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(Color online) (a) Streptavidin (SA)-coated microspheres are incubated with the target protein (SA is illustrated by diamonds). The analyte (biotinylated antibody) creates agglutination by binding to two different SA receptors located on separate microspheres. (b) Optical micrograph of monomer, dimer, and multimer structures which are formed by agglutination of SA microspheres with of analyte. (c) Schematic of the suspended microchannel resonator (SMR) for counting aggregates by weighing them one at a time. The resonance frequency of the SMR is sensitive to the presence of particles whose mass density differs from that of the solution in the microfluidic channel. (d) The SMR is used to classify a monomer (right) and a dimer (left) and anything larger as a multimer (not shown). The transient frequency shift from each structure is converted to a mass by using a calibration factor (see Ref. 9 ) and accounting for the buffer density.
Mass histogram from aggregates weighed by the SMR. There are primarily two types of structures (monomers and dimers) that resulted from an analyte concentration of and a particle concentration of . In each experiment approximately 2000 peaks were counted in . Particle types were classified by integrating the number of agglomerates between 0 and for monomers, 566 and for dimers, and and above for multimers (thresholds marked by dashed lines).
(Color online) (a) Mass-based dose response curve obtained by weighing aggregates with the SMR. Each data point represents the mean obtained from three experiments, and at , five experiments. The dependence of structure proportion is shown vs analyte concentration (biotinylated antibody in solid lines and pure antibody in dashed lines). Each data point represents the mean percentage of structures obtained from three separate experiments at each concentration. In each experiment approximately 2000 aggregates were weighed, and error bars represent the standard deviation from the mean. (b) A dose response curve was also obtained by optically imaging approximately 1500 aggregates per analyte concentration.
Normalized standard deviation (SD) of the mean percentage of monomers versus the number of aggregates counted (normalized to the SD at 2000 counts). Aggregates were counted for in each of three experiments using the same concentration analyte ( biotinylated protein). The data point at 2000 counts includes data from two earlier experiments, representing five experiments total. The number of monomers was examined for the first 1000 counts and up to 3500 counts in increments of 500.
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