Impact of laser excitation intensity on deep UV fluorescence detection in microchip electrophoresis

Abstract

A high intensity 266 nm continuous wave (cw-) laser developed for material processing was utilised as an excitation source for sensitive native fluorescence detection of unlabelled compounds in MCE. This 120 mW laser was attached via an optical fibre into a commercial epifluorescence microscope. With this MCE set-up we evaluated the impact of laser power on the S/N of aromatic compounds as well as of proteins. Compared with a previous work which used a 4 mW pulsed laser for excitation, improved S/N for small aromatics and to a lesser extent for proteins could be attained. The LOD of the system was determined down to 24 ng/mL for serotonin (113 nM), 24 ng/mL for propranolol (81 nM), 80 ng/mL for tryptophan (392 nM) and 80 ng/mL for an aromatic diol (475 nM). Sensitive protein detection was obtained at concentrations of 5 mg/mL for lysocyme, trypsinogen and chymotrypsinogen (340, 208 and 195 nM, respectively). Finally, a comparison of the cw-with a pulsed 266 nm laser, operating at the same average power, showed a higher attainable sensitivity of the cw-laser. This can be attributed to fluorescence saturation and photobleaching effects of the pulsed laser at high pulse energies.

Label‐free analysis in chip electrophoresis applying deep UV fluorescence lifetime detection

“…At a concentration of 2.5 mg/L each, i.e. 175 nM lysozyme, 104 nM trypsinogen and 98 nM chymotrypsinogen, we obtained S/Ns of 26, 10 and 12, respectively, which is approximately four times better compared to our previous results utilizing steady-state fluorescence [27]. Although well above the detection limit, this value appears to be quite moderate compared to the reported single molecule detection on quartz slides by Seeger's group [49].…”

“…In further studies, the S/N of several analytes in chip electrophoresis was investigated applying different laser sources operating at 266 nm with various excitation intensities [27]. However, a challenge was the presence of rather high background noise in this spectral region.…”

Recent developments in instrumentation for capillary electrophoresis and microchip‐capillary electrophoresis

“…This system enabled the separation and detection of a sample composed of three single-stranded DNA samples labeled with Cy3, FITC, and Alexa647 fluorescence in a single channel. The impact of the excitation intensity of a deep-UV laser (266 nm continuous wave) on the signal-to-noise ratio (S/N ratio) of aromatic compounds as well as of proteins was also investigated [78]. Although LODs in the 113 – 475 nmol L −1 range (for serotonin and 3-phenoxy-1,2-propandiol, respectively) were achieved, fluorescence saturation and photobleaching effects were observed.…”

Optimization of Native Fluorescence Detection of Proteins Using a Pulsed Nanolaser Excitation Source

“…In the case where labeling is undesirable, native fluorescence of the aromatic amino acids, tryptophan (trp), tyrosine (tyr) and phenylalanine (phe), is an appealing alternative. Extensive research on the use of laser induced native fluorescence (LINF) for protein detection has been conducted and documented 1-15. In many cases, LINF is coupled to capillary electrophoresis (CE) 6-13, 16.…”

“…In many cases, LINF is coupled to capillary electrophoresis (CE) 6-13, 16. LINF has also been used more recently with microcapillary electrophoresis (μ-CE) devices 13-15. Using laser induced native fluorescence, researchers have achieved sensitivities adequate to detect low-abundance proteins 14…”