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Team:TU Delft-Leiden/Project/Gadget
From 2014.igem.org
Gadget Overview
The goal of synthetic biology is to turn biology from a science into a technology. In order to do this, it needs to be brought out of the lab to fulfill its full potential. A product designed around the electrace whole-cell biosensor would allow for real world application, outside of the confines of the lab.
Figure 1:
A concept which allowed for the meeting of the digital and biological worlds was imagined, with the Electrace cell at the heart. Electrace creates a modular system where biosensor "plugins" in the form of biobricks can be designed by "wetware developers". This would allow the electrical output of Electrace to be used with a range of different inputs - for example the DNT detection seen elsewhere in our project. Since each biosensor would have it's own set of requirements for data processing, software developers would create matching plugins for a digital Electrace platform. Via a web-based app then, the user would select the appropriate combination of software for the analyte in question. A wifi enabled potentiostat measures the electrical output of the given Electrace bio-sensor, which then sends this data to the mobile app for processing and display.
Figure 2: Visualisation of the functional domains involved in the gadget.
Voltammetry
In the ET module, electrons are anticipated to be shuttled to the extracellular environment if the induced pathway works according to expectation. The intention of the aforementioned device is to accurately measure and quantify the electrons being transferred to a certain degree of tolerance. The task can be accomplished by a commercial Potentiostat but is economically unviable due to its’ outrageous price tag (>2000 USD). Hence an attempt has been made towards developing a comparable device from scratch using indigenous resources.
The primary electronic circuits that form the measuring device are composed of standardized components (Op-Amp etc.) and are therefore available in any general purpose hardware store. As an interfacing device between the analog and the digital domains, a National Instruments (NI) myDaQ is used. In turn the myDaQ can be interfaced with a PC through the USB in order to store this data. This can then be compared with the prediction of the system model, whose quality can then be judged. Also, input-output data can be used to fit models with a predefined model structure, thereby aiding one in obtaining the values of the crucial reaction rates. Hence it is desirable that the measuring device is accurate and precise. Potentiostats which are readily available are expensive as they utilize specialized precision electronics in order to maintain high accuracy of the readings. This device will not be extremely accurate but is expected to perform satisfactorily.
Working Principle and Construction
The Potentiostat is the hardware required to control a 3 electrode system used for the analysis of electrochemical experiments. As mentioned, the system consists of three separate electrodes namely the Working Electrode (WE), Reference Electrode (RE) and the Counter electrode (CE). The generalized cell configuration of such a system has been illustrated below. Operational amplifiers in various negative feedback configurations are employed to replicate the functioning of a Potentiostat. This has been demonstrated in subsequent sections.
Figure 3: A Sophisticated Potentiostat
Figure 4: Three electrode cell configuration
In this case both the working and the counter electrodes are made out of smooth machined carbon. The RE is analogous to system ground i.e. all other voltages applied or measured at the working / counter electrodes are with respect to the voltage here. Consequently the term “Reference” has been adopted. This component usually consists of an Ag/AgCl or Pt/PtCl electrode. In principle, any metal which tends to be chemically inert and is therefore capable of maintaining a stable and fluctuation free voltage can be used for this purpose.
A varying potential is applied to the WE with respect to the RE and hence the electrons which are ferried out into the sample by the e-coli resumes motion under the effect of the applied electromotive force. The Counter Electrode can now detect the generated current and relay the information to the PC via the DaQ.
The general design of the electrolytic cell along with the 3 electrodes has been discussed in fair detail until now. In the next segment, the problem of electronic circuit design and its interfacing with the computer is tackled in a methodical manner.
Primary Circuit Design and Interfacing
As previously mentioned, the associated circuitry consists of both digital and analogue parts which are required to effectively transmit the measured data to a PC. Hence the myDaQ / myRIO will be used in conjunction with the designed analog circuitry. The DaQ already possesses inbuilt A/D (Analog to Digital) and D/A (Digital to Analog) converters so one needs to merely connect the wires from the analog domain into suitable ports of the device.
Figure 5: Schematic of Potentiostat
The schematic above illustrates the circuitry which has been adopted for this purpose. It consists of various OPAMPS intelligently connected in different negative and positive feedback configurations such that the desired functions are fulfilled. The entire network might seem daunting at first sight, but it actually is quite simple and elegant when evaluated using a modular approach. The hardware can be segregated into various modules namely:
- Voltage Adder (Summing Device)
- Polarity Invertor
- Voltage Buffers ( Load independent Voltage Regulators)
- Cell Components ( WE, RE and CE)
- myDaQ / myRIO – Analog to Digital ( A / D) and Digital to Analog (D / A) convertors. Interfacing with PC (USB & Lab-View)
The following figure shows these in the form of a block diagram
Figure 6: Block Diagram of Circuitry
The core idea of a Potentiostat is to excite the Working Electrode with an external voltage in order to generate the electromotive Force capable of ‘pushing’ the transported electrons into the counter electrode. Consequently, the presence of the voltage “generator” is crucial and the computer aids directly in the process. Due to the availability of software like LabView any waveform can be generated. Frequently a periodic waveform is used to excite the system for e.g in order to assess the inherent system hysteresis (non-linear behavior). Refer Gopinath and Russel et al for more details.
The Voltage which is to be applied to the WE is generated via the myDAQ/ myRIO itself. These devices often possess the capability to simultaneously detect as well as produce signals .Hence the DaQ has been programmed such that both triangular and constant voltage waveforms are generated. The frequency, amplitude and the phase of the waveforms can be adjusted by altering a few variables in the associated software code. More discussion on this matter is presented later.
It is to be noted that any excitation voltages applied to the Working Electrode is always with respect to (wrt) the Reference. For example, if a potential difference of 5V DC wrt to the RE is required, merely generating a 5V DC from the battery and feeding it into the WE will not suffice! If the Reference Electrode senses a voltage of 1V DC, then it is essential that a voltage of 5V + 1V = 6V is totally applied to the WE. Only then does the condition of 5V with respect to the RE become valid. If this concept is well grasped, then the necessity of the Voltage “Adder” becomes obvious.
One needs to take note that the voltage adders which are implementable using OPAMPs produce an inverted sum. That is, instead of producing 6 Volts it produces -6V. In electrochemical applications, the polarity of the signal being applied plays a significant role in system behavior. Consequently it is necessary that the summer produces the signal with the correct sign. To facilitate this property a polarity “invertor” is cascaded with the summer (This device reverses the sign of the input voltage fed to it). In the block diagrams above/below, the Polarity Invertor is embedded in the Voltage adder and is considered to be it’s integral part.
Figure 7: Illustration of Polarity Invertor cascaded with the summer.
Figure 8: Voltage adder used for summing the 2 Voltages
The Voltage Buffer is a circuit comprising of an OPAMP in a unity negative feedback configuration. This has something to do with the output voltage of the OPAMP being fed-back in a manner such that it opposes the applied input voltage that generated this output in the first place. When in such a configuration, the amplification factor of the OPAMP become close to 1 and hence the Output Voltage perfectly “follows” the input Voltage irrespective of the output load conditions (this circuitry is also known as a voltage follower).
Figure 9: Usage of Voltage Buffers 1 & 2
Once the electrons are pushed into the counter electrode, the current generated is to be sensed. Since the myRIO /myDAQ require voltages to work with, the current is passed through a Current – to – Voltage convertor to obtain a voltage calibrated wrt the sensed current. Based on the range of the current to be sensed, the value of a certain precision Resistance is to be altered suitably. This has been discussed explicitly in the next section.
The circuitry discussed till now belongs completely to the domain of analog electronics. In order to store the sensed data onto a computer, the information has to be “digitized”. This is done with the help of an Analog to Digital converter (A to D). Similarly when random waveforms are produced on a PC, a digital version is initially created. Only when this digital data is passed through a Digital to Analog convertor (D to A) does one obtain the actual waveform suitable for exciting the W.E. The common job of a D to A and A to D convertor can be accomplished either by the DAQ or myRIO. Moreover, as these devices readily interface with a PC, real-time measurement data can be easily relayed to the PC screen.
Figure 10: Digital and Analog Domains
A modular overview of the associated circuitry has been presented until now. The next section deals with the detailed analysis and functioning of the implemented circuitry. Before continuing, it is advised that the reader is familiar with the basic working of electrical /electronic circuits and especially OPAMPS. He /she must be familiar with basic principles namely Ohms’ law, Kirchhoff’s laws, Norton – Thevnin equivalence etc. Before the next section begins, the same daunting schematic has been presented again but now with the different modules demarcated. It should feel easier to digest at this stage.
Figure 11: Schematic with demarcated modules
Detailed Circuit Analysis
Once the overall understanding of the system is achieved, the detailed working of the hardware can now be investigated. Operational Amplifiers (OPAMPs) form ‘the most’ important component of the designed circuitry as it is the only active element. Ideal OPAMPS are amplifiers with an amplification factor A = infinity. Also, the input resistance of the amplifier is ideally infinite and is depicted in the figure below as Rin. Rout is finite and hence a finite current can flow through it .There are two input terminals of the OPAMP – the positive and negative terminals.
Figure 12: An Operational Amplifier (OPAMP)
The OPAMP in principal is supposed to ideally amplify the difference in voltage between the two, which is called the differential input voltage. The terminals in the figure are as follows:
- V+ → Non inverting (Positive) input terminal
- V - → Inverting (negative) input terminal
- Vin → Net input Voltage into OPAMP = V+ - V-
- Vs+ / Vs- → Positive and Negative Power supplies
- Vout – Voltage output produced by OPAMP
- Rin /Rout → Input / Output Resistances
- A – Amplification factor of the OPAMP
Practical OPAMPs have finite but high gains and input resistances of the order of 10^6. The relationship between the Input and Output Voltages in an open loop setting is Vout = A( V+ – V-). Such massive gains are achieved by cascading various amplifiers with low gains in series with one another. These devices are capable of power amplification and termed active devices as they need an external power source to operate ( Vs+ and Vs-). The external power is required to bias the individual transistors that make up the OPAMP thereby preserving the law of conservation of energy.
Voltage Buffer/ Follower
A voltage buffer as mentioned before is used to regulate the output voltage around a certain fixed value, irrespective of the current drawn/ load attached => (Value of downstream resistance RL). Hence the most appropriate device for such a situation is the OPAMP in an unity negative feedback configuration. This is as illustrated below:
Figure 13: OPAMP in negative feedback
From the above figure it is clear that the entire Output voltage Vo is ‘fed back’ into the ‘negative’ input terminal. Consequently, the term “Negative Feedback” is utilized to describe such an interconnection. In such a configuration, the Output Voltage Vo perfectly follows the input Voltage fed into the Vi + terminal irrespective of any downstream activity. Unity refers to the situation of 100% Output voltage being fed back.
The relationship for negative feedback is: Output Voltage Vo = A /(1 + A) x Vi . If A is very high (≥ 10^6), then Vo = Vi as A/(1+A) ≅ 1
Two voltage buffers have been used in the circuit at strategic locations where downstream activity may potentially alter the line voltage and are expected to improve the overall performance.
Voltage Summer & Polarity Invertor
Figure 14: A two port summer
This device is required to maintain the WE voltage at a specified voltage with respect to the RE Voltage. This also consists of an OPAMP in a modified negative feedback configuration in order to generate the virtual ground and thereby converting all incoming voltages (V1, V2) into currents corresponding to the resistance used (R). The added current is now forced to pass through the resistor in the feedback loop to generate back the entire summed Voltage V1 + V2. The current is pushed into the feedback resistance (Note: also R) due to the existence of infinite input resistance between the two input terminals. Hence the current would prefer to flow through a path providing lesser hindrance.
Vo = V1 + V2. Due the existence of Virtual Ground, the voltage here = 0. Hence, I1 = (V1 - 0)/R1 = V1/R. Similarly I2 = V2/R . Consequently, net current flowing through the feedback resistor = I1 + I2 . Hereafter the Voltage created at the output is: Vo = 0 – R(I1 + I2) = - (V1 + V2). Note that the inverted sum is produced at the output.
Figure 15: Polarity Invertor
Current to Voltage Convertor
Figure 16: Current to Voltage Converter
The incoming current from the Counter electrode Iin needs to be converted into a corresponding voltage which can be sensed by the D to A convertor. This measured voltage is proportional to the current being sensed. By varying a precision Resistance placed in the feedback loop R, the range of the current to be sensed can be adjusted. As evident from the figure this is also a case of negative feedback where the current pushed into the feedback loop gets converted into the inverted voltage.
Vout = Iin x Rf. Vout can be made to drive an auxiliary load Rl. This is not the case in our situation!
Practical Implementation / Software
Once the signals have been digitized, the acquired signals must be charted on some kind of graph in the Signal (Voltage / Current) vs Time format. It is intended that this graph will get updated in real time as increasing no of samples are acquired. In order to achieve this, the associated software - LabView needs to be programmed accordingly.
In general, any general purpose computation & Data Acquisition software like Matlab, Maple etc can be interfaced with the DaQ provided the associated toolboxes are present. If required the software can be coded on C / C++ such that a “*.exe” is created, which is a standalone program and needs no third party software to run. As the myDaQ is a National Instruments product and so is LabView , the interfacing between the two has already been accomplished to a certain degree.
LabView provides an advantage over other conventional script based programming software like C etc as it works on Graphical Code (G Code). One simply needs to have the logic ready in his/her mind, which can be easily implemented by dragging and droping various functional blocks and interconnecting them via “wires”. These “wires” exhibits the signal flow within the digital domain in a software setting. This corresponding graphical code for this application has been represented in the figure below:
Figure 17: Potentiostat Software Block Diagram
The above wiring diagram is equivalent to the software code one writes using scripts but the advantage here is that the logic flow can be graphically seen and error can be easily troubleshooted. The DAQ Assistants boxes are the functional blocks which enables interfacing with the analog domain. The “DAQ Assistant” block is responsible for converting the digitized version of the WE Voltage to an actual analog signal ( step / triangular) which is fit for excitation.
Likewise the block “DAQ Assistant2” plays the crucial role of receiving the analog domain signals (like the sensed CE current) and converting them into a digitized version for use on the PC. The code embedded within these blocks has been supplied by NI. The charts are used for plotting the information in real time as the data vectors grow with time. The LabView interface consists of two main windows. One is the Block Diagram where the G code is implemented. The other is known as the “front panel” and is used to simulate the front panel of an actual instrument (in this case the Potentiostat). This is the window where the charts actually plot the values and one can read them off directly! This is shown as follows:
Figure 18: Front Panel
As mentioned earlier, the front panel plays an essential role. It forms the interface between the user and the device which has been presented here. Two different kinds of excitation signals are possible – Either a step DC Voltage or a triangular waveform for Cyclic Voltametry. The value of the DC Step can be adjusted via the knob. Other waveform settings for the cyclic voltammetry mode like signal phase, amplitude and frequency can also be adjusted via suitable input options on the left.
The various charts show the different signals being applied and sensed. This image was captured when an ongoing test with a dummy cell was being performed. Crucial settings such as Sampling Rate and no of Samples (Buffer size) should be carefully adjusted. The sampling rate must be chosen in accordance with Shannon’s Theorem. Or else the signal being sensed / generated would not be accurate due to probable aliasing issues.
Specifications
- Sensitivity range - >20 micro amps
- Excitation modes – Step and Triangular
- Step Max Amplitude – +1 V
- Triangular Amplitude - +/- 1V
- Triangle Frequency Adjustable ( Analogous to Scan Rate) – (100Hz max)
- Sampling Rate/ buffer size Adjustable – (100 KHz max)
- Displays : Applied voltage at Working electrode - WE + RE
- Displays : Sensed CE current & calibrated Voltage
- Data can be saved directly onto Microsoft Excel in a vector-wise format
Timeline - Photos , Videos and Disasters
Still a concept. Just back from the hardware store.
Building the modules bit by bit
Modules being tested as they are assembled
Sim is delighted. Important modules work individually
Testing with a dummy cell. Results not according to expectation. Wiring ERROR somewhere‼
The Close-up
DISASTER has struck. Potentiostat in the making falls on the ground ( Courtesy : Debu).
We don’t give up do we? Sim rebuilding from scratch. Now the components are more efficiently placed to minimize wiring length. This reduces confusion if the wiring has to be altered.
Performing Voltammetry tests with KCL. Results match expectation! Testing is done with a multimeter at this stage. Software environment is not yet ready.
Finalized system being tested using a Dummy Cell. Software environment confirmed as LabView. Data can now be measured in real-time. Graphs on the screen display the measured current and other variables.
