Light emitting diode, photodiode‐based fluorescence detection system for DNA analysis with microchip electrophoresis

Abstract

Electrophoretic separation of fluorescently end-labeled DNA after a PCR serves as a gold standard in genetic diagnostics. Because of their size and cost, instruments for this type of analysis have had limited market uptake, particularly for point-of-care applications. This might be changed through a higher level of system integration and lower instrument costs that can be realized through the use of LEDs for excitation and photodiodes for detection--if they provide sufficient sensitivity. Here, we demonstrate an optimized microchip electrophoresis instrument using polymeric fluidic chips with fluorescence detection of end-labeled DNA with a LOD of 0.15 nM of Alexa Fluor 532. This represents orders of magnitude improvement over previously reported instruments of this type. We demonstrate the system with an electrophoretic separation of two PCR products and their respective primers. We believe that this is the first LED-induced fluorescence microchip electrophoresis system with photodiode-based detection that could be used for standard applications of PCR and electrophoresis.

Microchip capillary electrophoresis dairy device using fluorescence spectroscopy for detection of ciprofloxacin in milk samples

“…To allow for future potential of mass-market adoption of the microchip capillary electrophoresis dairy device, cost minimization is an important design factor that is considered. Therefore, the fluorescence spectroscopy technique is implemented with low-cost components, being an ultraviolet (UV) light emitting diode (LED) for the fluorescence excitation and a photodiode for the fluorescence detection, as in previous work 35 . As ciprofloxacin emits fluorescence at an excitation wavelength of 270 nm 36 , the UV LED for fluorescence excitation (VLMU60CL00-280-125 Vishay USA) is selected for its wavelength range of 270-290 nm and sufficient optical power of 2.4 mW.…”

Droplet-based μChopper device with a 3D-printed pneumatic valving layer and a simple photometer for absorbance based fructosamine quantification in human serum

“…Alternative light sources such as light-emitting diodes (LEDs) have shown great promise in replacing more expensive flash based lamps in analytical chemistry applications. 48 Several works have explored the use of LEDs in capillary electrophoretic separations of ions, 49 fluorescence based electrophoretic DNA analysis, 50 and in photometric determination of hemoglobin in human blood or fluorometric determination of quinine or calcium ions. 51 In this work, inspired by the optical system developed by Cecil et al, 49 we adapted our µChopper to a low-cost optical system that uses an LED source and photodiode detector, both of which are much less expensive than conventional microscopes.…”

“…Alternative light sources such as light-emitting diodes (LEDs) have shown great promise in replacing more expensive flash based lamps in analytical chemistry applications. 48 Several works have explored the use of LEDs in capillary electrophoretic separations of ions, 49 fluorescence based electrophoretic DNA analysis, 50 and in photometric determination of hemoglobin in human blood or fluorometric determination of quinine or calcium ions. 51…”

Combining Affinity Selection and Specific Ion Mobility for Microchip Protein Sensing

“…However, typical capillary electrophoresis-based methods are non-continuous, with analyses subsequently limited in throughput. [20][21][22] The application of electric fields perpendicular to the direction of flow enables free-flow electrophoresis (FFE), with experiments conducted in a continuous manner. [23][24][25][26][27] Isoelectric focusing, a special case of FFE, utilises this approach and can achieve excellent separation of biomolecules.…”

“…For example, in microchip or capillary electrophoresis, molecules are separated via differential migration along a microchannel under an electric field. However, typical capillary electrophoresis-based methods are noncontinuous, with analyses subsequently limited in throughput. − The application of electric fields perpendicular to the direction of flow enables free-flow electrophoresis (FFE), with experiments conducted in a continuous manner. − Isoelectric focusing, a special case of FFE, utilizes this approach and can achieve excellent separation of biomolecules. ,, Nonetheless, this approach is complicated by the requirement of a suitable pH gradient, solution-phase stability of analyte species at their isoelectric point (pI), and sufficient differences in pI between analytes for separation to be successful. Therefore, FFE-based fractionation methods that utilize differences in analytes’ electrophoretic mobilities alone present several advantages, including the potential to operate in native, unperturbed environments.…”