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Stanford University

Stanford Microfluidics Laboratory

Microbarcodes for Multiplexed Binding Assays

Multiplexed hybridization assays are an important tool for detecting DNA oligomers and antigen-antibody interactions in applications such as biological warfare detection or clinical diagnostics.  Solution-based sandwich assays offer the ability to use high-sensitivity fluorescence imaging to detect target molecules that are not fluorescently labeled. By using identifiable particles as substrates for these hybridization reactions, many reactions can be multiplexed into a single batch reaction zone. This allows for a single-color, fluorescent marker to be used in detecting the presence of many identifiable targets.  The use of barcoded microparticles is one demonstrated method to effectively identify a large number of targets. An important example is the Nanobarcodes© particles fabricated by Nanoplex Technologies (Menlo Park, CA).  These particles are cylindrical metal rods typically 6 mm long by 250 nm in diameter.  The rods are electroplated in multiple steps to achieve “stripes” of metals with distinct reflective coefficients.


Figure 1. Schematic of a two-plexed assay using microbarcodes as the substrate by attaching different capture anitbodies to corresponding barcode patterns.  The stripes along the particle, typically silver and gold, define the binary barcode for each particle (silver-1, gold-0).  In this example, the antibody for analyte B is attached to particle flavor ‘00100’ and the antibody for analyte A is attached to particle flavor ‘01010’.  If analytes A or B are present in solution, they will bind to their respective antibodies and are then “sandwiched” with a fluorescently tagged antibody.  This technique is described in further detail by Nicewarner-Pena et al.




Figure2. Images of a multiplexed assay with microbarcodes.  (a) The barcode pattern of each particle is identified using elastic scatter optical microscopy from 430-500nm light.  (b) A corresponding fluorescence image identifies particles with bound antigen.  The ovals show the location of particles in the elastic scatter image that do not appear in the fluorescence image, denoting an absence of antigen on those particles.  Walton et al. provide an in depth explanation of particle identification.

Physics of colloidal metal rods and microbarcodes

We are developing physical models for the effects of electric field on nano-barcode alignment and motion.  We have shown experimentally the ability to align particles parallel to the field line of both AC and DC applied electric fields.  First, as shown below, particles have a “native” surface charge in solution which leads to a uniform electrical double layer of the form depicted in the schematic below.

Immediately upon application of an electric field, electric field lines around the particle are those of a conducting medium, perpendicular to particle surface as shown in the figure below. 
However, for small particles, the potential drop across the particle is too low to generate electrochemical reactions at the surface.  Therefore, after a characteristic charging time (see figure), charges migrate to the surface and induce a polarization of the particle as shown below.  The polarization process ends when the charges within the particle reorient to establish an equipotential state on the particle’s surface in the applied field.



Figure 3. Electric fields lines and charge diagram for a metal cylindrical particle in an applied field.  (a) When the electric field is first turned on, the particle acts as a pure conductor.  Charges within the particle reorient to maintain the equipotential surface, polarizing the particle.  (b) The current along the field lines in the conductor model will drive ions to the surface of the particle, eventually shielding the surface charge.  Once all of the surface charge has been shielded, the particle can be modeled as a pure insulator.  The charges that have been driven to the surface create a polarized double layer that leads to interesting second order effects.  Squires and Bazant have described this charging of the double layer for the case of spheres and infinite cylinders.

Finally (say, microseconds later!), induced surface charges result in a non-uniform induced electric double layer (EDL).  This non-uniform EDL induces non-uniform electroosmotic flow along the surface of the particle in a process which has been called induced-charge electro osmosis.

Figure 4. Streamlines of induced charge electroosmotic flow around a metal particle for a DC (or AC) electric field applied along the length of the particle.  The flow pattern is a cross-section of two ring vortices induced by the interaction of the polarized double layer and the field.

Experimental setup and results

To validate our particle alignment model, we have measured the orientation of homogenous and striped particles in a microchannel with AC electric fields ranging from 10 to 90 V/cm and ranging in frequency between 50 and 1000 Hz.  Our experimental setup uses pattern recognition and particle tracking to simultaneously track the translation, diffusion, and alignment of cylindrical particles 6 microns long and 0.25 microns in diameter under settling conditions and in both DC and AC electric fields. 

Figure 4. Schematic of the setup for particle alignment experiments.  The flow cell is oriented along the gravitational vector to allow particles to settle with negligible interaction with sidewalls.



(Mouseover figure to begin movie)




(Mouseover figure to begin movie)


Pure silver particles in DI water imaged as they settle through the flow cell.  (a) The particles settle in random orientations without an applied field.  Typical settling velocities are on the order of a few microns per second and vary depending on orientation.   (b) An AC field of 100 V/cm at 100 Hz is applied along the gravity vector direction to align the particles as they settle.  Both movies are shown at 10 times the real speed.

The orientation statistics of a group of particles in solution is a function of particle size and shape, the properties of the native electric double layer of the particle (due to native surface charge), the induced surface charge, and the Debye length and physical properties of the electrolyte. These parameters determine the electrokinetic/hydrodynamic forces which couple with the entropic force of Brownian motion to determine particle motion and orientation.

Figure. 6 Comparison of model and experiments showing the orientation distributions of particles in electric fields.  A 100Hz electric field was applied (fields shown above) and the orientation distribution of the particles in 1000 images was calculated.  The theory accurately predicts increased alignment in increased field strength and the greater alignment of the thin double layer particles versus the thick double layer particles.  The E=30 V/cm legend applies to all .  The “thin” and “thick” double layer thicknesses were approximate 135 nm and 25 nm.



This work was performed under the auspices of the U.S. Department of Energy by University of California, Lawrence Livermore National Laboratory under Contract W-7405-Eng-48.


1. Nicewarner-Pena, S.R., et al., “Submicrometer Metallic Barcodes,” Science, 294, 137-141 (2001).

2. Walton, I.D., et al., “Particles for multiplexed analysis in solution: detection and identification of striped metallic particles using optical microscopy,” Anal. Chem., 74, 2240-2247 (2002).

3. Squires, T.M. and M.Z. Bazant, “Induced-charge electro-osmosis,” J. Fluid Mech., 509, 217-252 (2004).

4. Rose, K.A., Santiago, J.G., "Flow-based Detection of Bar Coded Particles," Ninth International Symposium on Micro Total Analysis Systems (uTAS) Boston, Massachusetts, 2005


This document was prepared as an account of work sponsored by an agency of the United States. Government. Neither the United States Government nor the University of California nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or the University of California. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or the University of California, and shall not be used for advertising or product endorsement purposes.



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receives through a portal vein the dialytically separated elements of precedent decomposition for the verypet-
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