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 2 - 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 2 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 2 -, 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 3). 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 3 – 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 4 – 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 5 – 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 6 – 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.