Follow the links to explore how we overcame the hurdles of photocaging

Introduction

In the past, only the photographers and radiographers were in need of dark rooms for film processing. Nowadays, outburst of new applications in the fields of photochemistry, photobiology and photolithography forced even researchers to work in such light controlled environments. But, not all institutions are equipped with dark room facilities because of spatial and economic constraints. This serves as the motivation for developing an affordable and less-space-demanding light-proof workbench called “Dark Bench”.

Description

As mentioned above, the Dark Bench is a light controlled space which facilitates the handling of the photo-sensitive materials which undergoes photo-damage in the UV and blue region of the electromagnetic spectrum. The Dark Bench was entirely made using laser-cut parts and readily available resources to make it a Do-It-Yourself and inexpensive device. As all the parts are assembled with help of angles and hinges, the Dark Bench can be dismantled and assembled with little effort. The very first Dark Bench design was adapted from an anaerobic chamber design where the arm holes are located in the front. Since the Dark Bench is a rigid structure, it would be inconvenient to work with the hands in right angles. In order to avoid this, we modified the body design so that the hands can be inserted in the natural arm inclination thereby increasing the convenience.

Sliding Drawer

Another challenging task in building a dark chamber was to maintain the darkness even while transporting materials. In the photographic darkrooms, this is achieved by double door system or using a revolving cylindrical chamber. But both designs were unrealistic in our Dark Bench because the double door system needs more space for operation and one of the doors must remain closed; while the revolving doors can’t be implemented with the laser cutter. So we modeled a sliding drawer with a single door and a roller plate for smooth operation.

Optical window and Safelight

As the entire working space is covered with opaque Plexiglas sheet, one has to think of integrating an optical window and a light source in the device to look through it. The optical window is a light filter that eliminates the undesirable part of the spectrum which would alter the compound. For the optical window, we covered the transparent Plexiglas with UV protection foil on both sides for better absorption. The protection foil not only absorbs the UV region of spectrum but also the wavelengths less than 500 nm which makes the Dark Bench more versatile. Instead of using an expensive fluorescent lamp and then filter the undesirable spectrum out, we used the red LED(Light Emitting Diode) strips for illuminating the Dark Bench. As both of our light sensitive compounds are insensitive to wavelengths around 625 nm and also because two-photon excitation is not possible under such low intensities, the LED lightings we chose were ideal.

Characterization

The parts that needed to be characterized in the Dark Bench are the optical window and safelights. The spectrumand chromaticity of red LED elements are studied extensively and reported in the datasheet of the product. From the spectrum given in the datasheet, it is clear that the range is restricted well below 500 nm. The performance of the optical window was evaluated by acquiring the absorption spectrum of the window. A specimen of size 1X4 cm² was cut in transparent Plexiglas and the absorption profile was studied with and without protection foil.

As evident from the graph above, even the transparent Plexiglas has an intrinsic UV rejection property. We further improved the performance by adding two layers of filters, which strongly attenuate the UV and blue regions of the spectrum. The absorbance of the specimen in the UV-A region has a value of 4 units. Or to make it sound more convincing, we could say that it allows only 0.01% or block 99.99% of incident intensity. Similarly in the blue region, the two-layer filter blocks almost 99% of radiation which can be improved further by adding more layers of the filter.

Discussion

Even though we could manage to build a fully functional Dark Bench, there is always room for further improvements. The rubber foam sheet we used instead of thick rubber gloves needs some refinement as it wears off with long term usage. To add functionality, we can include temperature and humidity sensors to standardize the working environment.

Introduction

In the final stage of the project, we were in need of a device with which we can study the photo cleavage reaction of caged amino acid and activate the inhibited enzymes by irradiating it with light of a specific wavelength. In most cases, a suitable light source with filter, inside the concealed box will be sufficient. But the real-world application of our project is to activate the light inducible proteases (LIPs) in the liquid detergent which demands the development of a new compact exposure device called “LIPs-Stick”.

Description

The LIPs-Stick is an irradiating device for liquid samples, which allows you to set the volume of sample to be activated, radiation intensity and exposure time. The device can handle up to a volume of 100 ml at once.

Light source

The caged amino acids O-(2-Nitrobenzyl)-L-tyrosine (ONBY) require exposure to a particular wavelength of 365 nm for photo cleavage [1]. The other factors which affects the efficiency and rate of uncaging are the light intensity and exposure time. So, as to have a quick uncaging, we used 365 nm high-power UV-A LED. The LED was mounted at a height of 5.5 cm from the base of activation chamber and the maximum intensity at base is found to be 15 mW/cm².

Pumps

The two peristaltic pumps powered by DC motors are used to pump the samples into the activation chamber for irradiation and to pump out the activated samples. The simple peristaltic pump was preferred over the high accuracy pump, which was realized by the stepper motor like our last year’s team, for the cost, size, and switching simplicity. The outlet pump also pneumatically stirs the sample by pumping in air during irradiation step to have uniform exposure. The pumping rate of the pumps was calibrated with respect to the duty cycle of the PWM signal and found to be 8-54 ml/min.

Control circuit

The control circuit is constructed around the Arduino Nano development board which gets the input from the user through the keypad and actuates the pumps and UV LED accordingly. It also displays the user settings and status of the activation process through a graphic LCD module. In addition to controlling all components, the control circuit comprises of constant voltage supply for the power LED. As the difference between LED forward voltage and source voltage is large, the power dissipation of almost 6 W makes it impossible to work with the Linear voltage regulators. This overheating issue is brought under control by employing a switching mode power supply(SMPS). Since the output voltage of the regulator can be varied from 2.5 V to 6 V, almost any wavelength LED up to 3 W can be powered with the circuit. Furthermore, we designed a PCB (Printed Circuit Board) to miniaturize the circuit and for easy prototyping.

3D structures

We designed and printed a cylindrical activation chamber, on which we can connect the pumps and mount the LED. Since the photon flux density decreases away from the centre, the diameter of the chamber was restricted to 3 cm to have uniform and high intensity irradiation, which in turn reduce the effective volume of the container to 15 ml. So we modelled another collector container to store the unexposed solution while the sample is getting activated in parts of 15 ml. The circuit, pumps and the 3D structures are housed inside a laser-cut case.

Microcontroller firmware

The main task of the microcontroller firmware is to translate user inputs like pump rate, time, etc. to corresponding machine level variables to sequentially energize the actuators and constantly updates the status to the user. One of the thing that is worth noticing is that the PWM(Pulse Width Modulation) frequency is increased from 490 Hz to 1 kHz to save the H-bridge motor driver from current surges. Instead of activating the samples in 15 ml batches, it is also possible to continuously pump it at a much slower rate and collect it simultaneously by uploading another firmware.

Characterisation

The intensity of the high-power LED is modulated by changing the current that flows through it with the PWM technique. But the intensity is also a function of distance between the source and irradiating surface. In order to assess the intensity, we measured the intensity of the UV light at 365 nm by thermal power sensor (S302C, Thorlabs, Germany). As the sensor didn’t fit inside the chamber, we simulated the real conditions by fixing the LED at the same height as in activation chamber from the sensor surface. The following plot shows that the intensity is linearly related to the current and the maximum intensity recorded was 15.42 mW/cm²

Next, we wanted to evaluate whether our device can effectively activate the caged amino acid. For this experiment, three samples were prepared by dissolving 10 mg of O-(2-Nitrobenzyl)-L-tyrosine (ONBY) in 1 ml of acetonitrile and 200 µl of 1M HCL excluding the blank and then it was exposed to three different intensities 9.4 mW/cm², 12.7 mW/cm² and 15.4 mW/cm² respectively. As the photo cleavage reaction is associated with a color change, the visible spectrophotometry was done to track the rate of reaction for different radiation intensities at various time points. The graph below proves that the photo uncaging is possible with our device and reveals its rate dependency on intensity. However, the cleavage product couldn’t be quantified due to the non-availability of standard 2-nitroso benzaldehyde.

Discussion

As the constant voltage source is used to power the high-power LED, thermal breakdown of the LED may occur if it is operated at its full rating for a long duration. A current limiter or constant current source could be used for such long exposure applications. We also had an idea of miniaturizing it further so that it can be incorporated on the washing machine detergent drawer.

References

[1]  M. S. Kim and S. L. Diamond, “Photocleavage of o-nitrobenzyl ether derivatives for rapid biomedical release applications,” Bioorg. Med. Chem. Lett., vol. 16, no. 15, pp. 4007–4010, Aug. 2006.