Short Summary
LED panel
Purification Column EluX
To verify the elution as easily as possible, we created a fusion protein (BBa_K2201321) containing GFP for a visible fluorescence signal and streptavidin to immobilize the protein in our column using biotin. The two protein parts are connected through a flexible glycine-glycine-serine-linker of eleven amino acids, in which the sixth amino acid is our 2-NPA. This ncAA will then induce the cleavage of our target protein (GFP) from the streptavidin compound in the purification process.
LED panel: Overview
Background
Design Criteria
-
Multifunctionality
The device should be modifiable to emit light of different wavelengths, ensuring that it is applicable for many different purposes. -
Modularity & Compact Size
It should be compatible with other instruments processing microtiter plates.Therefore, the outer dimensions of the device shall not exceed those of commonly used microtiter plates. -
Narrow Irradiation Spectrum
A narrow distribution in the irradiation spectrum around the desired wavelength is preferred to prevent the occurrence of unintended reactions. This is especially important concerning highly energized ultraviolet light that could lead to protein degradation.Regarding this criterion, light emitting diodes (LEDs) are the best choice as the source of light. They are small, have a good energy conversion efficiency and emit light in a very narrow distribution around their irradiation maximum. -
Illumination Uniformity
To ensure reproducibility of the experiments and comparability between different wells of the microtiter plate, a uniform illumination of all wells is essential.Our approach is to provide an individual LED for every single well of the microtiter plate, providing equal conditions in all wells. In contrast, a single source of light creates a cone of light with decreasing intensities towards the outer regions. Additionally, individual LEDs allow for a more compact size and a higher operation flexibility. -
Intuitive & Flexible Operation
The operation of the device should be intuitive while offering the possibility for advanced settings such as time and position dependent variations in illumination strength.Therefore, we decided on two possible interfacing options: A convenient operation via a single turning knob and an LCD display which is suitable for simple applications. For more complex tasks, the device can be operated via Bluetooth by a self‑programmed application for android. -
Accessibility
To enable other iGEM teams to use this hardware tool for their experiments, all schematics and designs of the device need to be accessible and its construction feasible. Additionally, the software used in this project needs to be available and easily comprehensible.We proudly provide everything you need to build your own LED panel, including detailed instructions and commented software. This way, the whole LED Panel can be adjusted to perfectly fit the individual requirements of your experiments! -
Safety
The device can be fitted with ultraviolet LEDs which are harmful but invisible to the human eyes. Therefore, the operator must be warned by an indication light showing that the LEDs are operational.While the simplest option is a software controlled statues LED, we decided on a hardware solution which is failsafe even in case of a malfunctioning software.
LED panel: Development
Prototyping
Different through‑hole LEDs.
The TLC5947 PWM controller on an adafruit breakout board.
Two 7‑segment displays connected via two 8 bit shift registers.
A rotary encoder and the I2C controlled LCD‑display showing our team logo.
HC05 Bluetooth dongle that receives a series of advanced illumination settings from our android application.
PCB Design & Circuit Diagram
Circuitry overview. A compact representation of all circuitry that is underlying the circuit board design. The circuit layout was created with the Autodesk EAGLE CAD Software package. Electrical connections are represented by green lines or by labels for distant connection points. A detailed and structured version of the circuit diagram can be downloaded in our Downloads section.
Evolution of circuit board design. This animated graphic shows a few snapshots of the design process that led to our final PCB layout. The design was created with the Autodesk EAGLE CAD Software package. Every color represents a different layer that needs to be considered in the manufacturing process. For instance, red and blue indicate copper on the upper and lower side of the board respectively, green stands for pads and vias and gray lines represent outer dimensions of parts as well as their names and values.
The current flow through the LEDs is easily set by adjusting the reference resistance.
Safety Features
HC05 Bluetooth dongle that receives advanced settings from our android application.
Manufacturing & Assembly
After the final PCB design was completed and the circuitry tested, the manufacturing process began. Normally, it is necessary to search for a board manufacturer first and design the PCB in accordance with their manufacturing specifications and limitations. Because of that, we were very conservative in terms of critical design decisions like conductor line distances and diameter and the minimal drill size. This way we ensured that the PCB can be manufactured by most board houses. In our case, the PCB was kindly provided by Beta LAYOUT GmbH.
Circuit Board Manufactoring Process. The circuit board was manufactured by Beta LAYOUT GmbH. The process involves the following steps: (A) The holes and cavities are drilled into the 0.062 inch thick base material. After intensive cleaning, a photosensitive dry resist layer is laminated evenly onto the surfaces under extreme temperatures and pressure. By using an individually manufactured photoplot, the areas that shall remain free of copper are exposed to UV light. (B) Afterwards, nonexposed parts of the resist layer are removed through development in a 1 % sodium carbonate solution. Now, the areas free of resist layer are coated with approximately 35 μm of copper, overlayed by a thin tin film. The residual photoresist is stripped away with a 2.5 % potassium hydroxide solution. In the following etching step, excess copper is removed by applying ammonia solution. Afterwards, the tin that protected the copper layer is removed with a nitric acid based tin‑stripper. (C) Finally, a green solder mask is applied and developed through light exposure. It covers and protects most of the PCB except for solder pads and vials. These are covered with protective tin by a process called Hot Air Leveling (HAL) at 270 ℃ and 5 bar and immersion in liquid tin.
For the subsequent assembly of the PCB with the surface mounted components, we decided for reflow soldering. Nevertheless, if a reflow oven is not available, the design is suitable for soldering by hand too. First, a thin layer of solder paste is applied onto the solder spots on the PCB. For this purpose, stencils were used that are supplied by most PCB manufacturers. Stencils are thin metal sheets with cut-outs at the positions were solder shall be applied, ensuring that the perfect amount of solder is used. Afterwards, the components are placed on the solder paste with tweezers. The viscosity of the paste holds the components in place until they are soldered inside the reflow oven. For soldering, a two-stepped program sequence was used, consisting of a 70 s preheat step at 210 ℃ followed by 40 s at approximately 300 ℃.
Solder paste is applied to the PCB using a metal stencil.
SMD components are placed on the solder paste.
Exact positioning of ICs is controlled with a magnifier lens.
The components were soldered onto the PCB in two soldering rounds. First, the LEDs were soldered onto the bottom side of the PCB. Afterwards, the second stencil was used to apply solder paste on the top side to mount the remaing components. While soldering the second time, the LEDs stick to the PCB due to adhesive forces of the remelted solder, even though they hang freely on the bottom side of the PCB.
The PCB with the SMD components is placed inside the reflow oven.
Preset soldering profile and actual temperature is observable with the software controlling the reflow oven.
PCB after first reflow soldering round, with soldered LEDs facing upwards.
After reflow soldering of the SMD components, all other components such as the DC socket, pinheaders and the status LED were soldered to the PCB by hand. Afterwards, the LED panel was assembled with the 3D printed case, the Bluetooth adapter, the display, the microcontroller and the fans.
3D Printed Case
In terms of flexibility and accessibility, a 3D printed case is the optimal solution for prototyping and low cost product development. For the creation of our 3D models, we used the Google SketchUp Make 2017, which is free to use. The SketchUp files containing the 3D models of all printed parts are available in the Downloads section.
The 3D printer used for printing the LED panel cases.
3D model of the PCB with mounted components.
The cases were printed on an Original&nsbp;Prusa i3 MK2 3D printer with the help of Marco Radukic from the Cellular and Molecular Biotechnology Group at Bielefeld University. As thermoplastic, we used polylactic acid due to its low costs and easy handling. In order to achieve the desired precision, we started by creating a 3D model with the exact dimensions of the PCB and all its mounted components.
Model of 3D printed case, designed to fit the 3D model of the PCB. The upper and lower part of the case (red) are held together by bolts in each corner of the LED panel.
The upper part of the 3D case. The upper part holds all wires, fans and interfacing components in place, without the need for screws or glue.
Based on this model, the 3D printed case was designed, consisting of only two parts: The upper part holds all the wires, interfacing components and fans in place. The case is designed with to be easily assembled, without the need for any screws or glue. The components are held by the case on one side and by the PCB on the other. The upper part of the case has four bolts, positioned at each corner of the rectangular design. These staves hold the PCB and the lower part of the case in place. The lower part is responsible for defining the optimal distance of the LED to the wells of the microtiter plate. It is easily interchangeable, for example the microtiter plate shall be illuminated from above or from beneath. We designed and printed lower parts for both use cases that ensure for minimal lightspill and optimal sample illumination.
LED panel with adapter for illumination from above the microtiter plate.
LED panel with adapter for illumination from beneath the microtiter plate. The integrated tilt sensor detects the absolute orientation of the device and the display content is automatically rotated in accordance.
Equal illumination of all wells of a microtiter plate with transparent lower surface.
Because we wanted to enable users to illuminate a microtiter plate from beneath and from above, we integrated a tilt sensor into our PCB design. Together with the software of the microcontroller, this sensor detects the orientation of the device. When the LED panel is turned over, the content of the display is automatically tilted too, making the usage of our LED panel very intuitive for every application.
Ordered interior of the LED panel. The interior of the LED panel is neatly ordered, without the need for screws or glue. All components and cables are held in place tightly by the 3d printed case.
Software & Bluetooth Application
Nowadays, an ambitious hardware project is always closely connected to software. Apart from in silico desgin of the electronics, the PCB and the 3D printed case, we needed to write the software running the microcontroller. Without the microcontroller, which comtrols the LED drivers and manages the user interface, this project would not have been impossible.
The program for the teensy microcontroller was written in the Arduino integrated development environment (IDE). The Android IDE provides a very easy interface for compiling and transmitting code for micro-controllers. The teensy microcontrollers are fully compatible with this development environment, making it especially easy to adapt the code running the LED panel for your own requirements. To further simplify software adaptations of our LED panel, we created our own Arduino Library that contains all necessary functions for controlling the LED panel in an intuitive way. For example, instead of accessing every LED individually, the following lines of code are sufficient for configuring the LED panel and turning on all LEDs at the desired intensity:
#include "Bielefeld-CeBiTec_LED_panel.h"
LED_panel led_panel = LED_panel();
led_panel.begin();
led_panel.setPWM( INTENSITY );
All code, including the Arduino Library, are available for download in the Downloads section.
Apart from the software for the microcontroller, we developed an Android application that allows users to operate the LED panel via Bluetooth. This is especially helpful for advanced illumination protocols which would not be possible by controlling the LED panel with the rotary encoder alone. The software is written in Android Studio, which is free to use for everyone. On startup, the application searches for accessible Bluetooth devices in its range and automatically identifies the LED panel. It then establishes a serial connection and waits for the user to create a program sequence of illumination settings.
The Program Sequence activity of our Bluetooth application. The program sequence contains entries for each change in the illumination profile. At a glance, the whole illumination sequence is comprehensible to the user. In the given example, the LED panel is first illuminated evenly for 10 seconds at 20 % intensity. Afterwards, the intensity is linearly increased from 20 % to 80 % over 1:15 minutes, followed by an additional, constant illumination at 100 % intensity for 1 minute.
Creating and editing program sequence entries. Controlled via a single, clearly arranged interface, the user can create and edit entries of the program sequence. At the top, the user can choose between contant illumination and time dependent linear, exponential or logarithmic change in illumination intensity. Beneath, the user can set the illumination intensity before and after the specified period of time. If the optional gradient option is selected, the intensities before and after the time period can be adjusted for the leftmost and rightmost wells of the microtiter plate individually.
The edit menu allows the user to create and edit very complex illumination settings. First, it can be chosen between constant illumination intensity or time dependent linear, exponential or logarithmic change in illumination intensity. Afterwards, the illumination intensity before and after a specified period of time can be chosen. Even gradient illumination profiles are possible, setting the illumination intensities individually for the leftmost and rightmost wells of microtiter plate.
Hashing for control of data integrity. A hash function is used to ensure the data integrity of illumination protocols transmitted from the Bluetooth application to the LED panel. Only if the hash1, which is created by the Bluetooth application, is matching hash2 created by the LED panel after data transfer, the LED panel starts the illumination.
After the desired illumination protocol is created, the data is submitted to the LED panel via Bluetooth. Especially for sensitive experimental setups, it is extremely important that no data gets lost or corrupted during the transmission to the LED Panel. Otherwise, the illumination protocol could differ from the one created in the Bluetooth application. In order to prevent this from happening, we implemented a hashing algorithm to the transfer protocol. The hash function creates an individual identifier for a given input String, in this case the complete program sequence that is going to be submitted to the LED panel. This "fingerprint" is added to the transfered data stream. When the Teensy receives a program sequence via Bluetooth, it applies the same hashing function as the Bluetooth app to the received data and compares the resulting identifier with the transmitted fingerprint. Only if both are the same, the data transmittance was successful, and the LED panel begins the illumination protocol.
Circuit Board Manufactoring Process. The circuit board was manufactured by Beta LAYOUT GmbH. The process involves the following steps: (A) The holes and cavities are drilled into the 0.062 inch thick base material. After intensive cleaning, a photosensitive dry resist layer is laminated evenly onto the surfaces under extreme temperatures and pressure. By using an individually manufactured photoplot, the areas that shall remain free of copper are exposed to UV light. (B) Afterwards, nonexposed parts of the resist layer are removed through development in a 1 % sodium carbonate solution. Now, the areas free of resist layer are coated with approximately 35 μm of copper, overlayed by a thin tin film. The residual photoresist is stripped away with a 2.5 % potassium hydroxide solution. In the following etching step, excess copper is removed by applying ammonia solution. Afterwards, the tin that protected the copper layer is removed with a nitric acid based tin‑stripper. (C) Finally, a green solder mask is applied and developed through light exposure. It covers and protects most of the PCB except for solder pads and vials. These are covered with protective tin by a process called Hot Air Leveling (HAL) at 270 ℃ and 5 bar and immersion in liquid tin.
Solder paste is applied to the PCB using a metal stencil.
SMD components are placed on the solder paste.
Exact positioning of ICs is controlled with a magnifier lens.
The PCB with the SMD components is placed inside the reflow oven.
Preset soldering profile and actual temperature is observable with the software controlling the reflow oven.
PCB after first reflow soldering round, with soldered LEDs facing upwards.
The 3D printer used for printing the LED panel cases.
Model of 3D printed case, designed to fit the 3D model of the PCB. The upper and lower part of the case (red) are held together by bolts in each corner of the LED panel.
The upper part of the 3D case. The upper part holds all wires, fans and interfacing components in place, without the need for screws or glue.
LED panel with adapter for illumination from above the microtiter plate.
LED panel with adapter for illumination from beneath the microtiter plate. The integrated tilt sensor detects the absolute orientation of the device and the display content is automatically rotated in accordance.
Equal illumination of all wells of a microtiter plate with transparent lower surface.
Ordered interior of the LED panel. The interior of the LED panel is neatly ordered, without the need for screws or glue. All components and cables are held in place tightly by the 3d printed case.
led_panel.begin();
led_panel.setPWM( INTENSITY );
The Program Sequence activity of our Bluetooth application. The program sequence contains entries for each change in the illumination profile. At a glance, the whole illumination sequence is comprehensible to the user. In the given example, the LED panel is first illuminated evenly for 10 seconds at 20 % intensity. Afterwards, the intensity is linearly increased from 20 % to 80 % over 1:15 minutes, followed by an additional, constant illumination at 100 % intensity for 1 minute.
Creating and editing program sequence entries. Controlled via a single, clearly arranged interface, the user can create and edit entries of the program sequence. At the top, the user can choose between contant illumination and time dependent linear, exponential or logarithmic change in illumination intensity. Beneath, the user can set the illumination intensity before and after the specified period of time. If the optional gradient option is selected, the intensities before and after the time period can be adjusted for the leftmost and rightmost wells of the microtiter plate individually.
Hashing for control of data integrity. A hash function is used to ensure the data integrity of illumination protocols transmitted from the Bluetooth application to the LED panel. Only if the hash1, which is created by the Bluetooth application, is matching hash2 created by the LED panel after data transfer, the LED panel starts the illumination.
LED panel: Applications
GFP-Excitation
GFP excitation with our LED panel. Microwell plate with an E.coli DH5α culture (middle left) and a GFP producing culture (middle, right) irradiated with light of 465 nm with the LED panel. The fluorescence was captured using an appropriate filter to reduce background signal.
Photoswitching of azobenzene‑phenylalanine (AzoF)
Photoswitching of AzoF with our LED panel. Changes in the absorption spectrum of AzoF when irradiated with light of 367 nm or 465 nm. Naturally, cis and trans conformations are mixed (black) but can be primed by irradiation to the cis‑conformation (red) if irradiated with UV‑light or to the trans‑conformation (blue) by irradiating with blue light. The cis‑primed AzoF can be primed to trans again (green) and then back to cis (purple) until the amino acid degrades.
Photochanging of 2‑nitrophenylalanine (2‑NPA)
Photoswitching of AzoF with our LED panel. Changes in the absorption spectrum of AzoF when irradiated with light of 367 nm or 465 nm. Naturally, cis and trans conformations are mixed (black) but can be primed by irradiation to the cis‑conformation (red) if irradiated with UV‑light or to the trans‑conformation (blue) by irradiating with blue light. The cis‑primed AzoF can be primed to trans again (green) and then back to cis (purple) until the amino acid degrades.
Photobleaching & Destruction
Photobleaching of GFP. Documentation of the decrease in fluorescence signal of GFP over time when irradiated with light of 367 nm (cyan) and 465 nm (dark blue).
Reversible photobleaching of GFP. Documentation of the decrease in fluorescence signal of GFP over time when irradiated with light of 465 nm (light green). Dark green: Regeneration of the fluorescence 180 minutes after the 1 hour irradiation process.
LED panel: Build Your Own!
Safety Disclaimer
Downloads
File | MD5 Sum |
---|---|
T--Bielefeld-CeBiTec--LED_panel_circuit_diagram.pdf.zip | e10f4775a16967f3485edc9cbd0b589c |
T--Bielefeld-CeBiTec--LED_panel_EAGLE_files.zip | 88ba0101ac809950c75b18c9cad86090 |
T--Bielefeld-CeBiTec--LED_panel_3D_case.zip | b5310d9585eb36529b5497cab6735da7 |
T--Bielefeld-CeBiTec--LED_panel_Bluetooth_Application.zip | 4704eb97674884cf41c34696f21267d2 |
Parts & Components
Type | Value | Package | Quantity | p.p. | Total | Shop |
---|---|---|---|---|---|---|
capacitor | 100n | C1206K | 8 | 0.05 € | 0.36 € | Farnell |
capacitor | 10u/16V | C1206K | 5 | 0.27 € | 1.34 € | Farnell |
capacitor | 100u/6V3 | C1206K | 1 | 0.57 € | 0.57 € | Farnell |
resistor | 0R | R1206 | 4 | 0.01 € | 0.05 € | Farnell |
resistor | 520R | R1206 | 1 | 0.04 € | 0.04 € | Farnell |
resistor | 1K | R1206 | 6 | 0.01 € | 0.07 € | Farnell |
resistor | 10K | R1206 | 4 | 0.03 € | 0.11 € | Farnell |
trimm resistor | 5K | RTRIM4G/J | 4 | 1.29 € | 5.16 € | Farnell |
zener diode | 3V3 | DO-214AC | 1 | 0.34 € | 0.34 € | Farnell |
p-channel MOSFET | IRF7416PbF | SO-08 | 1 | 0.79 € | 0.79 € | Farnell |
PNP transistor | BC807-16SMD | SOT23-BEC | 1 | 0.02 € | 0.02 € | Farnell |
PWM controller | TLC5947-DAP | HTSSOP32DAP | 4 | 3.80 € | 15.20 € | Farnell |
tilt switch | 15°, 0.25A | E2,9-5,94 | 1 | 2.23 € | 2.23 € | Farnell |
jack plug | MJ-179PH | SPC4078 | 1 | 1.24 € | 1.24 € | Farnell |
pin header | female 5 pins | FE05 | 4 | 0.26 € | 1.04 € | Reichelt |
pin header | female 16 pins | FE16 | 2 | 0.84 € | 1.68 € | Reichelt |
heat sink | 6.3x10mm | SOIC-16 | 1 | 0.61 € | 2.44 € | Farnell |
heat transmissive tape | 1 | 10.70 € | 10.70 € | Reichelt | ||
rotary encoder | KY-040 | 1 | 1.88 € | 1.88 € | Amazon | |
LCD display | KY-040 | 1 | 4.93 € | 4.93 € | Amazon | |
fan | 20x20x10mm | 2 | 5.45 € | 10.90 € | Reichelt | |
jumper cables | 1 | 3.99 € | 3.99 € | Amazon | ||
microcontroller | Teensy 3.2 | 1 | 24.50 € | 24.50 € | Reichelt | |
mains adapter | 5V 4.5A stabilized | 1 | 14.40 € | 14.40 € | Reichelt |
Elux: Design & Development
The six steps in the development process of the purification column A) BSA-coated micro well plate, B) Hollow cylindrical column, C) Biotinylated microscope slides, D) First flow model, E) second flow model, F) future concept of a microfluidic like column.
Concept of a microwell plate coated with biotinylated BSA.
Concept of our cylindrical column 3D-Modell of a hollow column with an LED-rod in the middle and a carrier material coated with biotinylated BSA as first concept of a purification system using 2-NPA.
Three microscope glass slides as basis for biotinylated surfaces for the purification column.
EluX prototype one. First flow model of a purification column with parallel biotinylated glass slides inside and UV-permeable acrylic glass plates in front and back to enable the radiation with light of 365 nm wave length.
EluX prototype two. Second flow model of a purification column as basis for a workshop and a 3D-printed model to test, evaluate and improve the concept of light induced elution.
Future concept of EluX. Concept of a microfluidic like (a) and a bundle (b) purification column designed with the support of Dr. Benjamin Müller and Prof. Dr. Dirk Lütkemeyer as future prediction of how the procedure of light induced elution could be used.
Elux: Construction
Purification Column EluX
3D-Printed Show Model
3D-printed parts of the purification column.
Preparation of the non-3D-printed parts at the home of team member Markus.
Inside and outside of the EluX prototype.
Comparison of the build and presented show model and the 3D-Model.
Elux: Theoretical basics
Affinity Purification columns
Workflow of the common procedure of an affinity purification process.
Biotinylation of Silicon
Hydroxylation of a silicon surface with piranha acid and treatment with APTES do get free amine-residues on the surface, followed by biotinylation with NHS-biotin.
References
Lapin, N. A., & Chabal, Y. J. (2009). Infrared characterization of biotinylated silicon oxide surfaces, surface stability, and specific attachment of streptavidin. Journal of Physical Chemistry. 113(25): 8776–8783. Mersha, F. and England, N. (1997). Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein. Gene. 192(2): 271–281.