Focus

Abstract

†In this article SMD refers to detection of single fluorescent molecules in solution, and reference will not be made to single molecule detection techniques incorporating alternative optical phenomena.

Microfluidic means of achieving attomolar detection limits with molecular beacon probes

“…[1][2][3] However, the use of small sample volumes in these platforms also requires highly sensitive analyte detection schemes and it is the development and integration of these detection approaches, which remains one of the main challenges for the practical application of microfluidic devices. 4,5 Traditionally, laser-induced fluorescence (LIF) and methods for electrochemical detection provide the workhorse detection schemes for microanalysis, although recently there has been considerable progress in alternate detection techniques, such as, surface plasmon resonance (SPR), chemiluminescence, Raman, infrared, and absorbance-based detectors. 5,6 The original detection technique LIF is most often used in conjunction with micro-capillary electrophoresis (CE) platforms, and this combination of separation and sensitive fluorescence detection remains one of the most represented classes of analytical microsystems.…”

“…4,5 Traditionally, laser-induced fluorescence (LIF) and methods for electrochemical detection provide the workhorse detection schemes for microanalysis, although recently there has been considerable progress in alternate detection techniques, such as, surface plasmon resonance (SPR), chemiluminescence, Raman, infrared, and absorbance-based detectors. 5,6 The original detection technique LIF is most often used in conjunction with micro-capillary electrophoresis (CE) platforms, and this combination of separation and sensitive fluorescence detection remains one of the most represented classes of analytical microsystems. 6 In parallel to these micro-CE platforms several researchers concentrate on the development of target-specific, amplificationand separation-free fluorescent biomolecular detection methods.…”

“…Secondly, nearly all of the successful applications of these SMD platforms utilize traditional means of analyte delivery, that is, fluorescently-labeled biomolecules are delivered to the focused laser observation volume through continuous flow within a micro-capillary or microfabricated channel. 5,7,11,[13][14][15] In this case, the potential for assay miniaturization is confounded by inefficient fluidic couplings, reliance on external pumping systems, and size mismatch between the observation volume and flow cell. Indeed, these drawbacks restrict the use of homogenous, single molecule probe strategies, relegating them to isolated, large sample volume platforms with low mass detection efficiency 5,13,15 However, use of a closed-loop, rotary pump 22 eliminates the extra fluid couplings associated with traditional SMD platforms and provides repeated, random sampling of probe-target interactions from nanoliter chambers 23 thus, enabling new analyte delivery schemes tailored for discrete, lowvolume SMD assays and specific biosensing strategies.…”

“…5,7,11,[13][14][15] In this case, the potential for assay miniaturization is confounded by inefficient fluidic couplings, reliance on external pumping systems, and size mismatch between the observation volume and flow cell. Indeed, these drawbacks restrict the use of homogenous, single molecule probe strategies, relegating them to isolated, large sample volume platforms with low mass detection efficiency 5,13,15 However, use of a closed-loop, rotary pump 22 eliminates the extra fluid couplings associated with traditional SMD platforms and provides repeated, random sampling of probe-target interactions from nanoliter chambers 23 thus, enabling new analyte delivery schemes tailored for discrete, lowvolume SMD assays and specific biosensing strategies. Herein, we describe a microfluidic coupling to deliver and concentrate targets to nanoliter-sized SMD chambers 23 from otherwise undetectably low concentrations of sample DNA.…”