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Home > News & Events > Powerful New Fluorescence Option

Powerful New Fluorescence Option

 

Simple, cost-effective detection of fluorescent tags and tracers offers great value in biomedical applications, point-of-care testing and medical diagnostics. With a compact, low-cost spectral sensing device such as Spark, fluorescence measurements can be managed with great effectiveness. To evaluate the Spark spectral sensor for fluorescence, we measured different concentrations of fluorescein diluted in water.

Background

Aggressive competition, increasingly sophisticated technologies and the ever-changing landscape of research challenges — from health issues associated with aging populations to the emergence of more virulent diseases — have created a growing demand for analytical instrumentation that’s smaller, faster and more sophisticated with each successive generation. Simple, cost-effective setups are desired for lab, industry and OEM requirements, with applications ranging from blood and biological sample monitoring to cancer detection and tissue analysis.

With devices such as Spark — a solid-state spectral sensor — and an extensive line of modular spectrometers, embedded systems and optical sensors, Ocean Optics provides tools to solve research, development and point-of-care diagnostics measurement challenges. Sparks fills the price-performance gap between diode filter-based devices and spectrometers, adding a new dimension both in size – which is about the same as a small microcontroller — and in cost reduction, but with the ability to perform full spectral analysis from 380-700 nm.

Fluorescence Measurements with Spark Spectral Sensor

Fluorescence typically requires high sensitivity for measurements at low signal levels. The large aperture of the Spark makes it well suited for fluorescence measurements, as the increased throughput from its optical design allows low levels of light to reach the detector.

To test Spark’s fluorescence measurement capabilities, we measured aqueous (di)sodium fluorescein at different concentration levels. Fluorescein is an organic compound often used as a tracer in microscopy, forensics and medical diagnostics applications. Samples were prepared in aqueous solutions ranging from 10 nM to 100 µM, with a stock solution of 0.01 M sodium fluorescein in water.

Figure 1: Spark with cuvette holder

Figure 1: A cuvette holder is useful for fluorescence measurements of solutions. The Spark cuvette holder at lower left in this image is in development and will attach via an adapter base to the Spark unit.

Our fluorescence setup comprised the Spark-VIS and a 1 cm pathlength attachable cuvette accessory (Figure 1), with excitation from the Ocean Optics 470 nm LLS-470 LED light source. (Please note that the attachable cuvette holder used for this experiment is a prototype and not yet available. Currently, Spark fluorescence measurements require the use of a discrete cuvette holder like the CUV-ALL-UV.)

Spectral data were recorded at 100 ms integration time in a simple experimental setup using plastic, disposable 10 mm cuvettes (quartz cuvettes would be preferable for most fluorescence measurements). No optical filters or mirrors were used to enhance the signal. Cuvettes were covered to eliminate ambient light.

Figure 2: At 100 ms integration time, Spark measures the fluorescence of fluorescein in the concentration ranges of 1e-4 to 1e-8 Molar.

Figure 2: At 100 ms integration time, Spark measures the fluorescence of fluorescein in the concentration ranges of 1e-4 to 1e-8 Molar.

Fluorescein has a maximum absorption peak at 494 nm and a maximum emission peak at 521 nm. As Figure 2 shows, Spark measured a clear peak at 100 nM fluorescein and a shoulder (rounded) peak at 10 nM. Also, a “tail” peak (not shown in the graph) at 1 nM was captured. When the same graph is zoomed in at 20x (Figure 3), additional spectral features are revealed.

Figure 3: By using the zoom feature in OceanView spectroscopy software, users can discern more subtle spectral features.

Figure 3: By using the zoom feature in OceanView spectroscopy software, users can discern more subtle spectral features.

Conclusions

Our measurements suggest that under optimal fluorescence conditions (using filters, quartz cuvettes and a strong excitation source), the Spark-VIS can be used for analysis to <50 nM fluorescein solutions, and potentially even lower with a suitable calibration curve. This makes the Spark useful for a range of diagnostics, both medical and environmental, yielding high sensitivity measurements. For fluorescence measurements at longer integration times, we recommend selecting a Flame, Maya2000 Pro or other Ocean Optics spectrometer.

Fluorescence, either native or induced from special reagents, is a useful measurement technique for medical diagnostics, trace materials analysis and bacterial assay measurements. Also, many biological applications use fluorescent molecules as markers for other non-fluorescing compounds. With an inexpensive fluorescence sensor like the Spark, users can reduce measurement costs, incorporate the sensor more easily into applications and effectively combine Spark with other devices and techniques such as flow injection analysis and liquid chromatography.