Electronic Packaging for Bio-Diagnostic Microfluidics Application

U. Mastromatteo, A. Alzati, R. Brioschi, S. Conoci, M. De Fazio,

P. Magni, A. Maiema, M. Marchi, M. Palmieri, M. Shaw, M. Suardi, F. Ziglioli

ST Microelectronics - Agrate Brianza (ME) - Italy

Amadeo.Maierna@st.com

Abstract

Silicon based semiconductors techniques are creating a solid base for a new category of micro electro
mechanical systems (MEMS) with reliable structures for microfluidic funtions, handling either electrical
signals or mechanical/physical parameters. A particular category of these early developed devices is
constituted by Bio-MEMS, providing electro-microfluidic features in order to integrate in one system all
the functional elements needed for a complete bio-assay response. New packaging architectures are then
coming much more a reality and in particular some simple concept-structures for DNA analysis on a single
silicon device are involved in this bio-tech environment. Some notes on core biological characterizations of
peR and Matrix-Spot detection structures are also present in this paper.

Introduction Bio-Mems Packaging Architecture

The most common definition of Micro-Fluidics The main purposes of an electro-fluidic

physical range is related to the study of
structures that do not permit fluid dynamics
description with conventional theories of fluids,
due to the small dimensions of the involved
elements. This limit is conventionally intended
for channels of a minimum diameter from 5 to
100 11m, for reservoirs in the range from 10
picolitres to 10 microlitres (1). These structures
present dimensional ranges usually involved in
silicon-based MEMS dedicated to handle liquids,
which flows are controlled by the application of
electrical signals to the IC electronic elements,
like Power MOS or simple power-resistors.
Typically a generic microfluidic chip is
characterized by very small dimensions, so an
external fluidic system has to be designed with
the function of facilitate the "fluidic I/O's"
handling, in terms of filling, transfer and
washing of liquid samples and reagents. Some
different techniques can be used in order to
realise the microfluidic package of silicon
devices, but only few are considered applicable
for a good cost-effective result for an under
development product.
packaging architecture is to provide a reliable
and viable system for electrical connections of
the electronic IC, facilitate the fluidic
interconnection with the surrounding lab
environment and prevent the functional device
from handling damage. In the bio-MEMS field
these two features are not sufficient and there is a
third key aspect to face: the bio-protection of the
analysis micro system from the external
environment, in order to prevent cross­
contamination by different source of biological
samples. For DNA handling this constitutes one
of the main challenges for package designers and
assembly process engineers. Surfaces of the
package elements dedicated to fluid transfer have
to face the micromachining from 50 to 200 11m
channels depth. Typically, at cartridge level, the
filling volumes for samples are in the range of
0.5 - 5.0 microliters, therefore the creation of
"dead volumes" is strictly to be avoided. Inlet
holes have to match the pipette shapes with a
exactly, with the goal to avoid openings and
fillets smaller than approx of 1-3 microns,
furthermore DNA handling all the materials
involved in chambers dedicated to PCR
(Polymerase Chain Reaction) or "DNA
replication" needs to be made by means of

Authorized licensed use limited to: STMicroelectronics international NV. Downloaded on July 16,2020 at 08:29:23 UTC from IEEE Xplore. Restrictions apply.
 materials with high PCR biocompatibility (4).
The basic structure we approached for DNA
analysis is shown in the following drawing:

Pout

Silicon Chip PCR Chamber Fig. 3 Flow through Chambers

Fig. I Schematic Lab on chip Inside the microfluidic package four single inlets
will be reached by different pressure flow. In
DNA prob s pot matrix order to evaluate Pout considering channels with a
S
r
defmed section we shall have to combine only

I [111
few equations but with a complex structure like
�
1! i� ++-tt-
.l
_ �� ��:n _
t
S

Fluidi
Area

c
Outlets
the one shown in Fig. 4b, large numbers of
equations are involved, so the easiest way for
design is the simulation approach with CFD
00 techniques, applied to filling channel system

o0 Integrated Heating
shown.

roo 0 0 _������
Resistor

II I
L-J ---,t-+I+_'_'=
'--- _._

Fig. 2 Lab on chip Silicon Chip

All the operations, starting from samples filling,

are usually done manually. Sealing of the inlets,
outlets and other processing volumes are
provided by clamping elements or by cartridge­
level solutions. In the above image is shown the
silicon detection area and readings probe layout
of the lab-on-chip device involved in these Fig. 4a Cartridge schematic
considerations. The silicon device includes an
IC with very simple semiconductor technology;
this will ensure reliability and low-cost process _----..,./ Fluidic Inlet
for the MEMS on high production volumes. Channel Design
-'-U
Micro-Fluidic Channel Dimensioning

The overall structure of the fluidic package is

strongly driven by the geometrical dimensions of
the involved structures, like tanks, reservoirs,
channels and fluidic I/O. In the following
drawing we can identify the inlet filling pressure
Pin, and the outlet pressure Pout or the input value Fig. 4b Fluid inlet design
into the DNA probe detection area, both
parameters are important in order to defme the In order to improve the performance of the
fluid dynamics of the system: package, the fluidic loading flow due to the

Authorized licensed use limited to: STMicroelectronics international NV. Downloaded on July 16,2020 at 08:29:23 UTC from IEEE Xplore. Restrictions apply.
 sample and reagent insertion into the cartridge
have to be facilitated and it is important to Inlet Filling Simulations
recognize the key role of the contact angle of the
liquid on the walls of the fluidic channel. For
lab-on-chip application, involving a procedure
for manual insertion of liquids inside the
package, we consider only low-velocity models,
since the pressures at inlets are substantially
controlled by the pipette system, tunable in the
range of microliters.

Fig. 5 Fluid Inlet Fig. 6a Fig. 6b

Contact Angle = 110° Contact Angle = 30°
Also the parameter for contact angle referred to
aqueous compounds is an important factor in
Results of fluidic simulations shows that the
order to have a good evaluation of filling
filling inside inlets channel with plasma-treated
phenomena. By example, for silicon and glass
surfaces could improve by about 30% the filling
(4), the reference contact angle can be measured,
time, but with manual handling of the fluids this
and considered quite constant from 1 up to 40
will not constitute a real advantage, so the
PCR cycles (standard samples of 25 JlI). In
plasma cleaning from this point of view should
following Fig. 6a is the fluidic simulation for
be avoided, simplifying in this way the
untreated polycarbonate, and in Fig. 6b for the
production flow assembly steps.
same material after 02 plasma treatment,
From the thermal management point of view the
considering the difference introduced in terms of
structure of PCR chamber built by means of
contact angle.
glass structure (shown in fig. 1) is to be intended
. I Wettmg ang e in the polycarbonate package (as shown in fig.
Table 1 MaterIa
4a). The power-on transition for T(set) at 94°C,
Material Untreated With
can be also simulated with different P(diss) on
surface
the heaters, obtaining the following transient
treatment
family. The graphs shows a transient completion
Slicon 61° ± 7.7° 117° ± 5.2° in about 6.5 sec to a value of T(set) ± 1°C.
Glass 49° ± 8.1° 120° ± 6.4°
Polycarbonate 90° ± 9.0° 25°-30°
Makrolon®
PDMS 110° ± 10° 28°-30° ( * )
( * ) evaluated wIth 02 plasma

Authorized licensed use limited to: STMicroelectronics international NV. Downloaded on July 16,2020 at 08:29:23 UTC from IEEE Xplore. Restrictions apply.
 AN For this concept-package the silicon assembly
IEP 20 100S
T�" III.' l' 03 40 solution is the same as a simple chip-on-board
TtMP:::2ct assembly, with the only request of good thermal
TP:l'IY'
TElII'_Oe
..
contact from silicon to the PCR polycarbonate
TE.IlIp_)e - -
" - chamber, (fig8), involving the possibility to
.,
"
include a flexible printed circuit.
VAl)) '"
..
..
..
�-- Flexible Printed Circuit

Elastomer Membrane
Fluidic Module Body

..
OS

.................�Silicon Chip
" " ..
T11'1.E ..�....
Electrical Contact Array
/
Flex Circuit Rigidizer
With Heaters

Fig. 8 section cartridges design

Graph 1 Temperature Transient
As previously mentioned one of the main aspect
Further experimental data confirmed that the for fluidic handling inside electronic packages is
temperature performance of this system in the constituted by the dynamics of the inlet filling.
range 60°C - 95°C is effectively operating a For the system with 2 inlets for reagents we had
control inside T(set) O.5°C, in line with the then to face the 2-liquids mixing. Approaching
performance of a good PCR chamber heating once more with CFD simulation, some
system. This good Temperature range control evaluations have been done (Fig 9a,b,c),
constitutes one of the main advantage of the describing also the dead-volumes evolution
silicon based PCR chamber, built starting from during the reagents loading inside the system.
MEMS techniques. The main fluidic handling phases are as follows:

Cartridge Structure for DNA-PCR Fluid

1. Starting To condition: PCR Chamber is fully
Handling
loaded (Fig 9a light grey volume)

The Polymerase Chain Reaction (PCR) is a

thermal-induced bioprocess for the DNA
denaturation and amplification. Many research
groups and MEMS manufacturer worked around
the improvements of the performance of PCR
chambers in terms of temperature stability and
volume uniformity. The approach used for the
concept device package under development was
designed as in the following draft, showing a
concept fluidic package with 2 inlet on PCR
chamber and 2 inlets for reagents loading.

Valve Housing

Fig 9a

Reagents Loading Inlets

2. By washing liquid insertion into dedicated
inlet, start the liquid flux for PCR Chamber
cleaning (Fig 9b dark grey )

Fig. 7 Cartridge design

Authorized licensed use limited to: STMicroelectronics international NV. Downloaded on July 16,2020 at 08:29:23 UTC from IEEE Xplore. Restrictions apply.

Fig 9b
3. Fluidics transition complete after 600 msec.
with standard pipette pressure simulation,
residual liquids presence at chamber edge result
still present fig 9c
Fig 9c
After the above simulations and design
considerations, the dimensioning of the mixing
chamber can be considered correct in order to
have a good mixing in the time order of about 3
seconds.
Authorized licensed use limited to: STMicroelectronics international NV. Downloaded on July 16,2020 at 08:29:23 UTC from IEEE Xplore. Restrictions apply.
Fig. 9d complete assembly of the fluidic
concept-cartridge and housing on thermo-cycles
equipment.
Assembly Process
From the manufacturing point of view the
introduction of particular surface preparation
process and the DNA matrix spotting on the
dedicated detection area of the chip constitutes a
real difference from the conventional packaging
procedure for microelectronics system:
Packing and Oelivery
Fig 10 Assembly Flow
Biological Characterisation
The p-globin gene from human genomic DNA
was used as target of this model. Both
oligonucleotide primers (BgloF and BgloR) and
probes were designed with the Oligo primer
analysis software, version 6.65, using the p­
globin sequence (Accession no. AY260740). The
Microarray Layout employed for the present
study is reported in fig. 11.
 solution was prepared in 500ml containing O.lX
SSC and 0.1% of SDS..
After the PCR- hyb experiment the chip was read
at the optical reader at 250 ms and analysed. For
each probe have been calculated the following
parameters:
F635 Median: Foreground Median FMed -7 the
median of pixel intensity of fluorescence signal;
B635: Background Mean BM -7 the mean of
LEGEND

• Empty Position

• Sl»tificPr'obe pixel intensity of local background;

• H�idizallion Negallive ContrQI Probe

• Orientmion Probe
Distribution plot and box plot were used for
• H:tbridizllllion Control Probe

11. Microarray Layout on ST's Lab-on-Chip

graphical output.
Fig. The interpretation of results for a positive test of
a qualitative (yes/no) detection of the
PCR mixture was prepared in 50ul of volume betaglobine target gene was done according to
containing the following concentration of DNA the rules reported in the table 1.
template:
1) Experiment a: 0.25 ng/ul (�2xl03 copies/rx). The results obtained for a Limit of Detection of
2) Experiment b: 0.025 ng/ul (�2xlO2 In-Check system through PCR-Hybridization
copies/rx). integrated test by using as DNA target a
3) Experiment c: 0.0025 ng/ul (�2xl01 fragment of Betaglobin gene are below reported
copies/rx).
4) Experiment d: 0.00025 ng/ul (�2 copies/rx). Experiment A: [Input DNA]: 0.25ng/ul
The PCR thermal cycling consisted in: (estimated 2000 copies/rx)
• Step initial: 5 min 95°C
• Cycle Program (35 cycles): 20 sec 95°C, Table 3
45 sec 61°C, 30 sec noc Value Value
Probe Parameter Found Expected Result
Table 2 (a.u.) (a.u.)
Microarray F635
Function Results LoC + 56384
Probe AT683
2/3 probe passed
Median
F635
AT683 F635
AT730 61524 Median passed
Hyb
Median
>50.000
2/3 probe F635
AT 730 Control F635 a.u.
Median> 50.000 a.u. AT776 51579 passed
Probe Median
AT776 F635
MedianBglol -
Bgiol 17681 >lO28 passed
(F635 MedianBglol - F635
Bgiol F635 MedianEMPTY) > MedianEMPTY
lO28 (Cut-off) F635
(F635 MedianBglo2 - MedianBglo2 -
Specific Bglo2 11499 >1028 passed
Bglo2 F635 MedianEMPTY) > F635
Probe
lO28 (Cut-off) MedianEMPTY
(F635 MedianBglo3 - F635
Bglo3 F635 MedianEMPTY) > MedianBglo3 -
lO28 (Cut-off) Bglo3 4lO73 >lO28 passed
F635
MedianEMPTY
The hybridization master mix was prepared in 30
ul of total volume. The Hybridization Step was
Final Result: Positive Test
carried out at 55°C for 3600sec. The washing

Authorized licensed use limited to: STMicroelectronics international NV. Downloaded on July 16,2020 at 08:29:23 UTC from IEEE Xplore. Restrictions apply.
 Experiment B: [Input DNA]: 0.02Sng/ul Final Result: Positive Test
(estimated 200 copies/rx)

Table 4 Experiment D: [Input DNA]: 0.0002Sng/ul

Value Value (estimated 2 copies/rx)
Probe Parameter Found Expected Result
(a.u.) (a.u) Table 6

F635 Value Value

AT683 59764 2/3 probe passed Probe Parameter Found Expected Result
Median
F635 (a.u.) (a.u)
F635
AT730 57267 Median passed F635
Median AT683 52022 passed
>50.000 Median 2/3 probe
F635 a.u. F635
AT776 45225 passed F635
Median AT730 51129 Median passed
Median
F635 >50.000
MedianBglol - F635 a.u.
Bgiol 8322 >lO28 passed AT776 43353 passed
F635 Median
MedianEMPTY F635
F635 MedianBglol -
Bgiol 2347 >lO28 passed
MedianBglo2 - F635
Bglo2 5634 >lO28 passed
F635 MedianEMPTY
MedianEMPTY F635
F635 MedianBglo2 -
MedianBglo3 - Bglo2 2126 >lO28 passed
15984 >lO28 F635
Bglo3 passed
F635 MedianEMPTY
MedianEMPTY
F635
MedianBglo3 -
Final Result: Positive Test Bglo3 7863 >lO28 passed
F635
MedianEMPTY
Experiment C: [Input DNA]: 0.002Sng/ul Final Result: Positive Test.
(estimated 20 copies/rx)

From the above reported data, it can be noticed

Table 5 that the detection of the betaglobine gene target
Value Value (Genebank AY260740 from 72 to 311) is
Probe Parameter Found Expected Result achieved up to [Input DNA] of estimated a Limit
(a.u.) (a.u) of Detection attested in 2 copies/rx.
AT683 F635 Median 50519 2/3 probe passed
F635 Conclusions
AT730 F635 Median 56298 Median passed
>50.000 Microfluidic packaging will become in few next
AT776 F635 Median 42280 a.u. passed years a considerable and actual application for
F635 the bio-diagnostic field, taking origin from main
MedianBglol - concepts peculiar in microelectronics
Bglol 4330 >1028 passed environment. A reliable and cost-effective set of
F635
MedianEMPTY solutions is then mandatory to be faced in order
F635 to make the fluidic packaging a reality for device
MedianBglo2 - production. As a [mal consideration we need to
Bglo2 2538 >lO28 passed
F635 underline that the manufacturing steps for
MedianEMPTY
MEMS devices and related assembly procedures
F635 are compatible with bio-diagnostic concepts and
MedianBglo3 - a complete integration between semiconductor
Bglo3 11530 >lO28 passed
F635 assembly technologies and main DNA bio­
MedianEMPTY process will be possible since the near future.

Authorized licensed use limited to: STMicroelectronics international NV. Downloaded on July 16,2020 at 08:29:23 UTC from IEEE Xplore. Restrictions apply.
 Flow Control" University of Sheffield,
Acknoledgements United Kingdom.
[10]A.K.Henning, "Microfluidics MEMS" IEEE
We want to tank for micro-fluidic simulations Aerospace Conference, NJ, USA, 1998.
Dr. Gaetano Panvini and Ing. Christophe Canales [11]A. Han, O. Wang, M. Graff, S. K. Mohanty,
and Mr. Wolfgang Stoeters (Boehringer T. L. Edwards, KI-Ho Kan and B. Frazier,
Ingelheim) for supporting the basic development. "Multi-layer plastic/glass microfluidic
Furthermore the support of AB Engineering Italy system containing electrical and mechanical
was also appreciated in 3D-Flow software functionality", Georgia Institute of
simulations. Special thanks to Dr. Patrizia Di Technology, Atlanta, GA, USA, 2003.
Pietro and Floriana San Biagio for the support in
the biological characterization.

Bibliography
Articles from conference proceedings
[1] N.T.Nguyen, S.T.Wereley, "Foundamentals
and Application of Microfluidics", Artech
House, Boston (USA), 2006.
[2] I. D. Johnston, M. C. Tracey, J B Davis, C.
K. L. Tan, "Micro Throttle pump employing
displacement amplification in an elastomeric
substrate", University of Hertfordshire,
Hatfield, UK, lOP Publishing LTD, 2005.
[3] Paul Galambos, Gil Benavides, "Electrical
and Fluidic Packaging of Surface
Micromachined Electro-Microfluidic
Devices", Sandia National Labs,
Abuquerque, NM, USA.
[4] T.B. Christensen, C.M. Pedersen, K.c.
Grondahl, T.G. Jensen. A. Sekulovic,
D.D.Bang and A.Wolf "PCR
biocompatibility of lab-on-chip and MEMS
materials", Journal of Micromechanics and
Microengineering, 17 (2007).
[5] Imants R. Lauks, "Microfabricated
Biosensors And Micro-Analytical Systems
for blood Analysis", i-STAT Co., Ontario,
Canada, 1997.
[6] P.Galambos, G.L.Benavides, M.Okandan,
M.W.Jenkins D.Hetherington, "Precision
Alignment Packaging for Microsystems
With Multiple Fluid Connections", ASME,
New York, USA, Proceedings 2001.
[7] K.L.Tan, M. C.Tracey, J. B. Davis, I.D.
Johnston, Continuously variable mixing­
ratio micromixer with elastomer valves",
Science and Technology Research Institute,
University of Hertfordshire, Hatfield, UK,
18 Aug. 2005.
[8] Cliff Henke "DNA Chip Technologies"
Medical Devices Link, USA 1998.
[9] V. Tesar, R.W.K. Allen, lR.Tippetts, "
Microfluidics - The Challenge of Low Re

Authorized licensed use limited to: STMicroelectronics international NV. Downloaded on July 16,2020 at 08:29:23 UTC from IEEE Xplore. Restrictions apply.