400µm 12mm Steps Towards Mass Production - Stackspx491tp4561/SJ Osterfeld...Layer A: PDMS...
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Layer A: PDMS Microfluidics Testing and Protocol Optimization Testing and Protocol Optimization Steps Towards Mass Production Steps Towards Mass Production Sebastian J. Osterfeld Sebastian J. Osterfeld 1 1 , , Shu Shu - - Jen Han Jen Han 1 1 , Stefano Caramuta , Stefano Caramuta 2 2 , , Liang Liang Xu Xu 1 1 , , Heng Heng Yu Yu 2 2 , Jin Xie , Jin Xie 3 3 Shouheng Shouheng Sun Sun 3 3 , , Nader Nader Pourmand Pourmand 2 2 , Shan X. Wang , Shan X. Wang 1 1 1. Stanford University 1. Stanford University 2. Stanford Genome Technology Center 2. Stanford Genome Technology Center 3. Brown University 3. Brown University (Project funded by DARPA) (Project funded by DARPA) Testing and Fabrication of the MagArray Testing and Fabrication of the MagArray ® ® Biochip Biochip Our aim is to repeatedly test prototype chips with a variety of Our aim is to repeatedly test prototype chips with a variety of nanoparticles nanoparticles , biological molecules, and various , biological molecules, and various experimental protocols, to determine and improve the performance experimental protocols, to determine and improve the performance characteristics of the MagArray. For maximum characteristics of the MagArray. For maximum flexibility, each chip needs to be individually functionalized w flexibility, each chip needs to be individually functionalized w ith ith biomolecules biomolecules . As a result, the prototype chips employ . As a result, the prototype chips employ sensors on a freely accessible surface, and macroscopic fluidics sensors on a freely accessible surface, and macroscopic fluidics are employed instead of are employed instead of microfluidics microfluidics to deliver the to deliver the reagents during the measurement. reagents during the measurement. Left: A prototype MagArray4 chip, made Left: A prototype MagArray4 chip, made at Stanford. Inside the well are two freely at Stanford. Inside the well are two freely accessible arrays, each containing 16 accessible arrays, each containing 16 individually addressable sensors. At the individually addressable sensors. At the Stanford Genome Technology Center, the Stanford Genome Technology Center, the 16 sensors on the left are manually 16 sensors on the left are manually functionalized with a different functionalized with a different biochemistry than the 16 sensors on the biochemistry than the 16 sensors on the right. Additional sensors, in this case 32, right. Additional sensors, in this case 32, are covered with epoxy and serve as an are covered with epoxy and serve as an absolute control. absolute control. 25mm Right: After functionalization, the Right: After functionalization, the chip is returned to Stanford chip is returned to Stanford University for measurements. During University for measurements. During the prototype experiments, the prototype experiments, macrofluidics macrofluidics are employed to are employed to repeatedly transport the reagents to repeatedly transport the reagents to and from the chip. Not shown are the and from the chip. Not shown are the readout electronics and magnetic readout electronics and magnetic field generators for the chip. field generators for the chip. While the prototype chips reach maturity, we also work to While the prototype chips reach maturity, we also work to to to ensure that the functionalization protocol and experimental ensure that the functionalization protocol and experimental procedure can be quickly migrated to a mass procedure can be quickly migrated to a mass - - produced version of the MagArray. This effort encompasses three produced version of the MagArray. This effort encompasses three critical critical areas: Whole areas: Whole - - wafer robotic biofunctionalization, alignment wafer robotic biofunctionalization, alignment - - and topography tolerant and topography tolerant microfluidics microfluidics for reagent transport, for reagent transport, and mass and mass - - producible producible microfluidic microfluidic interconnects that do not require the drilling of holes. All th interconnects that do not require the drilling of holes. All th ese three areas are designed ese three areas are designed to become integral parts of the manufacturing process. to become integral parts of the manufacturing process. Whole Whole - - Wafer Robotic Biofunctionalization Wafer Robotic Biofunctionalization Mass Mass - - Producible Producible Microfluidic Microfluidic Interconnects Interconnects Sensors 1-16: DNA or Antigen1 Sensors 49-64: Bare or Antigen2 The Prototype MagArray4 Chip The Prototype MagArray4 Chip Representative Functionalization and Representative Functionalization and Experimental Protocol Experimental Protocol Magnetic nanoparticle w/ Streptavidin Antigen 1: Ferritin Biotin Antibody (Anti-Ferritin) Antigen 2: BSA Nanoparticle Solution Sensors 17-48 Sensors 49-64 Sensors 1-16 Epoxy Antibody Solution Sensors 17-48 Sensors 49-64 Sensors 1-16 Epoxy BSA Solution Ferritin Solution Sensors 17-48 Sensors 49-64 Sensors 1-16 Epoxy Functionalization 1. The antigens are applied selectively Measurement Results Measurement Results Shown on the right is a measurement curve that was Shown on the right is a measurement curve that was recorded as MACS recorded as MACS nanoparticles nanoparticles , coated with , coated with streptavidin streptavidin , , were binding to a MagArray4 chip. Some sensors on the were binding to a MagArray4 chip. Some sensors on the chip were bare. These resulted in the yellow signal curve chip were bare. These resulted in the yellow signal curve with a final value of ca. 1 with a final value of ca. 1 μ μ V. V. Other sensors on the same chip were functionalized with Other sensors on the same chip were functionalized with single stranded single stranded biotinylated biotinylated DNA. These sensors, in red, DNA. These sensors, in red, show a rapid increase in signal to ca. 38 show a rapid increase in signal to ca. 38 μ μ V as soon as the V as soon as the nanoparticles nanoparticles are applied around t = 25 minutes. Thus, the are applied around t = 25 minutes. Thus, the nanoparticles nanoparticles are clearly revealing the presence of DNA on are clearly revealing the presence of DNA on the chip with a signal to noise ratio of ca. 31 dB. The signal the chip with a signal to noise ratio of ca. 31 dB. The signal rise time of the red curve can also give insights into the rise time of the red curve can also give insights into the biotin biotin - - streptavidin streptavidin binding kinetics. binding kinetics. Note that the washing the chip with Note that the washing the chip with deionized deionized water (H2O) water (H2O) leads to temporary shifts in the signal, but it does not leads to temporary shifts in the signal, but it does not remove the remove the nanoparticles nanoparticles . The wash at t = 73 minutes only . The wash at t = 73 minutes only minimally reduces the signal minimally reduces the signal - - MACS are bound strongly. MACS are bound strongly. In the second figure, a different functionalization based on In the second figure, a different functionalization based on antibodies was used. The red curve shows the binding of antibodies was used. The red curve shows the binding of nanoparticles nanoparticles to the to the ferritin ferritin - - functionalized sites. The large functionalized sites. The large signal increase of these sites reveals the presence of an anti signal increase of these sites reveals the presence of an anti - - ferritin ferritin antibody. On the other hand, the lack of a distinct antibody. On the other hand, the lack of a distinct signal increase of the BSA signal increase of the BSA - - functionalized sensors reveals functionalized sensors reveals that no anti that no anti - - BSA antibodies were present. BSA antibodies were present. The rise time of the anti The rise time of the anti - - ferritin ferritin signal is slower than in the signal is slower than in the experiment using DNA, possibly indicating a lower density experiment using DNA, possibly indicating a lower density of available biotin sites on the chip surface. The exact of available biotin sites on the chip surface. The exact relation of signal and binding kinetics is being investigated relation of signal and binding kinetics is being investigated in more detail. in more detail. The electron microscope images on the right, taken after the The electron microscope images on the right, taken after the experiment, show clearly that signal differences correspond experiment, show clearly that signal differences correspond to to nanoparticle nanoparticle coverage differences. coverage differences. Substrate SV SiO 2 SiO 2 Au Au lead Au lead Spotting Needle Upper Right: Upper Right: Shown here is the result of a sample Shown here is the result of a sample run of our functionalization robot. run of our functionalization robot. The robot has placed different The robot has placed different functionalizations functionalizations on different on different sensors as programmed with good sensors as programmed with good accuracy. accuracy. Lower Right: Lower Right: Shown here is a Shown here is a microfluidic microfluidic MagArray4 chip. Instead of the open MagArray4 chip. Instead of the open well that the prototype has, this chip well that the prototype has, this chip features closed features closed microfluidic microfluidic structures that carry the reagents structures that carry the reagents across the chip. Some of the larger across the chip. Some of the larger microfluidic microfluidic features are visible in features are visible in the center of the chip. Note also the the center of the chip. Note also the three three “ “ DNA Align DNA Align ” ” marks that aid marks that aid the robot the robot - - to to - - chip alignment. chip alignment. The robotic functionalization is The robotic functionalization is applied first, and then encapsulated applied first, and then encapsulated by the by the microfluidic microfluidic structures. structures. 400μm 12mm Left: Left: A schematic representation of the A schematic representation of the chip and the robotically controlled chip and the robotically controlled spotting needle used for the spotting needle used for the functionalization. functionalization. In a custom In a custom - - build application, the build application, the functionalization for each of the 64 functionalization for each of the 64 (or more) sensors can be individually (or more) sensors can be individually programmed. A video camera is used programmed. A video camera is used to control the functionalization and to to control the functionalization and to aid in the alignment of the robotic aid in the alignment of the robotic applicator to either a single chip, or applicator to either a single chip, or to an entire wafer. The program is to an entire wafer. The program is highly flexible and adaptable to a highly flexible and adaptable to a variety of chips. It can apply up to variety of chips. It can apply up to 384 different 384 different functionalizations functionalizations – – e.g. e.g. antibodies or genes of interest antibodies or genes of interest – – to an to an arbitrarily sized and positioned arbitrarily sized and positioned substrate. substrate. Shown in the lower image is the Shown in the lower image is the robotic applicator and video camera robotic applicator and video camera during a test run. during a test run. Topography Topography - - and Alignment and Alignment - - Tolerant Tolerant Microfluidics Microfluidics Optical microscope and electron microscope images of the two microfluidic layers combined and separated. The reagents are guided towards the sensor in the large and, since they are on a separate wafer, coarsely aligned PDMS channels. The reagents then traverse the actual sensor in the much narrower and precisely aligned SiO2 channel. Layer B: SiO2 Microfluidics Flow 20 μm To be suitable for high To be suitable for high - - yield mass production, it is important that yield mass production, it is important that the the microfluidics microfluidics are somewhat tolerant to small alignment and are somewhat tolerant to small alignment and process variations. This is achieved with a 2 process variations. This is achieved with a 2 - - layer process. Layer A layer process. Layer A carries large channels and is fabricated on a separate wafer fro carries large channels and is fabricated on a separate wafer fro m m polydimethylsiloxane polydimethylsiloxane (PDMS). Layer B is fabricated right on the (PDMS). Layer B is fabricated right on the MagArray4 substrate wafer from SiO2 with photolithographic MagArray4 substrate wafer from SiO2 with photolithographic precision. Layer B contains the small precision. Layer B contains the small microfluidic microfluidic features, such as features, such as sub sub - - micron channels over the active sensing elements. The overlap micron channels over the active sensing elements. The overlap of the layers is generously sized to accommodate alignment error of the layers is generously sized to accommodate alignment error s s (see finished sample structure on the right). (see finished sample structure on the right). After the MagArray4 substrate wafer has been robotically After the MagArray4 substrate wafer has been robotically functionalized, the support wafer carrying layer A is cold seale functionalized, the support wafer carrying layer A is cold seale d to d to the substrate wafer. Since PDMS is the substrate wafer. Since PDMS is elastomeric elastomeric , it can seal over , it can seal over uneven topography and even accommodate an occasional particle uneven topography and even accommodate an occasional particle – – this would be impossible with the standard procedure of anodic this would be impossible with the standard procedure of anodic bonding. bonding. Sensor Electric lead Connecting the Connecting the microfluidic microfluidic structures to the outside world is one of the engineering structures to the outside world is one of the engineering challenges. We have developed an automatable approach which does challenges. We have developed an automatable approach which does not require the drilling not require the drilling of holes into any of the wafers, which would be laborious and un of holes into any of the wafers, which would be laborious and un wanted for wafer processing. wanted for wafer processing. The main feature consists of cleavable edges that, once removed, The main feature consists of cleavable edges that, once removed, provide openings to the provide openings to the microfluidics microfluidics . These openings can be robotically sealed to a complementary, s . These openings can be robotically sealed to a complementary, s elf elf - - centering centering microfluidic microfluidic holder, which can be a cheap mass holder, which can be a cheap mass - - producible plastic part. On the right, a producible plastic part. On the right, a MagArray prototype with working two MagArray prototype with working two - - layer layer microfluidics microfluidics and and microfluidic microfluidic holders is shown. holders is shown. Microfluidic Holder Microfluidic Channel IV Tubing Large channel, Ca. 10μm deep Ca. 2-100μm wide Sensor Shallow trench, Ca. 200nm deep Ca. 1-5μm wide Layer B SiO2, 200nm thick PDMS Sub-micron channel, Ca. 180nm deep Layer A Layer B Layer A PDMS 10μm thick Support Wafer (glass) Large channel, Ca. 10μm deep Ca. 2-100μm wide Substrate Wafer (silicon) Sensor Shallow trench, Ca. 200nm deep Ca. 1-5μm wide Layer B SiO2, 200nm thick PDMS PDMS Support Wafer (glass) Substrate Wafer (silicon) Sub-micron channel, Ca. 180nm deep Layer A Layer B Layer B Layer B Layer B Microfluidic Layer B PDMS Support Wafer (glass) Substrate Wafer (silicon) Layer A Layer B Partial Cut Layer B PDMS Support Wafer (glass) Substrate Wafer (silicon) Layer A Layer B Insert blade and twist edge off Layer B Microfluidic PDMS Support Wafer (glass) Substrate Wafer (silicon) Layer A Layer B Microfluidic opening Layer B Layer B PDMS Support Wafer (glass) Substrate Wafer (silicon) Layer A Layer B Adhesive Microfluidic Holder Inlet / Outlet DNA Detection (ssDNA w/ Biotin) on the MagArray4 Wafer RB2, Chip 2-2, Nov-29-2005, using Streptavidin Nanoparticles (MACS) -10 0 10 20 30 40 50 0 10 20 30 40 50 60 70 Timeline, Minutes Signal Amplitude, μV ssDNA-Biotin Bare Sensor H2O H2O H2O H2O Nanoparticles <-- SNR ~ 31 dB --> Figure 1: DNA Detection Antibody Detection (Anti-Ferritin w/ Biotin) on the MagArray4 Wafer RB2, Chip X-1, Jan-18-2006, using Streptavidin Nanoparticles (MACS) -10 0 10 20 30 40 50 0 10 20 30 40 50 60 70 Timeline, Minutes Signal Amplitude, μV Ferritin BSA PBS H2O PBS PBS PBS PBS PBS PBS PBS PBS H2O H2O Nanoparticles <-- SNR ~ 21dB --> Figure 2: Antibody Detection The measurement is carried out while the nanoparticles bind Functionalization 2. The biotinylated antibody is applied globally
Transcript of 400µm 12mm Steps Towards Mass Production - Stackspx491tp4561/SJ Osterfeld...Layer A: PDMS...
Lay
er A
: PD
MS
Mic
roflu
idic
s
Test
ing
and
Prot
ocol
Opt
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d Pr
otoc
ol O
ptim
izat
ion
Step
s To
war
ds M
ass
Prod
uctio
nSt
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Tow
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Mas
s Pr
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Seba
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1. S
tanf
ord
Uni
vers
ity1.
Sta
nfor
d U
nive
rsity
2. S
tanf
ord
Gen
ome
Tec
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ogy
Cen
ter
2. S
tanf
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Gen
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hnol
ogy
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3. B
row
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(Pro
ject
fund
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y D
AR
PA
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roje
ct fu
nded
by
DA
RP
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Tes
ting
and
Fabr
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e M
agA
rray
Tes
ting
and
Fabr
icat
ion
of th
e M
agA
rray
®®B
ioch
ipB
ioch
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Our
aim
is
to r
epea
tedl
y te
st p
roto
type
chi
ps w
ith a
var
iety
of
Our
aim
is
to r
epea
tedl
y te
st p
roto
type
chi
ps w
ith a
var
iety
of
nano
part
icle
sna
nopa
rtic
les ,
bio
logi
cal
mol
ecul
es,
and
vario
us
, bi
olog
ical
mol
ecul
es,
and
vario
us
expe
rimen
tal
prot
ocol
s, t
o de
term
ine
and
impr
ove
the
perf
orm
ance
expe
rimen
tal
prot
ocol
s, t
o de
term
ine
and
impr
ove
the
perf
orm
ance
char
acte
ristic
s of
the
Mag
Arr
ay.
For
max
imum
ch
arac
teris
tics
of t
he M
agA
rray
. Fo
r m
axim
um
flexi
bilit
y, e
ach
chip
nee
ds t
o be
ind
ivid
ually
fun
ctio
naliz
ed w
flexi
bilit
y, e
ach
chip
nee
ds t
o be
ind
ivid
ually
fun
ctio
naliz
ed w
ith
ith b
iom
olec
ules
biom
olec
ules
. A
s a
resu
lt, t
he p
roto
type
chi
ps e
mpl
oy
. A
s a
resu
lt, t
he p
roto
type
chi
ps e
mpl
oy
sens
ors
on a
fre
ely
acce
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le s
urfa
ce,
and
mac
rosc
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flu
idic
sse
nsor
s on
a f
reel
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sur
face
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d m
acro
scop
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luid
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are
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are
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tead
of
mic
roflu
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sm
icro
fluid
ics
to d
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he
to d
eliv
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he
reag
ents
dur
ing
the
mea
sure
men
t.re
agen
ts d
urin
g th
e m
easu
rem
ent.
Left:
A p
roto
type
Mag
Arr
ay4
chip
, mad
e Le
ft: A
pro
toty
pe M
agA
rray
4 ch
ip, m
ade
at S
tanf
ord.
Ins
ide
the
wel
l are
two
free
ly
at S
tanf
ord.
Ins
ide
the
wel
l are
two
free
ly
acce
ssib
le
arra
ys,
each
co
ntai
ning
16
ac
cess
ible
ar
rays
, ea
ch
cont
aini
ng
16
indi
vidu
ally
add
ress
able
sen
sors
. A
t th
e in
divi
dual
ly a
ddre
ssab
le s
enso
rs.
At
the
Stan
ford
Gen
ome
Tech
nolo
gy C
ente
r, th
e St
anfo
rd G
enom
e Te
chno
logy
Cen
ter,
the
16
sens
ors
on
the
left
are
man
ually
16
se
nsor
s on
th
e le
ft ar
e m
anua
lly
func
tiona
lized
w
ith
a di
ffer
ent
func
tiona
lized
w
ith
a di
ffer
ent
bioc
hem
istry
tha
n th
e 16
sen
sors
on
the
bioc
hem
istry
tha
n th
e 16
sen
sors
on
the
right
. Add
ition
al s
enso
rs, i
n th
is c
ase
32,
right
. Add
ition
al s
enso
rs, i
n th
is c
ase
32,
are
cove
red
with
epo
xy a
nd s
erve
as
an
are
cove
red
with
epo
xy a
nd s
erve
as
an
abso
lute
con
trol.
abso
lute
con
trol.
25m
m
Rig
ht:
Afte
r fu
nctio
naliz
atio
n,
the
Rig
ht:
Afte
r fu
nctio
naliz
atio
n,
the
chip
is
re
turn
ed
to
Stan
ford
ch
ip
is
retu
rned
to
St
anfo
rd
Uni
vers
ity fo
r mea
sure
men
ts. D
urin
g U
nive
rsity
for m
easu
rem
ents
. Dur
ing
the
prot
otyp
e ex
perim
ents
, th
e pr
otot
ype
expe
rimen
ts,
mac
roflu
idic
sm
acro
fluid
ics
are
empl
oyed
to
ar
e em
ploy
ed
to
repe
ated
ly t
rans
port
the
reag
ents
to
repe
ated
ly t
rans
port
the
reag
ents
to
and
from
the
chip
. Not
sho
wn
are
the
and
from
the
chip
. Not
sho
wn
are
the
read
out
elec
troni
cs
and
mag
netic
re
adou
t el
ectro
nics
an
d m
agne
tic
field
gen
erat
ors f
or th
e ch
ip.
field
gen
erat
ors f
or th
e ch
ip.
Whi
le t
he p
roto
type
chi
ps r
each
mat
urity
, we
also
wor
k to
W
hile
the
pro
toty
pe c
hips
rea
ch m
atur
ity, w
e al
so w
ork
to t
otoen
sure
tha
t th
e fu
nctio
naliz
atio
n pr
otoc
ol a
nd e
xper
imen
tal
ensu
re t
hat
the
func
tiona
lizat
ion
prot
ocol
and
exp
erim
enta
l pr
oced
ure
can
be q
uick
ly m
igra
ted
to a
mas
spr
oced
ure
can
be q
uick
ly m
igra
ted
to a
mas
s --pr
oduc
ed v
ersi
on o
f th
e M
agA
rray
. Thi
s ef
fort
enc
ompa
sses
thr
ee
prod
uced
ver
sion
of
the
Mag
Arr
ay. T
his
effo
rt e
ncom
pass
es t
hree
crit
ical
cr
itica
l ar
eas:
Who
lear
eas:
Who
le-- w
afer
rob
otic
bio
func
tiona
lizat
ion,
alig
nmen
tw
afer
rob
otic
bio
func
tiona
lizat
ion,
alig
nmen
t --an
d to
pogr
aphy
tol
eran
t an
d to
pogr
aphy
tol
eran
t m
icro
fluid
ics
mic
roflu
idic
sfo
r re
agen
t tr
ansp
ort,
for
reag
ent
tran
spor
t, an
d m
ass
and
mas
s --pr
oduc
ible
pr
oduc
ible
mic
roflu
idic
mic
roflu
idic
inte
rcon
nect
s th
at d
o no
t req
uire
the
drill
ing
of h
oles
. All
thin
terc
onne
cts
that
do
not r
equi
re th
e dr
illin
g of
hol
es. A
ll th
ese
thre
e ar
eas
are
desi
gned
es
e th
ree
area
s ar
e de
sign
ed
to b
ecom
e in
tegr
al p
arts
of t
he m
anuf
actu
ring
proc
ess.
to b
ecom
e in
tegr
al p
arts
of t
he m
anuf
actu
ring
proc
ess.
Who
leW
hole
-- Waf
er R
obot
ic B
iofu
nctio
naliz
atio
nW
afer
Rob
otic
Bio
func
tiona
lizat
ion
Mas
sM
ass --
Prod
ucib
le
Prod
ucib
le M
icro
fluid
icM
icro
fluid
icIn
terc
onne
cts
Inte
rcon
nect
s
Sens
ors 1
-16:
D
NA
or A
ntig
en1
Sens
ors 4
9-64
:B
are
or A
ntig
en2
The
Prot
otyp
e M
agA
rray
4 C
hip
The
Prot
otyp
e M
agA
rray
4 C
hip
Rep
rese
ntat
ive
Func
tiona
lizat
ion
and
Rep
rese
ntat
ive
Func
tiona
lizat
ion
and
Expe
rimen
tal P
roto
col
Expe
rimen
tal P
roto
col
Mag
netic
nan
opar
ticle
w/ S
trept
avid
inA
ntig
en 1
: Fer
ritin
Bio
tinA
ntib
ody
(Ant
i-Fer
ritin
)A
ntig
en 2
: BSA
Nan
opar
ticle
Solu
tion
Sens
ors 1
7-48
Sens
ors 4
9-64
Sens
ors 1
-16
Epox
y
Ant
ibod
y So
lutio
n
Sens
ors 1
7-48
Sens
ors 4
9-64
Sens
ors 1
-16
Epox
y
BSA
Sol
utio
nFe
rriti
nSo
lutio
n
Sens
ors 1
7-48
Sens
ors 4
9-64
Sens
ors 1
-16
Epox
y
Func
tiona
lizat
ion
1. T
he a
ntig
ens a
re a
pplie
d se
lect
ivel
y
Mea
sure
men
t Res
ults
Mea
sure
men
t Res
ults
Show
n on
the
rig
ht i
s a
mea
sure
men
t cu
rve
that
was
Sh
own
on t
he r
ight
is
a m
easu
rem
ent
curv
e th
at w
as
reco
rded
as
MA
CS
reco
rded
as
MA
CS
nano
parti
cles
nano
parti
cles
, coa
ted
with
, c
oate
d w
ith s
trept
avid
inst
rept
avid
in, ,
wer
e bi
ndin
g to
a M
agA
rray
4 ch
ip.
Som
e se
nsor
s on
the
w
ere
bind
ing
to a
Mag
Arr
ay4
chip
. So
me
sens
ors
on t
he
chip
wer
e ba
re.
Thes
e re
sulte
d in
the
yel
low
sig
nal
curv
e ch
ip w
ere
bare
. Th
ese
resu
lted
in t
he y
ello
w s
igna
l cu
rve
with
a fi
nal v
alue
of c
a. 1
w
ith a
fina
l val
ue o
f ca.
1 µµ
V.
V.
Oth
er s
enso
rs o
n th
e sa
me
chip
wer
e fu
nctio
naliz
ed w
ith
Oth
er s
enso
rs o
n th
e sa
me
chip
wer
e fu
nctio
naliz
ed w
ith
sing
le s
trand
ed
sing
le s
trand
ed b
iotin
ylat
edbi
otin
ylat
edD
NA
. Th
ese
sens
ors,
in r
ed,
DN
A.
Thes
e se
nsor
s, in
red
, sh
ow a
rapi
d in
crea
se in
sig
nal t
o ca
. 38
show
a ra
pid
incr
ease
in s
igna
l to
ca. 3
8 µµ V
as
soon
as
the
V a
s so
on a
s th
e na
nopa
rticl
esna
nopa
rticl
esar
e ap
plie
d ar
ound
t =
25 m
inut
es. T
hus,
the
are
appl
ied
arou
nd t
= 25
min
utes
. Thu
s, th
e na
nopa
rticl
esna
nopa
rticl
esar
e cl
early
reve
alin
g th
e pr
esen
ce o
f DN
A o
n ar
e cl
early
reve
alin
g th
e pr
esen
ce o
f DN
A o
n th
e ch
ip w
ith a
sig
nal t
o no
ise
ratio
of c
a. 3
1 dB
. The
sig
nal
the
chip
with
a s
igna
l to
nois
e ra
tio o
f ca.
31
dB. T
he s
igna
l ris
e tim
e of
the
red
cur
ve c
an a
lso
give
ins
ight
s in
to t
he
rise
time
of t
he r
ed c
urve
can
als
o gi
ve i
nsig
hts
into
the
bi
otin
biot
in-- s
trept
avid
inst
rept
avid
inbi
ndin
g ki
netic
s. bi
ndin
g ki
netic
s.
Not
e th
at th
e w
ashi
ng th
e ch
ip w
ith
Not
e th
at th
e w
ashi
ng th
e ch
ip w
ith d
eion
ized
deio
nize
dw
ater
(H2O
) w
ater
(H2O
) le
ads
to t
empo
rary
shi
fts i
n th
e si
gnal
, bu
t it
does
not
le
ads
to t
empo
rary
shi
fts i
n th
e si
gnal
, bu
t it
does
not
re
mov
e th
e re
mov
e th
e na
nopa
rticl
esna
nopa
rticl
es. T
he w
ash
at t
= 73
min
utes
onl
y . T
he w
ash
at t
= 73
min
utes
onl
y m
inim
ally
redu
ces t
he si
gnal
m
inim
ally
redu
ces t
he si
gnal
--M
AC
S ar
e bo
und
stro
ngly
.M
AC
S ar
e bo
und
stro
ngly
.
In th
e se
cond
fig
ure,
a d
iffer
ent f
unct
iona
lizat
ion
base
d on
In
the
seco
nd f
igur
e, a
diff
eren
t fun
ctio
naliz
atio
n ba
sed
on
antib
odie
s w
as u
sed.
The
red
cur
ve s
how
s th
e bi
ndin
g of
an
tibod
ies
was
use
d. T
he r
ed c
urve
sho
ws
the
bind
ing
of
nano
parti
cles
nano
parti
cles
to t
he
to t
he f
errit
infe
rriti
n --fu
nctio
naliz
ed s
ites.
The
larg
e fu
nctio
naliz
ed s
ites.
The
larg
e si
gnal
incr
ease
of t
hese
site
s re
veal
s th
e pr
esen
ce o
f an
anti
sign
al in
crea
se o
f the
se s
ites
reve
als
the
pres
ence
of a
n an
ti --fe
rriti
nfe
rriti
nan
tibod
y. O
n th
e ot
her
hand
, the
lac
k of
a d
istin
ct
antib
ody.
On
the
othe
r ha
nd, t
he l
ack
of a
dis
tinct
si
gnal
inc
reas
e of
the
BSA
sign
al i
ncre
ase
of t
he B
SA-- f
unct
iona
lized
sen
sors
rev
eals
fu
nctio
naliz
ed s
enso
rs r
evea
ls
that
no
anti
that
no
anti --
BSA
ant
ibod
ies w
ere
pres
ent.
BSA
ant
ibod
ies w
ere
pres
ent.
The
rise
time
of th
e an
tiTh
e ris
e tim
e of
the
anti --
ferr
itin
ferr
itin
sign
al is
slo
wer
than
in th
e si
gnal
is s
low
er th
an in
the
expe
rimen
t usi
ng D
NA
, pos
sibl
y in
dica
ting
a lo
wer
den
sity
ex
perim
ent u
sing
DN
A, p
ossi
bly
indi
catin
g a
low
er d
ensi
ty
of a
vaila
ble
biot
in s
ites
on t
he c
hip
surf
ace.
The
exa
ct
of a
vaila
ble
biot
in s
ites
on t
he c
hip
surf
ace.
The
exa
ct
rela
tion
of s
igna
l and
bin
ding
kin
etic
s is
bei
ng in
vest
igat
ed
rela
tion
of s
igna
l and
bin
ding
kin
etic
s is
bei
ng in
vest
igat
ed
in m
ore
deta
il.in
mor
e de
tail.
The
elec
tron
mic
rosc
ope
imag
es o
n th
e rig
ht, t
aken
afte
r the
Th
e el
ectro
n m
icro
scop
e im
ages
on
the
right
, tak
en a
fter t
he
expe
rimen
t, sh
ow c
lear
ly th
at s
igna
l diff
eren
ces
corr
espo
nd
expe
rimen
t, sh
ow c
lear
ly th
at s
igna
l diff
eren
ces
corr
espo
nd
to
to n
anop
artic
lena
nopa
rticl
eco
vera
ge d
iffer
ence
s.co
vera
ge d
iffer
ence
s.
Subs
trate
SVSi
O2
SiO
2
Au
Au
lead
Au
lead
Spot
ting
Nee
dle
Upp
er R
ight
:U
pper
Rig
ht:
Show
n he
re is
the
resu
lt of
a s
ampl
e Sh
own
here
is th
e re
sult
of a
sam
ple
run
of o
ur f
unct
iona
lizat
ion
robo
t. ru
n of
our
fun
ctio
naliz
atio
n ro
bot.
The
robo
t ha
s pl
aced
di
ffer
ent
The
robo
t ha
s pl
aced
di
ffer
ent
func
tiona
lizat
ions
func
tiona
lizat
ions
on
diff
eren
t on
di
ffer
ent
sens
ors
as p
rogr
amm
ed w
ith g
ood
sens
ors
as p
rogr
amm
ed w
ith g
ood
accu
racy
.ac
cura
cy.
Low
er R
ight
:Lo
wer
Rig
ht:
Show
n he
re is
a
Show
n he
re is
a m
icro
fluid
icm
icro
fluid
icM
agA
rray
4 ch
ip. I
nste
ad o
f the
ope
n M
agA
rray
4 ch
ip. I
nste
ad o
f the
ope
n w
ell t
hat t
he p
roto
type
has
, thi
s chi
p w
ell t
hat t
he p
roto
type
has
, thi
s chi
p fe
atur
es c
lose
d fe
atur
es c
lose
d m
icro
fluid
icm
icro
fluid
icst
ruct
ures
that
car
ry th
e re
agen
ts
stru
ctur
es th
at c
arry
the
reag
ents
ac
ross
the
chip
. Som
e of
the
larg
er
acro
ss th
e ch
ip. S
ome
of th
e la
rger
m
icro
fluid
icm
icro
fluid
icfe
atur
es a
re v
isib
le in
fe
atur
es a
re v
isib
le in
th
e ce
nter
of t
he c
hip.
Not
e al
so th
e th
e ce
nter
of t
he c
hip.
Not
e al
so th
e th
ree
thre
e ““ D
NA
Alig
nD
NA
Alig
n ””m
arks
that
aid
m
arks
that
aid
th
e ro
bot
the
robo
t -- toto
-- chi
p al
ignm
ent.
chip
alig
nmen
t.
The
robo
tic fu
nctio
naliz
atio
n is
Th
e ro
botic
func
tiona
lizat
ion
is
appl
ied
first
, and
then
enc
apsu
late
d ap
plie
d fir
st, a
nd th
en e
ncap
sula
ted
by th
e by
the
mic
roflu
idic
mic
roflu
idic
stru
ctur
es.
stru
ctur
es.
400µ
m
12m
m
Left:
Le
ft:
A
sche
mat
ic
repr
esen
tatio
n of
th
e A
sc
hem
atic
re
pres
enta
tion
of
the
chip
and
the
rob
otic
ally
con
trolle
d ch
ip a
nd t
he r
obot
ical
ly c
ontro
lled
spot
ting
need
le
used
fo
r th
e sp
ottin
g ne
edle
us
ed
for
the
func
tiona
lizat
ion.
func
tiona
lizat
ion.
In a
cus
tom
In a
cus
tom
-- bui
ld a
pplic
atio
n, th
e bu
ild a
pplic
atio
n, th
e fu
nctio
naliz
atio
n fo
r eac
h of
the
64
func
tiona
lizat
ion
for e
ach
of th
e 64
(o
r mor
e) se
nsor
s can
be
indi
vidu
ally
(o
r mor
e) se
nsor
s can
be
indi
vidu
ally
pr
ogra
mm
ed. A
vid
eo c
amer
a is
use
d pr
ogra
mm
ed. A
vid
eo c
amer
a is
use
d to
con
trol t
he fu
nctio
naliz
atio
n an
d to
to
con
trol t
he fu
nctio
naliz
atio
n an
d to
ai
d in
the
alig
nmen
t of t
he ro
botic
ai
d in
the
alig
nmen
t of t
he ro
botic
ap
plic
ator
to e
ither
a si
ngle
chi
p, o
r ap
plic
ator
to e
ither
a si
ngle
chi
p, o
r to
an
entir
e w
afer
. The
pro
gram
is
to a
n en
tire
waf
er. T
he p
rogr
am is
hi
ghly
flex
ible
and
ada
ptab
le to
a
high
ly fl
exib
le a
nd a
dapt
able
to a
va
riety
of c
hips
. It c
an a
pply
up
to
varie
ty o
f chi
ps. I
t can
app
ly u
p to
38
4 di
ffer
ent
384
diff
eren
t fun
ctio
naliz
atio
nsfu
nctio
naliz
atio
ns––
e.g.
e.
g.
antib
odie
s or g
enes
of i
nter
est
antib
odie
s or g
enes
of i
nter
est ––
to a
n to
an
arbi
traril
y si
zed
and
posi
tione
d ar
bitra
rily
size
d an
d po
sitio
ned
subs
trate
.su
bstra
te.
Show
n in
the
low
er im
age
is th
e Sh
own
in th
e lo
wer
imag
e is
the
robo
tic a
pplic
ator
and
vid
eo c
amer
a ro
botic
app
licat
or a
nd v
ideo
cam
era
durin
g a
test
run.
durin
g a
test
run.
Topo
grap
hyTo
pogr
aphy
-- and
Alig
nmen
tan
d A
lignm
ent --
Tole
rant
To
lera
nt M
icro
fluid
ics
Mic
roflu
idic
s
Opt
ical
mic
rosc
ope
and
elec
tron
mic
rosc
ope
imag
es o
f the
two
mic
roflu
idic
laye
rs
com
bine
d an
d se
para
ted.
The
rea
gent
s are
gui
ded
tow
ards
the
sens
or in
the
larg
e an
d,
sinc
e th
ey a
re o
n a
sepa
rate
waf
er, c
oars
ely
alig
ned
PDM
S ch
anne
ls. T
he r
eage
nts t
hen
trav
erse
the
actu
al se
nsor
in th
e m
uch
narr
ower
and
pre
cise
ly a
ligne
d Si
O2
chan
nel.
Lay
er B
: SiO
2 M
icro
fluid
ics
Flow 20
µm
To b
e su
itabl
e fo
r hi
ghTo
be
suita
ble
for
high
-- yie
ld m
ass
prod
uctio
n, it
is im
porta
nt th
at
yiel
d m
ass
prod
uctio
n, it
is im
porta
nt th
at
the
the
mic
roflu
idic
sm
icro
fluid
ics
are
som
ewha
t to
lera
nt t
o sm
all
alig
nmen
t an
d ar
e so
mew
hat
tole
rant
to
smal
l al
ignm
ent
and
proc
ess
varia
tions
. Thi
s is
ach
ieve
d w
ith a
2pr
oces
s va
riatio
ns. T
his
is a
chie
ved
with
a 2
-- laye
r pro
cess
. Lay
er A
la
yer p
roce
ss. L
ayer
A
carr
ies
larg
e ch
anne
ls a
nd i
s fa
bric
ated
on
a se
para
te w
afer
fro
carr
ies
larg
e ch
anne
ls a
nd i
s fa
bric
ated
on
a se
para
te w
afer
fro
m
m
poly
dim
ethy
lsilo
xane
poly
dim
ethy
lsilo
xane
(PD
MS)
. Lay
er B
is
fabr
icat
ed r
ight
on
the
(PD
MS)
. Lay
er B
is
fabr
icat
ed r
ight
on
the
Mag
Arr
ay4
subs
trate
waf
er f
rom
SiO
2 w
ith p
hoto
litho
grap
hic
Mag
Arr
ay4
subs
trate
waf
er f
rom
SiO
2 w
ith p
hoto
litho
grap
hic
prec
isio
n. L
ayer
B c
onta
ins
the
smal
l pr
ecis
ion.
Lay
er B
con
tain
s th
e sm
all m
icro
fluid
icm
icro
fluid
icfe
atur
es, s
uch
as
feat
ures
, suc
h as
su
bsu
b --m
icro
n ch
anne
ls o
ver t
he a
ctiv
e se
nsin
g el
emen
ts. T
he o
verla
p m
icro
n ch
anne
ls o
ver t
he a
ctiv
e se
nsin
g el
emen
ts. T
he o
verla
p of
the
laye
rs is
gen
erou
sly
size
d to
acc
omm
odat
e al
ignm
ent e
rror
of th
e la
yers
is g
ener
ousl
y si
zed
to a
ccom
mod
ate
alig
nmen
t err
ors s
(see
fini
shed
sam
ple
stru
ctur
e on
the
right
).(s
ee fi
nish
ed sa
mpl
e st
ruct
ure
on th
e rig
ht).
Afte
r th
e M
agA
rray
4 su
bstra
te
waf
er
has
been
ro
botic
ally
A
fter
the
Mag
Arr
ay4
subs
trate
w
afer
ha
s be
en
robo
tical
ly
func
tiona
lized
, the
sup
port
waf
er c
arry
ing
laye
r A is
col
d se
ale
func
tiona
lized
, the
sup
port
waf
er c
arry
ing
laye
r A is
col
d se
ale d
to
d to
th
e su
bstra
te w
afer
. Si
nce
PDM
S is
th
e su
bstra
te w
afer
. Si
nce
PDM
S is
ela
stom
eric
elas
tom
eric
, it
can
seal
ove
r ,
it ca
n se
al o
ver
unev
en to
pogr
aphy
and
eve
n ac
com
mod
ate
an o
ccas
iona
l par
ticle
un
even
topo
grap
hy a
nd e
ven
acco
mm
odat
e an
occ
asio
nal p
artic
le ––
this
wou
ld b
e im
poss
ible
with
the
sta
ndar
d pr
oced
ure
of a
nodi
c th
is w
ould
be
impo
ssib
le w
ith t
he s
tand
ard
proc
edur
e of
ano
dic
bond
ing.
bond
ing.
Sens
or
Elec
tric
lead
Con
nect
ing
the
Con
nect
ing
the
mic
roflu
idic
mic
roflu
idic
stru
ctur
es t
o th
e ou
tsid
e w
orld
is
one
of t
he e
ngin
eerin
g st
ruct
ures
to
the
outs
ide
wor
ld i
s on
e of
the
eng
inee
ring
chal
leng
es. W
e ha
ve d
evel
oped
an
auto
mat
able
app
roac
h w
hich
doe
sch
alle
nges
. We
have
dev
elop
ed a
n au
tom
atab
le a
ppro
ach
whi
ch d
oes
not r
equi
re th
e dr
illin
g no
t req
uire
the
drill
ing
of h
oles
into
any
of t
he w
afer
s, w
hich
wou
ld b
e la
borio
us a
nd u
nof
hol
es in
to a
ny o
f the
waf
ers,
whi
ch w
ould
be
labo
rious
and
un w
ante
d fo
r waf
er p
roce
ssin
g.
wan
ted
for w
afer
pro
cess
ing.
Th
e m
ain
feat
ure
cons
ists
of
clea
vabl
e ed
ges
that
, on
ce r
emov
ed,
The
mai
n fe
atur
e co
nsis
ts o
f cl
eava
ble
edge
s th
at,
once
rem
oved
,pr
ovid
e op
enin
gs t
o th
e pr
ovid
e op
enin
gs t
o th
e m
icro
fluid
ics
mic
roflu
idic
s . T
hese
ope
ning
s ca
n be
rob
otic
ally
sea
led
to a
com
plem
enta
ry, s
. The
se o
peni
ngs
can
be r
obot
ical
ly s
eale
d to
a c
ompl
emen
tary
, sel
fel
f --ce
nter
ing
cent
erin
g m
icro
fluid
icm
icro
fluid
icho
lder
, w
hich
can
be
a ch
eap
mas
sho
lder
, w
hich
can
be
a ch
eap
mas
s --pr
oduc
ible
pla
stic
par
t. O
n th
e rig
ht,
a pr
oduc
ible
pla
stic
par
t. O
n th
e rig
ht,
a M
agA
rray
pro
toty
pe w
ith w
orki
ng tw
oM
agA
rray
pro
toty
pe w
ith w
orki
ng tw
o --la
yer
laye
r mic
roflu
idic
sm
icro
fluid
ics
and
and
mic
roflu
idic
mic
roflu
idic
hold
ers i
s sho
wn.
hold
ers i
s sho
wn.
Mic
roflu
idic
Hol
der
Mic
roflu
idic
Cha
nnel
IV T
ubin
g
Laye
r A
P
DM
S
1
0µm
thic
k
Supp
ort W
afer
(gla
ss)
Larg
e ch
anne
l,C
a. 1
0µm
dee
pC
a. 2
-100
µm w
ide
Subs
trate
Waf
er (s
ilico
n)
Sens
or
Shal
low
tren
ch,
Ca.
200
nm d
eep
Ca.
1-5
µm w
ide
Laye
r B
Si
O2,
200
nm th
ick
PDM
S
Supp
ort W
afer
(gla
ss)
Subs
trate
Waf
er (s
ilico
n)
Sub-
mic
ron
chan
nel,
Ca.
180
nm d
eep
Laye
r ALa
yer B
Laye
r A
P
DM
S
1
0µm
thic
k
Supp
ort W
afer
(gla
ss)
Larg
e ch
anne
l,C
a. 1
0µm
dee
pC
a. 2
-100
µm w
ide
Subs
trate
Waf
er (s
ilico
n)
Sens
or
Shal
low
tren
ch,
Ca.
200
nm d
eep
Ca.
1-5
µm w
ide
Laye
r B
Si
O2,
200
nm th
ick
PDM
S
Supp
ort W
afer
(gla
ss)
PDM
S
Supp
ort W
afer
(gla
ss)
Subs
trate
Waf
er (s
ilico
n)
Sub-
mic
ron
chan
nel,
Ca.
180
nm d
eep
Laye
r ALa
yer B
PDM
S
Supp
ort W
afer
(gla
ss)
Subs
trate
Waf
er (s
ilico
n)
Laye
r ALa
yer B
Parti
al
Cut
PDM
S
Supp
ort W
afer
(gla
ss)
Subs
trate
Waf
er (s
ilico
n)
Laye
r ALa
yer B
Inse
rt bl
ade
and
twis
t edg
e of
f
PDM
S
Supp
ort W
afer
(gla
ss)
Subs
trate
Waf
er (s
ilico
n)
Laye
r ALa
yer B
Mic
roflu
idic
op
enin
g
PDM
S
Supp
ort W
afer
(gla
ss)
Subs
trate
Waf
er (s
ilico
n)
Laye
r ALa
yer B
Parti
al
Cut
PDM
S
Supp
ort W
afer
(gla
ss)
Subs
trate
Waf
er (s
ilico
n)
Laye
r ALa
yer B
Parti
al
Cut
PDM
S
Supp
ort W
afer
(gla
ss)
Subs
trate
Waf
er (s
ilico
n)
Laye
r ALa
yer B
Inse
rt bl
ade
and
twis
t edg
e of
f
PDM
S
Supp
ort W
afer
(gla
ss)
Subs
trate
Waf
er (s
ilico
n)
Laye
r ALa
yer B
Inse
rt bl
ade
and
twis
t edg
e of
f
PDM
S
Supp
ort W
afer
(gla
ss)
Subs
trate
Waf
er (s
ilico
n)
Laye
r ALa
yer B
Mic
roflu
idic
op
enin
g
PDM
S
Supp
ort W
afer
(gla
ss)
Subs
trate
Waf
er (s
ilico
n)
Laye
r ALa
yer B
Mic
roflu
idic
op
enin
gPD
MS
Supp
ort W
afer
(gla
ss)
Subs
trate
Waf
er (s
ilico
n)
Laye
r ALa
yer B
Adh
esiv
eM
icro
fluid
ic H
olde
r Inle
t / O
utle
t
PDM
S
Supp
ort W
afer
(gla
ss)
Subs
trate
Waf
er (s
ilico
n)
Laye
r ALa
yer B
Adh
esiv
eM
icro
fluid
ic H
olde
r Inle
t / O
utle
t
PDM
S
Supp
ort W
afer
(gla
ss)
Subs
trate
Waf
er (s
ilico
n)
Laye
r ALa
yer B
Adh
esiv
eM
icro
fluid
ic H
olde
r Inle
t / O
utle
t
DN
A D
etec
tion
(ssD
NA
w/ B
iotin
) on
the
Mag
Arr
ay4
Waf
er R
B2,
Chi
p 2-
2, N
ov-2
9-20
05, u
sing
Str
epta
vidi
n N
anop
artic
les
(MA
CS)
-1001020304050
010
2030
4050
6070
Tim
elin
e, M
inut
es
Signal Amplitude, µV
ssD
NA
-Bio
tinB
are
Sens
or
H2O
H2O
H2O
H2O
Nan
opar
ticle
s
<-- SNR ~ 31 dB -->
Figu
re 1
: DN
A D
etec
tion Ant
ibod
y D
etec
tion
(Ant
i-Fer
ritin
w/ B
iotin
) on
the
Mag
Arr
ay4
Waf
er R
B2,
Chi
p X-
1, J
an-1
8-20
06, u
sing
Str
epta
vidi
n N
anop
artic
les
(MA
CS)
-1001020304050
010
2030
4050
6070
Tim
elin
e, M
inut
es
Signal Amplitude, µV
Ferr
itin
BSA
PBS
H2O
PBS
PBS
PBS
PBS
PBS PBS
PBS
PBS
H2O
H2O
Nan
opar
ticle
s
<-- SNR ~ 21dB -->Fi
gure
2: A
ntib
ody
Det
ectio
n
The
mea
sure
men
t is c
arrie
d ou
t whi
le th
e na
nopa
rticl
esbi
ndFu
nctio
naliz
atio
n 2.
The
bio
tinyl
ated
antib
ody
is a
pplie
d gl
obal
ly
