The fluorescence probe is a robust and highly sensitive approach for detecting trace amounts of substance owing to its simplicity and non-invasiveness. Moreover, the use of optical methods which is usually low cost, light-weight, high throughput and can be easily deployed in large scale for on-field or point-of-care applications.
The Elveflow® OptoReader® is an optical device taking the most of detection power out of the use of LED source and only one optical fiber for both excitation and detection of fluorescence.
In this tutorial, we illustrate how to employ the OptoReader® to measure the concentration of tiny amount of Fluoresein dye in water.
The fluorescein is obtained from Sigma-Aldrich (46955). Solutions of different concentration are obtained as follows:
The optical fiber probe (right figure) is made from a step-index optical fiber of 400 µm-diameter core, 425 µm diameter cladding and of numerical aperture 0.5.
Manipulation of optical fibers:
Note that using an optical fiber probe submerged in the measured solution is the simplest measurement scheme. Detection with higher sensitivity can be achieved using lenses mounted at the end of the optical fiber.
The numerical aperture (NA) determines the exit angle of the illumination and the angle of acceptance of the fiber. Only light impinging on the flat cleaved surface at an angle of incidence smaller than the acceptance angle can propagate inside the fiber.
Using high NA optics in the OptoReader’s design is critical for two reasons:
The core diameter of 400 µm is chosen since it is approximately the size of the light spot entrance of the optical fiber. A smaller core fiber will accept less light by roughly a factor of the ratio between the fiber’s cross section to the spot’s area.
The OptoReader® is used to measure the fluorescence of the Fluorescein solutions. This instrument employs only one optical fiber for both illumination and detection. The detected light is separated into reflection and fluorescence, therefore offering a duplex measurement on a medium. In this application note, only fluorescence measurement is performed. An example of the simultaneous use of the two detection channels can be found here.
The OptoReader® runs on the Elveflow® Smart Interface, therefore can be smoothly integrated with other Elveflow® devices. SDK is also provided for simple interfacing with the popular programming languages (Matlab, Python, Labview, …).
We take advantage of the synchronous detection feature of the OptoReader® to substract the disturbance from ambient lighting.* This feature consists of modulating the light source and substracting the signals detected when the source is ON and OFF.
Here, the sampling frequency is 10 kHz, the modulation frequency of the source is set to 845.79 Hz and the integration time to 1 s. The OptoReader® is set to the Fluorescence only mode.
To measure the Fluorescein concentration, the optical fiber probe is dipped into the solution. The optical fiber emits blue light and captures green fluorescence light from the Fluorescein diluted in the sample of interest. The fluorescence intensity is translated into Fluorescein density. Between successive measurements, the fiber is rinsed with isopropanol.
The measured data is reported on the figure on below. The readout voltage on the photo-detector varies linearly with the concentration of Fluorescein.
The OptoReader® is shown here to be a suited tool for applications with fluorescence probe. Detection sensitivity of 100 nM is demonstrated and 1 nM.* Higher sensitivities can be obtained by increasing fiber diameter, by using focusing optics or with the option of high sensitivity cooled sensor of the OptoReader.
The combination of fluorescence probe with microfluidics can give rise to powerful tools in the development of lab-on-a-chip systems. The later offers many advantages over conventional analysis, including low fluid volume consumption, better process control, massive parallelization and compact system design.
*The ambient light removal algorithm consumes computational resources. For high speed measurement or measurement requiring both the reflection and the fluorescence channels, conducting the experiment in dark conditions is recommended.
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