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Deterministic bead-in-droplet ejection utilizing an integrated plug-in bead dispenser for single bead – based applications

View Article: PubMed Central - PubMed

ABSTRACT

This paper presents a deterministic bead-in-droplet ejection (BIDE) technique that regulates the precise distribution of microbeads in an ejected droplet. The deterministic BIDE was realized through the effective integration of a microfluidic single-particle handling technique with a liquid dispensing system. The integrated bead dispenser facilitates the transfer of the desired number of beads into a dispensing volume and the on-demand ejection of bead-encapsulated droplets. Single bead–encapsulated droplets were ejected every 3 s without any failure. Multiple-bead dispensing with deterministic control of the number of beads was demonstrated to emphasize the originality and quality of the proposed dispensing technique. The dispenser was mounted using a plug-socket type connection, and the dispensing process was completely automated using a programmed sequence without any microscopic observation. To demonstrate a potential application of the technique, bead-based streptavidin–biotin binding assay in an evaporating droplet was conducted using ultralow numbers of beads. The results evidenced the number of beads in the droplet crucially influences the reliability of the assay. Therefore, the proposed deterministic bead-in-droplet technology can be utilized to deliver desired beads onto a reaction site, particularly to reliably and efficiently enrich and detect target biomolecules.

No MeSH data available.


Related in: MedlinePlus

Bead-based streptavidin–biotin binding test.(a) Schematic of the collision and binding phenomena between streptavidin-coated beads and biotin molecules depending on the number of beads; (b) representative fluorescence images with (i) single, (ii) 12.4 ± 3.6, and (iii) 79.4 ± 33.0 beads in a dispensed droplet; (c) MFI of single beads at different biotin molecule concentrations after reactions with different numbers of streptavidin beads (square, upper triangle, and lower triangle indicate single, 12.4 ± 3.6, and 79.4 ± 33.0 beads, respectively). All scale bars are 50 μm.
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f6: Bead-based streptavidin–biotin binding test.(a) Schematic of the collision and binding phenomena between streptavidin-coated beads and biotin molecules depending on the number of beads; (b) representative fluorescence images with (i) single, (ii) 12.4 ± 3.6, and (iii) 79.4 ± 33.0 beads in a dispensed droplet; (c) MFI of single beads at different biotin molecule concentrations after reactions with different numbers of streptavidin beads (square, upper triangle, and lower triangle indicate single, 12.4 ± 3.6, and 79.4 ± 33.0 beads, respectively). All scale bars are 50 μm.

Mentions: As a practical application of our bead dispensing system, affinity-based capturing and enrichment of biotin molecules was demonstrated using streptavidin-coated beads in an evaporating droplet. During droplet evaporation, collision and binding between biotin molecules and streptavidin beads occur effectively through diffusion and internal flow28. Here, we discuss the effect of the number of streptavidin beads in an evaporating droplet on the detection limit and sensitivity. We assumed that collision of a streptavidin bead against biotin molecules would be more frequent with decrease in the total number of streptavidin beads loaded in a droplet; this led to strong enrichment of the biotin molecules (Fig. 6a). Fluorescence images of beads in pipetted PBS solution were captured after the interaction between the Cy3-labeled biotin solution and the streptavidin bead in an evaporating droplet (Fig. 6b). Complete evaporation of 200 nL of biotin solution required approximately 10 min under ambient conditions (temperature 25 °C and humidity 40%). Figure 6c shows the mean florescent intensity (MFI) per bead at different bead numbers. At 128 nM, the capturing performance (i.e., MFI) was reasonably similar. MFIs are in saturation for beads in a dispensed droplet because an adequate number of biotin molecules are spread around each bead. However, at concentrations less than 128 nM, a decline in the capturing performance per bead was observed, because the predispensed number of beads increases even under the same concentration, which validates the foregoing assumption. We anticipate that this feature would help enhance the detection limit and sensitivity in bead-based assays, such as the Luminex technology, based on single-bead detection29. In addition, highly reliable results are expected through precisely controlled bead numbers and repeatable experiments.


Deterministic bead-in-droplet ejection utilizing an integrated plug-in bead dispenser for single bead – based applications
Bead-based streptavidin–biotin binding test.(a) Schematic of the collision and binding phenomena between streptavidin-coated beads and biotin molecules depending on the number of beads; (b) representative fluorescence images with (i) single, (ii) 12.4 ± 3.6, and (iii) 79.4 ± 33.0 beads in a dispensed droplet; (c) MFI of single beads at different biotin molecule concentrations after reactions with different numbers of streptavidin beads (square, upper triangle, and lower triangle indicate single, 12.4 ± 3.6, and 79.4 ± 33.0 beads, respectively). All scale bars are 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5385560&req=5

f6: Bead-based streptavidin–biotin binding test.(a) Schematic of the collision and binding phenomena between streptavidin-coated beads and biotin molecules depending on the number of beads; (b) representative fluorescence images with (i) single, (ii) 12.4 ± 3.6, and (iii) 79.4 ± 33.0 beads in a dispensed droplet; (c) MFI of single beads at different biotin molecule concentrations after reactions with different numbers of streptavidin beads (square, upper triangle, and lower triangle indicate single, 12.4 ± 3.6, and 79.4 ± 33.0 beads, respectively). All scale bars are 50 μm.
Mentions: As a practical application of our bead dispensing system, affinity-based capturing and enrichment of biotin molecules was demonstrated using streptavidin-coated beads in an evaporating droplet. During droplet evaporation, collision and binding between biotin molecules and streptavidin beads occur effectively through diffusion and internal flow28. Here, we discuss the effect of the number of streptavidin beads in an evaporating droplet on the detection limit and sensitivity. We assumed that collision of a streptavidin bead against biotin molecules would be more frequent with decrease in the total number of streptavidin beads loaded in a droplet; this led to strong enrichment of the biotin molecules (Fig. 6a). Fluorescence images of beads in pipetted PBS solution were captured after the interaction between the Cy3-labeled biotin solution and the streptavidin bead in an evaporating droplet (Fig. 6b). Complete evaporation of 200 nL of biotin solution required approximately 10 min under ambient conditions (temperature 25 °C and humidity 40%). Figure 6c shows the mean florescent intensity (MFI) per bead at different bead numbers. At 128 nM, the capturing performance (i.e., MFI) was reasonably similar. MFIs are in saturation for beads in a dispensed droplet because an adequate number of biotin molecules are spread around each bead. However, at concentrations less than 128 nM, a decline in the capturing performance per bead was observed, because the predispensed number of beads increases even under the same concentration, which validates the foregoing assumption. We anticipate that this feature would help enhance the detection limit and sensitivity in bead-based assays, such as the Luminex technology, based on single-bead detection29. In addition, highly reliable results are expected through precisely controlled bead numbers and repeatable experiments.

View Article: PubMed Central - PubMed

ABSTRACT

This paper presents a deterministic bead-in-droplet ejection (BIDE) technique that regulates the precise distribution of microbeads in an ejected droplet. The deterministic BIDE was realized through the effective integration of a microfluidic single-particle handling technique with a liquid dispensing system. The integrated bead dispenser facilitates the transfer of the desired number of beads into a dispensing volume and the on-demand ejection of bead-encapsulated droplets. Single bead–encapsulated droplets were ejected every 3 s without any failure. Multiple-bead dispensing with deterministic control of the number of beads was demonstrated to emphasize the originality and quality of the proposed dispensing technique. The dispenser was mounted using a plug-socket type connection, and the dispensing process was completely automated using a programmed sequence without any microscopic observation. To demonstrate a potential application of the technique, bead-based streptavidin–biotin binding assay in an evaporating droplet was conducted using ultralow numbers of beads. The results evidenced the number of beads in the droplet crucially influences the reliability of the assay. Therefore, the proposed deterministic bead-in-droplet technology can be utilized to deliver desired beads onto a reaction site, particularly to reliably and efficiently enrich and detect target biomolecules.

No MeSH data available.


Related in: MedlinePlus