Issue 8, 2009
From the journal:

Lab on a Chip

Microfluidic means of achieving attomolar detection limits with molecular beacon probes†

Christopher M. Puleoa  and  Tza-Huei Wang*b  

* Corresponding authors

a Johns Hopkins University, Department of Biomedical Engineering, N. Charles St., Clark Hall Rm. 123, Baltimore, MD, USA
E-mail: cpuleo@jhu.edu
Fax: +1-410-516-4771
Tel: +1-410-516-7576

b Johns Hopkins University, Departments of Mechanical Engineering and Biomedical Engineering, N. Charles St., Latrobe Hall Rm. 108, Baltimore, MD, USA
E-mail: thwang@jhu.edu
Fax: +1-410-516-4316
Tel: +1-410-516-7086

Abstract

We used inline, micro-evaporators to concentrate and transport DNA targets to a nanoliter single molecule fluorescence detection chamber for subsequent molecular beacon probe hybridization and analysis. This use of solvent removal as a unique means of target transport in a microanalytical platform led to a greater than 5000-fold concentration enhancement and detection limits that pushed below the femtomolar barrier commonly reported using confocal fluorescence detection. This simple microliter-to-nanoliter interconnect for single molecule counting analysis resolved several common limitations, including the need for excessive fluorescent probe concentrations at low target levels and inefficiencies in direct handling of highly dilute biological samples. In this report, the hundreds of bacteria-specific DNA molecules contained in ∼25 microliters of a 50 aM sample were shuttled to a four nanoliter detection chamber through micro-evaporation. Here, the previously undetectable targets were enhanced to the pM regime and underwent probe hybridization and highly-efficient fluorescent event analysis via microfluidic recirculation through the confocal detection volume. This use of microfluidics in a single molecule detection (SMD) platform delivered unmatched sensitivity and introduced compliment technologies that may serve to bring SMD to more widespread use in replacing conventional methodologies for detecting rare target biomolecules in both research and clinical labs.

Graphical abstract: Microfluidic means of achieving attomolar detection limits with molecular beacon probes

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Article information

DOI
https://doi.org/10.1039/B819605B
Article type
Paper
Submitted
04 Nov 2008
Accepted
20 Feb 2009
First published
06 Mar 2009

Lab Chip, 2009,9, 1065-1072

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Microfluidic means of achieving attomolar detection limits with molecular beacon probes

C. M. Puleo and T. Wang, Lab Chip, 2009, 9, 1065 DOI: 10.1039/B819605B

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