target – biological receptor – transducer – signal – recording 
An immunosensor uses the specificity of an antigen – antibody interaction for its recognition. The antibodies are immobilized on the surface of the biosensor. When a liquid containing the antigen is flooded over the biosensor the antibodies bind to their antigen. This interaction can be detected in different ways. In our case, the binding leads to a mass shift which can be detected using the SAW technique. Biosensors offer the possibility of a label-free detection by which even small molecules can be identified.
Figure 1 General design of an immunosensor. An analyte-specific antibody catches the analyte and triggers a signal at the detector.
Setup of our next generation biosensor
Figure 2 SAW sensor components. Quartz piezoelectric crystal, IDTs (interdigital transducer) are built on the crystal, the sensing film is located between the IDTs, the SAW Chip is inserted in a chip case which establish the connection to the electronics, the electronics are connected to the app to evaluate the data.
Surface acoustic wave technique
Video 1 Piezoelectric effect. The crystal changes his form when pressure is applied. This leads to a displacement of the negative and positive charges. That displacement leads to an electric potential on the surface of the crystal.
But how does SAW work?
Figure 3 - Design of a SAW Chip: consists of piezoelectric crystal, IDTs, gold sensing film.The central components of a SAW chip is the piezoelectric and two interdigital transducers (IDTs), the s-shaped red lines in Figure 3 These are pairs of two electrodes with interlocking "fingers", applied as a layer of gold on the surface of the crystal. One of the IDTs is used to induce surface acoustic waves in the piezoelectric crystal by applying a high frequency, electric sine wave to one of its electrodes. The other IDT is measuring the acoustic waves that travel across the surface of the crystal by transducing them back into an oscillating electrical potential. Between these two IDTs, there is a so-called sensing film – the central red square in the Figure 3 -, where the antibodies are immobilized. When adding a sample on this film, the antigen is binding to its antibody leading to a mass shift and consequently to a phase shift between the input and output IDTs. Both analog electrical signals are transferred to the detection unit where the phase shift is detected and compared with a standard curve, leading to an estimate of the analyte concentration.
The SAW technique can be used in any scenario where a sensitive biosensor based on mass changes is needed. In that case, our setup can be used as a model for different applications. If target-specific antibodies already exist our SAW sensor can be used for sensitive measurement. In summary, SAW technology can function as an alternative to already existing biosensors, but it should also be kept in mind when developing new biosensors because of the already mentioned advantages. Therefore, it contributes to the whole iGEM community as an innovative, othogonal approach for biosensors.
Video 2 Simplified, schematic presentation of the SAW sensor Displayed in red is the piezoelectric crystal, on its surface is in the middle the gold sensing film visualized in green and the 2 green squares on the edges visualize the IDTs. After that the antibodies are immobilized on the sensing film, followed by putting on a sample represented by red dots, which represent the hormons. The antibody-antigen binding leads to a phase shift on the surface of the crystal represented by a slower wave.
DDS - Direct Digital SynthesisThe SAW technique is dependent on specific frequencies to induce surface acoustic waves in the piezoelectric crystal. In our case we decided to use a horizontal frequency of 250 MHz to detect substantial differences in phase shift through antibody-antigen binding of hormones. This requires an electronic device that can reliably generate such high frequencies. Initially, we considered three possible options to generate these frequencies::
2. an LC resonant circuit
3. a DDS
The second possible implementation we have considered was the use of an LC resonant circuit. It consists of a capacitor (C) which is connected in parallel with an inductor (L). By applying a voltage, the LC resonant circuit can be stimulated to electromagnetic self-oscillation, whereby sine waves are generated. However, this circuit requires an exact coordination of all components and is inflexible in the sense that these components need to be dimensionalized for a specific output frequency. At the beginning of the project we could not make any statement about individual frequency parameters (such as amplitude etc.), which is why this type of implementation proved to be unfavorable in this project stage.
In order to stay on the path of a portable measuring device, we have decided to take a closer look at DDS – direct digital synthesis. But what exactly are DDS?
DDS - The goal of microelectronics
The main advantage of direct digital synthesis (DDS) is its digital operation which allows fast switching between different frequencies. Even though in our case, we only need to generate a single, fixed excitation frequency, this allows us to test several different frequencies in fast succession. Because of the possibility to build very small integrated circuits (IC), a DDS is not much bigger than a 5- cent coin from Europe (Figure 4). DDS-Chips offer an incredibly high range of settings and with that many possibile applications which would go far beyond our project. In our case, the DDS should only generate a constant frequency of 250 MHz. However, this flexibility with respect to the frequency range and modulation greatly increases the reusability of our setup for similar applications of future iGEM teams.
Figure 4 – a DDS (black chip with “Analog Devices” compared to a 5-cent coin from Europe
The Arduino Shield of GRA & AFCH with the AD9910 At first, we decided to test our SAW chip with an already developed DDS-Board, operated by an Arduino and later to develop our own evaluation board (with all the necessary components, e.g. a phased locked loop (PLL), comparator and the phase shift detection circuit). After a little research, we found the Arduino Shield of GRA & AFCH with the DDS AD9910 from Analog Devices. This evaluation board is a shield for the microcontroller Arduino Mega. It can reliably generate frequencies up to 400 MHz at a clock speed of 1 GHz and frequencies up to 600 MHz at a clock speed of 1.5 GHz. Furthermore, there is only a small amount of interference frequencies which can likely be neglected, because the amplitudes are always lower than -50 dB (until frequencies of 400 MHz). We measured frequency spectra for several output frequencies in the range of 25 MHz up to 600 MHz generated with the AD9910 shield to evaluate its performance characteristics.
The results are described and analyzed in the results section of our wiki, see Hardware.
Figure 5 – The AD9910 Arduino Shield for the Arduino Mega developed by GRA & AFCH
SchematicsUnfortunately, we cannot publish the schematics of the Arduino shield, because it is still on sale. Furthermore, the developers asked us to use the schematic only for our further development of subsequent circuitry (comparator and shift detection circuits). However, the missing schematic will not affect a reproduction of our project in any way. It only served us to verify the functionality of the shield. The datasheet of the Analog Devices AD9910 DDS IC contains schematics of common use cases and can be downloaded from the manufacturer’s website.
Figure 6 – Texas Instruments Amplifier LMH2832EVM-50
We have decided to operate with the Texas Instruments' LMH2832EVM-50. This device allows an amplification in the range of 10-9 to 1030 which ensures that even very low amplitude values can be amplified. This range was neccessary because there has adaptability, allowing future iGEM teams to utilize our hardware setups for their own projects, independent of their specific damping factor. The amplifier can be controlled via a Windows GUI from Texas Instruments.
The results of the first parameterization and testing of the amplifier are shown under the tab "Results/Hardware".
The phase shift detection PCB - operating principle
We first thought about the possibilities to measure a phase shift. Research on the Internet has shown that there are barely any fully developed devices that can measure a phase shift. Therefore, the only possibility for us was to develop a new board that could measure a phase shift.
Various electronic components which are needed to compare the reference and measured frequency are located on this PCB. The following chapter will briefly explain the signal path on the board. After the excitation frequency has been generated and passed through the SAW chip, it is fed into the PCB as the reference signal, together with the amplified output signal of the SAW chip. The reference signal is led into input A and the measured output signal is led into input B.
Each of the two signals is high-pass filtered and reach a ADCMP572 comparator. These devices compare the input signal using the so-called Zero Crossing Detection and them to a current mode logic (CML) standard signal. If the voltages are above 0V, the comparator outputs a logical HIGH signal of 3.3 V at the output Q. If the voltages are below 0V, the comparator switches to a logical LOW output signal of 2.7 V at the output.
The output signals are directed to a multiplexer (MUX), which is mainly used for calibration and simulation of a phase shift of 0° and 180°. However, it has no further significance for the measurement of the phase shift.
The most important component is the XOR gate that processes the signals that arrive from the comparators or multiplexers. The XOR circuit is a digital circuit that only operates on the logical states 0 and 1 (see Table 1), that, in our case, correspond to the LOW and HIGH levels of the CML standard, 2.7 V and 3.3 V respectively. The output is a logical 1 if the two comparators have different output signals. The duration of XOR output being at logical 1 correlates with the amount of phase shift between the inputs of the XOR gate. In addition to the non-inverted output, the XOR gate also outputs an inverted output signal that is always at the opposite logical level as the non-inverted output. Both the inverted and non-inverted output of the XOR gate are subsequently low-pass filtered by an LC filter, averaging the previously oscillating signals to analog DC signals between 2.7 V and 3.3 V. These signals are passed to an IN333 instrumentation amplifier operating as a differential amplifier with gain adjustable via a potentiometer, amplifying the voltage difference between the filtered XOR outputs. The resuting analog output between 0 V and 5 V is is converted into a digital signal by the 10 Bit analog-to-digital-converter of the Arduino Mega (10 Bit means that 210 = 1024 different analog values are distinguishable by the converter). The converted signal can be monitored on the serial console of the Arduino IDE.
Table 1 - XOR Truth Table
Figure 7 – simplified model of the signal flow chart of the PCB
Shows the most important components of the PCB: comparator, XOR-gate, amplifier, low pass filter and the instrumentation amplifier. Reference and SAW-signal led to a comparator and after that to the XOR-gate, which creates impulses of either 0 or 1. The instrumentation amplifier generates a voltage with the impulses of the XOR.
-  A. A. Vives, Piezoelectric transducers and applications. 2008.
-  K. Länge, B. E. Rapp, and M. Rapp, “Surface acoustic wave biosensors: A review,” Analytical and Bioanalytical Chemistry. 2008.
-  E. Murphy, C. Slattery, "All About Direct Digital Synthesis", analog.com/analog-dialogue . 2004.
-  GRA & AFCH, "RF Signal Generator DDS Arduino Shield AD9910 600 MHz", create.arduino.cc/projecthub . 2020.
Figure 8 SAW sensor components. Quartz piezoelectric crystal, IDTs (interdigital transducer) are built on the crystal, the sensing film is located between the IDTs, the SAW Chip is inserted in a chip case which establish the connection to the electronics, the electronics are connected to the app to evaluate the data.
Figure 9 Dimensions of the piezoelectric quartz SAW crystal with IDTs and gold sensing film.
Table 1 Measurements IDTs: finger pairs, material, frequency .
|measurements||aperture A: 1 mm|
lambda λ: 20 um
The aperture is the height of the IDT fingers, from one end to the end of the adjacent finger. The wavelength λ is the distance (center-to-center) between two adjacent, connected fingers. The width of one finger as well as the free space between interlocking fingers is equal to a quarter of λ. A schematic overview of the design and dimensions of the IDTs is given in figure 9.
Figure 10 Dimensions of IDTs, with commonly used symbols and selected values
The fabrication should have been done via a so-called lift-off procedure due to the equipment at hand. The protocol is based on the “conventional lithographic and lift-off technique” described by Illa et al. . The setup requires a mask for the electrodes based on the values in table 1. The crystal needs to be cleaned and dried thoroughly before applying a UV photoresist is spun on followed by an image reversal process. Finally, the crystal is developed. The exact protocol can be found on the protocols page.
Connection SAW chip to electronics
Figure 11 Chip equipment, light grey: case, green: circuit board, gold: sensing film, screws: SMA connectors, dark grey: quartz crystal.
Figure 12 Chip equipment, plan view, light grey: case, green: circuit board, gold: sensing film, screws: SMA connectors, dark grey: quartz crystal.
Figure 13 Chip equipment, light grey: case, green: circuit board, gold: sensing film, screws: SMA connectors, dark grey: quartz crystal.
Figure 14 Chip equipment, side view, light grey: case, green: circuit board, gold: sensing film, screws: SMA connectors, dark grey: quartz crystal.
Analyzing the DDS
During our research we came across the direct digital synthesis (DDS) technology. This is described in the theoretical background to our hardware. To briefly explain DDS: A direct digital synthesizer (which is a microelectronic component with integrated circuits) is able to generate frequencies in any form. This could be e.g. a sine wave or a pulse wave. The possible settings for a DDS vary from model to model, but for nearly all devices these settings reach far beyond our project. First, the sine wave oscillations are of interest for us.
-PCB, needed to control the DDS, fit perfectly to the programmable pins of the Arduino Mega. Another advantage of the shield is the ability to adjust the frequencies directly on the shield with two buttons as well as to change certain settings of the synthesizer with an additional button. A little LCD shows the graphical user interface of the PCB.
To verify the functionality of the DDS and also the quality of the generated frequencies, we connected the DDS to an oscilloscope and visualized the frequencies. Afterwards we analyzed the frequency spectra of the DDS to determine if and how intense interference frequencies appear. The results are explained in the following section.
Frequency Spectra of the DDS
The frequency spectra of the AD9910 Arduino shield presented in the figures below are dependent on the clock frequency. We used a clock speed of 1.5 GHz which reduces the abundance and relative amplitude of interference in the frequency range of interest for our application. As one can see in Fig. 15 – Fig. 20 there is an amount of interference, small enough to be neglectable.
In comparison, at a clock speed of only 1 GHz, the frequency spectra of generated frequencies over 400 MHz comprise multiple disturbance peaks which could have a substantial impact on the excitation of the SAW chip’s piezoelectric crystal and thus on the reliability of the measurement. We therefore recommend using the shield’s option to set the clock frequency to 1.5 GHz. The difference in disturbance with the respect to the clock frequency is also visible in the following scope images.
Fig. 15 - Frequency spectra of a target frequency of 100 MHz (actual 103.5 MHz)
Fig. 16 - Frequency spectra of a target frequency of 200 MHz (actual 206.7 MHz)
Fig. 17 - Frequency spectra of a target frequency of 250 MHz (actual 258.3 MHz)
Fig. 18 - Frequency spectra of a target frequency of 400 MHz (actual 412.3 MHz)
Fig. 19 - Frequency spectra of a target frequency of 500 MHz (actual 514.6 MHz); one can see the disturbance peak in the low MHz range with approximately -50 dB.
Fig. 20 - Frequency spectra of a target frequency of 600 MHz (actual 617.0 MHz); one can see the disturbance peak at approx. 360 MHz with a level of approximately -47 dB.
To examine the functionality of the Arduino Shield we measured frequencies from 25 MHz up to 500 MHz. The frequencies can be set directly at the DDS. We observed a nearly linear factor which increases the target frequencies during this measurement (factor a1 = 1,028 at 1.5 GHz and factor a2 = 1.185 at 1 GHz). The factor is probably caused by the settings of the DDS, as it varies significantly or even disappears completely when selecting other clock frequencies. We decided to keep these settings anyway, because they create the most stable frequencies. This was important for the evaluation of the measuring data regarding the phase-shift. Therefore we used the derived factors to correct our measurement. The dependencies of the target and the actual frequencies generated by the AD9910 Arduino Shield are shown in figure 21.
Fig. 21 - Dependencies of the target and actual frequencies of the AD9910 Arduino Shield
Gain adjustmentThe amplifier can be adjusted by a software tool from the manufacturer which allows to select and change the gain of each channel separately. It is important to identify the amplitude generated by the DDS. In order to be able to give a reliable statement about the phase shift, it is necessary to work with an amplitude of approximately 0.5 V, since the components on our PCB operate with this voltage. Since the signal is doubled once for a reference measurement and once for our sensing chip, the amplitude decreases due to this operation and has to be amplified to approx. 0.5 V again.
To control the amplifier, it first needs to be powered by a 5 V supply voltage. We recommend exchanging the already soldered plugs for the supply voltage with pin headers, otherwise there is the risk of a short circuit. Moreover, the pin headers offer a direct connection to the GPIO of the Arduino, which delivers the supply voltage for the amplifier. Next the included USB cable can be connected to the computer.
After installing the software we first checked if the amplifier really increases the signal or whether there are difficulties with the configuration. In our case there was an incorrect configuration in the "Low Level View" menu. Two states required to be switched on 0, afterwards the amplifier could be controlled. The proper settings can also be found in the data sheet.
Fig. 22 - Low Level View of the LMH2832 Control Panel.
Fig. 23 - High Level View of the LMH2832 Control Panel.
In order to achieve an amplitude of at least 0.5 V, we have to adjust the gain to 13 which results in an amplification by the factor 1013. The settings are saved in the device's buffer memory, meaning that this step only requires a single adjustment. The images below illustrate the effect of the amplifier on the amplitudes.
Fig. 24 - 3D-Model of the PCB, created with Altium Designer. The model is free to download below.
As already mentioned in the human practice part, we worked together with the CITEC (Cluster of Excellence Cognitive Interaction Technology) of the Bielefeld University to define the technical implementation of our project idea and discussed the practical realization. The design of the board as well as the schematics were created by the working group "Cognitronics and Sensor Systems".
Various circuits would be suitable for measuring a phase shift. We discussed two possible realizations of the PCB.
One solution was to use an analog mixer. An exclusion criterion is the amplitude dependence of an analog mixer. This is problematic in the sense that at the time the circuit diagrams were developed, we did not have any knowledge of the amplitude of the frequency passing through the SAW chip. Therefore, this option was out of consideration. One can find more about the problems of an analog mixer in the Model section.
A second possibility, which was finally chosen, was the use of an XOR-gate. The functionality of the XOR is described in the Project/Hardware section. We attempted to capture the signal curve of the XOR at the contact points to visualize it on the scope. The signal curves of the XOR are shown in Fig. 25 and 26.
Fig. 25 - XOR curve on the scope.
Fig. 26 - XOR curve on the scope.
The Artificial Phase ShiftSince no real antibody-based phase shift could be created in the laboratory so far, it could not be evaluated with our SAW-technique. Therefore we had to create artificial phase shifts via the pin headers directly on the PCB. The areas marked in red in Fig. 24 can be connected in bridges, allowing a total of 16 different time shifts (in ps) to be simulated.
In order to cover the entire bandwidth of the DDS, we have generated the frequencies from 50 MHz to 600 MHz in 50 MHz steps for all 16 time shifts. We could detect the analog to digital converted voltage values on the serial console of the Arduino IDE. The results can be found in a table available for download below and in figures 25 and 26 underneath the text.
Fig. 27 - PCB Pinheader Marked which could be connected with bridges to create an artificial time shift.
Fig. 28 - Matlab Plot of the derived phase shift and time shift in 2D and 3D illustration.
We could prove by our measurement that our PCB delivers a voltage change at the output in case of a phase shift on the input, which we could detect by reading the analog output. However, it must be clarified that our PCB model is not able to detect a perfectly valid phase shift as well as voltage change.
To measure the precision of our board, further tests are necessary. The maximum mistake must be calculated to receive the percentage deviation. Moreover, the DDS generates the frequencies with a certain interference factor as well, which has already been analyzed in the section "Frequency spectra of the DDS".
Looking again at Figure 28, it becomes clear that e.g. there is supposed to be a minimum with a phase shift of 180°. But this point is located at a phase shift of 150°, which results a deviation of approximately 30°.
In conclusion, it can be said that even though there have been a number of systematic measurement deviations, we have developed a functional evaluation board that allows us to determine a phase shift from the analog output voltage value.
Files to downloadTo allow this project to be executed by further iGEM teams and possibly expanded, we have uploaded the files for the design of the PCB into our github repository. These files are available to download for free. Furthermore, one can find the source code for the Arduino Mega, which is used to read the PCBs analog output and additionally simulate phase shifts. We also uploaded the table of the results of the measurement as well as the matlab-plot so that the 3D plots can be reconstructed and viewed in three-dimensional.
The mobile device
3D Model of the Case
One can recognize that there are honeycomb-like structures in the cover as well as in the actual box. These have not only an visual but also a functional reason. Because the DDS as well as the Arduino Mega, which operates the DDS, can get extremely warm (we measured 90°C surface temperature in some areas), the warm air needs to leave the case. The openings on the top and bottom of the case ensure sufficient air circulation.
The stl files of the 3D-modelled case can also be downloaded from the github.
Fig. 29 - 3D Model of the case for the various electronic components
Fig. 30 - 3D Model of the case for the various electronic components (Case)
Fig. 31 - 3D Model of the case for the various electronic components (Cover)
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