Spectrophotometers have a long history in the scientific laboratory, primarily as devices for analyte quantification in solution. Myriad spectrophotometry applications use either the analyte’s inherent ability to absorb light or a reporter molecule, where its light absorption is directly proportional to the analyte concentration.
Spectrophotometer evolution includes improved workflows and conservation of precious sample. An example of this is microvolume instrumentation for nucleic acid quantification, which uses a mere 1–2 µL of sample. As the measurement is non-destructive and a minimal amount is used, sample is conserved or even fully recovered.
Conversely, most microvolume instruments are not useful for reporter molecule applications like colorimetric protein assays because reagent addition to quantify the analyte must be performed offline, and a small aliquot then used for analysis. This does not simply workflow or conserve sample, and the short pathlength defined by the detection volume requires that the analyte or reporter molecule have high absorptivity.
In this article, we describe a flexible, evolved spectrophotometer system using an accessory to provide microvolume analysis of analyte with innate absorptivity, and also reporter molecule-based assays requiring sequential sample/reagent additions. We will provide examples of microvolume direct nucleic acid quantification and in situ microvolume protein quantification using the colorimetric agent bicinchoninic acid (BCA) and a cytotoxicity assay in a standard microplate using the colorimetric reagent MTT.