCG is made by the placenta during
pregnancy. HCG may also be made abnormally by certain tumors, especially those that come
from an egg or sperm (germ cell tumors). HCG levels are generally tested in a woman who may
have abnormal tissue growing in her uterus, a molar pregnancy, or a cancer in the uterus
(choriocarcinoma) rather than a normal pregnancy. Several hCG tests may be done after a
miscarriage to be sure a molar pregnancy is not present. In a man, hCG levels may be measured
to help see whether he has cancer of the testicles.
A biosensor extends the range of human senses, making more precise observations and
measurements possible. A biosensor has two basic functions-recognition of the analyte, the
substance being detected, and production of a signal. If you were making a peroxide biosensor,
you might use the enzyme peroxidase because it recognizes the molecule peroxide. But
peroxidase alone does not produce a visible signal. You would need to use an indicator
molecule, such as sodium luminol, along with peroxidase to provide a visible signal. Thus, by
taking sample from the flask and adding peroxidase and sodium luminol to them, you could
determine which flask contains which liquid. In this biosensor, the amount of light produced
37
correlate with the amount to peroxide in each flask. (Here we won’t measure concentration, just
presence)
In this design, the enzyme peroxidase recognized the analyte to be measured (peroxide) and
catalyzes the reaction that oxidizes the indicator molecule (sodium luminol). The oxidized
indicator molecule gives off light, signaling the presence of peroxide. In this activity, you’ll be
making and testing a similar peroxide biosensor, using peroxidase and the indicator molecule
4CN.
ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)
Enzyme linked immunosorbent assay (ELISA) tests were originally developed for antibody
measurement. These immunoassays have also been adapted to successfully detect samples that
contain antigens.
This ELISA simulation experiment has been designed to detect a hypothetical patient's
circulating level of hCG. ELISAs are done in microtiter plates or strips which are generally made
of polystyrene or polyvinyl chloride. The plates or strips contain many small wells which are
somewhat transparent and in which liquid samples are deposited. First, the antibody against hCG
is added to the wells where some remain adsorbed
by hydrophobic association to the walls after washing away the excess. There is no specificity
involved in antigen or antibody adsorption although some substances may exhibit low binding to
the microwell walls. In certain cases the antigens can be covalently cross-linked to the plastic
using UV light. After washing away unadsorbed material, the unoccupied sites on the walls of
the plastic wells are blocked with proteins, typically gelatin or bovine serum albumin.
In this simulation experiment, hCG present in the urine sample will bind to the adsorbed
antibody in the well and remain there after washing. If hCG has remained bound to the anti-hCG
antibody in the well, then the secondary antibody will bind to another antigenic determinant on
the hCG. This complex will remain attached after washing. The second antibody to hCG is
purified and covalently cross linked to an enzyme with a high turn over number such as
horseradish peroxidase. This modification does not significantly affect the binding specificity and
affinity of the antibody or the enzymatic activity of the peroxidase.
After washing the well to remove unbound secondary antibody, a solution containing hydrogen
peroxide and aminosalicylate is added to each well. Peroxidase possesses a high catalytic activity
and can exceed turnover rates of 106 per second. Consequently, amplification of a positive
sample can occur over several orders of magnitude. Many hydrogen donor co-substrates can be
used by peroxidase. These co-substrates include aminodiansidine, aminoantipyrine,
aminosalicylic acid and numerous phenolic compounds that develop color upon oxidation. The
substrate solution added is nearly colorless. Peroxidase converts the peroxide to H2O + O2 using
the salicylate as the hydrogen donor.
It should be noted that polyclonal antibody preparations to a given antigen can have variable
binding affinities due to differences in the immunological responses between animals. Different
immunizations with the same antigen in the same animal can also produce variable binding
affinities. The use of monoclonal antibodies directed against a single epitope eliminates this
variability and makes the ELISA highly specific of hCG detection. In this experiment, students
will use the ELISA test to determine pregnancy of four hypothetical patients.
38
EXPERIMENT OBJECTIVE:
The objective of this simulation experiment is to introduce concepts and experimental procedures
involved with enzyme linked immunosorbent assays (ELISA) in the context of testing for
pregnancy.
LABORATORY SAFETY
Gloves and goggles should be worn routinely as good laboratory practice.
GENERAL INSTRUCTIONS AND PROCEDURES
Labeling the Microtiter Strip:
• Place the microtiter strip as shown in Figure 1.
• Carefully mark the strip with your initials or lab group number and
number the wells 1-4 down the side.
• Do not separate the reaction wells.
Labeling the Plastic Transfer pipets:
Label 5 transfer pipets as follows:
• ( - ) (negative)
• (+ ) (positive)
• USP 1 (Urine Sample Patient 1)
• USP 2 (Urine Sample Patient 2)
• PBS (Phosphate Buffered Saline)
Use the appropriately labeled plastic transfer pipet for sample additions, removals, and washes as
outlined in the experimental procedures. If using transfer pipets to add reagents to the wells, label
3 additional pipets "hCG Ab", "hCG Ab2" (hCGAb-HRP conjugate), and "substrate".
INSTRUCTIONS FOR ADDING LIQUIDS AND WASHING WELLS
Adding Reagents to Wells:
For adding reagents to the wells, use the labeled transfer pipets or use an automatic micropipet
and disposable tips.
Liquid Removal and Washes:
When instructed in the experimental procedures, remove liquids with the appropriately labeled
transfer pipet, and then wash the wells as follows:
A. Use the transfer pipet labeled "PBS", to add PBS buffer to each of the wells. Add PBS buffer
until each well is almost full.
39
The capacity of each well is approximately 200 l. Do not allow the liquids to spill over into
adjacent wells.
B. With the appropriately labeled transfer pipet, remove all the liquid (PBS buffer) from each of
the wells. Dispose the liquid in the beaker labeled "waste".
ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)
1. To all 4 wells, add 50 l or 3 drops of “hCG Ab” (human chorionic gonadotropin antibody). If
using a transfer pipet, use the one labeled "hCG Ab".
2. Incubate for 5 minutes at room temperature.
3. Remove all the liquid (hCG antibody) with a transfer pipet.
4. Wash each well once with PBS buffer as described above ("Liquid Removal and Washes").
In research labs, following this step, all sites on the microtiter strip are saturated with a
blocking solution consisting of a protein mixture, such as BSA. We have designed this experiment
to eliminate this step to save time.
5. To Test Patient 1, add reagents as outlined below:
Remember to use the appropriately labeled transfer pipets provided with this experiment for
adding a new reagent. If you are using automatic micropipets, use a clean micropipet tip for
each reagent.
• Add 50 l or 3 drops of PBS Buffer to the first well. (This is the negative control.)
• Add 50 l or 3 drops of "+" (positive) to the second well. (This is the positive control.)
• Add 50 l or 3 drops Urine Sample from Patient 1 “USP 1” to the third well.
• Add 50 l or 3 drops Urine Sample from Patient 2 “USP 2” to the fourth well.
6. Incubate at 37oC for 15 minutes.
7. Remove all the liquid from each well with the appropriately labeled transfer pipet.
8. Wash each well once with PBS buffer (as described under "Instructions for Adding Liquids
and Washing Wells" on page 8).
-hCG peroxidase conjugate (hCG Ab2) to all 4 wells of each
strip. If using a transfer pipet, use the one
At this time you can obtain the substrate to be used in step 13. Since the substrate must be
prepared just prior to use, your instructor will prepare it towards the end of the incubation in
step 10.
40
11. Remove all the liquid from each well with the appropriately labeled transfer pipet.
12. Wash each well once with PBS buffer (as described under "Liquid Removal and Washes").
r pipet, use the one
labeled substrate".
15. Remove the strip for analysis.
LABORATORY NOTEBOOK RECORDINGS:
Address and record the following in your laboratory notebook or on a separate worksheet.
Before starting the experiment:
• Write a hypothesis that reflects the experiment.
• Predict experimental outcomes.
During the Experiment:
• Record (draw) your observations, or photograph the results.
Following the Experiment:
• Formulate an explanation from the results.
• Determine what could be changed in the experiment if the experiment were repeated.
• Write a hypothesis that would reflect this change.
STUDY QUESTIONS
Answer the following study questions in your laboratory notebook or on a separate worksheet.
1. Why is the ELISA reaction so sensitive?
2. Why is it important to have a positive control?
3. What is being detected in the standard home pregnancy test?
4. What is the difference between polyclonal and monoclonal antibodies
41
Activity 6(EA)
USE OF BIOTECHNOLOGY TO ASSESSING ENVIRONMENTAL IMPACT OF
BIODEGRADATION ON ECOSYSTEM TOXICITY
Reporter genes or screenable markers have proven very useful in biotechnology research as they
provide a visual means to identify and monitor the effects of harmful compounds released into
the environment using genetically engineered cells in organisms such as, transgenic plants,
animals and microorganisms. This technology has been proven useful in assessing the harmful
effects of degradable materials, agricultural and industrial runoffs into the environment, as well
as assessing the adverse effect of degrading commonly used household items.
Cells transformed with the green fluorescence protein gene exhibit bright fluorescence under
ultraviolet or blue light. The proposed experiment is one that uses bacterial cells containing the
pGLO plasmid that was genetically engineered to express the gene for green fluorescence protein
(GFP) from jellyfish. This gene was placed next to the promoter that regulates expression of
bacterial genes needed to break down, or digest, the simple sugar arabinose. This promoter is
activated in the presence of arabinose but not in its absence. Bacterial cells containing this
plasmid, pGLO, when grown in the presence of arabinose, the GFP gene is turned on and the
bacteria fluoresce a brilliant green color under long-wave ultraviolet light as shown below.
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Obtained from BIO-RAD
pGLO can also be used to introduce students to the concept of genes and their most basic
function — to code for proteins.
Content Focus
42





Harmful compounds are released into the environment from degradable materials,
agriculture, industry and even from our own households.
Importance of detecting and predicting the effects of compounds released from this
process on the environment.
Released in small amounts that are disperse into soil and water, therefore difficult assess
long-term effect.
Use of harmless bacteria as biological monitors to determine whether a
compounds/chemicals released from degradation are toxic to the environment.
Suitable field-test and cost effective when compared to tests done with mice, rabbits, and
rats.
Activities
1. Bioluminescence. The use of biological engineered bacteria to produce green
fluorescence protein (GFP) to produce light.
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Obtained from Carolina Biological


Demonstrate potential mutagenic/toxic effects of biodegradable compounds in
using genetically engineered bioluminescent bacteria
Recording the level of light produced when cells are grown in the presence of a
test compound
Protocol
 Culture GFP expressing bacteria overnight in presence of arabinose and ampicillin
(This can be done in10ml test tubes or a flask)
 Aliquot samples of overnight culture into 96-well microtiter plate containing of
nutrient broth with arabinose and ampicillin (Help select for GFP expressing
bacteria)
 Add varying concentrations of compound in the lane of wells for each compound
tested
a. Here, students will perform a serial dilution of a control compound know
to cause mutations and affect cell growth, positive control.
43
b. There will be two control lanes, one lane containing the control compound
and another containing no compound to serve as a negative control.
c. Other lanes can be used to test material from biodegradation.


Incubate overnight at 370C.
Examine the effect of each compound on the growth and fluorescence by
recording degree of fluorescence detected in each lane for each compound tested
or counting the number of cells/ml of culture after plating cells on selective agar
medium.
Obtained from BIO-RAD
GFP Quantification.
The fluorescence of GFP-producing cells that are grown in culture can be quantified
by exposing culture to long-wave ultraviolet light. The mutagenic/toxic effect of
compounds can be determined by one of two methods.
1. Visually accounting for the lost of fluorescence when cells are exposed to
compounds tested.
2. The process can be quantified by plating cells from cultures illustrating a visual
effect of the compound(s) being tested, i.e., lost of fluorescence. Under these
44
conditions, a serial dilution can be performed on cultures showing a lost in
fluorescence to, (1) determine toxicity of the compound (lost in number of
cells/ml) and/or (2) mutagenicity of the compound (lost in number of cells
fluorescing). This can be accomplished by:




Performing a serial dilution of the culture
Plate and incubate cells overnight
Count number of cells per plate and multiply by dilution factor to determine
number of cells/ml in culture
Demonstrating the effect of the compound on cell growth by grafting effect
of increasing concentration of compound on number of cells in culture.
2. Assessment the toxicity/mutagenicity of compounds released from biogradation and/or
commonly used household products.
 Serial dilutions of compounds and household products to determine the
concentration needed to be toxic/mutagenic.
 Use of light producing bacteria to assess toxicity of compounds released from
degradation process.
 Use light producing bacteria to determine toxic effect of commonly used
household products.
 Test effect (toxicity/mutagenicity) of compounds based on lost or intensity of
luminescence from bacterial exposure to household compounds or compounds
resulting from biodegradation.
 Effect of compound on cell growth could also be determined by performing a
serial dilution of cells and plating an aliquot of the culture on selective agar plates
a counting the number of cells on plate as a result of exposure to degradable
compound
 Students develop ability to use statistical analysis, graphing, and tabulation of
findings
Discussions Points:
 Scientific method
 Biological molecules (monomers, polymers, synthesis and hydrolysis, enzymes)
45




Microbiology (prokaryotes – structure, metabolism)
Ecology (pollution)
Mutagenicity/toxicity
Cancer
46
Demonstration #1(JW)
Chemiluminescence
Although exothermic reactions usually liberate energy as heat, there are occasional reactions in
which energy is emitted as light at or below room temperature. These chemiluminescent
processes can occur when the energy given off in a reaction is capable of producing molecules in
an electronically excited state. As these excited molecules drop to the ground state, the light
emitted is sometimes visible. Many such reactions occur in nature (for example, in the firefly),
where they are referred to as bioluminescence.
In this experiment, luminol (3aminophthalhydrazide) will be prepared and oxidized under basic conditions causing the
emission of light. Although the mechanism of this chemiluminescent process in aqueous
solution is not entirely understood, the singlet (all electrons paired) excited state of the 3aminophthalate ion is know to be the light-emitting species.
NO2
NO2 O
COOH
+
COOH
3-Nitrophthalic Acid
NH2
NH2
NH
-H2O
NH
O
3-Nitrophthalhydrazide
Na2S2O4
NH2
O
NH
NH
O
Luminol
47
A. 3-Nitrophythalhydrazide
Pour 15 mL of water into a small beaker or flask and heat it on a steam bath or hot plate. Place
1.00 g (4.74 mmol) of 3-nitrophthalic acid and 2 mL of an 8% (by weight) solution of aqueous
hydrazine into a large test tube (20 x 150 mm or 25 x 200 mm). Add a boiling chip, mount the
tube at a slight angle on a support track, and heat the mixture with a small burner to dissolve the
solid. Add 3 mL of triethylene glycol and insert a thermometer so it rest on the bottom of th test
tube. Boil the solution to remove the excee water, allow the temperature to rise rapidly (over
several minutes) to 215oC, and maintain a temperature of 210-22- oC for 3-4 min. Let the
reaction mixture cool to 100 oC, add 15 mL of the hot water prepared previously, and cool the
test tube in running water. Collect the light yellow granular product and use it without
purification or drying in the next step.
B. 3-Aminophthalhydrazide (Luminol)
Rinse the test tube used in Part A and place the wet 3-nitrophthalhydrazide into it, together with
5 mL of 3M sodium hydroxide and 3.0 g (14 mmol) of sodium hydrosulfite dihydrate (also called
sodium dithionite), Na2S2O4.2H2O. Heat the mixture to boiling, stir, and maintain boiling 5
minutes. Cool the reaction mixture and in a hood slowly add 2 mL of glacial acetic acid.
Collect the precipitated luminol by suction filtration, wash with a little water and allow it to dry
or dry it with gentle heating (heat lamp for above). The moist material can be used in the
chemiluminescence step next.
C. Chemiluminescence
Mix 50 mg of luminol with 5 mL of 3 M sodium hydroxide in a 250 mL Erlenmeyer flask and
dilute the solution to about 150 mL. Add 10 mL of 3% hydrogen peroxide solution. Read ahead
to be certain you will have everything you will need and then go to an area that can be darkened
for the next part of the experiment. (Complete darkness in not necessary.) Add approximately
250 mg of small crystalline chunks of potassium hexacyanoferrate (III) also know as potassium
ferricyanide to the solution, swirl it and note what happens.
Pour some of the luminescent solution into a small beaker and add some 3 M hydrochloric acid.
Record the result. Put one or two pellets of sodium hydroxide into another small beaker and add
some of the luminescent. Record what happens in the vicinity of the pellets. To each solution,
place in a warm water bath and report your observations.
48
Proposed Emission of Light (h)
Intersystem
crossing
S1
T1
Emission
-h
Peroxide
-N2
Luminol
Dianion
+
O2
Photon
absorption
+h
S0
3-Aminophthalate dianion
Fluorescence emission spectrum of the 3-aminophthalate dianion
Intersystem
crossing
S1
T1
Emission
-h
Peroxide
-N2
Luminol
Dianion
+
O2
3-Aminophthalate dianion
Photon
absorption
+h
S0
49
Demonstration #2(JW)
Testing Know Concentrations of Peroxide
In this activity, you will see whether or not your peroxide biosensor can distinguish different
concentrations of peroxide. Then you’ll use your biosensor to try to identify unknown
concentrations of peroxide.
Predictions:
You will be testing samples that contain different concentrations of peroxide. You will test 1%
and 0.00003% peroxide solutions and six other peroxide concentrations of your choice. Predict
what will happen when you test each sample. How will the results differ for different
concentrations? Record your predications in a data table.
Make a data table with the following categories:




The concentrations of peroxide your are testing
Your predictions about what will happen to the sensor strip placed in each concentration
Your observations of what actually happened to the sensor strip placed in each
concentration
Your quantification of the data
Gather these materials:









8 different concentrations of peroxide (including 1% and 0.00003%)
8 test-tube caps (or other small containers)
Self-stick labels
Graduated droppers
Peroxidase enzyme solution
Tweezers
Sensor strips
Paper towels
Centimeter ruler
50
Procedure
1) Get 1 mL of each of the peroxide concentrations you will test
2) Label a container for each of the peroxide concentrations you will be testing
3) Using a graduated dropper, measure 0.1 mL of peroxidase into each container
4) Use another dropper to aid 0.3 mL of the appropriate peroxide concentrations to each
container. To avoid contamination of the sample, star with the least concentrated sample and
continue I order to the most concentrated sample. Gently tap each container to mi the contents
5) Use tweezers to add a sensor stripe to each container
6) When the entire strip is wet, use tweezers to remove it from the solution and place it on a
paper towel. Record your observations for each concentration in you data table.
51
Glossary (ALL)
Aerobe - An organism requiring free atmospheric oxygen for normal activity.
Anaerobe - An organism that derives energy for life from chemical changes that do not require
oxygen. Some anaerobes cannot tolerate the presence of free oxygen.
Bacteria - A group of microscopic one-celled organisms.
Bioluminescence - An emission of light by living organisms.
Chemiluminescence – is the emission of light (luminescence) without emission of heat as the
result of a chemical reaction.
Electron - A stable elementary particle which is the negatively charged constituent of ordinary
matter.
Enzyme - A protein that acts as a catalyst.
Exothermic Reactions – A reaction in which heat is released.
Flagella - Whip-like projections of cytoplasm used in locomotion by certain simple organisms.
Fluorescence – is a luminescence that is mostly found as an optical phenomenon in cold bodies,
in which the molecular absorption of a photon triggers the emission of another photon with a
longer wavelength
Luciferase - An enzyme that catalyzes the oxidation of luciferin.
Luciferin - A species-specific pigment in many bioluminescent organisms that emits heatless
light when combined with oxygen.
Mutagenicity – the act by which a physical or chemical agent changes the genetic information
(usually DNA) of an organism and thus increases the frequency of mutations above the natural
background level.
Photobacteria - Bacteria that can synthesize light.
Plasmid – A plasmid is a DNA molecule separate from the chromosomal DNA and capable of
autonomous replication. It is typically circular and double-stranded
Protein – Large organic compounds made of amino acids arranged in a linear chain and joined
together by peptide bonds between the carboxyl and amino groups of adjacent amino acid
residues
Symbiotic - The living together of two or more different organisms in close association.
52
53
Activity #5: Are we expecting?
54
Activity Outline: Electrochemical Glucose Sensor (BL)
Introduction
 Background information about Type I/II Diabetes: Insulin dependence and why?
 Feedback mechanisms: Insulin/Somatostatin
Teams and Materials Needed
 Sugars (Sucrose, Glucose, Fructose, Starch, Cellulose)
 Common beverages (milk, soda, beer, etc.)
 Electrochemical cell components (most likely microscale)
 Still looking for some “self-sampling” technique that doesn’t involve finger pricking, although this would
be an interesting option
The Concepts Needed
 Electrochemistry
o Nernst Equation
o Redox Reactions: Balancing of half-cell reactions
 Polymers
o Synthetic (isotactic, syndiotactic, and atactic)
o Natural (isoprene rubber)
 Organic polymers (basic concepts of sugar polymerization)
o Biopolymers and Biocolloids—Cellulose, Starch, Collagen, Insulin, Mucus, Lipids
 Electricity concepts/Circuitry
o Basic understanding of voltage, current, and resistance
o Kirchoff’s Laws
 Colligative properties
o Ionization
o Conductivity of solutions
Procedure
 Something to do with testing of different mono- and disaccharides as well as cellulose and starch from pure
compounds to mixtures of liquids with KNOWN glucose concentrations.
o Accuracy of sugar analysis—Blood glucose metering
o Selectivity of sugar analysis
o Tolerance
 Affects of conditions on accuracy of measurements (pH, temperature, concentration)
Analysis
 Something to do with analysis of accuracy of detecting glucose present different mono- and disaccharides as
well as cellulose and starch
Conclusions and Considerations
 Accuracy of detection v. insulin dosing
 How to make a “better” (i.e. prickles) monitor
55