Proof of Concept
We developed a novel detection mechanism that innovatively integrates hardware components and electronic circuitry. The Saptameter is a microcontroller-based biosensor that detects small molecules for use within the maple syrup industry. Our hardware consists of several different modules, which will be explained below.
Module #1: Saptameter
Our first module is the creation of the Saptameter. The Saptameter is a microcontroller-based biosensor that can quantify different invert sugar percentages found in maple syrup. To do this, a printed circuit board (PCB) shield was developed and attached to the top of our microcontroller. The PCB shield gives the microcontroller its functionality so that it can properly detect, read, and output the appropriate values of invert sugar. Our results show that the Saptameter is accurately able to discern the difference between various concentrations of glucose found in maple syrup (Figure 1). For example, the Saptameter produces a voltage reading that increases with higher concentration of glucose, allowing the different concentrations to be distinguished from each other quantitatively (Figure 1).
In addition to characterizing the efficacy of the Saptameter, we were also able to conduct user-testing of our device from students at our University, but most importantly, maple syrup farmers and producers themselves (Figures 2 and 3). After testing the Saptameter, many of the maple producers were highly enthusiastic about the design and functionality of the product as it addresses the pressing challenge of properly measuring invert sugars in syrup for downstream production of confectionery products.
Module #2: Glucose Strip Modifications
Our second module involves the creation of a novel glucose sensor. Currently, sugar makers dilute their maple syrup samples before placing them on glucose strips that are then inserted into a blood glucose meter. The primary reason for doing this dilution is to lower the high glucose concentrations in order for commercial devices to accurately read concentrations. This dilution process, however, is challenging for sugarmakers, as they often lack the proper equipment and techniques to accurately measure small volumes. To address this challenge, we developed a glucose test strip that maple syrup producers can use without diluting their sample. We made the test strip using a carbon screen printed electrode, and modified it by placing a small solution containing our glucose oxidase enzyme and pyrrole that was electrochemically deposited on the electrode followed by a chronoamperometric cycle using a potentiostat. After completing these modifications, we placed samples of varying concentrations of glucose solutions and maple syrup on our newly created electrode strips before running another chronoamperometric cycle. Our data indicates that our glucose strips are able to accurately detect high levels of glucose concentrations without the need of a dilution step, which is ultimately what maple syrup producers desire (Figure 4). More precisely, our strips are able to accurately detect glucose and maple syrup solutions up to 500mM. Our data shows that the 1mM glucose solution has the lowest current output at 3µA and the 500mM solution has the highest current output of 121µA. The data for glucose solutions between 2.5mM and 25mM, however, seemingly decreases. This could be due to experimental error, such as insufficient cleaning or faulty electrodes. 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). While it would be preferable for us to solve the inconsistent intermediate readings, our goal for this project was to develop a glucose test strip that can accurately sense glucose at high concentrations, which is something current commercial devices are unable to do. 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.
Module #3: Sarcosine Aptasensor
Our final hardware-related module is our sarcosine aptasensor. 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. Following this, each electrode had a small sample of methylene blue placed on top of the working electrode to help amplify signal.
The first experimental 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 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 5 and Figure 6). 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 show that there is more variance and more inconsistencies (standard deviation value of 83.25) between the various experimental trials involving the gold-electrode based aptasensor (Figure 6). This could be due to the methylene blue not fully washing off the electrode thus interfering with the resulting current output.
After modifying our carbon electrodes with a chitosan and a reduced graphene oxide (rGO) layer, we followed a very similar protocol used for the gold electrodes involving methylene blue. After running our controlled experiments involving sarcosine and our aptamers through differential pulse voltammetry, we saw that the electrodes that had sarcosine bound to the aptamers produced a higher current (µA) reading compared to our controls and non-sarcosine bound aptamer electrodes (Figure 7). More specifically, when looking at the peak values, the sarcosine bound aptamers had a five fold increase in initial current (µA) values compared to controls (Figure 8). The aptamer-only and control trials are similar to each other as neither of them have sarcosine attached. 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. In addition, the standard deviation (54.14) for the carbon-based aptasensor is 42% lower than the gold aptasensor, which shows the carbon electrode has more consistent results.
Importance to Synthetic Biology
All of our modules and results have great importance for the world of synthetic biology. 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, making them more widespread and accessible. In addition, our aptasensor showcases that we are effectively able to immobilize aptamers onto a screen printed electrode and sense our amino acid of interest in an accurate manner.