Envision and Create New Assays for the Qubit® 2.0 Fluorometer
The Qubit® 2.0 Fluorometer is a pioneering benchtop instrument—small, personal, and operated by simple and inexpensive components, yet capable of generating data with unprecedented accuracy, sensitivity, and selectivity. Integrated into each instrument are assay methods for DNA, RNA, and protein quantitation that utilize highly optimized algorithms to help ensure accurate results from only a 2- or 3-point calibration (Figure 1). Every sample read by the device is reported as an analyte concentration—no math or separate comparison to a standard curve is required—providing the perfect solution for researchers in a low- to mid-throughput setting.
Figure 1. The Qubit® workflow. The Qubit® 2.0 Fluorometer enables easy data collection with a seamless workflow, color touch screen, graphical display, and USB drive compatibility.
MyQubit Firmware Is Now Available
Although the preprogrammed algorithms make the Qubit® 2.0 Fluorometer extremely user-friendly, individual users have been unable to develop new Qubit® assays—until now! We have just released firmware called MyQubit that allows you to create applications in minutes using parameters that can easily be uploaded to the instrument using a USB drive, without changing the appearance or workflow of existing assays.
All Qubit® 2.0 instruments are now preloaded with the MyQubit firmware, and existing Qubit® 2.0 owners can download the firmware. Because the Qubit® 2.0 Fluorometer is operated by simple components, creation of additional applications is as straightforward as matching spectral properties of the assay product with the proper LEDs and emission filters. The instrument operates with two LEDs: blue (maximum wavelength ~470 nm), and red (maximum wavelength ~635 nm); excitation filters are 430–495 nm and 600–645 nm, respectively. Two bandpass emission filters are available at 510–580 nm and 665–725 nm. A new Qubit® assay can use existing Life Technologies kits and reagents as a starting point, or develop out of novel ideas altogether.
How to Make Your Own Assay Using MyQubit
To create new assays using MyQubit, several key parameters must first be defined. A simple .txt file generated with a standard PC allows fields such as assay name, units, light source, and dynamic range to be identified. In addition, the constants that determine the shape of the standard curve must also be resolved, which may require the collection of new data. In this case, the Qubit® 2.0 Fluorometer can be used as a mini-fluorometer in Qubit® Raw mode. This function allows you to manually select between the blue and red excitation sources while reading fluorescence in both emission channels.
The Qubit® 2.0 Fluorometer will guide you through the process of uploading your new assays. In a single .txt file, you can upload the parameters for up to four new assays, and a total of 20 new assays can be permanently uploaded to the device. The existing Qubit® assays for DNA, RNA, and protein quantitation remain unaffected by the presence of any new applications created using MyQubit. Likewise, the workflow and appearance of new assays remain consistent, as does the ability to store and access data. The only thing that changes is the addition of the new assay, increasing the flexibility of your Qubit® instrument. For detailed instructions on creating a new MyQubit assay file, and to download the Qubit® Raw application, visit lifetechnologies.com/qubit.
A Practical Example: The Amplex® Red Cholesterol Assay
Molecular Probes® Amplex® Red assays enable simple, straightforward quantitation of many biological analytes—including glucose, glutamic acid, peroxide, and phosphate—through a coupled enzymatic pathway. For this example, we have chosen to create a new Qubit® assay based on the Amplex® Red Cholesterol Assay Kit, which accurately measures cholesterol and cholesteryl esters in complex mixtures.
The Amplex® Red Cholesterol Assay Kit has been designed for use with a traditional fluorescence microplate reader. However, the spectral properties of the fluorescent reaction product make this assay a suitable candidate for optimization using the MyQubit firmware. To this end, replicate samples were run on a fluorescence microplate reader using the existing protocol and compared against equivalent samples run on the Qubit® 2.0 Fluorometer. After confirming equivalent performance based on sensitivity, dynamic range, and precision, the standard curve’s algorithm constants were optimized using a Microsoft Excel® template. The remaining assay parameters (e.g., units, dynamic range) were defined based on existing assay specifications, and the new assay was permanently uploaded to the instrument in a manner of minutes (Figure 2). As with any assay created with MyQubit firmware, assay parameters can be tailored for custom applications, even within the same class of analyte (e.g., cholesterol in biological vs. dairy samples).
Figure 2. Creation of a new Qubit® assay for cholesterol. (A) The Amplex® Red cholesterol assay was used to analyze triplicate samples of cholesterol standards from 0.32 μM to 20 μM using a Molecular Devices SpectraMax® M5 fluorescence microplate reader and the Qubit® 2.0 Fluorometer. The plot shows the line corresponding to the curve-fitting algorithm used in the calculation of concentration data. For reference, the positions of the standards and a set of data points from an actual experiment are shown superimposed onto the line. This plot demonstrates that the curve-fitting algorithm gives accurate values for the quantitation of free cholesterol in solution. (B) A new Qubit® assay for cholesterol can be easily uploaded to the instrument, sharing the same look and feel of existing assays.
The Qubit® 2.0 Fluorometer: Upgradable and Adaptable
Any reagent or assay that is spectrally compatible with the Qubit® hardware can be adapted for use with the Qubit® 2.0 Fluorometer. MyQubit brings your favorite fluorescence assays right to your benchtop, providing a reliable platform for many quantitation needs—from laboratory research and quality control to process monitoring and beyond.
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