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Fluorescence Detector (Lightbringer)
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Our pathogen detection approach relies on Cas13a digesting RNA. A common way of monitoring RNase activities is using commercially available RNaseAlert, consisting of a fluorescent RNA beacon. This is impractical for in-field applications because commercial fluorescence detectors are expensive and inconveniently large. We therefore make our pathogen detection system fit for in-field applications by developing a cheap and handy fluorescence detector. Although many previous iGEM teams constructed fluorescence detectors, we could not find any that had a high enough sensitivity or the ability to measure fluorescence quantitatively. We therefore constructed a detector matching our requirements 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 below.
Time lapse measurement of Cas13a digesting RNaseAlert on paper using our detector. The
positive control contains RNaseA and RNaseAlert. The negative control contains only RNaseAlert. Data points are
connected with lines for the convenience of the eye. Error bars represent the measurement uncertainties of the detector.
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.
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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 fluorophores 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 below show this overall design and the operational detector.
Overall design.
Operational detector.
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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.
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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.
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Circuit for Resistance Measurements
A voltage divider as shown in the figure below is the simplest way to measure resistance.
Voltage divider.
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.
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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 used LED has a luminous intensity of 12 cd, a maximum current of 20
mA and a forward voltage of 3.2 V.
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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.
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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 below.
Control circuit for the blue LED.
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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 below, 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.
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Transmission spectra of the chosen filter foils of our detector. The transmission spectra for the
orange filter foil(orange graph) has nearly no overlap with the transmission spectra of the blue filter foil(blue graph).
The transmission spectra of two blue and one filter foil(black graph) is therefore nearly 0 up to 700 nm.
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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 autofluorescent and causes a high background
signal.
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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 and 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 autofluorescent 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. We aso designed a paperstrip that matches our sample holder, to provide a low-cost and durable platform to store reaction mix. An
explosion drawing of the sample holder is shown in the figure below.
Explosion drawing of the sample holder.
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. An explosion drawing of the detection slot is shown in the figure below.
Explosion drawing of detection slot.
STL files of the 3D printed parts can be found here.
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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.
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Calibration |
Derivation of a Calibration Function
We want to find an equation that relates fluorophore concentration c and the corresponding resistance RLDR. The light intensity I at the LDR during a fluorescence measurement is a sum of signal intensity 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
 (12)
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,
 (13)
and that the background intensity Ib depends also linearly on I0,
 (14)
Therefore
 (15)
where k is a constant that depends on the transmission spectra of the filter foils, the spectra of the fluorophore, the
spectra of the LED and light scattering effects of the filter paper,but not on I0. k can be assumed to be constant for one specific measurement set-up.
Equation 15 inserted into equation 12 gives the final equation relating c to RLDR
 (16) |
Determination of the Calibration Parameter
We want to find a value for k from equation 16.
We therefore prepared 10-fold dilutions of fluorescein from 100 nM to 1 mM. For each measurement we pipetted 30 µl of sample on
a fresh filter paper, placed it in the detector and turned on the LED. We measured the resistance RLDR directly with a multimeter. After each measurement the detector was cleaned gently with ethanol. The first and last measurement
of each series was conducted with plain water to determine Rb and to confirm the absence of contaminations. A plot of the normalized resistances is shown in the figure below. We fitted the data with equation 16 to determine a value for k using a value of 0.8 for γ gives
 (17)
Normalized resistances RLDR/Rb vs fluorescein concentration
c and corresponding fit function.
To determine that k does not depend on the intensity I0 we made two measurement series. We changed the resistance R3 in series to the LED to dim the light Intensity I0. We used a 39 Ω resistor and a 60 Ω resistor. For the set-up with the 39 Ω resistor we additionally measured a 200 nM sample because this is the expected final working condition of the detector.
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Automatized Data Collecting and Processing
We want to verify if a reaction with a fluorescence product is taking place on a filter paper. Therefore we need to
measure the resistance RLDR over a certain time certain period. We choose to measure a data point
every 5 min over a time period of 1.5 h to take trace of the reaction kinetics. Before such a measurement series a blank
measurement with plain water is performed to determine the resistance Rb.
As a first step the exact value of the supply voltage U0 needs to be measured. The supply voltage is connected via an additional voltage divider with an analog pin. The voltage divider consists of a 100 Ω resistor and a 910 kΩ resistor. The analog pin measures still a correct value for the supply voltage U0 because the first resistor is negligible small compared to the second resistor. We did not connect the analog pin directly with the power supply to prevent the micro controller from damage in case of a short circuit or a peak voltage caused by an other component of the overall device. The micro controller measures the supply voltage 50 times with a delay time of 50 ms between measurements.
It calculates the average of U0 and the relative standard error σU0
, which is used as measurement
uncertainty for further calculations.
To initiate a resistance measurement of the LDR the LED needs to be turned on by setting a digital output pin to 5
V. An additional green LED on the device is turned on as well to indicate that a measurement is taking place. After
a waiting time of 30 s the actual measurement starts. This waiting time was determined empirically and is required
because of the slow response of the LDR. ULDR is measured in the same way as U0. The average of ULDR and the
relative standard error σULDR are calculated. Equation 5 is used to calculate RLDR from the average
of ULDR. We derived an equation for the propagation of the relative systematic and the relative statistical uncertainty
of U0 and ULDR. For the relative statistical uncertainty σstat of RLDR we get
 (18)
We used a value of 1 digit for the absolute systematic uncertainty for a voltage measurement. The relative systematic
uncertainty is 1/U for a measured voltage U. For the relative systematic uncertainty σsys of RLDR we therefore get
 (19)
The equation for the total uncertainty σRLDR is then
 (20)
RLDR, Rb and their uncertainties are calculated by the micro controller, read by the computer and saved for further analysis in a text file.
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Data Analysis and Final Result |
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With equation 16 and the fitted value for k the measured resistances can be translated into fluorescein concentrations c. Equation 16 solved for c is
 (21)
The equation for the relative uncertainty σc of the fluorescein concentration c is
 (22)
where σk is the relative uncertainty from the fit of k. We are now enabled to measure fluorescence in units of equivalent fluorescein concentrations c. We analysed data of a first experiment with these equations. The resulting figure is shown in the beginning of this documentation.
With these quantitative data it is now an easy task to verify if the collateral RNase-activity of Cas13a got activated by trRNA. For the convenience of the end user we choose to extract two informations from the time traces produced with our detector.
We check if a reaction has taken place on the filter paper by evaluating if the first 5 data points are monotonous rising. And we check if a threshold of 3 µM equivalent fluorescein concentrations was crossed by looking at the last 3 data points.
The two informations are computed in the following way,
if the reaction has taken place and the threshold was crossed the detector software will raise the message "Pathogen detected", if the reaction has not taken place but the threshold was crossed the detector software will rise the message "False positive".
The other two combinations will lead to the message "no Pathogen detected".
The software will also create a graph similar to the graph in the beginning of this documentation to provide a deeper insight for the enduser if needed.
We think that our detector is a more reliable read out system compared to other possibilities because our detector provides deep insight into the reaction dynamics.
With an exact knowledge of Cas13a's reaction kinetics and further analytic tools a nearly fail-safe read out can be achieved.
A colorimetric readout, a change in color, in contrast is way more primitive and provides no insight in any dynamics.
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