Difference between revisions of "Team:Munich/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 R_LDR with increasing light intensity I. The dependence of the resistance R_LDR 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)

R_LDR and U_LDR are the resistance and voltage drop at the LDR. R_ref and U_ref 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 R_LDR from U_LDR, which can be measured with the micro controller.

We need to find an equation to choose an optimal resistor R_ref . We want a maximum change of U_LDR for a certain detection range of R_LDR. Therefore equation 5 is solved for U_LDR giving

(6)

The change ∆U_LDR of U_LDR between a maximum value R_max and a minimum value R_min of R_LDR is

(7)

which has a maximum for

(8)

We expect an R_max of approximately 2 MΩ and an R_min of approximately 1 MΩ. By using equation 8 as a guideline we choose R_ref 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.