54#ULTRASONIC TRAPPING IN CAPILLARIES FOR TRACE-AMOUNT BIOMEDICAL ANALALY

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.