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Whispering gallery mode resonators for rapid label-free biosensing in small volume droplets.

Wildgen SM, Dunn RC - Biosensors (Basel) (2015)

Bottom Line: WGM resonances are sensitive to the effective refractive index, which changes upon analyte binding to recognition sites on functionalized resonators.Droplet evaporation leads to potentially useful convective mixing, but also limits the time over which analysis can be completed.We show that active droplet mixing combined with initial binding rate measurements is required for accurate nanomolar protein quantification within the first minute following injection.

View Article: PubMed Central - PubMed

Affiliation: Ralph N. Adams Institute for Bioanalytical Chemistry, University of Kansas, 2030 Becker Drive, Lawrence, KS 66047, USA. swildgen@ku.edu.

ABSTRACT
Rapid biosensing requires fast mass transport of the analyte to the surface of the sensing element. To optimize analysis times, both mass transport in solution and the geometry and size of the sensing element need to be considered. Small dielectric spheres, tens of microns in diameter, can act as label-free biosensors using whispering gallery mode (WGM) resonances. WGM resonances are sensitive to the effective refractive index, which changes upon analyte binding to recognition sites on functionalized resonators. The spherical geometry and tens of microns diameter of these resonators provides an efficient target for sensing while their compact size enables detection in limited volumes. Here, we explore conditions leading to rapid analyte detection using WGM resonators as label-free sensors in 10 μL sample droplets. Droplet evaporation leads to potentially useful convective mixing, but also limits the time over which analysis can be completed. We show that active droplet mixing combined with initial binding rate measurements is required for accurate nanomolar protein quantification within the first minute following injection.

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(A) For rapid protein quantification, biotin-streptavidin binding rates are measured as a function of streptavidin concentration. Each plot represents multiple, separate assays carried out in a 10 μL PBS droplet using rapid stirring. 1 μL injections of streptavidin/PBS solutions (0–7.6 μM) were quickly made in 10 μL PBS droplets containing BSA-biotin functionalized 38 μm spheres. Continuous measurement of WGM resonant wavelength shifts enables initial binding rates to be accurately quantified as a function of streptavidin concentration. Control experiments using unfunctionalized resonators showed no shifts following injections with 1 μL of 1.9 μM streptavidin/PBS (data not shown). (B) Initial binding rates measured from (A) are plotted versus streptavidin concentration showing the linear trend (R2 = 0.991). Initial binding rates are determined within the first minute following protein injection, enabling rapid quantification in a small volume. Error bars represent inter assay variability at each streptavidin concentration (N = 3).
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biosensors-05-00118-f007: (A) For rapid protein quantification, biotin-streptavidin binding rates are measured as a function of streptavidin concentration. Each plot represents multiple, separate assays carried out in a 10 μL PBS droplet using rapid stirring. 1 μL injections of streptavidin/PBS solutions (0–7.6 μM) were quickly made in 10 μL PBS droplets containing BSA-biotin functionalized 38 μm spheres. Continuous measurement of WGM resonant wavelength shifts enables initial binding rates to be accurately quantified as a function of streptavidin concentration. Control experiments using unfunctionalized resonators showed no shifts following injections with 1 μL of 1.9 μM streptavidin/PBS (data not shown). (B) Initial binding rates measured from (A) are plotted versus streptavidin concentration showing the linear trend (R2 = 0.991). Initial binding rates are determined within the first minute following protein injection, enabling rapid quantification in a small volume. Error bars represent inter assay variability at each streptavidin concentration (N = 3).

Mentions: Having established a rapid refractive index sensing scheme for 10 μL droplets, initial binding rate studies were carried out using biotin-streptavidin as a model system. For each assay, 38 μm diameter resonators functionalized with biotin were immersed in a 10 μL droplet of PBS. The droplets were mixed with the rotating rod and 1 μL spikes of streptavidin/PBS stock solutions (0–7.6 μM) were quickly injected into the droplet. Figure 7A compares the time evolution of WGM resonances as a function of streptavidin concentration. Each trace in Figure 7A represents multiple, separate assays using different functionalized WGM resonators to compare response times with streptavidin concentration. As expected, the initial rate of change and final equilibrium shift in the WGM resonance increases with increasing concentration of streptavidin added.


Whispering gallery mode resonators for rapid label-free biosensing in small volume droplets.

Wildgen SM, Dunn RC - Biosensors (Basel) (2015)

(A) For rapid protein quantification, biotin-streptavidin binding rates are measured as a function of streptavidin concentration. Each plot represents multiple, separate assays carried out in a 10 μL PBS droplet using rapid stirring. 1 μL injections of streptavidin/PBS solutions (0–7.6 μM) were quickly made in 10 μL PBS droplets containing BSA-biotin functionalized 38 μm spheres. Continuous measurement of WGM resonant wavelength shifts enables initial binding rates to be accurately quantified as a function of streptavidin concentration. Control experiments using unfunctionalized resonators showed no shifts following injections with 1 μL of 1.9 μM streptavidin/PBS (data not shown). (B) Initial binding rates measured from (A) are plotted versus streptavidin concentration showing the linear trend (R2 = 0.991). Initial binding rates are determined within the first minute following protein injection, enabling rapid quantification in a small volume. Error bars represent inter assay variability at each streptavidin concentration (N = 3).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4384086&req=5

biosensors-05-00118-f007: (A) For rapid protein quantification, biotin-streptavidin binding rates are measured as a function of streptavidin concentration. Each plot represents multiple, separate assays carried out in a 10 μL PBS droplet using rapid stirring. 1 μL injections of streptavidin/PBS solutions (0–7.6 μM) were quickly made in 10 μL PBS droplets containing BSA-biotin functionalized 38 μm spheres. Continuous measurement of WGM resonant wavelength shifts enables initial binding rates to be accurately quantified as a function of streptavidin concentration. Control experiments using unfunctionalized resonators showed no shifts following injections with 1 μL of 1.9 μM streptavidin/PBS (data not shown). (B) Initial binding rates measured from (A) are plotted versus streptavidin concentration showing the linear trend (R2 = 0.991). Initial binding rates are determined within the first minute following protein injection, enabling rapid quantification in a small volume. Error bars represent inter assay variability at each streptavidin concentration (N = 3).
Mentions: Having established a rapid refractive index sensing scheme for 10 μL droplets, initial binding rate studies were carried out using biotin-streptavidin as a model system. For each assay, 38 μm diameter resonators functionalized with biotin were immersed in a 10 μL droplet of PBS. The droplets were mixed with the rotating rod and 1 μL spikes of streptavidin/PBS stock solutions (0–7.6 μM) were quickly injected into the droplet. Figure 7A compares the time evolution of WGM resonances as a function of streptavidin concentration. Each trace in Figure 7A represents multiple, separate assays using different functionalized WGM resonators to compare response times with streptavidin concentration. As expected, the initial rate of change and final equilibrium shift in the WGM resonance increases with increasing concentration of streptavidin added.

Bottom Line: WGM resonances are sensitive to the effective refractive index, which changes upon analyte binding to recognition sites on functionalized resonators.Droplet evaporation leads to potentially useful convective mixing, but also limits the time over which analysis can be completed.We show that active droplet mixing combined with initial binding rate measurements is required for accurate nanomolar protein quantification within the first minute following injection.

View Article: PubMed Central - PubMed

Affiliation: Ralph N. Adams Institute for Bioanalytical Chemistry, University of Kansas, 2030 Becker Drive, Lawrence, KS 66047, USA. swildgen@ku.edu.

ABSTRACT
Rapid biosensing requires fast mass transport of the analyte to the surface of the sensing element. To optimize analysis times, both mass transport in solution and the geometry and size of the sensing element need to be considered. Small dielectric spheres, tens of microns in diameter, can act as label-free biosensors using whispering gallery mode (WGM) resonances. WGM resonances are sensitive to the effective refractive index, which changes upon analyte binding to recognition sites on functionalized resonators. The spherical geometry and tens of microns diameter of these resonators provides an efficient target for sensing while their compact size enables detection in limited volumes. Here, we explore conditions leading to rapid analyte detection using WGM resonators as label-free sensors in 10 μL sample droplets. Droplet evaporation leads to potentially useful convective mixing, but also limits the time over which analysis can be completed. We show that active droplet mixing combined with initial binding rate measurements is required for accurate nanomolar protein quantification within the first minute following injection.

Show MeSH
Related in: MedlinePlus