Biosensors and Instrumentation: Tutorial 3

Transcription

1 Biosensors and Instrumentation: Tutorial A schematic cross section of an ion sensitive field effect transistor (ISFET) is shown in figure 1. Vref Solution eference Electrode Encapsulation SiO2 nsi Source nsi Drain psi 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. ΔΨ = 2.3α T [1] F ΔpH bulk where 1 =! (2.3kT/q 2 )(C dl / int )1 [2] Define the term Cdl and describe how it will be affected by changes in the measured solution Define the term βint and why it should be maximised If α=1 what will be the sensitivity δψ/δph at 25 C (298 K)? What type of response does the result of (iii) resemble? 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? V 2 DS I DS = (V GS V T )V DS 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?

2 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. 2 ef. electrode 3 ISFET D S V 1 in 2 V out 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. Vdd I in V 1 V ef. electrode 1 ISFET D S DS 1 V 5 V ref out V out V 2 V S I f 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 1 = 2 = 3 =.

3 Vsource (vs. ef) [mv] 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 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 S and out to give an output voltage change of 1V ph 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 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?

4 4 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 In the Goldman equation, shown below, which parameters are principally affected by the the opening and closing of ion channels? E m = T F ln PK [K ] out P Na [Na ] out P Cl [Cl ] in P K [K ] in P Na [Na ] in P Cl [Cl ] out 4.3. Suggest two possible ways in which a neuron could be excited into an action potential 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? Vin V p 1 V in V in Vp 2 V p 3 I out Cell I Figure 6. Current clamp circuit for neuro-electrophysiology

5 4.5. Given that all resistors marked 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 out 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 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? 5 I V p 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 Derive an expression for the output Vout If Ich is in the pa range suggest a suitable value for f giving reasons for your answer 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. f V out Diff. Amp. V in Cell Bath Electrode Figure 8. Patch Clamp Circuit for Electrophysiology

6 6 6. The quenching of a u based fluorescent molecule by oxygen is described by the Stern-Volmer Equation which describes the kinetics of the quenching process. This is as follows: I 0 I = 0 =1K SV [O 2 ] 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: I 0 I = 0 =1k q 0 [O 2 ] 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 (a) 100% N 2 100% N 2 Fluorescence Intensity (mv) % O 2 100% O time (s) Figure 9. Intensity data from a fluorescent oxygen sensor exposed to saturated atmospheres of O2 and N 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: tan[ ]=2 f Estimate the values of τ0 and τ[100% O2] from the data in figure 10, given that the input frequency is 75 khz

7 (b) 100% O 2 100% O 2-10 φ d (degrees) % N 2 100% N time (s) Figure 10. Phase data from a fluorescent oxygen sensor exposed to saturated atmospheres of O2 and N 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 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. (a) % 100% φ d (degrees) % 12,3% 20,6% 39,1% 60% -18 0,4% time (s)

8 8 (b) 0,11 0,10 0,4% Fluorescence intensity (mv) 0,09 0,08 0,07 0,06 0,05 0,04 8% 12,3% 20,6% 39,1% 60% 80% 100% 0, time (s) Figure 11. Sensor response to step variations of O2 concentration: (a) phase response; (b) intensity response.