|
|
|
|
|
|
|
Hardware
|
|
Our pathogen detection approach relies on Cas13a digesting RNA. A common way of monitoring RNase activityis using commercially available RNaseAlert consisting of a fluorescent RNA beacon. This is impractical for in-fieldapplications because commercial fluorescence detectors are expensive and inconveniently large. We therefore makeour pathogen detection system fit for in-field applications by developing a cheap and handy fluorescence detector. Al-though many previous iGEM teams constructed fluorescence detectors, we could not find any that had a high enoughsensitivity or the ability to measure fluorescence quantitatively. We therefore constructed a detector matching ourrequirements and compared it to others in a cost vs sensitivity diagram.
Our detector is paper-based and can detect fluorescein concentrations down to 200 nM. The detector is able to automatically
measure fluorescence in units of equivalent fluorescein concentrations. It fits in a pipette box and costs less
than 15 $. We were able to measure a time trace of Cas13a digesting RNaseAlert with our detector. For comparison
we also measured a positive control containing RNase A and a negative control containing only RNaseAlert. The
data are displayed in the figure bellow.
The time traces show an enzymatic reaction taking place on filter paper. This proves that our detector is sensitive
enough and meets our requirements. However the detector is not limited to our specific application but can be used
for the detection of any fluorescence signal in biological or chemical systems. We therefore think that our detector
can benefit other iGEM teams and research groups that want to make fluorescence based detection fit for in-field
applications.
|
Overall Design
Light from a blue LED is filtered by a blue filter foil and excites fluorophores on a filter paper. The excitation light
is blocked by an orange filter foil while the emission light from the fluoroscopes passes through the orange filter foil
and illuminates a light dependent resistor (LDR). The LDR changes its resistance corresponding to the intensity
of the fluorescence light.Finally an Arduino Nano measures the resistance via a voltage divider and calculates the
fluorophore concentration. The two figures bellow show this overall design and the operational detector.
|
Components |
Micro Controller
We used an Arduino Nano for automatized data collection. This micro controller has analog pins that can measure
voltages from 0 to 5 V and gives an integer from 0 to 1023 as output. The micro controller is connected via an USB port with a computer or smart-phone where the data can be processed further.
|
Light dependent resistor (LDR)
For the detection of fluorescence light we used a light depending resistor (LDR). A LDR decreases its resistance RLDR with increasing light intensity I. The dependence of the resistance RLDR on the light intensity I is
 (1)
where γ is a parameter depending on the type of resistor being used and can even differ for LDRs with the same type
designation.
Equation 1 is motivated from the equation
 (2)
which is given in the data sheet of the LDR. The denominator is the decadic logarithm of the fraction of two light
intensities of 100 Lx and 10 Lx. R10 and R100 are the corresponding resistances at these light intensities. The used resistor with the type designation GL5516 NT00183 has a parameter γ of 0.8.
The response of a LDR depends on the wavelength λ of the incoming light. The data sheet provides information on
the relative response normalized to the maximal response. The relative response is maximum for a wavelength of 540
nm and is therefore appropriate for detection of green fluorophores.
|
Circuit for resistance measurements
A voltage divider as shown in the figure bellow is the simplest way to measure resistance.
Applying Kirchhoff’s laws we get
 (3)
and
 (4)
RLDR and ULDR are the resistance and voltage drop at the LDR. Rref and Uref are the resistance and voltage drop at a reference resistor. U0 is the supply voltage which we choose to be 5V. This gives
 (5)
an equation to calculate RLDR from ULDR, which can be measured with the micro controller.
We need to find an equation to choose an optimal resistor Rref . We want a maximum change of ULDR for a certain detection range of RLDR. Therefore equation 5 is solved for ULDR giving
 (6)
The change ∆ULDR of ULDR between a maximum value Rmax and a minimum value Rmin of RLDR is
 (7)
which has a maximum for
 (8)
We expect an Rmax of approximately 2 MΩ and an Rmin of approximately 1 MΩ. By using equation 8 as a guideline we choose Rref to be 1.5 MΩ.
|
Light Emitting Diode (LED)
To detect green fluorophores we choose a blue LED with peak emission at 470 nm. For optimum performance we
choose the brightest LED we could find. The LED used has a luminous intensity of 12 cd, a maximum current of 20
mA and a forward voltage of 3.2 V.
|
NPN Transistor
To provide a stable illumination, it is crucial to supply the LED with a constant voltage. We therefore control it via
a NPN transistor with type designation BC635. Its base-emitter-on voltage is 2 V.
|
Control Circuit for the LED
The digital output pin is connected to the base of the transistor via a voltage divider, consisting of the resistor R1 with a resistance of 1 kΩ and the resistor R2 with a resistance of 9.1 kΩ. When the output pin is set to 5 V a voltage of 4.5 V is present at the base of the transistor. This is above the base-emitter-on voltage and the LED is turned on.
The resistor R3 with resistance 39 Ω was chosen empirically to limit the LEDs working current and to power it at
maximum brightness. The control circuit is illustrated in the image bellow.
|
Light Filter Foils
For filtering the emission and excitation light we used filter foils from LEE filters. The producer provides transmission
spectra for every filter foil. For the excitation filter we choose the filter color "TOKYO BLUE" and for the emission
filter we choose the filter color "RUST". We used our UV-Vis spectrometer to measure the spectra of different combinations
of filter foils. As shown in the figure bellow, a combination of one orange and two blue filter foils blocks
nearly all light up to 700 nm. This combination is therefore ideal for blocking the excitation light of the blue LED
from reaching the LDR.
|
Filter Paper
We choose glass fiber filter paper from Whatman Laboratory Products with type designation "934-AH" to detect
fluorescence on. In contrast, cellulose or nitrocellulose filter paper is auto fluorescent and causes a high background
signal.
|
3D Printed Parts
We intended to put the LED,the fluorescence sample and the LDR in direct proximity, to ensures a maximum use
of excitation light and emission light. We therefore chose a sandwich-like design for our sample holder that can be
placed into a slot where the detection system snaps in keeps the sample in position.
In detail, two blue filter foils are stacked and glued with tape in front of two excitation windows of the upper half of
the sandwich. One orange filter foil is glued to the lower half of the sandwich. The two detection windows enable us
to measure a blank sample and an actual sample with the exact same set-up. We avoid using scotch tape to cover
the detection windows because tape is usually auto fluorescent and causes a high background signal. A piece of filter
paper is placed between the two halves of the sandwich. The upper and lower part of this sandwich are pressed
together with magnets to hold the filter paper in position and ensure an user-friendly exchange of filter papers. A
explosion drawing of the sample holder is shown in the figure bellow.
The sandwich can now be inserted into the slot of the detection device. The LED and the LDR are mounted onto
beams at opposite sites of the detection device. The sandwich snaps in when the LED and the LDR are at the right
position under the excitation and over detection window. Four magnets apply an additional force to the beams and
press the LED and the LDR close together. This design ensures that the distance between filter paper, LED and
LDR is only limited by the thickness of the filter foils.
|
List of materials and cost calculation
| Used item |
Cost in EUR |
| Geekcreit ATmega328P Arduino compatible Nano |
2.51 |
| USB cable |
1.50 |
| LDR |
0.15 |
| Blue LED |
0.10 |
| 2 NPN Transistors |
0.12 |
| Green LED |
0.10 |
| Hole raster board |
0.42 |
| 8 resistors |
0.48 |
| Filter foils |
0.10 |
| 10 Magnets |
4.80 |
| ca. 100 g PLA |
3.00 |
| 4 M3 screws |
0.04 |
| 4 M3 nutts |
0.04 |
| Total cost |
13.36 |
We kept an eye on using only low cost and easy available items for the construction of our detector.
|
Calibration |
Derivation of a calibration function
We want to find an equation that relates fluorophore concentration i>c and the corresponding resistance RLDR. The light intensity I at the LDR during a fluorescence measurement is a sum of signal intensity i>Is and background intensity Ib:
 (9)
We assume that Ib is the intensity for a water sample. Importantly, wet samples give a different background signal than dry ones, suspectedly due to different light scattering on the filter paper. With equation 1 this gives an equation for the resistance Rb of a water sample and for the resistance RLDR for a fluorescence sample,
 (10) (11)
Then, the normalized resistance can be expressed as
We assume that the intensity Is of fluorescence light depends linearly on the concentration c of fluorophores and the
light intensity I0 produced by the LED,
|
|
