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Abstracts: Lectures
LECT-19
Detection of unlabelled oligonucleotide targets using
whispering gallery modes in single, fluorescent microspheres
Edin Nuhiji, Paul Mulvaney
School of Chemistry & Bio21 Institute Level 2 North, 30 Flemington Road, University of Melbourne
Parkville, VIC, 3010 (Australia). E-mail:
Sequence specific nucleic acid detection has become an important goal in an array of biotechnology and
biomedical disciplines as well as in forensic analysis. However a disadvantage of most current nucleic acid
detection systems is the necessity to label the analyte. [1, 2] Labelling the target molecule can be costly,
laborious and may interfere with the binding affinity of the target molecule. In this work we demonstrate
the development and characterization of an innovative, highly-sensitive, label-free oligonucleotide specific
biosensor. [3]
Whispering gallery modes (WGM) have emerged as a powerful signal transduction mechanism that could
be utilized in ultra-sensitive biosensors. [4] In dielectric microspheres WGM are produced when incident
radiation is internally trapped as standing waves, resulting in a scattering spectrum composed of numerous,
sharp peaks. Light is introduced into the microsphere by coupling through an optic fibre or more simply by
attaching a fluorescent molecule to a microspheres surface and illuminating with a UV lamp. The
fluorescent microspheres within this work comprise a silica microsphere functionalised with a fluorophore
and a dense monolayer of 5’ tethered, single-stranded oligonucleotides. The adsorption of the unlabelled
complementary (cDNA) probe then causes nanometre shifts in the emission spectrum of the microsphere.
Intensity/ a.u.
9000
8000
7000
6000
5000
4000
550 560 570 580 590 600 610 620 630 640
λ/ nm
Pre-Hybridized
Post-Hybridized
WGM spectra from a single microsphere
hybridization assay using 7.5μm
oligonucleotide (70 bases) specific microspheres.
WGM emission spectra of the same
tetramethylrhodamine (TMR) modified
microsphere were acquired pre and post cDNA
probe treatment using an optical microscope
coupled to a CCD detector. The peak
wavelengths exhibit red-shifts (in brackets) at
575 (1.1nm), 585 (1.5nm), 595 (1.1nm) and
605nm (1.1nm) following a 90s exposure of the
target microspheres to the cDNA probe.
These highly sensitive deviations in the emission signal can be registered using an optical microscope
coupled to a CCD detector. The assays are run using a gridded array plate system that enables the relocation
and scrutiny of a single particle. This capability alleviates the need for statistical data analysis and laborious
data processing of samples which is associated with current micro-array and microfluidic DNA based
systems. The assay is capable of detecting sub-picomolar levels of unlabelled-oligonucleotide targets and
delineating between control reagent and non-specific oligomeric sequences. The spectral shifts can be used
to monitor both the hybridisation kinetics and the denaturation of duplex DNA at elevated temperatures.
Oligonucleotides with more than 30 bases are most readily detected, while a complete assay takes only a
few minutes. This inexpensive and highly sensitive nucleic acid-specific microsphere assay system provides
an alternative bio-molecular recognition format with tunable specificity (proteins, anti-bodies) and easy
implementation in high-throughput screening and point of care systems.
References: [1] M. Y. Han et al., Nature Biotechnology 19 (2001) 631; [2] B. J. Battersby et al., Chem. Comm. 2002,
1435; [3] E. Nuhiji and P. Mulvaney, Small, 2007, in press. [4] F. Vollmer et al., Biophys. J. 84 (2003 295A.
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