Whole Cell Based Biosensor

(BBL4100)

Dr. Sudipta Bhattacharyya

Department of Bioscience and Bioengineering
What is a biosensor?
Biosensor is a sensor device that is made up of the combination
of biological component with physicochemical detector.
Biosensors are analytical devices which can be used for
detection/monitoring of particular analytes.

Basically biosensor is a probe which combines biological

elements like whole cell or tissues/purified
enzymes/proteins/antibodies to selectively bind and/or
transform the analyte present in the experimental sample and
tranduces the binding signal to a physicochemical device to
obtain quantifiable/semi-quantifiable response

Main parts of a typical biosensor

(Ref: Bin Fan and Tim Zeyl)
Example of commonly used biosensors

Principle of Electro Chemical Glucose Strip:

Enzyme (Glucose oxidase) based biosensor (Quantitative)

Example of commonly used biosensors

Principle of Pregnancy Test Kit:

Antibody based biosensor (Qualitative)

Example of whole cell based biosensor

Immobilized whole cell (Nocardia erythropolis) based biosensor

(Quantitative)
Types of Biosensor
Whole cell based biosensors
 Biosensors can be classified into three types based on differences in their molecular,
 cellular and tissue sensing components.

 The molecular-based biosensors use biological active substances such as enzymes,

DNA, antigens, antibodies, and biofilms as the reporter elements. The significant
advantage of these molecular-based biosensors is their high selectivity.

 The applicability of this type of biosensor has been limited by shortcomings such as
expensive macromolecule isolation costs, limited detection capability and the short
useable lifetime of the identifying molecules.

 In contrast to molecular-based biosensors, sensors that are formed from cells or

intact tissue are cheaper and have seen rapid development in novel methods of their
microfabrication and immobilization, and recent changes have provided these types
of biosensors with unique and unexploited advantages. However, till now, their
performance is not as great as molecular based biosensors.

 The main issues regarding the performance of a whole cell-based biosensor are: (1)
the selection of reporter gene, as well as (2) the selectivity and sensitivity of the
molecular recognition that occurs when regulator proteins bind to their target
analytes.
Gene expression regulation in prokaryotes
Basic terminologies:

Constitutive gene expression: When the gene expression is always ON at fixed rate
. Generally observed in housekeeping genes

Regulatable gene expression: When the gene expression is regulated when their
expression is needed.

Operon: Operons are regions of DNA that contain clusters of related genes, whose
expression is regulated together by means of a common promoter region and
operator region. For example lac operon, trp operon.

Regulon: A regulon is a group of genes that are regulated as a unit, generally

controlled by the same regulatory gene that expresses a protein acting as a
repressor or activator. Sometimes, a regulon may constitute of few operons, for
example lexA regulon.
Gene expression regulation in prokaryotes
Cis-regulatory elements: Cis-regulatory elements (CREs) or Cis-regulatory modules (CRMs) are
regions of non coding DNA which regulate the transcription of neighboring genes. CREs are
vital components of genetic regulatory networks. For example, Promoter sequences and
Operator sequences.

Trans-acting elements: The trans-acting elements are transcription factors or other DNA-
binding proteins which recognize and bind to specific sequences in the cis-acting elements to
initiate, enhance or suppress transcription. For example RNA polymerase, repressor proteins,
enhancer proteins, transcription factors

Repressor proteins: A repressor is a DNA- or RNA-binding protein that inhibits

the expression of one or more genes by binding to the operator or associated silencers. A
DNA-binding repressor blocks the attachment of RNA polymerase to the promoter, thus
preventing transcription of the genes into messenger RNA. An RNA-binding repressor binds to
the mRNA and prevents translation of the mRNA into protein. This blocking or reducing of
expression is called repression.

Activator proteins: A transcriptional activator is a protein (transcription factor) that

increases transcription of a gene or set of genes. Most activators function by binding
sequence-specifically to a regulatory DNA site located near a promoter and making protein–
protein interactions with the general transcription machinery (RNA polymerase and general
transcription factors), thereby facilitating the binding of the general transcription machinery
to the promoter.
Reporter genes:
Reporter genes are genes that enable the detection or measurement
of gene expression. They can be fused to regulatory sequences or genes of interest to
report expression location or levels.

So, the promoter element should be specific for the analyte what we would like to
measure and there should be some qualitative/quantitative ways to check the
expression of reporter genes.

Example:
Antibiotic resistance markers: These genes, when expressed,
provide the ability of the host cell to degrade/ inactivate certain
type of antibiotics to become resistant against that antibiotic.
Antibiotic resistance can be easily quantifiable.

Metabolic markers: Few metabolic enzyme genes can also

serve as the good reporter genes by virtue of their ability to
catalyse chromogenic product conversion reaction when
expressed. As for example β-galactosidase enzyme can convert
X-gal substate into a chromogenic product.
 Whole Cell Based Biosensor
Part-II
(BBL4100)

Dr. Sudipta Bhattacharyya

Department of Bioscience and Bioengineering
 Green fluorescent protein: A Swiss army
knife to design biosensors

Aequoarea victoria (Crystal Jelly fish)

Absorption max= 395 nm

Emission max= 475nm

Heterologous expression of GFP
Use of Green fluorescent protein as a reporter

GFP as the gene expression reporter:

Use of Green fluorescent protein to track double
stranded DNA breaks caused by mutagens

GFP fusion to track real time protein

dynamics/recruitment:
Use of Green fluorescent protein as a Chemical
Biology tool to track effect of xenobiotics
GFP fusion to assay effect of xenobiotics on cellular protein
degradation machinary
C

+I

-I

Proteasome inhibitor screening (effect of xenobiotics) Decrease in GFP

fluorescence over time
Development of other fluorescent proteins: YFP

Yellow fluorescent protein: The red shift variant of GFP

GFP YFP
Absorption max= 395 nm Absorption max= 514 nm

Emission max= 475 nm Emission max= 527 nm

T203Y substitution
causing increased
pi-pistacking
Development of other fluorescent proteins
Use of fluorescent protein as a Chemical Biology tool

FRET: (Fluorescence Resonance Energy

Transfer)

A B

RF
P
P
GF
FRET (Fluorescence Resonance Energy Transfer): Basics
Use of fluorescent protein as a Chemical Biology tool

Strategy 1
Split auto fluorescent protein (AFP) based biosensor:
Use of fluorescent protein as a Chemical Biology tool

Strategy 2
FRET based biosensor:
Use of fluorescent protein as a Chemical Biology tool

Strategy 3
FRET based biosensor:
Use of fluorescent protein as a Chemical Biology tool

High throughput in vivo screening of chaperone activity:

 Whole Cell Based Biosensor
Part-III
(BBL4100)

Dr. Sudipta Bhattacharyya

Department of Bioscience and Bioengineering
Common genetic circuitry in prokaryotes

Lac operon:
Common genetic circuitry in prokaryotes
Lac operon:
Environmental Signal Processing by Bacteria: Two-
component systems (TCS)
• Two component systems are
Phospho-relay system mediated signal
transduction systems

• Two different type of protein

components are involved in these
systems – A. Sensor Histidine Kinase
(HK) B. A repose regulator (RR) [A
transcription factor]

• Ligand binding to sensor HK caused

conformation change leading to auto-
phosphorylation by ATP.

• Autophosphorylated HK can recruit

RRs and transfer phosphate group to
RRs.

• Phosphorylated RRs bind respective

enhacer region to increase cogante
gene synthesis
Typical Two-component system (TCS) and how can we use it

PhoR = Membrane histidine Kinase; PhoB/PhoP = Response regulator

(Transcription factor); PhoA/PhoB = Target genes

In a bioprocess where maintainance of higher concentration of Phosphate is

necessary we can use this TCS in a whole cell biosensor and replace PhoA/PhoD
genes with any reporter genes to give us quantitative response
Other important Two-component system (TCS) and how
can we use it

Can be manipulated to design Fe3+ detection Whole cell based biosensor

Can be manipulated to design Cu2+ detection Whole cell based biosensor

Can be manipulated to design low oxygen tension detecting Whole cell based
biosensor
DNA manipulation for reporter gene fusion

In our case, the target gene will

be the reporter gene

Reporter gene needs to be cloned

downstream of the cis acting
elements

cis acting elements are responsive

elements for signal trasduction in
response to external analyte
binding

The reporter gene needs to be

cloned in frame

Basic steps for recombinant DNA Technologies

Basic enzymes needed for DNA manipulation:

DNA polymerase :
DNA polymerase catalyze the amplification of the target
genes (reporter) from heterologous sources. For example
amplification of GFP gene from A. victoria (jellyfish)
genome

The DNA polymerase in use should have 3’-5’

exonuclease (proof reading) activity along with 5’ – 3’
polymerization activity

Generally DNA polymease from hyperthermophilic

organisms are used for cloning. (For example: Pyrococcus
furiosus DNA polymerase (Pfu DNA pol)

The forward and reverse primers used in this cloning

carry restriction enzyme recognition sites (which are to
be used
Taq DNA pol lacks 3’-5’
exonuclease domain
Basic enzymes needed for DNA manipulation:
Restriction endonuclease :
Restriction endonucleases are site specific
endonucleases which recognizes and cleaves dsDNA to
produce blunt or cohesive DNA ends (termini)

Restriction endonucleases can also be classified by the

length of their DNA recognition and way of recognition
and DNA digestion (eg. hexacutter will recognize and cut
the 6bp long DNA recognition site ; Some restriction
endonucleases recognize the DNA substrate far apart
from their cutiing site)

Generally for rDNA application restriction enzymes

which recognize and cut in between the chosen DNA
strand is preferred. Restriction enzymes which produces
cohesive termini are preferred over blunt end producers MCS

Restriction endonucleases are chosen in such a way so

they do not cut the target gene but their cut site is
present in the vector DNA sequence (MCS = Multiple
cloning site/sequence)
Basic enzymes needed for DNA manipulation:
DNA Ligase :
DNA ligase enzymes are involved in
ligation of the phosphodiester bonds

DNA ligases need either ATP or NAD+ as

their cofactor

ATP dependent DNA ligases are from

bacteriophage origin and they are most
efficient compared to the NAD+
dependent bacterial enzymes

ATP dependent DNA ligases can ligate

both cohesive and blunt ended DNA
fragments (but cohesive ends ligates
more efficiently)

NAD+ dependent DNA ligases only

ligate the blunt ended DNA fragments
Immobilization of Biosensors on inert matrices:
Some important points to remember during/before immobilization:

• Biosensors always have an associated biorecognition element/component

(protein/nucleic acid/whole cell). Three dimensional structure of the biorecognition
component is highly important for precise identification of the the analyte

• Three dimensional structure of the macromolecules (protein/nucleic acid) are

stabilized by the weak non-covalent interactions which are too sensitive to their
surrounding environment

• During the cross-linking/immobilization steps care must be taken to minimize the three
dimensional structural changes of the bio-recognition component of a particular
biosensor

• Protein based biosensors are most sensitive towards the surrounding environment
compared to nucleic acid based sensors which is more sensitive compared to whole cell
based sensors, To it is easier to immobilize whole cells>Nucleic Acids>proteins

• Immobilization often needs surface activation

Immobilization of Biosensors on inert matrices:
General Strategies for Biomolecule Immobilization:
A.

Piranha solution is the mixture (3:1) of H2SO4 and

H2O2 (30%): It is used to clear the solid surface
from organic substrates
Immobilization of Biosensors on inert matrices:
General Strategies for Biomolecule Immobilization:
B.
Immobilization of Biosensors on inert matrices:
General Strategies for Biomolecule Immobilization:
C.

In this case the antigen or the counter strand is the signal you want to detect
Mode of Biomolecule attachment to the inert surfaces:
Attachment of the sensor biomolecules to the inert surfaces can be attained by
three principal methods –

• through hydrophobic interactions

• through ioninc interactions

• through covalent bond formation

Hydrophobic interaction

Ionic interaction Covalent interaction

Mode of Biomolecule attachment to the inert surfaces:

Hydrophobic immobilization:
Mode of Biomolecule attachment to the inert surfaces:

Ionic immobilization:

If purified protein molecules are to be used then knowledge of overall charge of the
protein molecule at working pH is necessary.
Mode of Biomolecule attachment to the inert surfaces:

Covalent immobilization:

Most stable immobilization method, unaffected by sample pH, ionic strength or

presence of organic hydrophobic molecules
Cross linking strategies for protein immobilization:
Cross linking strategies for DNA immobilization:
Whole Cell immobilization:
Whole cell immobilzation is an alternative to purified biocomponent
(protein/nucleic acid ) immobilization
It is cost effective method –

 When isolation and immobilization of purified biocomponent is too expensive

When individual purified biocomponent looses activity during immobilization

technique

Where multiple protein/enzyme is needed to work togather to produce

effective readout

In whole cell immobilzation the biocomponents will be stable in active states for
longer period of time

The encasement of biocomponents inside the cell will ensure their protection
from harsh conditions

Other than the whole cell, cell organelles like mitochondria and choloroplasts
can be also effectively immobilized to serve our purpose
Advantage and disadvantage of whole cell immobilization:

Advantages:
Expensive isolation of purified biocomponents are not required

Multiple biocomponents can be introdiced in a single cell

Biocomponents such as proteins retain their biological activity for much longer time

Excahnge of analye/product from the sample is fast due to high surface/volume ratios of
immobilized microbial cells or organelles

Disadvantages:
Effective concentration of biomolecules are much lower

Presence of other contaminating biomolecules which might interfare with the

biosensor signal transduction and/or quench the signal by presence of other
enzymes/cell metabolites
Methods of whole cell immobilization:
Nucleic Acid Based Biosensors:

Principle of Nucleic Acid Based Biosensors:

Immobilization of Nucleic acid probes:
Example of Nucleic acid immobilization and their
principle of action:
Example of Nucleic acid immobilization and their
principle of action:
Molecular Beacons :

The Principle is to separate Fluorophore (F)/Quencher(Q) pair to get fluorescence

signal
A. Hybridization of complementary nucleic acid pair (analyte) causes the
separation of F and Q
B. Binding with a target protein (analyte) to the nucleic acid causes the separation
of F and Q
C. Cleavage of one strand of the nucleic acid in presence of a metal ion (analyte)
causes the seperation of F and Q
D. Use of PNA (peptide nucleic acid) to probe protein peptide interaction which
causes separation of F and Q.
Electrochemical DNA Hybridization Biosensors:
 Whole Cell Based Biosensor
Part-IV
(BBL4100)

Dr. Sudipta Bhattacharyya

Department of Bioscience and Bioengineering
Whole cell biosensor to detect ground water arsenic:
arsR
P O arsB arsC

Wild type ars operon E.coli

Concentration of arsenic (µM) Concentration of arsenic (µM)

mcherry = reporter protein which gives red

fluorescence
ArsR = Arsenic responsive trascriptional repressor;
Biosensor genetic circuit A.
looses DNA binding after binding As(III)
without positive feed back B.
LuxR = Transcriptional activator for luxI promoter
With positive feedback
(PluxI)
Whole cell biosensor to detect free tyrosine in urine:
Presence of amino acid tyrosine or elevated levels of tyrosine in urine is a hallmark of few
metabolic diseases(). The presence of tyrosine in human urine is necessary for such disease
surveillance

[Tyrosine]
TyrR is the transcription factor sensitive to tyrosine
binding
In absence of Tyrosine ParoF promoter binds RNApol
and RFP expression is on whereas PtyrP is a weak
promoter needs some transcriptional activator so it is
off
In presence of tyrosine TyrR behaves as the repressor
for ParoF promoter (inhibiting the expression of RFP)
and activator for PtyrP promoter (enhancing the
expression of GFP).
Bacteriophage based biosensor to detect pathogenic bacteria:
Strategy 1:

Impedance is measured
between the working electrode
and the reference electrode

Capture of the bacteria on top

of the phage layer causes
higher impedance (lower
current flow)

The difference in the electrical

signal is counted
Bacteriophage based biosensor to detect pathogenic bacteria:
Strategy 2:
Phage mediated Incorporation and expression of reporter genes in target bacteria

Lytic cycle Lysogenic cycle

We can use a pathogenic bacteria specific phage

carrying a reporter gene (eg. GFP) in its own genome
and set the infection condition as such that the
lysogenic cycle is induced. In presence of bacteria the
phage will transfer its repoter gene inside bacerial
genome and we can get a readout from the reporter
expresion.
Bacteriophage based biosensor to detect pathogenic bacteria:

Strategy 3:
Yeast (an unicellular eukaryote) based whole cell biosensor to
detect fungal pathogens in body fluids
• The yeast whole cell biosensor, selectively detect fungal pathogens was devised by Ostrov,2017

• This biosensor is based on yeast (fungal) signal transduction mechanism during sexual
reproduction
Overview of Yeast reproductive mechanism:

In sexual reproduction (meotic

cell division results haploid (n)
progeny cells of opposite sex
(a/α) these cells can grow
vegetatively. They secrete mating
peptide (species specific) which
is recognized by opposite sex
type receptors and mating
happens to produce deploid cells
(2n) Asexual reproduction through
budding (mitotic cell division)

Mating of opposite sex types requires recognition of mating peptide by receptor proteins followed by
signal transduction to express downstream gene products required for mating
Yeast (an unicellular eukaryote) based whole cell biosensor to
detect fungal pathogens in body fluids
Most of the fungal pathogen will have their own type of mating peptide and corresponding
cognate receptors

The biosensor yeast cell will be genetically engineered to produce the receptors for
pathogenic mating peptide. The cell will also incorporate a reporter gene (here biosynthesis
genes of red lycopene production) in the down stream signaling cascade as a function of
receptor/mating peptide binding.

In presence of a certain fungal pathogen in body fluid samples the cognate yeast biosensor
will show red pigmentation.

Ca peptide = Candida albicans (a fungal

pathogen) mating peptide
Mammalian cell based biosensors to detect toxins/adverse
effect of xenobiotics

Advantages:
Cell based biosensors response to the toxins/xenobiotics mimicking the physiological condition

These toxin/xenobiotic-cell interaction reveal functional information such as mode of action of

the toxin/xenobiotic, bioavailability, target cell/tissue type as well as the effect of antidote
Organ on a chip
What is organ on a chip? And why it is needed?
In simple terms Organ on a chip is a 3D microfluidic cell culture device (chip) which
simulates the activities and mechanisms of entire organs and/or organ systems

The chip is small and can be visualized under microscope (fluorescent/TIRF etc). This
provides direct visualization of cellular activities of a particular organ un real time
Why it is needed?
Classical in vitro cell cultures are devoid of physiological niche and observations based
on those do not reflect the physiological effect of compounds/conditions under study
(e.g effect of drug or attack by pathogen)

In vivo animal studies do not fully mimic human physiological conditions (e.g. the
perfect animal model to study SARS-CoV2 is still under investigation)

Often human models do not fully mimic the effect of certain drugs/conditions
applicable to other group of humans (most of the clinical studies are conducted on
adults but the effect of any drug to be taken by infants or older people cannot be
judged in that way; again some people are allergic to certain drugs compared to
others
Organ on a chip can provide personalized effects of drugs or other compounds in
lesser time by growing personalized stem cells to grow an organ on a chip
Example of an Organ on a Chip
Lung on a chip:

Size of the Chip

Operation of the chip

The upper channel and lower channel are separated by a
semi permeable membrane (Fig.a)

The upper channel has epithelial cells and lower channel

has blood cells; Air and airborne objects are passed
through upper channel blood cells and blood growth
factors are passed through lower channel (Fig. b left panel)

The microfluidic chamber Vacuum pressure leads the expansion and shrinkage of the
upper and lower chambers (like lungs alveoli) (Fig. b right
Huh D. D. et al., 2010 panel)
Advantages and Current Limitations of Organ on a Chip Tools
Advantages
1. Efficiently replaces the animal models for several disease mechanism studies including
interaction of pathogen with organ cells, modeling viral infection studies
2. Highly useful for pharmacological actions of drugs, for toxicological studies, to assess the
effects of cosmetics etc.
3. Efficient system to study cancer cells and their microenvironment and to screen
anticancer drugs
Limitations
4. Every property of the organ cannot be mimicked or measured. For example cgnition in the
brain, mechanical function in bone, ligaments, tendons, drugs actions during pregnancy
cannot be modeled on chips.
5. It is not always to possible to represent the complete organ
6. The system is highly prone to bacterial or fungal contamination
7. Chronic diseased conditions cannot be modeled.
8. The devices needs specialized micro-engineering capabilities.
Further readings:
Eye on a Chip (Seo & Huh, 2014); Gut on a Chip (Kim et. al., 2012); Liver on a chip (Lee et.
al, 2007); Kidney Glomerulus on a chip (Musah et. al., 2017); Heart on a chip (Zhang et. al,
2015); Human on a chip: Soohee Cho & Jeong-Yeol Yoon, 2017.

Useful link: https://youtu.be/CpkXmtJOH84

Whole organism based biosensor to detect environmental
pollution

Zebra fish (Danio rerio)

Vital organs of Zebrafish and Ion balance mechanism in
Zebrafish
Preparation of the transgenic construct sensing the heavy metal
ions in water

Creation of Transgenic Zebrafish

Transgenic Zebrafish biosensor sensing aquatic heavy metal ions
Transgenic Zebrafish biosensor sensing aquatic heavy metal ions
Plant Cell Based Impedemetric Biosensor to detect heavy
metal pollution
Plant Cell Based Impedemetric Biosensor to detect heavy
metal pollution

Aim and Principle of operation:

• Heavy metals present in soil or irrigation water causes physilogical stress upon
plant cells cultiminating into death of economically important plants

• Heavy metal induced plat stress needs to be monitored carefully

• Vitronectin like proteins expressed on the cell surface are plant biomarkers for
heavy metal stress

• Quantitative assessment of cell surface vitronectins can monitor heavy metal

stress in real time.

• A cell surface expressed vitronectin detecting impedometric biosensor is

developed to monitor plant heavy metal stress
Vitronectin binding to heavy metal ions (Cadmium and Lead ions)

Vitronectin active site for metal binding

Cadmium bound Vitronectin active site

Lead bound Vitronectin active site

Principle of Biosensor Design:
Change of circuit impedence upon different concentrations of
Vitronectin:

Each point in plot A represents circuit impedence at a given AC frequenccy plot B

depicts Impedence at a fixed AC frquency with incresing conc. of vitronectin .

NB: Impedence is concept of resistance extends to alternative current circuit

E=IZ=I|Z|ejθ Where Z = Z’ (real part) + jZ’’ (imaginary part)
Equivalent to V = IR in DC
Change of circuit impedence upon different concentrations of
heavy metal ions as function of increased surface Vitronectin:
For Cadmium:

For Lead: