NANOSENSORS:
MICROTUBULES
By Mary Coan
Chemical Engineering
Outline





Nanosensors
Introduction to Microtubules (MT’s)
Formation of MT’s
Dynamic Instability of MT’s
Applications of MT’s
 Bio-Nanosensor

Transporter
Assessment of Performed Work
Nanosensors



Detect very small particles or quantity of something
Chemical, Mechanical or Biological sensors
Amongst other applications they can be used:
 To
detect various chemicals in gases for pollution
monitoring
 For medical diagnostic purposes either as blood borne
sensors or in lab-on-a-chip type devices
 To monitor physical parameters such as temperature,
displacement and flow
 As accelerometers in MEMS devices like airbag sensors
http://www.azonano.com/details.asp?ArticleID=1840 1) http://thewere42.files.wordpress.com/2009/11/500x_iphonetricorder.jpg 2) http://www.medgadget.com/archives/img/nanosensor.jpg 3) http://www.bu.edu/eng/newscms/photos/soundlight thumbnail2.jpg 4) http://www.ck12.org/ck12/images?id=127659
Nanosensors

Bio-nanosensors
 Single
molecule or molecular complex based
 Proteins,
antibodies, enzymes, DNA, and RNA
 Cell-based
 Whole

cells, tissue or whole organisms
Problems with bio-nanosensors
 Human
interaction causes errors
 Low signals (dim fluorescence)
http://www.rpi.edu/dept/chem-eng/Biotech-Environ/BIOSEN/biosen.htm,
Thorsten Fischer, et.al. “A smart dust biosensor powered by kinesin motors” Nature Nanotech Letters, 2009
Outline

Nanosensors

Introduction to Microtubules (MT’s)



Formation of MT’s
Dynamic Instability of MT’s
Applications of MT’s
 Bio-Nanosensor

Transporter
Assessment of Performed Work
Introduction to Microtubules (MT’s)


A principal component of the cytoskeleton (cellular
scaffolding/skeleton made out of protein, present in
all cells)
Rigid hollow rods approximately 25 nm in diameter
 Single
type of globular protein called tubulin
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=cooper&part=A1820 ,
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mcb&part=A5406
Introduction to Microtubules (MT’s)

Tubulin
Polymerize to form MT’s
 Dimers made up of 2 closely related polypeptides
called αβ-tubulin
 Polar structures, fast-growing plus end and slowgrowing minus end
 Bind to GTP (Guanasine triphosphate), source of
energy for protein synthesis



α
β
GTP functions analogously to the ATP (adenosine
triphosphate) bound to actin to regulate polymerization
Υ-tubulin localized to the centrosome (main MT
organizing center (MTOC) of the animal cell as well
as a regulator of the cell-cycle progression)

Initiates MT assembly
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=cooper&part=A1820 ,
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mcb&part=A5406
Introduction to Microtubules (MT’s)
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=cooper&part=A1820 ,
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mcb&part=A5406
Introduction to Microtubules (MT’s)


Are dynamic structures that undergo continual
assembly and disassembly within the cell
Function both to determine cell shape and in a
variety of cell movements
 cell
locomotion, the intracellular transport of organelles,
and the separation of chromosomes during mitosis
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=cooper&part=A1820 ,
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mcb&part=A5406
Outline

Nanosensors
Introduction to Microtubules (MT’s)

Formation of MT’s



Dynamic Instability of MT’s
Applications of MT’s
 Bio-Nanosensor

Transporter
Assessment of Performed Work
Formation of Microtubules (MT’s)


Complexes of γ-tubulin form ring structures that
contain 10 to 13 γ-tubulin molecules and have
diameters similar to those of MT’s
γ-tubulin rings
 nucleation
 May

sites for the assembly of microtubules
remain bound to their minus ends
MT’s extend outward from the MT organizing center
(MTOC)
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=cooper&part=A1820 ,
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mcb&part=A5406
Formation of Microtubules (MT’s)
α
β
α
β
α
β
Outline





Nanosensors
Introduction to Microtubules (MT’s)
Formation of MT’s
Dynamic Instability of MT’s
Applications of MT’s
 Bio-Nanosensor

Transporter
Assessment of Performed Work
Dynamic Instability of MT’s


Each αβ-tubulin is bound to a GTP molecule
GTP is hydrolyzed to GDP when the tubulin dimers are added to
the plus end of the growing microtubule

GTP hydrolysis is not mandatory for microtubule formation

only GDP-bound tubulin molecules are able to depolymerize

GTP-bound tubulin serve as a cap at the tip of microtubule to
protect from depolymerization
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=cooper&part=A1820 ,
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mcb&part=A5406
Dynamic Instability of MT’s

GTP bound tubulin are
hydrolyzed to GDP during or
shortly after polymerization




weakens the binding affinity of
tubulin for adjacent molecules
favors depolymerization
resulting in the dynamic behavior
of microtubules
GTP hydrolysis results in the
behavior known as dynamic
instability

Alternate between cycles of
growth and shrinkage

Determined by the rate of tubulin
addition relative to the rate of
GTP hydrolysis
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=cooper&part=A1820 ,
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mcb&part=A5406
Dynamic Instability of MT’s

If new GTP-bound tubulin molecules are added more
rapidly than GTP is hydrolyzed


microtubule retains a GTP cap at its plus end and
microtubule growth continues
Rate of polymerization slows, the GTP bound to tubulin
at the plus end of the microtubule will be hydrolyzed to
GDP
GDP-bound tubulin will dissociate
 Results in rapid depolymerization and shrinkage of the
microtubule.


Dynamic instability results in the continual and rapid
turnover of most MT’s
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=cooper&part=A1820 ,
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mcb&part=A5406
Dynamic Instability of MT’s

MT’s can be stabilized
 Chemotherapy
Taxane Drug Class, e.g. Docetaxel
 Stabilizes
GDP bound tubulin in the MT’s
 Hydrolysis of the GTP cap does not result in
depolymerization
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=cooper&part=A1820 ,
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mcb&part=A5406
Outline

Nanosensors
Introduction to Microtubules (MT’s)
Formation of MT’s
Dynamic Instability of MT’s

Applications of MT’s



 Bio-Nanosensor

Transporter
Assessment of Performed Work
Application of MT’s

Typical target applications of MT’s
 Drugs
that affect microtubule assembly are useful not
only as experimental tools in cell biology but also in the
treatment of cancer
 Some
experimental drugs bind tubulin and inhibit microtubule
polymerization, thus stops mitosis
 Selectively inhibit rapidly dividing cells
 Stabilizes microtubules rather than inhibiting their assembly,
blocks cell division

Newer application
 Transporters
for bio-nanosensors
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=cooper&part=A1820 ,
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mcb&part=A5406
Thorsten Fischer, et.al. “A smart dust biosensor powered by kinesin motors” Nature Nanotech Letters, 2009
Bio-Nanosensor Transporter


Traditional double-antibody-sandwich (DAS) assays
Dr. Fischer replaced the wash step with a transport
step powered by adenosine triphosphate (ATP)
Thorsten Fischer, et.al. “A smart dust biosensor powered by kinesin motors” Nature Nanotech Letters, 2009
Bio-Nanosensor Transporter
Thorsten Fischer, et.al. “A smart dust biosensor powered by kinesin motors” Nature Nanotech Letters, 2009
Bio-Nanosensor Transporter
Thorsten Fischer, et.al. “A smart dust biosensor powered by kinesin motors” Nature Nanotech Letters, 2009
Positive End
Minus End
α
ADP
Kinesin
ATP
P
α
β
PP
β
PP
Bio-Nanosensor Transporter

Dr. Fischer tested the new device
 Two
different unlabeled analytes were tested using
different tags
 Fluorescent
Nanospheres functionalized to attach to the
analyte Quantum Nanodots conjugated to an antibody

New device accounts for the time required for the
 Analyte/tag
to diffuse to the functionalized MT
 MT to move to the boundary of the cell
Thorsten Fischer, et.al. “A smart dust biosensor powered by kinesin motors” Nature Nanotech Letters, 2009
Bio-Nanosensor Transporter

Speed of the MT’s was modified by controlling the
ATP concentration depending on which analyte was
to be captured
 Based

on diffusion rates
Concentrations of the analyte, tags and antibodies
functionalized to the MT’s were varied
 Increased
signal (brighter flouresence)
Thorsten Fischer, et.al. “A smart dust biosensor powered by kinesin motors” Nature Nanotech Letters, 2009
Bio-Nanosensor Transporter

Six hours after analyte introduction, microtubules
have aggregated at the wall, depositing along with
them a ring of nanospheres
MT’s
Nanospheres
Thorsten Fischer, et.al. “A smart dust biosensor powered by kinesin motors” Nature Nanotech Letters, 2009
Bio-Nanosensor Transporter

Imaging the peripheral region at higher
magnification 3 h after the start of the experiment
reveals microtubule and nanosphere accumulation
at the wall
Nanospheres
MT’s
Thorsten Fischer, et.al. “A smart dust biosensor powered by kinesin motors” Nature Nanotech Letters, 2009
Bio-Nanosensor Transporter

After experimentation Dr. Fischer decided that the
distinction between the zones for analyte pick-up
and tagging should be abolished:
 First,
it is acceptable for the analyte to bind to the
antibody on the tag first and then to the antibody on
the gliding shuttle
 Second, a sufficient number of encounters between
shuttles and tags still occurs
 Third, the device assembly process is dramatically
simplified
Thorsten Fischer, et.al. “A smart dust biosensor powered by kinesin motors” Nature Nanotech Letters, 2009
Outline





Nanosensors
Introduction to Microtubules (MT’s)
Formation of MT’s
Dynamic Instability of MT’s
Applications of MT’s
 Bio-Nanosensor

Transporter
Assessment of Performed Work
Assessment of Performed Work

Very innovative
 First

biologically motorized detecting device
Failure to mention on the MT’s were stabilized
 Potential

effects from non-stabilized MT’s
Future work
 Different
analytes need to be tested
 Strategies to manufacture, package and store
 Performance needs to be optimized and matched to
specific applications
 Signal strength needs to be improved for remote
detection
Questions ?
G3
Rebuttal: Nanosensors Microtubules
Mary Coan
Rebuttal
• “An additional interesting point not discussed during the presentation is
how can we exploit the ‘dynamic’ self-assembling properties intrinsic to
microtubules for nanosensing and nano-engineering.”
– This was not discussed due to time. However it was briefly mentioned that
research into the topic was done but many of the papers found only
discussed the targeting the MT’s instead of using the MT’s as nanosensors.
For an example, a lot of research has been conducted to inhibit the dynamic
instability of MT’s.
• “Nonetheless, it was perhaps too much material for a short time” and
“There was a lot of material covered in the slides, perhaps to fast to
comprehend in the amount of time given. It became hard to follow at
some points.”
– You are correct, there was a lot of information presented in a short time. I
wanted the audience to understand the dynamic instability of MT’s, which
is one of the most important details about MT’s. Without MT’s mitosis
would not occur. I also wanted the audience to fully understand how the
MT’s transported nanosensors, which is the first nanotransporter using only
biological materials.
– I did limit some of the information presented. This may have caused the
audience to become confused or lost at certain points in the presentation.
Rebuttal
• “I could see that the time frames relevant to the technique are
quite large, which would create inconveniences for real time
measurements and monitoring and quick action expected
from point-of-care approaches.”
– You are correct. However, I did mention that the authors proposed
removing the separate regions, ie capture and tag regions, to
decrease the amount of time needed to detect the analyte.
Another point to mention is that the same amount of time, if not
more, is required to perform the traditional double-antibodysandwich (DAS) assays.
• “The graphics and text were well balanced, and the graphics
aided a great deal in trying to understand the content of the
presentation.”
– Thank you. I try to include motion into my presentation to help the
audience.
Rebuttal
• “The speaker covered all relevant topics from an
introduction of the research to how it can be
furthered. It might have been nice to have heard
some of the opposition to such research and or
more research in the same field, but overall the
presentation was well balanced and informative.”
– Thank you, I was struggling to keep the information
too excessive and thus did not include more research
in the same field or others points of view
G1
Review Nanosensing, Microtubules
by Edson Bellido
The presenter described what are bio-nanosensors and microtubules. She
described how the microtubules are formed within the cell. She explained what is
the dynamic instability and what molecules affect this behavior of the
microtubules. She explained how the microtubules are being used as bionanosensor transporter. She explained how the kinase moves the microtubule to
the cell boundaries and allow us to detect analytes captured within the cell.
Something missing was the details about the
experimental procedures, most likely because of the
short time for the presentation. those details are
very important factor to analyze and determine the
accuracy and reproducibility of the system. Other
factor to study will be how the addition of those
functionalized MTs and the quantum dots will affect
the cellular response. If it affects; quantitatively how
this response change the accuracy of the measure?. http://www.cancerquest.org/images/microtubules.gif
G2
Review Nanosensing, Microtubules
by Alfredo Bobadilla
Review of Nanosensing & Microtubules
• Microtubules are biological nanostructures. They are part of the
cytoskeleton which actually look like the nervous system of the
biological cell and they indeed transmit vibrational signals, perform
mechanotransduction, own a highly dynamic behavior because of
self-assembly dynamic process (dynamic instability) and have been
found to be involved in cell decision-process mechanisms as well as
in memory & learning functions.
• It was shown in the lecture how functionalized microtubules can be
used for nanosensing purposes and as a nanotransporter.
• An additional interesting point not discussed during the presentation
is how can we exploit the ‘dynamic’ self-assembling properties
intrinsic to microtubules for nanosensing and nano-engineering.
Alfredo D. Bobadilla
G4
Microtubules Summary and review
Diego A Gomez-Gualdrón
Microtubules
•Microtubules (MT) are part of the
cytoskeleton and posses a diameter of ~ 25
nm, and a length of ~ 250 nm
•Microtubules (MT) can transmit signals as
well as transport substances within the cells
•They are formed by a ‘polymerization
mechanism’
Functionalizing a microtubule can make it
suitable as a fully-biologically-constitued
nanosensor
Sensing Mechanism
• It was shown that functionalizing a MT with an antibody
allows for specific binding of the analyte.
• The interaction of kinesin with the MT enables the transport
of the analyte to tagging and detection stages
MT
Detection occurs through
fluorescence
Comments
• The presentation used a lot of didactic elements
to convey the information and it was general a
very good presentation. Nonetheless, it was
perhaps too much material for a short time
• I could see that the time frames relevant to the
technique are quite large, which would create
inconveniences for real time measurements and
monitoring and quick action expected from pointof-care approaches.
G5
Review Nanosensing, Microtubules
by Norma Rangel
Nanosensors: Microtubules
By Mary Coan
• Mary presented the use of microtubules as
nanosensors, basic concepts, production of MTs,
stability and alternative applications were shown.
Also
• Mary used great animations that helped for a
better understanding of the Bio-Nanosensor
Transporter concept.
• I particularly like the criticism given to the paper,
where she mentioned some missing details in the
paper about the stability of the MTs and of course
the toxicity of the MTs, but also including
potential future work to further develop portable,
cheap and more sensitive and stronger sensors.
G6
Review Nanosensing, Microtubules
by Jung Hwan Woo
Questions
• What is the mechanism to the selective inhibition of
the rapid dividing cells?
• Is there a way for the MT’s to differentiate a cancer
cell from a normal cell that is rapidly dividing, for
example, cells on a wounded skin?
• Has there been animal tests on the effectiveness of
the MT’s on cancer cells?
• What was the method used to determine that the
MT’s selectively inhibited rapidly dividing cells?
Jung Hwan Woo
U6
Review Nanosensing, Microtubules
by Group U6
By Mary Coan
Chemical Engineering
Reviewed by: Group U6 - Pavitra Timbalia,
Michael Trevathan, Jared Walker



The speaker maintained a appropriate tone which was easily
understandable.
She maintained eye contact with the audience and didn’t not
read off the slides.
She seemed confident and knowledgeable of the subject of
the presentation.



There was a lot of material covered in the slides, perhaps to
fast to comprehend in the amount of time given. It became
hard to follow at some points.
The graphics and text were well balanced, and the graphics
aided a great deal in trying to understand the content of the
presentation.
The speaker covered all relevant topics from an introduction
of the research to how it can be furthered. It might have been
nice to have heard some of the opposition to such research
and or more research in the same field, but overall the
presentation was well balanced and informative.