Description

At iGEM Bolivia, we emphasize the synergy between microbial engineering (biosensors) and hardware/software integration. To that end, we developed an electronic device capable of measuring the fluorescence signals produced by our biosensor in response to mercury in water. Our focus was to make this device user-friendly, ensuring that the general public can easily access and operate it in the future.
The device uses fluorometer technology and applies a calibration curve through microcontroller software to quantify mercury concentrations. It features an intuitive interface that allows users to initiate readings and view mercury levels in parts per billion (ppb). Additionally, the device provides alerts when mercury levels reach hazardous thresholds. With its compact, portable design, it can be easily transported to target areas, such as communities in the Bolivian Amazon, for practical use and implementation.

Operating Principle

Fluorometers are commonly used in laboratories to measure the fluorescent properties of biological and mineral samples. These devices detect the intensity and wavelength distribution of emitted fluorescence after the sample is excited by a specific light spectrum. The data collected is used to determine the presence and concentration of specific molecules in a medium.
Fluorometers are commonly used in laboratories to measure the fluorescent properties of biological and mineral samples. These devices detect the intensity and wavelength distribution of emitted fluorescence after the sample is excited by a specific light spectrum. The data collected is used to determine the presence and concentration of specific molecules in a medium.

Filters were added during both the excitation and emission stages to transmit the appropriate spectra for accurate fluorescence measurements. To detect the fluorescence, an LDR (light-dependent resistor) sensor was used, which varies in resistance based on the intensity of light it receives. This signal was amplified to improve measurement precision and then converted into a voltage signal readable by a microcontroller.
The signals received by the LDR sensor were compared within the microcontroller to a pre-established calibration curve based on samples with known concentrations. The mercury concentration result was displayed on a screen for easy visualization by the user. For validation, we replaced mercury with riboflavin, a readily available component with an emission spectrum similar to that of the mChartreuse protein, which is used as a fluorescent reporter for mercury detection.

Use Diagrams

We developed a quick-use diagram for the fluores10cence measurement device to help our target audience easily understand the basic operation of the biosensor in conjunction with the electronic device.

Demostration video

Diagram of Blocks

Design Considerations

Given the application environment and the biological agents involved, certain prerequisites were considered to ensure the basic functionality of the device. These include:

With these requirements in mind, the electronic and mechanical design of the device was developed.

Electronic Components

Emisor

A light source within the appropriate excitation spectrum was selected to interact with the simulation compound (Riboflavin) and generate fluorescence. To determine the correct range, fluorescent protein databases were consulted to obtain data on the excitation wavelength of riboflavin and the mChartreuse protein.

Excitation and emission spectrum of the mChartreuse protein (Lambert, T. 2024).

Excitation and emission spectrum of riboflavin (AAT Bioquest, Inc. 2024)

Riboflavin has an excitation peak at 349 nm, which falls within the UV light range. A UV LED diode with a spectrum range of 353–360 nm was chosen to generate sufficient fluorescence in the riboflavin sample.

Receptor

To measure the fluorescence signal, a light-dependent resistor (LDR) was used, varying in resistance based on the intensity of received fluorescence. This was connected to an ADC module, which improved signal sampling, and the data was transmitted to the ESP32 microcontroller using the I2C communication protocol.

To prevent UV light from reaching the LDR sensor and affecting the fluorescence measurement, a UV and blue light high-pass optical filter was added inside the device, positioned between the riboflavin sample and the sensor. Tests were conducted to verify the filter's effectiveness by measuring analog signals detected by the microcontroller. The results showed that UV light was fully blocked, confirming the filter's efficiency.

Microcontroller

The ESP32 microcontroller was used to power the system components, activate the emitting LED, receive data from the LDR sensor via the ADC module, and display the results on the OLED screen.

OLED Screen

A 1.3-inch OLED screen was chosen to display icons and text to the user with minimal energy consumption.

Battery Module

A Li-ion ICR18650 battery pack was implemented, along with a charging module and an SX1308 voltage regulator, to ensure portability.

ICR18650 Battery
Source : (Tacbattery.com, 2024)

By calculating the consumption of the device’s main components against the battery capacity, it was determined that the system can operate continuously for up to 37 hours.

Mechanical Design

The mechanical design of the device was created using SolidWorks software and divided into two main sections: the fluorescence circuit and the housing.

The fluorescence circuit design was based on the model from the paper “Simple and Inexpensive 3D Printed Filter Fluorometer Designs” (Journal of Chemical Education) [1], with modifications to fit the size of the water sample container. Holes were designed for the LED light and LDR sensor, positioned at a 90° angle to allow the excitation light to pass through the sample without interfering with the fluorescence reading. Receptacles were included for bandpass and UV filters to block unwanted signals from the UV LED emitter that are unrelated to the sample’s fluorescence.

The housing was divided into two sections. The left side houses the control electronics, including the charging circuit and batteries. The second section is designed to hold the fluorescence circuit and features a sliding lid to insert samples while preventing ambient light from entering.

Calibration

Development of Standard Solutions

Standard samples with measured concentrations of riboflavin were prepared in tubes, creating a calibration curve based on the fluorescence data obtained from each sample.

Calibration Curve

Once the fluorescence data from the standard solutions was obtained, it was plotted on a graph, with fluorescence signals on the X-axis and riboflavin concentrations on the Y-axis. A slope was generated from this data and entered into the microcontroller’s code, allowing it to automatically calculate riboflavin concentrations in future samples based on their fluorescence signals.

User's Manual and Biosafety

The ESP32 microcontroller was used to power the system components, activate the emitting LED, receive data from the LDR sensor via the ADC module, and display the results on the OLED screen.

Production Cost

To optimize accessibility, the electronic components were sourced from the national market. 3D printing was used for the housing and fluorescence circuit assembly, ensuring efficient assembly and facilitating large-scale replication of the device for cost-effective mass production.

Appendices

Bibliography