Hardware

Team Saptasense has been nominated for the Best Hardware!



Background


Maple sap consists of several compounds that contribute to the quality of the maple syrup produced from it. As maple trees progress towards bud break (when a flower or plant finally emerges), there are several metabolic changes that result in elevated levels of amino acids and its derivatives in late season sap. Higher levels of amino acids, such as sarcosine, can produce off-flavors known as “buddy” syrup and result in an unsellable product for farmers. There are currently no early detection methods for maple farmers to measure sarcosine. Farmers only come to realize the negative effects of these molecules, such as poor taste and smell, after they finish producing a full batch of maple syrup. This can leave maple syrup batches unsuitable for sale and consumption resulting in millions of dollars lost for maple syrup producers [1].

Based on our discussions with maple syrup producers for our Human Practices initiative, we found that sugar makers also have difficulty accurately measuring invert sugar levels in maple syrup. Invert sugars are six carbon sugars made up of glucose and fructose. While they have the same number of carbon, oxygen, and hydrogen atoms, they differ slightly in their arrangement [2]. The term “invert” refers to the way these sugars bend polarized light. In general, all grades of maple syrup contain some level of invert sugar. It is important for sugar makers to test levels of invert sugar, as different percentages alter whether a sample of syrup is used for confections or varying grades of syrup [2].

Currently, sugar makers dilute their maple syrup samples before placing them on glucose strips that are then inserted into a blood glucose meter. This dilution is performed to reduce the thickness of the syrup sample and allow it to properly enter the test strips for readout. In addition, the glucose concentration from undiluted maple syrup is typically higher than the range of most commercial meters, which are tailored to measure human blood glucose levels. To address this problem, sugar makers need to conduct a 1:10 dilution with water. This dilution process, however, is challenging for sugar makers, as they often lack the proper equipment and techniques to accurately measure small volumes.

Our hardware solutions, innovatively address these challenges while also utilizing important facets of synthetic biology and wet lab research. Through our hardware work, we worked to improve the detection limit of typical glucometers to measure invert sugars more easily, as well as the detection of small molecules such as sarcosine to detect buddiness in sap rather than syrup.

Device Synopses


The Saptameter is an affordable and easy-to-use biosensor that maple syrup producers can utilize to examine invert sugar levels of maple syrup. The Saptameter is built on a microcontroller along with a printed circuit board (PCB) shield that can enable successful detection and readout. These components allow the Saptameter to have a production cost around 30 USD, cheaper than any alternative techniques currently used by sugar makers [2]. To test the efficacy of our hardware components, we created a modified glucose strip with a higher glucose detection threshold and performed several rounds of modifications and testing using electrochemical techniques such as cyclic voltammetry, chronoamperometry, and differential pulse voltammetry. We were also able to conduct user-testing on our Saptameter and received valuable feedback from maple syrup producers and sugar makers. Overall, they loved our product and cannot wait for it to be introduced into the industry.

We also created a novel sarcosine aptasensor that can detect the amino acid sarcosine in solution. We modified gold and carbon electrodes by immobilizing aptamers designed to bind to sarcosine onto our electrodes. After this modification, we deposited sarcosine onto the electrode to test if there was an observable change in the potential of the electrode when sarcosine bound to the immobilized aptamer. Our data shows that there is a change in signal especially with regards to the carbon electrode-based aptasensor. This change in potential would allow for the quantification of sarcosine levels in the sap and would be a valuable predictor of buddy sap.

Saptameter


Background

One of the hallmarks of our project is the development of the Saptameter, a novel and cost-effective, hand-held biosensor that is capable of detecting various molecules in maple syrup. The Saptameter is based on an Arduino UNO microcontroller, which is a small computer on a single integrated chip. The various electrodes and test strips that we modified to detect specific molecules such as sarcosine and invert sugar, as explained above, can be inserted into the Saptameter for quick and easy digital readout. To make the bare microcontroller tailored towards its use as a functional biosensor, we developed a printed circuit board (PCB) that has several modifications thus enabling it to help detect different molecules in syrup. The PCB simply clips on top of the microcontroller and using a computer, the subsequent readout is displayed after a sample of solution is placed on the electrode.



Engineering Technique and Circuitry

Prior to manufacturing the PCB shield, it is vital to first ensure that the Saptameter is feasible both with regards to its internal circuitry and electronic components. To conduct the preliminary prototype testing to ensure proper functionality, the device was first developed using a breadboard with a series of wires and components attached to it. Since our Saptameter is based on a microcontroller, we needed to ensure that the electrical energy throughout the system was properly regulated and efficiently stored to avoid overloading the system. To do this, we conducted research on how to calculate and position components such as resistors and capacitors so that they can effectively supply the needed energy to the system without it being overloaded. Based on the calculated parameters along with the limitation of components available to us, we created a wiring sketch of our prototype using a breadboard [3] (Figure 1).

Figure 1. The initial wiring diagram that we set up through the Fritzing software of how the Saptameter is set up on a breadboard

After assessing the validity of the breadboard sketch, the Fritzing software helped us convert the breadboard sketch into a PCB design. At a rudimentary level, the PCB design incorporates the same fundamental wiring approach as the breadboard, but simply makes the system easier to use since there is a lack of wires running across the system and instead a series of copper lines (known as traces) allowing electrical charge to flow (Figure 2) [3]. Following this, the corresponding PCB design files were then sent to a JLCPCB, a PCB manufacturer, which were then produced and sent back to us (Figure 3).

Figure 2. A virtual sketch of the PCB shield of the Saptameter that was uploaded to a PCB manufacturer for fabrication


Figure 3. The fully printed out PCB shield that adds functionality to the Saptameter

From the manufactured PCB shield, the board was populated with the components such as transistors, capacitors, and voltage regulators that help regulate electricity and current flow to produce the final shield that directly attaches to the Arduino microcontroller. The soldering process for this particular circuit board was more difficult than a traditional circuit as the components we used are on a microscale compared to surface mount components. A 0.3mm diameter soldering tip and a magnifying lens were necessary to solder the various components onto the PCB shield. Following this step, we added a set of female-header pins that easily allow the PCB shield to clip onto the Arduino board itself. By doing this, the Arduino now has all the functionalities that are provided to it by the PCB shield (Figure 4 and 5).

Figure 4. A diagram of the different components that were soldered on to the Saptameter


Figure 5. The fully modified and soldered PCB shield that clips directly onto our microcontroller to turn it into a functional Saptameter

Table 1. Financial breakdown of the Saptameter components that were soldered on to our PCB shield
Component Quantity Cost per Unit Cost
Arduino Uno 1 15 USD 15 USD
PCB Shield 1 2 USD 2 USD
Voltage Regulator 1 1 1.34 USD 1.34 USD
Voltage Regulator 2 1 1.12 USD 1.12 USD
Amplifier 1 1 4.78 USD 4.78 USD
Amplifier 2 1 0.49 USD 0.49 USD
Electrolytic capacitor 1 1 0.19 USD 0.19 USD
Electrolytic capacitor 2 1 0.037 USD 0.037 USD
Ceramic capacitor 1 2 0.039 USD 0.078 USD
Ceramic capacitor 2 2 0.10 USD 0.20 USD
Ceramic capacitor 3 1 0.27 USD 0.27 USD
Thick film resistor 1 1 0.71 USD 0.71 USD
Thick film resistor 2 1 0.10 USD 0.10 USD
Thick film resistor 3 1 0.19 USD 0.19 USD
Thick film resistor 4 1 0.10 USD 0.10 USD
Mini pushbutton 1 1.05 USD 1.05 USD
Stackable header set 1 2.65 USD 2.65 USD
SPE connector 1 0.95 USD 0.95 USD

Total Budget for Saptameter: 31.25 USD*
Average Commercial Glucometer Price: ~50.00 USD
Total Savings: 37.5% cheaper
* Numbers are based on prices from United States companies

3D Printed Casing

Using the measurements from our device, we created a casing that houses our Arduino along with the PCB shield. There are specific cut outs which allow for the cables needed to connect the Arduino to the computer, connect the Arduino to a power source, and also a place to insert the test strips we developed. After creating the case design and transforming it into this file, we printed the casing using Acrylonitrile Butadiene Styrene (ABS) plastic from a printer in our university's fabrication studio (Figure 6, 7, 8).

Figure 6. The 3D printed top of our case.


Figure 7. The 3D printed bottom of our case.


Figure 8. The fully combined 3D printed case.


Software Code


We used the Arduino Integrated Development Environment (IDE) to code the Arduino UNO, which serves as the microcontroller for this device. The software for the Arduino was coded in Java and does three primary things:

  1. Detects the presence of screen printed electrode into the device
  2. Requests the user to place a sample of syrup on the electrode and detects the presence of a solution on the electrode
  3. Outputs the corresponding voltage based on the solution’s concentration by doing a mathematical calculation involving reading the baseline voltage

The code can be found in the GitLab

Screen Printed Electrode (SPE)

SPEs are an electrochemical measurement device that allows for rapid in situ detection of analytes. The three most important components of an SPE are the counter, working, and reference electrodes. The counter electrode (CE) allows for the passage of current and electronic transfer to occur. The working electrode (WE) is modified to be sensitive towards the analyte’s concentration. The reference electrode (RE) maintains a constant electric potential that the working electrode is measured and compared against it [4]. For this project, the working electrode has been modified to detect the concentration of the specific analytes of invert sugar (Figure 9).

Figure 9. The different components of a screen printed electrode.

Throughout the development of our device, we primarily focused on three types of screen printed electrodes. The first was a hyper value carbon electrode that contains a carbon counter electrode and working electrode along with a silver/silver chloride (Ag/AgCl) reference electrode. These specific electrodes are very low cost (0.99 USD) and are printed on paper composite material. The second type of electrode we selected was a higher quality and higher cost carbon electrode that contains a 4 mm diameter carbon WE, carbon CE, and Ag/AgCl RE. While this specific electrode is slightly more expensive, we wanted to assess if there is a difference in terms of accuracy when sensing our selected biomarkers. The final electrode we chose to experiment with was a gold SPE with a gold 4mm WE diameter, gold CE, and Ag RE. Since the electrode contains gold, it reportedly has a higher sensitivity [5]. Here too, we wished to test if there is a noticeable and significant difference in sensitivity among the various electrodes. We primarily chose to focus on these three types of electrodes due to their lower cost and widespread use within electrochemical tests in similar fields of research [6].


Electrochemical Techniques

Cyclic Voltammetry (CV)


Cyclic Voltammetry (CV) is a widely used electroanalytical technique for acquiring qualitative information about electrochemical activity. CV gives insight on various parameters such as electron-transfer reactions and electrochemical reactions [7]. The principle of CV involves sweeping the potential back and forth (positive to negative and negative to positive) between the chosen limits and recording the changing current during the sweeps. The cycle of switching voltages from positive to negative and vice versa repeats until the system reaches an equilibrium state with the help of redox reactions (Figure 10) [8]. This information obtained by the CV is then analyzed to provide data about the electrochemical behavior of a material.

Figure 10. This graph shows a standard CV “duckbill” curve and represents how the voltage changes throughout the duration of the experiment. The y-axis is in current (µA) while the x-axis is in voltage [9].


Chronoamperometry

Chronoamperometry is a time-dependent technique where a potential is applied to the working electrode to help with the deposition of an organic compound. The current of the electrode, which is listed as a function of time, fluctuates as the molecule being deposited diffuses from the bulk solution toward the sensor’s surface. Generally speaking, the working electrode is stepped from a potential at which there is no electrode reaction to one where the solution fully reacts with a redox couple (Figure 11) [10].

Figure 11. A representation of chronoamperometry and shows how the current varies over time [11].


Differential Pulse Voltammetry


In addition to CV and chronoamperometry, differential pulse voltammetry was used. DPV is a pulse technique that is designed to minimize background charging currents by running a series of pulses increasing over a linear baseline under voltage conditions (Figure 12). The base potential value is chosen and applied to the electrode and is increased with equal increments over time, while the current is immediately measured before and after the pulse application, forming a pulse wave by recording the difference between the start and the end of the pulse [12].

Figure 12. A representation of differential pulse voltammetry (DPV) that shows how the current varies over time [13].


Saptameter Results


In order to test the functionality of the Saptameter, we used five different hyper value carbon electrodes to test the Saptameter’s ability to detect five different glucose concentrations. The primary rationale behind choosing hyper value electrodes was due to their lower cost, which is more accessible for downstream application by local sugarmakers. To modify these electrodes, 75µL of our glucose enzyme solution was electrochemically deposited on the working electrode and run under chronoamperometry with an applied potential of 0.7 V and a run time of 600 seconds. Figure 13 shows the polypyrrole layer (PPy) deposition curves detected by our potentiostat. Since there were no curves that had an abnormal result such as straight line or inverted graph, we concluded that our electrodes were clean and functional and that the PPy layer was evenly deposited.

Figure 13. The chronoamperometry data from the five hyper value electrodes. This data is representative of the electropolymerization of several hyper value carbon electrodes with polypyrrole and glucose oxidase enzyme-solution. Each electrode is labeled with different colors for simplicity of viewing. The x-axis represents time in seconds while the y-axis represents current in µA.


Following this and a wash step with phosphate saline buffer (PBS), 4µL of the Fe(CN)63−/Fe(CN)64−redox couple was placed on top of the electrode. Once the electrodes were dry, glucose solutions of varying concentrations were deposited on the electrodes for 20 minutes afterwards (Figure 14). We tested 4 different glucose concentration stock solutions along with a sample of maple syrup with a known glucose concentration, which is indicative of what sugar makers will test in reality (Figure 15). The very first sample (near 1mM) is an actual syrup sample, while the remaining 4 samples of 25mM, 50mM, 100mM, and 500mM are all glucose stock solutions. The obtained data shows that the Saptameter produces different voltage readings that appropriately correspond to different concentrations of glucose. As the glucose concentration increases, the resulting voltage reading increases (Figure 15). The 1mM syrup sample has the lowest voltage output, while the 500mM syrup sample has the highest with every intermediate value slowly increasing.

Figure 14. An image of our team member Alec modifying a glucose strip.


Figure 15. This illustrates the efficacy of the Saptameter by showing that the increase in glucose concentration (mM) on the x-axis results in a higher average voltage (V) that is displayed on the y-axis.


User Testing

One of the most rewarding aspects of our project was presenting the Saptameter to maple syrup producers and receiving valuable feedback from them. We first had an in-person meeting with Merle Maple located in Attica, New York (Figure 16). Here, they gave us suggestions on how to improve the physical attributes of the Saptameter such as creating a fully covered top enclosure, which we then incorporated into creating our final 3D printed casing. In addition, they also gave us important pointers on how to commercialize our device. They mentioned that we should conduct testing on the temperature sensitivity of the device and electrodes, since in practice, samples of sap and syrup would be tested at different temperatures, including samples that are freshly boiled. They also mentioned that integrating a display screen onto the device would make it more user friendly.

Figure 16. Our team presenting the Saptameter and its case design to Merle Maple

We also had the opportunity to have Gordon Putman, a lead sugar maker from Whispering Brook Farms in New York, test our device and provide feedback on its accessibility and functionality (Figure 17 and 18). Gordon enjoyed the clean design of the product and encouraged us to look into different readout methods that can be adjusted by the user.

Figure 17. An image of our team member, Sudarshan, presenting the glucometer to Gordon from Whispering Brooks.


Figure 18. Gordon from Whispering Brooks testing the Saptameter on a sample of syrup.


We also conducted user feedback from students who attend the University of Rochester who provided input on the Saptameter’s usability (Figure 19). Students expressed that they wished the product had its own display and standalone battery to power it. They also wanted the physical material of the casing to be more robust so that it could withstand higher degrees of wear and tear. Overall, students really enjoyed the product and found it easy to use.

Figure 19. A fellow student from the University of Rochester conducting user testing on our Saptameter.


Product Implementation

Implementation of our glucometer will largely resemble the current practices for measuring invert sugar levels in syrup. Sugar makers will only need to purchase two things: the Saptameter device and our modified test strips. After boiling down the sap to syrup, the sugar maker will let their syrup cool to approximately room temperature, then apply a small amount of their syrup to their test strip. This can be done in several different ways, such as by dipping the strip in a small sample of syrup, using an eyedropper to transfer a small amount of syrup to the strip, or dipping their finger into the syrup and gently pressing it onto the test strip. The sugar maker will then place the test strip in the port of the Saptameter device, which will display the sample’s percentage of invert sugar. This process is simple, intuitive, and importantly does not involve the tricky dilution steps that typically leads to improperly made ancillary maple syrup products.


Future Directions

Looking to the future, our goal is to increase the accessibility of the Saptameter. We would like to add and integrate features such as a Liquid Crystal Display (LCD) screen with easy visual readout and a standalone battery for portability and ease of use. All of these improvements were suggestions provided to us by maple farmers when we conducted user testing with them. We also wish to incorporate different colored light-emitting diode (LED) lights and a piezo buzzer or a speaker that would visually and audibly indicate information about the quality of the sample to the user. This would make the device more accessible to sugar makers who are unable to hear or see clearly.

To make the device more commercially viable, we could also produce a kit that includes the Saptameter along with various strips of the different targets for buddy sap and detect different small molecules and amino acids. Ideally, we could take it a step further to produce a test strip that is capable of detecting multiple molecules at once and a modified device that can take in several inputs at once. This would negate the need for the use of different strips for different analytes, streamlining the detection process. In addition, we also wish to conduct more long-term testing of the modified screen printed electrodes (i.e. 1+ years) to identify the ideal storage conditions and longevity of our test strip SPEs. This was in fact an important consideration posed to us by sugar makers during our meetings with them.

Invert Sugar Detection - A Human Practices and Hardware Collaboration

Background

Our preliminary research identified preventing buddy defects in maple syrup or repurposing maple syrup with ropy defects as promising targets for our project. Yet after meeting with sugar makers, we uncovered an additional problem in the industry: difficulties in accurately measuring invert sugar levels in maple syrup. Traditionally, maple producers measure invert sugar concentrations in their syrup using commercial glucometers, which typically range from about 5mM to 12mM [14, 15]. Shelf-stable maple creams require syrup with invert sugar levels of approximately 190mM to 220 mM, a value about ten times greater than glucose levels in human blood [16]. These values are far outside the reliable detection range of commercial glucometers, so sugaring standard practice is to dilute a small amount of syrup. However, the dilution is deceivingly difficult for sugarmakers, as they often lack the proper equipment to accurately measure small volumes of syrup and water which is necessary for the dilution to occur. In response to the requests of the sugarmakers, we decided to create a new glucometer specifically made to measure glucose levels found in maple syrup without requiring the tricky dilution step. This component was added to our project somewhat late, so we decided to make this an ancillary supplement to our current hardware and human practices work. Our new modified glucose strips can be inserted into our Saptameter device for easy and effective readout.

Our glucometer largely resembles traditional glucometers, but it will have one key difference: the ability to measure higher glucose concentrations than normally present in human blood. Traditional glucometers consist of an electrochemical test strip inserted into an electronic sensor (Figure 20). Glucometer test strips contain glucose oxidase, an enzyme from Aspergillus niger that catalyzes the oxidation of glucose to gluconic acid. This redox reaction involves the transfer of electrons to a series of electrodes on the test strip, drawing a current across the electrodes. The magnitude of current is directly dependent on the amount of glucose administered to the test strip. The electronic device quantifies the electric signal and calculates the corresponding amount of glucose on the strip [18]. Our glucometer has much of this same framework with additional modifications that will increase the limit of detection.

Figure 20. Description of how a glucometer works. In the first step, a drop of sample is applied to the test strip. In the second step, the oxidation of glucose generates a current across the test strip. In the third step, the glucometer calculates the corresponding glucose concentration based on the magnitude of the current [19].

Carbon Screen Printed Electrodes

The primary goal of devising our own glucose strips is to increase the glucose detection range at higher concentrations so that maple producers do not need to dilute their syrup sample (Figure 21). To increase the range detection of our test strip, which are based on carbon SPEs, we electrodeposited an organic compound on top of the working electrode called pyrrole [20]. We selected pyrrole as our organic compound as it is characterized by a ring structure containing four carbon atoms and one nitrogen atom. It is this arrangement that ultimately enables it to become a useful conductive polymer. In addition, its carrier material characteristics significantly improves its ability to immobilize enzymes and can be easily prepared in the lab. When the pyrrole is deposited onto the working electrode, it is oxidized to form polypyrrole (PPy). Polypyrrole (PPy) can be easily deposited onto an electrode’s surface from aqueous solutions [21]. In our pyrrole solution, we also included the glucose oxidase enzyme so that the pyrrole layer and the enzyme are both deposited onto the working electrode at the same time. This addition of glucose oxidase to the working electrode makes our electrodes capable of specifically testing for the glucose found in invert sugar.

Figure 21. Several series of electrodes including hyper value, carbon, and gold screen printed electrodes that our team tested with different concentrations of glucose solutions throughout our project

Using chronoamperometry, we set up our system using the following parameters. The potential applied during measurement was 0.7 V with a run time of 600 seconds. In addition, the time for equilibration at which the potential is applied was set at 1 second. These parameters enable the layer of PPy with the enzyme to form on top of the working electrode (Figure 22). Most of the curves in this graph line up over each other, which indicates that the PPy layer is reacting uniformly with the redox couple across various electrodes. The main intention behind these curves is to ensure that no electrodes have any abnormally different modifications than the other electrodes. We also review each electrode under a light microscope to ensure that the electrode is fully covered with PPy (Figure 23).

Figure 22. The chronoamperometry data is representative of the electropolymerization of several carbon electrodes with polypyrrole and glucose oxidase enzyme-solution. Each electrode is labeled with different colors for simplicity of viewing. The x-axis represents time in seconds while the y-axis represents current in µA.


Figure 23. One of our lab members examining the deposition of the pyrrole layer under a light microscope.


Figure 24. Comparison of the working electrode of a modified (left) and unmodified (right) carbon electrode. Images were taken under an optical microscope.


When examining the electrodeposition of PPy under an optical microscope, the electrode with the PPy layer (left electrode) displayed a slightly darker working electrode compared to the bare electrode on the right (Figure 24). This visual change can be seen with the naked eye and allows us to verify that the PPy has been appropriately deposited onto the SPE.

Given the chemistry of how a three electrode system works, it is also crucial to carefully select a redox couple, which is a combination of oxidized and reduced form of a compound so that the electron transfer process is done efficiently and thoroughly. To do this, we created a solution of ferricyanide/ferrocyanide Fe(CN)63−/Fe(CN)64− [22]. 12µL of the selected redox couple solution was placed on top of the working electrode for 20 minutes. This is to ensure that the redox couple fully reacts with the PPy and enzyme layer. Following this, the redox couple was washed off with deionized water and the electrode was left to dry. After drying, 60µL of a glucose sample was placed on top of the electrode for 20 minutes. Different concentrations of glucose solutions ranging from 1mM to 100mM were used throughout the experiment with multiple trials being run on different prepared electrodes as these electrodes can only be used once. A 120 second cycle of chronoamperometry was run with slightly altered parameters including a lower applied potential of 0.3 V and a run time of 120 seconds. The subsequent results were recorded and summarized in Figure 25.

Figure 25. The chronoamperometry scan results of various carbon electrodes with different concentrations of glucose solutions. T1 refers to trial 1 while T2 refers to trial 2. Multiple trials were conducted to gain robust data. The x-axis represents time in seconds while the y-axis represents current in µA.


Results

The data shows that the 1mM solution has the lowest current output. While there is an increase between the 1mM and 2.5mM solution, the current output slowly decreases, which could be due to experimental error, such as insufficient cleaning or faulty electrodes, between the 2.5mM and 25mM concentration solutions (Figure 26). With that being said, there is an increase in current with samples between 25mM and 500mM, which shows that our carbon-based glucose strips are more accurate at higher concentrations (Figure 26). This is not an issue for us since our goal for this project was to develop a glucose test strip that can accurately sense glucose at high concentrations unlike current commercial devices. Therefore, our Saptameter will be able to help local sugar makers avoid the difficult and error prone dilution process in testing glucose levels within their syrup samples.

Figure 26. Converts the chronoamperometry data into a more visually understandable format. Here the x-axis represents the glucose concentrations in mM while the y-axis represents the average current (µA) over multiple trials. The data shows that our glucose strips are successful at detecting higher concentrations of glucose.


Connections to the Wet Lab

Due to the late addition of this facet of our project, we used purchased glucose oxidase for our biosensor rather than purify our own enzyme. Future work on this biosensor would involve expressing and purifying our own glucose oxidase from Escherichia coli cells. When we talked with Merle Maple, we found out that invert sugar levels are tested soon after the syrup comes out of the evaporator while the syrup is still quite hot. For this reason, we sought to design glucose oxidase biobricks with maximal thermostability in order to allow for accurate testing of these hot syrup samples. We therefore conceptualized several variants of the A. niger glucose oxidase biobrick that was originally created by Team Manhattan College Bronx 2017. This initial part contains the coding sequence for the enzyme plus a 6x Histidine tag on the C-terminal end for protein purification. We created three new parts that contain point mutations that have been computationally predicted in literature to improve the thermostability of glucose oxidase [23][24]. One such biobrick variant contains 2 mutations (P192C and H201C) and was referred to throughout the project as “Glucose Oxidase 2” or “GOx 2” [23]. Both point mutations result in the substitution of an amino acid for cysteine. This change will, in theory, promote the formation of an additional disulfide bond in the protein structure, increasing stability of the enzyme [23]. Another of the glucose oxidase variants contains 5 mutations (T10K, A36M, R145N, G274S, and E374Q) and was known throughout the project as “Glucose Oxidase 5” or “GOx 5” [24]. These mutations were computationally predicted to improve the stability of glucose oxidase, though they had not yet been tested in the wet lab [24]. We also designed a glucose oxidase variant containing both sets of mutations. Because this version contains 7 total mutations, it was referred to as “Glucose Oxidase 7” or “GOx 7.” Due to the presence of the mutations for an additional disulfide bond in both GOx2 and GOx7, these enzymes would need to be expressed in an E. coli strain that contains an oxidizing cell environment such as the E. coli strain SHuffle.

In the future, we would work towards purifying our own glucose oxidase, as this process is cheaper than buying the enzyme and allows for easy production of our thermo-stabilizing glucose oxidase variants. This will allow us to decrease the price of our glucometer, making the device more accessible to all sugar makers. To maximize protein expression, the parts have a strong T7 promoter (BBa_I712074) and a strong ribosome binding site (BBa_B0034). Our parts also contain a TAA double terminator (BBa_B0015). Finally, we designed the parts with restriction sites for EcoRI, XbaI, SpeI, and PstI corresponding with the pSB1C3 plasmid so we could easily insert the part into the plasmid using 3A assembly.

Sarcosine Aptasensor


Background

Sarcosine is another amino acid that when found in elevated levels in maple sap can have negative effects on the quality of maple syrup produced. More specifically, these compounds act as methyl donors and therefore alter a tree's metabolic profile, impacting the flavors of the syrup boiled from the sap, when present in high concentrations [25]. In maple sap, sarcosine is found in increasing concentrations from the beginning of the sugaring season to the end of the season when the trees start budding [25]. There are currently no methods to test levels of sarcosine in sap. Our sensor is aimed at detecting this molecule to sense levels of “buddiness” in sap, preventing the creation of buddy syrup. In order to sense sarcosine, we immobilized sarcosine-binding aptamers, which are short, single-stranded DNA molecules that can selectively bind to a specific target (in our case sarcosine), such as proteins and molecules on both carbon and gold screen printed electrodes [26]. The electrodes were modified and tested using chronoamperometry in order to assess any differences in signal output between electrodes that have sarcosine and those that do not.

Gold Screen Printed Electrodes

In order to test the validity of our aptasensors, we set up two different experiments involving two types of screen printed electrodes. The first set involves the use of gold electrodes. The gold electrodes had a layer of a chitosan deposited onto the working electrode. Each electrode was then placed in a small bath of glutaraldehyde, which is a cross-linking solution that can help our polymers and molecules cure each other [29]. Following this, each electrode had a small sample of methylene blue placed on top of the working electrode. The first set included our control gold electrodes with no aptamers bound. The second set included our aptamers immobilized onto the electrode. Finally, we had our experimental set where the aptamers are deposited on the electrode with sarcosine bound on top. The electrodes were thoroughly washed off with a 1M Tris buffer solution. They were then run with 100µL of PBS solution.

The results from the gold electrode trials show that the lowest current outputs are from the control electrodes with the aptamer only electrodes producing a slightly higher output (Figure 27 and Figure 28). While there is one data point showing a high signal for the gold electrode that has the aptamer with sarcosine bound, the other replicate of this same sarcosine bound aptamer produced an output that was on-par with the controls. In addition, the error bars displayed in Figure 28, show that there is more variance and more inconsistencies between the various experimental trials involving the gold-electrode based aptasensor. In addition, the standard deviation value of 83.25 for the gold aptasensor (Figure 28). This could be due to the methylene blue not fully washing off the electrode thus interfering with the resulting current output.

Figure 27. Shows the corresponding curves for the gold screen printed electrode aptasensor comparing a total of 6 trials. There are 2 trials that have the aptamer with sarcosine (sar) bound. There are another 2 trials with the aptamer alone and no sarcosine deposited. Finally, there are 2 more trials with no aptamers and no sarcosine bound to the electrode. The y-axis represents the current in µA and the x-axis represents the potential (V) of the run.


Figure 28. Compares the average current (µA) between different experimental trials of the gold-electrode based aptasensor. The control does not have sarcosine or the aptamer attached. The aptamer only electrode does not have any sarcosine bound to it. The sarcosine + aptamer has both sarcosine and the aptamer present. Error bars are also included to show statistically significant differences. The standard deviation for these tests is 83.25.

Carbon Screen Printed Electrodes

To modify our carbon-based aptasensors, a layer of a chitosan and reduced graphene oxide (rGO) was developed and deposited onto the working electrode of a carbon electrode. This is done to increase the sensitivity of the electrode itself [28]. Each electrode was then placed in a small bath of glutaraldehyde similar to the gold electrode approach [29]. Following this, each electrode had a small sample of methylene blue placed on top of the working electrode. This step helps amplify the signal of the molecule of interest binding to the aptamer on the electrode [30]. In order to test the efficacy of our carbon aptasensor, we had three different trials, each made up of two electrodes. First, we had our experimental set where the aptamers were on the electrode with sarcosine bound to the top. The second set included our aptamers immobilized onto the electrode without any sarcosine. Finally, we had our control set where there were no aptamers (Figure 29).

Figure 29. Shows the corresponding curves for the carbon screen printed electrode aptasensor comparing a total of 6 trials. There are 2 trials that have the aptamer with sarcosine (sar) bound. There are another 2 trials with the aptamer alone and no sarcosine deposited. Finally, there are 2 more trials with no aptamers and no sarcosine bound to the electrode. The y-axis represents the current in µA and the x-axis represents the potential (V) of the run.

The results from the carbon electrode trials show that the lowest current outputs are from the control electrodes with the aptamer only electrodes producing a signal slightly above the control. Both carbon electrodes that have the aptamer with sarcosine bound give close to a fivefold increase signal compared to the controls when comparing peak current values (Figure 30). This observable increase clearly shows that the carbon electrode-based aptasensor is able to successfully sense when the amino acid, sarcosine, is present in a sample.

Figure 30. Compares the average current (µA) between different experimental trials of the carbon-electrode based aptasensor. The control does not have sarcosine or the aptamer attached. The aptamer only electrode does not have any sarcosine bound to it. The sarcosine + aptamer has both sarcosine and the aptamer present. Error bars are also included to show statistically significant differences. The standard deviation for these tests is 54.15.

We originally planned to focus our apta-sensing technology on gold electrodes due to their sensitivity. However, when the results for the gold electrode did not come as anticipated, we swiftly adjusted to using carbon electrodes instead. It is clear that the carbon electrodes do a much better and more consistent job at sensing the presence of sarcosine in solution in comparison to gold electrodes. In addition, the standard deviation values for the carbon electrode aptasensor is lower (roughly 42%) than the gold electrode aptasensor, meaning the carbon electrode has more consistent data.

Why is our hardware important for the world of Synthetic Biology?


Our novel detection mechanism innovatively integrates our hardware device along with the potential of various synthetic biological parts (biobricks) that our team created. The biobricks we created, involving glucose oxidase, for example, would theoretically allow us to modify screen printed electrodes and tailor them to detect specific molecules. In the future we can also develop new testing strips that include different enzymes such as choline oxidase to use for detecting different molecules. Through our unique approach, we open up a new avenue for researchers within the field of synthetic biology to easily assess specific molecules and integrate biological components into hardware devices thus making them more widespread and accessible.

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