INTRODUCTION
Nonintrusive manipulation by standing-wave acoustics
has been widely used for levitation and trapping of macroscopic
objects.1 The standing-wave field creates an acoustic
radiation force on the object that depends on its size and
acoustic parameters.2 In the present article we demonstrate
size-selective separation and retention of latex spheres inside
a small-diameter flow-through capillary by use of an ultrasonic
radiation trap. The work aims at rapid in-flow detection
and separation of specific molecules via antibody-coated latex
spheres.
Acoustic radiation forces have been used for nonintrusive
manipulation of macroscopic as well as microscopic objects.
Basically, objects with higher acoustic impedance than
the surrounding medium are trapped in the velocity antinodes
of the standing-wave acoustic field. The theory of
acoustic levitation and trapping is well understood.2,3 In
aqueous solutions the method has been applied for studies of
mechanical properties of mm- to mm-sized liquid droplets
and biological cells.4,5 These experiments typically operate at
low frequencies ~kHz up to a few 100 kHz! using a closed
cylinder levitation vial. Similar systems have been used for
cell concentration,6 cell filtering,7 and for enhanced rate and
sensitivity of latex agglutination tests by increasing particle
collision rates.8 Here few-mm-diam tubes or chambers are
combined with a transverse acoustic field ~propagation perpendicular
to the length of the tube!, resulting in a higher cell
concentration in several velocity antinodes parallel with the
tube. In-flow separation and fractionation of suspended particles
are performed on the basis of size and/or acoustic properties
with a liquid flow in combination with flow
splitters.9,10 Electric fields have been employed in a similar
arrangement to transversely separate charged particles by exploiting
the competition between acoustic radiation forces
and electrostatic forces.11 At higher ultrasonic frequencies
~.10 MHz! acoustic traps based on a standing-wave confocal
ultrasonic cavity have demonstrated that longitudinal
forces of similar magnitude or higher than those of optical
traps can be achieved.12 Finally, it should be noted that lowfrequency
airborne acoustic traps have been used for improved
biomedical analysis.13
In biomedical analysis, great effort is made to develop
detection techniques of trace amounts of specific proteins
and other biomolecules. Very small sample volumes (pl-nl)
may be analyzed with narrow-bore sub-100 mm capillaries in
combination with electrophoresis, capillary electrophoresis
~CE!, or capillary electrochromatography ~CEC!.14 CE/CEC
separates analytes according to charge, size, and chemical
characteristic with high selectivity and high throughput.
Typically, the limit of detection using fluorescent analytes is
femtomols, which may be improved by several orders of
magnitude in special cases.15,16 The use of antibody-coated
microspheres in CE/CEC may improve the selectivity and
the detection limit of specific macromolecules.17
In the present article we introduce the combination of
flow-through small-diameter capillaries and a high-frequency
longitudinal ultrasonic trap, which allows microspheres to be
separated according to size with high selectivity inside the
capillary. This capillary ultrasonic trap utilizes the competition
between acoustic radiation forces and viscous drag
forces. Inside the capillary, these forces act in opposite directions
on the spheres, resulting in size-selective trapping
and in-flow separation. In the present article the separation
efficiency of the system is theoretically analyzed. Furthermore,
in a first proof-of-principle experiment the capillary
ultrasonic trap is applied on differently sized fluorescent la-
a!Electronic
mail: Martin.Wiklund@biox.kth.se
tex spheres suspended in water inside 20–75 mm quartz capillaries.
The experiment demonstrates the size-selective trapping
and verifies the applicability of the theory inside the
capillary.
Longitudinal acoustic trapping has previously been combined
with liquid flow in large-diameter tubes for fluid–
particle separation and selection in, e.g., slurries.18 The use
of small-diameter capillaries allows smaller sample volumes
and higher sensitivity, which is of importance for biomedical
applications. The long-term goal of the present article is to
improve the limit of detection of specific proteins or other
sample molecules compared to current CE/CEC technology.
Here two different monoclonal antibodies against two different
antigenic sites on the analyte molecule will be covalently
bound to different latex spheres. When the sample molecules
are mixed with the two different latex spheres, a sandwich
assay ~latex-sample-latex complex! is formed with high
specificity.19 The size difference between the sandwich assay
and single latex spheres may then be employed for separation
and enrichment with the capillary longitudinal
ultrasonic-trap system, allowing detection of minute concentrations
of the target molecule. Compared to laser-optical
trapping for similar purposes,20 a longitudinal ultrasonic trap
has the potential to provide a much more compact arrangement,
and to produce a more uniform trapping field in
smaller-diameter capillaries, which is important for high selectivity
and sensitivity.