Lab-on-Microsphere-FRET-Based Multiplex Sensor Platform

Abstract

Here we report on the development and investigation of a novel multiplex assay model based on polymer microspheres (PMS) encoded with ternary AIS/ZnS quantum dots (QDs). The system was prepared via layer-by-layer deposition technique. Our studies of Förster resonance energy transfer (FRET) between the QD-encoded microspheres and two different cyanine dyes have demonstrated that the QD photoluminescence (PL) quenching steadily increases with a decrease in the QD-dye distance. We have found that the sensitized dye PL intensity demonstrates a clear maximum at two double layers of polyelectrolytes between QDs and Dye molecules on the polymer microspheres. Time resolved PL measurements have shown that the PL lifetime decreases for the QDs and increases for the dyes due to FRET. The designed system makes it possible to record spectrally different bands of FRET-induced dye luminescence with different decay times and thereby allows for the multiplexing by wavelength and photoluminescence lifetimes of the dyes. We believe that PMS encoded with AIS/ZnS QDs have great potential for the development of new highly selective and sensitive sensor systems for multiplex analysis to detect cell lysates and body fluids' representative biomarkers.

Keywords: AgInS2; FRET; cyanine dyes; microspheres; sensing; ternary quantum dots; time-resolved fluorescence spectroscopy.

Figure 1

Absorption (dotted lines) and PL (solid lines) spectra of aqueous solutions of AgInS₂/ZnS quantum dots (QDs) (green) and the cyanine dyes: Cy3 (purple) and Cy5 (red). PL excitation wavelengths: QDs—405 nm, Cy3—500 nm, Cy5—600 nm.

Figure 2

Confocal image of polystyrene microspheres with AIS QDs (PMS-AIS) and PMS-AIS-Cy5. PL (a) and transmission electron microscopy (TEM) (b) images of PMS-AIS; PL excitation at 405 nm, detection at 470–600 nm region. (c,e) correspond to the PL images of PMS-AIS-Cy5 excited by 405 nm radiation with PL detection at 470–600 nm and 650–750 nm, respectively and their superposition (f). (d) TEM images of PMS-AIS-Cy5.

Figure 3

Extinction spectra of MS, PMS-AIS and PMS-AIS-Cy5. Inset: Cy5 absorption is revealed in PMS-AIS-Cy5 after the subtraction of the PMS-AIS background.

Figure 4

(a) Photoluminescence (PL) spectra of PMS-AIS, PMS-AIS-Cy3 L2 and deconvolution of PMS-AIS-Cy3 L2 by AIS and Cy3 PL band. (c) Dependence of QD PL and Cy3 FRET-induced PL on number of polymer layers L. (b) PL spectra of PMS-AIS, PMS-AIS-Cy5 L2 and deconvolution of PMS-AIS-Cy5 L2 by AIS and Cy5 PL band. (d) Dependence of QD PL and Cy5 FRET-induced PL on number of polymer layers L.

Figure 4

(a) Photoluminescence (PL) spectra of PMS-AIS, PMS-AIS-Cy3 L2 and deconvolution of PMS-AIS-Cy3 L2 by AIS and Cy3 PL band. (c) Dependence of QD PL and Cy3 FRET-induced PL on number of polymer layers L. (b) PL spectra of PMS-AIS, PMS-AIS-Cy5 L2 and deconvolution of PMS-AIS-Cy5 L2 by AIS and Cy5 PL band. (d) Dependence of QD PL and Cy5 FRET-induced PL on number of polymer layers L.

Figure 5

PL spectra of (a) PMS-AIS-Cy3 and (b) PMS-AIS-Cy5 with 1-5 double layers of polyelectrolytes. Excitation wavelength is 520 and 600 nm, respectively.

Figure 6

PL decay curves of (a) PMS-AIS (green) and PMS-AIS-Cy3 L2 (red) registered at the 580 nm and (b) PMS-AIS (blue) and PMS-AIS-Cy5 L2 (red) recorded at the 665 nm.

Figure 7

Fluorescence lifetime images of the microspheres doped by QDs and QDs with Cy3 or Cy5 dyes measured at maxima of QD PL and the FRET-induced dyes PL. (a) PMS-AIS (${\lambda }_{PL}$ = 665 nm, $<\tau >$ = 245 ns), (b) PMS-AIS-Cy5 L2 (${\lambda }_{PL}$ = 665 nm, $<\tau >$ = 160 ns) and (c) PMS-AIS-Cy3 L2 (${\lambda }_{PL}$ = 580 nm, $<\tau >$ = 80 ns). PL wavelengths (λ_PL) and corresponding average PL decay times <τ> are shown. The color scales of PL lifetimes are shown.