Team:CLS CLSG UK/Hardware Design

Hardware design

Hardware Design

Aptasensors

Biosensors are analytical devices that contain two important functional components: a target recognition element (e.g. enzyme, protein, nucleic acid) and a signal transduction element (where the signal from the target recognition element is converted into a measurable chemical or physical signal). Aptasensors are biosensors which use aptamers as the biological target recognition element.

Aptamers are short (usually 30 to 100 nucleotides), single stranded oligonucleotide or peptide molecules that can specifically bind to a wide range of target molecules[1], for example, nucleic acids, proteins, metal ions, other small molecules and even whole cells. They display a high level of sensitivity, selectivity, affinity and reproducibility against these targets[2].

Aptasensors can utilise almost any functional signal transduction element, of which the main categories are electrochemical, optical and mass transduction. Aptasensors can also use label-based (fluorescent tag) or label-free techniques[1].

Types of aptasensors[3]

There are, amongst others, 6 very widely used aptasensors. This section will briefly describe each, and explain why we chose the aptasensors we did:

    Quartz crystal microbalance (or thickness shear mode) aptasensors[4]
  • The aptamer is attached to a quartz crystal, which is connected to an oscillation circuit. This induces crystal oscillation, and the oscillation frequency can be measured. When the aptamer binds with the target, the combined mass of the crystal and aptamer increases, and the resonant frequency decreases. The changes in frequency of crystal oscillation are indirectly proportional to the mass changes, which can be calculated using the Sauerbrey equation.
  • These aptasensors do not need labels
  • However, target molecules must be relatively massive in order to cause measurable changes in frequency.
    • Micromechanical cantilever arrays
  • The binding of the aptamer to the target results in steric crowding that causes it to bend. This bending can be detected optically or electronically.
    • Colloid gold nanoparticle (AuNP) -based colorimetric aptasensors
  • Aptamers are initially bound to the surface of the AuNPs. When they bind with the target, the aptamers change from a random coil to a folded rigid structure, and detach from the AuNPs. The AuNPs then aggregate, which causes a detectable colour change.
  • A downside to this method is that AuNPs may aggregate randomly in the presence of salt and other molecules.
    • Fluorometric aptasensors
  • The aptamers are attached to a substrate (graphene oxide is often used), and are labelled with a fluorophore and a quencher. On binding with the target, the aptamers change shape and either bring the fluorophore and quencher into contact (causing fluorescence).
    • Surface plasmon resonance aptasensors
  • Polarised light is incident on an electrically-conducting surface (e.g. gold) parallel to the incidence plane. At the interface, the light interacts with oscillating electrons, creating electron charge density waves (surface plasmons). These are totally reflected. When the aptamer binds with the target, the film’s refractive index and resonance angle changes. The intensity of reflected light at the resonance angle changes in proportion to the increase of mass due to binding.
  • No elaborate sample preparation is needed, and no radioactive or enzyme-labelled reagents are necessary.
    • Electrochemical aptasensors
  • The aptamer is immobilised on an electrode surface and labelled with a redox probe (e.g. methylene blue). When the aptamer binds with the target, the conformational change of the aptamer causes the distance between the probe and the electrode surface to decrease. When they are close together, electron transfer occurs which causes an electrochemical reading.
  • There are 4 main types of electrochemical aptasensors - conductometric-based (sense the change of electrical change in solution under constant voltage), potentiometric-based (sense changes in electrical potential difference when the binding occurs), amperometric-based (sense the difference in current potentials during the redox reactions), and impedimetric-based (sense changes in impedance upon interaction).

We chose to use an electrochemical aptasensor because they are:
  • Fast
  • Very sensitive (the antibiotic aptasensor we used had a limit of detection of 10pM)
  • Efficient
  • Require no extra lab work at site of detection - no labels need to be added, and no special reagents need to be used or prepared.
  • More specifically, we decided to design a potentiometric based aptasensor as they are much more sensitive and are much easier to automate.

Aptasensor design specifics

We designed a sensitive aptasensor device capable of taking regular in-situ samples for detection of cocaine, antibiotics and other potential drugs. Our electrochemical aptasensor is designed to allow for fully automated analysis of cocaine concentrations in water sources. This device would allow data to easily be recorded even in potentially toxic environments (such as the sewer), a great advantage in comparison to paper based or handheld aptasensors which require user-based operation. In addition to the effect of cocaine in the river thames, we discovered the devastating effect of antibiotic resistance upon aquatic ecosystems [5] [6], and so designed our system to use an aptamer capable of detecting ampicillin concentrations as low as 10 pM [7]. We also planned to use a chip-sized potentiostat capable of square wave voltammetry, allowing our device to have the same processing power of any other benchtop analysis tool [8].

Our design would involve the immobilisation of an aptamer (a short DNA strand with specific functional groups) onto a gold working electrode. Gold electrodes are inert but have a great ability to detect extremely low concentrations of compounds through electrochemical analysis [9]. We planned on using 2 different aptamers, one for cocaine and the other for ampicillin. Our cocaine aptamer works by changing its conformational shape in the presence of cocaine which in turn changes the current in the electrodes (please see animation of electrochemical aptasensor above). Our antibiotic detection is very similar but works slightly differently. This aptamer used a signalling probe displacement mechanism. In the presence of a target, the signaling probe would be displaced and released from the electrode surface, leading to the decrease of current signal.

As part of our design, gold electrodes (upon which aptamers would be immobilised) would be placed in a voltammetry cell. If water contains a concentration larger than the limit of detection for cocaine or antibiotics, the difference in voltage between working and reference electrodes would be measured by a potentiostat connected to the system. These results would be suitable for retrieval every 3 days, and would provide reliable information on the changing concentrations of cocaine and other drugs within the targeted water source. In addition, every 3 days, electrodes would be polished, cleaned and aptamers re-immobilised. As a final tool for automation, we designed a peristaltic pump under a specific time setting (every 30 mins) so water flowing from the water source could be pumped into our voltammetry cell.

How it works

  • Potentiostat instructs peristaltic pump to push water through voltammetry cell for 30 seconds in order to hello w-completely replace sample
  • Potentiostat sets timer for 30 minutes, allowing aptamers to equilibrate to new concentrations
  • Potentiostat suns SWV in order to get readings for concentration
  • Readings are inputted into SD card
  • Sequence repeats from step 1

    • Electrode treatment protocol

    During our research, we also found that there were no standardised protocols for such aptamer immobilisation and so designed a standard procedure based on a number of previous studies: [7] [10] [11] [12]

    Electronics

    To find out the levels of concentration of cocaine in our samples, we needed to run an electrochemical technique known as voltammetry. In order to take readings, we would require a potentiostat, a complex electrochemical analysis device which is used to control voltage input of the electrodes. Traditional potentiostats are extremely bulky and expensive which makes usage a large issue for low budget research projects (especially on a high school level). Our solution would need to be cost effective, portable and low maintenance. In addition, while there are some portable solutions, almost all can only run cyclic voltammetry (CV), a voltammetry technique almost 1000 fold less sensitive than Square Wave voltammetry. As a result, we planned on using a solution made by Orlando Hoilett and his team at Purdue University - Kickstat.

    Kickstat is an extremely small potentiostat that utilises Texas Instruments’ LMP91000 chip, an analog front end IC designed for use in electrochemical sensing applications, with an Arduino based SAMD21G MCU. Although in previous uses the LMP91000 has not been capable of the complex SWV technique, thanks to it’s high level of configurability over the I2C bus and some clever usage of the hardware capabilities of the powerful Atmel SAMD21 MCU series, the creators were successfully able to carry out SWV to a high degree of accuracy - thousands of times more accurate than CV could be.

    In order to further increase portability of the potentiostat, we modified the arduino so that all software programs could be run through only an SD card. In addition to this, we coded timed instructions for the use of a peristaltic pump, allowing the Kickstat potentiostat to fully automate our aptasensor design.

    Although we did have PCBWay manufacture two Kickstats using gerber files already provided, we wanted further expandability for SD cards, pumps and potentially more, as mentioned. Therefore, although we did not have time to do lab work, we designed a potentiostat that still uses the LMP91000 chip and Kickstat’s firmware but instead uses a conventional SAMD21G development breakout board and an LMP91000 breakout so that we can easily access the hardware pins of the MCU to incorporate the other elements into our project. The battery included below is a standard 18650 Lithium Ion 3.7v cell connected to the built in charging circuitry of the development board. Other batteries (such as a 3 cell lithium battery for 12v output) could potentially be used depending on the type of pump chosen but would need other components for charging (eg multiple TP4056). This is a wiring diagram of how we would create such a device:

    Furthermore, as the Kickstat code was designed to output data via the Serial Monitor of the Arduino IDE, mandating a computer to obtain the data, we modified the code to save the necessary information on an SD card instead, allowing the device to record data remotely. While we were not able to test this on the kickstats, we did test this code on other boards with an SD card and proved that it does work. Please see our GitHub for the code.

    References

    1. Di Pietrantonio, F., Cannatà, D., & Benetti, M. (2019). Biosensor technologies based on nanomaterials. Functional Nanostructured Interfaces for Environmental and Biomedical Applications, 181–242. https://doi.org/10.1016/b978-0-12-814401-5.00008-6
    2. Moreno M. (2014) Aptasensor. In: Amils R. et al. (eds) Encyclopedia of Astrobiology. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-27833-4_5167-1
    3. Mehlhorn, A., Rahimi, P., & Joseph, Y. (2018). Aptamer-Based Biosensors for Antibiotic Detection: A Review. Biosensors, 8(2), 54. https://doi.org/10.3390/bios8020054
    4. Jha, S. N. (2016). Biosensor. Rapid Detection of Food Adulterants and Contaminants, 125–145. https://doi.org/10.1016/b978-0-12-420084-5.00005-6
    5. Michael, I., Rizzo, L., McArdell, C. S., Manaia, C. M., Merlin, C., Schwartz, T., Dagot, C., & Fatta-Kassinos, D. (2013). Urban wastewater treatment plants as hotspots for the release of antibiotics in the environment: A review. Water Research, 47(3), 957–995. https://doi.org/10.1016/j.watres.2012.11.027
    6. Rizzo, L., Manaia, C., Merlin, C., Schwartz, T., Dagot, C., Ploy, M. C., Michael, I., & Fatta-Kassinos, D. (2013). Urban wastewater treatment plants as hotspots for antibiotic resistant bacteria and genes spread into the environment: A review. Science of The Total Environment, 447, 345–360. https://doi.org/10.1016/j.scitotenv.2013.01.032
    7. Yang, Z., Ding, X., Guo, Q., Wang, Y., Lu, Z., Ou, H., Luo, Z., & Lou, X. (2017). Second generation of signaling-probe displacement electrochemical aptasensor for detection of picomolar ampicillin and sulfadimethoxine. Sensors and Actuators B: Chemical, 253, 1129–1136. https://doi.org/10.1016/j.snb.2017.07.119
    8. Hoilett, O. S., Walker, J. F., Balash, B. M., Jaras, N. J., Boppana, S., & Linnes, J. C. (2020). KickStat: A Coin-Sized Potentiostat for High-Resolution Electrochemical Analysis. Sensors, 20(8), 2407. https://doi.org/10.3390/s20082407
    9. Gold Electrodes from Recordable CDs. (2020). Acs.Org. https://doi.org/10.1021/ac000437p
    10. Liu, R., Yang, Z., Guo, Q., Zhao, J., Ma, J., Kang, Q., Tang, Y., Xue, Y., Lou, X., & He, M. (2015). Signaling-Probe Displacement Electrochemical Aptamer-based Sensor (SD-EAB) for Detection of Nanomolar Kanamycin A. Electrochimica Acta, 182, 516–523. https://doi.org/10.1016/j.electacta.2015.09.140
    11. Schoukroun-Barnes, L. R., & White, R. J. (2015). Rationally designing aptamer sequences with reduced affinity for controlled sensor performance. Sensors (Basel, Switzerland), 15(4), 7754–7767. https://doi.org/10.3390/s150407754
    12. Tavakkoli, N., Soltani, N., & Mohammadi, F. (2019). A nanoporous gold-based electrochemical aptasensor for sensitive detection of cocaine. RSC Advances, 9(25), 14296–14301. https://doi.org/10.1039/c9ra01292c