Supplementary Figure 1. The normalized absorption and emission spectra of 605QD

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1 Q Absorbance 65Q Emission Cy5 Absorbance Cy5 Emission 1..8 Extiction Absorption Coefficient Wavelength (nm) Fluorescence Emission Intensity Supplementary Figure 1. The normalized absorption and emission spectra of 65Q and Cy5. The selection of 65Q/Cy5 as a FRET pair permits only minimal spectral cross-talk. The broad absorption spectrum of the Q allows excitation at 488 nm, which is near the minimum of the absorption spectrum of Cy5, thereby nearly eliminating direct acceptor excitation.

2 2R R 5-75Å Q 69.4 Å NA Å Streptavidin Biotin Supplementary Figure 2. A schematic depicting the Förster distance (R ) of an idealized streptavidin-functionalized 65Q donor and a Cy5 acceptor. The radius of this streptavidin-functionalized Q 1 is 5-75 Å. The Förster distance R (69.4 Å) is calculated according to the Förster formalism 2 as R 2 9(ln1) κ pq = I N A π n 1/ 6, where Q is the quantum yield of the 65Q donor (~.6) 3 and 2 κ p is the orientation factor (2/3 for randomly oriented dipoles); N A is the Avogadro s number and n is the refractive index of the medium (~1.4 for biomolecules in aqueous solution 4 ). I is the normalized overlap 4 integral defined as I = PL corrε A ( λ) λ d λ, where PL corr is the normalized donor emission spectrum and ε A (λ) is the acceptor absorption spectrum (expressed as an extinction coefficient, ε A (647) = 25, cm -1 M -1 for Cy5). The average Q-Cy5 separation distance (117.1±3.7 Å) is estimated according to the formalism 2 as r n = R n(1 E) E 1/ 6, where n is the average number of acceptor molecules interacting with one donor and E is the FRET efficiency. The FRET efficiency can be

3 FA τ A experimentally obtained from E = 1 = 1, where F A is the integrated F τ fluorescence intensity of donor in the presence of the acceptors and F is the integrated fluorescence intensity of donor alone in absence of the acceptors; τ A is the lifetime of donor in the presence of the acceptors and τ is the lifetime of the donor in the absence of the acceptors 2. References [1] Qdot TM streptavidin conjugates user manual, PN 9-3 Rev 5, Quantum ot Corp., Hayward, CA. [2] Clapp, A. R. et al. Fluorescence resonance energy transfer between quantum dot donors and dye-labeled protein acceptors. J. Am. Chem. Soc. 126, (24). [3] Technical supporting information from Quantum ot Corporation, Hayward, CA. [4] Lakowicz, J. R. Principles of fluorescence spectroscopy (Kluwer Academic/Plenum, New York, 1999).

4 12 PL (AU) R= R=12 R=24 R=54 Control Wavelength (nm) Supplementary Figure 3. Photoluminescence was measured at each acceptor/donor ratio (R) as shown, using a USB2 spectrometer from Ocean Optics Inc. (unedin, FL). The 65Q concentration was fixed at 1nM, while the arrows indicate the increasing ratio R. The control is the direct excitation of Cy5 alone (at 1nM) with 488 nm argon laser.

5 a Biotin Capture probe Reporter probe Cy5 Perfect match target One-base mismatch target Taq NA Ligase Nick repaired b Excitation enaturing & Capturing ligation products by Qs c Q Q FRET No FRET Supplementary Figure 4. Procedure for OLA-based point mutation detection with Q nanosensors. a, Left: A Cy5-labeled discrimination reporter probe and a biotinylated common capture probe are sandwiched by a perfect match target. Right: These two probes are sandwiched to a one-base mismatch target. b, Left: Taq NA ligase repairs the nick between two probes. Right: Ligation does not take place. c, After the targetprobe hybrids are denatured by heat, streptavidin-functionalized Q donors are added to capture the ligation products. Left: Cy5 acceptors are linked to a Q through ligation products. Upon illumination on the Q, emission from Cy5 acceptors occurs. Right: Cy5 acceptors are not associated to a Q due to the absence of ligation products.

6 R= Number of Events R=12 R=24 R=54 FRET Factor Supplementary Figure 5. The FRET histograms using 525Q/Cy5 as the FRET donor/acceptor pair at different acceptor/donor ratios (R) as shown. In these experiments, 525Q (catalog number #1-4, also having 15~25 Streptavidins/Q and overall size of 1~15 nm in diameter) was used as a control. Compared to Fig. 3a, where 65Q/Cy5 was used as the FRET pair, FRET histograms above showed no sign of Cy5 FRET signals even with an increase in R. This was due to the greatly reduced overlap of 525Q emission and Cy5 absorption spectra. In addition, no Cy5 signals were seen even at R=54, suggesting the direct excitation of multiple Cy5 dyes on Q to be minimal in all of our experiments.