Difference between revisions of "Team:Munich/Measurement"

Although fluorescence measurements are a fundamental tool to characterize biochemical circuits, suitable measurement instruments are usually very expensive and therefore not available for all iGEM teams. We therefore constructed a portable low-cost fluorescence detector that can be assembled for less than 15$ and can measure time traces with the sensitivity of a commercial plate reader. The assembly of the detector only requires standard electronic components and a 3D printer. We provide equations to calibrate the data from the detector including a complete consideration of the propagation of uncertainties and wrote program code to automatize data analysis.

As a first proof of principle we measure a time trace of Cas13a digesting RNaseAlert with our detector. RNaseAlert is a commercially available fluorescent beacon consisting of a reporter fluorophore and quencher connected by an RNA-linker, bringing them in close proximity. While in the intact state, the fluorophore is quenched, cleavage of the RNA-linker separates the two labels and restores fluorescence. For comparison, we also measured a time trace of the highly active RNaseA digesting RNaseAlert and a time trace of a sample containing only RNaseAlert. The time traces measured with our detector are shown 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 data show typical curves of enzyme kinetics. It can be seen that RNaseA is more active than Cas13a. This shows that our detector is in fact able to quantitatively measure different levels of enzyme activity and can therefore be used to characterize biobricks.

A detailed description, including derivations of all equations and all necessary information to rebuild our detector can be found on our hardware page. Here we provide a brief description of the functionality of our detector and a summary of all equations needed to process and analyse data with our detector.

Overall Design

The conceptional design of our fluorescence detector is illustrated in the figure below. 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 R_LDR corresponding to the intensity of the fluorescence light. Finally an Arduino Nano measures the resistance via a voltage divider and calculates the fluorophore concentration.

Overall design.

Framework of Equations for Calibration and Data Analysis

An Arduino Nano can measure voltages in integers from 0 to 1023. To measure R_LDR we use a voltage divider to translate the voltage drop U_LDR at R_LDR via

(1)

where R_ref is the resistance of a reference resistor and U₀ is supply voltage measured in the same way as U_LDR. To measure time traces, we chose to acquire a data point every 5 minutes by measuring U_LDR 50 times in a time interval of 2 s. From that, we calculate the average and the relative standard error σ_ULDR. The relative uncertainty σ_RLDR of R_LDR can be calculated with

(3)

To calibrate our detector, we measured R_LDR for 10-fold dilutions of fluorescein from 100 nM to 1 mM. We derived an equation to fit the resistances R_LDR

(4)

where R_b is the resistance measured for plain water, γ is a parameter depending on the type of LDR used and c is the fluorescein concentration. k is the fit parameter and is a constant for a measurement set up. Fitting the data for our setup using γ = 0.8 gives

(5)

The calibration is shown in the figure below.

Normalized resistances R_LDR/R_b vs. fluorescein concentration c and corresponding fit function.

We are now able to measure fluorescence in equivalent fluorescein concentrations c. Equation 4, solved for c gives

(6)

The equation for the relative uncertainty σ_c of c is

(7)

where σ_Rb is the relative uncertainty of R_b and σ_k is the relative uncertainty of k given by the result from the fit of k.

The detailed documentation and our framework of equations provide an excellent template for future iGEM teams to rebuild and use our detector for their applications. The compact design of the detector makes it an excellent alternative to a commercial fluorescence detectors for in field applications.