Revista Facultad de Ingeniería Universidad de Antioquia 0120-6230
Revista Facultad de Ingeniería Universidad de Antioquia 0120-6230
Revista Facultad de Ingeniería Universidad de Antioquia 0120-6230
Antioquia
ISSN: 0120-6230
revista.ingenieria@udea.edu.co
Universidad de Antioquia
Colombia
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Rev. Fac. Ing. Univ. Antioquia N.º 72, September, 2014
Abstract
Biosensor devices have applications in a variety of fields as environmental
analysis, biomedical, bio-defense, food and agriculture. On this kind of
sensors, a biological material (known as biomediator) reacts with target
analytes and an appropriated transduction system converts that reaction to
an electrical signal that can be processed, saved and transmitted by using
electronic systems.
In this article, two transduction methods used for biosensing applications are
described: amperometry that is based on the measurement of the electron
transfer occurring inside the biomediator and fluorescence, that is based on
the measurement of the re-emitted light. Emphasis has been done on the
electronics design, including component selection, useful circuit topologies
and common problems and solutions. Electronics has been validated for the
development of biosensor-based instruments characterized by low production
costs and portability.
----------Keywords: Biosensors, amperometry, fluorescence,
transduction methods, electronic design
* Corresponding author: Juan Bernardo Cano, e-mail: jbcano@gmail.com, phone number: + 57 4 2198558 (J. Cano)
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Transduction methods used on biosensors: amperometry and fluorescence
Resumen
Los biosensores tienen aplicación en una gran variedad de campos, incluyendo
el análisis ambiental, biomedicina, biodefensa, alimentación y agricultura,
entre otros. En este tipo de sensores, un material biológico (conocido como
biomediador) reacciona con el analito y un sistema de transducción apropiado
transforma dicha reacción en una señal eléctrica que puede ser procesada,
almacenada o transmitida usando sistemas electrónicos.
En este artículo, se describen dos métodos de transducción usados en
aplicaciones bio-sensoriales: la amperometria que consiste en la medida del
transferimento electrónico (corriente) del biomediador y fluorescencia que es
basada en la medida de la luz re-emitida. Se enfatiza en el diseño electrónico
(selección de componentes, topologia de los circuitos, problemas comunes y
soluciones). Estos diseños han sido utilizados en el desarrollo de instrumentos
comerciales para biosensores, caracterizados por bajos costes de producción
y portabilidad.
----------Palabras clave: Biosensores, amperometria, fluorescencia,
métodos de transducción, diseño electrónico
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Rev. Fac. Ing. Univ. Antioquia N.° 72, September, 2014
between the electrodes, and the correct potential the reference electrode circuit presents a high
depends on the characteristics of the reaction impedance to block the current flow (Ire=0). To
occurring and the electrode’s materials. set the reference electrode potential a special
control circuit is necessary (potensiostat circuit
Two types of configurations are commonly used
on Figure 2).
in amperometry for the electrodes: two and three
electrodes systems. A two electrodes system is The electrodes systems for biosensors are normally
composed by a reference (RE) and a working implemented on screen-printed electrodes (S.P.E)
(WE) electrode, and, in this case, the current is [7]. These are built by the deposition of different
measured on the working electrode (Irw) whereas “ink” layers over a PVC, ceramics or aluminum
the potential is set between working and reference base. There are one or more layers of electrical
electrodes (Vrw) (Figure 1, up). conductive surfaces for electrodes conformation
and a contact line for external electronics
interface. S.P.E’s main advantages are low cost
(intended to be disposable), easy automated
production and small size.
Figure 2 shows the electronics circuit
implemented for amperometric detection on
three electrodes S.P.E. This circuit is discussed
on the next paragraphs.
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Transduction methods used on biosensors: amperometry and fluorescence
offset voltage and finite gain) that is important for decoupling, star ground and ground planes
precision of electrochemical potential (Vrw) [8]. are important [9, 10].
Considering that the input current on to the • Residues of soldering process can lead to
amplifier inputs is zero, the measurement current losses, appropriated cleaning is
current travels through the RGAIN resistor, advised [8].
giving an output voltage of minus RGAIN times
• Induced noise from AC power and other
measurement current. A feedback capacitor, in
electromagnetic devices is always present.
parallel with RGAIN is necessary for stability
Shorter cable (and PCB tracks) length and
reasons (especially if large gains are desired) and
proper cable shielding (in some cases even
forms a single pole low pass filter.
amplifier shielding) helps to mitigate this.
In biosensor applications, the intensities are
normally in the range of some nano-ampere up Potensiostat
to tenths of micro-ampere. These low currents
require special considerations to obtain accurate Potensiostat circuit must keep the required
measurements: reference potential on three electrodes systems
(Fig, upper, A1 and A2 amplifiers) [11]. The
• The operational amplifier must be potensiostat circuit uses the high input impedance
characterized by low input bias current (Ibias), of operational amplifier (A2 on figure 2) to block
this bias is subtracted (or added depending on the current flow through the reference electrode.
his sign) from the WE current before being The A2 amplifier is configured as voltage follower
amplified, becoming an important error and his output can be used to monitor the actual
source. By this, F.E.T (field effect transistor) status of the reference potential (Vsense signal).
based amplifiers are recommended for this
type of applications. It’s also important to The Vref input is used for setting the desired
control the Ibias variation with temperature reference potential (from a digital to analog
when choosing the right amplifier. converter). If the A1 amplifier is on his linear
region (possible only if exists a feedback path
• As already discussed, low input offset between RE and CE electrodes -Z1-, due to
amplifiers are required. The electrochemical the chemical substances on contact with the
potential can be low as few tents of millivolts electrodes) is possible to demonstrate that the
for some applications. By this amplifiers voltage on RE electrode is kept equal (with
with input offset lower than one millivolt opposite sign) to the Vref signal.
or with an offset adjustment mechanism are
recommended. Proper potensiostat operation strongly depends on
the conditions of the substances on contact with
• Special attention must be taken on the the electrodes (on Figure 2 modeled as Z1 and
printed circuit board (P.C.B) layout. Current Z2 impedances). A poor conductivity between
losses trough board isolation are possible if CE and RE or/and an excessive WE current will
“high” voltage signals crosses near to the require a higher CE to RE voltage and can put
amplifier input (depending on measurement the A1 amplifier on saturation condition. Also
range, isolation between signals and amplifiers stability is not guaranteed because
distance is possible to have considerable feedback impedances are unknown. By this a
losses with voltages as low as five volts). constant monitoring is necessary on Vsense
Use of guard rings is recommended. signal, warning the user when the potensiostat is
Proper P.C.B distribution is necessary, out of operation in order to modify the analysis
digital and switching signals must be keep conditions.
away from low current signals. Supply line
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Rev. Fac. Ing. Univ. Antioquia N.° 72, September, 2014
Considerations done for current measurement are Finally, the control and data processing is
also applicable to this circuit. The RE electrode carried out by an 8-bit Atmel microcontroller
signal is particularly sensible to coupled noise due (C51 family). A friendly user interface -trough
to the higher input impedance, by this additional graphical LCD and keyboard- and a PC
shielding on this signal is recommended. communications interface were also provided.
The interconnection of the different electronic
System Integration systems is shown on figure 3.
Development of an integrated, low cost, portable From a mechanical point of view, the instrument
instrument for amperometric detection has consist in two measurement cells, each one
taken as basis the circuits described on Current with space for S.P.E insertion and flow lines for
Measurement and Potensiostat. sample and measurement buffers. The cells are
also provided with light sources (Light Emitting
A digital to analog converter has been used to Diodes, LEDs at 670nm and 470nm wavelengths)
generate the voltage reference for the potensiostat for stimulation of photo-sensible materials.
circuit. This converter can be configured (by the
central processing unit) to generate a constant
Transduction system validation
DC voltage (necessary on current vs time
analysis –chronoamperometry-) or a different Validation of the potensiostat and current
waveform (by example a triangle waveform used measurement circuits was conducted by
on cyclical voltammetry analysis). This converter connecting these to a dummy load circuit (Figure
has 12 bits resolution and a conversion rate of up 2). This circuit guarantees the feedback path
to 1M samples/second. necessary for proper potensiostat operation.
It consists of two impedances: Z1 connected
Instead, an analog to digital converter is used
between CE and RE and Z2 connected between
to sample the Vsense signal and the output of
RE and WE. Z1 is a resistor in parallel with a
the current amplifier. This converter is 16 bits
capacitor (capacitance is necessary for stability
resolution and up to 64ksample/s (Figure 3).
reasons) while Z2 is pure resistive.
108
Transduction methods used on biosensors: amperometry and fluorescence
Validation of the potensiostat circuit was done Linearity and accuracy shown on instrument
by setting -on the user interface menus- a potential and current allows using it on real
desired potential and measuring the true voltage sample measurements, as illustrated on next
between the reference and working electrodes. paragraphs.
That measurement was conducted using a
Keithley 2700 Digital Multimeter (DMM)/data Real sample instrument validation
acquisition system. Figure 4 (up) shows the
data points obtained and his linear regression. Instrument operation was validated using
Offset and gain calibration factors were software photosynthetic bio-mediators immobilized
implemented on the instrument to diminish the on screen printed electrodes, an unicellular
effect of electronics tolerances. green algae (Chlamidomonas reinhardtii)
was used [12].
Validation was also conducted on current
measurement. To do this, Z2 was replaced by a Operation whit this type of bio-mediator is based
variable resistor (in order to vary the working on successive dark/light periods. During the dark
electrode current while maintaining a constant period the biological material enters on relaxation
reference electrode potential) and the current was status, after that it’s stimulated by a light pulse
measured using the Keithley DMM (in series with producing an electronic transport due to the
the working electrode) and the instrument itself. photosynthesis process. This process is repeated
Figure 4 (Down) shows the plot of the obtained cyclically.
data, with the measurement of Keithley DMM on In the dark period exists a constant “base line”
X axis and instrument on Y axis. current on the working electrode. During the
light time, the current rises to a peak value and
then decreases until returning to the base line
level when the light is turned off. That light-
induced current peak depends on photosynthesis
efficiency and if an external factor (by example
a photosynthesis blocking herbicide) is stressing
the bio-mediator the current peak will be lower.
Thus, measuring the magnitude of the current peak
(peak minus baseline) when the bio-mediator is
in non stress conditions (Ibuffer) and when is in
contact with a sample (Isample), then calculating
the inhibition percentage (Eq. 1) is possible to
obtain an estimate of herbicide presence on sample
[12].
(1)
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Rev. Fac. Ing. Univ. Antioquia N.° 72, September, 2014
110
Transduction methods used on biosensors: amperometry and fluorescence
Photo-diode response speed is an important factor transient fluorescence analysis. Figure 6 shows
on applications where interest is on fluorescence PSPICE simulation results for the figure 5 circuit.
transient. On this cases use of PIN photo-diodes It shows the transient response at a photo-diode
is recommended. Speed depends on internal current step (ie: a light suddenly turning on)
diode capacitance that can be further reduced by at time 1 ms. Simulation has been built using
using a reverse bias [16]. LTC6081 operational amplifier model from Texas
Instruments, a gain of one million (1Mohm), and
Figure 5 shows a circuit used for photo-diode
a feedback capacitance varying from 1pF to 15
conditioning based on an operational amplifier in
pF. The photo-diode simulated was the Osram
trans-resistance configuration. This configuration
BPW34 which model was built using data-sheet
has already been discussed on Current
information.
Measurement title.
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Rev. Fac. Ing. Univ. Antioquia N.° 72, September, 2014
Figure 8 Example of fluorescence transient (Kautsky curve), X axis is time in ms (log) and Y axis is fluorescence
on arbitrary units
112
Transduction methods used on biosensors: amperometry and fluorescence
This transient fluorescence is due to a series adaptation period, using the maximum light
of electron transfer process internal at the intensity possible by the instrument (aprox.
chloroplasts (tilakoid membrane). When the 550Umol/m2/s2). The algae were in solution
bio-mediator is on contact with a photosynthesis in a volume of 500uL, Optical Density (OD) =
blocking substance (by example some types of 1. Measurements where done with and without
herbicides) the shape of this transient is changed. presence of Linuron pesticide.
The fluorescence instrument has been tested Figure 9 (left) shows results. The fluorescence
by measuring the fluorescence transient of curves (measured on arbitrary units) where
C. reinhardtii algae. These measurements normalized by subtracting the F0 level and
were conducted after fifteen minutes of dark dividing by the Fm level.
Figure 9 Left: Kauskty curves of the IL mutant with linuron pesticide. Each curve corresponds at a pesticide
concentration (molar). Right: V10m parameter vs pesticide concentration
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Rev. Fac. Ing. Univ. Antioquia N.° 72, September, 2014
The instruments that have been developed were 5. M. Giardi, E. Pace. “Photosynthetic proteins for
capable of detecting the biomediator response technological applications”. Trends in Biotechnology.
Vol. 3. 2005. pp. 257-263.
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design and implementation of the transduction 6. G. Rea, G. Pezzottiand, M. Giardi. Portable optical
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20th 2006. Available on: https://brevetti.cnr.it/brevetti/
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This work has been supported by Cristallography 9. P. Brokaw. An IC Amplifier User’s Guide to
Institute of Italian National Research Council IC- Decoupling, Grounding, and Making Things Go Right
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