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Biosensors and Instrumentation: Tutorial 3

1. A schematic cross section of an ion sensitive field effect transistor (ISFET) is shown in figure 1.

�Figure 1. Schematic cross section through an ISFET

1.1. In this device, how does the value of pH affect the potential at the oxide/solution interface and the effective threshold voltage of the ISFET?

1.2. Equations [1] and [2] define the change the surface potential, and therefore VT, with pH for an ISFET.

[1]

where

! [2] 1.2.1. Define the term Cdl and describe how it will be affected by changes in the measured

solution1.2.2. Define the term βint and why it should be maximised

1.2.3. If α=1 what will be the sensitivity δΨ/δpH at 25 °C (298 K)?1.2.4. What type of response does the result of (iii) resemble?1.2.5. Suggest a gate oxide material which has high sensitivity with reasons for your choice

1.3. Figure 2 shows an alternative circuit for biasing and measuring the output of an ISFET pH sensor. Define the ISFET current and voltage IDS and VDS.

1.4.The source-drain current through a MOSFET (or ISFET) in the linear region of operation is given by the equation below. Given that the source drain voltage (VDS) and current (IDS) of an ISFET are kept constant by the op-amp feedback circuit shown in figure 3, what will be the dependence of VGS on VT?

�1.5. Given that the effective value of VG is controlled by the reference electrode what will

happen to the output voltage Vout if the threshold voltage increases due to a change in the pH of the measured solution?

p−Si

n−Si n−Si

DrainSource

SiO2

Encapsulation

Vref

ReferenceElectrode

Solution

€

ΔΨ = −2.3αRTFΔpHbulk

↵ =1

(2.3kT/q2)(Cdl/�int) + 1

IDS = �✓

(VGS � VT )VDS �V 2DS

2

◆

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1.6.If the ISFET used has a threshold voltage VT with a pH sensitivity of -58mV/pH at 298K and has been set up so that the output voltage Vout is -1V at pH = 7 what will the output be for a pH of 1 and 12? Design an amplifier to give an output with a pH dependence of 1V/pH.

�Figure 2. Source-Drain Follower ISFET circuit

2. Figure 3 shows the ISFET amplifier circuit we looked at in the lecture. We’re now going to analyse this in a little more detail.

�Figure 3. ISFET Amplifier

2.1. The important voltages are numbered V1...V5 in figure 4. Define each of these in terms of the known voltages and currents in the circuit. Assume that R1 = R2 = R3 = R.

−

+

D

SISFET

+−

RRef. electrode

V

V1 in

2

out

R

R

3

D

SISFET

Ref. electrode

−

+

−

+

−

+

R

R R

R2

2 3

3

VoutR1

Vdd

R1

RDS

−

+

refVRout

RS

Iin

I f

V1

V2

V3

V4

V5

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2.2. What is the voltage VDS and current IDS of the ISFET?2.3. Explain how the current IDS is set by controlling the input Vref.2.4. The calibration routine suggests that the ISFET be placed in a buffer of a known pH (pH 7

typically) while the voltage Vref is adjusted to zero the output voltage Vout. What does this imply about the voltage V2 at this point? What about the output of the final op-amp comparator?

2.5. If the VT of the ISFET is dependent on pH with a sensitivity of –50 mV pH–1 choose appropriate values of RS and Rout to give an output voltage change of 1V pH–1.

3. The “O2-FET” mentioned in lecture 11 can potentially measure both the pH and oxygen concentration in a solution. Please try to read reference [7] mentioned in the notes for lecture 11 before attempting this question. Figure 4 shows the response of an O2-FET used to investigate a cell culture.

�Figure 4. O2-FET measurements

3.1. When the circulating pump is operating the conditions in culture chamber are kept constant. In the graph above, what is the difference between the two measurement times when the pump been turned off?

3.2. Estimate the change in pH for the first measurement time if the ISFET has a sensitivity of 55 mV pH-1.

3.3. There is an additional change in the ISFET output of 10 mV between the change in pH measured with the oxygen transducer switched off, and with it switched on. What is the source of this?

3.4. Can you think of reasons why an ISFET is better suited to measuring short term changes in pH rather than long term measurement of absolute pH?

M. Lehmann et al. / Biosensors & Bioelectronics 16 (2001) 195–203 199

Fig. 7. Fig. 9.

not have a significant effect on the output signal of theISFET. It only produces a constant offset of less than 1mV, which does not influence the measurements. Thefollowing negative potential causes a basification signalof the ISFET due to the generation of hydroxyl ions inthe surrounding of the ISFET. The application of thebiphasic potentials is repeated seven times and producesthe ‘new’ acidification–respiration signal. When thepositive potential is applied, the ISFET measures theacidification (Fig. 7). However, while applying the neg-ative potential the ISFET measures both the acidifica-tion and respiration. For the evaluation of therespiration the following method is applied: substrac-tion of two absolute values of the basification peakswhen the potential is changed from positive to negativesignals. Due to possible side effects caused by fluidicmovement when the pump is turned off [see also (Sohnand Kim, 1996)], the two chosen values for our evalua-tion were the second and the last peak (Fig. 7).

The respiration of the cells before the addition ofTriton-X-100 was 10 mV (Fig. 7). After addition of

0.3% Triton-X-100, both the acidfication and respira-tion disappeared (Fig. 8).

The first potential steps before and after the Triton-X-100 addition have a comparable value of approxi-mately 40 mV (Fig. 7 and Fig. 8). This can be explainedby the same oxygen content in the media at the begin-ning of the pump–off period.

The aim of the second experiment was to show thatthe sensor is able to measure opposite trends in acidifi-cation and respiration which may occur in the course ofan experiment. In earlier experiments with iodoacetate(Brischwein, 1997; Lehmann et al., 2000), it was shownthat iodoacetate, a potent inhibitor of glycolysis, byblocking the activity of glycerinaldehyde-3-phosphate-dehydrogenase (a key enzyme of glycolysis), causes thiseffect. The second experiment was therefore made withiodoacetate. Fig. 9 depicts the whole measurement.Both the respiration and acidification are present. Bysubtracting the slope of the acidification from theacidification–respiration signal there is a respirationvalue of 3.35 mV on average (Fig. 10). The acidification

Fig. 8. Fig. 10.

V sou

rce

(vs.

Ref)

[mV

]

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4. Figure 5 shows a plot of membrane potential against time for a neuron being activated and delivering an action potential.

�Figure 5. Schematic plot of an action potential in a typical neuron cell

4.1. Describe the changes in the cell, in terms of ion channels and the flow of charge, that cause the action potential.

4.2. In the Goldman equation, shown below, which parameters are principally affected by the the opening and closing of ion channels?

� 4.3. Suggest two possible ways in which a neuron could be excited into an action potential.4.4. An example of a current clamp circuit which can inject a current I into a cell is shown in

figure 6. What natural process does this attempt to emulate?

�Figure 6. Current clamp circuit for neuro-electrophysiology

Em

=RT

Fln

✓PK[K+]out + PNa[Na+]out + PCl[Cl�]inPK[K+]in + PNa[Na+]in + PCl[Cl�]out

◆

−

+−

+

−

+

V R

R

R

R

R

R

in

V pV p

V +Vin p

V inI

I

out

Cell

1 2

3

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4.5. Given that all resistors marked R are equal, and the voltage input (Vin) and output Vp are referenced to ground, what are the voltages at points 1-3?

4.6. Choose a resistor value for Rout that will give a maximum current output of 1nA. Assume that the resistance of the probe is 1 GΩ, and the op-amps have ±15V power supplies.

4.7. When a step change of current is injected the output voltage Vp shown in figure 7 is recorded. Explain how this differs from the actual membrane voltage Vm and what the source of the error is. Can you suggest a way of correcting this?

�Figure 7. Input current and output voltage traces from current clamp electrophysiology experiments

5. Figure 8 shows a circuit that could be used in patch clamp measurements of the ion current (Ich) through a single ion channel.5.1. Derive an expression for the output Vout.5.2. If Ich is in the pA range suggest a suitable value for Rf giving reasons for your answer.5.3. Given the above information why should the operational amplifier have FET based input

terminals?5.4. Assume that the resting value of the transmembrane potential is -70mV and the patch clamp

has been successfully located over a voltage gated potassium ion channel. Describe how this ion channel can be activated, and what the effect will be on the output of this circuit.

�Figure 8. Patch Clamp Circuit for Electrophysiology

3.2. Intracellular Recording Current Clamp

The Axon Guide - 2500-0102 Rev. C 31

Figure 3.3: The

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6. The quenching of a Ru based fluorescent molecule by oxygen is described by the “Stern-Volmer Equation” which describes the kinetics of the quenching process. This is as follows:

�I0 is the intensity when there is no quenching while I is the intensity at the given oxygen concentration [O2]. In a similar way, τ0 is the fluorescence lifetime with no quenching and τ is the lifetime at oxygen concentration [O2]. KSV is the Stern-Volmer constant which is a measure of the quenching efficiency and is often expanded to be the product of the baseline lifetime τ0 and kq, a rate coefficient for the quenching process such that:

�

6.1. The intensity data from an oxygen sensor made from a fibre optic cable tipped with an immobilised fluorescent material is shown in figure 9. Suggest why this data would make it difficult to use for a sensor and explain a possible source for the observed drift.

�Figure 9. Intensity data from a fluorescent oxygen sensor exposed to saturated atmospheres of O2 and N2

6.2. The lifetime, which is generally unaffected by issues causing drift in the intensity, is commonly measured through modulating the light input with some frequency (f) and measuring the phase shift in the resulting fluorescence. The phase shift (φ) is related to the lifetime (τ) by the following equation:

�Estimate the values of τ0 and τ[100% O2] from the data in figure 10, given that the input frequency is 75 kHz

I0I

=⌧0⌧

= 1 + KSV [O2]

I0I

=�0�

= 1 + kq�0[O2]

294 P.A.S. Jorge et al. / Sensors and Actuators B 103 (2004) 290–299

case where larger fiber core diameters are used. Indeed fora fiber with a 1mm core diameter light coupling efficienciesof ≈20% were achieved.In the experimental set-up of Fig. 1 the butt-coupling con-

figuration was used with a 550!m core fiber. In such situa-tion, an optical power of 70!Wwas available in each outputof the fiber coupler.Preliminary tests, in gaseous environments, were per-

formed using the coated glass slides in order to characterizethe sol gel thin films. The parameters Q and !τ were eval-uated as a function of the solution aging time, from 2 h tomore than 48 h at 20 ◦C. Both these parameters increasedwith increasing aging time. Values for Q parameter between30 and 40% were obtained. Values for!τ varied between 77and 176 ns. As stated in the literature, this behavior results

0 60 120 180 240 300 360 42040

60

80

100

120

140

160

180

(a)

100% N2100% N2

100% O2100% O2

Fluo

resc

ence

Inte

nsity

(mV)

time (s)

0 60 120 180 240 300 360 420-20

-18

-16

-14

-12

-10

-8

-6(b)

100% N2

100% O2

100% N2

100% O2

φ d (d

egre

es)

time (s)

Fig. 5. Response of fiber taper to O2/N2 saturation cycles: (a) fluorescence intensity response; (b) phase response.

from increased film porosity and easier oxygen diffusioninto the matrix [8]. This traduces in higher quenching ef-ficiency and increased oxygen sensitivity. The unquenchedlifetime, τ0, decreased with increased aging time, 630 and545 ns, for 2 and 24 h aging time, respectively. With a dipcoating speed of 3mm/s, film thickness between 600 and800 nm were obtained (measured with a perfilometer). Theresponse time of the sensing films when they undergoneO2/N2 saturation cycles varied between 9 and 11 s (framedto the 10 and 90% reference levels). In the slide configu-ration the detected fluorescence emission was very weak(Pd ≈ 0.36 nW), and due to a low signal to noise ratio (SNR)the phase signal was unstable. The resulting Stern–Volmerplots were non-linear indicating that the sensing complexhad a heterogeneous distribution inside the porous silica

tan[�] = 2⇡f⌧

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�Figure 10. Phase data from a fluorescent oxygen sensor exposed to saturated atmospheres of O2 and N2

6.3. The data shown in figure 11 is phase and intensity data for step changes in the atmosphere being sensed. Estimate values of I0/I and τ0/τ for each oxygen concentration, assume that the results for [O2] = 0.4 % are effectively the same as for 0% oxygen. Why don’t you actually require the value of the input modulation frequency?

6.4. Explain the discrepancy between the two sets of measurement results. Plotting the results on a graph can help demonstrate this difference.

6.5. Describe a possible method of miniaturising the sensor and light source and integrating it with a microfluidic system, perhaps with the aim of measuring the oxygen partial pressure in blood samples.

�

294 P.A.S. Jorge et al. / Sensors and Actuators B 103 (2004) 290–299

case where larger fiber core diameters are used. Indeed fora fiber with a 1mm core diameter light coupling efficienciesof ≈20% were achieved.In the experimental set-up of Fig. 1 the butt-coupling con-

figuration was used with a 550!m core fiber. In such situa-tion, an optical power of 70!Wwas available in each outputof the fiber coupler.Preliminary tests, in gaseous environments, were per-

formed using the coated glass slides in order to characterizethe sol gel thin films. The parameters Q and !τ were eval-uated as a function of the solution aging time, from 2 h tomore than 48 h at 20 ◦C. Both these parameters increasedwith increasing aging time. Values for Q parameter between30 and 40% were obtained. Values for!τ varied between 77and 176 ns. As stated in the literature, this behavior results

0 60 120 180 240 300 360 42040

60

80

100

120

140

160

180

(a)

100% N2100% N2

100% O2100% O2

Fluo

resc

ence

Inte

nsity

(mV)

time (s)

0 60 120 180 240 300 360 420-20

-18

-16

-14

-12

-10

-8

-6(b)

100% N2

100% O2

100% N2

100% O2

φ d (d

egre

es)

time (s)

Fig. 5. Response of fiber taper to O2/N2 saturation cycles: (a) fluorescence intensity response; (b) phase response.

from increased film porosity and easier oxygen diffusioninto the matrix [8]. This traduces in higher quenching ef-ficiency and increased oxygen sensitivity. The unquenchedlifetime, τ0, decreased with increased aging time, 630 and545 ns, for 2 and 24 h aging time, respectively. With a dipcoating speed of 3mm/s, film thickness between 600 and800 nm were obtained (measured with a perfilometer). Theresponse time of the sensing films when they undergoneO2/N2 saturation cycles varied between 9 and 11 s (framedto the 10 and 90% reference levels). In the slide configu-ration the detected fluorescence emission was very weak(Pd ≈ 0.36 nW), and due to a low signal to noise ratio (SNR)the phase signal was unstable. The resulting Stern–Volmerplots were non-linear indicating that the sensing complexhad a heterogeneous distribution inside the porous silica

P.A.S. Jorge et al. / Sensors and Actuators B 103 (2004) 290–299 297

difference is expected to be maximum around 188 kHz,which is the ideal modulation frequency [5]. This way, an in-crease in modulation frequency is expected to improve sen-sor performance. In Fig. 5(a) and (b) the phase and the fluo-rescence intensity response of the sensing system to O2/N2saturation cycles can be observed. The phase signal showssome instability indicating the need for SNR improvement.In order to demonstrate the phase insensitivity to opticalpower drift a simple test was performed. With the sensinghead in a 21% O2 atmosphere, the optical power injectedinto the fibre system was changed up to 25%. Fig. 6 showsthe consequence of this variation in the intensity and phaseof the fluorescence signal. Although a significant change in

0 60 120 180 240 300 360 420 480-20

-18

-16

-14

-12

-10

-8(a)

80%

100%

39,1%

60%

20,6%

12,3%8%

0,4%

φ d (d

egre

es)

time (s)

0 60 120 180 240 300 360 420 4800,03

0,04

0,05

0,06

0,07

0,08

0,09

0,10

0,11(b)

100%80%

60%

39,1%

20,6%

12,3%8%

0,4%

Fluo

resc

ence

inte

nsity

(mV)

time (s)

Fig. 8. System response to step variations of O2 concentration level: (a) phase response; (b) fluorescence intensity response.

the intensity response occurs, the phase response remainsessentially unchanged. This confirms the ability of thephase detection scheme to avoid power fluctuations inducederrors.The Stern–Volmer plots obtained from phase measure-

ments with all configurations are clearly non-linear, indicat-ing that the dopant is not homogeneously distributed withinthe silica matrix. Instead, the ruthenium complex occupiesenvironments with different oxygen accessibilities. In thissituation, the standard Stern–Volmer equation (Eq. (1)),based on a single exponential decay, no longer describesaccurately the quenching behavior. Alternatively a dual ex-ponential model, corresponding to two different dominant

�8

�Figure 11. Sensor response to step variations of O2 concentration: (a) phase response; (b) intensity response.

P.A.S. Jorge et al. / Sensors and Actuators B 103 (2004) 290–299 297

difference is expected to be maximum around 188 kHz,which is the ideal modulation frequency [5]. This way, an in-crease in modulation frequency is expected to improve sen-sor performance. In Fig. 5(a) and (b) the phase and the fluo-rescence intensity response of the sensing system to O2/N2saturation cycles can be observed. The phase signal showssome instability indicating the need for SNR improvement.In order to demonstrate the phase insensitivity to opticalpower drift a simple test was performed. With the sensinghead in a 21% O2 atmosphere, the optical power injectedinto the fibre system was changed up to 25%. Fig. 6 showsthe consequence of this variation in the intensity and phaseof the fluorescence signal. Although a significant change in

0 60 120 180 240 300 360 420 480-20

-18

-16

-14

-12

-10

-8(a)

80%

100%

39,1%

60%

20,6%

12,3%8%

0,4%

φ d (d

egre

es)

time (s)

0 60 120 180 240 300 360 420 4800,03

0,04

0,05

0,06

0,07

0,08

0,09

0,10

0,11(b)

100%80%

60%

39,1%

20,6%

12,3%8%

0,4%

Fluo

resc

ence

inte

nsity

(mV)

time (s)

Fig. 8. System response to step variations of O2 concentration level: (a) phase response; (b) fluorescence intensity response.

the intensity response occurs, the phase response remainsessentially unchanged. This confirms the ability of thephase detection scheme to avoid power fluctuations inducederrors.The Stern–Volmer plots obtained from phase measure-

ments with all configurations are clearly non-linear, indicat-ing that the dopant is not homogeneously distributed withinthe silica matrix. Instead, the ruthenium complex occupiesenvironments with different oxygen accessibilities. In thissituation, the standard Stern–Volmer equation (Eq. (1)),based on a single exponential decay, no longer describesaccurately the quenching behavior. Alternatively a dual ex-ponential model, corresponding to two different dominant