Application note Measurement of fluorophore concentration using OptoReader

Fluorescence probe measurement

Fluorescence probe is a robust and highly sensitive approach for detecting trace amount 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.

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Samples preparation & Notes

Fluorescein solution

Fluorescein moclecule

The fluorescein is obtained from Sigma-Aldrich (46955). Solutions of different  concentration are obtained as follows:

  • Preparation of pH buffer solution containing 10 mM of Hepes in deionized water.
  • Dilution of 0.026 g Fluorescein into 100 ml of the above pH buffered solution. This solution is next diluted 20 times (with pH buffered water) to obtain 33.4 µM Fluorescein solution which serves as the master solution.
  • Solutions of different Fluorescein concentration are obtained by diluting the master solution, each time with pH buffered solution obtained as in the first step.
Optical fiber probe

The optical fiber probe (right figure) is made from a step-index optical Un-stripped optical fiber showing the step-index structure: glass core (1) and cladding (2) make up the wave guide channel by total internal reflection effect. They are usually protected by a plastique jacket (3) which is removed at the extremity of the fiber.fiber of 400 µm-diameter core, 425 µm diameter cladding and of numerical aperture 0.5.

Manipulation of optical fibers:

  • Remove the protection layers using cutting tools to reveal the plastique jacket (3) which covers the internal glass fiber composed of glass core (1) and cladding (2). They make up the wave guide channel by total internal reflection effect.
  • Remove the plastic jacket at the extremity of the fiber using an appropriate un-stripping tool.
  • Cleave the fiber to obtain a flat opening perpendicular to the optic axis. This step is critical and requires some practice. Thorlabs provides a useful guide on the manipulation of optical fiber.

Note that using 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.

Note on the numerical aperture

NA
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 LED source emits light in a large conic angle. The light is next focus at one end of the optical fiber for the excitation. The light power is channeled through the optical fiber scales as square of the focusing optics.
  • At the other end of the optical fiber, the detected fluorescence signal also scales as square of the fiber’s NA. In fact, since the fluorescence is isotropic in space, the higher the numerical aperture of the fiber is, the larger is the angle of acceptance, hence more fluorescence photons are captured.
Note on the core diameter of the fiber

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.

OptoReader Duplex fiber based high sensitivity optical detection

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.

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Measurements

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 OptoReader® to substract the disturbance from ambient lighting.* This feature aconsists of modulating the light source and substracting the signals detected when the souce 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 Fluorescein diluted present in the sample of interest. The fluorescence intensity is translated into Fluorescein density. Between successive measurement, the fiber is rinsed with isopropanol.

Results

The measured data is reported on the figure on below. The readout voltage on the photo-detector varies linearly with the concentration of Fluorescein.

Conclusion

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 resource. For high speed measurement or measurement requiring both the reflection and the fluorescence channels, conducting the experiment in dark condition is recommended.

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