# A Dependable Microelectronic Peptide Synthesizer Using Electrode Data

Authors: H. G. Kerkhoff; X. Zhang; F. Mailly; P. Nouet; H. Liu; A. Richardson
Journal: VLSI Design
 (2008)

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## Abstract

The research in the area of microelectronic fluidic devices for biomedical applications is rapidly growing. As faults in these devices can have serious personal implications, a system is presented which includes fault tolerance with respect to the synthesized biomaterials (peptides). It can employ presence and purity detection of peptide droplets via current (charge) tests of control electrodes or impedance (phase) measurements using direct sensing electrodes near the peptide collector area. The commercial multielectrode array performs better in pure and impure detection of peptides in impedance and phase. Our two-electrode X-MEF case shows slightly poorer results. In both cases the phase is the best choice for contents detection. If there are presence or purity problems, the location is marked, and repeated peptide synthesis at another collector site is initiated.


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## Body

## 1. Introduction

There
is a rapid increase in  with the complex
control and massive signal processing nowadays available in advanced CMOS technology [1]. This development is
especially of importance in life-science applications. However, in these
applications, usually strict regulations are applied in terms of patient safety
and the quality of materials used.In
a previous paper, we have suggested the implementation of a new droplet-based
peptide synthesizer for point-of-care diagnostics using a new advanced
heterogeneous technology, combining automotive SoC and fluidics technology [2].
As defects in these devices can have serious personal implications, like an
incorrect initial diagnosis of cancer or virus, a system is presented in this
paper which includes fault tolerance with respect to the quality of synthesized
(peptide) biomaterials.Our
approach employs new presence and purity detection of peptide droplets via current
or impedance measurements using eithercontrol or directsensing electrodes near the peptide collector area. In the
case of droplet presence or purity problems, the location is marked in the
embedded RAM-based database, and repeated synthesis at the same or another site
is initiated.


## 2. Peptide Synthesis

Our
previously presented microelectronic fluidic (MEF) device [2] is able to
synthesize many peptides via the conventional Fmoc method, but at a microscale. This new device is fully
software programmable, via a dedicated on-chip processor tailored for massive
parallel fluidic operations and calculations.As
a demonstrator, we have studied a peptide consisting of nine amino acid
elements (9-chain).AMLDLLKSV
This particular antigenic peptide is being
used for the detection of soft tissue sarcoma (STS), a form of cancer [3].
Because
in the normal process of peptide synthesis some amino acids may be less
efficient in peptide coupling reactions (between 5% and maximum 20%), we have
compared the previous “pure” peptide with the followingpotential side-product, the 8-mer peptide.AMLDLLKSV
This
8-mer potential impurity may be considered as an altered peptide ligand (APL)
[4], a nonimmunogenic modification of the original antigenic peptide. The immuneresponse of a T-cell (category of white blood cells in blood) is
crucial for cancer detection as it recognizes the presented antigenic peptide.
For details is referred to the general biomedical literature. An APL will only
rarely cause this immune response and together with the requirement of
sufficient volume, it is hence unlikely to result in a false alarm with regard
to cancer detection.
In
the normal case, peptide binding and cancer cell detection takes place in two
steps: first, the MHC tetramers (set of molecules displayed on cell surfaces
that are responsible for antigen presentation) have to be charged with peptide,
and after a sufficient amount of MHC tetramers have been loaded withantigenic peptide, they will interact
with the T-cell receptor of cancer cell specific T-cells indicating the presence
of cancer cells that present this particular antigen.
As
a result, it is not important to determinewhich APLs are involved (test all kind of chain permutations) or how much (volume
percentage), but rather take care that sufficient (10 nmol) pure peptides are
present to guarantee correct immune response detection. Any deviation exceeding
the pure peptide measurement error (<1%) will result in the setting of a
flag in our software. This flag will start up our fault tolerance procedure as
discussed in Section 6. Subsequent proper action can be choosing
another peptide collector site and start afresh, or enhancing the current site
with more pure peptides and remeasure.


## 3. Peptide Presence and Purity Tests Via Control and Direct Sensing Electrode Measurements

It
has been shown in the past, that there is a direct relationship between the
degree of binding of amino acids in a peptide using solid-phase synthesis and the
peptide conductivity [5]. Hence, one approach to detect the purity of a peptide
is to measure the conductivity of the
fluid containing the peptide by using direct sensing electrodes. The latter means there is a direct
contact between the electrodes and the biofluid.   In [5], the electronics for measuring this conductance have been suggested; it uses discrete operational
amplifiers and passive components to achieve its goal.As
an alternative approach, the change in dielectric value of a pure/impure
peptide droplet could be used for this purpose [6, 7]. This employs capacitive control electrodes (not in direct
contact with the fluid) and further requires RF sources. However, this would
result in a complex implementation on a chip.We will discuss our two new test methods: one
using (capacitive)control electrodes
and transient current sensors as test infrastructure, while in the other approach, direct sensing electrodes measuring
the phase at low frequencies is being used. Together, they form a powerful
evaluation set in order to obtain a dependable MEF device.


## 4. Tests Based on Control Electrode and Direct Sensing Electrode Infrastructures

At
the end of the peptide synthesis cycle, it has been shown to be of key
importance to be able to detect the presence and purity of the produced peptides
on the MEF top substrate [2]. Instead of the traditional chemical method in macrosynthesis, we intended to use some
electrical way for on-chip peptide presence and purity detection. Two methods are
proposed in this paper: using the droplet control electrodes or the direct sensing electrodes; they can also be used simultaneously.Both
methods have been evaluated by actual measurements on our MEF, and for direct sensing,
our MEF device and a commercial multielectrode array (MEA) device [8, 9] have
been used for comparison. The base solution for dissolving the peptides was
0.1% TFA. This is Triflouroacetic acid, usually employed for peptide cleavage
at the last step of peptide synthesis. The droplets
containing these target peptides for the pure and
impure cases have a concentration of 0.5 mmol/L.During the measurements, three droplet cases
have been considered. They are 0.1% pure TFA,
0.1% TFA with pure peptide, and 0.1% TFA with impure peptide; hence only the
solution contents changes, from pure TFA to a TFA mixture with peptide. The
measurement methods and results will be separately discussed for both types of
electrodes.


### 4.1. Control Electrode Measurements

Our
microfluidic X-MEF chip, wire-bonded on a PCB which has been used in the test experiments, is shown in Figure1(a) (top
right).experimental PCB including our fluidic chip (X-MEF) for tests on direct sensing
electrodes and control electrodes [1, 2].
(a)
Our X-MEF chip
and PCB (top), and the commercially available MEA (bottom)(b)
Cross-section of
the combined fluidic SoC chipThe top left of the figure shows both the
control (serrated edged rectangles) and direct sensing electrodes (two
white dots) in that chip. They are
accessible from the interface ports on the PCB.The
bottom of Figure1(a) shows the second object of our measurements, being the multielectrode
structure of a commercially available MEA device [8, 9].This
is a commercial design and not directly linked to our X-MEF design. However, it
can serve to show if it is worthwhile to extend ourtwo-direct sensing electrodes to many electrodes for the sake of increased measurement sensitivity.
Figure 1(b) shows a cross-section of the combined fluidic SoC chip [2].
Basically, the fluidic part is built on top of an SoC using our proven
interconnect technology. The SoC contains all (driver and measurement)
electronics and digital signal processing (DSP) hardware. The control
electrodes are made of Platinum, while the direct sensing electrodes use TiN. A
photomicrograph of the layout of the fluidic part in the right top of Figure 1(a)
shows two-droplet input channels (left & right), which merge in a third
channel forming a cross. At the bottom end of this third channel, the green
arrow indicates the location of the two-direct sensing electrodes of which a
detailed view is seen on the top left of Figure 1(a).For
droplet movement control, a DMOS-CMOS level shifter circuit was designed to “amplify” the digital control signals
from a 3.3 V SoC control system into a
higher voltage (40 V–80 V). This
circuit
is suitable to drive the control electrodes on the device and thus
facilitate the transport and other operations of droplets. Besides their usage
for transporting the droplets, the control electrodes can also bereused for detection purposes: capacitive sensing for the presence and
contents of droplets.Figure2(a) shows the principle
of the double-diffused MOS (DMOS) high-voltage driver used for droplet
motion. The voltage Vdrive is controlled by the control voltage Vctr.
When Vdrive is applied, the electrode capacitance is charged through
the P-DMOS transistor. It is subsequently discharged through the N-DMOS
transistor when Vdrive is switched off. The charge (or discharge)
current Idrive is directly linked to the capacitance by
(1)Q=C⋅Vdrive=∫Idrive⋅dt=∫(VRw/Rw)⋅dt,
where Rw denotes the wire and itanium tin
oxide (ITO) glass resistance from the top ITO plate (peptide collector site) to
ground (Figure 1(b)) [2]. Its value is around 350 Ω. The transient voltage VRw across Rw
was measured and recorded using an oscilloscope in order to deduce the
transient of current along the output path of the DMOS driver. The complete schematic of the DMOS-CMOS circuit,
which we designed and implemented, is shown in Figure 2(b).The DMOS-CMOS interface circuit schematics. (a) Basic principle of the DMOS-CMOS high-voltage
driver.  Red parts indicate
the additional test hardware required for the peptide presence and purity
detection. The capacitor represents the control electrode impedance. (b) Complete
implemented DMOS-CMOS interface circuit scheme.
(a)(b)Therefore,
our proposed architecture for presence and purity detection is based on the
monitoring of the(dis)charging current.
The latter can be duplicated by using a P-DMOS current mirror, while the
discharge equivalent may be copied by an N-CMOS current mirror (red parts in Figure 2(a)).The
discharge current has been converted into a voltage via a transimpedance
circuit and subsequently integrated; this result is then converted into a
digital word via an 8-bit ADC. These parts have currently been implemented in a
field-programmable analog array (FPAA). As for the purity test by impedance
measurements later, the digital output value is then compared to the typical
values of the pure peptides stored in an SRAM in the SoC.Figure3 shows part of the measurement
results based on using the control electrodes in the case of only pure peptide
droplets, according to Figure 2(a). Similar measurements were carried out with
only the impure peptide droplets. These measurements of the transient voltage VRw have shown indeed that the charge current is linked to the purity of the
peptides. Moreover, as the area under the purple curve is different in the case
of pure and impure peptides, one can state that the electrode capacitance
increases with the peptide purity.Figure 3
Actual transient measurement: rising edge control voltageVctr (yellow, 2 V per grid), Vdrive (green, 20 V per grid), and VRw (purple, 50 mV per grid) for droplet with pure peptide.Table1 provides the results of using the structure in Figure 2 for
purity detection, based on measurement data similar to Figure 3. The time
interval (X-axis) was about 3 microseconds, and the charge through the top and
bottom mirror parts of the driver circuit (Figure 2) at the rising and falling edge
of the control voltage was compared for different droplet situations.Table 1
Percentage differences in measured values compared to pure
peptide solution for top mirror (m12, m13) and bottom mirror (m23, m24) for
rising and falling edge of the control voltage.
ConditionTop mirror,Bottom mirror,rising edgefalling edgePure peptide00Impure peptide5,5%4,1%TFA solution8,9%7,0%No solution18,1%14,3%Table1 shows that the top mirror under a
rising edge of the control voltage resulted in the largest changes between pure
and impure peptides. The results were reproducible and well within 1%
tolerance. Presence detection (no solution) is very simple to implement.Hence our new structure based on control
electrodes and transient current measurements could be used for our purposes.
In the remaining part of this paper, we will investigate the merits ofdirect sensing electrodes in this
respect.


### 4.2. Direct Sensing Electrode Measurements (X-MEF)

Our second approach for presence and purity
detection uses the direct sensor electrodepair of the X-MEF (Figure 1). A cross-section of this pair is presented in Figure 4;
notice that the fluidic droplet flow, in contrast to Figure 1(b), is in this
case perpendicular to the drawing. Impedance measurements have been carried out
between the nodes 1 and 2 (Figure 4).Figure 4
The direct sensing electrode pair infrastructure. The impedance
is measured between nodes 1 and 2.One can model this direct sensing electrode
infrastructure as two-interfacial capacitances (CEDL) in series with
a solution resistance (RSOL) and a solution capacitance (CSOL)
as shown in Figure 5. CEDL represents the electrical double layer in
the electrode-electrolyte interface dominating the interfacial impedance; for
more details on direct sensing electrode modelling, especially for the MEA, [9]
is useful.Figure 5
Simple component model of direct sensing electrode pair.The direct sensing electrode tests used a standard
impedance setup. The test infrastructure consists of two Pt/TiN electrodes with
dimensions of50×50 μm, making direct contact with an on-top
droplet. Since the sensing electrodes have direct contact with the droplet, impedance
spectroscopy theories using the X-MEF can be applied [6, 7]. Hence we carried out
impedance measurements instead of the (dis)charging method as discussed
previously.We used a Wayne Kerr precision impedance analyzer
6500 to analyze the impedance variation of the droplets containing different
materials; the sweep frequency range was set from 1 kHz to 50 MHz.In Figure6(a), the actual measurement results
are shown in case of a pure peptide.
The top plot shows the impedance (10 Ω–1 MΩ scale), the lower plot depicts the phase (−80 deg–+40 deg scale) versus the frequency (1 kHz–50 MHz). Figure 6(b) presents the results of the impedance in all droplet content
cases versus the frequency. If no fluid (e.g., peptide droplet) is present at
the direct sensing electrode pair location, an (expected) pure capacitive
behavior was observed and hence peptide droplet presence detection is extremely
simple. A detailed graph in the case of droplet purity differences derived from
Figure 6(b) is shown in Figure 6(c). Note that the frequency range was chosen
here on the basis of the value of the phase most close to zero degrees because
in that case the dropletsolution content (Figure 5) is dominating
other effects. At a frequency of 33 kHz, only a 1% impedance variation was
observed between pure and impure peptides. This is in the range of the
measurement tolerance (<1%) and can therefore not be used for purity
detection.X-MEF measurement
results from the direct sensing electrode pair infrastructure.
(a)
X-MEF direct-sensing
electrode pair measurement in the case of a pure peptide droplet. Impedance and
phase versus frequency(b)
Measured X-MEF direct-sensing electrode
pair impedance in the case of droplet absence (empty), TFA, pure (R3), and
impure (R4) peptide droplets(c)
Previous
measurement zoomed in for the last three droplet cases under phase condition(d)
Measurement
of X-MEF direct sensing electrode pair phase in the case of droplet absence,
TFA, pure (R3) and impure (R4) peptide droplets(e)
Previous measurement zoomed in for the last three droplet cases under phase
conditionFigures6(d) and
6(e) show the same approach, but now in the case of the phase. The largest
variations (17%, at 33 kHz) are found via the phase as compared to the previous
impedance test with regard to purity/impurity detection.This is therefore the preferred approach for
impurity tests. The results were reproducible and well within acceptable
tolerances (<1%) and hence usable for purity detection using the X-MEF
direct sensing method.An alternative multielectrode layout (now
onlytwo electrodes are used, Figure 1(a) top (left) is expected to enhance the measurement variations and accuracy
even more; it will be investigated in future research work.


### 4.3. Direct Sensing Electrode Measurements (MEA)

As a first step in that direction, we have
also carried out similar tests with regard to a direct sensing electrode
infrastructure of the multielectrode array (MEA) which is commercially
available [8, 9].This electrode infrastructure is shown in
Figure1(a) at the bottom. Although our tests are currently confined to two
electrodes only, the multielectrode potential is clearly available.The X-MEF
cross-section in Figure4 is easily converted into a two-electrode MEA
infrastructure, by removing both the Si-SoC and A part.Similar measurements using the same contentfluids (not droplets!) were carried out
on a direct sensing electrode pair in the MEA [8] (see Figure 1,
bottom). The modelling of this infrastructure has been discussed in [9]. This
experiment was carried out in order to see whether any similarities exist
between the MEA and X-MEF, and in longer term, whether more electrode pairs
could provide more reliable data on presence and contents of fluids/droplets.Although the devices differ in amount of
fluid used, distance between electrodes (layout) and used materials and electronics,
trends should be similar.Figure7(a) presents the measurement
screenshot of impedance and phase in the case of a pure peptide fluid over a large frequency range.
Figure 7(b) shows the measurements of the impedance for all possible fluid
cases. It shows that the presence detection for a fluid (e.g., peptide) is very
easy. A zoomed in representation of the impedance in Figure 7(c) reveals that
the variation in pure and impure peptide contents is around 5.3% in the area of
interest. Figure 7(d) shows the phase versus frequency for all fluid contents cases
which indicates also a very simple detection of fluid presence. Figure 7(e)
presents a zoomed in version of Figure 7(d); calculations show a variation of
20% in phase at 29 kHz between a pure and impure peptide fluid. Clearly, also
in the case of the MEA, the largest variations are found via the phase with
regard to purity/impurity detection. The results were reproducible and the
measurement tolerances less than 1%.MEA direct sensing electrode pair (electrode # 62 and # 63) measurements
of impedance and phase.
(a)
MEA direct sensing electrode pair (62-63) measurement
in the case of a pure peptide fluid. Impedance (10 Ω–100 kΩ scale) and phase (−120 deg–40 deg scale) versus frequency (1 kHz–50 MHz
scale)(b)
Measured
MEA direct sensing electrode pair impedance in the case of droplet absence
(empty), TFA, pure, and impure peptide fluids (1 kHz–407 kHz frequency range)(c)
Previous
measurements zoomed in for the last three droplet cases in the 11 kHz–70 kHz
range(d)
MEA
direct sensing electrode pair phase measurements in the case of droplet absence,
TFA, pure, and impure peptide droplets (1 kHz–1.2 MHz frequency range)(e)
Previous
phase measurements zoomed in for the last three droplet cases in the 11 kHz–70 kHz frequency rangeWhen comparing the data in detail of Figures6 and 7 of the XMEF and MEA, respectively, one can draw the following
conclusions: the MEA performs better in pure and impure detection of peptides
in impedance and phase, and the phase is the best parameter to be used. The
X-MEF case shows somewhat poorer results, but also the phase is the best choice
for contents detection. The difference in test frequencies and absolute values
of impedance and phase stems from the difference in structure between the X-MEF
and MEA.In the next two paragraphs will be elaborated
how the previous measurements can be used to increase the dependability of the
peptide synthesis process. First, the construction and location (collector) of
the sensors are discussed in detail. After that, the results of these
measurements are used to start up a new peptide synthesis sequence. This is
implemented in software.


## 4.1. Control Electrode Measurements

Our
microfluidic X-MEF chip, wire-bonded on a PCB which has been used in the test experiments, is shown in Figure1(a) (top
right).experimental PCB including our fluidic chip (X-MEF) for tests on direct sensing
electrodes and control electrodes [1, 2].
(a)
Our X-MEF chip
and PCB (top), and the commercially available MEA (bottom)(b)
Cross-section of
the combined fluidic SoC chipThe top left of the figure shows both the
control (serrated edged rectangles) and direct sensing electrodes (two
white dots) in that chip. They are
accessible from the interface ports on the PCB.The
bottom of Figure1(a) shows the second object of our measurements, being the multielectrode
structure of a commercially available MEA device [8, 9].This
is a commercial design and not directly linked to our X-MEF design. However, it
can serve to show if it is worthwhile to extend ourtwo-direct sensing electrodes to many electrodes for the sake of increased measurement sensitivity.
Figure 1(b) shows a cross-section of the combined fluidic SoC chip [2].
Basically, the fluidic part is built on top of an SoC using our proven
interconnect technology. The SoC contains all (driver and measurement)
electronics and digital signal processing (DSP) hardware. The control
electrodes are made of Platinum, while the direct sensing electrodes use TiN. A
photomicrograph of the layout of the fluidic part in the right top of Figure 1(a)
shows two-droplet input channels (left & right), which merge in a third
channel forming a cross. At the bottom end of this third channel, the green
arrow indicates the location of the two-direct sensing electrodes of which a
detailed view is seen on the top left of Figure 1(a).For
droplet movement control, a DMOS-CMOS level shifter circuit was designed to “amplify” the digital control signals
from a 3.3 V SoC control system into a
higher voltage (40 V–80 V). This
circuit
is suitable to drive the control electrodes on the device and thus
facilitate the transport and other operations of droplets. Besides their usage
for transporting the droplets, the control electrodes can also bereused for detection purposes: capacitive sensing for the presence and
contents of droplets.Figure2(a) shows the principle
of the double-diffused MOS (DMOS) high-voltage driver used for droplet
motion. The voltage Vdrive is controlled by the control voltage Vctr.
When Vdrive is applied, the electrode capacitance is charged through
the P-DMOS transistor. It is subsequently discharged through the N-DMOS
transistor when Vdrive is switched off. The charge (or discharge)
current Idrive is directly linked to the capacitance by
(1)Q=C⋅Vdrive=∫Idrive⋅dt=∫(VRw/Rw)⋅dt,
where Rw denotes the wire and itanium tin
oxide (ITO) glass resistance from the top ITO plate (peptide collector site) to
ground (Figure 1(b)) [2]. Its value is around 350 Ω. The transient voltage VRw across Rw
was measured and recorded using an oscilloscope in order to deduce the
transient of current along the output path of the DMOS driver. The complete schematic of the DMOS-CMOS circuit,
which we designed and implemented, is shown in Figure 2(b).The DMOS-CMOS interface circuit schematics. (a) Basic principle of the DMOS-CMOS high-voltage
driver.  Red parts indicate
the additional test hardware required for the peptide presence and purity
detection. The capacitor represents the control electrode impedance. (b) Complete
implemented DMOS-CMOS interface circuit scheme.
(a)(b)Therefore,
our proposed architecture for presence and purity detection is based on the
monitoring of the(dis)charging current.
The latter can be duplicated by using a P-DMOS current mirror, while the
discharge equivalent may be copied by an N-CMOS current mirror (red parts in Figure 2(a)).The
discharge current has been converted into a voltage via a transimpedance
circuit and subsequently integrated; this result is then converted into a
digital word via an 8-bit ADC. These parts have currently been implemented in a
field-programmable analog array (FPAA). As for the purity test by impedance
measurements later, the digital output value is then compared to the typical
values of the pure peptides stored in an SRAM in the SoC.Figure3 shows part of the measurement
results based on using the control electrodes in the case of only pure peptide
droplets, according to Figure 2(a). Similar measurements were carried out with
only the impure peptide droplets. These measurements of the transient voltage VRw have shown indeed that the charge current is linked to the purity of the
peptides. Moreover, as the area under the purple curve is different in the case
of pure and impure peptides, one can state that the electrode capacitance
increases with the peptide purity.Figure 3
Actual transient measurement: rising edge control voltageVctr (yellow, 2 V per grid), Vdrive (green, 20 V per grid), and VRw (purple, 50 mV per grid) for droplet with pure peptide.Table1 provides the results of using the structure in Figure 2 for
purity detection, based on measurement data similar to Figure 3. The time
interval (X-axis) was about 3 microseconds, and the charge through the top and
bottom mirror parts of the driver circuit (Figure 2) at the rising and falling edge
of the control voltage was compared for different droplet situations.Table 1
Percentage differences in measured values compared to pure
peptide solution for top mirror (m12, m13) and bottom mirror (m23, m24) for
rising and falling edge of the control voltage.
ConditionTop mirror,Bottom mirror,rising edgefalling edgePure peptide00Impure peptide5,5%4,1%TFA solution8,9%7,0%No solution18,1%14,3%Table1 shows that the top mirror under a
rising edge of the control voltage resulted in the largest changes between pure
and impure peptides. The results were reproducible and well within 1%
tolerance. Presence detection (no solution) is very simple to implement.Hence our new structure based on control
electrodes and transient current measurements could be used for our purposes.
In the remaining part of this paper, we will investigate the merits ofdirect sensing electrodes in this
respect.


## 4.2. Direct Sensing Electrode Measurements (X-MEF)

Our second approach for presence and purity
detection uses the direct sensor electrodepair of the X-MEF (Figure 1). A cross-section of this pair is presented in Figure 4;
notice that the fluidic droplet flow, in contrast to Figure 1(b), is in this
case perpendicular to the drawing. Impedance measurements have been carried out
between the nodes 1 and 2 (Figure 4).Figure 4
The direct sensing electrode pair infrastructure. The impedance
is measured between nodes 1 and 2.One can model this direct sensing electrode
infrastructure as two-interfacial capacitances (CEDL) in series with
a solution resistance (RSOL) and a solution capacitance (CSOL)
as shown in Figure 5. CEDL represents the electrical double layer in
the electrode-electrolyte interface dominating the interfacial impedance; for
more details on direct sensing electrode modelling, especially for the MEA, [9]
is useful.Figure 5
Simple component model of direct sensing electrode pair.The direct sensing electrode tests used a standard
impedance setup. The test infrastructure consists of two Pt/TiN electrodes with
dimensions of50×50 μm, making direct contact with an on-top
droplet. Since the sensing electrodes have direct contact with the droplet, impedance
spectroscopy theories using the X-MEF can be applied [6, 7]. Hence we carried out
impedance measurements instead of the (dis)charging method as discussed
previously.We used a Wayne Kerr precision impedance analyzer
6500 to analyze the impedance variation of the droplets containing different
materials; the sweep frequency range was set from 1 kHz to 50 MHz.In Figure6(a), the actual measurement results
are shown in case of a pure peptide.
The top plot shows the impedance (10 Ω–1 MΩ scale), the lower plot depicts the phase (−80 deg–+40 deg scale) versus the frequency (1 kHz–50 MHz). Figure 6(b) presents the results of the impedance in all droplet content
cases versus the frequency. If no fluid (e.g., peptide droplet) is present at
the direct sensing electrode pair location, an (expected) pure capacitive
behavior was observed and hence peptide droplet presence detection is extremely
simple. A detailed graph in the case of droplet purity differences derived from
Figure 6(b) is shown in Figure 6(c). Note that the frequency range was chosen
here on the basis of the value of the phase most close to zero degrees because
in that case the dropletsolution content (Figure 5) is dominating
other effects. At a frequency of 33 kHz, only a 1% impedance variation was
observed between pure and impure peptides. This is in the range of the
measurement tolerance (<1%) and can therefore not be used for purity
detection.X-MEF measurement
results from the direct sensing electrode pair infrastructure.
(a)
X-MEF direct-sensing
electrode pair measurement in the case of a pure peptide droplet. Impedance and
phase versus frequency(b)
Measured X-MEF direct-sensing electrode
pair impedance in the case of droplet absence (empty), TFA, pure (R3), and
impure (R4) peptide droplets(c)
Previous
measurement zoomed in for the last three droplet cases under phase condition(d)
Measurement
of X-MEF direct sensing electrode pair phase in the case of droplet absence,
TFA, pure (R3) and impure (R4) peptide droplets(e)
Previous measurement zoomed in for the last three droplet cases under phase
conditionFigures6(d) and
6(e) show the same approach, but now in the case of the phase. The largest
variations (17%, at 33 kHz) are found via the phase as compared to the previous
impedance test with regard to purity/impurity detection.This is therefore the preferred approach for
impurity tests. The results were reproducible and well within acceptable
tolerances (<1%) and hence usable for purity detection using the X-MEF
direct sensing method.An alternative multielectrode layout (now
onlytwo electrodes are used, Figure 1(a) top (left) is expected to enhance the measurement variations and accuracy
even more; it will be investigated in future research work.


## 4.3. Direct Sensing Electrode Measurements (MEA)

As a first step in that direction, we have
also carried out similar tests with regard to a direct sensing electrode
infrastructure of the multielectrode array (MEA) which is commercially
available [8, 9].This electrode infrastructure is shown in
Figure1(a) at the bottom. Although our tests are currently confined to two
electrodes only, the multielectrode potential is clearly available.The X-MEF
cross-section in Figure4 is easily converted into a two-electrode MEA
infrastructure, by removing both the Si-SoC and A part.Similar measurements using the same contentfluids (not droplets!) were carried out
on a direct sensing electrode pair in the MEA [8] (see Figure 1,
bottom). The modelling of this infrastructure has been discussed in [9]. This
experiment was carried out in order to see whether any similarities exist
between the MEA and X-MEF, and in longer term, whether more electrode pairs
could provide more reliable data on presence and contents of fluids/droplets.Although the devices differ in amount of
fluid used, distance between electrodes (layout) and used materials and electronics,
trends should be similar.Figure7(a) presents the measurement
screenshot of impedance and phase in the case of a pure peptide fluid over a large frequency range.
Figure 7(b) shows the measurements of the impedance for all possible fluid
cases. It shows that the presence detection for a fluid (e.g., peptide) is very
easy. A zoomed in representation of the impedance in Figure 7(c) reveals that
the variation in pure and impure peptide contents is around 5.3% in the area of
interest. Figure 7(d) shows the phase versus frequency for all fluid contents cases
which indicates also a very simple detection of fluid presence. Figure 7(e)
presents a zoomed in version of Figure 7(d); calculations show a variation of
20% in phase at 29 kHz between a pure and impure peptide fluid. Clearly, also
in the case of the MEA, the largest variations are found via the phase with
regard to purity/impurity detection. The results were reproducible and the
measurement tolerances less than 1%.MEA direct sensing electrode pair (electrode # 62 and # 63) measurements
of impedance and phase.
(a)
MEA direct sensing electrode pair (62-63) measurement
in the case of a pure peptide fluid. Impedance (10 Ω–100 kΩ scale) and phase (−120 deg–40 deg scale) versus frequency (1 kHz–50 MHz
scale)(b)
Measured
MEA direct sensing electrode pair impedance in the case of droplet absence
(empty), TFA, pure, and impure peptide fluids (1 kHz–407 kHz frequency range)(c)
Previous
measurements zoomed in for the last three droplet cases in the 11 kHz–70 kHz
range(d)
MEA
direct sensing electrode pair phase measurements in the case of droplet absence,
TFA, pure, and impure peptide droplets (1 kHz–1.2 MHz frequency range)(e)
Previous
phase measurements zoomed in for the last three droplet cases in the 11 kHz–70 kHz frequency rangeWhen comparing the data in detail of Figures6 and 7 of the XMEF and MEA, respectively, one can draw the following
conclusions: the MEA performs better in pure and impure detection of peptides
in impedance and phase, and the phase is the best parameter to be used. The
X-MEF case shows somewhat poorer results, but also the phase is the best choice
for contents detection. The difference in test frequencies and absolute values
of impedance and phase stems from the difference in structure between the X-MEF
and MEA.In the next two paragraphs will be elaborated
how the previous measurements can be used to increase the dependability of the
peptide synthesis process. First, the construction and location (collector) of
the sensors are discussed in detail. After that, the results of these
measurements are used to start up a new peptide synthesis sequence. This is
implemented in software.


## 5. The Peptide Collector Site

In
a previous paper [2], several electrode configurations have been suggested for
monitoring droplet presence and contents. The situation in our case is
different, as the purity of the peptide has to be determined at a collector site.
This collector site consists of a deep-etched trench in the top ITO glass, and
filled with a porous hydro gel. This approach is required to obtain sufficient
peptide material for accurate cancer cell detection. Although a single pair of
direct sensing electrodes can be used (shown in Figure 8, and used in the previous tests), also other, more complex
options are open, as for example, like the ones used in an MEA as employed  by QinetiQ [8]. Our current approach is
depicted in Figure 8. The lower figure
is the cross-section of A-A’.Figure 8
The direct sensing electrodes at the
peptide collector site. Usage is made of special test droplets for purity
detection.Our
system uses a special test droplet, which is smaller in dimensions than the
ones during synthesis and consists of an acid solution, which cuts part of the
peptide from the collector site. The droplet now contains the peptide; for
enhancing the sensitivity three measurements are taken subsequently. The previously
discussed tests at the chosen frequency (e.g.,flo=33 kHz) can be carried out by for instance a
bridge structure [9].


## 6. Fault Tolerant Peptide Synthesis

The
electronics in the SoC implementing the previous measurement system will send
the droplet presence and contents information (based on control-electrode
transient current or/and direct sensing electrode(s) phase) in digital form to our
on-chip general processor (ARM), fluidic-specific coprocessor, and embedded RAM
(Figure9). Here, it will be decided if the deviation in measured peptide purity
(or in worst-case absence) jeopardizes the correct (cancer or virus) detection.
If so, the collector site is flagged and its location stored in the database. The
digital information in terms of charge or phase of a specific pure peptide can
be loaded in advance by the manufacturer, together with the measurement
tolerance band and required boundary conditions (e.g., the frequency).Figure 9
The handling of pure and impure peptide
collector sites, employing the previous measurement techniques.After
flagging, a new round for synthesis will be initiated by the processor of that
peptide, at either thesame location or
another site. Which decision is made depends on the measurement results. If no
presence of peptides has been detected, probably the original collector site is
unreachable or faulty and hence another free site will be allocated.  If a large deviation (>10% margin, but
software adjustable) from the pure peptide data has been detected, it is also
decided to go to another site as the site can only contain a limited volume of
fluids and a certain minimum is required for immune response. Only if a slight
deviation (<5% margin, but software adjustable) is noticed from pure
peptides, the same site will be used to add additional pure peptide via
synthesis. This location information is also automatically stored and this
means that a direct link exists between the original collector site, and the
correct one; this is essential in the next optical step for cancer detection
[10]. The simplified procedure is illustrated in Figure 9.The
user (or manufacturer) indicates which peptides have to be synthesized. These
are specific to the cancer cells to be potentially detected. Currently, one can
maximally provide 96 peptides on a single substrate. Multiple peptides are
synthesized in parallel. By using our approach, adependable MEF system results, which is a prerequisite for the
application in a biomedical environment. All software has been written in C++ and
runs on the on-board processor.


## 7. Conclusions

In
this paper, two new test techniques have been investigated to determine the presence
and purity of synthesized peptide droplets in our MEF and a commercial MEA via
measuring the charge and phase of the biomaterial. Use is made of either direct
sensing electrode pairs or control electrodes. It has been shown that detection
is possible in both cases. The result is used to mark collector sites of
insufficient pure peptides which could obscure cancer detection, and relate
them to (a) pure site(s). In this way, the quality of cancer detection in this
type of device is significantly increased, resulting in adependable point-of-care device. Multielectrodes for sensing and
additional DSP are seen as a possibility to further enhance the quality of
measurements. A very strong point of our SoC-MEF approach is that the system is
scalable in the future to offer fully automatic dependable diagnosis in life
sciences by on-the-fly specific peptide generation depending on previous diagnostic
measurement results of the sample.



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*Source: 437879-2008-08-05.xml*