Surface stress cantilever sensing used for biological and chemical species

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15 Νοε 2013 (πριν από 3 χρόνια και 8 μήνες)

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Surface stress

cantilever sensing used for
biological and chemical species


L. Magnan.

Department of Mechanical Engineering (REU), Iowa State University, Ames, Iowa
50011, USA


Abstract:

In this report, we present a cantilever sensor capable of static and dynamic sensing with
high sensitivity and one that can be used in a liquid environment without
drastically
altering the setup of the system. A Mach
-
Zehnder type interferometer is combine
d in the
device to allow alteration of the divided light waves
used in determining the
measurable
interference signal. Detection of biological and chemical species in a liquid medium was
determined using the
deformation of the sensing cantilever

from a
st
atic

standpoint. The
corresponding relationship among

cantilever

deformation

and the
path length difference

was also verified.
Static deformation was also
confirmed

from
the concepts of optics and
computer
technology
.
Utilizing the concepts of cantilever deflection and interferometry,
we demonstrate sensing capabilities of surface stress sensing
in and out of a liquid
medium.

The deformation of the sensing cantilever with respect to the reference
cantilever creates a m
easured response which is insensitive to most environmental
instabilities
for enhanced accuracy
.


Introduction:

Mi
c
r
oc
antilever sensi
ng and the search for the ideal
cantilever sensor have been an important
topic among researchers.
Highly

sensitive and simple means of measuring cantilever deflections
is important
mainly
because of its applications. Cantilever deflections are used
for

Atomic Force
Microscopy (
AFM
)
,
Scanning Tunneling Microscopy (
STM
)
, various drug detections, DNA
recogniti
on, as well as the indicat
ion of proteins, antibodies,

antigens
, and cancerous cells
.
New
sensing innovations generally
lead to an increase in sensitivit
y

and a smaller means of measuring
cantilever beam deflection.
W
e present
an interferometry system th
at consists

of various optical
components with

dual cantilevers
, a sensing and referencing pair. This
allows

a compact
sensitive
setup capable
of static and dynamic sensing
in and out of a liquid medium.

Currently, there are dozens of ways to appl
y
micro
cantilevers as transducers to

measure
deformation or
deflection.
Lavrik et al [1]
used
a single cantilever

that

could be used where a
single beam of light is reflected and measured.
Figure 1 provides a visual depiction of the setup.


Figure
1
: Single cantilever beam deflection [1]

The single cantilever sensor consists of a cantilever beam and a means of recording the
deformation that occurs within the cantilever. To determine that deformation, a
single
laser and
a position sen
sitive photodetector are used. Essentially, the laser shoots a beam of light toward
the reflective coated cantilever where that light then reflects toward the photodetector. As the
cantilever deforms, the light
changes

positions from where the photodetec
tor initially sensed the
beam of light. That change
provides

the

needed results which can then be used to determine the
change in deflection of the cantilever beam. The advantages of this single cantilever and optical
be
am deflection technique are a
good

dynamic response
with high

precision, as well as a

high
reliability. Additionally, there is an
enhanced

readout efficiency and reduction of size of the
system. On the contrary, the disadvantages are that the system may easily be affected by
electric, el
ectromagnetic, or acoustic fields such as a person talking. Likewise, the system may
also be affected by thermally induced noise and temperature
variations
. The other problem with
the single cantilever

technique is that the distance between the cantileve
r and position sensitive
pho
todetector is relatively large.
Since the sensitivity is proportional to the distance between the
photodetector and the cantilever components, the greater the distance the more sensitive the
system is to nanoscale cantilever de
formation.
The system is becoming smalle
r, but still not
small enough
where
a great amount of sensitivity

exists
.
Although the cantilever and its
components are small, the m
eans of measuring the
deformation of the beam is not. This large
distance also c
ontributes to a loss of intensity and directionality of the reflected optical beams,
providing imprecise

and inaccurate

results.


Second, a single cantilever could be used with a reference and sensing beam of light as
presented in Cunningham et al [2].
As

in Fig.

2, a
fter the coherent beam of light is split in two,
one beam reflects off the base of the cantilever and the other reflects
off the tip of the cantilever.


Figure
2
:
Dual cantilever beam deflection [2]

On the cantilever base, there will be no measurable deflection which establishes a reference
point. However, on the cantilever tip, there will be measurable deflection which establishes a
sensing point. Through interferometry, the measurable phase change

between the reflected
reference beam and the sensing beam can be determined and results can be
established
. The
advantages of this technique include less sensitivity to outside noise, an overall smaller device,
as well as a resolution increase.
In oppos
ition
, the primary disadvantage is that this concept is
hard to apply to commercial microcantilevers. This set up works well with larger cantilevers, but
not with

the micromachined, commercially

mass produced,

cantilevers.

Third, a
differential cantilever

system may

be used where two beams of light are aimed at a
reference and sensing cantilever pair as explained by Shrotriya et al [3]. The differential surface
stress sensor consists of two similar cantilevers: a reference cantilever and a sensing cantile
ver.

Details

of the setup

can be seen in Fig
.

3
.


Figure
3
:
Optical differential surface stress sensor [3]

This technique requires full understanding of path length difference as well as phase changes and
constructive/destructive

interference. This technique measures the cantilever deflection using
concepts of
fiber optic interferometry
. The
advantages of using dual cantilever beams essentially
enhance

the results. It decreases accuracy among unwanted factors and yet increases
accuracy
among the wanted, sought
-
after, results. Basically the main improvements eliminate
environmental
instabilities

and disturbance, any changes in pH, and atmospheric fluctuations
such as temperature change. Sensitivity and resolution are also incre
ased with this technique.
On the contrary, the disadvantages of using this two beam technique with fiber optics are
difficult
lens alignment factors and an overall more complicated system. Measurements
associated with

this technique and

DNA hybridization, however, are not dependent of the
distance between the cantilever components and the photodetector. This provides an overall
simplified mircocantilever sensing device applicable to read world situations.


Principle:

There are two main
types of cantilever sensing: static sensing and dynamic sensing. Surface
stress sensing results in a static or fixed deflection of the cantilever beam. The cantilever is
coated with
receptor

molecule
s that are

attracted

to analyte

molecule
s

being sensed.

As the
number of molecules on the cantilever increases, and whether or not the molecules share an
attractive force, the cantilever will deflect. The coated surface may decrease or increase in
surface area

due to the binding of chemicals or biological sp
ecies

and cause the measurable fixed
beam deflection.

Tensile surface stress
,

due to

the

attraction
among

molecules
,

leads to upward

de
formation

of
the cantilever beam
and can be seen in Fig. 4A
.

In contrast,

c
ompressive surface stress
,

due to
the repulsion
among

molecules
,

leads to downward def
ormation

of the cantilever beam

and is
provided in Fig. 4B

[5].
Regardless of the deflection of the cantilever beam, the deformation
of
this type
remains

fixed.


Figure
4
:
Stati
c mode de
flection (A) Tensile

(B)
Compressive

[4]

Mass sensing results in a dynamic or vibrant deflection of the cantilever beam. Similar to the
static mode, the cantilever during the dyn
amic mode is again coated with
receptor

molecule
s that
are

attracted to
analyte

molecule
s

be
ing

sensed.
Here the beam vibrates at its resonant frequency
and while molecules attach themselves to the cantilever, the frequency will decrease.
A
schematic of the dynamic mode is provided in Fig. 5.
That decrease in
the frequency is
proportional

to the addition of the mass of the molecules. If performed in a liquid medium, the
frequency will be dampened and the sensitivity may decrease as compared to an air medium.

Although the frequency is significantly dampened, t
he differential bending of the sensing and
reference cantilevers are expected to overcome this problem.


Figure
5
:
Dynamic mode deflection [4]

The two novel

types of interferometry include Michelson and Mach Zehnder interferometry
.
The

Michelson, as provided in Fig.6
, consists of a coherent light s
ource, a half
-
silvered mirror,
two

reflective
mirrors, and a photodetector.
Basically, the light waves split, recombine with a
phase change, and reach the photodetector for sensing.
Wh
en the two beams reach the
detector,
the
y

interfere and create a fringe pattern of light and dark rings or, in some cases,
a linear
pattern
. This fringe pattern correlates precisely with the additional wavelength created by
moving mirror A or mirror B forward or backward.
Constructive interference will occur when
the separated light waves differ by a whole number of wavelengths

and will res
ult in the bright
patterns
. In contrast, destructive interference will occur when the two waves differ by a half
wavelength

and will show up as the dark areas on the fringe
. For most purposes, the geometry of
the Mach

Zehnder interferometer is more usefu
l and more applicable to experiments.


Figure
6
:
Michelson type interferometer [
8
]

For the Mach
-
Zehnder interferometer, the collimated light source is split and two beams are
sent to two detectors. Unlike the Michelson technique,

the Mach
-
Zehnder is not folded upon
itself and each individual be
am may be manipulated however

desire
d
. This manipulation can
exist as light wave plane changes as well as a change in speed of the wave

and is an important
factor for our technique. The in
terferometer provided in Fig. 7, is used to measure the phase
shift of a thin opaque sample such as glass. The glass would be placed before either mirror and a
relative amount of light entering the detectors would allow a calculation of the resulting phas
e
shift. This technique requires precise alignment and understanding of
refraction indices as well
as phase

shifts.

Our setup essentially uses one mirror or the reflective surface of the cantilevers,
but the concepts of the device remain identical.


Figure
7
:
Mac
h
-
Zehnder type interferometer [9
]

Implementation:

The
experimental setup

is
provided in Fig. 8

along with a simplified schematic
.




Figure
8
:
Experimental Mach
-
Zehnder type interferometer

(L. Magnan 2011)

The first step in the alignment process was to secure the
collimation

lens

(Thorlabs: F230FC
-
8)

and the
fiber coupled laser source (Thorlabs: S1FC635)

for a consistent
steady
light source.
Once secure, and the placement of the light beam

was marked on the back of the bookshelf

for
precise
alignment
,
the polarized lens

(Model unknown
)

and the beam displacer
(
Thorlabs:
BD27
)
were added. The purpose of the polarized lens is to polarize the light into one plane while the
beam displacer

separ
ates the incoming beam into two beams on different planes 90 degrees
apart.

The next step was to cancel out one of the two beams produced by
BD27
. This was
accomplished by manipulating and rotating the polarized lens.

Once one axis was cancelled out,
the

Soleil
-
Babinet Compensator (
Thorlabs: SBC
-
VIS) was placed on the rail. The rotation
locking thumb screw
dial was then
loosened and the SBC
-
VIS was turned
to
a horizontal
position keeping

only one beam of light on the bookshelf.

This was performed to establish an
axis on the polarizer.

Finally, the polarized lens was turned 45 degrees from the original position

to allow equal
intensity beams out of the BD27

and the other parts were placed on the
main rail
. The liquid cell

(Manu
factured at ISU)
, the Wollaston Prism (
Thorlabs: WP10
),
the concave lens (Model
unknown),
and the photodetector
s

(Thorlabs: DET110)
were added respectively.

These
components, among the aforementioned parts, were all

mounted on
Newport
po
sts,
rails
, and
ra
il
-
to
-
post attachments
.
A few
Thorlabs
rail
-
to
-
post attachments were modified for this setup

as
well
.

The WP
10

was oriented the same as the polarized lens, 45 degrees with respect to the SBC
-
VIS

and
BD27
. The purpose of this orientation is to take the
interfered l
ight waves and separate
them into two

interfered beams which reach each photodetector
. Since the tilt of the prism is 45
degrees, it will take the
combine the
horizontal and vertical components of the incoming beam

which is where the interfere
nce takes place.


The main significance of the Soleil
-
Babinet Compensator is to change the plane of the light
waves as well as create a slow wave axis with respect to the fast axis. Additionally, the
rotational dial on the end may

be manipulated to determ
ine the length of the wave length of the
laser light.

The alignment procedure for our experiments consisted of several steps. With the orientation
of the optical components correct, the first step was to insert the cantilevers and align the two
light beam
s on the tip of the cantilevers.
This was performed with the laser intensity at










. Figure 9

shows the alignment of the cantilevers.


Figure
9
: 250x magnification of cantilever alignment

(L. Magnan 2011)

If the canti
levers were not centered on the light beam and roughly





apart, the cantilevers

were pulled out and remounted on the cantilever holder.

Once in place, the cantilevers were
lower
ed

to the cantilever base and the laser intensity was turned to approximately





.
Next, a concave lens (Thorlabs: LMR05) was placed in one of the detector posts to truly align
both reflected beams for a fringe pattern.
To find the fringe pattern, the

cantilever mounts had to
be moved side to side and forward and backward respectively until
the desired fringe appeared
.
The
next

step consisted of r
ising the cantilev
ers back onto the tips and, with the same intensity as
the previous step, the SBC
-
VIS was

turned approximately one wavelength and verified with
LabView.

For the final alignment step, the buffer was added into the liquid cell and the
wavelength was again checked with the SBC
-
VIS movement.

The rest of the experimental
procedure is explained in

the second validation.


Validation:

To validate this
arrangement
, test
s

were performed in a laboratory setting.
The first
validation was
to test the SBC
-
VIS

movement

and
check
whether

one wavelength, as determined
previously
by
the
calibration

steps followed in the manual (Thorlabs: SBC User Guide)
,
corresponds to one w
avelength on the computer imaging program

(LabView)
.
To begin the
experiment,
the beam was focused
on a flat mirror, repres
enting the reflective surface of

the
cantilevers. The
n, in .
5
00
mm increments,

starting from 0.000mm,

the SBC
rotational dial

was
rotated outward until 19.000mm. By recording the SBC values with respect to the values on the
computer, a graph of intensity vs. SBC movement could be constructed

shown in Fig. 10
.

T
his
experiment was performed 3
times

from 0.000
-
19.000mm and 3 times from 19.000
-
0.000
.


Figure
10
:
Wavelength verification of SBC
-
VIS movement with respect to intensity output

with mirror

A second validation was performed usi
ng the cantilevers, rather than a mirror

and the buffer
was inserted in the liquid cell
.
Again, in .500mm increments, the SBC rotational dial was rotated
outward until 19.000mm.
It was also performed once rotating the dial inward from 19.000mm to
0.000mm

so an average could be determined. Fig
ure 11

is the average of the two

computer
determined results.


Figure
11
:
Wavelength verification of SBC
-
VIS movement with respect

to intensity output in buffer

With the reflection being off

the cantilevers, the unfiltered intens
ity reading on LabV
iew
fluctuated so hand recordings were not possible. Rather, a different technique was attempted
where the SBC value was changed quickly and held constant for 5 seconds to establish a pattern
on th
e computerized graph. From the graph, the results could be picked out and a new graph
could be constructed.

0
1
2
3
4
5
6
7
0
5
10
15
20
Intensity Output (V)

SBC
-
VIS Dial Movement (mm)

Left Output
Right Output
0
0.1
0.2
0.3
0.4
0.5
0.6
0
5
10
15
20
Intensity Output (V)

SBC
-
VIS Dial Movement (mm)

Left Output
Right Output
Once all necessary results were obtained and recor
ded, the SBC was rotated to a

position
where the

intensity of the both waves was

equal. At this
point, the cantilever deflection will be
the most sensitive and responsive

to the sensing cantilever’s deflection.

Finally, the setup was ready for introduction of
mutant
protein solution

(MLCN2
-
R103A/K147A)
.
The first injection contained




and produ
ced a solution with a final
concentration of
5nM
mutant

protein
. The second injection contained
100µL
again and created a
solution with a final concentration of

50nM
mutant
protein
.

Likewise, the

third injection
contained
100µL

and produced a final concentrated solution

of 300nM
mutant protein. 30
minutes were allotted after each injection to ensure a reaction if capable. See Fig. 12 and Fig. 13
for the results on the mutant protein and non
-
specific binding. The small surface

stress change
toward the end of the experiment is solely due to the negligible intermolecular reactions taking
place on the cantilevers surface.

After the
non
-
specific binding

experiment

was finished
, the sensing and reference cantilevers
were heated in d
eionized water at





to regenerate the protein sequence. The regeneration
allows the sensing cantilevers to be used a number of times (Baker, Lai et al. 2006) and each
cantilevers could be used for at least three sensing experiments.

For the
specific
binding

experiment, we introduced a wild
-
type protein into the
liquid cell
solution (MLCN2)
. The first injection contained




and produced a solution with a final
concentration of
5nM
wild
-
type
protein. The second injection contained 100µL
again and
created a solution with a final concentration of

50nM
wild
-
type
protein.

Likewise, the

third
injection contained 100µL

and produced a final concentrated solution

of 300nM
wild
-
type
protein. 30 minutes were allotted after each injection to ensure a reacti
on if capable. See Fig. 12
and Fig. 13 for the results on the wild
-
type protein and specific binding. Notice the reaction and
cantilever deformation happens soon after the injection

and then remains relatively steady
.
After
the

each 30 minute interval
,
the SBC was rotated
back to the
position where the intensities
are
equal.
This was done to ensure maximum sensitivity of the sensing cantilever’s deformation.

From these results
, the path length difference, or th
e amount of beam deflection, that

took
place from

the reaction can be determined. In addition, a relationship between surface stress and
concentration can be composed.



Figure
12
:
Experimental data for specific and non
-
specific protein binding



Figure
13
:
Experimental data for wild
-
type and mutant protein

Discussion/Conclusion:

In sum, the search for the ideal transducer continues.
N
ew innovations generally lead to
better sensitivity and a smaller means of measuring the beam defl
ection. In our case, static and
dynamic responses may be measurable with the same device. A measurement may also be taken
from a liquid medium where the dampening of the frequency has little to no effect.

From the
two experiments in this report, we are
able to conclude that the injection of the mutant protein
caused little to no cantilever deformation. However, the injection of the wild
-
type protein caused
relatively a good amount of cantilever deformation. Also the specific binding of the wild
-
type
pr
otein causes tensile and compressive stress on the cantilever surface.
The Mach
-
Zehnder type
interferometer keeps the system compact while the dual cantilevers hinder surrounding
vibrations and thermal noise. This system may also remain highly sensitive
and useful toward
further applications

and is amendable toward insertion in a MEMS device
.


Future Work:

Although the surface stress sensing was included in this report, mass detection
was not. In
the fut
ure, this device will test

mass detection from

a dy
namic standpoint. The

concepts
explaining mass detection
are
available
, but
results regarding

mass
addition are

difficult

to obtain

in a liquid environment due to the dampening effect. Our setup, composed of the differential
cantilevers,

should gain back

some of the sensitivity
lost through dampening
.

Additional
substances and molecules may also be tested with this device such as DNA hybridization and
other protein sequences.


Ethical Implications:

Throughout this research, there were many contemporary
ethical issues and obstacles
addressed.

The novel principles such as voluntary participation, informed consent, and risk of
harm were taken into consideration.
Due to the equipment in the laboratory, such as the laser
used in this report, consideration f
or my safety as well as the safety of others was always in
mind.
Another important consideration during our experiments was computer usage. It would
be great if everyone could have their own computer, programs, and laboratory equipment. Since
the equipm
ent is expensive, sharing was necessary

and sometimes difficult. During our
experiments, the lights in the laboratory had to be shut off to reduce error in the results. This
was also difficult because of the others in the lab. In the end, everyone worke
d out a system that
worked for
all
.
Since all research is unique, n
o set of ethical standards can be established or

can
possibly
prelude

every ethical implication.
That is why it is important to take all research
differently and consider all possible eth
ical implications in a serious manner

as done for this
research
.


Acknowledgements:

This research was supported by the Iowa State University REU Program along with K. Kang
and P. Shrotriya. The author would like to thank the program and its members for
the materials,
helpful discussions, and assistance in design.


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