Introduction

Why an UltraViolet Spectrometer?

High-Performance-Liquid-Chromatography (HPLC) is expensive. It's like. Really. Really. Expensive. The need for a reliable and cheap (at least in comparison) PET degradation assay has led to many solutions, including titrimetry, fluorimetry, chromatography, and spectrometry. While many of these methods are valid an effective, their implementations are often limited by their discrete measurement. Out hardware aims to provide an accessable form of spectrometery that will allow for continuous data collection during reactions. By continuously monitoring the degradation progress, we are also able to more effectively visualize and analyse the different characteristics of our variants. This would be especially significant for us to iterate and improve our variants by providing more feedback into the efficacy of our rational designs.

What is a UV Spectrometer

The hardware is placed on a hot plate set to the degradation temperature. Since the quartz cuvettes are immersed in a water bath, the system requires time to reach thermal equilibrium. A quartz collimator lens is used to parallelize the light emitted from the UV LED as it passes through the quartz cuvette. Because of their benzene ring, many of the byproducts from PET depolymerization absorb UV light in the 240–260 nanometer range. Our sensor specifically measures the light intensity at 254 nm. By monitoring changes in light intensity at this wavelength, we can estimate the concentrations of these byproducts.

Design Iterations

Throughout development, we went through multiple iterations to refine our UV spectrometer design. Below are entries for each iteration:

• Iteration 1
• Iteration 2
• Iteration 3
• Iteration 4
• Iteration 5

Design

Our first iteration attempted to minimize the cost and increase accessibility by using an inexpensive 28byj 48 stepper motor to power the spectrometer. The design uses 2 3d printed spur gears. One for the 28byj stepper motor and one for the sensor and UV-LED. The sensor spins on the outside around the cuvette and the LED spins in the middle.

The cuvettes sit inside as shown, with water on both sides between each cuvette and the next. We included the water between the cuvettes because we planned on putting the whole hardware in the air convection oven. While it is generally decent at temperature control, it may fluctuate too much for optimal PET degradation. The addition of water and its high specific heat capacity meant that it would act to stabilize the temperature of the hardware.

Build

We were able to quickly 3d print a prototype to test outside of the lab.

Test

The prototype was made as a proof of concept for the rotating mechanism for the spectrometer. After spinning up the prototype for a few minutes, it became evident that the 28byj 48 stepper motor was not nearly powerful enough to accurately spin the spectrometer wheel. The stepper began to heat up and skip steps.

Learn

From this prototype, we were able to gain a better understanding of what electronics we would need to be able to accurately run the spectrometer for the time required to degrade PET.

Design

After the first prototype failed because of the weak stepper motor we had used, we decided to swap it out for a more accurate servo motor. But because most positional servo motors could only rotate 90-180 degrees, which wouldn’t work unless we geared the servo up, we had to use continuous servos instead.

Build

For the prototype, we once again printed it out in PLA.

Test

We didn’t get to comprehensively test this version of the hardware because we realized the mistake we made while we were wiring the system.

Learn

One big mistake that we had made was not realizing that continuous servo motors could not accurately move to specific angles. This was a huge problem because the spectrometer would need to move with incredibly narrow margins of error for the UV readings to be accurate. Workarounds like encoders would either require an absolute encoder (expensive) or rotary encoder (prone to cumulative error). This was probably the most avoidable mistake we made in the entire iteration process.

Design

For the third iteration, we switched to a more powerful motor. After the first two attempts, we realized overkill was, in fact, just right. We used a beefy stepper motor for its torque and accuracy. We also replaced gears with belts, which offer nearly zero backlash.

We raised the cuvettes slightly to allow water to flow around and underneath them, improving temperature uniformity.

Build

The electronics required a 24V power supply to drive the stepper motor. We continued to use PLA for quick prototyping.

Test

This version worked wonderfully. The stepper motor maintained accurate positioning for hours without requiring a closed-loop system.

Learn

We identified two issues:

• The belts were too finicky and difficult to work with, making the design overly tall and complex.
• The water between the cuvettes seemed unnecessary and added complexity without clear benefit.

Design

The previous iteration had the large stepper motor standing upright, which caused two problems: it took up too much space, and the vertical design made the structure wobbly. In this version, we used beveled gears with helical teeth, allowing the motor to lie flat and reducing both height and backlash.

Build

Everything was 3D printed in PLA. Mounting the motor horizontally required some experimentation to get the alignment right.

Test

The bevel gear system had minimal backlash and took up much less space. The alignment between sensor and LED remained consistent after hours of spinning.

Learn

Although this version performed well, the top bevel gear attached to the sensor/LED assembly felt slightly wobbly, which raised concerns. We also realized that testing sensors and LEDs in a simplified, fixed setup (see Iteration 5) would be easier.

Design

This was a simplified proof-of-concept test, not a full iteration. The goal was to reduce moving parts and test only the LED and sensor alignment with a static cuvette in between. Balancing ease of assembly with accuracy was the main challenge.

Build

This was both the easiest and hardest iteration to assemble. Although it was a single piece of PLA, installing the LED and sensor was time-consuming due to tight access areas.

Test

Initially, the electronics worked well on a breadboard. But once we tried attaching Dupont cables to the LED legs, connections became unstable. Debugging was difficult due to unreliable breadboards and resistors not seating properly. Eventually, after multiple connection issues and hours of tinkering, one of the LED’s legs broke off completely.

Learn

We learned several lessons—both technical and personal. Cheap <$1 wires and breadboards are not worth the risk. Most importantly: patience matters. If we had slowed down and handled components more carefully, we might have avoided the damage.

Materials

Components and Functions

Our design of UV-Vis Spectrometer includes multiple components, including:

Results

While we initially planned to run multiple rounds of degradation assays with our hardware, time constraints and unexpected complexities with the electronics prevented us from obtaining consistent and repeatable data. Our biggest obstacle was integrating the UV-LED with the rest of the spectrometer due to faulty wires and breadboards. This significantly set us back since we had spent hours debugging the electronics to no end.

We had planned to perform several experiments as a proof of concept. However, just as we were setting up the hardware, we accidentally bent the legs of our only UV-LED. Normally, this wouldn’t be a major issue, but after a week of frequent adjustments and testing, the LED’s legs had already been weakened. This final bend caused one of its legs to snap off, abruptly halting our experiments until we can order a replacement after the wiki freeze.

Despite significant setbacks, especially near the end, we will continue to iterate and improve our hardware. We plan to perform 4 rounds of experiments and assays:

1. Assay Validation: Testing the effectiveness and accuracy of our sensor using varying concentrations of TPA in HEPES buffer.
2. Initial PET Degradation: Running continuous PET degradation data with the wild-type TfCut using the smaller proof-of-concept hardware.
3. Assay Validation: Testing on the same standards but using the 8-slot hardware.
4. Full-Scale Assay: Performing the assay validation round on the final 8-slot hardware to collect continuous data simultaneously on the wild-type TfCut, six variants, and a blank control.

Our goal is to be able to consistently obtain accurate and continuous data on wild-type TfCut, our 6 variants, and a blank at the same time. In spite of numerous setbacks, we will continue to design, build, test, and improve our hardware so that we will be able to provide a more thorough analysis of the characteristics of our mutants.